Physiology of the Gastrointestinal Tract, Volume 1-2, Fourth Edition

PHYSIOLOGY OF THE GASTROINTESTINAL TRACT FOURTH EDITION

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Physiology of the

Gastrointestinal Tract FOURTH EDITION

Volume 1 Editor-in-Chief Leonard R. Johnson Department of Physiology University of Tennessee College of Medicine Memphis, Tennessee

Associate Editors

Kim E. Barrett Department of Medicine University of California, San Diego, School of Medicine University of California San Diego Medical Center San Diego, California

Fayez K. Ghishan Department of Pediatrics Steele Children’s Research Center University of Arizona Health Sciences Center Tucson, Arizona

Juanita L. Merchant Departments of Internal Medicine and Molecular and Integrative Physiology University of Michigan Ann Arbor, Michigan

Hamid M. Said Departments of Medicine and Physiology/Biophysics University of California Irvine, California, and VA Medical Center-151 Long Beach, California

Jackie D. Wood Departments of Physiology and Biology and Internal Medicine The Ohio State University College of Medicine Columbus, Ohio

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Library of Congress Cataloging-in-Publication Data Physiology of the gastrointestinal tract / editor-in-chief, Leonard R. Johnson ; associate editors, Kim Barrett ... [et al.].-- 4th ed. p. ; cm. Includes bibliographical references and index. ISBN 0-12-088394-5 (set : alk. paper) -- ISBN 0-12-088395-3 (v.1 : alk. paper) ISBN 0-12-088396-1 (v.2 : alk. paper) 1. Gastrointestinal system--Physiology. I. Johnson, Leonard R., 1942[DNLM: 1. Gastrointestinal Tract--physiology. WI 102 P578 2006] QP145.P492 2006 612.3′2--dc22

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library

Volume 1: ISBN 13: 978-0-120883950 • ISBN 10: 0-12-088395-3 Volume 2: ISBN 13: 978-0-120883967 • ISBN 10: 0-12-088396-1 Two Volume Set: ISBN 13: 978-0-120883943 • ISBN 10: 0-12-088394-5

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Contents Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Preface to the First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii VOLUME 1

Section I. Basic Cell Physiology and Growth of the GI Tract 1. Transcriptional and Epigenetic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Juanita L. Merchant and Longchuan Bai

1

2. Translation and Posttranslational Processing of Gastrointestinal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cheryl E. Gariepy and Chris J. Dickinson

31

3. Transmembrane Signaling by G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claire Jacob and Nigel W. Bunnett

63

4. Gastrointestinal Hormones: Gastrin, Cholecystokinin, Somatostatin, and Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . Graham J. Dockray

91

5. Postpyloric Gastrointestinal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Ella W. Englander and George H. Greeley Jr. 6. Gastrointestinal Peptide Hormones Regulating Energy and Glucose Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Daniel J. Drucker 7. Growth Factors in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 John A. Barnard and Kirk M. McHugh 8. Developmental Signaling Networks Wnt/β-Catenin Signaling in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . 247 Guido T. Bommer and Eric R. Fearon 9. Hedgehog Signaling in Gastrointestinal Morphogenesis and Morphostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Gijs R. van den Brink, Maikel P. Peppelenbosch, and Drucilla J. Roberts 10. Developmental Signaling Networks: The Notch Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Guy R. Sander, Hanna Krysinska, and Barry C. Powell 11. Physiology of Gastrointestinal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Alda Vidrich, Jenny M. Buzan, Sarah A. De La Rue, and Steven M. Cohn 12. Apoptosis in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Leonard R. Johnson 13. Molecular Aspects and Regulation of Gastrointestinal Function during Postnatal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 James F. Collins, Liqun Bai, Hua Xu, and Fayez K. Ghishan 14. Effect of Aging on the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Adhip P. N. Majumdar and Marc D. Basson 15. Regulation of Gastrointestinal Normal Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Mark R. Hellmich and B. Mark Evers

v

vi / CONTENTS 16. Mucosal Repair and Restitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Mark R. Frey and D. Brent Polk 17. Mechanisms of Gastrointestinal Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 John Lynch and Anil K. Rustgi

Section II. Neural Gastroenterology and Motility 18. Development of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Michael D. Gershon and Elyanne M. Ratcliffe 19. Cellular Physiology of Gastrointestinal Smooth Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523 Gabriel M. Makhlouf and Karnam S. Murthy 20. Organization and Electrophysiology of Interstitial Cells of Cajal and Smooth Muscle Cells in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 Kenton M. Sanders, Sang Don Koh, and Sean M. Ward 21. Functional Histoanatomy of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Simon J. H. Brookes and Marcello Costa 22. Physiology of Prevertebral Sympathetic Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Joseph H. Szurszewski and Steven M. Miller 23. Cellular Neurophysiology of Enteric Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629 Jackie D. Wood 24. Integrative Functions of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 Jackie D. Wood 25. Extrinsic Sensory Afferent Nerves Innervating the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 Michael J. Beyak, David C. E. Bulmer, Wen Jiang, C. Keating, Weifang Rong, and David Grundy 26. Processing of Gastrointestinal Sensory Signals in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727 Anthony Hobson and Qasim Aziz 27. Enteric Neural Regulation of Mucosal Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Helen Joan Cooke and Fedias Leontiou Christofi 28. Effect of Stress on Intestinal Mucosal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763 Johan D. Söderholm and Mary H. Perdue 29. Effect of Stress on Gastrointestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Michèle Gué 30. Central Corticotropin-Releasing Factor and the Hypothalamic-Pituitary-Adrenal Axis in Gastrointestinal Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791 Yvette Taché and Mulugeta Million 31. Neural Regulation of Gastrointestinal Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817 Peter Holzer 32. Neural Control of the Gallbladder and Sphincter of Oddi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841 Gary M. Mawe, Gino T. P. Saccone, and María J. Pozo 33. Brainstem Control of Gastric Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851 Richard C. Rogers, Gerlinda E. Hermann, and R. Alberto Travagli 34. Neural and Hormonal Controls of Food Intake and Satiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877 Timothy H. Moran

CONTENTS / vii 35. Pharyngeal Motor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 Reza Shaker 36. Motor Function of the Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913 Ray E. Clouse and Nicholas E. Diamant 37. Neurophysiologic Mechanisms of Gastric Reservoir Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jan Tack

927

38. Small Intestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William L. Hasler

935

39. Function and Regulation of Colonic Contractions in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sushil K. Sarna and Xuan-Zheng Shi

965

40. Neural Control of Pelvic Floor Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David B. Vodusek and Paul Enck

995

41. Pathophysiology Underlying the Irritable Bowel Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009 Jackie D. Wood VOLUME 2

Section III. Gastrointestinal Immunology and Inflammation 42. Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033 Lars Eckmann 43. Biology of Gut Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067 Finn-Eirik Johansen, Elizabeth H. Yen, Bonny Dickinson, Masaru Yoshida, Steve Claypool, Richard S. Blumberg, and Wayne I. Lencer 44. Mechanisms of Helicobacter pylori–Induced Gastric Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091 Dawn. A. Israel and Richard M. Peek Jr. 45. Mechanisms and Consequences of Intestinal Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1115 Wallace K. MacNaughton 46. Recruitment of Inflammatory and Immune Cells in the Gut: Physiology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137 D. Neil Granger, Christopher G. Kevil, and Matthew B. Grisham 47. Physiology of Host–Pathogen Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163 Kim Hodges, V. K. Viswanathan, and Gail Hecht

Section IV. Physiology of Secretion 48. The Cell Biology of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189 Curtis Okamoto, Serhan Karvar, and John G. Forte 49. Regulation of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223 Arthur Shulkes, Graham S. Baldwin, and Andrew S. Giraud 50. Gastroduodenal Mucosal Defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259 Marshall H. Montrose, Yasutada Akiba, Koji Takeuchi, and Jonathan D. Kaunitz 51. Genetically Engineered Mouse Models of Gastric Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293 Linda C. Samuelson 52. Structure–Function Relations in the Pancreatic Acinar Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313 Fred S. Gorelick and James D. Jamieson

viii / CONTENTS 53. Stimulus-Secretion Coupling in Pancreatic Acinar Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1337 John A. Williams and David I. Yule 54. Cell Physiology of Pancreatic Ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1371 Barry E. Argent, Michael A. Gray, Martin C. Steward, and R. Maynard Case 55. Regulation of Pancreatic Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1397 Rodger A. Liddle 56. Bile Formation and the Enterohepatic Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1437 Paul A. Dawson, Benjamin L. Shneider, and Alan F. Hofmann 57. Mechanisms of Hepatocyte Organic Anion Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463 Allan W. Wolkoff 58. Mechanisms of Hepatocyte Detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483 Karen F. Murray, Donald J. Messner, and Kris V. Kowdley 59. Physiology of Cholangiocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505 Anatoliy I. Masyuk, Tatyana V. Masyuk, and Nicholas F. LaRusso 60. Gallbladder Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1535 Sum P. Lee and Rahul Kuver

Section V. Digestion and Absorption 61. Tight Junctions and the Intestinal Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559 Thomas Y. Ma and James M. Anderson 62. Protein Sorting in the Exocytic and Endocytic Pathways in Polarized Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . 1595 V. Stephen Hunt and W. James Nelson 63. Physiology of the Circulation of the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627 Philip T. Nowicki 64. Sugar Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 Ernest M. Wright, Donald D. F. Loo, Bruce A. Hirayama, and Eric Turk 65. Protein Digestion and Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667 Vadivel Ganapathy, Naren Gupta, and Robert G. Martindale 66. Role of Membrane and Cytosolic Fatty Acid Binding Proteins in Lipid Processing by the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693 Nada Abumrad and Judith Storch 67. Genetic Regulation of Intestinal Lipid Transport and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711 Zhouji Chen and Nicholas O. Davidson 68. Digestion and Intestinal Absorption of Dietary Carotenoids and Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1735 Alexandrine During and Earl H. Harrison 69. Vitamin D3: Synthesis, Actions, and Mechanisms in the Intestine and Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1753 J. Wesley Pike, Makoto Watanuki, and Nirupama K. Shevde 70. Vitamin E and Vitamin K Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1773 Ronald J. Sokol, Richard S. Bruno, and Maret G. Traber 71. Intestinal Absorption of Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1791 Hamid M. Said and Bellur Seetharam 72. Water Transport in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827 Jay R. Thiagarajah and A. S. Verkman

CONTENTS / ix 73. Na+-H+ Exchange in Mammalian Digestive Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1847 Pawel R. Kiela and Fayez K. Ghishan 74. Intestinal Anion Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1881 Pradeep K. Dudeja and K. Ramaswamy 75. Ion Channels of the Epithelia of the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917 John Cuppoletti and Danuta H. Malinowska 76. Integrative Physiology and Pathophysiology of Intestinal Electrolyte Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . 1931 Kim E. Barrett and Stephen J. Keely 77. Molecular Mechanisms of Intestinal Transport of Calcium, Phosphate, and Magnesium . . . . . . . . . . . . . . . . . . . . 1953 James F. Collins and Fayez K. Ghishan 78. Iron Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1983 Nancy C. Andrews 79. Trace Element Absorption and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1993 Robert J. Cousins

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Contributors Nada Abumrad, Departments of Medicine and Nutritional Sciences, Washington University, Campus Box 8031, 660 South Euclid Avenue, Saint Louis, Missouri 63110

Marc D. Basson, John D. Dingell Veterans Affairs Medical Center, Department of Surgical Service, Wayne State University, Detroit, Michigan 48201 Michael J. Beyak, Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario, Canada, and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

Yasutada Akiba, Brentwood Biomedical Research Institute, Building 114, Suite 217, West Los Angeles VAMC, Los Angeles, California 90073 James M. Anderson, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 6312 MBRB, 103 Mason Farm Road, Chapel Hill, North Carolina 27599-7545

Richard S. Blumberg, Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Hepatology, and Endoscopy, Harvard Digestive Diseases Center, Laboratory of Mucosal Immunology, Thorn 1419, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115

Nancy C. Andrews, Department of Basic Sciences and Graduate Studies, Harvard Medical School, and Department of Pediatrics, Children’s Hospital, Howard Hughes Medical Institute, Gordon Hall, 25 Shattuck Street, Boston, Massachusetts 02115

Guido T. Bommer, Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan School of Medicine, LSI 5-183A, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216

Barry E. Argent, Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom

Simon J. H. Brookes, Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042

Qasim Aziz, University of Manchester, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom

Richard S. Bruno, Department of Nutritional Sciences, University of Connecticut, 3624 Horsebarn Road Ext, Unit 4017, Storrs, Connecticut 06269-4017

Liqun Bai, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073

David C. E. Bulmer, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

Longchuan Bai, Department of Internal Medicine, University of Michigan, 1150 West Medical Center Drive, 3510 MSRB I, Ann Arbor, Michigan 48109

Nigel W. Bunnett, Departments of Surgery and Physiology, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, California 94143-0660

Graham S. Baldwin, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia

Jenny M. Buzan, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708

Kim E. Barrett, Department of Medicine, University of California, San Diego, School of Medicine, University of California San Diego Medical Center 8414, 200 West Arbor Drive, San Diego, California 92103-8414

R. Maynard Case, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Zhouji Chen, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, Campus Box 8046, 660 South Euclid Avenue, Saint Louis, Missouri 63110

John A. Barnard, Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, 700 Children’s Drive WA2011, Columbus, Ohio 43205

xi

xii / CONTRIBUTORS Fedias Leontiou Christofi, Departments of Anesthesiology and Physiology and Cell Biology, The Ohio State University, 226 Tzagounis Medical Research Facility, Columbus, Ohio 43210

Bonny Dickinson, Department of Pediatrics, Research Institute for Children, Children’s Hospital, Research and Education Building Room 2231, 200 Henry Clay Avenue, New Orleans, Louisiana 70118

Steve Claypool, Laboratory of Mucosal Immunology, Thorn 1419, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115

Chris J. Dickinson, Division of Pediatric Gastroenterology, University of Michigan, D3252 MPB, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718

Ray E. Clouse, Departments of Medicine and Psychiatry, Washington University, 660 South Euclid Avenue, Campus Box 8124, Saint Louis, Missouri 63110

Graham J. Dockray, Department of Physiology, School of Biomedical Sciences, Crown Street, University of Liverpool, PO Box 147, Liverpool, L69 3BX United Kingdom

Steven M. Cohn, Digestive Health Center of Excellence, University of Virginia, 2 Jefferson Park Ave, Room 2091, Charlottesville, Virginia 22908-0708 James F. Collins, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Helen Joan Cooke, Department of Neuroscience, The Ohio State University, 4066D Graves Hall, 333 West Tenth Avenue, Columbus, Ohio 43210 Marcello Costa, Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042 Robert J. Cousins, Boston Family Professor of Nutrition, Center for Nutritional Sciences, University of Florida, PO Box 110370, Gainesville, Florida 32611-0370 John Cuppoletti, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way ML 0576, Cincinnati, Ohio 45267-0576 Nicholas O. Davidson, Division of Gastroenterology, Washington University School of Medicine, Campus Box 8124, 660 South Euclid Avenue, Saint Louis, Missouri 63110 Paul A. Dawson, Department of Internal Medicine, Section of Gastroenterology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157 Sarah A. De La Rue, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708 Nicholas E. Diamant, Departments of Medicine and Physiology, University of Toronto, 6B Fell 6-176, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8

Daniel J. Drucker, Banting and Best Diabetes Centre, Toronto General Hospital, University of Toronto, 200 Elizabeth Street MBRW 4R-902, Toronto, Ontario, Canada M5G 2C4 Pradeep K. Dudeja, Department of Medicine, University of Illinois at Chicago College of Medicine, Research and Development, Jesse Brown VAMC, MP151, 820 South Damen Avenue, Chicago, Illinois 60612 Alexandrine During, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 Lars Eckmann, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0665 Paul Enck, Department of Internal Medicine VI, University Hospitals Tuebingen, Osianderstr. 5, 72076 Tuebingen, Germany Ella W. Englander, Department of Surgery, The University of Texas Medical Branch, 815 Market Street, Galveston, Texas 77550 B. Mark Evers, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536 Eric R. Fearon, Division of Molecular Medicine and Genetics, Departments of Internal Medicine, Human Genetics, and Pathology, University of Michigan School of Medicine, LSI 5-183A, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216 John G. Forte, Department of Molecular and Cell Biology, University of California, 241 LSA, MC 3200, Berkeley, California 94720 Mark R. Frey, Department of Pediatric Gastroenterology, Hepatology, and Nutrition, S4322 MCN Vanderbilt University, Nashville, Tennessee 37232-2576

CONTRIBUTORS / xiii Vadivel Ganapathy, Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1459 Laney-Walker Boulevard, Augusta, Georgia 30912-2100 Cheryl E. Gariepy, Division of Pediatric Gastroenterology, University of Michigan, D3252 MPB, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718 Michael D. Gershon, Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, 630 West 168 Street, New York, New York 10032 Fayez K. Ghishan, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Andrew S. Giraud, Department of Medicine, University of Melbourne, Western Hospital, Footscray 3011, Australia Fred S. Gorelick, Department of Medicine, VA Healthcare CT, and Yale University School of Medicine, 950 Campbell Avenue, West Haven, Connecticut 06516 D. Neil Granger, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 Michael A. Gray, Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom George H. Greeley Jr., Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0725 Matthew B. Grisham, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 David Grundy, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Michèle Gué, Department of Physiology, Université Paul Sabatier, IFR31, Institut Louis Bugnard, BP 84225, INSERM U388, Laboratoire de Pharmacologie Moléculaire et Physiopathologie Rénale, 31432 Toulouse, Cedex 4, France Naren Gupta, Department of Surgery, University of Virginia Health System, Charlottesville, Virginia 22908

Earl H. Harrison, Phytonutrients Laboratory, United States Department of Agriculture Human Nutrition Research Center, Building 307 C, Room 118 BARC-East, Beltsville, Maryland 20705 William L. Hasler, Department of Internal Medicine, Division of Gastroenterology, University of Michigan Health System, 3912, Taubman Center, Box 0362, Ann Arbor, Michigan 48109 Gail Hecht, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 Mark R. Hellmich, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536 Gerlinda E. Hermann, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808 Bruce A. Hirayama, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 Anthony Hobson, University of Manchester, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom Kim Hodges, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 Alan F. Hofmann, Department of Medicine MC 0813, Division of Gastroenterology, University of California, San Diego, La Jolla, California 92093-0813 Peter Holzer, Department of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, A-8010 Graz, Austria V. Stephen Hunt, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5435 Dawn A. Israel, Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Room 1012A MRB IV, 2215 Garland Avenue, Nashville, Tennessee 37232 Claire Jacob, Departments of Surgery and Physiology, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, California 94143-0660

xiv / CONTRIBUTORS James D. Jamieson, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Wen Jiang, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Finn-Eirik Johansen, Institute of Pathology, University of Oslo, Department of Pathology, Rikshospitalet University Hospital, Sognsvannsveien 20, N-0027 Oslo, Norway Leonard R. Johnson, Department of Physiology, University of Tennessee College of Medicine, 894 Union Avenue, Memphis, Tennessee 38163 Serhan Karvar, Department of Molecular and Cell Biology, University of California, 245 LSA, MC 3200, Berkeley, California 94720 Jonathan D. Kaunitz, Department of Medicine, Division of Digestive Diseases, West Los Angeles VAMC, and UCLA School of Medicine, Building 114, Suite 217, Los Angeles, California 90073 C. Keating, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Stephen J. Keely, Department of Medicine, University of California, San Diego, School of Medicine, University of California San Diego Medical Center 8414, 200 West Arbor Drive, San Diego, California 92103-8414 Christopher G. Kevil, Department of Pathology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 Pawel R. Kiela, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Sang Don Koh, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Kris V. Kowdley, University of Washington, Box 356174, 1959 NE Pacific Street, Seattle, Washington 98195 Hanna Krysinska, Child Health Research Institute, North Adelaide, 72 King William Road, South Australia 5006, Australia Rahul Kuver, Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Box 356424, 1959 NE Pacific Street, Seattle, Washington 98195

Nicholas F. LaRusso, Departments of Medicine, Biochemistry, and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Sum P. Lee, Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Box 356424, 1959 NE Pacific Street, Seattle, Washington 98195 Wayne I. Lencer, Department of Pediatrics, Harvard Medical School, and Division of Pediatric Gastroenterology and Nutrition, Harvard Digestive Diseases Center, Gastrointestinal Cell Biology Laboratories, Enders 720, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115 Rodger A. Liddle, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 Donald D. F. Loo, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 John Lynch, Division of Gastroenterology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 600 Clinical Research Building, Philadelphia, Pennsylvania 19104 Thomas Y. Ma, Division of Gastroenterology and Hepatology, Departments of Medicine, Cell Biology and Physiology, Inflammatory Bowel Disease Program, University of New Mexico, MSC10 5550, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001 Wallace K. MacNaughton, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada Adhip P. N. Majumdar, John D. Dingell Veterans Affairs Medical Center, Department of Medicine, Karmanos Cancer Center, Wayne State University, Research Service, Room-B-4238, 4646 John R, Detroit, Michigan 48201 Gabriel M. Makhlouf, Department of Medicine, Virginia Commonwealth University Medical Center, Sanger Hall, Room 12-003, 1101 East Marshall Street, Richmond, Virginia 23298 Danuta H. Malinowska, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576

CONTRIBUTORS / xv Robert G. Martindale, Department of Surgery, Oregon Health and Science University, Portland, Oregon 97239 Anatoliy I. Masyuk, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Tatyana V. Masyuk, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Gary M. Mawe, Department of Anatomy and Neurobiology, The University of Vermont, D403A Given Building, 89 Beaumont Avenue, Burlington, Vermont 05405

Karnam S. Murthy, Departments of Medicine and Physiology, Virginia Commonwealth University Medical Center, Sanger Hall, Room 12-003, 1101 East Marshall Street, Richmond, Virginia 23298 W. James Nelson, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5435 Philip T. Nowicki, Departments of Pediatrics and Physiology, Center for Cell and Vascular Biology, Columbus Children’s Research Institute, College of Medicine and Public Health, The Ohio State University, Children’s Hospital, 700 Children’s Drive, Columbus, Ohio 43205

Kirk M. McHugh, Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, 700 Children’s Drive WA2011, Columbus, Ohio 43205

Curtis Okamoto, Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033

Juanita L. Merchant, Departments of Internal Medicine and Molecular and Integrative Physiology, University of Michigan, 1150 West Medical Center Drive, 3510 MSRB I, Ann Arbor, Michigan 48109

Richard M. Peek Jr., Division of Gastroenterology, Departments of Medicine and Cancer Biology, Vanderbilt University Medical Center, Room 1012A MRB IV, 2215 Garland Avenue, Nashville, Tennessee 37232

Donald J. Messner, Bastyr University, 14500 Juanita Drive, NE, Kenmore, Washington 98028

Maikel P. Peppelenbosch, Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Steven M. Miller, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905

Mary H. Perdue, Intestinal Disease Research Program, HSC-3N5C, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N3Z5, Canada

Mulugeta Million, CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, CURE Building 115, Room 117, 11301 Wilshire Boulevard, Los Angeles, California 90073

J. Wesley Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706

Marshall H. Montrose, Department of Molecular and Cellular Physiology, University of Cincinnati, Medical Science Building, Room 4253, 231 Albert Sabin Way, Cincinnati, Ohio 45267

Barry C. Powell, Child Health Research Institute, North Adelaide, and School of Pharmacy and Medical Sciences, University of South Australia; Department of Pediatrics, University of Adelaide, 72 King William Road, South Australia 5006, Australia

Timothy H. Moran, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Avenue, Baltimore, Maryland 21205 Karen F. Murray, Children’s Hospital and Regional Medical Center, University of Washington School of Medicine, 4800 Sand Point Way, NE, PO Box 5371/A5950, Seattle, Washington 98105

D. Brent Polk, Departments of Cell and Developmental Biology and Pediatric Gastroenterology, Hepatology, and Nutrition, Digestive Disease Research Center, S4322 MCN Vanderbilt University, Nashville, Tennessee 37232-2576

María J. Pozo, Department of Physiology, Nursing School, University of Extremadura, Avda Universidad s/n, 10071 Cáceres, Spain K. Ramaswamy, Department of Medicine, University of Illinois at Chicago College of Medicine, 840 South Wood Street (m/c 716), Chicago, Illinois 60612

xvi / CONTRIBUTORS Elyanne M. Ratcliffe, Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Columbia University, 3959 Broadway, CHN 702, New York, New York 10032

Reza Shaker, Division of Gastroenterology and Hepatology, Digestive Disease Center, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, Wisconsin 53226

Drucilla J. Roberts, Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114

Nirupama K. Shevde, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706

Richard C. Rogers, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808

Xuan-Zheng Shi, Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, 9.138E Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1064

Weifang Rong, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Anil K. Rustgi, Division of Gastroenterology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 600 Clinical Research Building, Philadelphia, Pennsylvania 19104 Gino T. P. Saccone, Department of General and Digestive Surgery, Flinders Medical Centre, Flinders Drive, Bedford Park, South Australia 5042, Australia Hamid M. Said, Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California, and VA Medical Center-151, Long Beach, California 90822 Linda C. Samuelson, Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109-0622 Guy R. Sander, Child Health Research Institute, North Adelaide, and Department of Pediatrics, University of Adelaide, 72 King William Road, South Australia 5006, Australia Kenton M. Sanders, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Sushil K. Sarna, Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, 9.138C Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1064 Bellur Seetharam, Departments of Medicine and Biochemistry, Medical College of Wisconsin and Clement Zablocki VA Medical Center, Research 151, 5000 National Avenue, Milwaukee, Wisconsin 53295

Benjamin L. Shneider, Department of Pediatrics, Box 1656, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029 Arthur Shulkes, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia Johan D. Söderholm, Department of Biomedicine and Surgery, Linköping University Hospital, Linköping SE-581 85, Sweden Ronald J. Sokol, Department of Pediatrics, Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Pediatric General Clinical Research Center, University of Colorado School of Medicine, The Children’s Hospital, 1056 East 19th Avenue, Box B290, Denver, Colorado 80218 Martin C. Steward, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Judith Storch, Department of Nutritional Sciences, Rutgers University, 96 Lipman Drive, New Brunswick, New Jersey 08901-8525 Joseph H. Szurszewski, Department of Physiology and Biomedical Engineering, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905 Yvette Taché, CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, CURE Building 115, Room 117, 11301 Wilshire Boulevard, Los Angeles, California 90073

CONTRIBUTORS / xvii Jan Tack, Department of Gastroenterology, University Hospitals Leuven, Center for Gastroenterological Research, University of Leuven, Herestraat 49, 3000 Leuven, Belgium Koji Takeuchi, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan Jay R. Thiagarajah, Departments of Medicine and Physiology, University of California San Francisco, 1246 Health Sciences East Tower, San Francisco, California 94143-0521 Maret G. Traber, Department of Nutrition and Exercise Sciences, Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, Oregon 97331-6512 R. Alberto Travagli, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808 Eric Turk, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 Gijs R. van den Brink, Laboratory for Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands A. S. Verkman, Departments of Medicine and Physiology, University of California San Francisco, 1246 Health Sciences East Tower, San Francisco, California 94143-0521 Alda Vidrich, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708 V. K. Viswanathan, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 David B. Vodusek, Division of Neurology, University Medical Center, Ljubljana 1525, Slovenia Sean M. Ward, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

Makoto Watanuki, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706 John A. Williams, Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0522 Allan W. Wolkoff, Marion Bessin Liver Research Center and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 Jackie D. Wood, Departments of Physiology and Biology and Internal Medicine, The Ohio State University College of Medicine, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210-1218 Ernest M. Wright, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, 10833 Le Conte Avenue, 53-263 Center for Health Sciences, Los Angeles, California 90095-1751 Hua Xu, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Elizabeth H. Yen, Harvard Medical School Fellowship in Pediatric Gastroenterology and Nutrition, Gastrointestinal Cell Biology, Enders 720, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115 Masaru Yoshida, Frontier Medical Science in Gastroenterology, International Center for Medical Research and Treatment, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan David I. Yule, Departments of Pharmacology and Physiology, University of Rochester Medical School, Rochester, New York 14642

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Preface to the First Edition As with any publishing venture and especially one of this magnitude, one must first ask, “Why?” The Associate Editors and I were motivated primarily to collect in one set of volumes the most up-to-date and comprehensive knowledge in our field. Nothing comparable has been attempted in the area of gastrointestinal physiology during the past fourteen years. During this time, there has been a rapid expansion of knowledge and many new areas of investigation have been initiated. More than fifty leading scientists—physiologists, clinical specialists, morphologists, pharmacologists, immunologists, and biochemists—have contributed chapters on their various areas of expertise for these volumes. Our original goal was to review the entire field of gastrointestinal physiology in one work. After examining all of the chapters, however, it was apparent that the final product encompassed more than physiology. The chapters reflect the backgrounds of the authors and the approaches of their different disciplines. As such, these volumes contain information for not only the investigator working in these fields but for the clinician or graduate student interested in the function of the gastrointestinal tract. Anyone involved in teaching gastrointestinal physiology of pathophysiology can readily find the latest and most pertinent information on any area in the discipline. This work is divided into five sections. The first consists of topics such as growth, the enteric nervous system, and gastrointestinal peptides, each of which relates to all areas of the gastrointestinal tract. The second section contains material describing smooth muscle physiology and gastrointestinal motility. The third section presents treatment of the functions of the stomach and pancreas. The fourth series of chapters treats the entire field of digestion and absorption. These chapters vary from basic electrophysiology and membrane transport to reviews of mechanisms leading to clinical conditions of malabsorption. The final section contains chapters on areas peripheral to physiology (such as immunology, parasitology, and prostaglandins) yet necessary for a comprehensive understanding of the subject. No one person can presume to organize and edit a scientific work of this scope. I was fortunate to enlist the aid of four preeminent scientists whose expertises cover the entire field. James Christensen was primarily responsible for the chapters on smooth muscle and motility. Eugene D. Jacobson solicited and edited most of the chapters dealing with secretory mechanisms as well as those covering many of the general topics. Chapters relating to regulation were primarily handled by Morton I. Grossman, and those covering aspects of digestion and absorption were organized and reviewed by Stanley G. Schultz. I am exceedingly grateful to these four men without whom this work would not have been possible. L.R.J.

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Preface This fourth edition of Physiology of the Gastrointestinal Tract follows 12 years after the third edition. The delay was mainly due to buyouts and mergers of the involved publishing houses, certainly not to a lack of new information. On the contrary, the explosion of information at the cellular level, made possible, in part, by the continued emergence of powerful molecular and cellular techniques, has resulted in a greater degree of revision than that of any other edition. Section I, now titled “Basic Cell Physiology and Growth of the Gastrointestinal Tract,” contains numerous new chapters on topics such as transcriptional regulation, signaling networks in development, apoptosis, and mechanisms in malignancies. Most of the chapters in the first section have been edited by Juanita L. Merchant. Section II has been renamed “Neural Gastroenterology and Motility” and has been expanded from 7 chapters with rather classic titles to more than 20 chapters encompassing not only the movement of the various parts of the digestive tract but also cell physiology, neural regulation, stress, and the regulation of food intake. Almost all of the chapters in the second section have been recruited and edited by Jackie D. Wood. Section III is entirely new and contains chapters on “Gastrointestinal Immunology and Inflammation,” which were edited by Kim E. Barrett. Section IV, “Physiology of Secretion,” consists of chapters with familiar titles but with completely updated information to reflect the advances in our understanding of the cellular processes involved in secretion. Section V, “Digestion and Absorption,” contains new chapters on the intestinal barrier, protein sorting, and ion channels, together with those focusing on the uptake of specific nutrients. These chapters have been recruited and edited by Hamid M. Said and Fayez K. Ghishan. The original purpose of the first edition of this textbook—to collect in one set of volumes the most current and comprehensive knowledge in our field—was also the driving force for this edition. As mentioned earlier, this edition includes completely new chapters that cover many new areas. Although the number of chapters has increased by 15, some chapters from the previous edition have been eliminated, some with identical titles have been written by different authors, and a few have been updated by the original authors. The final product again encompasses more than physiology. The information provided is relevant not only to the researcher in the various specialized areas but also to the clinical gastroenterologist, the teacher, and the student. The authors have done an excellent job of presenting their knowledge in a style that is readable and understandable. Much of the effort in organizing and editing these volumes has come from five preeminent scientists whose interest and expertise cover the entire field. Drs. Barrett, Ghishan, Merchant, Said, and Wood met with me to decide on chapter topics, authors, and the overall organization of the material. They were responsible for recruiting authors and for the scientific editing of most chapters. The enthusiasm and abilities of these individuals simplified my task as editor, and without them this work would not have been possible. I also am especially grateful to Philip Carpenter of Elsevier, who contacted authors, tracked submissions, and assisted me in many ways. My Associate Editors and I are all grateful to the contributing authors who were generous enough to make their expert knowledge available. Their efforts have made this work more than a mere review of past contributions to a field. The various chapters synthesize and criticize this accumulated knowledge and identify voids in it, pointing out future directions for research; many of them are superb presentations of information in fields that have been reviewed nowhere else. L.R.J.

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Acknowledgments From the organization stage to actual production the following people provided invaluable assistance, helpful suggestions, and a great deal of support. Their role and efforts have been much more than what is normally provided by a publisher, and I express my thanks to them. Jasna Markovac (Senior Vice President, Global Academic & Customer Relations) Julie Eddy (Publishing Services Manager) Lisa Royse (Production Editor) Mara Conner (Publishing Editor) Judy Meyer (Publishing Editor) Tari Broderick (Senior Publishing Editor) Cate Rickard Barr (Design Manager) Andrea Lutes (Book Designer) Patricia Howard (Senior Marketing Manager) Trevor Daul (Senior Marketing Manager) Philip Carpenter (Developmental Editor)

xxii

CHAPTER

1

Transcriptional and Epigenetic Regulation Juanita L. Merchant and Longchuan Bai Overview of Gene Organization, 1 Gene Composition, 1 Epigenetic Influences, 5 Histone Modifications, 5 DNA Methylation, 8 Chromatin-Binding Proteins, 8 Epigenetics and Development, 9 Epigenetics and Cancer, 9 Anatomy of the Promoter, 9 DNA Elements, 9 DNA-Binding Proteins, 12 Coregulatory Proteins, 15

Methodology, 16 Functional Methods, 16 Structural Methods, 17 Transcriptional Control of Gastrointestinal Peptides, 19 Posttranscriptional Processing, 19 Polyadenylation, 19 RNA Splicing, 20 Transport across the Nuclear Membrane, 21 Conclusion, 22 Acknowledgments, 22 References, 22

With the completion of the human genome sequencing project at the dawn of the third modern millennium, we have come to appreciate that we are only at the start of a new era of genomic enlightenment. Perhaps the most important piece of information that we have learned is that the clues to our genetic destiny are contained in more than just the primary sequence of DNA. Apparently, what distinguishes humans from other life-forms, and most interestingly, other mammals, lies in the complex modifications and function of the 20,000 to 30,000 genes. Not only are these 25,000 or so genes alternatively spliced, but these products are chemically modified to change their function. Therefore, as opposed to our genetic template being composed of a mere 25,000 genetic units, we are actually controlled by 25,000 to the nth power. The latter value has yet to be determined, but likely results in an enormous combination of genetic events.

This chapter reviews what has led us to reformulate our notions of gene expression in the postgenomic era.

OVERVIEW OF GENE ORGANIZATION Gene Composition The molecular definition of a eukaryotic gene is complex, but in the simplest terms, it is a nucleic acid sequence that encodes one polypeptide or messenger ribonucleic acid (mRNA) molecule (1). Genes are composed of two intertwining polymers of DNA that are noncovalently attached to a variety of proteins, including histones and specialized proteins (e.g., polymerases and various accessory proteins). The association of DNA, histones, and specialized nuclear proteins collectively is called chromatin. Chromosomes are composed of continuous strands of chromatin that have been compacted by supercoiling and looping to fit into the nucleus. Most importantly, they are the basic heritable unit in the mammalian cell. In humans, there are 46 chromosomes, or 23 pairs. The smallest unit of the DNA polymer is a nucleotide, a base attached to the first carbon of a five-carbon sugar phosphorylated at its fifth carbon (Fig. 1-1). Nucleosides do not contain phosphates; thus, they differ from nucleotides, which contain one, two, or three

J. L. Merchant: Departments of Internal Medicine and Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109. L. Bai: Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1

2 / CHAPTER 1

Base

P

OCH2

Nucleoside Nucleotide

FIG. 1-1. Nucleic acid structure. A nucleoside consists of a purine or pyrimidine base covalently linked to the first carbon of the pentose ring. The addition of one, two, or three phosphate groups yields a nucleotide monophosphate, diphosphate, or triphosphate, respectively. The type of sugar determines the type of nucleic acid: ribose in ribonucleic acid (RNA) and deoxyribose in DNA.

phosphate groups. The four nucleotides are distinguished by the type of base that they contain: adenine (A), thymine (T), cytosine (C), or guanine (G). DNA contains the sugar deoxyribose, whereas RNA contains the sugar ribose and the base uracil (U) instead of thymine. Polymers of nucleotides or nucleic acids (also called nucleoside monophosphates, diphosphates, or triphosphates) are formed when the free phosphate group attached to the fifth carbon of an adjacent nucleotide of the pentose sugar condenses with the hydroxyl group on the third pentose carbon to produce two ester bonds and water (phosphodiester bond). Accordingly, the proximal end of each DNA strand (5′ end) contains a phosphate group in the 5 position of the deoxyribose sugar residue. The terminal nucleic acid at the 3′ end of each DNA strand contains a free hydroxyl group in the 3 position of the deoxyribose ring. By convention, nucleotide sequences are written from 5′ to 3′, reading from left to right, with the sense strand presented as the upper strand. The antisense strand, written on the bottom, is antiparallel and complementary to the sense strand so that the 5′ to 3′ direction proceeds from right to left. Each nucleotide within the polymer is base paired with a particular nucleotide on the opposing strand by hydrogen bonds; adenine pairs with thymine, and guanine pairs with cytosine. The DNA strand containing the same sequence as the mRNA is designated the sense strand, and the strand that it pairs with is designated the antisense strand. The antisense strand becomes the template sequence that will be transcribed by RNA polymerase II (Pol II) into mRNA and subsequently translated into amino acids.

Most studies on transcriptional control focus on genes transcribed by the seven-subunit enzyme Pol II, and thus are designated as class II genes (2). It is Pol II that is responsible for transcribing gene sequences into protein-encoding mRNA. Only 4% of the total RNA in the cell is mRNA. Many of these initial primary transcripts (heterogeneous nuclear RNA [hnRNA]) are further processed as discussed later. Nine percent of cellular RNA is hnRNA, the bulk of which are small nuclear RNA (snRNA; e.g., U2 involved in RNA splicing, 4%) and small nucleolar RNA (e.g., U22 snoRNA comprising 1%). The other 4% of hnRNA is mRNA. An additional 1% of total cell RNA is called guide RNA, which edits mature mRNA transcripts (3). RNA polymerase I (Pol I) transcribes all of the ribosomal genes except for the 5S gene. Ribosomal RNA represents about 75% of the RNA in the cell. RNA polymerase III (Pol III) transcribes the 5S ribosomal gene and the genes encoding transfer RNA. Transfer RNA represents about 15% of the total RNA in the cell. Pol I and III transcribe genes that will not be further translated into peptides, although their primary transcripts are also processed before reaching the cytoplasm. Because Pol II transcribes genes encoding proteins and peptides, Pol II–regulated genes are the primary focus of this chapter. One may conceive of a gene as being analogous to a long sentence read from left to right and composed of letters organized into words separated by spaces and marks of punctuation. Specific DNA sequences “punctuate” the gene with important start and stop signals for transcription and translation. One gene may comprise several hundred to several thousand DNA base pairs. These base pairs (the alphabet) are organized into functional groups (phrases) based on whether a particular sequence is untranscribed, only transcribed, or both transcribed and translated (Fig. 1-2). Exons are DNA sequences that are transcribed into mRNA by Pol II and exit the nucleus. Within the cytoplasm, exons may or may not be translated into peptides. Those exons that are transcribed and translated form the coding sequences (coding exon). In general, the term intron is used to describe the intervening DNA sequence that is transcribed but is removed from the primary transcript by RNA splicing (RNA processing) before it exits the nucleus as a mature transcript (see Posttranscriptional Processing later in this chapter and also Chapter 2). DNA sequences or elements that regulate transcription and are not transcribed into mRNA usually reside in the 5′ portion of a gene upstream (to the left of) of the promoter. The promoter is a group of DNA sequences that binds Pol II in concert with accessory proteins to initiate the synthesis of mRNA. Accessory proteins control the accuracy and rate of polymerase binding. The first nucleotide transcribed into mRNA is assigned the number 1 with subsequent nucleotides (downstream or to the right of the promoter) assigned positive numbers as transcription proceeds toward the 3′ end. Nucleotides preceding the promoter (upstream or 5′) are assigned negative numbers. DNA sequences that encode a polypeptide (open reading frame) begin with the translational start site codon ATG (encoding methionine) and end with one of the three stop codons: TAA, TAG, or TGA.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 3 Intron

3′ Exon 2

Exon 3

Exon 2

Exon 3

AATAAA sla ted

lat ed

Exon 1

tra n

tra

un

Transcription

3′

5′

un

Untranscribed

Intron

ns

5′ TTCCAATGACTCAAGTATAAGTCTC −30 +1

Primary Transcript of hnRNA (Nucleus)

m7Gppp

Exon 1

Cap

AAUAAA AAAAAAn Polyadenylation

Posttranscriptional Processing

Processed Transcript (Cytoplasm)

m7Gppp

Exon 1

Exon 2

Exon 3

AAUAAA

AAAAAAn

FIG. 1-2. Gene structure, transcription, and posttranscriptional processing. A gene is composed of several hundred to several thousand base pairs, subdivided into functional elements. The location of 5′ and 3′ untranslated sequences, exons, and introns are shown. The 5′ flanking sequences contain specific DNA elements (e.g., TATA box). Ribonucleic acid (RNA) polymerase II transcribes DNA into heterogeneous nuclear RNA (hnRNA) during transcription. Twenty base pairs after the sequence AATAAA is transcribed to AAUAAA, messenger RNA (mRNA) are cleaved and the polyadenylate tail is added to the 3′ end. A methylated guanylate residue is added to the 5′ end of the mRNA through a triphosphate linkage. Before exiting the nucleus, intron segments are removed by splicing factors during posttranscriptional processing.

(The translational start and stop codons, respectively, are transcribed into mRNA as AUG, UAA, UAG, and UGA.) Because one amino acid is encoded by three nucleotides or a triplet (codon), two or three peptides may be encoded by overlapping codons simply by shifting the reading frame by one or two nucleotides. Regulatory sequences that are transcribed but not translated reside at both the 5′ and 3′ ends of the mature RNA transcript. Both 5′ and 3′ untranslated regulatory sequences, which range from 10 to several thousand nucleotides, are thought to participate in the fidelity of translation and mRNA stabilization or destabilization. RNA molecules that encode proteins (except most histone proteins) are distinguished from ribosomal and transfer RNA by the series of adenosines added to the 3′ end of the molecule (poly(A) RNA; see Fig. 1-2). This feature is a useful means to isolate mRNA from other, more abundant RNA species (transfer and ribosomal RNA) and also designates the functional termination of the protein-encoding portion of the gene. During transcription, the primary RNA transcript is cleaved 20 bp downstream of the AAUAAA site at the 3′ end, and ~150 to 200 adenine nucleotides are added to form the poly(A) tail (4–6). The 5′ end of the mRNA transcript receives a protective “cap” after synthesis of the first 30 nucleotides, which consists of a guanylate residue methylated at the 7 position and linked to the first nucleotide of RNA by three phosphates. The RNA cap is a high-affinity binding site for ribosomes (7,8). Notably, the element

AATAA that signals the site of the poly(A) tail is not necessarily the functional end of the gene. Rather, the 3′ untranslated region (3′UTR) and 3′ untranscribed regions may still contain regulatory elements that can modulate gene expression. Therefore, just as the 5′ end of a gene must be determined empirically, so must the 3′ end of the gene. The 5′ border of a gene is identified by the promoter region (functionally determined) and structurally by the first nucleotide transcribed into mRNA (cap site) as determined by various reverse transcriptase methods—for example, primer extension analysis or anchored polymerase chain reaction (PCR) (9). These techniques use reverse transcriptase to synthesize complementary or copy DNA (cDNA; Fig. 1-3). Radiolabeled primers complementary to the 5′ end of the DNA sequence to be copied are allowed to anneal to mRNA. Reverse transcriptase then adds deoxynucleotides to the primer in the 3′ to 5′ direction. Synthesis of the cDNA will terminate when the 5′ end of the mRNA is reached. Template mRNA molecules are removed by ribonucleases (RNases), and the synthesis of a double-stranded cDNA is completed through the action of DNA polymerase. Because the newly synthesized cDNA is radiolabeled at the 5′ end, the length of the cDNA (and hence the transcriptional start site) is determined by resolving the fragments on a denaturing polyacrylamide gel and comparing the length observed in base pairs to the known cDNA sequence. cDNA is also a useful tool for making probes to detect complementary nucleotide

4 / CHAPTER 1 1

5′

2

3

AATAAA

3′ Gene

Transcription/Processing

5′

1

2

3

AAAAAAAA 3′ mature mRNA

3′

TTTTTTTT 5′ nucleotide primer

Reverse Transcriptase + dNTPs

2

3

AAAAAAAA 3′ mature mRNA

2

1

3′

1

3

5′

TTTTTTTT 5′ Primer Extended Product/single strand copy DNA RNase DNA Polymerase + dNTPs

1

2

5′

3

3′

1

2

3

TTTTTTTT 5′ Copy of Complementary DNA AAAAAAAA 3′ (cDNA)

FIG. 1-3. Complementary DNA (cDNA). Primers complementary to a portion of the messenger ribonucleic acid (mRNA) are allowed to anneal. For unknown sequences, as in the synthesis of cDNA libraries, a primer complementary to the poly(A) tail is used, that is, poly (dT). Reverse transcriptase added together with all four deoxynucleotides (dNTPs) will transcribe mRNA in the 3′ to 5′ direction to make cDNA. The mRNA template is removed by RNases, and double-stranded cDNA is made using DNA polymerase. In primer extension analysis, the 5′ end of mRNA (the cap site) is identified by annealing primers of a known sequence near the 5′ end of mRNA.

sequences and for making cDNA libraries that reflect the spectrum and relative abundance of specific mRNA within a given cell. These cDNA libraries must be contrasted with genomic phage libraries in which the DNA sequences in the phage heads reflect the number of times that a particular gene sequence is represented in the host genome, which is usually once. The 5′ sequences flanking the gene are defined functionally by various methods other than simple structural information. These sequences direct the developmental, tissue-specific, and inducible expression of the gene and can range from a few hundred to several thousand base pairs (10). It is possible to identify the sequences conferring these regulated gene activities by using methods such as DNA transfer into cell lines (11,12) and transgenic mouse models (13,14). For example, the expression of gastrin in the adult occurs in the antrum of the stomach and in the first portion of the duodenum (15–17). However, gastrin is never expressed in skin or kidney. Thus, if 1000 bp of 5′ flanking sequence permits the expression of gastrin in a fibroblast or kidney cell line, but 20,000 bp do not, it may be concluded that the untranscribed sequences between −1000 and −20,000 bp from the promoter are important in shutting off expression of gastrin in skin and kidney, sites where gastrin is never expressed in vivo. Thus, the 5′ regulatory sequences important in normal expression of the gastrin gene may extend as far upstream as −20,000 bp from the start site

of transcription. Alternatively, the 5′ or even 3′ borders may extend even further if functional data indicate that a larger sequence is required for the appropriate tissue and temporal expression to be observed with the native gene. Recently, it has been found that there are specific DNA elements called Insulator elements that mark the boundary of genes (18). These elements, originally identified on the globin gene, bind a transcription factor called CTCF and are capable of preventing the spread of histone acetylation between adjacent genes (19). Specific examples of tissue-specific elements have been reported within the promoters of several gastrointestinal (GI) peptides (e.g., gastrin and secretin), as well as for specific intestinal proteins (e.g., sucrase-isomaltase) (20–26). Similar experiments may also be performed in transgenic mice with constructs containing various lengths of 5′ flanking sequences regulating reporter gene expression. Instead of transferring these reporter constructs into cell lines, they are injected into fertilized eggs and reimplanted into ovulating female mice to be expressed in the mouse germ line (13,14). The expression of these constructs in the offspring is analyzed by cytochemical detection of reporter gene products in various organs or in response to physiologic inducers (27). The transgenic approach to gene expression, like the experiments described earlier, permits anatomic, environmental, and developmental analysis in the whole animal (28,29). This approach is particularly valuable in understanding the

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 5 regulatory sequences important for the tissue-specific expression of the genes in different cell types of the same organ such as the small intestine (30,31). Given the requirement for larger and larger pieces of DNA to recapitulate native expression in transgenic mouse models, techniques have been developed to clone and manipulate large pieces of DNA (more than 50 kilobases; e.g., yeast artificial chromosomes [YACs] and bacterial artificial chromosomes [BACs]) (32,33). Recombineering is a powerful technique performed in bacteria that permits introduction of foreign DNA or point mutations into these large plasmids that are eventually introduced into transgenic mice (34–37).

EPIGENETIC INFLUENCES Epigenetics literally means “outside of or beyond genetics,” and it refers to the “study of genetic modifications that are mitotically and/or meiotically heritable yet do not change the DNA sequence” (38). Thus, mutations or deletions alter the character or length of the sequence that, in turn, alters the primary sequence of the protein. By contrast, epigenetic influences chemically modify the nucleotide or amino acid structure that, in turn, changes how that particular residue is recognized by nuclear proteins, without changing the sequence itself. Although it is now clear from the completed sequence of the human genome that there are only about 20,000 to 30,000 gene loci, the complexity of the genetic information encoded in human chromosomes must enlist other features of chromatin (39). The epigenetic influences on chromatin appear to be one of the critical features that enhance genomic complexity. Major targets of epigenetic changes are histones, basic proteins coating the naked DNA double helix. The N-terminal tails of histones (H1, H2A, H2B, H3, H4) are positively charged because of the basic amino acid lysine. The positively charged histones attach to DNA because of the negatively charged phosphate backbone of DNA. The ionic interaction is reduced if the positive charge on the lysines is removed. Specific enzymes called histone acetyltransferases (HATs) acetylate the lysine side group, effectively eliminating the positive charge (Fig. 1-4). The loss of the ionic interaction between the histones and phosphate groups on DNA permit greater access to the DNA helix by accessory proteins such as polymerases, transcription factors, and coactivators or repressors. DNA in the form of chromatin becomes open, accessible, and readily transcribed. By contrast, there are enzymes that will “close” chromatin by removing the acetyl groups from the lysines at the N-terminal tails of histone proteins. These enzymes are called histone deacetylases (HDACs). Removal of the acetyl group restores the positive charge to the histones allowing the ionic interaction between histones and DNA to be restored. The nonhistone proteins such as polymerases and transcription factors become excluded from DNA, transcription is silenced, and chromatin is inactive. Collectively, the histones and accessory proteins associated noncovalently with DNA are what forms chromatin.

Closed Chromatin

Open Chromatin

Transcription OFF

Transcription ON Ac

K K K K

K K

HAT p300, CBP

K K

Ac K K

Ac K K K Ac K

K K HDAC

Butyrate

FIG. 1-4. Nucleosome structure. The double-strand DNA helix winds twice around a complex of the four core histones assembled as dimers. Unacetylated histones are positively charged and adhere tightly to the negatively charged DNA, preventing access by transcription regulatory proteins. Histones that are acetylated are less positively charged and do not adhere as tightly to chromatin, allowing access of regulatory proteins to the DNA. The addition or removal of acetyl groups to the ends of histones is regulated by histone acetyltransferase (HATs) and histone deacetylase enzyme complexes (HDACs). The short-chain fatty acid butyrate inhibits the activity of HDACs.

Chromatin exists in two forms: euchromatin and heterochromatin (40). Euchromatin contains the actively transcribed genes and becomes decondensed during DNA replication. Euchromatin is also centrally located in the nucleus. By contrast, heterochromatin contains transcriptionally silent genes that remain condensed at the periphery of the nucleus. The DNA sequences within heterochromatin are repetitive, and only 15% of nuclear chromatin is heterochromatin. The major forms of epigenetic modifications in mammalian cells occur on DNA and histones and include such covalent modifications as acetylation and methylation, but also via the addition of other organic residues. These epigenetic changes affect such events as chromatin folding, gene expression, X-chromosome inactivation, and genomic imprinting (41). Epigenetic events are essential for development and differentiation, during which clusters of genes must be activated or silenced at precisely timed intervals to allow for the organism’s growth and maturation.

Histone Modifications The basic repeating unit of chromatin is the nucleosome. Each nucleosome is composed of 147 bp of DNA wrapped twice around a histone protein octamer consisting of 2 molecules of each of the 4 core histones (H2A, H2B, H3, and H4). The linker histone H1 sits alone between each core nucleosome, facilitating further compaction (42). Each histone contains a structured globular domain with a histone-fold motif important for nucleosome assembly and a highly charged unstructured amino-terminal tail of 25 to 40 residues,

6 / CHAPTER 1 which protrudes from the body of the nucleosome to latch onto the phosphate backbone. The amino termini are the major sites for histone modifications (43). Histones can be modified by acetylation, methylation, phosphorylation, adenosine diphosphate (ADP)-ribosylation, ubiquitination, and sumoylation (44). The mixture of these covalent modifications creates a “code” on the surface of the histone molecule that is subsequently recognized by bromo and chromo domain–containing proteins mediating chromatin compaction, transcription, and DNA repair (45). Acetylation, methylation, ubiquitination, and sumoylation occur on the lysine residues, whereas methylation also occurs on arginine residues. Phosphorylation occurs on serines and threonines, and ADP-ribosylation occurs on glutamic acids. Most of these modifications, particularly acetylation, alter the charge distribution on the amino terminus and also alter nucleosome structure, which may, in turn, regulate chromatin structure (46,47). Some covalent modifications act as molecular switches, enabling or disabling subsequent covalent modifications, which explains the functional complexity of epigenetic modifications (48). Therefore, each modification correlates with a specific physical status of chromatin. Histone Acetylation Acetylation of histones occurs at the ε-amino side group of specific lysines within the N termini of histones. HATs transfer an acetyl group from acetyl-coenzyme A as a donor to the histone terminal lysines (49). In hypoacetylated chromatin, the positive charges on unacetylated lysines are attracted to the negatively charged DNA, producing compact, closed chromatin thereby repressing transcription (50). In contrast, acetylation of the lysines removes their positive charges, resulting in a less compact, open chromatin structure, which facilitates gene transcription. Therefore, HAT activity, and subsequently histone acetylation, is linked mainly to transcriptional activation (51) (see Fig. 1-4). Removal of the acetyl group (deacetylation) by HDACs restores the positive charge on lysines, and chromatin becomes compacted and less accessible to regulatory proteins required for transcription. Thus, HDACs and deacetylation are primarily associated with transcriptional repression (see Fig. 1-4). HATs are divided into five families. These include the p300/CBP (cyclic 3′,5′-adenosine monophosphate [cAMP] response element binding [CREB] protein) HATs (p300 and CBP), Gcn5-related acetyltransferases (GNATs; including Gcn5, p300/CBP-associated factor [PCAF], etc.), MOZ, Ybf2, Sas2, and Tip60 (MYST) (monocytic leukemia zinc finger protein [MOZ], Ybf2/Sas3, Sas2, and Tip60)-related HATs, the general transcription factor (GTF) HATs (TFIID subunit TAF250 and TFIIIC), and the nuclear hormonerelated HATs (SRC1 and ACTR) (52). The most consistent functional characteristic of the HATs is that they are transcriptional coactivators. These proteins are components of large multisubunit complexes that do not bind DNA directly, but instead form protein–protein interactions with DNAbinding transcription factors (53).

The more numerous mammalian HDACs have been grouped into three protein classes (54). Class I includes HDACs 1, 2, 3, and 8. Class II includes HDACs 4, 5, 6, 7, 9, and 10. The class III HDAC family consists of the conserved nicotinamide adenine dinucleotide (NAD)–dependent Sir2 family of deacetylases. Like HATs, HDACs do not bind directly to DNA but rather are recruited by large multisubunit complexes to function primarily as corepressors of transcription (55). The function of HATs and HDACs are of particular relevance in the GI tract because of the effect of butyrate, a by-product of colonic bacterial fermentation, on histone acetylation (see Fig. 1-4). Epidemiologic studies uniformly concur that a diet high in fiber is protective against colon cancer (56). The short-chain fatty acid butyrate is one of several fiber-derived fermentation products capable of maintaining epithelial cell differentiation (57). The differentiation effects were initially demonstrated after treatment of erythroleukemic cells with butyrate (58). Subsequently, it was discovered that the induction of differentiation by butyrate correlated with histone hyperacetylation (59–61) due to suppression of HDACs (62–66). Thus, the HDAC effects of butyrate and resulting histone hyperacetylation may, in fact, be one mechanism by which dietary fiber exerts its anticancer effects (67). Reviews support the viewpoint that butyrate is a potent anticancer agent (68–70). Collectively, early studies emphasized the global effects of butyrate on chromatin remodeling, but the molecular basis for the gene-specific effects of butyrate remains poorly defined. HDAC inhibitors regulate less than 10% of actively transcribed genes. Most of those are up-regulated through GC-rich sites (71,72). In addition to histone acetylation, it is now known that DNA-binding proteins can become acetylated (52). Thus, a possible mechanism by which hyperacetylation induced by butyrate might target specific genes is through acetylation of specific transcription factors. The proposed function of acetylated transcription factors varies and includes increased or decreased DNA binding, as well as protein stability (73). In many instances, the genetic targets of butyrate are GC-rich sequences that bind Sp1 and Sp3. Gamma glutamyl transferase (74), insulin-like growth factor (IGF) binding protein 3 (75), G α(i2) (76), galectin (77), Cox1 (78), and intestinal alkaline phosphatase (79) are all up-regulated by butyrate through Sp1 sites. Sp1 binding sites are also implicated in the butyrate induction of p21WAF1 gene expression (80). HAT p300, recruited to the p21WAF1 promoter, cooperates with Sp1 and Sp3 to mediate the effects of butyrate (81). However, Sp1 does not cooperate directly with p300, but instead binds HDAC1 (82,83). The Sp1-HDAC1 complex, in turn, forms complexes with other corepressors such as Sin3A (84). Thus, Sp1 appears to be the factor that confers p21WAF1 promoter repression by recruiting HDACs and corepressor complexes. HDACs can have opposing functions, especially in cancer. HDACs can prevent the activation of tumor suppressor genes and block the ability of a cancer cell to undergo

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 7 apoptosis (85). However, HDAC2 silencing can trigger apoptosis (86). Another important feature of HDACs is their interaction with DNA methylation. HDACs cooperate with DNA methyltransferases (DNMTs) by removing the acetyl groups that would otherwise block methylation targets on histones or DNA (87,88). Histone Methylation There are two types of histone methylation, targeting either lysine or arginine residues. Histone methyltransferases perform these modifications using S-adenosyl-methionine as the methyl group donor. Lysine methylation is implicated in changes in chromatin structure and gene regulation, whereas arginine methylation correlates with the active state of transcription, such as acetylation (89). Histone Methylation at Lysines Methylation of lysines residues (K) occurs on histone H3 primarily at K4, K9, and K27 and on H4 at K20 (Fig. 1-5). The lysine residues can be monomethylated, dimethylated, or trimethylated at the ε-amino group. The methylation of H3 is associated with an open chromatin configuration and gene activation (90,91). In contrast, the methylation of H3 at K9 is associated with condensed, repressed chromatin (92). In general, there are at least four families of lysine methyltransferases. All of the lysine methyltransferases are distinguished by the presence of Su(var)3-9, Enhancer of Zeste, and Trithorax (SET) domains. The fourth family of these methyltransferases contains other protein domains aside from the SET domain. SET protein domains are approximately 130 residues homologous to amino acid segments in SET, three Drosophila proteins with intrinsic methyltransferase activity (93,94). The mammalian form of Su(var)3-9 is SUV39H and is involved in stabilizing heterochromatin by trimethylation of histone H3 at lysine K9. The trimethyl

N -S --K --K -- K -K ----K ---- H2A 1 5 9 13 15 36

N --K -- K-S -K --K --K -K ---S --S ---- H2B 5 12 1415 20 23 24 32 36

--R -T - K- K- S -T - K --R - K ----K --R - K --S --- K ---- H3 2 3 4 9 1011 14 1718 23 26 27 28 36

N -S -R -K --K --K -- K --- K ---- H4 1 3 5 8 12 16 20

FIG. 1-5. Histone modifications on histone tails. Shown are the amino-terminal histone residues modified by acetylation (filled circles), methylation (filled squares), and phosphorylation (filled triangles).

group creates an atomic feature or imprint on H3 that, in turn, is recognized by HP1, a chromatin organization modifier (chromo domain proteins) (95). The methylated or acetylated imprints on DNA or histones are recognized by two classes of proteins: those with chromo domains that recognize methyl group imprints and those with bromo domains that recognize acetyl group imprints. Transcriptional coactivators such as CBP, p300, and PCAF are HATs that contain bromo domains. They acetylate histones and other nuclear proteins; thus, not surprisingly, they also recognize an acetyl group imprint. These proteins are discussed in greater detail later in this chapter in Chromatin-Binding Proteins. Histone Methylation at Arginines Methylation at arginines occurs within the tails of histones H3 (R2, R17, and R26) and H4 (R3) and is catalyzed by coactivator-associated arginine methyltransferase 1 (CARM1) and protein arginine N-methyltransferase 1 (PRMT1), respectively, in mammalian cells (see Fig. 1-5). Like lysines, arginines can be either monomethylated or dimethylated (asymmetric or symmetric) on the guanidino nitrogen, and this process is antagonized by human peptidylarginine deiminase 4 (PADI4), which converts methyl-Arg to citrulline (96,97). Less is known about the fate of histones methylated at arginines. However, initial studies indicate that the methylated arginines create an imprint recognized by coregulatory molecules, for example, p300 and switching/sucrose nonfermenting (SWI/SNF) (98,99). Histone Phosphorylation Histone phosphorylation occurs on all four core histones: H2A (S1), H2B (S14), H3 (S10 and S28), and H4 (S1) (see Fig. 1-5). The phosphorylation of S10 in H3 is associated with transcriptional activation (100) and chromosome condensation during mitosis (101). In addition, phosphorylation of S10 in H3 is also associated with the transduction of external signals to chromatin, leading to the transient expression of immediate early genes (102,103). The phosphorylation of H3 is mediated by several specific kinases, activated by distinct pathways. For example, mammalian mitotic H3 phosphorylation is associated with Aurora B kinases (104,105), H3 phosphorylation by IKKα is important for the activation of nuclear factor (NF)-κB (106), and the immediate early gene response is mediated mainly by mitogen and stressactivated kinases MSK1 and MSK2 (107). Histone H2B phosphorylation condenses the chromatin and is involved in apoptosis (108,109). The downstream effects of phosphorylation of H2A and H4 are unknown. Of the histone modifications, acetylation and phosphorylation are reversible. Consequently, if the presence of a modification influences transcription in a particular way, its removal may have the opposing effect. In this way the cell could effectively respond to changes in environmental cues. Different histone modifications may be linked mechanistically. For example, phosphorylation of S10 on H3 enhances

8 / CHAPTER 1 histone acetylation by Gcn5 (110,111), whereas H3 K9 methylation inhibits phosphorylation at H3 S10 (93). Given the number of sites and the variety of possible modifications, the combinatorial possibilities are extremely large. The combinatorial pattern of N-terminal modifications results in a heterogeneous identity for each nucleosome that the cell interprets as a readable code from the genome to the cellular machinery directing various processes to occur. This concept is commonly referred to as the “histone code hypothesis” (45). The precise modification status of a given histone tail on a given gene can also change during the process of transcriptional regulation and each of these different constellations of histone modifications may elicit distinct downstream transcriptional signals (45).

DNA Methylation DNA methylation is a postsynthesis modification that normal DNA goes through after each replication. This modification is catalyzed by DNMTs and occurs on the C-5 position of cytosine residues within CpG dinucleotides located primarily in the promoter of a gene. There are three major DNMTs (DNMT1, DNMT3a, and DNMT3b). Each DNMT plays a distinct and critical role in cells. Murine knockouts of DNMT1 and DNMT3b exhibit embryonic lethality (112). The DNMT3a homozygous knockout mouse appeared normal at birth but died by aged 4 weeks (112, 113). In humans, mutations of DNMT3b are linked to ICF syndrome (immunodeficiency, centromere instability, facial anomalies) (112,114). Sixty percent of human genes contain a CpG island (115). Although methylation can also occur in other parts of the gene, CpG dinucleotides tend to be underrepresented in the genome, and when they are found, they appear in clusters ranging from 0.5 to several kilobases with a GC content greater than 55% (116). These clusters are known as CpG islands (117). Methylation of CpG islands is a late evolutionary development and functions to maintain genome stability by repressing transposons and repetitive DNA elements (118). DNA methylation is an important player in many processes, including transcriptional repression, X-chromosome inactivation, and genomic imprinting. CpG islands located in the promoter region of genes are normally hypomethylated about 40% of the time (116). Their hypermethylation causes stable heritable transcriptional silencing. As observed with HDACs and deacetylation, the methylation status in cancers may seem contradictory. Aberrant de novo hypermethylation of CpG islands is a hallmark of some human cancers and is found early during carcinogenesis (119–121). Tumor suppressor genes are locally hypermethylated in some cancers to silence their expression, whereas oncogenes may be hypomethylated (116). Tumor cells globally demonstrate an overall hypomethylation of DNA, a process that has more recently been linked to nutrition (122). S-adenosylmethionine is the primary methyl donor in the cell and is reduced in conditions predisposed to cancer (123).

Genomic imprinting occurs in gametogenesis and is necessary for development. One of the X chromosomes in female individuals is not expressed because of the heavy methylation of the inactive X chromosome. The epigenetic phenomenon whereby expression of a gene depends on whether it is inherited from the mother or the father is called imprinting, and is caused by differential methylation of specific cytosine bases on the maternal versus the paternal genes.

Chromatin-Binding Proteins The remaining histone methyltransferases also recognize methyl groups on other regulatory proteins; therefore, they are discussed here. The second family of SET domain proteins is related to the Drosophila protein Enhancer of Zeste, with the prototypical mammalian protein named EZH2. EZH2 is part of a complex of proteins called the Polycomb group (PcG). Two variants of these complexes have been designated Polycomb repression complexes 1 (PRC1) and 2 (PRC2). EZH2 belongs to the PRC2 complex that also includes EED and SUZ12; whereas PRC1 includes the proteins RNF2, HPC, EDR, and BMI1. BMI1 has received increased attention because it is an important marker of normal and cancerous hematopoietic stem cells (124–126). The Polycomb group of proteins with their SET domains not only participates in histone lysine methylation, but the complexes that they form (PRC1, PRC2) are also important in recognizing the methylated protein imprint. A human homolog of Drosophila Trithorax is the mixed leukemia gene 1 (MLL1). There are four human MLL homologs. MLL1 has been shown to be a specific methyltransferase for H3 at K4 (127). In turn, it forms protein– protein interactions with coactivators, for example, CBP and corepressors chromatin remodelers (e.g., SWI/SNF) (128,129). Other Trithorax homologs (e.g., Ash1, Trx) form complexes with different coregulatory complexes. Collectively, members of the Trithorax group (TrG) of proteins can either activate or repress transcription depending on the coregulator with which they associate. Retinoblastoma protein-interacting zinc finger protein (RIZ), SMYD3, and MDS-EVI1 form a fourth family of SET domain proteins because they have two isoforms that exhibit opposing functions. The isoform containing the SET domain has tumor suppressor function, whereas the isoform missing the SET domain is cancer promoting. This “yinyang” theory put forth by Huang (123) is especially true for RIZ and MDS-EVI1, in which by an unclear mechanism, the cancer disturbs the normal ratio between the two isoforms. The SMYD3 protein contains another DNA-binding domain called MYND, in addition to a SET domain, and is overexpressed in colorectal and hepatocellular carcinomas (130). Cross talk between DNA methylation and the histone modifications exists. These interactions were shown by the observation that HDAC1 forms a complex with DNMT1 and 5-methyl-cytosine binding protein (MBP) on a methylated promoter to silence gene expression (131). Similar cross talk

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 9 occurs between the HDACs SUV39 and HP1, the HDACs PRC2 and PRC1, and the HATs MLL1 and BRM (47).

Epigenetics and Development The epigenetic control of gene expression is a fundamental feature of mammalian development, as indicated by the occurrence of developmental arrest or abnormalities in mutants deficient in methylation or acetylation. X-chromosome inactivation is an example of sequence-identical alleles being maintained stably in different functional states. In humans, X-linked inactivation serves to normalize the level of expression of X-linked genes in female (XX) and male (XY) individuals. Mutations in genes that affect global epigenetic profiles can cause human diseases. For example, the Fragile X syndrome results when a CGG repeat in the Fragile X Mental Retardation gene 1 (FMR1) 5′ regulatory region expands and becomes methylated de novo, causing the gene to be silenced and creating a visible “fragile” site on the X chromosome under certain conditions (132). On a more global level, mutations in the DNMT3b gene (which regulates the DNA methylation) lead to ICF syndrome (112,114), and CBP (with acetyltransferases activity) mutations cause Rubinstei–Taybi syndrome (133).

Epigenetics and Cancer Epigenetic changes play an important role in tumorigenesis. The major epigenetic changes that take place during the development of cancer are generally the aberrant DNA methylation of tumor suppressor genes and histones. Chapter 17 covers in greater detail the role of epigenetic influences in cancer, but a few highlights are mentioned here to conclude this section. Genomic methylation patterns are frequently altered in tumor cells, with global hypomethylation accompanying region-specific hypermethylation events. When hypermethylation events occur within the promoter of a tumor suppressor gene, this can silence expression of the associated gene and provide the cell with a growth advantage in a manner similar to deletions or mutations. Although cancer cells are hypomethylated in the genome compared with normal tissues, many tumor-suppressor genes are silenced in tumor cells because of hypermethylation. This aberrant methylation occurs early in tumor development and increases progressively, eventually leading to the malignant phenotype. For example, a high percentage of patients with sporadic colorectal cancers with a microsatellite instability phenotype show methylation and silencing of the gene encoding MutL protein homolog 1 (MLH1) (134). Other methylated tumor suppressors include p16CDKN2A, p14ARF, Rb, E-cadherin, and breast cancer gene-1 (BRCA1). Deregulation of genomic imprinting can also play a role in cancer development, as exemplified by loss of imprinting of the IGF2 gene in Wilms’ tumor (135).

Chromatin remodeling also plays an important role during tumorigenesis. Loss or misdirection of HATs has been linked to embryonic aberrations in mice (136,137) and to human cancers (138,139). Misdirection of HAT activities as a result of chromosomal translocations is associated with multiple human leukemias (140–142). In acute promyelocytic leukemia, the oncogenic fusion protein promyelocytic leukemia-retinoic acid receptor-α (PML-RARα) recruits an HDAC to repress genes essential for the differentiation of hematopoietic cells (143). Similarly, in acute myeloid leukemia (AML), AML1-eight-twenty-one (ETO) fusions recruit the repressive N-CoR-Sin3-HDAC1 complex that, in turn, inhibits normal myeloid development (144). That many human diseases, including cancer, have an epigenetic cause has encouraged the development of a new therapeutic option called “epigenetic therapy” (145). Many agents have been discovered that alter methylation patterns on DNA or the modification of histones, and several of these agents currently are being tested in clinical trials.

ANATOMY OF THE PROMOTER DNA Elements RNA Pol II and its accessory factors bind to a DNA sequence called the promoter, which is located upstream of protein-coding sequences to direct RNA transcription (146). Without the promoter, the genetic sequences that encode the information to make a functional peptide product will not be transcribed. Other 5′ flanking sequences or DNA elements that participate in transcription are sequence-specific binding sites for proteins that regulate the fidelity, rate, and timing of Pol II binding, formation of the preinitiation complex (PIC), and initiation of transcript elongation under basal and regulated conditions (147–149). These sequences are defined as cis-acting elements because they are a part of the same (cis) gene (150–153). DNA elements are categorized according to their ability to regulate transcription as a function of their distance and orientation from the promoter. Sequences that are contained within the first 30 to 100 bp of the promoter and operate in one orientation are considered promoterdependent, cis-acting elements. If they are positive-acting elements and increase the rate of transcription, they are considered activating DNA elements, whereas if they are negative-acting DNA elements and decrease or repress the rate of transcription, they are repressor elements (154–156). The structure of the promoter includes several critical elements that include the TATA element, which lies upstream of the transcription start site, the initiator sequence (Inr) that spans the start site, upstream regulatory elements that bind either transcriptional activators or repressors, and finally downstream poly(dA-dT) elements (157). The TATA element, or “TATA box,” is an element with a DNA sequence that is TATA or variants thereof (151,158–161). This sequence resides at a fixed distance 25 to 30 bp upstream from the transcriptional start site in many Pol II promoters, and its

10 / CHAPTER 1 location relative to the start site is dependent on position and distance (162–164). However, it became apparent that many genes did not have TATA sequences. These “TATA-less promoters” still remain dependent on assembly of the TATAbinding protein (TBP) at the promoter to form the PIC, but the recruitment of TBP is not rate limiting (165). Inr elements, although initially identified at the “TATAless promoters” (166,167), have subsequently been found in both TATA-containing and TATA-less promoters. Their role appears to be in directing the accuracy of Pol II initiation (168). These Inr elements reside within the first 60 bp of the transcriptional start site and directly overlap the start site itself, but they do not have a clearly defined consensus sequence (169). Many of the genes encoding GI peptides (e.g., gastrin, somatostatin, cholecystokinin [CCK], glucagons, and secretin) contain TATA elements (170–174); however, the gene encoding the growth factor, transforming growth factor-alpha (TGF-α), does not (175). Regulatory elements are generally sequence-specific DNA elements that bind transcription factors. In the case of transcriptional activators, there are two variations, upstream activating sequences (UASs) and enhancers. Both elements are orientation and distance independent. However, UAS elements do not function downstream of the TATA box. Thus, their function is restricted by their location relative to the TATA box (176,177). UAS elements, which bind transcription factors, facilitate assembly of the PIC directly by forming protein–protein interactions with GTFs, or indirectly by complexing with coactivators. Upstream repressor sequences (URSs) use several approaches to disrupt formation of the PIC. They can interfere with the activation domain of the activator complex, disrupt interaction with the core promoter factors, or recruit corepressors (e.g., Sin3-Rpd3, HDACs). Homopolymeric dA-dT sequences are required for normal levels of transcription. The repetitive dA-dT sequence has intrinsic structural ability to impair nucleosome assembly or stability (178,179). Models describing the formation of the Pol II initiation complex are constantly evolving and essentially involve the convergence of information gathered from biochemistry, structural biology, and genetics, particularly yeast genetics (148,180,181). Elucidation of the three-dimensional crystal structure of the TBP has advanced our understanding of preinitiation assembly complexes (182,183). Protein folding of TBP into a β-sheet forms a “saddle-shaped” concave surface of sufficient size to contact helical DNA (Fig. 1-6A). On the opposing convex surface are potential binding sites for various regulatory proteins, for example, TBP-associated factors (TAFs), GTFs, and Pol II (see Fig. 1-6A). At least 10 to 14 different human TAFs have been identified from HeLa cells, with their molecular weights ranging from 18 to 250 kDa (167,184,185). TAFs are multiple subunit proteins that associate with TBP to form the essential transcription factor TFIID. The proteins are conserved from yeast to humans with the bulk of our understanding of these factors coming from experiments in yeast and Drosophila. An interesting finding is that TAFs are not universally required for

transcription, but each one is required for only a subset of genes. Thus, for example, one TAF is required for transcription of 8% of genes, whereas three different TAFs are required for 60% of transcribed genes. In addition, TAFs are found in protein complexes other than with TBP. In fact, some TAFs have HAT activity, whereas others are similar to histones. Still other TAFs (e.g., TAFII250) have numerous enzymatic features including ubiquitin-conjugating activity (186). The conclusion from these studies is that TAFs are involved in promoter selection through yet to be defined mechanisms (185). TBP is not specific to Pol II promoters, but also forms PICs at the start site of Pol I and III promoters, as well as Inr promoters that do not contain TATA elements (167,187,188) (see Fig. 1-6B). For example, in Pol I promoters, TBP does not bind DNA directly in a sequence-specific manner, but instead forms protein–protein interactions with the selectivity factor complex (SL1) and the upstream binding factor (UBF) (189). In Pol III promoters, TBP complexes with TFIIIB and TFIIIC (190). In TATA-dependent and -independent Pol II promoters, TBP forms protein–protein interactions with spatially constrained upstream activators that bind DNA; for example, CCAAT-enhancer binding protein (C/EBP) and Sp1. Thus, TBP forms the core of the PIC through both DNA– protein and protein–protein contacts in TATA-dependent promoters but primarily protein–protein interactions in nonTATA promoters (see Fig. 1-6). Apparently, the selection of a promoter by TBP preceding the assembly of the PIC is determined by the type of accessory factors recruited (TAFs, SL1, Sp1, TFIIIC) (188,190,191). Moreover, this recruitment may be regulated by temporal and tissue-specific influences. Inhibition of transcription (repression) may occur simply by preventing one of these general TAFs from participating in the assembly of the PIC (182,187). Like Pol II itself, many TAFs and GTFs are composed of multiple subunits. Thus, there is an enormously complex pattern of assembly of proteins (TBP + TAFs = TFIID, other GTFs, and upstream activators) on specific DNA elements at the promoter (e.g., TATA, INR, UAS) that results in the initiation and elongation of mRNA (182,188,192). Other GTFs besides the TFIID complex include TFIIA, TFIIB, TFIIE, TFIIF, TFIIG, TFIIH, TFIII, TFIIJ, and TFIIK (160,167,193–195). There appears to be a strict requirement for these factors to assemble at the promoter in a specific order (181,182,192). TFIID binds to the TATA elements first, followed by protein–protein interactions of TFIID with TFIIA and TFIIB. The 12-subunit Pol II binds next. TFIIF is then recruited to the TFII-diaminobenzidine complex and facilitates binding of other general (basal) transcription factors E, J, H, and K. Many of these basal factors do not bind DNA directly (e.g., TFIIB, TFIIE, TFIIF), but instead form bridging complexes between the general Pol II transcriptional machinery and TAFs with specific upstream regulators. GTFs are required for the basal activity of the promoter, whereas UAS enhancers are dispensable. Specific functions of some of the GTFs have been elucidated. For example, the larger subunit of TFIIF (Rap74)

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 11

TBP

TAF

AC

T GF

TBP TAF

GF DNA (TATA)

POL

ACT

TAF

POL

GC-RICH

A

B FIG. 1-6. Schematic diagram of a polymerase (POL) II initiation complex. The saddle-shaped TATA-binding protein (TBP) (A) binds DNA directly at the TATA sequence and (B) is tethered between TBP-associated factors (TAFs) in non-TATA promoters. Thus, TBP forms the core of a complex consisting of TAFs, general transcription factors (GF), upstream activators (ACT), and ribonucleic acid (RNA) POL I, II, and III. (Modified from Comai and colleagues [187], by permission.)

functions as an ATPase-dependent helicase to unwind DNA ahead of the transcription complex (196). TFIIF appears to play a role in promoter stability rather than selectivity. TFIII, a helix-loop-helix (HLH) protein, binds preferentially to Inr promoter with or without TATA elements and cooperates with upstream regulatory factors and the general transcription complex (197). TFIIH is one of several C-terminal domain (CTD) kinases that phosphorylates the CTD of Pol II to signal elongation of the nascent mRNA chain (192,194). Other kinases are now known to phosphorylate CTD (198). Certainly, all genes are not transcribed concurrently; thus, the cell must have various mechanisms for silencing genes either permanently or in response to extracellular cues. The mechanisms for repressing genes may be general (e.g., DNA methylation [199,200]; see also #1375 in Bird [201]) or sequence-specific (202). Alternatively, loss of the ability to inhibit transcription of a gene (derepression) may permit certain cellular functions to proceed unchecked. Examples of the interaction between positive and negative regulators occur during cellular proliferation and differentiation (203). During fetal development, most cells are in the process of rapid proliferation. This period is followed by one of regulated differentiation during which the genes controlling proliferation are repressed. However, proliferative pathways may be derepressed (reactivated) during periods of organ repair or during neoplastic transformation (203,204). Examples include the reexpression of fetal proteins during liver regeneration and neoplasia (e.g., α-fetoprotein) or GI mucosal neoplasia (e.g., carcinoembryonic antigen) (205–207). Negative promoter elements or repressors may serve as the binding sites for proteins that sterically hinder the binding of GTFs (e.g., TFIID) or upstream activators (e.g., Sp1) critical in the formation of the Pol II transcription PICs (DNA–protein interactions). Alternatively, proteins

responsible for gene repression may act by preventing the recruitment of required general or accessory factors (e.g., TFIIB or TAFs) to the bound PIC (protein–protein interactions) (202). The DNA elements CCAAT and GGGCGG, which bind the nuclear proteins C/EBP and Sp1, respectively, are examples of promoter-activating elements that are distinct from the TATA box (151). These upstream promoter elements are distinguished from the TATA element in that mutation or removal of these UASs reduces basal promoter activity without completely eliminating it, whereas mutation or elimination of the TATA sequence completely abolishes transcription. DNA elements that function independently of their position on the gene or their orientation (3′ to 5′ or 5′ to 3′) are called enhancers if they bind nuclear proteins that activate transcription and silencers if they bind nuclear proteins that inhibit transcription (208–210). Many of these enhancer and silencer elements occur far upstream within the 5′ flank, but they may also occur within introns, exons, or 5′ or 3′ untranslated sequences. To identify cis-acting enhancer elements, constructs are made by ligating the regulatory elements to be studied in front of a functional promoter expressing a gene encoding a protein or enzyme that is easily assayed. Typical reporter genes encode proteins that are not normally expressed by the transfected cell. By systematically deleting portions of 5′ flanking sequence, the transcriptional activity of the promoter under various conditions is altered and the regulatory elements of interest are identified. DNA elements responsible for tissue specificity can be identified by transfecting (transferring DNA into eukaryotic cell lines) cell lines derived from different tissues. Transcriptional initiation from a promoter that requires a particular cis-acting sequence for expression in a specific cell type is diminished or abolished if this sequence is eliminated or mutated.

12 / CHAPTER 1 Cis-acting sequences conferring inducible responses are also identified by this method. Alternatively, elements that are only active during development must be identified in eukaryotic systems in which differentiation of a cell line can be controlled, or in transgenic animal models.

DNA-Binding Proteins DNA-binding proteins are also referred to as trans-acting factors by virtue of their ability to bind to the 5′ flanking regions of genes in a sequence-specific manner and regulate transcription (211–213). The term trans was coined to acknowledge that the protein product of one gene regulates the transcription of a different gene. With the genes for several hundred trans-acting factors now cloned, the study of their primary and secondary amino acid structures has demonstrated characteristic protein domains (214,215). In general, these proteins contain specific DNA-binding, transactivation, and oligomerization domains (Fig. 1-7). The amount of a transcription factor binding to a particular sequence initially is considered to be the primary mechanism of control. However, it is now clear that the proteins themselves are regulated by a variety of mechanisms in addition to controlling their levels in the nucleus and include activation or inactivation by proteolysis (e.g., NF-κB), covalent modification (e.g., phosphorylation, acetylation), and ligand binding (e.g., steroid receptors), in addition to regulating translocation

ACTIVATION

to and from the nucleus and transcriptional induction or repression of the trans-acting factor (216). The DNA-binding domain is the portion of the protein that contacts DNA in a sequence-specific manner. However, flanking amino acids may also influence DNA-binding through noncovalent interactions. Examples of four major designs for DNA-binding domains are proteins with a helix-turn-helix domain, “zinc finger” domains, amphipathic helices (e.g., basic-zipper [bZip], HLH), and β-ribbon (prokaryotic proteins) (215) (Fig. 1-8). Most of the proteinDNA contacts occur in the major groove through noncovalent interactions (e.g., hydrogen bonds, hydrophobic interactions, and van der Waals interactions). An α-helical structure appears to be a common motif used in the formation of the DNA-binding domain. The helix-turn-helix motif was initially identified in prokaryotic DNA-binding proteins, but similar motifs have now been identified in the homeodomains of eukaryotic transcription factors (217–219) (see Fig. 1-8). Homeobox factors are a class of DNA-binding proteins that predominantly play a role in the developmental expression of genes. Their discovery arose from the idea that developmental regulation involves control of gene expression by a few regulatory transcription factors called “master switch genes” (220). These DNA regulatory proteins initially were identified in simpler organisms such as the roundworm Caenorhabditis elegans (C. elegans) or Drosophila, in which the genetic development from the single-cell stage to maturity is well defined.

DNA BINDING

OLIGOMERIZATION

VA TI AC

IZA

Methylation

ER OM

Acetylation

TI

Glycosylation

Amphipathic helices e.g., leucine zippers helix-loop-hellix helix-span-helix

IG OL

ON

Phosphorylation

TIO

Sumoylation

N

Glutamine-rich proline-rich acidic domain

DNA BINDING e.g., helix-turn-helix, zinc fingers, basic domains

FIG. 1-7. DNA-binding protein domains. DNA-binding proteins have three functional domains: (1) a surface to bind DNA, (2) a surface to interact with other proteins containing similar oligomerization (dimerization) motifs, and (3) a surface to interact with the signal transduction pathways or the preinitiation complex. Examples of motifs associated with these domains are listed.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 13

DNA-binding domain 22 15 8 1

Leucine 5

4

7

Helix-turnHelix

2

3

A

Leucine-zipper domain

6

bZip

B

C

DNA-binding domain

++++

++++

Helical regions

Loop

Activation

Basic

L

F C Helix Span Helix

H

C

H

C

Zn

Dimerization

C

L

F

H Zn H

DNA binding bHSH

bHLH

D

E

Zing finger

F

FIG. 1-8. Example of DNA-binding motifs. (A) Helix-turn-helix. (B) Amphipathic helix formation. An α-helical structure is formed so that all leucines or hydrophobic amino acids line up at one surface. Shown are (C) a leucine zipper adjacent to basic DNA-binding domain (bZip); (D) a helixloop-helix adjacent to a basic DNA-binding domain (bHLH); (E) and a helix-span-helix adjacent to a basic domain (bHSH). (F) A zinc finger motif of the C2H2 type with tetrahedral coordination of a zinc ion between two histidines and two cysteines. (Modified from Berg [241], Falke and Juliano [255], Ellenberger and colleagues [266].)

Through site-directed mutagenesis studies, a specific protein domain required to effect developmental progression of these organisms was identified. This domain shared significant homology with a region within proteins controlling cell lineage in the pituitary (Pit-1) and immune system (B-cell octamer proteins, Oct-1 and -2) (221–225). These proteins also shared significant homology with the C. elegans “homeotic gene,” unc-86. Thus, the ~60- to 75-amino-acid region of shared homology was renamed the “POU” domain after the three proteins Pit-1, Oct-1, and unc-86. Initially, the POU domain was named without knowledge of function or the ability to form specific secondary protein structure. Although some bind similar AT-rich consensus DNA-binding sites (octamer proteins bind an eight-nucleotide sequence ATTTGCAT; Pit-1 [also called GHF-1] binds a nine-nucleotide consensus site TATATATNCAT), others do not (Drosophila eve protein recognizes TCAGCACCG) (217). Mouse homeobox genes have nomenclature based on their similarity to

Drosophila homeobox genes (e.g., caudal, forkhead) and have been associated with control of gut development (226–228). In fact, homeobox genes have emerged as critical regulatory factors in the development of both the luminal GI tract and pancreas (229,230). Homeobox genes in the luminal GI tract are related to the 39-member Hox gene family of transcriptional regulators that control anterior-posterior patterning, and they are related structurally to the Drosophila Antennapaedia gene (229). Hox genes are so strongly conserved in evolution that this cluster of genes has been repeated four times in mammals on different chromosomes (231). Collectively, the replicated genes are called Hox clusters and are expressed primarily in either the mesoderm or ectoderm (e.g., skin, muscle, neural tissue), but not in endodermal tissues. Rather, an evolutionarily related cluster of homeotic genes call the Para-Hox genes appear to play the more important role in endodermal tissue, and therefore gut patterning (232). These genes include Pdx1, which is

14 / CHAPTER 1 essential to the correct development of the pancreas and duodenum (233,234), and the genes related to the Drosophila caudal gene, Cdx1, Cdx2, and Cdx4 (229,235). Cdx2 is not only relevant to development of the luminal gut, but it also is an indicator of neoplastic transformation, especially in the upper GI tract (236,237). The forkhead family of homeotic genes is another group of transcriptional regulators with important implications in the gut because of their role in GI cancers (238,239). There are at least 43 members of the forkhead family spread over three chromosomes. The “winged helix” motif of the forkhead DNA-binding proteins is a variant of the 60-amino-acid homeodomain helix-turn-helix because it has additional peptide domains that have been described as “wings” (240). The forkhead transcription factors are downstream targets of the hedgehog pathway, which is an important developmental signaling cascade originally described in Drosophila (see Chapter 9 for a more detailed discussion). The zinc finger motif is distinguished by the occurrence of cysteine and histidine resides tetrahedrally coordinating a zinc ion (241–243) (see Fig. 1-8F). Two subcategories of zinc finger proteins have been identified: those regulatory proteins in which only cysteine contacts the zinc ion (e.g., the steroid receptor family, GAL4 [244,245]), and those in which both cysteine and histidine residues are involved (e.g., Sp1 and Zif 268 [245,246]). The X-ray crystallographic structures of several zinc finger and helix-turn-helix proteins have now been identified, with identification of more structures still to follow (247–253). Through crystallographic studies and computer modeling, investigators have been able to identify which amino acids within the DNA-binding domain contact particular nucleotides within the DNA element. It is anticipated that most of these interactions will be defined sufficiently well to predict protein–DNA contacts at the molecular level for other trans-acting factors. In the future, this will facilitate the targeting of specific transcription factors (natural or synthetic) to specific promoter sequences (248,254–257). Landschulz and coworkers (258) originally described the bZip/coiled-coiled DNA-binding motif as a dimerization domain (see Fig. 1-8). However, this motif, which consists of 55 to 65 amino acid residues, actually forms two domains: one for dimerization and a second for DNA binding (259). Seven repeating leucine residues forming an α-helical coil compose the dimerization domain (Zip domain) (see Fig. 1-8). Immediately adjacent to the Zip domain, toward the amino terminus, lies the basic/hydrophobic domain (b domain) (215). Thus, the bZip family of proteins, the first of the amphipathic helices to be described, must dimerize to form a complete DNA-binding domain (260,261). Other transcriptional regulatory proteins containing the same heptad repeat are able to dimerize with each other to form a “coiled coil” (262). For stable binding to DNA to occur, some bZip proteins prefer that each dimerization partner be the same (e.g., CREB, C/EBP, or general control of amino acid synthesis 4 [GCN4] homodimers [see #564 in Pu (260); 263,264]), whereas other bZip proteins form more stable complexes as heterodimers (e.g., Fos/Jun), although lower

affinity binding is also possible as homodimers (e.g., Jun/Jun) (265). The first report of a crystal structure for a bZip protein, the yeast transcription factor GCN4, confirmed the predicted model of two α-helical coils, which merge into diverging b domains that straddle and grip the major groove of DNA like “forceps” (266). Other amphipathic helices, which combine a dimerization domain with a basic DNA-binding domain, have been described; however, less is known about their threedimensional structure. The helical domains contain hydrophobic amino acid residues arrayed in an α helix so that they are clustered on one face of the helix, whereas hydrophilic residues reside on the opposing face (see Fig. 1-8). According to thermodynamic principles, the hydrophobic face is sequestered away from the aqueous environment by noncovalent interactions when they dimerize with similar domains on other proteins. In addition to the bZip model described earlier, the HLH and helix-span-helix (HSH) motifs were coined to describe other subclasses of amphipathic helices, albeit with longer linker sequences between the two α helices (267–269) (see Fig. 1-8). In the case of the leucine zipper, the hydrophobic face is formed by a series of leucine residues spaced seven amino acids apart (258). In contrast, the HLH and HSH proteins use a variety of different hydrophobic amino acids in addition to leucine to form two amphipathic α helices separated by a stretch of amino acids (“loop or span”) that do not form a helix. Like the bZip family, HLH and HSH regulatory proteins bind DNA through an adjacent basic domain. Thus, bZip proteins (e.g., CREB, activator protein 1 [AP1], activating transcription factor [ATF], Fos, Jun) are potentially interchangeable partners within homodimeric or heterodimeric complexes with the corresponding ability to recognize a greater repertoire of DNA-binding elements (270–272). For example, the Fos/Jun-binding site differs from the CREB/ ATF-binding site by 1 bp: CREB/ATF binds TGACGTCA, whereas Fos/Jun binds TGAGTCA. Likewise, the bHLH proteins that recognize the CANNTG consensus binding site are also able to complex with each other (273). Currently, there are three family members of the transcription factor AP2, which are the only members of the bHSH family (269, 274,275). An HLH protein without the basic DNA-binding domain called Id was cloned (276). This protein has been shown to combine with three bHLH proteins (MyoD, E12, and E47) and to prevent the formation of normal homodimers or heterodimers, thereby functioning as a dominant negative mutant. Similar types of negative regulatory proteins have been identified for bZip proteins (277,278). Therefore, the combinatorial ability of transcription factors permits flexibility in responding to extracellular signals at the level of DNA–protein and protein–protein interactions. The transactivation domains of regulatory proteins consist of predominantly acidic, basic (glutamine), or proline residues (152,279). These non-DNA-binding surfaces interface with signal transduction pathways and other proteins, but their specific function is not completely understood. Domains with a high degree of acidic charges are thought to represent important contact points for interaction with the Pol II PIC

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 15 (e.g., Gal 4, VP16). Ptashne (280) coined the phrase acidic blobs to describe such negatively charged trans-activating domains. Glutamine-rich (Sp1) and proline-rich (C/EBP, CTF) domains also presumably cooperate with the transcriptional machinery through protein–protein interactions (160,184,281–284). However, it has more recently been confirmed that transcription factors form protein–protein interactions with other transcription factors not within the same DNA-binding domain family. The most common transcription factor exhibiting this property is Sp1. Sp1 can interact directly with other transcription factors, for example, YY1, Smads, or Jun family members (285,286). A functional interaction between cJun and Sp1 has been shown to mediate epidermal growth factor activation of lipoxygenase gene expression (287). Presumably, the “acidic blob” in the transactivation domain of Sp1 creates a “sticky” surface on which new partnerships are formed at various promoters in response to a variety of extracellular signals. Likewise, Smad proteins, which mediate TGF-β signaling, are also promiscuous in their ability to partner with other transcription factor family members (288,289). Although at one time undetected, protein–protein interactions among transcription factors are now recognized as common occurrences, particularly because there are convenient means to identify the interactions genetically through two-hybrid cloning methods, or biochemically using affinity chromatography, immunoblot assays, and mass spectroscopy. Many of the mechanisms involving transactivation of transcription factors involve protein phosphorylation and dephosphorylation (290). Phosphorylation by protein kinases occurs at serine, threonine, or tyrosine amino acid residues. Several classes of protein kinases exist within the cell; however, the best studied are the protein kinase A (PKA) and C (PKC) pathways. PKA is activated indirectly by the catalytic subunit of adenylate cyclase. Signals that increase intracellular cAMP will activate PKA (291,292). In contrast, PKC is activated by calcium released from intracellular stores and by the phospholipid diacylglycerol (293). Phospholipase Cγ catalyzes the hydrolysis of phosphatidylinositol to diacylglycerol. The tumor promoter 12-0-tetra-decanoyl phorbol-13-acetate (TPA) is a lipid-soluble compound that mimics diacylglycerol and directly activates PKC. Hundreds of additional protein kinases within both the cytoplasm and the nucleus exist that may be implicated in the specific phosphorylation of transcription factors (294). Ligand binding triggers a variety of different activation pathways that appear to result in the direct phosphorylation of transcription factors by protein kinases other than PKC and PKA; for example, casein kinase II (CKII), glycogen synthase kinase III, and several DNA-dependent protein kinases (295–297). Direct phosphorylation of the DNA-binding protein may result in a conformational change that enhances its ability to induce transcriptional activation (e.g., CREB, cJun, C/ EBP-β) or inhibition (e.g., yeast protein A[298]DRI) (290). Alternatively, phosphorylation of an inhibitory subunit may release the transcription factor from an inactive state (e.g., NF–κB) (299–303). Phosphorylation can also regulate the

ability of a protein to dimerize, thereby broadening or narrowing the repertoire of DNA sequences that are recognized (e.g., signal transducer and activator of transcription [STAT] and Fos/Jun family) (304,305). The removal of phosphate groups by sequence-specific phosphatases is an additional mechanism by which the transcriptional activity of DNA-binding proteins may be altered (290,306,307). Interestingly, dephosphorylation appears to be a more common mechanism for regulating transacting factor binding than is kinase-mediated phosphorylation (295). Binding of the Jun family (bZip class), homeodomain proteins, and cMyb to DNA is regulated by dephosphorylation. Phosphorylation of sites within or adjacent to the DNA-binding domain of these proteins inhibits DNA binding, whereas removal of phosphates enhances binding. In contrast, activation of DNA binding by phosphorylation has fewer documented examples. One example is the serum-response factor (SRF) that binds to and activates the cFos promoter (308,309). SRF appears to be activated by phosphorylation at sites adjacent to the DNA-binding domain by CKII. This observation is supported by studies involving both mutational analysis of these phosphorylation sites and increasing cellular CKII kinase activity through microinjection of the enzyme into cells (310,311). Although glycosylated proteins are usually observed on the plasma membrane of cells or in the lumen of intracellular organelles, nuclear proteins have been shown to contain O-linked glycosylated residues as well (312). Sp1 represents the prototypical glycosylated transcription factor, the activity of which is enhanced by the presence of carbohydrate residues (312–315). Other eukaryotic transcription factors such as CTF, AP1, and AP4 are also known to be glycosylated, but the effect of the carbohydrate residues on their transcriptional activity is unknown. Glycosylation may regulate the transcriptional activity of individual transcription factors, perhaps by increasing their resistance to proteolysis, by targeting them to the nucleus, by blocking potential phosphorylation sites, or by facilitating their interaction with coactivators (316).

Coregulatory Proteins By the mid 1990s, it became clear that DNA-binding factors were working in a combinatorial manner, not only with other DNA-binding factors, but with non-DNA-binding proteins that were closely linked to chromatin structure and the PIC. These large molecular weight proteins were initially identified as factors interacting with the steroid hormone receptors, which are DNA-binding proteins that translocate to the nucleus after binding hydrophobic ligands in the cytoplasm (317–319). At about the same time, it was discovered that phosphorylation of the cAMP-activated transcription factor CREB induced its interaction with a 300-kDa coactivator protein called CBP. Subsequent to the discovery of CREB, the homologous transcriptional coactivator designated p300 was also identified (320). Coactivators were

16 / CHAPTER 1 found to facilitate transcriptional activation through intrinsic HAT activity, resulting in an “open” chromatin state at the start site of transcription. There are now several of this class of proteins that include PCAF and GCN5 (321). Conversely, the protein complexes that inhibited transcription were multiprotein complexes that recruited histone deacetylators, which, in turn, deacetylate histones returning chromatin to its closed, inactive state (322). The prototype corepressors were identified because of their ability to suppress activation by the retinoid and thyroid hormones (SMRT/N-CoR) (216). It is now known that there are transcriptional corepressors of a variety of signal transduction pathways, including Sin3A, a corepressor of the cMyc bHLH transcription factor family, and PIAS/SUMO, a corepressor of the STAT signaling pathway (323–328). Collectively, these proteins are considered to be coregulatory factors because they do not contact DNA directly as transcription factors do, but rather form protein bridges between the sequence-specific DNA-binding proteins and the Pol II assembly apparatus, bringing with them enzymatic activity, for example, acetylase and deacetylase activity involved in remodeling chromatin (329–331). Currently, there are three broad categories of coactivators (332). p300 and CBP are the prototypes of the HAT class of coactivators. The TRAP/DRIP/Mediator/ARC complex compose the second class and are proteins that bind transcription factors and recruit RNA Pol II without having intrinsic histone modification capabilities. The third class comprises the yeast SWI/SNF and their mammalian homologues BRG1/BRM. This third class of coactivators contains intrinsic ATPdependent DNA-unwinding activity required for efficient in vivo transcription. Coactivators increase the transcriptional activation of a promoter through its interaction with a sequence-specific DNA-binding protein, but it is not yet clear how the coactivator selects one group of promoters over another. Two concepts have been considered (332). For example, a promoter might need a “threshold level” of positive signals to be activated. Alternatively, some promoters might have a greater requirement for the presence of one coactivator than another. The precise mechanisms of transcriptional activation continue to evolve, and certain themes are emerging. In rare instances, positive or negative enhancer activity is dependent on a single DNA-binding protein that functions as a master switch to activate a family of related genes, for example, the myogenin MyoD family in muscle differentiation (333). However, further scrutiny of this model has indicated a large network of transcription factors that interact with non-DNA-binding complexes involved in chromatin remodeling, for example, histone acetyltransferase proteins p300 and CBP (334–337). Therefore, the more common mechanism implies that most cells respond to their environment by recruiting subsets of ubiquitous and promoter-specific transcription factors that combinatorially produce the desired cellular phenotype (204,338-341). Corepressors SMRT and N-CoR both recruit HDACs, yet they mediate activation downstream of different kinase cascades (342). In addition to the recruitment of classic HDAC-associated corepressors

(e.g., mSin3A and Groucho) (343–345), the runt-related transcription factor (RUNX) proteins exert gene silencing by associating with histone methyltransferases (e.g., SUV39H1) (346). Bifunctional attributes of transcription factors have been attributed to their regulated association with either coactivators or corepressors.

METHODOLOGY This section summarizes some of the molecular techniques used to study transcriptional control of genes. These methods are used to study either genetic structure or function. Three systems have been used to study function: reconstituted cell-free transcription assays, cell culture models, and whole-animal studies. Methods that analyze structural interactions include those techniques that assess DNA– protein interactions and those that assess protein–protein interactions.

Functional Methods Reconstituted Transcription Systems The most basic approach to the functional study of a gene is an in vitro transcription system in which the minimal components required for transcription are isolated and reconstituted to produce the gene product (347,348). mRNA is transcribed from cloned cDNA in the presence of radiolabeled nucleotides, RNA polymerase, and accessory factors isolated from nuclear extracts. The radiolabeled RNA synthesized in vitro is resolved by gel electrophoresis after extraction from the cell. Changes in basal levels of transcription are measured by quantifying the amount of newly synthesized RNA transcripts produced in the presence or absence of cloned or purified gene-specific DNA-binding proteins (349). In this way, differences in gene expression attributable to the activity of a purified transcription factor or enriched nuclear fraction may then be studied under tightly controlled assay conditions. Cell Culture Models The study of transcriptional regulation has been advanced greatly by the use of cell lines derived from the same tissues as the endogenous gene of interest. These cell lines have become the vehicles in which the study of gene expression is performed. Two major advantages of using cell lines are that they are homogeneous populations and they continue to divide in minimal culture conditions. However, in many situations, the cell lines are derived from neoplastic tissues, which may have lost the normal regulatory mechanisms that maintain the differentiated state. In a dedifferentiated state, cells tend to express a variety of genes outside of the repertoire expressed by their normal counterparts. Therefore, studies with cell lines always carry the caveat that they may not reflect activities of native cells.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 17 The use of cell lines permits the direct study of regulators of endogenous gene expression, avoiding the confounding effects of contaminating cell types. However, this approach does not permit alteration of the regulatory domains of genes to assess their contribution to transcription. Therefore, techniques have been developed to insert altered genetic material into cells by chemical, electrical, or viral mechanisms. In this way, specific elements controlling transcription can be isolated and studied. To tag the inserted gene, the promoter from which transcription will be initiated is ligated upstream of the coding sequences for a reporter gene, for example, chloramphenicol acetyltransferase, β-galactosidase, growth hormone, green fluorescent protein, or luciferase (9,350–353). The products of the reporter gene are easily measured, and spurious detection of reporter gene activity is kept to a minimum because their products are not normally expressed by most mammalian cells. Regulatory sequences to be analyzed are ligated upstream of a promoter with basal transcriptional activity in the test cell line. Taking advantage of various restriction sites, sequentially shorter 5′ flanking sequences are created, and each resulting construct is then tested by assaying the reporter gene product as an indicator of gene expression. Whole-Animal Models Whole-animal studies have been useful in assessing the contribution of transcriptional control to the regulation of several GI peptides, including gastrin, CCK, and somatostatin (354–356). Brand and Stone (357) showed that gastrin mRNA levels in the antrum increase under conditions of chemical or surgical achlorhydria and coincide with a reciprocal decrease in somatostatin mRNA. These observations are correlated with prior observations that gastrin plasma levels increase under conditions of achlorhydria (354, 358). Furthermore, infusion of the somatostatin analogue octreotide blocks the increase in gastrin mRNA (357). Walsh and coworkers (359,360) found that gastrin mRNA levels are predictably regulated by cycles of fasting and refeeding. Recently, infusion of the proinflammatory cytokine interferon-γ into mice has been used to recapitulate the effect of Helicobacter pylori infection on gastrin and somatostatin (361). Similarly, studies on the dietary control of CCK gene expression have been reported (355). Although such studies permit the linkage of transcriptional regulation to physiologic events, they do not allow dissection of the responsible regulatory elements.

genotypes on the overall phenotype may be amplified or abolished. In many situations, these alterations reproduce clinically relevant pathologic states (362–366). Chapter 53 provides specific details on transgenic technology including the powerful technique of homologous recombination. Cell-Based Knockout Strategies Once a genetic target is identified, whether DNA, RNA, or protein, the next step is to determine the significance of the molecule in a particular signaling, developmental, or neoplastic cascade. This usually is done by blocking, reducing, or removing the gene product at the cellular level before applying the extracellular signal. A change in the expected phenotype would confirm that the gene product makes a significant contribution. At the cellular level, the traditional approach has been to use small molecules, for example, pharmaceutical inhibitors. Once DNA vectors were developed in the early 1980s, antisense and dominant negative approaches to inhibit gene expression came into vogue (367). With the emergence of transgenic technology, it became apparent that one could remove the gene product through genetic manipulation specifically by homologous recombination to disrupt the gene in mice (362,368). With the discovery of snRNA molecules that interfere with either transcriptional initiation or translation, the commercial availability of synthetic “interfering” RNA molecules has emerged (369,370). High-throughput methods using RNA silencing are now being used to complement the gene discovery methods of DNA microarray technologies (371). Nevertheless, RNA interference technology, although relatively easy to use, does not eliminate the gene product as effectively as direct gene targeting. Therefore, genetic methods must be used to generate a complete null cell line. Cell lines are either created from a null mouse model (e.g., embryonic fibroblasts), or somatic cell gene targeting can be performed in the cell line of choice (372,373). The advantage of creating the null cell line from a mouse is that the cells will be from normal tissue and not a tumor cell line. However, unless molecules are introduced to immortalize the cells, the lines are not permanent. Gene targeting in a somatic cell line has not been as widely used because of the difficulty in performing the technique, but it is a powerful approach that permits the study of a null locus without incurring the expense of mice.

Structural Methods Transgenic Animals Through transgenic animals it is possible to introduce genetic information into the mouse genome such that there is permanent alteration of the genetic makeup in both the founder line and successive generations (13,14). Transgenic studies afford the opportunity to study the importance of specific genetic sequences in cell, organ, and whole-animal function. By breeding mice with different transgenic lineages, the interaction between these artificially produced

Once functional regulatory DNA elements have been identified, assays that assess DNA–protein interactions are performed (374). Indeed, in circumstances where a long sequence (>50 bp) must be analyzed, it is simpler to identify DNA–protein interactions first, and then determine whether these DNA elements are involved in transcriptional regulation. DNase I footprinting assays are used to identify DNAbinding elements that interact with crude or purified nuclear proteins by protecting them from chemical or enzymatic

18 / CHAPTER 1 cleavage (375,376). Such assays are particularly well suited for studying cooperative interactions among proteins bound to adjacent DNA elements. The technique can be performed in vivo or in vitro (9). However, in vivo footprinting has been superseded by chromatin immunoprecipitation (ChIP) assays (see the next section). Electrophoretic gel mobility shift assays (EMSAs; gel shift, gel delay, or band-shift assays) permit a more detailed analysis of the following: (1) the type of protein complexes that bind to individual DNA elements, and (2) the specificity of the protein interaction with a specific base pair (377–379) (Fig. 1-9). This assay system is also rapid and easier to use than footprinting assays. Methylation interference assays extend the power of the gel shift assay by identifying specific nucleotide contacts that are required for DNA binding (380). DNA affinity precipitation is a DNA– protein interaction assay that uses the biotinylated DNA binding site to identify the proteins that are recruited to the element (381). The assay uses the DNA element to isolate the protein factors, coupled with immunoblots to identify the proteins that form both the protein–DNA and protein– protein interactions. Southwestern blot analysis takes advantage of specific DNA elements that are used to detect nuclear proteins separated on a denaturing gel and transferred to nitrocellulose or produced by a phage expression library (382–384).

Chromatin Immunoprecipitation Assays ChIP analysis is now the most effective method to document an in vivo interaction at DNA (385–387). First, a fixative, usually formaldehyde, is used to cross-link proteins to DNA. Then antibodies are used to immunoprecipitate the DNA-binding proteins. After a series of extractions to remove the protein from DNA, specific primers are used to PCR amplify the DNA-binding element precipitated with the protein and antibody. Variations of this method are used to identify the in vivo preferred binding sites of known DNA-binding proteins. Alternatively, the immunoprecipitate is resolved on a sodium dodecyl sulfate gel, and mass spectroscopy can be used to identify the proteins that coprecipitate and are likely involved in protein–protein interactions with the DNA-binding proteins. The technique completely depends on the quality of the antibodies, the quantity and quality of genomic DNA precipitated, and primer specificity. ChIP assays complement in vitro DNA–protein interaction assays such as EMSAs or footprinting. Expression vectors or cell-based knockout strategies using dominant negative constructs, antisense technology, or RNA interference may be used to demonstrate functional significance (388). These approaches are rapid and useful to perform before using transgenic mouse approaches.

5 ′ GTCCCTCAGGGGCGGGGATCTAT

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FIG. 1-9. Electrophoretic mobility shift assay (EMSA, gel shift). A DNA element ~30 to 100 bp in length is labeled, and then is incubated with crude nuclear extract or purified protein. A band on the autoradiogram is detected if the radiolabeled probe is retarded and does not migrate to the bottom of the gel. The specificity of binding is determined by competing with unlabeled DNA sequences. Competitor 1 is related to the probe sequence, whereas Competitor 2 is unrelated to the probe sequence.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 19 Microarray Technology The latest method to comprehensively analyze gene expression is by microarray technology. At the transcription level, DNA array technology increases by several orders of magnitude the number of genes that can be examined simultaneously under different conditions (389–391). The number of genes that are either stimulated or inhibited under various conditions can be studied simultaneously with the limitations being the number of genomic sequences that are spotted on the glass slide. A glass slide is able to hold the genomic sequences of 25,000 to 30,000 genes, which is the current estimate of the total number of genes in the human genome. Two types of arrays are available: EST/cDNA and oligonucleotide (Affymetrix, [Santa Clara, CA]) based. The EST microarray chips use expressed sequence tags that are fragments of DNA corresponding to segments of the genome that encode mRNA. The Affymetrix gene chips spot commercially designed oligonucleotide sequences. These DNA fragments are subsequently “arrayed” onto glass slides. In most instances, several regions of the genomic sequence unique to that gene are spotted in multiple copies to ensure reproducibility. Different genetic domains are plated because of differences in hybridization affinity. RNA is isolated from cells or tissue after treatment with an extracellular molecule or from cells at different stages of development or transformation. cDNA are then generated and tagged fluorescently, then hybridized under stringent conditions to the DNA arrayed on the glass slide followed by analysis by a special plate reader. Computer-generated algorithms are required to interpret the fluorescent signals and rank the degree of change from baseline fluorescence. The technology is being used to study the gene expression pattern found in various tissues at designated stages, for example, developmental or transformation stages (392–394). The significance of the findings must be confirmed by alternative methods including Northern blot analysis or quantitative PCR. Proteomics Analogous high-throughput approaches have been developed to study protein modifications (395). However, the techniques used to detect protein posttranslational modifications are more complex and use more labor-intensive technology. Protein is extracted from the cell or organelle of interest and resolved by two-dimensional gel electrophoresis, in which proteins are separated by both size and ionic charge (along a pH gradient). The proteins are visualized with a dye either directly on the gel or after transfer to a paper substrate. Both substrates (gel or paper) can be used for further analysis. However, proteins transferred to a paper substrate permit several options for analysis. Resolved proteins that are transferred to paper can be submitted for analysis with an antibody (immunoblot) that might recognize phosphorylated or acetylated peptides. Differences in the size of the spot corresponding to the amount of a particular protein version (phosphorylated, acetylated) can be

quantified by computer. Proteins that cannot be identified by antibody can be analyzed by mass spectroscopy. Therefore, proteomic studies allow the monitoring of regulatory changes that occur because of posttranslational modifications and quantification for large numbers of proteins simultaneously. Taking advantage of the technology used to develop DNA arrays, companies are now developing protein arrays that will be applied to new drug discovery (396).

TRANSCRIPTIONAL CONTROL OF GASTROINTESTINAL PEPTIDES Although knowledge in the transcriptional control of GI peptides has accelerated over the last several years, the field is still hampered by the paucity of gut-derived cell lines that express regulatory peptides. The problem has been circumvented somewhat through the use of neural and endocrinederived hormone-producing cell lines, but application of data obtained with these models to the gut requires assumptions that may not be accurate. Future work in this field will be assisted greatly by the application of high-throughput and transgenic technologies and the development of immortalized and transformed cell lines using in vitro DNA transfer techniques. An overview of what has been accomplished with respect to specific GI peptides can be found primarily in Chapters 4 through 6. Nevertheless, a few peptides deserve brief mention. To date, most studies of the transcriptional control of peptide hormones have focused on somatostatin and vasoactive intestinal peptide because they are expressed in islet or neural-derived cell lines (397–400). The downside of this is that little is known about how somatostatin is regulated in gut-derived tissues; for this reason, the peptide should become a priority for future transcriptional control studies in the GI tract. Studies on the transcriptional control of gastrin have been slow for similar reasons and have been reviewed recently (401). Information on the transcriptional control of secretin and CCK has increased because of the use of transgenic mouse models (25,402–404).

POSTTRANSCRIPTIONAL PROCESSING Polyadenylation Three major events occur at the end of transcription: (1) The poly(A) tail is added, (2) adenine bases are methylated, and (3) hnRNA is processed by removing introns before exiting the nucleus (see Fig. 1-2) (405). All mRNA, except those encoding most histone proteins, have poly(A) tails. The length of the poly(A) tail that is added ranges from 200 to 250 bp and is quite uniform among eukaryotic organisms. Once the transcript reaches the cytoplasm, the length of the poly(A) sequence decreases with the age of the transcript (406). Thus, polyadenylation contributes to mRNA stability and translational activation, processes that also involve a synergistic interaction with the cap site (407–409).

20 / CHAPTER 1 Because there is no poly (dT) sequence within DNA, addition of the poly(A) tail represents a posttranscriptional modification of the newly synthesized mRNA. The AATAAA site in DNA is transcribed as AAUAAA and signals endonuclease cleavage of hnRNA ~20 bp after this RNA element (410). Several factors are required for specific recognition of the AAUAAA element before the addition of adenylate residues by poly(A) polymerase (411,412). Polyadenylation occurs in two phases: (1) an AAUAAA-dependent phase marked by addition of the first 10 residues, and (2) an AAUAAA-independent phase marked by rapid elongation and catalyzed by a poly(A)-binding protein (413). In addition, endonuclease cleavage of polyadenylated histone H1 transcripts have also been shown to require the presence of small nuclear ribonucleoproteins (U7 snRNP, pronounced “snurp”), which are trans-acting factors that participate in RNA splicing reactions (414). Transcription can proceed for up to 2 kb past the polyadenylation site and may terminate prematurely 30% of the time. Adenylate residues within exons are methylated at the sixth nitrogen and are thought to serve a protective role for those sequences that will eventually be translated (415). It is now known that formation of the PIC is linked to the assembly of factors involved in polyadenylation (416).

RNA Splicing The Spliceosome Soon after the termination of transcription, most vertebrate hnRNA (pre-mRNA) will be posttranscriptionally processed after exiting the nucleus into a form that can be translated (see Fig. 1-2). This involves removing intervening sequences that in some transcripts contain transcriptional regulatory signals (cis-acting elements). Splice sites are identified by comparing the genomic sequence with the cDNA prepared from an RNA template. The cis-acting elements within the intron that regulate RNA splicing are GU (GT in the genomic sequence) at the 5′ splice border, AG at the 3′ splice border, and a pyrimidine-rich element that defines the area of the branch point 20 bp upstream from the 3′ splice junction (Fig. 1-10). The branch point lies just upstream of the pyrimidine-rich region (PyPy)n and is a highly conserved sequence in yeast (UACUAAC) but much less so in vertebrates. Five snRNA-U1, U2, U5, U4, and U6-combine with subsets of about 10 different proteins to form small nuclear ribonucleoproteins (snRNPs) (417,418). The snRNA, ranging in size from 56 to 217 nucleotides, are quite abundant in the nucleoplasm and contain a trimethylguanylate cap. Some proteins are components of all five major snRNPs, whereas others are unique to one snRNP. The U7 snRNP, which is present in low concentrations, participates in the 3′ posttranscriptional processing of hnRNA [poly(A)] (419). The five major snRNPs assemble into large multicomponent complexes called spliceosomes to perform the splicing reactions (420). There reactions occur in three steps: cleavage

at the 5′ exon-intron border with formation of a branch point, excision of the branch point as a lariat, and joining of the exons. Splice site selection can be influenced by subtle changes in flanking exon sequences (421–423). The basic steps in RNA processing illustrated in Figure 1-10 are as follows (419): U1 snRNP binds in a sequence-specific manner to the 5′ exon-intron junction of capped pre-mRNA (424). An U2 snRNP accessory factor (U2AF) then binds to the pyrimidine-rich element before sequence-specific recognition of the branch point element by U2 snRNP (425,426). The 5′ exon is released by cleavage of the 5′ exon junction. This allows the freed 5′ guanylate residue to form a phosphodiester bond at the 2′ site of an adenylate residue within the branch point. U4 and U6 snRNPs are paired together by complementary bases and function as a single snRNP complex (427). The recruitment of the U4/U6 snRNPs to the spliceosome is essential to the last excision step and final removal of the intron from the pre-mRNA. U4/U6 snRNP cooperates with the U2 branch point complex without direct contact with RNA (428). U5 snRNP binds just upstream of the 3′ splice junction to initiate cleavage of the 3′ intron border. Finally, the intron is removed as a lariat and the two exons are joined. More recent evidence indicates that small RNA catalyze the splicing reactions without the presence of specific enzymes (429,430). As observed for polyadenylation, the splicing events coincide with transcriptional events (431). It is therefore somewhat surprising that the events involved in splicing are not better understood. Nevertheless, with the understanding that the complexity of the human genome lies beyond the DNA sequence and at the level of epigenetics and alternative splice products, the next decade will likely witness heightened attention to this additional nuclear process (431,432). Alternative Splicing Eukaryotic cells have applied the mechanics of RNA splicing to generate the protein diversity necessary to meet their multiple demands. Thus, in contrast with the original definition of a gene in which only one transcript is produced, complex genes can generate multiple protein isoforms from multiple RNA transcripts through alternative splicing (433). This can be achieved by altering which introns and exons are included in or excluded from the mature mRNA transcript that is used as the template for peptide chain elongation. Accordingly, the definition of introns and exons for each gene is actually a fluid concept because an intron for one gene product may become an exon within another transcript. Alternative splicing is a mechanism used by many protein classes, including muscle-related genes, hormones, and transcription factors (434–438). Regulated Posttranscriptional Mechanisms In addition to cis-acting DNA elements, the cis and trans models of regulation also occur at the posttranscriptional level (439). Ferritin and the transferrin receptor (TfR),

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 21 Pre-mRNA

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FIG. 1-10. Ribonucleic acid (RNA) splicing reactions. First, small ribonucleoproteins (snRNPs, pronounced “snurps”) and accessory factors (U2 accessory factor [U2AF]) bind in a sequencespecific manner to the branch point and intron-exon borders. Second, the 5′ exon-intron border is cleaved, and a “lariat” is formed by the free end of the intron at the branch point. Third, the 3′ intron-exon border is cleaved, the exons are joined, and the excised intron is removed in the form of a lariat.

which regulate the storage and uptake of iron, are the best known examples of regulated posttranscriptional control (440). Cis-acting RNA elements, responsible for conferring iron regulation on both proteins (iron-response elements [IREs]), reside in the 5′ UTR and 3′ UTR of ferritin and TfR mRNA transcripts, respectively. The same iron-binding protein (IRE-BP) that binds to the IRE in the 5′ UTR of ferritin to block translation can also bind to the 3′ UTR of TfR to block mRNA degradation (439,441,442). Therefore, regulation of iron homeostasis ultimately depends on posttranscriptional mechanisms that either block translation or increase mRNA stability.

TRANSPORT ACROSS THE NUCLEAR MEMBRANE As noted earlier, RNA is synthesized initially as a much larger primary transcript molecule that in many instances undergoes posttranscriptional modification (e.g., splicing,

degradation). However, for any mature RNA transcript to be translated, it must be transported from the nucleus to the cytoplasm. In contrast, nuclear regulatory proteins are translated in the cytoplasm and are eventually returned to the nucleus, either immediately after synthesis or after a dormant state from which they are activated in response to signals (443). This bidirectional shuttling of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex, a specialized compartment of the nuclear membrane regulated by a group of transport receptors called karyopherins. Both import and export processes through the nucleus require energy in the form of the Ras-related GTPase Ran and specific targeting signals on the cargo to be transported (nuclear localization and export signals) (444). The three-dimensional structure of the nuclear pore complex shows a doughnut-shaped structure comprising eight subunits (445). From the eight subunits emanate spokelike structures that radiate inward to form a central plug (446, 447). The cytoplasmic surface of the nuclear pore complex (NPC) is closely associated with ribosomes. Its nuclear

22 / CHAPTER 1 surface is thought to participate in the organization of the genome by binding to specific DNA sequences within transcribed genes with products that may be destined for export from the nucleus (gene-gating hypothesis) (448).

CONCLUSION With the dawn of the postgenomic era on us, our next challenge is to apply the volumes of available genetic, molecular, and cell biological information to tackle questions of GI physiology and development. To accomplish this task and make optimal use of past, ongoing, and future discoveries, physiologists will need to acquire the basic vocabulary of several disciplines including bioinformatics. It is our hope that this chapter has laid the initial foundation necessary to understand those aspects of physiology that pertain to transcriptional control.

ACKNOWLEDGMENTS The work was supported by Public Health Service National Institutes of Health grants DK 55732 and DK 45729 to Dr. Merchant. The authors thank Gail Kelsey and Colleen Hill for assistance with the preparation of both the manuscript and the figures.

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CHAPTER

2

Translation and Posttranslational Processing of Gastrointestinal Peptides Cheryl E. Gariepy and Chris J. Dickinson Translation, 31 Initiation, 32 Regulation of Initiation, 32 Elongation, 34 Termination, 34 Localized Translation Regulation, 34 RNA Silencing, 34 Other Regulators of Messenger RNA Stability, 35 Posttranslational Processing, 35 Transport into the Endoplasmic Reticulum, 35 Signal Peptides, 36 Signal Recognition Particle, 36 Signal Recognition Particle Receptor, 37 Endoplasmic Reticulum Membrane Protein Channel or Translocon, 37 Processing in the Endoplasmic Reticulum, 38 Signal Peptidase, 38 Disulfide Bond Formation, 38 Asparagine-Linked N-glycosylation, 39

Protein N-myristoylation, 39 Protein Folding, 39 Transport from the Endoplasmic Reticulum and through the Golgi, 39 Processing Reactions in the Golgi, 41 Serine- or Threonine-Linked O-glycosylation, 41 O-phosphorylation, 41 Tyrosine Sulfation, 41 Formation of Secretory Vesicles, 42 Processing Reactions in the Secretory Vesicle, 42 Acetylation, 42 Dibasic Cleavage, 42 Monobasic Cleavage, 44 Carboxypeptidases, 45 Aminopeptidases, 46 Glutaminyl Cyclase, 47 Amidation, 47 Posttranslational Processing of Preprogastrin, 48 References, 51

TRANSLATION

as well as energy in the form of guanosine triphosphate (GTP) and adenosine triphosphate (ATP). To be translated into protein, mRNA must contain, in addition to a string of codons, information that specifies nuclear export, translation, and stability. Much of this information is communicated by specific RNA-binding proteins. These proteins first associate with pre-mRNA (primary transcripts of genomic DNA-containing exons and introns) cotranscriptionally and undergo a dynamic series of rearrangements involving the binding and dissociation of numerous proteins throughout the life of mRNA. The mRNA nucleoprotein complex (mRNP) communicates information to the cytoplasm about the structure of the gene from which the mRNA was formed and the processing steps experienced by the mRNA. The mRNP therefore carries significantly greater information than the sequence of the mRNA itself (1).

Translation is the complex process by which a sequence of codons of messenger ribonucleic acid (mRNA) directs the synthesis of a polypeptide chain. Beyond the sequence of codons, the mRNA contains untranslated regions (UTRs) with structural and regulatory sequences that determine its translational fate. Translation involves hundreds of molecules including mRNA, transfer RNA (tRNA), ribosomal RNA, activation enzymes, and many RNA-binding proteins,

C. E. Gariepy and C. J. Dickinson: Division of Pediatric Gastroenterology, University of Michigan, Ann Arbor, Michigan 48109. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

31

32 / CHAPTER 2 Initiation

Regulation of Initiation

Translation requires the positioning of an elongationcompetent 80S ribosome at the initiation codon (AUG). Binding of the small (40S) ribosomal subunit at the 5′ end of the mRNA is rate limiting and requires energy in the form of ATP. It then forms a 43S preinitiation complex with eukaryotic initiation factors (eIFs) 3, 1, 1A, and 5, and a ternary complex including the methionine-loaded initiator tRNA that will recognize the AUG codon and eIF2 that is coupled to GTP. The preinitiation complex recognizes the mRNA by the binding of eIF3 to the eIF4 protein complex associated with the 5′ cap structure (eIF4F) (2). The eIF4F protein complex contains an enzyme (eIF4A) that unwinds RNA duplexes, allowing the 43S complex to bind and scan the mRNA, and a scaffold protein (eIF4G) that serves as a platform for the assembly of other proteins and interacts with the poly(A)binding protein. This interaction is thought to loop the mRNA and bring the 3′ UTR in close proximity to the 5′ end of the mRNA (3). This provides a means by which sequences in the 3′ UTR can regulate translation initiation. Most known translational regulatory sequences are found within the 3′ UTR. The 43S complex recognizes the initiation codon through the formation of base pairs (bp) between the initiator tRNA and the start codon. Subsequently, eIF2-bound GTP undergoes hydrolysis, a reaction that is necessary for the 60S ribosomal subunit to join the initiation complex. This appears to release most of the initiation factors from the small ribosomal subunit, leaving the initiator tRNA associated with the ribosome (in the P site). Formation of the 80S initiation complex capable of catalyzing the formation of a peptide bond occurs with the hydrolysis of a second molecule of GTP on eIF5B (Fig. 2-1A).

Although the specific translational regulatory mechanisms active in peptide hormone synthesis are not yet clear, translation is generally controlled at the initiation step where regulation may be global or mRNA specific. Global control of mRNA translation generally occurs through changes in the phosphorylation state of initiation factors or regulators that interact with them. Proteolytic cleavage of translation factors can also reduce translation of all mRNA species within the cell. mRNA-specific regulation of translation can be achieved by steric blockage, interference with the eIF4F complex, and cap-independent inhibition of the early initiation steps. Steric blockage refers to the binding of regulatory proteins to message-specific response elements that results in insufficient space for the binding of critical initiation complex proteins (4–7). Interference with the eIF4F complex is achieved by mRNA-specific binding proteins that block eIF4E recognition by eIF4G (8–11). Cap-independent inhibition of translation refers to proteins that bind to specific sites in both the 5′ and 3′ UTRs and recruit corepressors to the 3′ UTR. This affects stable association of the small ribosomal subunit with the mRNA (12–14). Translation can also be controlled later in the initiation process. RNA-binding proteins have been described that prevent the binding of the 60S ribosomal subunit to the 40S subunit at the initiation codon, apparently through interference with initiation factors (15). The existence of more than one open reading frame on an mRNA and the sequence distance between the open reading frames can also play a significant role in determining the likelihood of translation. For example, amino acid deprivation reduces global protein

FIG. 2-1. Translation of messenger RNA (mRNA) into protein, highly simplified. Some translation-initiation factors are omitted (see Preiss and Hentze [526], Ramakrishnan [527], and Hershey [528] for more complete descriptions). (A) Cap-mediated initiation: The methionine-containing ternary complex (methionine-loaded transfer RNA [tRNA], eIF2, and GTP) binds to the 40S ribosomal subunit and other initiation factors (eukaryotic initiation factor-1 [eIF1], 1A, 3, and 5) to form the 43S preinitiation complex. The preinitiation complex recognizes the mRNA through the binding of eIF3 to eIF4 in the cap-binding complex. The cap-binding complex contains eIF4A, an RNA helicase that unwinds the secondary structure of the mRNA during the subsequent scanning step. The cap-binding complex also contains eIF4G, which contacts the poly(A)-binding protein (PABP). This contact is thought to bring the 3′ region of the mRNA in close proximity to the 5′ cap. The 43S preinitiation complex scans the mRNA from 5′ to 3′ until the initiation codon, AUG, is encountered. Stable binding of the preinitiation complex to the AUG codon yields the initiation complex. Subsequent joining of the 60S ribosomal subunit results in the formation of the 80S initiation complex. AUG recognition and the joining of the 60S ribosomal subunit both trigger GTP hydrolysis. The 80S complex contains an aminoacylated initiator tRNA in the P site of the ribosome and an empty A site. It now is competent to catalyze the formation of the first peptide bond. (B) Elongation: A ternary complex containing aminoacylated tRNA and the correct anticodon is brought into the A site of the ribosome. Codon–anticodon recognition leads to guanosine triphosphate (GTP) hydrolysis. This allows for conformational changes within the tRNA and the ribosome. Peptide bond formation (deacylation of the P site tRNA and the transfer of the peptide chain to the A site tRNA) then occurs. Translocation of the tRNA and the mRNA is facilitated by a GTPase, eukaryotic elongation factor-2 (eEF2). The ribosome is then ready for the next round of elongation, with a deacylated tRNA in the E site, peptidyl tRNA in the P site, and an empty A site. (C) Termination: When a stop codon on the mRNA is encountered in the A site, eukaryotic release factor-1 (eRF1) binds to the ribosome A site and triggers the release of the peptide chain from the tRNA in the P site. eRF3 then binds GTP and promotes dissociation of eRF1 from the ribosome. Hydrolysis of GTP is required for subsequent release of eRF3. The ribosome is then left with mRNA and a deacylated tRNA in the P site. The ribosomal releasing factor, together with eEF2 and GTP, is required to disassemble the complex and prepare the ribosome for a new round of protein synthesis. Much of the mechanism of mRNA, translation factor, and subunit release after peptide chain termination remain to be determined. GDP, guanosine diphosphate.

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 33 Met

Met GTP

IF 1,1A,3,5

2

GTP 2

U A C

U A C

40S 43S pre-initiation complex 3′ AAAAAAAAAAAAAAAA ATP

PABP

Met

ADP + Pi GTP

IF4F U A C CAP

5′

AU GC G AU A G

ATP

Scanning

3′ AAAAAAAAAAAAAAAA

2 GDP +2Pi

2 GTP

ADP +Pi PABP

Met

Met

GTP

IF4F U A C CAP

5′

E site P site A site

AUG

U A C C G AU A G

eIF5B + 60S subunit

AUG

C G AU A G

80S initiation complex

A GDP

GTP

Arg

EF-1A

GTP

EF-2

EF-2

GDP +Pi

EF-1A

GCU

Met

Met Arg

Arg

Met Arg GTP

UA C GC U A U G CG AU

AG

EF-2

UA C GC U

UA C GC U

A U G CG A U A G

A U G CG A U AG

B Met Arg

RF-1

RF-1

Met Arg

Met Arg

GTP RF-1 RF-3

RF-1 RF-3 UA C GC U A U G C GA U A G

GC U

GC U

A U G C GA U A G

A U G C GA U A G

RF-3

GTP

RF-3 GDP +

Pi

Factor, tRNA and mRNA release GDP + Pi

GTP RF-3

A U G C GA U A G Ribosomal recycling factor GTP

C

EF-2

+ Pi

34 / CHAPTER 2 synthesis by phosphorylation of eIF2a, which blocks GDPGTP exchange and reconstitution of the functional ternary complex. Paradoxically, the same modification increases the translation of some mRNA that have upstream open reading frames. It appears that the 60S ribosomal subunit dissociates at the stop codon of the first open reading frame and the 40S subunits remain associated with the mRNA and resumes scanning. The 40S subunit must acquire an active ternary complex during scanning to translate downstream open reading frames. The probability of translating the most 3′ open reading frame therefore depends on the distance (scanning time) between the open reading frames and the availability of amino acids within the cell (16). Internal ribosome entry sites (IRESs) mediate translation initiation independent of the cap structure by recruiting the ribosome directly to an internal position of the mRNA (17). Both structural features and short-sequence elements appear to be involved in ribosome recruitment in eukaryotic IRESs. Exactly how these motifs combine to promote internal initiation remains to be determined. The IRES appears to be a complex RNA scaffold that contains multiple sites for interaction with components of the translational apparatus. Structural domains have been identified that interact with the initiation factors eIF4G and eIF4B (18,19), with eIF3 (20,21), or directly with the 40S ribosome subunit at multiple sites. An IRES is also described that can assemble an 80S ribosome at its initiation codon without the aid of any initiation factors or an initiator tRNA (22). A growing body of evidence exists to support the hypothesis that cellular IRESs are involved in the regulation of gene expression under physiologic conditions during which the efficiency of cap-dependent protein synthesis is greatly reduced. IRESs enable cells to respond to these conditions against the background of a general reduction in protein synthesis.

Elongation Each ribosomal subunit has three binding sites for tRNA: designated the A (aminoacyl) site, which accepts the incoming aminoacylated tRNA; P (peptidyl) site, which holds the tRNA with the nascent peptide chain; and E (exit) site, which holds the deacylated tRNA before it leaves the ribosome. The end of the initiation process leaves an aminoacylated initiator tRNA in the P site of the ribosome and an empty A site, which serves to start the elongation process. Aminoacylated tRNA is brought into the A site as a ternary complex with eukaryotic elongation factor-1A (eEF1A) and GTP. Correct codon-anticodon interactions result in conformational changes in the ribosome that stabilize tRNA binding and trigger GTP hydrolysis by eEF1A. This leads to the release of the aminoacyl end of the A site tRNA by eEF1A; the tRNA then swings into the peptidyl transferase site of the large subunit in a process called accommodation. The peptide bond is formed through deacylation of the P site tRNA and the transfer of the peptide chain to the A site tRNA. The ribosome then has a deacylated tRNA in the

P site and peptidyl tRNA in the A site. Translocation of tRNA and mRNA is facilitated by eEF2, which is also a GTPase. The ribosome is then ready for the next round of elongation, with deacylated tRNA in the E site, peptidyl tRNA in the P site, and an empty A site ready to receive the next cognate ternary complex (see Fig. 2-1B).

Termination Termination begins when a stop codon (UAA, UGA, or UAG) is encountered in the A site mRNA. Stop codons are recognized by eukaryotic release factor-1 (eRF1). The GTPase eRF3 then binds the complex of eRF1 bound to the ribosome. Binding of eRF1 to the ribosome at the stop codon A site triggers the hydrolysis and release of the peptide chain from the tRNA in the P site. Hydrolysis of peptidyl tRNA by eRF1 is required for binding of GTP to eRF3 on the ribosome. This, in turn, leads to a conformational change in eRF3 that has high affinity for ribosomes and the dissociation of eRF1 from the ribosome. Hydrolysis of GTP is required for subsequent dissociation of eRF3 from the ribosome (see Fig. 2-1C) (23,24).

Localized Translation Regulation In addition to regulation of the initiation process, mRNAspecific translation regulation also occurs regionally in polarized cells. This is clearly demonstrated in neural tissues where stimulation of synapses induce the polyadenylation and translation of cytoplasmic polyadenylation elementcontaining, but not cytoplasmic polyadenylation elementlacking, mRNA stored in dendrites (25). This allows the generation of protein gradients emanating from particular positions in cells or the restriction of protein expression to a specific region and is a potential mechanism by which a cell may modulate its response to repeated, directional stimuli.

RNA Silencing Small RNA molecules regulate mRNA-specific translation either by translational repression, in the case of microRNA (miRNA) (26–28), or by mediating the degradation of the target mRNA, in the case of small interfering RNA (siRNA) (29,30). The functional difference between miRNA and siRNA (both about ~22 nucleotides in length) depends on the degree of complementation between the small RNA molecule and the mRNA target (31,32). miRNA hybridize by incomplete base paring, usually to several sites in the 3′ UTR of target mRNA. siRNA show perfect complementation to the target mRNA. miRNA and siRNA have distinctly different origins within the nucleus, but they have common RNA-binding proteins (33). It is unclear whether a single type of small RNA-protein complex can mediate both

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 35 target mRNA cleavage and translational inhibition (see review by Sontheimer [34]).

Other Regulators of Messenger RNA Stability Regulation of the rate of decay is an important control point in determining the abundance of an mRNA species, and decay rates of individual mRNA differ widely and can be differentially affected by environmental cues. Several sequence elements can regulate the rate of turnover of a transcript by attracting specific binding proteins that can either destabilize or stabilize the transcript. The strength of the association of these binding proteins can be modified by changes in the cellular environment. The principal mRNAdegradation pathway begins with removal of the 3′ poly(A) tail. Interaction of the cap proteins and the poly(A)-binding proteins with the translational machinery likely protects the 5′ and the 3′ end of the mRNA from attack by deadenylases and decapping enzymes (35,36). This means that translation and mRNA decay are linked. Support for this comes from studies demonstrating that inhibition of translation initiation destabilized mRNA (37) and inhibition of translation elongation (with cycloheximide) promotes mRNA stability (38). The nonsense-mediated decay pathway further links translation to mRNA turnover. This pathway ensures that mRNA with premature stop codons are not translated. To be recognized as premature, a termination codon must lie upstream of the last intron (39–43). Exon-exon junctional complex proteins, which mark the position of exon-exon junctions in the mature mRNA, may play an important role in surveillance for potentially deleterious nonsense mutations (44).

POSTTRANSLATIONAL PROCESSING Although it would appear that the translation of polypeptide hormones is similar to that of other eukaryotic proteins, the posttranslational processing of prohormones is unique. Since the initial discovery of proinsulin (45), it has been evident that the synthesis of polypeptide hormones of the gut involves a series of modification steps after the initial translation of the gene product that are distinct from the biosynthesis of other cellular proteins. These modifications, achieved via a variety of posttranslational processing reactions, may enlarge or diminish the size of the peptide precursor, but, in general, they result in the formation of biologically active and physiologically relevant products. Efforts to determine the nature and mechanisms of peptide hormone posttranslational processing reactions were greatly facilitated by the development of molecular biological techniques that permitted the deduction of peptide precursor sequences. Information on precursor structure has led to the development of molecular probes that can be used to characterize individual processing reactions, as well as patterns of processing reactions for groups of related peptides. Application of these probes to ultrastructural studies has provided

information on the cellular compartments in which processing reactions take place. In vitro reconstitution experiments have led to the elucidation of some of the mechanisms responsible for the transport of peptide precursors between cellular compartments. Development of techniques to isolate and culture functionally intact peptide-secreting cells has permitted physiologists to examine the sequence and dynamics of the complete posttranslational modification and activation process for given peptides. Many of the enzymes responsible for prohormone processing have now been isolated. Coexpression or deletion, or both, of these enzymes within cells has allowed for elucidation of their activities for multiple prohormone substrates. Previously, it was thought that proteins exited from cells via two distinct pathways: the constitutive or the regulated secretory pathways (46,47). As has been the case with other biological systems, more recent evidence suggests that there might be overlap between these pathways (48). Generally, however, the constitutive pathway is reserved for those secreted proteins that are not stored in the cell and usually do not undergo extensive posttranslational processing, as seen with the products of fibroblasts and hepatocytes. Proteins secreted constitutively exit the cell soon after synthesis on the ribosome. Polypeptide hormones, however, enter the regulated pathway of secretion in most neuroendocrine cells. These cells are capable of storing secretory products for hours or days in electron-dense secretory vesicles and releasing them on stimulation. The intracellular pathways and organelles involved in this pathway were first defined in studies (49) in the exocrine pancreas demonstrating that polypeptides are initially synthesized on the rough endoplasmic reticulum (ER), transported to the Golgi apparatus, and finally placed into secretory granules (Fig. 2-2). On cell stimulation, these secretory granules or vesicles fuse in a calcium-dependent manner with the cell membrane to release their contents into the extracellular milieu. This chapter reviews the enormous progress made in recent years in elucidating the mechanisms for posttranslational processing of gastrointestinal peptide hormones, and then presents a detailed analysis of one hormone, gastrin.

TRANSPORT INTO THE ENDOPLASMIC RETICULUM Polypeptide hormones are synthesized ribosomally from the amino-terminal end and enter the secretory pathway via translocation into the ER. This process is of critical importance to both prokaryotes and eukaryotes. Thus, it has been thoroughly examined by several notable scientists including Gunther Blobel, who won a Noble Prize for his work in this area (50). In summary, the first few amino acids of the preprohormone, translated from the leader sequence of the specific mRNA, are called the signal peptide (Fig. 2-3) (51). This peptide (designated as the presequence in preprohormones) is not secreted under normal circumstances but serves as a means of translocating the newly synthesized and

36 / CHAPTER 2 CYTOPLASM Intiation of protein synthesis

ER Completion of precursor synthesis Cleavage of signal peptide Disulfide bond formation Initial glycosylation

GOLGI Further glycosylation Phosphorylation Sulfation

SECRETORY VESICLE Acetylation Dibasic cleavage Monobasic cleavage Carboxypeptidase Aminopeptidase Glutaminyl cyclase Amidation

SECRETION

FIG. 2-2. Intracellular location of posttranslational processing steps. ER, endoplasmic reticulum.

gradually elongating polypeptide chain into the ER (52–54). After emerging from the ribosome, the signal peptide binds to the signal recognition particle (SRP) in the cytoplasm after chain elongation has produced a preprohormone of approximately 50 to 60 amino acids (55–57). This binding results in an arrest of translation, and the SRP initiates the translocation of the nascent polypeptide by binding to the SRP receptor or docking protein located on the cytosolic side of the ER (58–60). The SRP is then released, and the translocation of the peptide continues through a protein channel across the ER membrane (53,59,61–63). The signal peptide is later cleaved by a specific enzyme (signal peptidase) located on the inner membrane of the ER (64). The individual components of these ER translocation events are described in more detail in the next section.

characteristics are shared: (1) a positively charged aminoterminal region of 1 to 10 amino acids, (2) a central hydrophobic region of 7 to 17 amino acids, and (3) a more polar region that often contains an α helix breaking proline or glycine residue, as well as uncharged residues that determine the cleavage site and complex pattern of amino acids adjacent to the site of cleavage between the signal peptide and the prohormone (65,66). The secondary structure of these peptides can assume several different conformations including α helices and β-pleated sheets, depending on the environment (67). Recently, analysis of the new, extensive protein databases has allowed investigators to accurately predict signal peptides (68). The positively charged amino terminus appears to be important in the release of the SRP once docking of the nascent peptide to the ER has occurred. Mutations in this area that result in a net negative charge interfere with both export and synthesis of secretory proteins in prokaryotes (69), although this does not appear to be the case in eukaryotic systems (70). Mutations that substitute polar or charged amino acids for the amino acids present in the hydrophobic region of the signal peptide result in impaired binding of the nascent peptide chain to the SRP (57). Thus, translation is complete, but export of newly synthesized protein is inhibited (71). Initially, it was thought that conservative substitutions of one hydrophobic amino acid for another (e.g., glycine for valine) did not alter the recognition between the SRP and the signal peptide (72). More recently, others have noted that even small changes in the central hydrophobic core can alter SRP binding (73,74). However, in these cases, translocation across the ER membrane still occurs through an unknown mechanism. SRP binding is not dependent on the presence of a net positive charge at the amino terminus or on any identifiable features at the carboxyl terminus (75,76). There do not appear to be any specific structural requirements for the site of signal peptide cleavage, although the carboxyl-terminal amino acid of the signal peptide usually has a small uncharged side chain such as alanine (65,77) (see Signal Peptidase later in this chapter). After translocation through the ER membrane pore, the signal peptide can loop back through the membrane. The signal peptide is then cleaved at its c terminus by signal peptidase. However, recent studies have shown that the signal peptide can be cleaved further by a signal peptide peptidase to release the amino terminal fragment into the cytosol (78). In the case of prolactin, this fragment then binds to calmodulin in a calcium-dependent manner (79). Currently, the full biological implications of this finding are unknown. In addition, it is not known if this applies to multiple other peptide hormones.

Signal Peptides The signal peptide, or presequence, constituted by the amino-terminal 20 to 30 amino acids of a newly synthesized polypeptide chain, directs the translocation of the polypeptide into the ER lumen. There seems to be little primary amino acid homology in the signal peptides of the known gastrointestinal hormone precursors. However, three general

Signal Recognition Particle SRP has three known functions: signal peptide recognition, elongation arrest, and promotion of translocation (55,56, 80–83). This particle consists of six polypeptide components with molecular weights of 72, 68, 54, 19, 14, and 9 kDa,

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 37 A

B

SRP

C SRP receptor

D

E

F

G

H mRNA

ER Cistem

Translocon

BiP

SP

SPP

FIG. 2-3. Signal peptides. (A) Ribosome binds to the messenger RNA (mRNA) and translation begins at the amino terminus. (B) Signal sequence emerges from the ribosome and binds to the signal recognition particle (SRP), which induces an arrest of translation. (C) SRP-ribosome complex binds to the SRP receptor or docking protein located on the endoplasmic reticulum (ER) membrane. (D) The translocon or protein pore binds to the ribosome, releasing the SRP and its receptor. (E) With binding of the ribosome to the cytosolic side of the translocon, BiP is released from the lumenal side of the translocon. (F) Translation resumes at the carboxyl terminus, and the signal peptide can be reinserted into the ER membrane. (G) Signal peptide is cleaved for the prohormone by signal peptidase (SP), and the signal peptide fragment is released into the cytosol via signal peptide peptidase (SPP) cleavage. (H) Translation continues until the entire precursor is located within the ER cistern and properly folded, often in association with chaperones such as BiP.

as well as a 7SL RNA (80,84). Each component is held together in a defined tertiary structure by Mg2+ ions and is essential for the functions of the SRP. The 19- and 54-kDa proteins exist as monomers, but heterodimers of the 9- and 14-kDa proteins and the 68- and 72-kDa proteins are formed (85). The 54-kDa protein contains a series of amphipathic helices with methionine residues, located predominantly on one face, that appear to be important for the binding to the hydrophobic region of the signal peptide (83,86–88). Interestingly, SRP will not bind to signal peptides that are not tethered to a ribosome, although the peptide region responsible for the SRP–ribosome interaction is not known. It appears that the 54-kDa protein binds to the 7SL RNA through the 19-kDa protein that binds directly to the middle of the RNA strand (88). In addition, the 7SL RNA contains 5′ and 3′ Alu-like elements that bind to each other and the 9/14-kDa protein heterodimer (89–91). The 9/14-kDa protein heterodimer mediates elongation arrest of translation, but plays no role in the translocation process (91,92). The 68/72-kDa heterodimer binds to the middle segment of RNA close to the 19-kDa binding site and appears to mediate the binding of SRP to its receptor (93,94). Thus, the 68/72-kDa heterodimer is not involved in elongation arrest but serves to aid in translocation. The 54-kDa protein binds GTP in concert with binding to the signal peptide (80,82,95–97). An additional GTP is required on binding of the SRP to the SRP receptor (96).

When the SRP/SRP receptor complex associates with the ER membrane or translocon there is a subsequent release of GDP (98). The hydrolysis of GTP releases the SRP from the signal peptide and allows translation to proceed.

Signal Recognition Particle Receptor The SRP receptor is located on the cytosolic side of the ER and binds to the SRP–ribosome complex, but not to free SRP as noted earlier. The SRP receptor plays an important role in termination of the elongation arrest and in the translocation of polypeptides into the ER lumen (59). The SRP receptor is a heterodimeric protein consisting of a 30-kDa integral membrane protein (β subunit) and 72-kDa α subunit that possesses domains that are homologous to GTP-binding proteins and the GTP-binding region of the 54-kDa SRP protein (88,95,99–104). The 72-kDa α subunit of the SRP receptor binds to SRP, and GTP is necessary to release SRP from the signal peptide–ribosome complex (95,105).

Endoplasmic Reticulum Membrane Protein Channel or Translocon There have been numerous theories about whether the nascent polypeptide chain is transported directly across the

38 / CHAPTER 2 lipid bilayer or in the aqueous environment of a protein channel. Although the initial thought was that the hydrophobic core of the signal peptide would allow for direct translocation across the membrane, there is a large transmembrane channel that is opened by the presence of signal peptides (106–108). The eukaryotic ER translocon is a heterotrimer (Sec61α, Sec62β, and Sec61γ) estimated to have a diameter of about 30 Å (109–111). The size of the pore is too small for folded proteins, thus ensuring that only nascent, unfolded proteins can enter the ER lumen. The lumenal side of the translocon is sealed by a protein, BiP, that aids in protein folding after passage through the membrane (112). On binding of the ribosome to the translocon, the SRP and its receptor disassociate from the complex, allowing resumption of translation (113). This process also seals the pore on the cytosolic side of the ER membrane, releasing BiP from the lumenal side.

PROCESSING IN THE ENDOPLASMIC RETICULUM Signal Peptidase During translocation, the signal peptide is cleaved from the propeptide by signal peptidase, an integral membrane protein complex on the lumenal surface of the ER. Signal peptidase has been purified from the dog pancreas as a complex of 5 polypeptides with molecular weights of 12, 18, 21, 22/23, and 25 kDa (114,115). The enzyme in the hen oviduct has only 2 subunits of 19 and 22/23kDa (116). The canine and hen 22/23-kDa proteins are glycosylated, and their amino acid sequences are 90% identical (117,118). cDNA encoding the canine 18 (119) and 21 kDa (120) are homologous to 2 yeast SEC11 proteins (121) that are components of the yeast signal peptidase, which contains 4 proteins in total (with molecular weights of 13, 18, 21, and 25 kDa). The 21kDa protein is absolutely required for enzymatic function in yeast (122,123), raising the question of the exact function of the other proteins. As is the case with processing enzymes, there are great similarities between the yeast and mammalian enzymes (124). Although it appears that the structure of eukaryotic signal peptidases are phylogenetically conserved, the Escherichia coli signal peptidase consists of only a single subunit of 323 amino acids (125). Nevertheless, there is some sequence homology between bacterial signal peptidases and subunits of the eukaryotic enzyme. Furthermore, the substrate specificity of the eukaryotic and prokaryotic signal peptidases is similar (126). Eukaryotic signal peptidase has a broad pH optimum and requires phosphatidyl choline as a cofactor (127,128). Determination of the amino acid sequences that define the substrate specificity of signal peptidase has been difficult because of the enormous structural diversity of signal peptides (129,130). However, there is clearly a hierarchy of preferred substrates for amino acids located at the carboxyl terminus of the signal peptide as follows: Ala>Cys>Gly>

Ser>>Thr>Pro>Asn>>Val, Ile, Leu, Tyr, His, Arg, Asp (65,131). Mutations of the signal peptide that increase the number of amino acids between the end of the central hydrophobic domain and the site of cleavage and mutations in the positively charged amino-terminal domain inhibit the cleavage reaction (132–134).

Disulfide Bond Formation After peptide prohormones are translocated into the ER lumen, they can undergo intermolecular or intramolecular disulfide bond formation (e.g., proinsulin) (Fig. 2-4). In the case of proinsulin, disulfide bonds are formed before cleavage of proinsulin into its component A and B fragments by removal of the C peptide. Thus, the disulfide bonds that are intramolecular on the prohormone are subsequently converted to intermolecular linkages that cannot be recreated easily after they are reduced. Although spontaneous formation of disulfide bonds of peptides such as somatostatin can occur in vitro over a few hours under optimal conditions, in vivo, the process occurs either cotranslationally or within seconds after translocation (135). The rapidity of this process suggests that it is catalyzed by an enzyme, the prime candidate being protein disulfide isomerase (PDI) (136–138). In solution, PDI exists as a homodimer (2 × 57 kDa) with a highly acidic isoelectric point (pI) (139). PDI has a broad substrate specificity encompassing relatively small proteins such as insulin, as well as large multidomain proteins such as immunoglobulins (137). PDI also forms the β subunit of a tetrameric enzyme (α2β2) denoted as prolyl-4-hydroxylase, which is responsible for hydroxylation of proline in the formation of procollagen (140). In tissues requiring both PDI and prolyl-4-hydroxylase activities, it appears that the β subunits of prolyl-4-hydroxylase are synthesized in large excess with a fraction being recruited into the prolyl-4-hydroxylase tetramers and the remainder as functional PDI homodimers (140). Previously, it was thought that glutathione provided the oxidizing equivalents for PDI (141,142). More recently, investigators have identified an ER membrane protein S

S

S

A-chain

S

S S

B-chain

Connecting peptide

FIG. 2-4. Structure of proinsulin. A and B peptides are linked by intramolecular disulfide bonds (asterisk), but after the connecting peptide is removed by endoproteolytic cleavage, disulfide bonds are intermolecular. On exocytosis, insulin (A and B peptides) is coreleased with the C peptide.

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 39 (Ero 1p) in yeast (143,144) that serves this function. Indeed, Ero 1p directly oxidizes PDI through disulfide exchange (141,145). The reoxidation of Ero 1 involves flavin adenine dinucleotide (FAD) (145).

N-glycosylated proteins (158). Because few prohormones are glycosylated, it is hypothesized that this pathway is not involved in prohormone processing. An important factor that should not be forgotten is the role that disulfide bond formation plays in maintaining the folded nature of many polypeptides such as proinsulin.

Asparagine-Linked N-glycosylation There are few examples of gastrointestinal peptides with N-linked glycosylation. The primary amino acid sequence of -Asn-X-Thr/Ser, where X can be any amino acid except proline, is obligatory for N-glycosylation of asparagine (146). The anterior pituitary glycoprotein hormone family (thyroid-stimulating hormone, follicle-stimulating hormone, and leuteinizing hormone) is the best example of glycosylated hormones. These are dimeric proteins with a common β subunit and different but homologous β subunits that confer specific biological activities. The glycosylation of both subunits is important for their correct assembly into dimers (147). Proopiomelanocortin (POMC) (148) and proenkephalin A (149) are also glycosylated, although the functional significance of this modification is unknown in these peptides. Secretogranin I (also known as chromogranin B) has a single glycosylation site, but it is uncertain whether it is glycosylated in vivo (150).

Protein N-myristoylation Protein N-myristoylation refers to the cotranslational linkage of myristic acid (C14:0) to the amino-terminal glycine of proteins; protein N-myristoylation is reviewed elsewhere (151). There are no known examples of myristoylated prohormones; however, this modification may play a role in the regulation of a variety of cellular events including posttranslational processing. Examples of N-myristoylated proteins include GTP-binding proteins and the catalytic subunit of cyclic 3′,5′adenosine monophosphate-dependent protein kinase A.

Protein Folding Polypeptides must be folded into a conformation that is compatible with exit from the ER (109,152). Misfolded proteins are tightly but noncovalently bound to a heavy chain binding protein or BiP and retained in the ER (153) until folding is complete and the polypeptide is released on hydrolysis of ATP (154,155). BiP, a member of the heat shock family of proteins (HSP70), binds newly translated and translocated aliphatic single polypeptides and prevents them from folding prematurely (156,157). It is currently unknown whether BiP or other folding proteins (158) are involved in the posttranslational processing of mammalian gastrointestinal prohormones, although BiP is clearly important in the translocation and folding of the yeast prohormone, pro-α factor in the ER (159). Another important folding chaperone is calnexin, but it interacts only with

TRANSPORT FROM THE ENDOPLASMIC RETICULUM AND THROUGH THE GOLGI The mechanisms responsible for protein sorting beyond the ER have been the subject of much investigation. Unlike the well-defined sorting of prohormones to the ER lumen through a signal peptide, there is no single unifying mechanism of prohormone transport from the ER and through the Golgi. Two types of sorting mechanisms have been hypothesized. The first is that prohormones are transported in the nonspecific “bulk flow” of contents from the ER to the Golgi in transport vesicles. An alternative hypothesis is that there is some signal contained in the prohormone structure that specifically directs their sorting through the intracellular compartments. This latter hypothesis is the case for resident soluble ER proteins such as BiP and PDI. Investigators noted in the structures of BiP and PDI a carboxyl-terminal consensus sequence KDEL (LysAspGluLeu) (160). Truncated forms of BiP lacking the KDEL sequence are not retained in the ER, but rather are secreted constitutively. In analogous fashion, prohormones destined for secretion but tagged with KDEL are retained in the ER in an unprocessed form (161). The homologous tetrapeptides DKEL, RDEL, and KNEL are all capable of directing ER retention in mammalian cells, whereas the HDEL sequence is used primarily in yeast (162,163). Although the KDEL-tagged proteins could be retained by a KDEL receptor in the ER membrane, it appears that these proteins initially exit the ER and are then recaptured in a salvage compartment at or near the cis-Golgi and returned to the ER (164). A mutant strain of yeast ERD2 (for ER retention defective) has been shown to have a defect in the KDEL/HDEL receptor (165). The structure of the ERD2 gene was then used to aid in the search for a mammalian homologue (166). This powerful technique of identifying genes of fundamental importance to the sorting of proteins in yeast and then using the yeast model to identify a mammalian homologue has been a fruitful approach in the study of peptide hormone processing. The ERD2 gene encodes a protein of 26 kDa that contains 7 membranespanning regions and is highly homologous to a putative human ERD2-like gene (167). The mammalian KDEL receptor cycles from the ER to the Golgi and back to the ER, thus retaining lumenal ER proteins within that compartment (168). Prohormones proceed from the ER to the Golgi stack where they undergo further posttranslation modification. Prohormone movement through the Golgi stack is by bulk flow (169,170) rather than a process mediated by a sorting signal. Bulk transport of soluble ER proteins to the Golgi and through the various Golgi compartments (cis, stack, and

40 / CHAPTER 2 trans-Golgi network [TGN]) was once thought to occur through transport vesicles (171). In this model, the Golgi was a series of stable, disconnected stacks through which proteins were progressively sorted, modified, or “distilled” toward their final destination. Although this model was attractively simple, it now appears that the Golgi is a much more fluid organelle (171–174). Although the transport vesicle model was indeed attractive, the described vesicles were too small (70 nm in diameter) to transport many secreted proteins. An alternative model is that newly synthesized proteins move from the ER to the cis-Golgi cisternae located near the ER. This newly formed cisternae progresses through the Golgi stack from the ER to the transGolgi (175). The transport vesicles in this case merely shuttle enzymes that characterize the various layers of the Golgi back through the cisternae (176–179). Thus, prohormones are transported from the ER to the cis-Golgi and are not transported out of this compartment, but rather are carried forward to the trans side as newer enzymes and proteins are added to the cis-Golgi. The nature of the ER to Golgi transport has been studied extensively (173). The ER membrane has a fixed number of exit sites from which proteins leave the lumen (180). The ER membranes cause buds that eventually become coated on their outer cytoplasmic surface with dispersed cytoplasmic proteins (coatamers, coat promoter, or COPs) (181–184). The budding ER vesicle is coated with COPII and traps prohormones together with other ER proteins (185–187). The COPII-coated vesicles then uncoat and fuse into a larger vesicular tubular complex (VTC) (185). It appears that the VTC is not continuous with the ER membrane. Eventually, the VTC combines with COPI (188). After fusing with the Golgi membrane (in a GTP-dependent manner) (189–191), the COPI-associated VTCs return ER proteins for recycling. Thus, nonhydrolyzable analogues of GTP such as GTP-γ-s interfere with fusion and block transport through the stack (192). VTCs move from the ER to the Golgi along a microtubular network that is powered by the dynein–dynactin motor (190,193). The fungal metabolite Brefeldin A, which is known to block protein transport through the Golgi stack, blocks the binding of the coatamer complex to budding Golgi membranes (194–197). The finding that Brefeldin A interferes with the posttranslational processing of progastrin suggests that this pathway is involved in the sorting of prohormones, as well as other soluble secretory proteins (198,199). Proteins secreted via either the constitutive or regulated secretory pathways share a common trail from the ER through the Golgi stack, but they diverge in the TGN where proteins are sorted according to their final destination (169,200–202). The sorting signal for enzymes destined for lysosomes involves a glycosylation reaction that occurs in the Golgi stack to attach mannose-6-phosphate residue proteins. A receptor protein in the TGN specifically binds mannose-6-phosphate–modified proteins (203,204) and directs their sorting to lysosomes. To date, searches for a common sequence (KDEL-like) or posttranslational modifications (mannose-6-phosphate–like) in the structure of

prohormones that might direct sorting to secretory vesicles in the TGN have not been successful. Although investigators have long sought to elucidate “the” Golgi sorting signal in neuroendocrine cells, none has been entirely successful. Indeed, it appears that three different mechanisms may be responsible for prohormone sorting to secretory vesicles. These include sorting signal motifs, aggregation, and membrane or lipid raft binding (205). Initially, the search for a sorting signal was pursued vigorously. In a fashion akin to the signal peptide (“pre” region of preprohormones), investigators sought sequences in the “pro” region of prohormones that, although lacking homology in their primary amino acid sequence, still contain sufficient structural information to direct sorting in the TGN. An α-helical motif with three leucine residues occupying one side of the helix was proposed as such a signal, but this hypothesis was not proved (206). In other studies, a chimeric protein containing the “prepro” region of somatostatin at the amino terminus and a constitutively secreted protein such as γ-globulin at the carboxyl terminus were sorted and processed in the secretory pathway (207). Studies with POMC and somatostatin precursors containing deletions in the “pro” region indicate the presence of sorting information at these sites, as well as in other portions of the peptide (208,209). In contrast, deletion of the “pro” sequence from trypsinogen and renin did not disrupt the routing of these proteins into the secretory pathway (210,211). A study expressed neuropeptide Y (NPY) fragments tagged with green fluorescent protein (GFP). GFP, a jellyfish protein not normally secreted, was correctly sorted, stored, and released from neuroendocrine cells when fused to half of the prepro-NPY sequence or only the signal sequence alone of pre-NPY (212). Thus, it appears that some prohormones are likely sorted by a specific signal found in their “pro” regions, but this does not appear to be a universal finding. A second sorting hypothesis is selective aggregation (213–215) of prohormones into acidic clathrin-coated secretory vesicles in the presence of high concentrations of divalent cations such as Zn2+ or Ca2+. Support for this hypothesis comes from observations that specific mutations in the structure of proinsulin that result in inhibition of hexamer formation with zinc also impede processing (48,216). Furthermore, in vitro studies have demonstrated that intravesicular conditions (pH 5.2 and 10 mM Ca2+) can result in selective precipitation of peptides that exit the cell through the regulated pathology of secretion. This applies to secreted proteins such as secretogranin II but not proteins that are constitutively secreted such as immunoglobulins (217). A heterodimeric protein in adrenal chromaffin granules, termed glycoprotein III, can selectively aggregate with two prohormone-processing enzymes carboxypeptidase E (CPE) and a dibasic endoprotease (218). Evidence contradictory to the selective aggregation hypothesis can be found in studies with guinea pig proinsulin, which does not form hexamers with zinc and yet is sorted and processed with high efficiency (219). In addition, in the marine mollusk Aplysia, the egg-laying hormone precursor is processed into two distinct mature hormone products that are sorted into different

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 41 secretory vesicles within a single cell (220). It is difficult to reconcile the selective aggregation model to explain this and other observations. However, aggregation clearly plays a role in the sorting of some prohormones. A third proposed prohormone sorting mechanism is the notion that prohormone-processing enzymes serve as a sorting receptor directing prohormones to secretory vesicles. CPE has been proposed as one putative sorting receptor (221–224). In this model, CPE is associated with Golgi membranes or “lipid rafts” (223,225–229). Others have found little evidence to support these findings with CPE (230–232). Other prohormone-processing enzymes or their activities also have been implicated in sorting. For example, a dibasic prohormone convertase (PC2) is associated with the lipid bilayer during sorting (233). Impairment of dibasic processing for proneurotensin (234), proneuromedin (234), proinsulin (235), and pro-NPY (236) all demonstrate impaired processing and sorting. However, other mutant prohormones are inefficiently cleaved yet sorted correctly (237). Again, it is difficult to reconcile these divergent findings and apply them generally to prohormone sorting. Most of the studies discussed earlier involve expressing a nonnative prohormone in a neuroendocrine cell (e.g., expressing prepro-NPY in insulin-producing β cells), or expressing mutated precursors in neuroendocrine cell lines such as AtT-20 or GH3. Thus, conclusions may be valid only for the studied prohormone or cell line and are not generally applicable. Indeed, prepro-NPY is synthesized, efficiently processed, and stored in cultured neurons (238). However, when these cells are infected with an adenoviral vector expressing POMC, POMC is poorly processed and stored despite that the pro-NPY processing is unaltered. Thus, many investigators now believe that prohormone sorting involves multiple processes that are cell and prohormone specific (48,175,213,239).

PROCESSING REACTIONS IN THE GOLGI Serine- or Threonine-Linked O-glycosylation Glycoproteins of the anterior pituitary such as the β subunit of human chorionic gonadotropin are O-glycosylated in the Golgi (240,241). Although there are potential sites for O-glycosylation in some gastrointestinal peptide hormone precursors, there is no evidence to suggest that they are functionally relevant. Confirmation that O-linked glycosylation occurs in the Golgi can be found by inhibiting transport through the Golgi with Brefeldin. Neuroendocrine cells treated with Brefeldin lead to O-linked glycosylation of prohormones not normally glycosylated (242).

O-phosphorylation Serine-linked O-phosphorylation is an uncommon posttranslational modification of peptide hormones that occurs

in the trans area of the Golgi stack just before entry into the TGN (243). Phosphorylation was first reported for POMC (244) and subsequently for proatrionaturetic factor (245,246), proenkephalin (247), and progastrin (248). The physiologic significance of peptide hormone phosphorylation is uncertain, although a hypothesis has been put forth for gastrin (249). At the carboxyl terminus of progastrin is a GlyArgArgSer processing site that eventually causes the amidated and biologically active site of the mature hormone. Varro and colleagues (250) suggest that the formation of amidated gastrin may be correlated with the degree to which the Ser moiety at that site is phosphorylated. Further clarification of the proposed regulatory function of O-phosphorylation on progastrin processing is required. Because there are few known phosphorylated hormone precursors, it has been difficult to identify a consensus sequence to signal phosphorylation. Although there are several phosphorylases capable of catalyzing the reaction, a candidate enzyme has not yet been clearly identified.

Tyrosine Sulfation Sulfation of carbohydrate residues and protein backbones is a common posttranslational reaction that all eukaryotic cells are capable of performing. Sulfation of prohormones at specific tyrosine residues is far less common, but nevertheless it is a critical step in the activation of some hormones. Examples of sulfated gut peptides are gastrin (251), cholecystokinin (CCK) (252,253), the chromogranin/secretogranin family (243), and Leu-enkephalin (254). Whereas sulfation is essential for the biological action of CCK at CCK-A receptors located in the gallbladder and pancreas (255,256), the acid secretagogue effect of gastrin is not influenced by the presence or absence of a sulfate moiety (257). Sulfation results in the inactivation of Leu-enkephalin (254). Some authors have hypothesized that sulfation may play a role in the sorting of prohormones to the secretory pathway; however, the sulfation of chromogranin A, the pancreastatin precursor, does not affect its sorting in the parathyroid (258). From the large number of sulfated secretory proteins it has been possible to identify the structural features that promote tyrosine sulfation (Table 2-1) (259,260). Some authors have examined a larger number of sulfated proteins TABLE 2-1. Structural criteria for tyrosine sulfation 1. Presence of an acidic residue at position −1 (the amino acid directly preceding the sulfated tyrosine). 2. Presence of at least three acidic residues from position −5 to +5. 3. Not more than one basic residue between positions −7 to −2 and +1 to +7. 4. Presence of β-turn-inducing residue (often serine or proline) between positions −7 to −2 and +1 to +7. 5. Not more than three hydrophobic residues between positions −5 and +5. 6. Absence of disulfide bridging or glycosylation sites nearby.

42 / CHAPTER 2 and modified the previous consensus sequence (261). For gastrin, these rules have been confirmed experimentally by mutational analysis (262). Tyrosyl protein sulfotransferase catalyzes the transfer of the sulfate moiety from the sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate, to the protein acceptor (263). The purified enzyme has a pH optimum of 6.5 consistent with its localization in the TGN, where the pH is substantially greater than that of the secretory vesicle (pH 5.5). The enzyme has an absolute requirement for Mg2+ or Mn2+ for biological activity. Two putative tyrosyl protein sulfotransferase genes (TPST-1 and TPST-2) have been identified (262,264–267). The enzymes are highly homologous and are expressed in a wide number of cell types. Homology searches for other family members have been unsuccessful. A TPST-1-deficient mouse has been generated with a reduced body weight phenotype (268). It is unknown if TPST-1 deficiency alters CCK or gastrin sulfation (268). Although other sulfotransferase activities have been identified in other tissues, it is unclear if they can sulfate tyrosine residues. Tyrosine sulfation of CCK and gastrin affects the processing of both peptides. Gastrin sulfation is variable between species and tissues. However, enhanced sulfation of gastrin increases the endoproteolytic maturation of progastrin (269). In contrast, sulfation of pro-CCK is not an absolute requirement for CCK processing, but a lack of sulfation does impair CCK secretion (270).

FORMATION OF SECRETORY VESICLES From early morphologic studies it is known that there is selective condensation of proteins in the TGN to form a dense core that is subsequently enveloped by a membranecontaining clathrin (271,272). Clathrin, which has been reviewed extensively elsewhere, is a 180-kDa polypeptide made up of three light and three heavy chains that coats both endocytic and immature secretory vesicles (273–275). The immature clathrin-containing vesicles contain the prohormones, as well as many of the enzymes responsible for their processing (276). As the secretory vesicles mature, they lose their clathrin coat and contain relatively more processed biologically active peptides and lesser quantities of prohormone precursors. Mutation of proinsulin so that it cannot be processed causes it to remain stored in immature clathrin-coated vesicles, suggesting that prohormone processing may be involved in the uncoating process (277). However, the mechanisms of secretory vesicle formation, clathrin coating, and uncoating remain a mystery. The pH of the secretory vesicle is maintained in the acidic range (pH 5.5) via a proton-transporting Mg2+-dependent ATPase on the membrane (278,279). A low intravesicular pH is believed to be essential for the activation of the processing enzymes contained within the secretory vesicle. Also associated with the acidification and uncoating of the secretory vesicle is an increase in intravesicular Ca2+ that plays a key role in the activation of prohormone dibasic cleavage enzymes (see later).

PROCESSING REACTIONS IN THE SECRETORY VESICLE Acetylation Of the processing reactions that take place in secretory vesicles, the least is known about acetylation because there are only a few examples of acetylated gut peptides. In the rat, acetylation is required to activate a melanocyte-stimulating hormone (280), but it inactivates β-endorphin (281); thus, acetylation is important in the physiologic regulation of these peptides (282,283). The enzyme responsible for acetylation has not been purified, but it has been characterized as a crude enzyme that requires acetyl coenzyme A as an acetyl donor (284,285). Much of the available information on opioid peptide acetylation is derived from experiments in the rat pituitary and brainstem; however, this reaction may be species and organ specific. Because acetylation does not seem to occur in the human pituitary or other nonbrain tissues of the rat, the significance of this reaction as an important step in the posttranslational processing of gut peptides has yet to be determined (286–288). With identification of the five subtypes of melanocortin (MC) receptors (MC1-5), investigators have attempted to determine the potential molecular mechanisms that might mediate the effects of acetylation on MC activity (289). Acetylation of α-MSH results in few, if any, differences in binding or receptor coupling to HEK293 cells expressing MC1 to MC5 receptors. However, native acetylated α-MSH is much more effective than deacetylated α-melanocyte stimulating hormone (MSH) in stimulating rat osteoblast growth (275). Thus, more work clearly is necessary to determine the physiologic implications of MC acetylation.

Dibasic Cleavage Selective endoproteolytic cleavage is perhaps the most important posttranslational processing step for gastrointestinal peptide hormones, because virtually all of them undergo this reaction in one form or another. Furthermore, this step results in the tissue-specific processing of prohormones, resulting in the formation of distinct patterns of mature peptides, often with markedly different biological activities. The most frequent cleavage reactions occur at pairs of basic amino acid residues, although tribasic and tetrabasic sites also serve as cleavage sites. Cleavages usually occur on the carboxyl-terminal side of a dibasic pair (Fig. 2-5). The LysArg pair is the most common dibasic cleavage site (60%), followed by ArgArg (25%), with LysLys and ArgLys sites being the least common (290). Support for the secretory vesicle localization of the cleavage enzymes was initially provided in peptide-release studies demonstrating that processed hormones (e.g., insulin) and prohormone fragments (e.g., C peptide) were cosecreted in equimolar amounts during vesicle exocytosis (291). In more direct immunohistochemical studies, intact proinsulin was shown to be abundantly present in the Golgi stack and in the immature clathrin-coated

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 43

-Lys-ArgTryptic-like cleavage

-Lys-Arg + Carboxypeptidase cleavage

+ Cleaved peptide(s)

FIG. 2-5. Endoproteolysis of prohormones involves tryptic-like cleavage on the carboxyl-terminal side of monobasic or dibasic residues. This is followed by the removal of the basic residues by carboxypeptidase E.

vesicles, whereas processed insulin was present primarily in the mature uncoated vesicles (292). Thus, the dibasic cleavage reaction occurs primarily in the Golgi-derived, clathrincoated, secretory vesicles. There does not appear to be a strict requirement for a specific primary amino acid sequence in the region surrounding the dibasic site for cleavage to occur. Mutation of one of the basic residues to a nonbasic amino acid will result in impaired processing (293). Not all dibasic sites in a given prohormone are cleaved, and different tissues may process the same prohormone at different dibasic sites. These observations may result differential expression of cleavage enzymes in various tissues or from the conformational characteristics of the precursors. Some investigators suggest that the secondary structure (β-turn) near the cleavage site is important for processing. They further hypothesize that dibasic sites buried in an α helix will be cleaved inefficiently (294–296). Although in vitro studies have shown that trypsin can perform dibasic cleavages, the exact nature of the enzymes responsible for cleavage of prohormones in situ had been difficult to ascertain because of their presence in low quantities compared with the relative abundance of proteases associated with cellular organelles (particularly lysosomes) that contaminate many secretory granule preparations (297,298). Although many enzymatic activities were purified and characterized (297,299), it was difficult to determine whether these enzymes were responsible for prohormone processing in vivo. Furthermore, because many enzymes were described from different neuroendocrine tissues, it was unclear whether their actions were specific for a given prohormone substrate in an individual endocrine cell or more generalized and applicable to other prohormones. Gene transfer studies that involve expression of prohormone cDNA in heterologous neuroendocrine cell lines that do not normally express or process a given hormone demonstrated that the posttranslational machinery and enzymes are common to a variety of cell types. For example, the processing of gastrointestinal peptide hormone cDNA encoding progastrin (300,301), pro-NPY (302), pro-CCK (303), pro-SS (304), proglucagon (305), and propancreatic polypeptide (306) has been demonstrated in various neuroendocrine cell lines. The posttranslational processing machinery appears to be specific to neuroendocrine cells because prohormones are processed inefficiently in nonendocrine cells (307). A breakthrough in the field came when Julius and colleagues (308) described a mutation in the kex2 gene of yeast.

This mutation prevented the synthesis of a specific killer toxin and a mating prohormone, both of which require cleavage at dibasic sites for biological activity. The kex2 gene was found to encode a Ca2+-dependent serine protease, not one related to trypsin, as many had expected, but rather one related to the bacterial subtilisins (309–312). In searching for a mammalian homologue for kex2, van den Ouweland and colleagues (313) demonstrated homology between the deduced kex2 protein sequence and the human c-fur gene, which was originally detected in an open reading frame immediately upstream of the fes/fps protooncogene (314). The c-fur gene product, termed furin or PACE (paired basic amino acid residue cleaving enzyme), has a conserved sequence in eukaryotes (315,316). The efforts to identify the mammalian neuroendocrine homologue of kex2 were rewarded with the identification of PC2 (317,318). Similar methods led to the discovery of a second related cDNA encoding a product that was named PC1 (317) or PC3 (319). Like kex2, PC2 and PC1/PC3 are both Ca2+-dependent serine proteases related to the bacterial subtilisins (320,321). The acidic pH optimum (pH 5.5) of both enzymes is consistent with their putative function in secretory vesicles. PC2 and PC1/PC3 transcripts are found only in endocrine and neural tissues (317,322,323). PC4 (324) is expressed only in the testis and not in other neuroendocrine tissues or cell lines. Similar to furin, PC5/PC6, PC7, and PC8 (325–328) are expressed in many tissues including neuroendocrine tissues. These convertases and furin generally are not involved in prohormone processing. Their active forms are localized to the TGN via cytoplasmic tails and in the small vesicles that characterize constitutive secretion (329). These proteases process many secreted protein from organs such as the liver. They also are responsible for the processing of growth factors such as transforming growth factor-β (330) and insulin-like growth factors (331). The cytosolic tail of these enzymes in the constitutive pathway contains motifs that allow for their recycling out of early secretory granules (332,333). The enzymes are not active in the ER inasmuch addition of the ER retention signal, KDEL, to the carboxyl terminus of furin abolishes proteolytic activity (334). Similar to kex2, all members of the PC family including furin are Ca2+dependent proteases. Furin has a pH optimum of 6.5, which is consistent with its localization to the Golgi (335). All members of the PC family are synthesized as a biologically inactive preconvertase. Autoactivation occurs for all

44 / CHAPTER 2 members except PC2 via an intramolecular cleavage (329,336,337). In the case of furin (337), the covalent dibasic bond in the “pro” region is cleaved, allowing exit from the ER. The propeptide remains associated with the protease until the slightly lower pH of the Golgi (pH 6.5) and its calcium-rich milieu remove the propeptide. A second cleavage in the released propeptide prevents its reassociation with the protease. Similar mechanisms have been described for PC1/PC3, PC4, PC5, and PC7 (312). The activation of pro-PC2 requires an accessory protein 7B2. PC2 and 7B2 associate in the ER. It is believed that 7B2 is necessary for pro-PC2 folding and subsequent activation in that PC2 will not autocleave and thus autoactivate in the absence of 7B2 (338). In the more acidic compartment of the TGN, a carboxyl-terminal fragment of 7B2 is cleaved by furin or another similar protease (339,340). This fragment remains near the catalytic site of the enzyme and a LysLys site in the fragment inhibits PC2 activation (341). As the pH in the TGN continues to decrease, the “pro” region of PC2 is removed by autocatalysis (342). Eventually, the partially activated PC2 cleaves the associated carboxyl-terminal fragment of 7B2, allowing for its full activation (340). Although PC1/PC3 is autoactivated, there is another protein that may regulate its activity. Pro-SAAS is a 26-kDa protein produced in neuroendocrine cells and sorted to the secretory pathway (343). Indeed, Pro-SAAS expression overlaps that of PC1/PC3 (344) and can inhibit prohormone processing when expressed in a neuroendocrine cell line (345). The earlier series of events has implications for prohormone processing (346). For example, PC2 is believed to be more efficient than PC1/PC3 at LysLys cleavage sites (347). However, PC2 activation occurs much later than that of PC1/PC3, which can alter the mixture of final peptide products from prohormones with multiple cleavage sites such as POMC and proglucagon (346,348). To examine the physiologic and biochemical effects of individual PCs on prohormone processing, investigators generated several PC-deficient mouse models. Because the motif for the substrate specificity of furin, Arg-X-Lys/Arg-Arg (349), is not present in many prohormone cleavage sites, it has been suggested that PACE/furin may be responsible for processing proteins secreted via the constitutive pathway but not involved in prohormone processing in the secretory pathway (311,349). In addition, the almost ubiquitous expression of furin and its involvement in the processing of many secreted proteins suggests that it is a protein critical to normal development. Indeed, furin-deficient mice all die by E11.5 (350). The mouse embryos fail to undergo axial rotation and heart looping (350,351). PC4 is expressed only in the testis (324). Disruption of PC4 expression results in markedly diminished male fertility without any noted spermatogenic abnormality (352). PC4 deletion also inhibits pituitary adenylate cyclase activating peptide (PACAP) processing in the testis (353). Gene disruption experiments for PC1/PC3 and PC2 are of considerable interest because both enzymes are involved in prohormone processing. Moreover, their substrate specificities

can overlap extensively. PC2-null mice survive but grow more slowly than wild-type mice (354). Proinsulin processing is maintained in these mice, suggesting that PC2 is not necessary for insulin production. However, proglucagon is processed into glucagon but not glucagon-like peptide-1 (GLP-1) or GLP-2 (355,356). Prodynorphin and prosomatostatin processing at its dibasic site are also affected in PC2-null mice (356,357). As anticipated with its role in PC2 activation, 7B2-null mice are phenotypically similar to PC2-null mice. However, 7B2 mice also have a dramatic defect in POMC processing and adrenocorticotropin hormone (ACTH) production, adding to an exaggerated Cushing’s syndrome (358). PC2- and 7B2-null mice also have alterations in pro-CCK and progastrin processing (359,360). PC1/PC3 gene disruption causes dwarfism and multiple peptide processing defects (361). Defects include processing of proinsulin to insulin, POMC to ACTH, proglucagon to GLP-1 and GLP-2, progrowth hormone-releasing hormone. This phenotype and spectrum of prohormone processing defects is similar to a reported human subject with a defect in PC1/PC3 activity (362,363).

Monobasic Cleavage Many gastrointestinal peptide hormones are processed to their biologically active forms by cleavage at single basic amino acids. Examples of such peptides include pancreatic polypeptide (364), gastrin-releasing peptide (365), and CCK (366). The monobasic cleavage reaction must be highly specific because cleavage does not occur at all basic amino acid residues. This specificity no doubt also contributes to the tissue specificity of peptide precursor processing (367). Arginine (81%) is the most commonly cleaved basic amino acid, but lysine cleavage sites also have been identified. Proline, which is thought to induce a β-turn in the prohormone secondary structure, is found in a position adjacent to approximately one third of the known cleavage sites. From the peptide precursors known to be processed, Devi (368) has proposed several principles that define the structural criteria for monobasic processing depicted in Table 2-2 (369). These principles are useful not only in predicting which single basic amino acid sites will be cleaved, but also in predicting which sites will not be cleaved. When the rules are applied to single arginines and lysines that are not cleaved in peptide precursors, approximately 90% are predicted not to be processing sites (368). To determine whether monobasic and dibasic cleavages are distinct phenomena, Schwartz and Tager (370) performed time-course biosynthetic studies involving the pulse-labeling of pancreatic islets producing propancreatic polypeptide, a hormone known to be processed at both monobasic and dibasic sites. In contrast with the dibasic cleavage reaction that usually occurs within 60 to 90 minutes, the monobasic reaction required 120 to 180 minutes for completion. Similar experiments with neurophysin have confirmed these findings (371). Thus, monobasic and dibasic cleavage reactions

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 45 TABLE 2-2. Structural features of monobasic cleavage sites Rules 1. Basic amino acid (Arg, Lys, or His) is always present at either the −3, −5, or −7 position (3, 5, or 7 amino acids preceding the cleavage site). 2. Aliphatic amino acids (Leu, Ile, Val, Met) are never present at the +1 position. 3. Cysteine is never present in the vicinity (−7 to +3) of the cleavage site. 4. Aromatic amino acids (Trp, Tyr, Phe) are never present in the −1 position. Tendencies 1. The monobasic cleavage is usually at a single arginine, occasionally at a single lysine, and never at a single histidine. 2. Short side-chain amino acids, predominantly Ser or Ala, tend to be at the +1 position in approximately 50% of cases. 3. Aliphatic and small side-chain amino acids occur primarily in the −2 position and the +2 to +8 positions. 4. Aromatic amino acids are rarely found at the +1 position.

appear to involve distinct enzymes. Further support for this notion includes the finding that PC2 appears to be unable to cleave peptide substrates containing a single basic amino acid (320). Prosomatostatin also requires processing at a dibasic site to generate SS-14 and is processed at a monobasic site to generate SS-28. Mackin and colleagues (372) have identified two distinct enzymatic activities in tissues that appeared to be responsible for pro-SS processing at monobasic and dibasic sites. The first is a serine protease that cleaved proSS at the dibasic site for SS-14, and a second aspartyl protease cleaved pro-SS and led to the formation of SS-28. There are numerous studies demonstrating the abilities of PC1/PC3 and PC2 to cleave pro-SS at its dibasic site to form S14 (373,374). Finally, others have suggested that SS-14 and SS-28 production occur in different secretory pathways (375), which is consistent with the notion of distinct monobasic and dibasic PCs. To determine whether members of the PC family were capable of monobasic pro-SS cleavage, investigators examined pro-SS processing in PC2-null mice (355). In PC2deficient mice, pro-SS was cleaved at the monobasic but not dibasic site leading to the formation of SS-28 and little SS-14. This suggested that PC2 is not a monobasic pro-SS– processing enzyme, despite that PC2 is capable of monobasic cleavages of other prohormones (357,376). It is not known if PC1/PC3 is the pro-SS monobasic protease in mammals. Although other investigators have identified possible monobasic cleavage activities in various tissues (372,377– 379), the yeast aspartyl protease (YAP3) is perhaps the most promising candidate (380,381). YAP3 has been implicated as an enzyme capable of monobasic processing in anglerfish pro-SS-II to yield S28 (382). Interestingly, both an anglerfish aspartyl protease (372) and a mammalian aspartyl protease not only have similar pH optima and molecular weights to YAP3, but they also share immunologic cross-reactivity

with YAP3 (383). Thus, it is quite possible that a mammalian YAP3 homologue is the monobasic pro-SS–processing enzyme. However, identification of this mammalian homologue has not been as forthcoming as was the identification of the mammalian dibasic PCs after the identification of the yeast kex2 enzyme. Beinfeld and colleagues (378,379) have described two distinct monobasic cleavage activities from brain and gut using a fragment of pro-SS as a substrate. They both have a broad range of pH optima with maximal activity at pH 8.0, but their inhibitor profiles are somewhat different. The activity from the gut was capable of cleavage at both monobasic Arg and dibasic sites. Finally, production of GLP-1 from proglucagon requires processing at a monobasic site. PC1/PC3-null mice cannot generate GLP-1, suggesting that proglucagon monobasic processing can be mediated via PC1/PC3 (384). Although several putative monobasic enzymatic activities have been characterized, final proof of their role in monobasic prohormone cleavage will await their cloning and expression in neuroendocrine cells. Criteria for evaluating candidate processing enzymes initially proposed by Docherty and Steiner (297) for characterization of the dibasic cleavage enzyme(s) are applicable for evaluation of monobasic enzymes (Table 2-3). Dynorphin converting enzyme has been characterized in rat brain extracts and is capable of cleaving the 29-amino-acid peptide leumorphin at a single arginine residue to Dyn B-13 (377). The purified enzyme is unable to cleave substrates at dibasic ArgArg sites (385–387). The enzymatic activity has a pH optimum of 8.0 and appears to be a thiol protease. The amino acid requirements appear to conform to the rules for monobasic enzymatic activity described in Table 2-2. Its tissue distribution and subcellular localization to the secretory vesicle are consistent with a putative role in peptide hormone processing.

Carboxypeptidases A frequently hypothesized mechanism for the formation of biologically active peptides from larger precursors involves TABLE 2-3. Criteria for identification of a prohormone monobasic cleavage enzyme 1. Protease must correctly cleave precursor to generate all known peptide products. 2. Enzyme must be resolved from other contaminating proteases before characterization. 3. Biochemical characterization should include studies of: Cleavage specificity pH characteristics (optimum and stability) Cleavage mechanism (serine, thiol, metalloenzyme) Susceptibility to known protease inhibitors 4. Tissue distribution and subcellular localization must be appropriate for its putative role. 5. Final proof of normal participation in prohormone cleavage requires demonstration that inhibition, inactivation, or mutation of the enzyme prevents cleavage in intact cells.

46 / CHAPTER 2 not only the endoproteolytic cleavages at basic residues, but also the subsequent removal of the remaining basic residues via the action of an aminopeptidase or carboxypeptidase (see Fig. 2-5) (388). This hypothesis arises from the observation that many gut peptides are missing the basic amino acids at their carboxyl termini after undergoing endoproteolytic cleavage. Other experiments demonstrated that addition of carboxypeptidase B to trypsin-induced cleavage of some prohormones in vitro results in the generation of correctly processed peptides (298). The eukaryotic enzyme thought to be responsible for this action in neuroendocrine cells has been extensively characterized and is alternatively known as CPE, carboxypeptidase H, or enkephalin convertase. CPE was partially purified from adrenal medullary secretory granules and found to be maximally active at pH 5.5 (389). CPE has a molecular weight of approximately 50 kDa and exists in both soluble and membrane-bound forms (390). The soluble form of the enzyme appears to be fully active, whereas the membrane-bound form exhibits less than 50% of the activity of the soluble form (391). Enzymatic activity is enhanced by the presence of Co2+ or Ni2+ ions, although it is presumed that the active site metal of the enzyme is Zn2+ (389,390). CPE removes a carboxyl-terminal arginine slightly faster than lysine and much faster than histidine from a variety of peptide substrates (392). Peptides with other amino acids at the carboxyl terminus are not hydrolyzed by CPE. An excess of free arginine and lysine inhibits CPE activity (393). Bovine (394), rat (395), and human (396) CPE share a 93% amino acid homology. The amino acid sequence deduced cloned CPE cDNA contain 41 amino-terminal residues not found in the purified active form of the enzyme. The first 17 to 22 amino acids represent a signal or presequence and the remainder is a prosequence that is endoproteolytically cleaved at five arginine residues that immediately precede the amino terminus of the active enzyme. This cleavage occurs rapidly after synthesis, presumably in the ER or cis-Golgi, or both, and the five consecutive arginine residues conform to the substrate motif necessary for cleavage by the endoprotease PACE/furin active in the cis-Golgi (397). Further analysis of the nucleotide sequences of CPE cDNA indicates that CPE is a member of the metallocarboxypeptidase gene family, although the amino acid homology is only 20 to 50%. Members of this family include carboxypeptidases A and B found in the exocrine pancreas (398), as well as carboxypeptidases M and N, which are thought to function in the processing of serum proteins such as the kinins (399,400). Interestingly, the yeast functional homologue of CPE, the kex1 gene product, has little or no homology to mammalian CPE (401). This is in contrast to the high degree of homology that exists between the yeast kex2 gene product and the mammalian PC1/PC3 and PC2. It is not currently known if a mammalian homologue of the kex1 gene product exists, although kex1 heterologously expressed in mammalian cells is able to process mammalian prohormones (402). The rat CPE gene has been identified and Southern blots indicate only a single copy of the CPE gene (403).

CPE activity and mRNA are widely distributed throughout neuroendocrine tissues of many species, and the tissue levels of CPE mRNA generally reflect the levels of enzymatic activity (404). Stimulation of adrenal chromaffin cells that synthesize and process proenkephalin results in an increase of both CPE activity and mRNA (405). In contrast, stimulation of GH4C1 cells increases CPE activity without altering CPE mRNA levels, suggesting that CPE is regulated differently in various tissues (406). CPE activity has been detected in early embryonic development of the brain, before the appearance of processed neuropeptides, as is expected given its proposed role in the posttranslational processing of prohormones (407). Hyperinsulinemia and obesity are found in mice with a single point CPE mutation that reduces enzymatic activity (408). This exciting discovery led to further examination in these mice of prohormone processing including gastrin (409). Because prohormone cleavages were also affected, investigators examined the role of CPE beyond that of basic amino acid residue removal, including PC activation and activity (225,410,411). Sorting of CPE to the regulated pathway of secretion requires its interaction with lipid rafts via a C-terminal membrane anchor (221,412). It is also hypothesized that CPE might serve as a sorting receptor for prohormones and other processing enzymes, although this hypothesis remains controversial (412).

Aminopeptidases An aminopeptidase capable of removing amino-terminal basic residues may have an important role in prohormone posttranslational processing. Although the endoproteolytic cleavage of most prohormones occurs at the carboxyl side of a dibasic site, there are some examples of gastrointestinal hormones that are characterized by one or more basic amino acids at the amino terminus. Gainer and colleagues (413) have characterized an enzyme from bovine pituitary secretory granules with specificity for amino-terminal arginine residues. The enzyme has a pH optimum of 6.0 and is inhibited by ethylenediaminepentaacetic acid in a fashion reversible with Co2+ and Zn2+, but it cannot be inhibited by serine or cysteinyl protease inhibitors. Although the enzyme can cleave the amino-terminal arginines from β-lipotropin 60-65 (leaving Met-enkephalin) and a variety of synthetic substrates, it does not remove the amino-terminal arginine from fully processed substance P. The enzyme appears to be distinct from lysosomal and degradative aminopeptidases, but its importance in gastrointestinal prohormone processing remains to be clarified. Dipeptidyl aminopeptidase IV sequentially removes two amino acids from the amino terminus of peptides and is clearly involved in the posttranslational processing of prohormones in the honeybee and frog (414,415), although its role in mammals is yet unknown. The STE13 mutant strain of Saccharomyces cerevisiae, which lacks an enzymatic activity present in wild-type yeast capable of hydrolyzing

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 47 mammalian substrates of dipeptidyl aminopeptidase IV, fails to process a mating factor at the amino terminus, thus producing peptides extended by two amino acids (416). Dipeptidyl aminopeptidase IV has been characterized in frog skin and has a molecular weight of 98 kDa with a pH optimum of 8.0 (417). Dipeptidyl aminopeptidase IV is capable of amino-terminal cleavages with an X-Ala, X-Gly, or X-Pro amino terminus wherein X is usually Glu. A variety of gastrointestinal peptides including gastric inhibitory polypeptide (418) and gastrin-releasing peptide (365) are potential substrates for dipeptidyl aminopeptidase IV, because these peptides have been recovered from tissues with the two or four amino acids (e.g., AlaProValPro) missing from their amino termini. The enzyme is expressed in high concentrations of hepatocytes and in nonendocrine epithelial cells of the small intestine (415), thus it may not function in prohormone processing before secretion from neuroendocrine cells, but it may be responsible for cleaving peptides after their release or during the extraction process (419).

Glutaminyl Cyclase N-terminal pyroglutamyl formation is necessary for the full biological activity of thyrotropin- and gonadotropinreleasing hormones (420). The modification also is found in other gastrointestinal peptides such as bombesin and gastrin, although it is not required for their classical functions (421). Other proteins constitutively secreted from nonendocrine cells such as immunoglobulins also contain this modification. The reaction involving the conversion of an amino-terminal glutamine to a pyrole ring structure with the release of ammonia is known to occur spontaneously in the presence of phosphate ions (422). A pituitary enzyme, glutaminyl cyclase (QC; Q is the single letter notation for glutamine), catalyzes this reaction at a rate approximately 70 times faster than the nonenzymatic reaction (423,424). QC has a molecular weight of approximately 40 kDa with a pH optimum of 8.0, despite its localization in the acidic secretory vesicles of neuroendocrine cells (423). The Km is about 100 µM, and the enzyme requires ATP for full activity. A cDNA-encoding QC has been isolated from a bovine (425) and human (426) pituitary. By Northern blot analysis, QC is expressed in many portions of the brain but not in the spleen where large amounts of immunoglobulins containing pyroglutamate residues are produced. It is possible that two forms of the enzyme might exist: one (425) to process prohormone substrates in neuroendocrine cells, and the other to process immunoglobulins and other constitutively secreted proteins. There is some evidence for multiple forms of the enzyme (427).

Amidation Many gastrointestinal peptides are characterized by the presence of a carboxyl-terminal amide moiety that is often essential for biological activity (428). Tatemoto and Mutt

(429) have used this property to identify many gut peptides. The first indication of the source of the amide group was provided by Kreil and coworkers (430,431), who noted that the precursor of melittin, an amidated honeybee venom, contained a glycine residue immediately following the carboxyl-terminal amino acid of the processed peptide. These findings prompted the hypothesis that glycineextended peptides may serve as substrates for the amidation reaction. Since the proposal of this hypothesis, the rapid generation of new prohormone peptide sequences from the cloned cDNA encoding them has confirmed that the carboxyl terminus of an amidated peptide is flanked in the structure of its precursor by a glycine residue followed by one or more basic amino acids. The basic residues are processed by cleavage enzymes, leaving a glycine-extended precursor that, in turn, serves as a substrate for the amidation reaction. Bradbury and colleagues (432) initially identified the amidating enzyme as a 60-kDa protein capable of converting the synthetic substrate D-TyrValGly to D-TyrValNH2. The pituitary form of this enzyme was characterized further by Mains, Eipper, and others (433–435), and initially was referred to as peptidyl-glycine alpha-amidating monooxygenase (PAM). PAM requires Cu2+ and molecular oxygen for function. Enzymatic activity is enhanced by the presence of ascorbic acid and catalase, and there is a biphasic pH optimum (pH 5–7). The requirements for copper, oxygen, and ascorbic acid suggest that PAM is similar to dopamine β-hydroxylase. Amidating activity has been identified in a variety of tissues, as well as in the systemic circulation and cerebrospinal fluid (436–439). The amidating enzyme appears to be specific for a glycine residue in the carboxyl terminus of peptide substrates (440). Peptides terminating in other L-amino acids do not serve as substrates, although D-alanine–containing peptides can serve as a substrate that reacts slowly (441). The enzyme appears to exhibit selectivity for neutral amino acids preceding the glycine residue (442). In vivo amidation enzyme activity can be regulated by the availability of Cu2+ ions as demonstrated by the finding that animals treated with the copper chelator diethyldithiocarbamate have decreased amidated gastrin and increased glycineextended gastrin in the gastric antrum (443,444). Although the importance of copper to peptide amidation was known for some time (444), the mechanisms by which copper reached PAM in cells were unknown. Investigators have identified the Menkes protein as an intracellular copper transport p-type ATPase, ATP7a (445–448). The Menkes protein is related to other copper transport genes such as the Wilson’s disease gene product. However, the expression of the Wilson’s gene is limited to the liver, whereas the Menkes gene is expressed in most tissues except the liver (446). Menkes kinky hair syndrome is an X-linked genetic copper deficiency that is usually fatal early in childhood (449). Children with Menkes syndrome usually do well for the first few months of life, and then develop progressive neurologic degeneration (450). The cause of the neurologic deterioration

48 / CHAPTER 2 is unknown, but it is believed to be a consequence of the low in vivo activities of copper-dependent enzymes such as cytochrome c oxidase, dopamine-β-hydroxylase, and PAM (450–453). The Menkes protein transports copper into the Golgi (454) and has been directly linked to PAM activity (455,456). PAM also requires ascorbate for optimal activity. The monooxygenation performed by PAM requires ascorbate in a reduced state that is subsequently oxidized to dehydroascorbate. The uptake of dehydroascorbate into cells is mediated by the glucose transporters, GLUT3 and GLUT4 (457), but at insufficient levels to account for the biological actions of ascorbate. Two cDNA-encoding ascorbate transporters (SVCT1 and SVCT2) have been isolated (458–461). SVCT1 is expressed at high levels in the kidney and to a lesser extent in the gut. SVCT2 is expressed in levels in neurons but not astrocytes (459,460). To date, there is no direct linkage between SVCT expression and PAM activity. Humans and guinea pigs are two of the more unusual mammals that require ascorbate in their diets. To examine the possible role of ascorbate deficiency (“scurvy”) and peptide amidation, investigators noted that antral extracts of guinea pigs deprived of ascorbic acid contain less amidated gastrin and more glycine-extended gastrin (462). Peptide amidation occurs in a two-step reaction with the initial formation of a hydroxyl intermediate via the action of the monooxygenase domain of PAM (463–468). Another distinct enzyme, with lyase activity, catalyzes the breakdown of hydroxyglycine to form the peptide amide and glyoxylate. The nitrogen of the peptide amide comes from the amino nitrogen of the carboxyl-terminal glycine (432). The oxygen involved in the hydroxylation step originates from molecular oxygen (469). Both enzymes are distributed in same neuroendocrine tissues and are encoded by a single gene. The amino-terminal portion of the mammalian PAM precursor contains a signal peptide followed by a putative “pro” region of unknown physiological significance. The peptidyl-α-hydroxylating monooxygenase (PHM) domain is separated by a linker containing a LysLys endoproteolytic cleavage site from the lyase or peptidyl-α-hyodroxyglycine α-amidating lyase (PAL) domain (470). This is followed by another dibasic endoproteolytic cleavage site and a putative transmembrane domain with a carboxyl-terminal cytoplasmic tail. In the rat, alternative RNA splicing appears to be responsible for the tissue-specific expression of several different mRNAencoding proteins lacking any one of the regions described earlier (470–472). Expression of the full-length PAM cDNA in an endocrine cell line led to increases in monofunctional PHM and PAL activities, although most of the activity was soluble, suggesting that there had been endoproteolytic removal of the carboxyl-terminal transmembrane domain and cleavage between the PHM and PAL domains. Expression of a truncated cDNA without the linker segment resulted in a single bifunctional protein with PHM and PAL activities. Expression of another truncated segment with only the PHM domain resulted in the expression of active soluble monooxygenase activity (471,472).

POSTTRANSLATIONAL PROCESSING OF PREPROGASTRIN Many of the principles of prohormone posttranslational processing can be exemplified in the production of a single gastrointestinal peptide hormone, gastrin. The mechanism of gastrin biosynthesis has long been a focus of interest because of its physiologic importance in the regulation of gastric acid secretion and gastrointestinal cell growth. From the earliest studies, multiple molecular forms of gastrin were characterized in the circulation and antral tissues (257, 473–476). The presence of gastrin heptadecapeptide (G17) in larger molecular forms of gastrin suggested that they shared a precursor-product relation (477). The isolation and sequencing of the gastrin cDNA allowed investigators to deduce many of the mechanisms of gastrin posttranslational processing (478). It has become apparent that preprogastrin undergoes several important modifications necessary for full biological activity, including signal peptide cleavage, tyrosine sulfation, serine O-phosphorylation, dibasic cleavage at two or more sites, carboxypeptidase removal of basic residues, carboxyl-terminal amidation, and amino-terminal pyroglutamate formation. The structure of human preprogastrin, as deduced from the nucleotide sequence of cDNA (479), consists of a 21amino-acid signal peptide, an amino-terminal extension of 35 amino acids, and a carboxyl-terminal extension of 9 amino acids (Fig. 2-6). Analysis of the gastrin precursor yields valuable information and is illustrative of the posttranslational processing steps outlined earlier. Preprogastrin contains a 21-amino-acid signal peptide (479–481) that possesses the three general characteristics shared by signal peptides: (1) a positively charged amino-terminal region of 3 amino acids, (2) a central hydrophobic region of 15 amino acids, and (3) a more polar pattern of amino acids adjacent to the site of cleavage. The alanine residue located at the carboxyl terminus of the signal peptide is one of the preferred amino acids at the signal peptide cleavage site (65,70). The 34-amino-acid “pro” region preceding the Arg57Arg58 site is homologous to progastrins and pro-CKKs in other species, but it does do not have significant homology in primary structure with the “pro” regions of other gastrointestinal hormones. Sorting studies of progastrin have suggested that removal of 30 amino acids within the “pro” does not impair sorting and subsequent processing (482). Indeed, the carboxyl-terminal amino acids including intact processing sites are required for progastrin sorting (482). Sulfation of gastrin at Tyr87 is known to occur in different tissues (483,484). Biosynthetic studies using 35S-sulfate have demonstrated that even large molecular forms of gastrin are sulfated at Tyr87 (251). This is consistent with the presumption that sulfation occurs in the Golgi before endoproteolytic cleavage. If one applies the known structural criteria (259,260) for tyrosine sulfation (see Table 2-1) to the sequence surrounding Tyr87 of gastrin, there is an acidic residue missing at position −1, but many acidic residues (Asp and Glu) are present in positions −5 to +5. Furthermore, there are no basic residues (Arg or Lys) and no more than

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 49 21

1 Signal peptide

Met Gln Arg Leu Cys Val Tyr Val Leu lle Phe Ala Leu Ala Leu Ala Ala Phe Ser Glu Ala

G34

24 26 40 Ser Trp Lys Pro Arg Ser Gln Gln Pro Asp Ala Pro Leu Gly Thr Gly Ala Asn Arg 57 58 Asp LEU Glu LEU Pro Trp Leu Glu Gln Gln Gln Gly Pro Ala Ser His His Arg Arg 59 74 75 Gln Leu Gly Pro Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys Lys

G17

76 87 Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp Phe

Amino-terminal "Pro" fragment

Carboxyl-terminal extension

93 94 95 96 Gly Arg Arg Ser Ala Glu Asp Glu Asn

FIG. 2-6. Deduced amino acid sequence of the human gastrin complementary DNA. The 21-aminoacid signal peptide is shown at the top with the central hydrophobic core underlined. The monobasic (Lys24, Pro25Arg26, and Arg40) and dibasic (Arg57Arg58, Lys74Lys75, and Arg94Arg95) cleavage sites are depicted in boldface. The underlined portion of the amino-terminal “pro” fragment from amino acids 40 to 51 is predicted to form an α helix with three leucine residues shown in capital letters that may be important in prohormone sorting. Gln59 and Gln76 at the amino termini of G34 and G17, respectively, serve as substrates for glutaminyl cyclase. Tyr87 is a site for gastrin sulfation, and Ser96 is a phosphorylation site. After cleavage of Arg94Arg95 and the removal of these residues by carboxypeptidase E, gastrin processing intermediates extended by Gly93 serve as substrates for the amidation enzyme.

three hydrophobic residues from in the adjacent region. On Chou-Fasman analysis there are putative β-turn sites present at −9 and +6. There are no glycosylation or disulfide bridging sites near Tyr87. On the basis of studies using a gastrin peptide fragment as a substrate for the sulfation reaction, it appears that the gastrin sulfation enzyme is similar to the tyrosine sulfotransferase described in other tissues (485). Gastrin conforms to these criteria, and this has been tested experimentally by expressing mutant progastrins in endocrine cell lines and observing the effects of the mutation on gastrin sulfation (262). Interestingly, these studies suggest that sulfation of Tyr87 enhances the subsequent endoproteolytic maturation of progastrin (262). Progastrin has been described to be phosphorylated at Ser96, and this modification can be blocked with Brefeldin in accordance with the localization of the phosphorylation reaction to the distal Golgi stack (198). Because of the proximity of the Ser96 residue to the carboxyl-terminal Gly93Arg94Arg95 amidation site, it has been suggested that phosphorylation of this site may influence the formation of the peptide amide (248,250,486). Indeed, fasting in rats leads to a relative decrease in Ser96 phosphorylation that coincides with decreased amidated gastrin, but with no effect on endoproteolysis at Arg94Arg95 (250). Similar results have been reported in dogs with a surgically excluded antrum (487). After signal peptide cleavage and passage through the Golgi, the 80-amino-acid progastrin molecule is packaged into the secretory vesicle. It appears that there is little endoproteolytic cleavage of gastrin before packaging in the secretory vesicle (488). On the amino terminus, progastrin contains three dibasic cleavage sites—Arg57Arg58, Lys74Lys75, and Arg94Arg95—and three potential monobasic cleavage sites—Lys24, Pro25Arg26, and Arg40 (see Fig. 2-6). Cleavage of

Arg57Arg58 results in the formation G34, whereas cleavage of Lys74Lys75 results in the formation of G17. G17 and G34 are the major molecular forms found in most gastrinproducing tissues, although small amounts of amidated gastrin22-93 or Component I are found in normal antral tissues and in greater concentrations in gastrinoma extracts (489). The initial step in the carboxyl-terminal processing of progastrin is cleavage at the Arg94Arg95 residues. These basic amino acids are subsequently removed by CPE, and the remaining glycine-extended peptide serves as a substrate for PAM, resulting in the formation of amidated gastrin. Sugano and colleagues (490,491) developed region-specific antisera that recognized glycine-extended gastrins (G-Gly), as well as progastrins extended by the Arg94 and Arg95 residues. With these antisera, they demonstrated that progastrin, G-Gly, and amidated gastrin were colocalized within G cells of the antral mucosa (492). Biosynthetic studies with G cells isolated from the canine antrum demonstrated that cells incubated in medium containing 35S-Met incorporated label sequentially into immunoreactive progastrin, G-Gly, and finally amidated gastrin (493–495). In accord with the hypothesis that these posttranslational processing reactions take place in a single enclosed cellular compartment such as the secretory vesicle, they could identify molecular forms of both progastrin and G-Gly that corresponded to heptadecagastrin (G17), tetratriacontagastrin (G34), and Component I (Fig. 2-7; see also later in this chapter). Further confirmation of the notion that G-gly is converted to amidated gastrin in the secretory vesicle is the finding that G-gly and gastrin are cosecreted from isolated G cells (496,497). Endoproteolytic cleavage at the Lys74Lys75 dibasic processing site of progastrin is the major determinant for the relative distribution G17 and G34 in tissues. Thus, we explored

50 / CHAPTER 2 G17 G34 Component I

Signal peptide

GlyArg94 Arg95Ser

Lys74 Lys75

ArgArg “Pro” fragment

Signal peptidase ArgArg

GlyArg94 Arg95Ser

Lys74 Lys75

Sultation and phosphorylation ?SO4

GlyArg94 Arg95Ser

Lys74 Lys75

ArgArg

PO4

Prohormone convertase (PC1/PC3) ?SO4 GlyArg94 Arg95

Lys74 Lys75

Carboxy peptidase E ?SO4 Lys74 Lys75 PAM Lys74 Lys75

Gly PC2

NH2

Gly ?SO4

PC2

NH2

PAM

FIG. 2-7. Posttranslational processing of progastrin.

the ability of two PCs—PC1/PC3 and PC2—to cleave this important site within progastrin. We expressed wild-type human gastrin cDNA in AtT-20 cells. Because AtT-20 cells express PC1/PC3 but not PC2 (319,498), we also coexpressed a cDNA encoding PC2. The Lys74Lys75 progastrin processing site was efficiently cleaved in AtT-20 cells only after coexpression of PC2 (499). These data suggest that PC1/PC3 is not responsible for the cleavage of LysLys sites in wild-type progastrin. Nevertheless, PC1/PC3 is expressed in the duodenum (500) where the predominant form of gastrin is G34. Thus, we sought to explore the possibility that PC1/PC3 may process progastrin at the C-terminal Arg94Arg95 site. We inhibited expression of PC1/PC3 in these cells with two different antisense PC1/PC3 constructs (full and partial length). Coexpression of either antisense construct resulted in a consistent decrease in G34-NH2. We concluded that PC1/PC3 is a progastrin-processing enzyme, suggesting a role for PC1/PC3 progastrin processing in gastric or duodenal G cells (501). Northern blot analysis of enriched canine antral G cells shows PC2 but not PC1/PC3 expression, whereas fundic D cells expressed both enzymes (499,502). Thus, we anticipated that PC2 was responsible for progastrin processing in antral G cells. Inhibition of PC2 expression in

canine antral G cells with antisense oligonucleotide probes resulted in diminished amidated gastrin formation and an accumulation of progastrin in gastric antral G cells. These studies are consistent with our previous studies in endocrine cell lines and confirm that PC2 is a progastrin cleavage enzyme in antral G cells (495). Gastrin biosynthetic studies in the rat (503) demonstrate that progastrin is processed to G34-Gly that undergoes further endoproteolytic cleavage and amidation, resulting in the formation of G17-NH2. Furthermore, it appears that most G17-NH2 arises from cleavage of G34-NH2 rather than amidation of G17-Gly via the action of a PAM, suggesting that G17-Gly is a distinct end-product of progastrin processing. These results suggest that other factors such as cofactor (perhaps ascorbate) availability may account for the presence of G17-Gly in some tissues. Complete posttranslational processing with carboxylamidation of G17 and G34 are required for full biological activity mediated by gastrin/CCKB receptors. Indeed, glycineextended gastrins (G-Gly) are at least four orders of magnitude less potent than amidated G17 in acutely stimulating gastric acid secretion (504,505). Although high concentrations of G-Gly can be found in tumors (506) and during

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 51

Stomach

of other well-characterized monobasic cleavage sites (see Table 2-2). Pro25Arg26 is preceded by a proline and contains a serine in the +1 position, suggesting that it may prove to be a monobasic cleavage site. However, the site lacks a basic amino acid in the −3, −5, or −7 positions, suggesting that it is likely to be a poor site for monobasic cleavage. The glutamine residue at the amino-terminal ends of G17 and G34 serve as substrates for GC after cleavage of Arg57Arg58 or Lys74Lys75, although the activity of this enzyme has not been examined in gastrointestinal tissues or with a gastrin substrate.

REFERENCES G-NH2

jun c-fos and c-jun kinase expression

G-Gly

phosphorylation of c-jun

↑ gene expression and growth

Proliferating gut cell FIG. 2-8. Interaction of amidated gastrin and G-Gly gut growth.

development (507), most investigators believed that the accumulation of G-Gly was of no biological significance. In addition to its well-known regulation of gastric acid secretion, G-NH2 has a proliferative action on normal and malignant gastrointestinal tissues (508–512); therefore, we questioned whether G17-Gly might also act as a trophic agent. To explore this possibility, we noted that both G17NH2 and G17-Gly stimulated DNA synthesis in several cell lines via distinct receptors and signal transduction mechanisms (513-516). Other groups have confirmed the trophic nature of G-Gly on the gut (516–522). In other studies, we noted that the ligand specificity of the G-Gly receptor on parietal cells was similar to that on cell lines (523). Furthermore, although acute administration of G-Gly had no effect on gastric acid secretion (504,505), prolonged treatment (48 hours) of parietal cells with G-Gly does increase histamine-stimulated acid secretion and gene expression of the proton pump, H+,K+-ATPase (523). These chronic effects of G-Gly on acid secretion were confirmed by others in vivo (524) and in gastrin-deficient mice (525). These studies underscore the importance of prohormone-processing mechanisms to gut biology (Fig. 2-8). Cleavage of any of three monobasic sites appears to a relatively rare event, but cleavage of Pro25Arg26 has been observed in gastrinoma tissues. Unlike the other monobasic sites, only the Pro25Arg26 site has any of the structural features

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526. 527. 528.

neutralize amidated and glycine-extended gastrin-17 and inhibit the growth of colon cancer. Cancer Res 1996;56:880–885. Kaise M, Muraoka A, Seva C, Takeda H, Dickinson CJ, Yamada T. Glycine-extended progastrin intermediates induce H+, K+ -ATPase αsubunit gene expression through a nevel receptor. J Biol Chem 1995; 270:11155–11160. Higashide S, Gomez G, Greeley GH Jr, Townsend CM Jr, Thompson JC. Glycine-extended gastrin potentiates gastrin-stimulated gastric acid secretion in rats. Am J Physiol 1996;270:G220–G224. Chen D, Zhao CM, Dockray GJ, Varro A, Van Hoek A, Sinclair NF, Wang TC, Koh TJ. Glycine-extended gastrin synergizes with gastrin 17 to stimulate acid secretion in gastrin-deficient mice. Gastroenterology 2000;119:756–765. Preiss T, Hentze MW. Starting the protein synthesis machine: eukaryotic translation initiation. Bioessays 2003;25:1201–1211. Ramakrishnan V. Ribosome structure and the mechanism of translation. Cell 2002;108:557–572. Calkhoven CF, Muller C, Leut A. Translational control of gene expression and disease. Trends Mol Med 2002;8(12):577–583.

CHAPTER

3

Transmembrane Signaling by G Protein–Coupled Receptors Claire Jacob and Nigel W. Bunnett Structure and Function of G Protein–Coupled Receptors, 64 G Protein–Coupled Receptors Share Structural Motifs, 64 Rhodopsin Is a Model G Protein–Coupled Receptor, 64 Agonists Interact with Different Receptor Domains, 65 G Protein–Coupled Receptors Can Form Dimers, 68 Accessory Proteins Are Required for the Function of Some G Protein–Coupled Receptors, 69 Mechanisms of Signal Transduction, 70 G Protein–Coupled Receptors Interact with Heterotrimeric G Proteins, 70 G Protein–Coupled Receptors and Heterotrimeric G Proteins Exist in Multiple States, 70 Monomeric G Proteins also Mediate G Protein–Coupled Receptor Signaling, 71 Organization of G Protein–Coupled Receptors, G Proteins, and Signaling Proteins into Microdomains Increases the Efficiency and Fidelity of Signal Transduction, 73 Receptor Tyrosine Kinases Are Signaling Partners for G Protein–Coupled Receptors, 74 Receptor Tyrosine Kinases Comprise a Family of SingleTransmembrane Domain Proteins with Intrinsic Kinase Activity, 74

Tyrosine Phosphorylation Recruits and Activates Receptor Tyrosine Kinase Signaling Molecules, 74 G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases, 76 Receptor Tyrosine Kinases also Signal through Heterotrimeric G Proteins, 76 Mechanisms That Regulate Signaling by G Protein–Coupled Receptors, 77 G Protein Receptor Kinases and Arrestins Mediate Homologous Desensitization, 77 Other Kinases Regulate G Protein–Coupled Receptor Signaling, 79 Clathrin, β-Arrestins, and Dynamin Mediate Endocytosis of Many G Protein–Coupled Receptors, 81 Receptor Recycling Mediates Resensitization of Signal Transduction, 82 Internalized G Protein–Coupled Receptors Can Continue to Signal, 83 Lysosomal Trafficking Permanently Arrests Signal Transduction, 83 Conclusion, 85 Acknowledgments, 85 References, 85

Cells are required to detect and respond to an extraordinarily diverse array of stimuli from the external environment or from intrinsic sources. Environmental stimuli include signals as diverse as photons, odorant and taste molecules, and both innocuous and noxious physical and chemical agents. Intrinsic stimuli include a range of structurally distinct hormones, neurotransmitters, and paracrine substances.

Receptors in the plasma membrane specifically detect these stimuli and are essential in determining the appropriate physiologic responses to environmental signals and regulatory molecules. Cell-surface receptors can be divided into several broad families based on their structure and mechanisms of action. The superfamily of G protein–coupled receptors (GPCRs) comprises by far the largest and most functionally diverse group of receptors. GPCRs, which include receptors for most hormones and neurotransmitters, are seven-transmembrane domain proteins that interact with heterotrimeric G proteins to initiate signal transduction. The general family of catalytic, enzyme-linked receptors are single-transmembrane domain proteins with cytosolic domains that either have intrinsic enzymatic activity or

C. Jacob and N. W. Bunnett: Departments of Surgery and Physiology, University of California, San Francisco, San Francisco, California 94143. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

63

64 / CHAPTER 3 associate directly with enzymes. They are subdivided into five families depending on the nature of their enzymatic activities: receptor tyrosine kinases (RTKs), tyrosine kinase– associated receptors, receptor tyrosine phosphatases, receptor serine-threonine kinases, and receptor guanylyl cyclases. This family includes receptors for growth factors, certain hormones, and immune mediators, and the receptors initiate signaling by activation of their intrinsic enzymatic activity or by association with enzymes. Certain ion channels are also directly regulated by extrinsic and intrinsic signals. The ligand-gated ion channels bind directly to neurotransmitters, which regulate their activity. Other channels, such as those of the transient receptor potential vanilloid receptor family, can directly bind regulatory molecules or are indirectly regulated by agonists of other receptors, which control their activity and function. All of these categories of cellsurface receptors play crucial roles in the regulation of the gastrointestinal tract. The purpose of this chapter is to review some of the major recent advances in our understanding of transmembrane signaling by GPCRs. It summarizes current knowledge of the mechanisms of activation, signal transduction, and regulation of these receptors. The RTKs are discussed briefly because of the recent appreciation that signaling by GPCRs and RTKs is inextricably linked: activation of many GPCRs results in stimulation of RTKs, and these receptors share many of the same mechanisms of signal transduction. This chapter does not discuss the roles of specific GPCRs and RTKs in the control of particular aspects of digestion; these topics are covered in other chapters.

disease, and it has been estimated that more than half of all therapeutic drugs target GPCRs, either directly or indirectly (6). The structure and function of GPCRs have been reviewed in detail previously (2,7).

G Protein–Coupled Receptors Share Structural Motifs GPCRs have similar amino acid sequences with some residues that are conserved between family members (2). They have an extracellular amino terminus, an intracellular carboxyl-tail, seven transmembrane domains of hydrophobic amino acids that are organized in α helices, three extracellular loops, and three intracellular loops (see Fig. 3-1A for the structure of rhodopsin, a prototypical GPCR). Consequently, they are also known as heptahelical or serpentine receptors. Sequences of the transmembrane domains are highly conserved among the different GPCRs, and receptors differ more in their loop structures and intracellular and extracellular tails, which vary considerably in length. Based on similarities in their amino acid sequences, the GPCRs have been divided into six families, each with more than 20% amino acid identity in the transmembrane domains (2,8,9). In mammals, the principal families are: A, or rhodopsin-like family (the largest); B, or secretin-like family; and C, or metabotropic family (Table 3-1). Additional families in mammals include the LNB-7TM, frizzled/ smoothened, vomeronasal 1 receptor, and taste 2 receptor; there are also other families of GPCRs that are found only in nematodes, fungi, and plants.

STRUCTURE AND FUNCTION OF G PROTEIN–COUPLED RECEPTORS

Rhodopsin Is a Model G Protein–Coupled Receptor

With at least 1,000 members, the superfamily of GPCRs comprises about 2% of the proteins encoded by the human genome (1). Many GPCRs sense environmental stimuli such as photons, odorants, and tastes. However, there are more than 360 nonsensory GPCRs, 200 of which respond to endogenous agonists including amino acid transmitters, biogenic amines, regulatory peptides, glycoproteins, proteases, nucleotides, nucleosides, prostanoids, phospholipids, and fatty acids (1,2). There are also approximately 160 “orphan receptors” that have been identified in the genome but for which there is no known ligand or function (3). GPCRs as a group participate in all aspects of physiologic regulation, including the sensory perception of light, odors, tastes, and painful stimuli, and they control cell motility, endocrine and exocrine secretion, neurotransmission, metabolism, inflammation, and immune responses (1). GPCRs also play a major role in human disease. Mutations in GPCRs can cause genetic diseases (4), and certain invading organisms, such as viruses, can hijack GPCRs, thereby making use of their intracellular signaling pathways to ensure their replication, which can contribute to their pathogenesis (5). The GPCRs are of great importance in the treatment of

Formidable technical obstacles have hampered attempts to purify GPCRs and study their structure by X-ray crystallography. Obstacles to purifying GPCRs include their low abundance, the instability of these hydrophobic proteins in environments lacking phospholipids, and the tendency of GPCRs to aggregate and precipitate. These obstacles have been overcome for one GPCR, rhodopsin. Rhodopsin is highly concentrated in the retina, allowing purification and investigation of its crystal structure (10) (see Fig. 3-1B). Consequently, much of our understanding about the structure, function, signal transduction, and regulation of GPCRs derives from studies of opsins. Opsins are photoreceptors of the retina. They include rhodopsin, the photoreceptor of retinal rod cells, and blue, green, and red opsins, the photoreceptors of cone cells. They differ from other GPCRs in that they form a covalent link with their ligand, 11-cis-retinal. The link takes the form of a Schiff base between the aldehyde of retinal and the ε amino acid of a lysine residue in transmembrane domain 7. A single photon converts 11-cis-retinal to all-trans-retinal, leading to a conformational change in the receptor and activation of its G protein, transducin, or Gt.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 65 C C-terminal

C-II Cytoplasmic side

C-III

C-I

E-III Extracellular side

E-I E-II

N-terminal N

A

B FIG. 3-1. The structure of bovine rhodopsin. (A) Two-dimensional model of bovine rhodopsin. Cylinders indicate the transmembrane helices. The disulfide bridge, which is conserved among G protein–coupled receptors, is at residue (110). (B) The three-dimensional model of rhodopsin; parallel view to the plane of the membrane. (Reproduced from [A] Teller and colleagues [223]; and [B] Palczewski and colleagues [10], by permission.)

There are several notable characteristics of the structure of rhodopsin (reviewed by Filipek and colleagues [11]), which has been analyzed with X-ray diffraction at 2.2-Å resolution (12). The transmembrane domains are arranged in an anticlockwise manner when viewed from the extracellular side of the membrane, in the order of 1-2-3-4-5-6-7. The amino acids of the transmembrane domains are in α helices, although there are certain kinks at proline and other residues, and there are also interactions among the helices. For example, helix 6 of rhodopsin interacts with helix 7 by hydrogen bonds and with helices 2, 3, and 5 by van der Waals forces, which together confer mobility to helix 6 that is probably important for receptor activation. The highly conserved motif NPXXY of helix 7 may allow interaction with helix 6, which could hold the receptor in its inactive state. The carboxyl-terminus of helix 3 of rhodopsin is of particular importance because it contains the E(D)RY motif implicated in the regulation of the receptor’s interaction with its G protein. Rhodopsin has an additional short helix in the cytoplasm, which, together with the cytoplasmic loops 2 and 3, constitutes a part of the binding site for Gtα subunit and is involved in the regulation of Gtγ binding (13) (see G Protein–Coupled Receptors Interact with Heterodimeric G Proteins later in this chapter). The cytoplasmic loops are not well defined and must be mobile in solution, and the residues of the carboxyl-tail of the receptor are probably flexible,

lacking a definite conformation (14,15). The amino-terminal extracellular region of rhodopsin consists of 33 residues, which form a glycosylated unit overlaying the extracellular loops. The residues 173 through 198 on the loop between helices 4 and 5 may form a “plug” for binding of retinal arrestin, an important regulator of GPCRs (see G-Protein Receptor Kinases and Arrestins Mediate Homologous Desensitization later in this chapter). Molecular modeling of other class A receptors based on the structure of rhodopsin suggests that they adopt similar conformations. However, information about GPCR structure comes from study of rhodopsin in its resting state, and little is known about how agonist binding alters the receptor conformation to initiate signal transduction.

Agonists Interact with Different Receptor Domains The sites at which agonists and antagonists interact with their receptors have been deduced by a combination of approaches that include molecular modeling, generation of chimeric and mutant receptors, and binding and signaling assays (Fig. 3-2). As may be expected, the sites of interaction between GPCRs and their agonists and antagonists vary, depending on the structures of the receptors and their ligands.

66 / CHAPTER 3 TABLE 3-1. A partial list of G protein–coupled receptors in classes A, B, and C Class

Examples

Class A: rhodopsin-like

Amines: acetylcholine muscarinic receptors, adrenoreceptors, dopamine receptors, histamine receptors, serotonin receptors Peptides: angiotensin, bombesin, bradykinin, chemokine, cholecystokinin, endothelin, galanin, melanocortin, neuropeptide Y, neurotensin, opioid, somatostatin, vasopressin Protease-activated receptors Glycoproteins: follicle-stimulating hormone; luteinizing hormonereleasing hormone; thyrotropin Rhodopsin Olfactory Prostanoids Nucleotides Cannabinoids Platelet-activating factor Lysosphingolipids Calcitonin Calcitonin gene-related peptide Corticotropin-releasing factor Gastric inhibitory peptide Glucagon Growth hormone-releasing hormone Parathyroid hormone Pituitary adenylyl cyclase activating polypeptide Secretin Vasoactive intestinal polypeptide Diuretic hormone Cadherin Metabotropic glutamate Extracellular calcium-sensing Pheromone γ-Aminobutyric acid receptor B Taste receptors (T1R)

Class B: secretin-like

Class C: metabotropic glutamate pheromone

Class A G Protein–Coupled Receptors Small endogenous agonists usually interact in a noncovalent manner within the transmembrane domains of class A receptors. The sites of interaction between the β2-adrenergic receptor (β2-AR) and its agonists have been investigated extensively. For example, the predicted binding site of the agonist epinephrine is deep within the receptor in between helices 3, 4, 5, 6, and 7 (16). Mutagenesis studies have identified some residues of the receptor that are of critical importance for this binding. For example, Asp113 of helix 3 interacts with amine groups of epinephrine. This residue is highly conserved among the receptors for biogenic amines. Ser203, Ser204, and Ser207 of helix 5 form a network of hydrogen bonds with the two catechol hydroxyl groups of epinephrine (17). Asn293 within helix 6 also interacts with the alkyl-hydroxyl group of epinephrine and with Asp113 of

helix 3. Ile169 (helix 4) interacts with the N-methyl group of epinephrine and might be responsible for the stronger affinity of epinephrine for the receptor than norepinephrine, which does not have this N-methyl group. Zn2+ ions act as a positive allosteric ligand for the β2-AR, enhancing agonist affinity for the receptor (18,19). The Zn2+ binding sites responsible for this effect have been identified by mutagenesis as the His269, Cys265, and Glu225 residues, which are localized on the third intracellular loop between helices 5 and 6. Thus, bridging the cytoplasmic extensions of helices 5 and 6 with Zn2+ facilitates agonist binding. Indeed, biophysical studies have demonstrated that agonist binding leads to the movement of helix 6 relative to helix 5 (20). Binding sites within the transmembrane domains have been identified for other small molecular weight agonists of GPCRs, including adenosine (21), nucleotides (22), and lipids such as anandamide, a cannabinoid receptor agonist (23). Receptors for peptide hormones, neurotransmitters, and paracrine agents are the largest subgroup of the A family of GPCRs. Most peptides interact with residues in the amino terminus and the extracellular loops of their receptors, including receptors for angiotensin II (24), bradykinin (25), and cholecystokinin/gastrin (26,27). However, residues in the superficial regions of transmembrane helices 2, 3, 5, 6, and 7 also contribute to binding of certain neuropeptides, such as neuropeptide Y, opioids, and somatostatin (see Gether [7] and references therein). The binding sites for agonists and antagonists of the neurokinin-1 receptor (NK1R) have been studied in considerable detail. Studies of mutant and chimeric neurokinin receptor indicate that natural peptide agonists bind to residues scattered throughout the extracellular regions, whereas nonpeptide antagonists interact with residues in the transmembrane domain (28–33). Cross-linking experiments using photoactivatable substance P (SP) indicate that the peptide interacts with domains in the amino tail and second extracellular loop of the NK1R (34). Different residues are important for interaction with different antagonists (32,33,35,36). For example, His197 in the fifth transmembrane domain of the human NK1R is critical for binding to CP-96,345 but not RP67580 (nonpeptide NK1R antagonists). However, His265 in the sixth transmembrane domain is required for binding of RP67580 but not CP-96,345. Although involving different residues, the binding pocket for epinephrine in the β2-AR is similarly located. Thus, there may be conservation of binding pockets for small molecules in GPCRs. The importance of other residues in the putative binding pocket for nonpeptide antagonists of the NK1R has been further explored by the systematic mutation of residues near to His, to create a metal ion–binding domain (37). Substitution of Glu193 and Tyr272 to His increases the affinity for Zn2+ by 794-fold by creating a pocket of 4 His residues to bind Zn2+. Remarkably, the creation of a high-affinity binding pocket for Zn2+ in this region inhibits binding and signaling of SP. Thus, binding of Zn2+ ions to the mutated receptor may cause an allosteric alteration that mimics that induced by nonpeptide antagonists, which stabilizes the receptor in a conformation that cannot bind SP.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 67 Competitive antagonist Opsin

Biogenic amine

Glycoprotein (family A)

Non-competitive antagonist

Neuropeptide (family A, B)

Glutamate (family C)

Agonist

Substance P

Protease-activated

Frizzled (F/S family)

FIG. 3-2. Models of ligand binding sites for various G protein–coupled receptors. Small molecules bind to sites within the transmembrane domains. Competitive antagonists and agonists interact with partially overlapping binding sites in biogenic amine receptors, whereas binding sites for allosteric antagonists have been identified in extracellular regions of muscarinic receptors. Peptides bind to extracellular sites in the amino tail and loops. Substance P binds to extracellular domains of the neurokinin-1 receptor. Proteases cleave receptors exposing tethered ligands that bind to sites in the second extracellular loop. Glycoprotein hormones interact with leucine-rich repeat motifs of the large amino tails of their receptors. Family C receptors possess binding sites for agonists and competitive antagonists in a cleft between two extracellular domains. The amino terminus of Frizzled receptors contains the binding site for their native ligands, the Wnt proteins. (Reproduced from Kristiansen [2], by permission.)

The protease-activated receptors (PARs) are a family of four GPCRs that are activated by proteolytic cleavage (reviewed by Ossovskaya and Bunnett [38]). Certain serine proteases (e.g., coagulation factors such as thrombin and factor VIIa and Xa, mast cell tryptase, trypsins) cleave the extracellular amino terminus of these receptors to expose an amino-terminal domain, which acts as a “tethered ligand” by binding to and activating the cleaved receptor. Synthetic peptides that mimic the tethered ligand domain can directly activate the receptors, which provide experimental support for this mechanism of activation. Thus, the PARs could be viewed as specialized peptide receptors–that is, ones in which the peptide is physically part of the receptor molecule and only exposed by proteolysis. Studies of chimeric and mutant receptors indicate that the tethered ligand interacts with the second extracellular domains of these receptors, and mutational analyses have identified residues in the tethered ligand and second extracellular loop that are critical for this interaction (39,40). Receptors for glycoproteins such as luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone are unusual in that they have large amino-terminal extracellular

tails of up to 500 residues. These domains contain multiple leucine-rich repeat or LRR motifs, which are involved in protein–protein interactions. These regions are essential for interaction of glycoproteins with their receptors (see Kristiansen [2] and references therein). Class B G Protein–Coupled Receptors Members of the class B family of GPCRs have extracellular tails of intermediate length (~120 residues), and the family includes receptors for functionally important peptides such as secretin, vasoactive intestinal polypeptide (VIP), pituitary adenylyl cyclase–activating polypeptide (PACAP), calcitonin gene–related peptide (CGRP), and corticotropin-releasing factor. Agonists of these receptors interact with several sites within the amino-terminal tail and the extracellular loops (41–43). Most B family GPCRs possess a conserved aspartic acid residue and a basic residue in extracellular loop I at the junction of transmembrane domain 2. Mutational analyses indicate that these are essential residues for agonist binding and receptor activation.

68 / CHAPTER 3 Class C G Protein–Coupled Receptors The class C family of GPCRs includes receptors for endogenous ligands such as glutamate (metabotropic glutamate receptors) and γ-aminobutyric acid (GABAB receptors), for Ca2+ ions (Ca-sensing receptors), for pheromones, and for certain tastes. Given the diverse nature of these agonists, it is not surprising that they interact with various regions of their receptors. Class C GPCRs such as the metabotropic glutamate, GABAB, and Ca-sensing receptors have long extracellular tails. This region of the metabotropic glutamate receptor contains a domain known as the “Venus flytrap module.” Mutational analyses indicate that agonists bind to a cleft between two lobes of this domain, resulting in a conformational change that traps the agonist in the cleft (reviewed by Pin and colleagues [44]). This conformational change may then expose amino acid sequences that serve as tethered ligands, which bind to extracellular loops to activate the receptors. Ca2+ ions also appear to interact with extracellular and transmembrane domains of Ca-sensing receptors, allowing the receptors to switch from an inactive to an active state over a narrow range of Ca2+ ion concentration.

G Protein–Coupled Receptors Can Form Dimers GPCRs were thought to function as monomers: a single agonist molecule binds to one receptor, which then associates with a G protein. However, it is now well established that some GPCRs are dimers (see reviews by Terrillon and Bouvier [45] and Milligan [46]). This view is based on a variety of biochemical studies of the patterns of GPCR migration on electrophoresis gels, coimmunoprecipitation of different receptors, and fluorescence or bioluminescence resonance energy transfer that allows observations of protein– protein interactions in living cells. Through these approaches, investigators have shown GPCRs to exist as homodimers and heterodimers in the endoplasmic reticulum, at the cell surface, and in endosomes. This dimerization has been suggested to contribute to the posttranslational processing and plasma membrane trafficking or “ontogeny” of GPCRs, to their interaction with agonists and antagonists or “pharmacologic diversity,” to their mechanisms of signal transduction, and to agonist-induced trafficking of GPCRs (Fig. 3-3A). The correct folding of proteins in the endoplasmic reticulum is essential for their sorting at the plasma membrane. Some proteins form oligomers in the endoplasmic reticulum, which facilitates this sorting by masking retention signals. The formation of GPCR dimers in the endoplasmic reticulum may be required for sorting to the plasma membrane. This is clearly the case for the metabotropic GABAB receptor, which is formed of two receptor subunits: R1 and R2. When expressed separately, GABABR1 is retained in the endoplasmic reticulum because it contains a retention signal, and although GABABR2 traffics to the cell surface, it is unable to signal (47). However, when coexpressed, the subunits form a heterodimer that masks the retention signal of GABABR1 so that both are translocated

to the cell surface where they form a functional receptor. Vasopressin and oxytocin receptors also form dimers in the endoplasmic reticulum (48). Notably, expression of truncated mutants of GPCRs can sometimes impede expression of wild-type receptors at the plasma membrane, possibly by preventing assembly of dimers in the endoplasmic reticulum (49,50). Although many receptors can form dimers at the cell surface, it is unclear whether they dimerize constitutively, or if agonist binding affects dimerization (see Terrillon and Bouvier [45] and references therein). However, it is well established that dimer formation can have a major effect on receptor pharmacology. For example, when expressed together, δ- and κ-opioid receptors form a stable heterodimer that has a low affinity for either δ- or κ-selective ligands alone, but a high affinity for a combination of ligands, suggesting positive cooperativity (51). Both positive and negative cooperativity have been observed for heterodimers of other GPCRs, including δ/µ-opioid (52) and somatostatin SSTR5/dopamine D2 receptors (53). Such diversity could have important implications for drug development, because the efficacy of agonists and antagonists may be influenced by the components of the heterodimers. Heterodimerization may also be important for signal transduction. In the case of the GABAB receptor, the GABABR1 subunit appears to bind GABA, whereas the GABABR2 subunit couples to heterotrimeric G proteins (47). The heterodimerization of taste receptors is also required for signal transduction. In addition, formation of heterodimers has been proposed to explain the potentiation of signal transduction that can occur when GPCRs are coexpressed, including δ/κ opioid receptors (51), δ/µ opioid receptors (52), and somatostatin SSTR5/dopamine D2 receptors (53). Dimerization could also affect receptor endocytosis, as there are reports that activation of one receptor can lead to endocytosis of a second receptor. For example, when the µ-opioid and NK1R are coexpressed, selective activation of either receptor induces endocytosis of the other receptor (54). There are many other examples of cointernalization, including somatostatin SSTR1/SSTR5 (55) and somatostatin SSTR2A/µ-opioid receptors (56). The functional relevance of this trafficking remains to be determined. Despite the aforementioned reports of GPCR dimerization, it remains uncertain whether dimerization plays an important role in physiologic regulation. Most studies of receptor dimerization are in heterologous expression systems, and, to be of physiologic relevance, the receptors should be naturally coexpressed in the same cell. Given the difficulty of specifically localizing GPCRs in tissues, such colocalization studies have been completed for only a few receptors. An obvious caution is that overexpression may promote dimerization and, indeed, certain receptors will only form heterodimers when they are highly expressed (57). However, formation of dimers has been observed in native cells and tissues and in transfected cells expressing physiologically relevant numbers of receptors, where dimerization can have a major influence on receptor pharmacology, signaling, and regulation.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 69

3 Endocytosis

2 Pharmacological diversity 1

Early endosome

Ontogeny

Endoplasmic reticulum-Golgi apparatus

A

2 Pharmacological diversity Adrenomedullin CGRP

3 RAMP1

RAMP2

RAMP3

Endocytosis

Early endosome

1 Ontogeny

CLR

B

Endoplasmic reticulum-Golgi apparatus

FIG. 3-3. The role of dimerization and of accessory proteins in the ontogeny (1), pharmacologic diversity (2), and trafficking (3) of G protein–coupled receptors (GPCRs). (A) (1) Certain GPCRs dimerize in the endoplasmic reticulum, and dimerization is required for receptor folding and translocation to the plasma membrane. (2) Once at the plasma membrane, dimerization can influence the ability of GPCRs to couple to heterotrimeric G proteins by both positive and negative cooperativity, resulting in pharmacologic diversity. (3) Some GPCRs internalize as dimers, but the function of this process is unknown. (B) (1) Receptor activity-modifying proteins (RAMPs) are chaperones that are required for trafficking of calcitonin receptor-like receptor (CLR) from the endoplasmic reticulum to the plasma membrane. (2) The presence of RAMPs at the plasma membrane leads to pharmacologic diversity as it determines the affinity of CLR for CGRP and adrenomedullin. (3) CLR and RAMP1 cointernalize.

Accessory Proteins Are Required for the Function of Some G Protein–Coupled Receptors The expression and activity of certain GPCRs is critically dependent on the existence of accessory proteins. Perhaps the best example is calcitonin receptor–like receptor (CLR), a receptor for several peptides including CGRP, adrenomedullin, and intermedin (58,59). The function of CLR depends on receptor activity–modifying proteins (RAMPs), a family of three single-transmembrane domain proteins (see Fig. 3-3B). When expressed alone, CLR is retained in the endoplasmic reticulum and does not traffic to the plasma membrane or

signal. RAMPs act as chaperones that mediate CLR trafficking to the plasma membrane and influence the glycosylation state of the receptor. In addition, the presence of RAMPs with CLR at the cell surface determines the affinity of the receptor for agonists. Thus, CLR/RAMP1 shows preference for CGRP over adrenomedullin, whereas CLR/RAMP2 or 3 binds to adrenomedullin with a higher affinity. CLR interacts similarly with intermedin when it is expressed with RAMP1, 2, or 3. RAMPs may also interact with other GPCRs of the B family, including the calcitonin receptor, the VPAC1 VIP/PACAP receptor, the glucagon receptor, and receptors for parathyroid hormone receptors (60).

70 / CHAPTER 3 MECHANISMS OF SIGNAL TRANSDUCTION Activated GPCRs interact with heterotrimeric G proteins. The subunits of these G proteins then regulate an array of enzymes and ion channels, resulting in both transient and sustained effects on cellular function. Many of these pathways are activated by other classes of receptors, such as RTKs, and there is accumulating evidence for cross talk between GPCR and RTK signaling (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases later in this chapter). However, scaffolding proteins can localize signaling molecules to particular intracellular domains and, in this manner, increase the specificity and fidelity of signal transduction.

G Protein–Coupled Receptors Interact with Heterotrimeric G Proteins Agonist-bound GPCRs adopt a conformation that promotes interaction with heterotrimeric G proteins. These G proteins play a major role in signaling by GPCR (see reviews by Cabrera-Vera and colleagues [61] and Hampoelz and Knoblich [62]). There are three G-protein subunits: α (39– 45 kDa), β (35–39 kDa), and γ (6–8 kDa). Currently, there are 28 α, 5 β, and 12 γ known subunits that can associate in many different combinations to provide great flexibility in the process of signal transduction. The heterotrimeric G proteins follow a common pathway of activation and inactivation after agonist interaction with a GPCR. When bound to guanosine diphosphate (GDP), the Gα subunit associates with Gβγ and the complex is an inactive heterotrimer. Agonist binding induces the change of conformation of a GPCR resulting in an increased affinity for the G protein. The activated GPCR functions as a guanine nucleotide-exchange factor (GEF) for G proteins, allowing dissociation of GDP and almost immediate replacement with guanosine triphosphate (GTP), which is present at greater concentrations than GDP. The GTP-bound Gα subunit dissociates from the Gβγ subunits, and the dissociated Gα and Gβγ regulate downstream effectors. The intrinsic GTPase activity of the Gα subunit terminates the activation, and the resulting GDPbound Gα reassociates with Gβγ to form the heterotrimer of the resting state. Based on structural similarities, there are four main families of Gα proteins: Gs (Gs, Golf), Gi (Gi1-3, Gt, Gg, Go, Gz), Gq (Gq, G11, G14, G15/16), and G12 (G12, G13) (61). The Gα subunits and the Gβγ subunits (Gβγ subunits are tightly associated and can be regarded as a functional unit) associate with a variety of signaling proteins including enzymes and ion channels to regulate multiple signaling pathways (Fig. 3-4). Each of the four main subtypes of heterotrimeric G proteins regulates particular downstream effectors (61). Gαs activates adenylyl cyclases 1 to 9, leading to formation of cyclic 3′, 5′-adenosine monophosphate (cAMP), and also regulates certain Ca2+ channels. Conversely, Gαi inhibits adenylyl cyclase 5 and 6 to suppress generation of cAMP, and also

controls Ca2+ and K+ channels. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase A. Protein kinase A is a serine/threonine kinase that is targeted to substrates by A kinase anchoring proteins. cAMP can also act by protein kinase A–independent mechanisms, for example, by controlling the GEF for the GTPase Rap-1 and by activating certain cAMP-gated ion channels. The Gαq family activates phospholipase Cβ1-4 (PLCβ), which hydrolyses phosphatidyl bisphosphate into diacylglycerol and inositol trisphosphate, which subsequently activate protein kinase C and mobilize Ca2+ ions from intracellular stores, respectively. Protein kinase C is a serine/threonine kinase that is targeted to numerous substrates by receptors for activated C kinase or RACKs (63), and regulates multiple events ranging from channel activities to mitogenic signaling. The G12 subfamily regulates the Ras homology (Rho) GEFs. Gβγ subunits contribute to the regulation of these pathways and also have distinct effects of their own (see Fig. 3-4) (64). For example, Gβγ subunits regulate K+ and Ca2+ channels, activate phospholipase A2 leading to release of arachidonic acid, stimulate phospholipase C β, inhibit or activate adenylyl cyclase, control activity of certain G-protein receptor kinases (GRKs), and contribute to Ras-dependent activation of mitogen-activated protein kinases (MAPKs). Receptors vary considerably in their specificity for activating or coupling to distinct G proteins. Some receptors interact specifically with one class of G proteins (Gq/11, Gs, Gi) to activate a limited number of signaling pathways, whereas others couple promiscuously to many G proteins to generate multiple intracellular signals (65). Different agonists that bind to the same receptor can activate different heterotrimeric G proteins (66), and the formation of GPCR homodimers or heterodimers can result in a complex series of signals (45,46).

G Protein–Coupled Receptors and Heterotrimeric G Proteins Exist in Multiple States Several models have been proposed to describe the interactions among GPCRs, their agonists, and heterotrimeric G proteins. In the most simplistic model, receptors are either unoccupied (off) or agonist-bound (on). The first refinement of this model arose from the report that receptor affinity for agonists depends on its interaction with guanine nucleotides (67). Thus, in the presence of GDP, interaction of an agonist with the β-AR induced assembly of a “ternary complex,” comprising agonist, GPCR, and G protein, in which the receptor was GPCR in a high-affinity state. In the absence of G proteins, the receptor was proposed to exist in a lowaffinity state. The second refinement was prompted by the discovery of constitutively active GPCRs, (i.e., GPCRs that are active even in the absence of agonists). The α1b-AR, with a substitution of part of the third intracellular loop of the β2-AR, or even with a mutation of a single amino acid residue, was observed to activate Gq/11 proteins in the absence of agonists (68). These and similar observations of other GPCRs led to the development of the “allosteric ternary

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 71

GPCR

αi

βγ

αs

RTK

Transactivation

α 12/13

βγ

βγ

α q11

βγ

Shc Grb2

AC

AC

PI3K

PLCβ

GEFs

Src

+

−

PLCβ

SRE

Rho

JAK

InsP3

Ras

DAG

PKA

Raf GRK ROK

STAT

Ca2+

PKC MEK

Ki Pyk2 TK

ERK

Morphology proliferation migration secretion adhesion transcription FIG. 3-4. A summary of G protein–coupled receptor (GPCR) signal transduction. GPCRs couples to heterotrimeric G proteins. The Gα subunits Gs, Gi, G12/13, and Gq11 regulate various enzymes. For example, Gs stimulates, whereas Gi inhibits adenylyl cyclase (AC) to increase or decrease cyclic 3′,5′-adenosine monophosphate (cAMP) levels, and cAMP regulates protein kinase A (PKA). Gq11 activates phospholipase Cβ (PLCβ) to generate inositol trisphosphate, which mobilizes Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). G12/13 couple to guanine nucleotide exchange factors (GEF), resulting in activation of Rho, Rho-kinase (ROK), and serum response elements (SRE). GPCRs can activate the mitogen-activated protein kinase (MAPK) cascade by transactivation of the EGF receptor, through activation of PKC, phosphatidyl inositol 3-kinase (PI3K), Pyk2, and other mechanisms. Gβγ subunits couple GPCRs to other pathways, such as activation of G protein receptor kinases (GRKs), potassium channels (Ki), and nonreceptor tyrosine kinases (TK). (Modified from Ossovskaya and Bunnett [38], by permission.)

complex model.” In this model, GPCRs are thought to exist in equilibrium between two allosteric states: R (inactive) and R* (active) (69). Intermolecular interactions between transmembrane helices are thought to maintain unstimulated GPCRs in the inactive R conformation. In the presence of agonists, the equilibrium shifts toward the R* state, where the agonist, activated receptor R*, and G protein exist as a complex. The advantage of the allosteric ternary complex model is that it can explain the actions of various agonists and antagonists. Full agonists stabilize the R* conformation, producing maximal responses. Partial agonists produce a submaximal response, and thus can attenuate responses to full agonists. Neutral antagonists interact similarly with R and R* forms of receptor, having no effect on constitutive activity. Inverse antagonists have a higher affinity for the R state, and thus can suppress constitutive activity. However, the allosteric ternary complex model cannot account for accumulating evidence that suggests GPCRs exist in multiple activated states. Any models that predict that GPCRs exist in

a single active conformation would predict that the response to an agonist should be the same. However, many GPCRs can interact with different heterotrimeric G proteins to activate several different signaling pathways (70–72). Moreover, the order of potencies of different agonists can vary between different signaling pathways, and receptor mutations can affect one pathway but leave others unaffected (73). Finally, the formation of homodimers and heterodimers of GPCRs can have major effects on their pharmacologic properties. Clearly, new and more sophisticated models are required to explain this increased complexity of GPCR activation and signaling.

Monomeric G Proteins also Mediate G Protein–Coupled Receptor Signaling In addition to the well-characterized mechanisms of GPCR signaling that depend on activation of heterotrimeric

72 / CHAPTER 3 G proteins, small G proteins also mediate GPCR signaling (see review by Bhattacharya and colleagues [74]). Small G proteins are 20- to 30-kDa monomeric GTPases, which together form a superfamily with 100 members. There are five subfamilies, depending on their structure: Ras GTPases (e.g., Ras, Rap, and Ral), Rho GTPases (Rho, Rac, and cdc42), Arf GTPases (Arf1-6, Arl1-7, and Sar), Rab GTPases (>60 members), and Ran GTPases. The members of each subfamily have a general task: The Ras family controls gene transcription, the Rho family also regulates transcription and modulates the actin cytoskeleton, the Rab and Arf families control intracellular vesicular trafficking, and Ran family members regulate microtubule organization and the transport of proteins between the cytoplasm and nucleus. Like the heterotrimeric G proteins, the monomeric GTPases are molecular switches that exist in two states: a GDP-bound inactive state and a GTP-bound active state. The interconversion of these states is closely regulated. Thus, interaction of GTPases with GEFs, which, in turn, are controlled by upstream signals such as those arising from the heterotrimeric G proteins, induces the dissociation of GDP to allow association of the more abundant GTP, resulting in activation. GTPase-activating proteins or GAPs bind to the GTP-bound form and enhance the intrinsic GTPase activity, which converts the GTP-bound form back to the GDP-bound inactive form. GPCRs activate monomeric GTPases by a variety of mechanisms. Ras GTPase Ras GTPase plays a major role in mitogenic signaling by growth factors (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases later in this chapter). Growth factors activate RTKs, leading to the tyrosine phosphorylation and activation of Ras. Ras, in turn, activates the MAPKs, which regulate numerous cytoplasmic and nuclear targets, thereby controlling multiple cellular events. GPCRs also activate the Ras/MAPK pathway by three general mechanisms, depending on the cell type, the GPCR, and the heterotrimeric G-protein involved (Fig. 3-5). The first general mechanism involves established G-protein effectors, such as second messenger kinases and their products. For example, GPCRs that couple to Gαq/11 mobilize intracellular Ca2+ and activate protein kinase C, both of which stimulate Pyk2 (proline-rich tyrosine kinase), a member of the focal adhesion kinase family (75,76). Pyk2 then interacts with Src, resulting in activation of Ras and the MAPK cascade. The second general mechanism involves transactivation of RTKs. Transactivation refers to the process by which GPCRs co-opt RTKs to transduce their signal. One process by which GPCRs transactivate RTKs involves shedding of ligands for RTKs from cells, which can then activate RTKs in an autocrine or paracrine manner (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases later in this chapter) (77). The third general mechanism involves arrestin-dependent interaction of

GPCRs with upstream components of the MAPK pathways (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases and also Internalized G Protein–Coupled Receptors Can Continue to Signal later in this chapter). Rho GTPases Agonists of many GPCRs, such as acetylcholine, lysophosphatidic acid, and thrombin, induce reorganization of the cytoskeleton, as well as proliferation (see Sah and colleagues [78] and references therein). The transforming ability of these receptors depends in large part on G12/13 proteins and activation of Rho GTPases. Rho activation involves the interaction of G12/13 with RhoGEF proteins, such as PDZ-RhoGEF and p115-Rho-GEF. Once activated, the RhoGTPases interact with a wide range of effectors to activate multiple signaling pathways controlling the cytoskeleton and the cell cycle. Arf GTPases The Arf GTPases control membrane trafficking that is associated with endocytosis and recycling (see Takai and colleagues [79] and references therein). Two GEFs, ARNO (ADP ribosylation factor nucleotide-binding site opener) and EFA6, regulate Arf6. Both Arf6 and ARNO contribute to the regulation of GPCR signaling and trafficking by direct interaction with receptors or important trafficking proteins such as β-arrestins (see discussion in Mechanisms That Regulate Signaling by G Protein–Coupled Receptors later in this chapter). One consequence of these interactions is enhanced coupling of GPCRs to phospholipase D activation, which is independent of Gq/11 and Gi/o proteins. β-arrestins may serve as scaffolds to promote ARNO-mediated activation of Arf6, thereby stimulating endocytosis of the β2-AR (80). Arf1 and phospholipase D2 also interact with the carboxyltail of the µ-opioid receptor to control endocytosis (81). Many GPCRs, including metabotropic glutamate receptors, can activate phospholipase D2, which is a regulator of vesicular trafficking, cytoskeletal organization, endocytosis, and exocytosis (82). Thus, phospholipase D2 may be a novel adaptor protein inducing GPCR endocytosis, independently or together with β-arrestin. Rab GTPases The Rab GTPases play a major role in vesicular trafficking (see Seabra and colleagues [83] and Rosenfeld and colleagues [84] and references therein; see also Mechanisms That Regulate Signaling by G Protein–Coupled Receptors later in this chapter). Rab1, Rab4, Rab5, Rab7, and Rab11 have been implicated in various steps of GPCR trafficking from the endoplasmic reticulum to the plasma membrane, and from there to early endosomes, sorting endosomes, recycling endosomes, or lysosomes. Rab1 controls the trafficking of β2-AR and angiotensin II type 1a

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 73 EGF-like ligand ADAM GPCR

EGFR 2

Gβγ Gα

AC

1

? PLC

Pyk2 Ca

Shc

2

DAG

cAMP

Grb2

PKC

PKA

Sos

1 Ras

Rap

Raf 3 β-arrestin scaffold

MEK

ERK

FIG. 3-5. A simplified view of some of the mechanisms by which G protein–coupled receptors (GPCRs) activate mitogen-activated protein kinases (MAPKs). Agonist binding to a GPCR activates heterotrimeric G proteins, which dissociate into Gα and Gβγ subunits. In general, GPCRs can activate the MAPK cascade by three mechanisms: (1) Gα-dependent activation of adenylyl cyclase (AC) or phospholipase Cβ (PLC), leading to activation of second messenger kinases such as protein kinases A (PKA) and C (PKC). (2) Activation of ADAMs (a d istintegrin and metalloprotease) by unknown mechanisms, resulting in shedding of ligands for receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR). The EGFR tyrosine kinases, in turn, activate Ras. (3) Formation of MAPK signaling modules, by which β-arrestins recruit and organize components of the MAPK cascade to endosomes containing activated receptors.

(AT1aR) from the endoplasmic reticulum and Golgi apparatus to the plasma membrane (85). Rab5a participates in endocytosis of the β2-AR, AT1aR, NK1R, and PAR2, and in the trafficking of receptors to a perinuclear sorting region (86–90). Both Rab11a and Rab4a contribute to receptor recycling (89,91), whereas Rab7 promotes trafficking of receptors to lysosomes (92). Certain Rabs can associate directly with GPCRs. For example, Rab5a interacts with a domain in the carboxyl-tail of the AT1aR, and agonist binding promotes GDP/GTP exchange, suggesting that the receptor itself can function as a GEF for this GTPase (87). After activation, the AT1aR is directed to early endosomes, which are its final destination. The direct interaction between AT1aR and Rab5 prevents the receptor from being recycled to the plasma membrane or targeted to the lysosomes (87, 92). However, the overexpression of Rab11 promotes AT1aR recycling, whereas Rab7 overexpression leads to AT1aR

degradation, suggesting that the receptor can bind to other Rab proteins and that its trafficking depends on the Rab proteins expressed in a given cell.

Organization of G Protein–Coupled Receptors, G Proteins, and Signaling Proteins into Microdomains Increases the Efficiency and Fidelity of Signal Transduction Although GPCRs, G proteins, and signaling molecules are expressed at low concentrations in most cells, these proteins must physically interact for effective signal transduction to occur. However, many different pathways of signal transduction make use of the same effectors, and these effectors must be spatially confined to ensure specificity. An established theme of GPCR signaling is that signaling molecules are

74 / CHAPTER 3 concentrated into particular microdomains at the plasma membrane or in the cytosol (see reviews by Ostrom and Insel [93] and Luttrell [94]). This compartmentalization enhances the efficiency of signaling by increasing the concentration of proteins in specific regions of the cell, thereby increasing the likelihood of their interaction. It also promotes the fidelity of signaling by confining signaling proteins to particular regions of the cell, thereby increasing the specificity of their effects. Lipid rafts and caveolae are examples of microdomains that are found at the plasma membrane (Fig. 3-6A) (93). Lipid rafts are formed by the coalescence of sphingolipids and cholesterol, and caveolae are pits in the plasma membrane of similar lipid composition, but with caveolin proteins 1 to 3 on the inner leaflet of the membrane. The concentration of GPCRs and their binding partners in lipid rafts and caveolae may be a general mechanism that promotes efficient signal transduction. For example, β1-AR, β2-AR, Gs, and adenylyl cyclase 6 are enriched in lipid rafts and caveolae in cardiac myocytes (95–97). The mechanisms that determine the organization of proteins in lipid rafts are not fully understood, although they may include interactions with caveolin and posttranslational modifications that favor interaction with lipids. For example, coupling of the 5-HT1A receptor to Gi and inhibition of adenylyl cyclase depends on the palmitoylation of two cysteine residues in the carboxyl-tail of the receptor, leading to its retention in lipid rafts (98). The existence of scaffolding proteins is another example of a mechanism that determines the subcellular location and function of activated GPCRs and associated signaling proteins. Scaffolding proteins are of established importance for MAPK signaling, where multiple different stimuli can activate the same MAPK cascade. Scaffolding proteins provide a mechanism by which agonists of particular receptors can activate a specific MAPK, thereby controlling a given cellular response. β-Arrestins are important scaffolds that couple GPCRs to the extracellular signal-regulated kinases 1 and 2 (ERK1/2; see Fig. 3-6B) (99–104). β-Arrestins serve as scaffolds that recruit GPCRs, Src, Raf-1, and MAPKs to clathrin-coated vesicles. In this manner, they can determine the subcellular location and function of activated ERKs. The role of β-arrestins as scaffolds for GPCR signaling is discussed later (see Internalized G Protein–Coupled Receptors Can Continue to Signal).

Receptor Tyrosine Kinases Comprise a Family of Single-Transmembrane Domain Proteins with Intrinsic Kinase Activity Approximately 60 RTKs have been identified in the human genome, and RTKs are classified into 20 subgroups on the basis of sequence similarities (see reviews by Schlessinger [105] and Blume-Jensen and Hunter [106]). All RTKs have certain common structural characteristics: They are singletransmembrane domain proteins with functionally important extracellular and intracellular domains. The agonist-binding domain is extracellular and is usually glycosylated. Various features of the extracellular domain, such as cysteine-rich motifs, immunoglobulin-like repeats, fibronectin type III repeats, and epidermal growth factor (EGF) motifs, determine receptor specificity for different agonists. An intracellular tyrosine kinase domain is highly conserved between different RTKs. The tyrosine kinase domain catalyzes the transfer of γ phosphate of adenosine triphosphate (ATP) to hydroxyl groups of tyrosine residues of the receptor itself, leading to autophosphorylation or to other target proteins. With the exception of members of the insulin receptor family, which exist as preformed dimers that are linked by a disulfide bridge, RTKs exist principally as monomers in the unstimulated state (see Schlessinger [105] and Blume-Jensen and Hunter [106] and references therein). Agonist binding promotes the dimerization of these monomers, and RTKs can homodimerize or heterodimerize with receptors from the same family, which provides increased flexibility of signaling. For example, there are four epidermal growth factor receptors (EGFRs), designated ErbB1 through ErbB4, and EGF can induce heterodimerization of different combinations of the receptors. Receptor–receptor interactions further stabilize the dimers. On agonist binding and dimerization, RTKs adopt a conformation that favors trans-phosphorylation of tyrosine residues in the activation loop by the dimeric receptor partner (see Schlessinger [105] and Blume-Jensen and Hunter [106] and references therein). This phosphorylation results in repositioning of the activation loop away from the active site, which promotes access to target proteins and ATP, thereby increasing phosphorylation of both the receptor and other proteins that mediate signal transduction. Autophosphorylation also occurs outside of the kinase domain and provides docking sites for downstream signaling molecules.

RECEPTOR TYROSINE KINASES ARE SIGNALING PARTNERS FOR G PROTEIN–COUPLED RECEPTORS

Tyrosine Phosphorylation Recruits and Activates Receptor Tyrosine Kinase Signaling Molecules

Once thought to engage distinct signaling pathways, it is now well established that GPCRs and RTKs can activate the same signal transduction pathways, and that GPCRs can signal by activating RTKs, and vice versa, a process known as receptor transactivation. In view of this functional relationship, it is pertinent to review briefly the RTK family, and then to discuss in detail functional interactions between GPCRs and RTKs.

Tyrosine autophosphorylation of noncatalytic domains in the cytoplasmic regions of RTKs allows recruitment and activation of signaling proteins (see Schlessinger [105] and Blume-Jensen and Hunter [106] and references therein). These tyrosine-phosphorylated sites interact with Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains of signaling proteins, which themselves may have enzymatic activity or act as adaptors that recruit other signaling proteins.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 75 GPCR

Endocytosis Clathrin-coated pit

P

G 1

Gβγ

P

Arrestin

3

GRK

Dynamin

Arrestin

2 Arrestin GRK

Raf-1 MEK1/2

B

Membrane targets

Clathrin 4

Arrestin

Raf-1

ERK1/2 MEK1/2

ERK1/2 Calveolae Early endosome

ERK1/2 signaling modules GPCR Calveolin Gα Cytoskeleton

Gβγ

Cytosolic retention OR Nuclear targets

5

Arrestin

Raf-1 MEK1/2

ERK1/2

A FIG. 3-6. Examples of the spatial organization of signaling by G protein–coupled receptors (GPCRs). (A) Some GPCRs and heterotrimeric G proteins have been identified to associate with lipid rafts of sphingolipid- and cholesterol-rich structures. Caveolae are pit-shaped depressions in the membrane that resemble lipid rafts but which are coated with caveolin proteins. They may associate with the cytoskeleton by poorly understood mechanisms. (B) The role of β-arrestins as molecular scaffolds that mediate activation and targeting of extracellular signal-regulated kinases 1 and 2 (ERK1/2). (1) Agonist-bond GPCRs adopt a conformation that promotes association with heterotrimeric G proteins. (2) G-protein receptor kinases (GRKs) phosphorylate activated GPCRs to increase their affinity for β-arrestins, which translocate to the plasma membrane and interact with GPCRs. (3) β-arrestins are adaptors for clathrin and AP2 to mediate endocytosis. β-Arrestins also recruit Src, Raf-1, MEK1/2 (mitogen-activated protein kinase kinase), and ERK1/2 to GPCRs in clathrin-coated pits (4) and early endosomes (5), thereby forming signaling modules. For some receptors, this recruitment retains activated ERK1/2 in pits or endosomes where they may regulate cytosolic- or membrane-associated targets.

In this manner, the RTKs promote assembly of multiprotein “signaling platforms.” These mechanisms of RTK signaling have been extensively studied (105,106). They include pathways that activate MAPKs, PLCγ, protein kinase C, phosphatidylinositol-3 kinase, and small GTPases such as Rho, Rac, and Cdc42. Activated RTKs induce GTP/GDP exchange on the small GTPase Ras by the following pathway (see Fig. 3-5). RTK autophosphorylation promotes binding of the adaptor proteins SH2-containing (SHC) and growth factor receptor–bound protein 2 (Grb2). Grb2 interacts with the GEF Sos (Son-ofsevenless), which activates Ras. Activated Ras then interacts with effector proteins such as Raf and phosphatidyl inositol 3-kinase (PI3K) kinase, which then regulate multiple targets. Prominent among these targets are the MAPKs. The MAPKs are a family of evolutionarily conserved serine/threonine kinases that play critically important roles in regulating growth, division, differentiation, and apoptosis of cells (see Chang and Karin [107] and references therein). In mammalian cells, there are three major MAPK modules: ERK1/2, the c-Jun N-terminal kinase/stress-activated protein kinases

(JNK/SAPK), and the p38/HOG1 MAPKs. The MAPKs are regulated by a series of parallel kinase cascades, comprising three kinases that successively phosphorylate and activate a downstream target. For example, in the ERK1/2 cascade, Raf-1 (MAPK kinase kinase) phosphorylates and activates MEK1/2 (MAPK kinase), which phosphorylates and activates ERK1/2. Similar cascades activate JNK and p38. Once activated, the MAPKs can translocate to the nucleus to phosphorylate transcription factors, traffic to the plasma membrane to regulate receptors and channels, or remain in the cytosol to phosphorylate a variety of substrates. Activated RTKs also stimulate PLCγ (108,109). PLCγ binds directly to the EGFR through interaction of its SH2 domains with phosphotyrosine residues. The EGFR phosphorylates and activates PLCγ, which then cleaves phosphatidyl inositol bisphosphate, generating inositol trisphosphate, which mobilizes Ca2+, and diacylglycerol. Both Ca2+ and diacylglycerol activate protein kinase C. Most RTKs also activate phosphatidyl inositol 3-kinase (108,109). The phosphatidyl inositol 3-kinases can interact with phosphotyrosine residues of the EGFR or its docking proteins. Activated

76 / CHAPTER 3 phosphatidyl inositol 3-kinase has multiple downstream effects, including protecting against apoptotic cell death and generation of hydrogen peroxide. RTKs can also phosphorylate and activate the STAT (signal transducer and activation of transcription) proteins, which translocate to the nucleus to regulate transcription.

G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases The mechanisms by which RTKs activate MAPKs are well established (see “Tyrosine Phosphorylation Recruits and Activates Receptor Tyrosine Kinase Signaling Molecules”). However, GPCRs can also activate MAPKs to induce effects on differentiation and proliferation of cells (see reviews by Luttrell [94] and Waters and colleagues [110]). There are three general mechanisms by which GPCRs can activate MAPKs: by activating effectors of heterotrimeric G proteins, such as protein kinase A, protein kinase C, and Ca2+ ions; by transactivation of RTKs; and by β-arrestin–dependent interactions between GPCRs and MAPKs (see Fig. 3-5; see also Internalized G Protein–Coupled Receptors Can Continue to Signal later in this chapter). In some cell types, such as those of neuronal and hematopoietic origin, Gαs activation results in protein kinase A-mediated activation of the Ras family GTPase Rap-1 and subsequent activation of B-Ras, resulting in ERK1/2 activation (111,112). The Gq/11-coupled GPCRs can activate MAPKs by Ras-dependent and -independent pathways. For example, protein kinase Cα directly phosphorylates Raf-1 (113), and α1B-adrenergic and muscarinic m1 receptors, which couple to Gq/11, activate ERK1/2 in fibroblasts by this Raf-1-dependent but Ras-independent process (114,115). In neuronal cells, Ca2+ and protein kinase C can activate Pyk2, leading to Rasdependent activation of ERK1/2 (75,76,116). GPCRs can transactivate many RTKs, including receptors for EGF, platelet-derived growth factor (PDGF), insulin-like growth factor, vascular endothelial growth factor, brainderived neurotrophic factor, and nerve growth factor (see reviews by Luttrell [94] and Waters and colleagues [110]). The general process entails RTK autophosphorylation and recruitment of RTK signaling molecules and has been described for GPCRs that couple to Gi, Gq, and G13 proteins. The mechanisms of RTK transactivation have been most thoroughly investigated for the EGFR. A prominent mechanism of EGFR transactivation is ectodomain shedding. Most EGFR ligands, such as EGF, transforming growth factor-α heparin-binding (HB)-EGF, amphiregulin, betacellulin, and epiregulin, are produced as transmembrane precursors. Cellsurface metalloproteases cleave these precursors to liberate soluble growth factors, which can signal to RTKs on the same (autocrine signaling) or nearby (paracrine signaling) cells. In ectodomain shedding, agonists of GPCRs activate metalloproteases to induce shedding of EGFR ligands, which then activate the EGFR in an autocrine or paracrine fashion to induce activation of the Ras/MAPK pathway (77,117).

Thus, inhibitors of metalloproteases can suppress GPCRinduced transactivation of RTKs. These metalloproteases are members of the ADAM (a distintegrin and metalloprotease) family of zinc-dependent metalloproteases (118). Some members of this family have been implicated in GPCRmediated transactivation of the RTKs. For example, ADAM10 mediates bombesin-induced shedding of HB-EGF (119), and ADAM12 is required for the shedding of HB-EGF in cardiomyocytes in response to angiotensin II and endothelin-1 (120). ADAM17 (also known as tumor necrosis factor-α– converting enzyme, or TACE) cleaves proamphiregulin in cells treated with carbachol or lysophosphatidic acid, to liberate soluble amphiregulin, which then binds to the EGFR, resulting in its phosphorylation and activation of the MAPKs (121). In tumor cells, ADAM17 also mediates cannabinoidinduced transactivation of the EGFR through release of HBEGF or amphiregulin (122). Transforming growth factor-α also has been implicated in carbachol-induced transactivation of the EGFR in colonocytes (123). However, there remains much to learn about the process of ectodomain shedding. In particular, the signaling mechanism by which activated GPCRs can stimulate cell-surface metalloproteases, many of which have small intracellular domains, is not well understood. Both Gi/o- and Gq/11-coupled receptors can induce ectodomain shedding, and there are various requirements for Gβγ and Gq/11 subunits in this process. The targets of these G proteins that mediate shedding are not known, although phosphatidylinositol 3-kinase and Src have been implicated as intermediates in EGFR transactivation (124). GPCRs can transactivate other RTKs, such as receptors for insulin-like growth factor, vascular endothelial-derived growth factor, and nerve growth factor, but the mechanisms of their transactivation are not completely understood (see review by Waters and colleagues [110]). However, in some cases, transactivation does not entail ectodomain shedding. For example, agonists of 5-HT2A receptors can transactivate EGFR and activate ERK1/2 by mechanisms that are unaffected by metalloprotease inhibitors (125). Lysophosphatidic acid induces transactivation of the PDGF receptor by a phospholipase D–dependent mechanism (126). Transactivation of the PDGF receptor may also involve heterotrimeric G proteins (127).

Receptor Tyrosine Kinases also Signal through Heterotrimeric G Proteins Although heterotrimeric G proteins are viewed principally as signaling partners for GPCRs, there is abundant evidence that they also mediate signaling by other types of receptors, including RTKs (see review by Patel [128]). For example, the EGFR can activate Gs, thereby stimulating adenylyl cyclase and generating cAMP. Indeed, the cytosolic juxtamembrane region of the EGFR is important for association with Gs. The EGFR can also phosphorylate tyrosine residues of Gs, resulting in its activation. There is also evidence that GPCRs and RTKs can directly associate. The EGFR may exist in a preformed complex with the β2-AR, and stimulation with isoproterenol (β2-AR

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 77 agonist) transactivates EGFR and leads to the internalization of the β2-AR-EGFR complex, which is required for the activation of ERK1/2 pathway (101). The internalization of EGFR after transactivation induced by GPCR agonists, such as lysophosphatidic acid, is dependent on G proteins and β-arrestin. However, EGF itself can stimulate the recruitment of β-arrestin-1 to EGFR (129). In addition, EGF ligandinduced EGFR internalization requires Gβγ subunits and β-arrestin-1 (130). It has been shown that EGFR can interact with, and be serine phosphorylated by, GRK2 (131), but the role of this interaction has not yet been elucidated.

MECHANISMS THAT REGULATE SIGNALING BY G PROTEIN–COUPLED RECEPTORS A common feature of GPCR signaling is that G-protein signals are transient even in the continued presence of agonist, and that repeated challenge with agonist results in a diminution of the signal to subsequent challenges, a phenomenon known as receptor desensitization (see reviews by Bohm and colleagues [132] and Gainetdinov and colleagues [133]). The diminution of a signal after repeated challenge with the same agonist is termed homologous desensitization, whereas diminution of a signal by prior activation of a different receptor is termed heterologous desensitization. Receptor desensitization is often transient and reversible, and with time some receptors resensitize. However, other receptors are degraded after activation, resulting in permanent arrest of their capacity to signal, a phenomenon called down-regulation. For some receptors, challenge with an agonist can also magnify or sensitize responses to subsequent stimuli, but the mechanisms of GPCR sensitization are not fully understood.

functions: the amino terminus contributes to receptor recognition, and the carboxyl-terminus is important for membrane targeting, although there are other domains that mediate interactions with many other proteins. The GRK1/7 family is primarily found in the retina, and thus mediates desensitization of opsins, and GRK4 is found principally in the testis. Thus, GRK2/3/5 and 6 account for desensitization of most GPCRs elsewhere in the body. There are four arrestins, two of which (visual arrestin or arrestin-1 and cone arrestin or arrestin-4) are present in the retina, where they regulate opsins (Fig. 3-7). β-arrestin-1 and -2 (arrestin-2 and -3) are widely expressed and mediate desensitization of other GPCRs (138–140). The crystal structure of visual arrestin reveals two major domains, an aminoterminal and a carboxyl-terminal domain, both of which are formed of a seven-stranded β sandwich, with an interdomain

Carboxyl-terminal domain Hinge

Carboxyl-tail

Amino-terminal domain

A N domain

G Protein Receptor Kinases and Arrestins Mediate Homologous Desensitization Homologous desensitization of GPCRs requires the coordinated actions of two families of proteins, the GRKs and arrestins in the following manner (see reviews by Kohout and Lefkowitz [134] and Luttrell and Lefkowitz [135]). On activation, many GPCRs adopt a conformation that favors interaction with GRKs. The GRKs phosphorylate serine and threonine residues in the carboxyl-tail and intracellular loops of GPCRs (136,137). This phosphorylation increases the affinity of the GPCR for arrestins, which interact with the receptor and uncouple its association with heterotrimeric G proteins, resulting in desensitization (138,139). The seven GRKs are serine-threonine kinases that can be divided into three subfamilies based on structural and functional similarities: GRK1/7, GRK2/3, and GRK4/5/6. The GRKs have a common general structure, with an aminoterminal RGS (regulators of G-protein signaling) domain, a middle protein kinase domain, and a variable carboxylterminal domain. These domains have several general

C domain

A

B

β-arrestin1 R1

R2

1 β-arrestin2 R1

R2

418 410

Src-SH3

B

SRC-SH1 Ask 1 (other MAPKKKs?)

IP6

Clathrin

JNK3 NSF (other MAPKs?) Mdm2

AP2 S412

FIG. 3-7. The structure of arrestins. (A) Ribbon diagram of rod arrestin showing the functionally important amino domain, carboxyl domain, and interdomain hinge. The circles highlight positively and negatively charged residues of the polar core. This core is the main phosphate sensor of arrestin. (B) The domain architecture of β-arrestin-1 and 2. The amino domain interacts with activated G protein–coupled receptors and the carboxyl domain is responsible for secondary receptor recognition. The phosphate sensor domain is illustrated by P, between the amino and carboxyl domains. Important binding and regulatory domains are also illustrated. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase. (Reproduced from [A] Gurevich and Gurevich [224]; and [B] Luttrell and Lefkowitz [135], by permission.)

78 / CHAPTER 3 Membrane targeting of GRK1, 4, 6, and 7 depends on posttranslational modification of the carboxyl-terminus. Thus, GRK 1 is farnesylated, GRK4 and GRK6 are palmitoylated, and GRK7 has a CAAX sequence that predicts a geranylgeranyl modification (see Kohout TA, Lefkowitz [134] and references therein). These lipid groups anchor the kinases to the plasma membrane. GRK5 is also mostly associated with the plasma membrane through interaction between a positively charged domain at its carboxyl-terminus and negatively charged head groups of membrane lipids such as phosphatidylinositol-4,5-bisphosphate. GRK-induced phosphorylation of GPCRs has little effect on G-protein receptor coupling. Rather, GPCR phosphorylation serves to increase the affinity of the receptor for arrestins, and it is arrestin binding that disrupts the interaction of receptors and heterotrimeric G proteins to mediate desensitization. For example, visual arrestin does not bind inactive, unphosphorylated rhodopsin, but it does interacts with phosphorylated rhodopsin with a high affinity. Crystallography and mutagenesis studies suggest that arrestins undergo a major conformational change to expose binding sites for phosphorylated GPCRs, which is made possible by the interdomain hinge. This increased affinity of arrestins for GPCRs promotes the translocation of arrestins

hinge region (141) (see Fig. 3-7). Studies of mutated arrestins indicate the existence of receptor binding domains on the concave surfaces of both the amino and carboxyl domains (142). However, in addition to binding to phosphorylated GPCRs to arrest signal transduction, arrestins interact with a large number of other proteins, and thereby participate in receptor trafficking and signaling (see Clathrin, β-Arrestins, and Dynamin Mediate Endocytosis of Many G Protein– Coupled Receptors and also Internalized G Protein–Coupled Receptors Can Continue to Signal later in this chapter) (135). Therefore, there are many additional protein binding and regulatory domains. Desensitization requires the presence of GRKs at the plasma membrane in close proximity to agonist-occupied receptors (Fig. 3-8). Whereas some GRKs are recruited to the plasma membrane, others are constitutively present at the plasma membrane. GRK2 and GRK3 are cytosolic proteins in unstimulated cells, but they are targeted to agonist-occupied GPCRs by several mechanisms. These mechanisms include interaction with phosphatidylinositol4, 5-bisphosphate through their plekstrin homology domains (143), interaction with free Gβγ subunits (144), and interaction with activated GTP-bound Gαq (145), all of which would target GRK2 and GRK3 to activated GPCRs. Uncoupling and desensitization

Resensitization

Endocytosis

GPCR

Gα 1

Clathrin-coated pit Dynamin 3

P

2

Arrestin

Gβγ GRK

Resensitization

Fast recycling

Gα P

Gβγ

Clathrin

Arrestin

Arrestin AP2

Slow recycling 6

Rab4 4

Rab5 Arrestin

Recycling endosome

H+ Pase

5

Rab11 Recycling endosome

Early endosome

Arrestin

Multivesicular body

Arrestin

P

FIG. 3-8. Mechanisms of agonist-induced desensitization and trafficking of recycling G protein– coupled receptors (GPCRs). (1) Agonist-occupied GPCRs couple to heterotrimeric G proteins, which transduce signals through Gα and Gβγ subunits. (2) G-protein receptor kinases (GRKs) phosphorylate agonist-occupied GPCRs to promote membrane translocation and binding of β-arrestins. (3) β-arrestins are clathrin and AP2 adaptors, and thereby mediate GPCR translocation into clathrincoated pits. The GTPase dynamin mediates the formation of the first formed vesicles. (4) GPCRs that form low-affinity associations with β-arrestin-2 rapidly dissociate from β-arrestins at the cell surface or in endosomes and follow a fast recycling pathway to the plasma membrane that is mediated by Rab4a and Rab11a. (5) GPCRs that form high-affinity interactions with β-arrestin-1 and -2 translocate to perinuclear sorting endosomes and multivesicular bodies. Translocation to sorting endosomes depends on Rab5a. Endosomal acidification promotes agonist dissociation, and endosomal phosphatases remove phosphates and promote dissociation of β-arrestins. (6) These GPCRs then follow a slow recycling pathway to the plasma membrane. Recycling by either pathway can mediate resensitization.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 79 from the cytosol to the plasma membrane, where they interact with GRK-phosphorylated receptors and hinder their association with heterotrimeric G proteins (see Fig. 3-8). Given that there are 7 GRKs and more than 1,000 GPCRs, it is a formidable task to determine which GRKs can regulate signaling of a particular GPCR. With the exception of GRK1 or rhodopsin kinase, which is confined to the retina and regulates opsins, most other GRKs are widely distributed and could regulate signaling of many different GPCRs. Observations made with heterologous expression systems indicate that, although some GRKs preferentially interact with specific receptors, in many cases, several GRKs can efficiently phosphorylate a single GPCR and induce its desensitization. For example, GRK2, 3, 4, 5, and 6 can phosphorylate and desensitize the β2-AR (136,146–149); GRK2, 3, and 5 phosphorylate and desensitize the secretin receptor (150); and both GRK2 and 3 phosphorylate and desensitize the NK1R (151). However, whereas overexpressed GRK2, 3, 5, or 6 phosphorylate the VIP type 1 receptor, only GRK2, 3, or 5 actually desensitize its signaling (152). Because several GPCRs are capable of phosphorylating particular receptors, at least in transfected cell lines, it is of considerable importance to determine the expression of specific GRKs in tissues and elucidate whether they are colocalized with the receptor of interest. β-Arrestin-1 and -2, like the GRKs, are also widely distributed, and therefore could interact with many different GPCRs. However, GPCRs can be subdivided into two classes on the basis of their affinity and consequent duration of interaction with β-arrestins. The “class A” GPCRs, such as β2- and β1b-AR, µ-opioid receptor, endothelin A receptor, dopamine D1A receptor, and NK3R, interact with low affinity with β-arrestin-2 but do not interact with β-arrestin-1 (153– 155). These receptors interact transiently with β-arrestin-2 at the plasma membrane, and then quickly dissociate from β-arrestin-2 near the plasma membrane; thus, they are largely excluded from endosomes (Fig. 3-9). The “class B” GPCRs, exemplified by AT1aR, neurotensin 1 receptor, vasopressin V2 receptor, thyroid-releasing hormone receptor, and NK1R, interact with both β-arrestin-1 and -2 with high affinity (153–155). These receptors form stable interactions with β-arrestin-1 and -2 at the plasma membrane, and then translocate with β-arrestin-1 and -2 to endosomes, where they remain associated with GPCRs for prolonged periods. (Note that these classes do not correspond to the major GPCR classes described earlier in G Protein–Coupled Receptors Share Structural Motifs). The affinity of interaction between GPCRs and β-arrestins is determined by the phosphorylation of serine and threonine residues in the third intracellular loop and carboxyl-tails of GPCRs, because exchange of these domains can predictably alter the duration of association of certain GPCRs with β-arrestins (155–157). There are no antagonists of GRKs and β-arrestins that can readily be used to study the function of these proteins in animals. However, recent studies of knockout and transgenic mice have provided important new information about the specificity and function of GRKs and β-arrestins under

physiologic circumstances (Table 3-2). In general, animals lacking or overexpressing GRKs and arrestins are phenotypically normal under resting conditions, but they reveal interesting phenotypes when appropriately challenged. Such studies illustrate the physiologic importance of receptor desensitization. For example, mice lacking GRK1 or visual arrestin exhibit exaggerated and sustained responses of retinal rod cells to light because of reduced desensitization of rhodopsin (158,159). Similarly, GRK2+/− mice (GRK−/− animals die in utero) and β-arrestin-1−/− mice show enhanced contractility of the heart to isoproterenol, which can be explained by impaired desensitization of adrenergic receptors (160,161). Although these former results could be predicted from experiments in cell lines, studies of genetically modified mice have identified unexpected new roles for GRKs and β-arrestins, notably in the nervous system (see Gainetdinov and colleagues [133] and references therein). For example, GRK6-deficient mice show remarkable supersensitivity of locomotion in response to psychostimulants such as cocaine and amphetamine (162). These drugs inhibit dopamine transporters, thereby increasing the levels of dopamine in the extracellular fluid, which, in rodents, results in locomotor hyperreactivity. Direct agonists of dopamine receptors also result in exaggerated locomotion in GRK6−/− mice, and there is increased coupling of striatal dopamine D2 receptors to G proteins in these animals (162). These studies indicate a major role for GRK6 in regulating dopamine signaling in the central nervous system, with possible implications for diseases linked to abnormalities in the dopaminergic system. Studies of knockout mice have also shown involvement of β-arrestin-2 in regulating µ-opioid receptors in the brain (163-165). Thus, β-arrestin-2-deficient animals exhibit enhanced and prolonged analgesic responses to acute administration of morphine, which correlates with enhanced coupling of µ-opioid receptors to G proteins. Together, these data suggest diminished desensitization of opioid receptors in β-arrestin-2-deficient animals. Given the capacity of GRKs and β-arrestins to regulate signaling by many GPCRs in heterologous systems, similar studies of genetically modified animals can be expected to reveal important roles for these GRKs and β-arrestins in many aspects of physiologic control. Because experimentally induced alterations in expression of GRKs and β-arrestins can have marked implications for physiologic control, it is also likely that altered expression of these proteins in diseases may contribute to functional abnormalities of many systems.

Other Kinases Regulate G Protein–Coupled Receptor Signaling In addition to GRKs, other kinases such as protein kinase A, protein kinase C, and MAPKs can phosphorylate GPCRs and decrease their capacity to signal, resulting in desensitization. For example, the β2-AR activates protein kinase A, which can phosphorylate β2-ARs and other GPCRs, leading to heterologous desensitization (166–168). Protein kinase C

80 / CHAPTER 3 β-arrestin2-GFP, 10 min

β-arrestin1-GFP, 10 min

[Ca2+]i (% control)

Alexa-MP-NKB β-ARR2

Alexa-SP

Alexa-MP-NKB β-ARR1

NK3R wt

β-ARR2

Alexa-SP

A

B 120

120

100

100

80

80

60

60

40

40

20

20

0

C

NK1R wt

NK3R wt

β-ARR1

NK1R wt

NK1R

0

NK1R/NK3RI3ct

Control 0 min

#

30 min

NK3R

NK3R/NK1Rct

D

FIG. 3-9. Differences in trafficking and resensitization of G protein–coupled receptors (GPCRs) depending on association with β-arrestins. (A, B) Localization of neurokinin-1 receptor (NK1R; detected by binding with the specific agonist Alexa-Sar-Met-substance P [SP]) and the NK3R (detected by binding of the specific agonist Alexa-Me-Phe-neurokinin B [NKB]) with β-arrestin 1 and 2 tagged with green fluorescent protein at 10 minutes after stimulation. Note that both NK1R and β-arrestin-1 (A) and β-arrestin-2 (B) are colocalized in endosomes, indicative of high-affinity, sustained interactions. In contrast, the NK3R is in endosomes, but β-arrestin-1 (A) and -2 (B) have returned to the cytosol. (C, D) Time course of desensitization and resensitization of Ca2+ responses to the NK1R agonist (SM-SP) and NK3R (MP-NKB) in cells expressing NK1R and a chimeric receptor composed of the NK1R with the third intracellular loop and carboxyl tail of the NK3R (C), and the NK3R and a chimera comprising the NK3R with the carboxyl tail of the NK1R (D). Cells were incubated with 10 nM agonist or vehicle for 15 minutes, washed and challenged immediately (0 minute) or after 30 minutes. Results are expressed as the percentage response to the second challenge compared with vehicle-treated controls. Note the slow resensitization of NK1R and the rapid resensitization of NK3R. Replacement of loop 3 and to a lesser extent C tail of NK1R with NK3R accelerates resensitization of NK1R. Conversely, replacement of these domains of the NK3R slows resensitization. *p < 0.05 compared with wild-type NK1R (C) and NK3R (D); #p < 0.05 compared with wild-type NK1R. Together, these results show that receptors that interact with high affinity with β-arrestins slowly resensitize, whereas those that interact with low affinity rapidly resensitize. Domains in the third intracellular loop and carboxyl-tail, which determine these interactions, also influence the rate of resensitization. (Reproduced from Schmidlin and colleagues [155], by permission.)

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 81 TABLE 3-2. Phenotypes of G-protein receptor kinase and arrestin-deficient mice GRK or arrestin

Target GPCR

Phenotype

References

GRK1−/− GRK2−/− GRK2+/− GRK3−/−

Rhodopsin Unknown β1-AR, β2-AR Odorant receptor Muscarinic M2 muscarinic

Prolonged responses of retinal cells to light Embryonic lethal Increased cardiac contractility to isoproterenol Olfactory supersensitivity Enhanced airway response to methacholine Enhanced hypothermia, hypoactivity, cholinergic supersensitivity Dopaminergic supersensitivity of striatal neurons Impaired lymphocyte chemotaxis Prolonged photo response in rods of retina Increased cardiac contractility to isoproterenol Potentiation and prolongation of morphine-induced analgesia Impaired development of morphine intolerance Impaired lymphocyte chemotaxis

Lyubarsky et al. (158) Jaber et al. (225) Rockman et al. (160) Peppel et al. (226) Walker et al. (227) Gainetdinov et al. (228)

GRK5−/− GRK6−/− Visual arrestin−/− β-Arrestin-1−/− β-Arrestin-2−/−

Dopamine D2 Chemokine CXCR4 Rhodopsin β1-AR, β2-AR µ-opioid CXCR4

Gainetdinov et al. (162) Fong et al. (229) Xu et al. (159) Conner et al. (161) Bohn et al. (163–165) Fong et al. (229)

β1-AR, β1-adrenergic receptor; GPCR, G protein–coupled receptor; GRK, G protein receptor kinase.

has also been implicated in desensitization of several receptors including the m1 muscarinic receptor, the vasopressin receptor, and AT1aR (169–171). Clathrin, β-Arrestins, and Dynamin Mediate Endocytosis of Many G Protein–Coupled Receptors On binding agonists, many GPCRs translocate from the plasma membrane to endosomes, a process that is also referred to as receptor internalization or sequestration. Internalized GPCRs can either recycle back to the plasma membrane to mediate resensitization of signaling, or are targeted to lysosomes or proteasomes for degradation, thereby mediating down-regulation, which irrevocably terminates signaling. These processes of agonist-induced trafficking of GPCRs play major roles in the regulation of signal transduction. The molecular mechanisms of GPCR endocytosis have been investigated extensively, and the precise mechanisms depend on the receptor and cell type in question (see Fig. 3-8). GPCRs mostly commonly internalize at sites of clathrincoated pits, but sometimes involve caveolae, depending on the receptor in question and the cell type. The contribution of clathrin to GPCR endocytosis has been studied by treating cells with agents that disrupt clathrin-mediated endocytosis, such as hypertonic sucrose, which causes abnormal clathrin polymerization, and monodansylcadaverine, which blocks invagination of clathrin-coated pits. An alternative approach is to localize GPCRs to clathrin-coated pits by microscopy. By these approaches it is well established that many GPCRs internalize by a clathrin-dependent mechanism, exemplified by the β2-AR (172), NK1R, gastrin-releasing peptide receptor (173), and PAR2 (174,175). By coupling activated GPCRs to clathrin and other endocytic proteins such as AP2, adaptor proteins also contribute to receptor endocytosis. β-Arrestins are prominent adaptors that couple

many GPCRs, such as β2-AR, NK1R, and PAR2 to clathrin and AP2 (176,177). The role of β-arrestins usually is examined by overexpression of wild-type or dominant negative β-arrestin mutants, or by study of cells from β-arrestindeficient mice. For example, overexpression of wild-type β-arrestins promotes endocytosis of GPCRs (176), whereas overexpression of a fragment of β arrestin (residues 319–418), which comprises a clathrin-binding domain and acts as a dominant negative mutant, impairs their endocytosis (176,178,179). Mutagenesis studies have identified the motifs of β-arrestins that interact with clathrin and AP2, and thus are required for function as adaptor proteins. A motif between residues 374 and 377 of β-arrestin interacts with a domain within the amino terminus of the clathrin heavy chain (180), and a RXR sequence between residues 394 and 396 of β-arrestin binds to the β2 adaptin subunit of the heterotetrameric AP2 adaptor complex (181,182). This complex also interacts with dynamin and clathrin, thereby playing an essential role in the formation of clathrin-coated pits. Just as GRK-mediated phosphorylation of GPCRs promotes their interaction with β-arrestins to mediate uncoupling and desensitization, this phosphorylation is also necessary for endocytosis of certain GPCRs, presumably by enhancing interactions of receptors with β-arrestins (183–185). However, β-arrestins do not participate in endocytosis of all receptors because dominant negative β-arrestin mutants do not inhibit agonist-induced endocytosis of the AT1aR or the m1, m3, and m4 muscarinic receptors (178,186). The overexpression of mutants of the GTPase dynamin, such as the GTPase-defective dominantnegative mutant, dynamin K44E, has revealed an important role for dynamin in the formation of endosomes at sites of clathrin-coated pits and caveolae (187,188). Overexpression of dynamin K44E inhibits constitutive endocytosis of the transferrin receptor, a model for receptor trafficking, and also disrupts agonist-induced endocytosis of numerous GPCRs, exemplified by the β2-AR; NK1R; δ-opioid; muscarinic m1,

82 / CHAPTER 3 m3, and m4; and dopamine D1 receptors (178,186,189–193). However, dynamin K44E does not affect agonist-stimulated endocytosis of the AT1aR, muscarinic m2 receptor, dopamine D2 receptor, and α2B-AR, which must internalize by dynaminindependent mechanisms (178,191,192,194). The receptor domains that are required for endocytosis have been determined for some GPCRs by mutagenesis and domain swapping approaches. These domains may be sites of phosphorylation that interact with adaptor proteins such as β-arrestins. For example, truncation of the carboxyl-tail of the NK1R reduces the rate of SP-induced endocytosis by up to 60% (195). Although the explanation of this effect is not firmly established, it is likely that truncated receptors show impaired GRK-mediated phosphorylation, and hence diminished interactions with β-arrestins (102). Thus, whereas SP promotes membrane translocation of β-arrestins in cells expressing wild-type NK1R, there is no β-arrestin translocation in cells expressing a truncated form of the receptor. Some receptors also possess tyrosine-containing endocytic motifs, including β2-AR (196) and NK1R (197). Thus, mutation of Tyr305 within the putative NPX2-3Y endocytic motif of the seventh transmembrane domain of the NK1R impairs SP-induced endocytosis of this receptor. It appears, therefore, that the SP-stimulated endocytosis of the NK1R relies on endocytic motifs in the carboxyl-tail and seventh transmembrane domain. Agonist-induced endocytosis of GPCRs theoretically could contribute to desensitization by removing receptors from the cell surface, and thus making them inaccessible to hydrophilic agonists in the extracellular fluid. However, detectable endocytosis occurs more slowly than desensitization of G-protein signaling. Furthermore, drugs that disrupt endocytosis or expression of mutants of β-arrestins and dynamin have minimal effects on desensitization of G-protein signaling. Thus, endocytosis is not considered to contribute substantively to desensitization. Instead, endocytosis plays a major role in resensitization and signaling of certain GPCRs.

Receptor Recycling Mediates Resensitization of Signal Transduction Once internalized, some GPCRs recycle to the plasma membrane where they can once again interact with agonists and heterotrimeric G proteins, and thereby mediate resensitization (see Fig. 3-8). However, the mechanisms and pathway of receptor recycling, and thus resensitization, vary among receptors and can also depend on the nature of the stimulus. Moreover, compared with our understanding of the molecular mechanisms and function of endocytosis, little is known about the mechanisms of receptor recycling. One factor that determines the rate of recycling is the interaction of GPCRs with β-arrestins. The class B GPCRs, which interact with β-arrestin-1 and -2 with high affinity, cointernalize with both β-arrestins in endosomes and remain colocalized until the receptor recycles and β-arrestins resume their steady-state distribution in the cytosol (153–155). These

receptors slowly recycle and resensitize. In contrast, the class A GPCRs interact only with β-arrestin-2, and do so transiently (153–155). These receptors rapidly dissociate from β-arrestin-2 at the plasma membrane or in endosomes, and they recycle and resensitize more rapidly. For example, the class B neurokinin 1, AT1aR, and vasopressin V2 receptor slowly recycle and resensitize, whereas the class A NK3R and β2-AR rapidly recycle and resensitize (see Fig. 3-9). The differences in the interaction of class A and B GPCRs with β-arrestins are probably attributable to the presence of GRK phosphorylation sites within their intracellular carboxyl-tails and third intracellular loops. For example, exchange of the carboxyl-tail and third intracellular loop of the NK1R with equivalent domains of the NK3R diminishes the duration of association with β-arrestins and promotes recycling and resensitization (155). Exchange of these domains of the NK3R with the same ones from the NK1R has the opposite effect. Another factor that determines the rate of recycling and resensitization is the nature of the stimulus. For example, moderate concentrations of SP (10 nM) induce the “long pathway” of recycling of the NK1R: The receptor traffics from superficial early endosomes to perinuclear sorting endosomes by a mechanism that depends on the GTPase Rab5a, and enters recycling endosomes to translocate to the plasma membrane over a period of hours (89). After stimulation with low concentrations of SP (1 nM), the NK1R follows the “short pathway” of recycling: The receptor is confined to endosomes located immediately beneath the plasma membrane, from which it rapidly recycles to the cell surface by Rab4a- and Rab11amediated mechanisms. The mechanism by which the NK1R enters the long and short recycling pathways remains to be determined. However, SP causes a concentration-dependent phosphorylation of the NK1R, which could affect association with β-arrestins (198). Perhaps low levels of receptor phosphorylation induce a lower affinity interaction with β-arrestins, which could be more rapidly reversed endosomal phosphatases to allow faster recycling. The importance of receptor endocytosis and recycling for resensitization of signal transduction has been investigated by using a variety of genetic and pharmacologic approaches. For example, inhibition of receptor endocytosis with drugs, receptor mutation, or expression of dominant negative mutants of dynamin and β-arrestin, and inhibition of trafficking and recycling by expression of mutants of Rab5a, Rab4a, and Rab11a, impede resensitization of β2-AR and NK1R (88,89,155,199–202) (Fig. 3-10). Recycling and resensitization can also be suppressed by pharmacologic inhibition of phosphatases and of vacuolar H+ ATPase, suggesting a role for endosomal phosphatases and endosomal acidification (203). Together, these findings suggest the following mechanism of recycling and resensitization. Receptors translocate from the plasma membrane to early endosomes, and then to sorting endosomes. Acidification promotes dissociation of agonist, and endosomal phosphatases dephosphorylate the receptors to allow dissociation of β-arrestins and recycling to the cell surface.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 83

Calcium mobilization (% control)

120 100

NK1R NK1R + DYN

80

NK1R + DYNK44E

60

NK1R + Rab5a NK1R + Rab5aS34N

*

40 *

20 0

control

0 180 Time between SP challenge (min)

FIG. 3-10. Resensitization of substance P (SP)-induced Ca2+ mobilization in cells expressing the neurokinin-1 receptor (NK1R) alone or with dynamin, dominant-negative dynamin K44E, Rab5a, or dominant-negative Rab5a S34N. Cells were exposed to 10 nM SP or vehicle (control) for 10 minutes, washed, and challenged with 10 nM SP 0 to 180 minutes later. Note that expression of dynamin K44E and Rab5a S34N had no effect on desensitization at 0 minute, but strongly inhibited resensitization at 180 minutes. *p < 0.05 compared with wild-type cells. Thus, endocytosis does not affect desensitization but is necessary for resensitization. (Reproduced from Schmidlin and colleagues [88], by permission.)

Internalized G Protein–Coupled Receptors Can Continue to Signal Although β-arrestins uncouple GPCRs from heterotrimeric G proteins, thereby terminating G-protein signaling at the plasma membrane, they also recruit various signaling molecules to activated receptors at the cell surface or in endosomes, thereby permitting receptors to continue to signal through these pathways. Thus, β-arrestins are molecular scaffolds that interact with GRK-phosphorylated receptors, Src family tyrosine kinases, and members of the ERK1/2 and JNK3 MAPK signaling modules (94). Evidence for the existence of this signaling mechanism was first provided by the finding that agonists of the β2-AR promote the assembly of a multiprotein signaling complex in clathrin-coated pits that includes the receptor, β-arrestins, and Src (see Fig. 3-6) (99). In a similar manner, SP promotes interaction of the NK1R with β-arrestin and Src (100,102). These interactions depend on the binding of β-arrestins to both the receptor and to Src. The Src homology (SH) 3 domain of Src interacts with proline-rich PXXP motifs in the aminoterminal domain of β-arrestins, and there are additional interactions between the SH1 domain of Src and β-arrestin epitopes. The assembly of this GPCR/β-arrestin/Src complex contributes to several signaling mechanisms, including tyrosine phosphorylation of dynamin, which may regulate receptor endocytosis, and activation of ERK1/2 (99,204). For example, disruption of assembly of the complex by expression of mutant β-arrestins or truncated receptors impedes

coupling of β2-AR and NK1R mediated to activation of ERK1/2 (99,102). The activation of various MAPK signaling cascades in cells often requires molecular scaffolds that recruit and organize components of the cascade (94,205). These scaffolds have several functions, including increasing efficiency of interaction between the various components of the cascade, minimizing cross talk between different cascades and thereby maintaining the fidelity of the signal, and targeting activated MAPKs to particular regions of the cell to determine their function and specificity. β-arrestins may serve as scaffolds for some signaling pathways. For example, activation of PAR2 promotes the assembly of a complex containing the receptor, β-arrestins, Raf-1 (MAPK kinase kinase), MEK1/2 (MAPK kinase), and ERK1/2 (103). This complex prevents nuclear trafficking of ERK1/2 and retains them in the cytosol where they may regulate cytosolic substrates. Agonists of the AT1AR also induce formation of protein complexes of the receptor, β-arrestin, Raf-1, and activated ERK1/2 (206–208). β-arrestins also interact with JNK, and thus may serve as scaffolds for several MAPK modules (209).

Lysosomal Trafficking Permanently Arrests Signal Transduction Some activated GPCRs, exemplified by PAR2 (174), δ-opioid receptor (210), and chemokine receptor CXCR4 (211), are invariably targeted for lysosomal degradation (Fig. 3-11). Other receptors that generally recycle can, under certain circumstances, traffic to lysosomes. For instance, although the NK1R usually recycles, after prolonged activation with high concentrations of SP (100 nM for several hours), the NK1R is degraded (N. W. Bunnett, unpublished observation). Given that some internalized receptors can continue to signal, this degradation irrevocably arrests signal transduction. It may also serve as an adaptive mechanism to prevent sustained stimulation of cells when there is a continuous release of a receptor agonist. However, the mechanisms that target receptors to degradation have only recently been understood, and many aspects of lysosomal targeting of GPCRs remain to be elucidated. Ubiquitination of GPCRs and associated proteins, such as β-arrestins, contributes to lysosomal trafficking of several GPCRs (see Fig. 3-11). The covalent attachment of the polypeptide ubiquitin to lysine residues alters the location and function of many proteins, including cell-surface receptors (212,213). Three classes of enzyme are responsible for this ubiquitination. First, an ubiquitin-activating enzyme (E1) uses ATP to form ubiquitin C-terminal adenylate, a substrate for formation of an E1-ubiquitin thiol ester. Second, activated ubiquitin is passed to ubiquitin-conjugating enzymes (E2). Third, ubiquitin ligases (E3) attach the C-terminus of ubiquitin to ε-amino lysines of target proteins. Selectivity of this process depends on the selective interactions between target proteins and the many hundreds of E3 ligases. Attachment of single ubiquitin molecules to lysines, or monoubiquitination,

84 / CHAPTER 3 GPCR

Recovery Endocytosis Clatrin-coated pit

Arrestin AP2 Arrestin

ubi

1

6

Clathrin

Clathrin 2

E3

Rab1

Arrestin AP2

E3

Rab5

3

Down-regulation

Golgi apparatus Endoplasmic reticulum

5

4

Rab7 Arrestin

Lysosome

Arrestin

ubi Early endsome Multivesicular body

FIG. 3-11. Down-regulation of G protein–coupled receptors (GPCRs). (1) Activated GPCRs are phosphorylated and interact with β-arrestins, as described for Figure 3-8, and accumulate in clathrincoated pits. (2) E3 ligases such as c-Cbl in the case of protease-activated receptor 2 (PAR2) and AIP4 in the case of CXCR4 attach activated ubiquitin molecules (ubi) to GPCRs at the plasma membrane or perhaps in endosomes. Other E3 ligases such as Mdm2 can ubiquitinate β-arrestins. (3) GPCRs cointernalize with β-arrestins into early endosomes and are then sorted to multivesicular bodies (4) from whence they are sorted to lysosomes for degradation (5). (6) Mobilization of preformed pools of receptors from the Golgi apparatus or synthesis of new receptors is required for recovery from this down-regulation process.

determines the trafficking and subcellular localization of proteins (212). However, chains of ubiquitin molecules can be attached to the three lysine residues of ubiquitin itself. Such polyubiquitinated proteins usually are destined for degradation in the proteasome (213). Agonist binding prompts the ubiquitination of a growing number of GPCRs, including Ste2p and Ste3p in yeast, and β2-AR, chemokine receptor CXCR4, and PAR2 in mammalian cells (211,214–217). These receptors contain several lysine residues in the carboxyl-tails and intracellular loops that could be ubiquitinated, but the precise sites of ubiquitination remain to be defined. Given the large number of E3 ligases that could mediate this ubiquitination, identifying the ligases involved is a formidable challenge. However, the E3 ligase AIP4 mediates ubiquitination of CXCR4 (218), and c-Cbl ubiquitinates PAR2 (217). Although the E3 ligase that ubiquitinates the β2-AR is unknown, GPCR agonists promote interaction between β-arrestin-2 and Mdm2, which ubiquitinates β-arrestin-2 (216,219). Ubiquitination of β-arrestin-2 determines the stability of its interaction with the β2-AR and affects receptor ubiquitination and trafficking (216,220). Ubiquitination can also affect the trafficking

of GPCRs. Although ubiquitination-defective mutants of β2-AR, CXCR4, and PAR2 internalize normally in response to agonists (211,216), ubiquitination contributes to endocytosis of Ste2p, Ste3p, and EGFR (214,215,221). Disruption of ubiquitination of CXCR4 and PAR2 causes their retention in early endosomes, suggesting that ubiquitination is required for the postendocytic sorting of these receptors (217,218). However, some receptors traffic to lysosomes by ubiquitinindependent mechanisms. For example, lysosomal targeting of the δ-opioid receptor is unaffected by mutation of lysine residues, and thus does not require receptor ubiquitination (210). Instead, lysosomal trafficking of the δ-opioid receptor requires its interaction with GPCR-associated sorting protein (GASP). GASP binds to the carboxyl-tail of the δ-opioid receptor but not to the µ-opioid receptor, which efficiently recycles. Therefore, GASP could preferentially interact with receptors that are destined for lysosomal degradation. Domains in the carboxyl-tail of other GPCRs are also required for lysosomal trafficking. PAR1 associates with sorting nexin 1, and disruption of this interaction impedes degradation (222). The interaction is mediated by domains in the carboxyl-tail of PAR1. Clearly, several mechanisms

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 85 can determine the lysosomal trafficking of GPCRs, and the factors that determine which mechanism is used remain to be discovered.

CONCLUSION The superfamily of GPCRs include receptors for an extraordinarily diverse array of stimuli from the external and internal environment. Given the diversity of their agonists— ranging from simple ions, through small molecules such as biogenic amines, to large proteins such as thrombin—it is remarkable that there are such similarities in the mechanisms of activation, signal transduction, and regulation of GPCRs. However, despite the great progress that has been made in understanding the biology of these receptors, there is still much to learn. There are well more than 100 orphan receptors, with no known ligand or biological function. Given that the gastrointestinal tract contains so many hormones and neurotransmitters that signal through GPCRs, and because many of them were first discovered in this organ, it is likely that these orphans, once adopted, will be found to play important roles in the gut. The mechanisms of activation of GPCRs are incompletely understood. To date, rhodopsin is the only GPCR that has been crystallized and examined by X-ray diffraction. Although may GPCRs have been modeled using information about the structure of rhodopsin, structural studies are required for other GPCRs, particularly in their active states. It is unknown how agonist binding alters the conformation of GPCRs to initiate signal transduction, although such information is invaluable for the development of antagonists. Most drugs exert their effects by affecting signaling by GPCRs either directly or indirectly, and GPCRs are thus tractable targets for the pharmaceutical industry. However, many GPCRs that are thought to play important roles in gastrointestinal diseases have been difficult or impossible to antagonize. Clearly, further studies of the mechanisms of activation and signaling of these receptors is required to facilitate the development of therapeutically useful antagonists. The signaling pathways by which GPCRs exert their effects are also incompletely understood. All cells express multiple GPCRs, either for the same or different ligands. Why do cells often express many distinct GPCRs for the same agonist? As certain GPCRs can exist as homodimers or heterodimers in heterologous expression systems, it is possible that they also dimerize when expressed at physiologic levels in cells, with a marked impact on their pharmacologic properties and mechanisms of signal transduction. The complexity of signaling is further amplified by the now established interactions between GPCRs and other cell-surface receptors, most notably the RTKs, where the interactions of these receptors can markedly affect their pharmacology, regulation, and function. The mechanisms that terminate receptor signaling are also incompletely understood. Although the processes of receptor desensitization, down-regulation, and trafficking have been thoroughly studied in heterologous expression system, little is known about

the role of GRKs and arrestins in vivo. A limited number of studies, most notably in the central nervous system, have shown that specific GRKs and arrestins play major roles in controlling responses to agonists of GPCRs in the intact animal, but nothing is known about this type of control in the gastrointestinal tract. Future studies of the basic mechanisms of activation, signal transduction, and inactivation of GPCRs are likely to have a large influence on our understanding of the role of these receptors and their agonists in the control of the digestive system.

ACKNOWLEDGMENTS Research in the authors’ laboratory is funded by National Institutes of Health grants DK39957, DK43207, and DK57480.

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CHAPTER

4

Gastrointestinal Hormones: Gastrin, Cholecystokinin, Somatostatin, and Ghrelin Graham J. Dockray Gastrin, 92 Overview, 92 Peptide Structure, 92 Gene Structure and Expression, 93 Posttranslational Processing, 93 Cellular Origins, 95 Assay, 95 Release Mechanisms, 95 Metabolism, 96 Receptors, Transduction, and Structure–Activity Relations, 96 Biological and Physiologic Actions, 97 Actions of Nonclassical Gastrins, 99 Conditions of Gastrin Excess, 100 Cholecystokinin, 100 Overview, 100 Peptide Structure, 101 Gene Structure and Expression, 101 Posttranslational Processing, 101 Cellular Origins, 101 Assay, 102 Release Mechanisms, 103 Metabolism, 103 Receptors, Transduction, and Structure–Activity Relations, 103 Biological and Physiologic Actions, 104 Conditions of Excess and Deficiency, 106

Somatostatin, 106 Overview, 106 Peptide Structure, 106 Gene Structure and Expression, 106 Posttranslational Processing, 107 Cellular Origins, 107 Assay, 107 Release Mechanisms, 107 Metabolism, 108 Receptors, Transduction, and Structure–Activity Relations, 108 Physiologic Actions, 108 Conditions of Somatostatin Excess or Deficiency, 108 Ghrelin, 109 Overview, 109 Peptide Structure, 109 Gene Structure and Expression, 109 Posttranslational Processing, 109 Cellular Origins, 109 Assay, 110 Release Mechanisms, 110 Metabolism, 110 Receptors, Transduction, and Structure–Activity Relations, 110 Biological and Physiologic Actions, 110 Conditions of Excess and Deficiency, 111 References, 111

Hormones are molecules carried through the bloodstream to transmit information from one cell to another by interacting with specific receptors on the target cell. The first hormone to be discovered was secretin, which is released from the

intestine by acid and stimulates the flow of pancreatic juice (1). The term hormone was then introduced to describe the general properties of substances such as secretin that were carried from their cells of origin (endocrine cells) to their targets through the general circulation. The gastrointestinal hormones are a subset of the hormonal system; they typically are produced in specialized cells of the gastrointestinal epithelium, the gut endocrine, or enteroendocrine cells, and act on other gut or associated cells to regulate the processes of digestion, control energy intake, and regulate

G. J. Dockray: Department of Physiology, School of Biomedical Sciences, University of Liverpool, Liverpool, L69 3BX United Kingdom. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

91

92 / CHAPTER 4 the secretion of other hormones concerned with nutrient utilization such as insulin. Some of the products of gut endocrine cells act locally rather than via the bloodstream, that is, through paracrine mechanisms. A good example is somatostatin; because somatostatin cells (or D cells) function in a similar way to other gut endocrine cells, and because there are close functional links between somatostatin and gastrin in the control of gastric function, this peptide is considered here. Other locally acting substances include growth factors and cytokines, and these are considered in Chapters 7, 42, and 43. The hormonal peptides produced in gut endocrine cells may also be found elsewhere in the body, most notably in neurons of the central or peripheral nervous systems. The role of neuropeptide transmitters in the enteric nervous system is also considered in other chapters. The term regulatory peptide is sometimes used as a general expression to encompass peptides that function either as hormones, paracrine factors, or neurotransmitters. Most of the currently known gut hormones had been characterized by the early 1980s. However, new hormones continue to be discovered; a good example is the gastric appetite-stimulating peptide ghrelin, which was first described in 1999 (2), although its cell of origin (the gastric A-like or Xcell) had been recognized for many years previously. At the time of the last edition of this book, excellent progress had been made in defining the molecular identities of the then known gut hormones, the receptors at which they acted, their structure–activity relations, and cellular signaling mechanisms. Since then, impressive progress has been made in the use of genetically modified animals to explore the biology of gut hormones. New insights into the physiology of the major gut peptides have come through the study of animals either overexpressing the genes encoding particular hormones or their receptors, or in which these genes have been deleted (3,4). This chapter is concerned with hormones produced in the upper gastrointestinal tract (gastrin, cholecystokinin [CCK], somatostatin, and ghrelin). The main functional links among these hormones are the control of acid secretion, the control of protein and fat digestion, and the regulation of nutrient delivery to the small intestine either through control of food intake or control of gastric emptying. In addition, there are evolutionary links between two of these peptides (gastrin and CCK). In keeping with the previous editions of this book, this chapter is organized around individual peptides. Each is discussed with respect to its structure, cellular origins and mechanisms of release, the relevant receptors, hormonal actions, and physiology, including data from transgenic and gene knockout (KO) models. Integrative actions of the gastrointestinal hormones with neurotransmitters, paracrine, and other humoral mediators are discussed in other chapters throughout this book.

GASTRIN Overview Gastrin is produced by G cells of the pyloric antral part of the stomach. Its main physiologic function is the control

of gastric digestion through the regulation of acid secretion and gastric epithelial cell proliferation. The idea that gastric acid secretion might be controlled by a gastric hormone was first introduced in 1905 by Edkins (5). There was subsequently some doubt whether the putative gastric secretagogue might not be histamine (6), which was eventually resolved by Komarov (7), who produced the first histaminefree extracts of stomach that stimulated acid secretion. The first preparations of pure gastrin were produced by Gregory and Tracy (8) in the early 1960s, and this material was used to elucidate the sequence of the main peptide found in gastric G cells, which is currently known as G17. Gastrin is an important regulator of postprandial acid secretion, parietal cell maturation, and gastric epithelial cell proliferation; it also influences the proportion of different cell types in the gastric epithelium, notably increasing the abundance of the histamine-secreting enterochromaffin-like (ECL) cell. Gastrin is released by food, particularly protein, in the stomach. Its release is inhibited by gastric acid, probably via the release of somatostatin, which then acts as a paracrine mediator in the antrum. Hypergastrinemia may result from gastrin-producing tumors (Zollinger–Ellison syndrome), which cause gastric acid hypersecretion, or from reduced acid inhibition of the G cell, for example, after therapeutic inhibition of acid secretion or autoimmune loss of parietal cells. In both cases, hypergastrinemia is associated with ECL cell hyperplasia. The gastrin gene is also expressed in some gastrointestinal tumors (9). Some of the products of gene expression in these cells lack the peptide structure required for stimulation of acid secretion but are nevertheless able to act as growth factors. These peptides (progastrin and the Gly-gastrins) can be considered “nonclassical gastrins,” in contrast with the “classical gastrins” such as G17, which are acid secretagogues.

Peptide Structure Gastrin is a linear peptide existing in multiple molecular forms. Gregory and Tracy first isolated two peptides consisting of 17 amino acid residues that were identical but for the presence or absence of a sulfated tyrosine residue (8); the unsulfated peptide was named gastrin I and the sulfated peptide was named gastrin II, based on their elution from ion-exchange chromatography columns. Subsequently, larger and less acidic peptides were identified; these were named at the time big gastrin, to distinguish them from the two heptadecapeptides (then called little gastrins I and II) (10). Gregory and Tracy (11) then characterized big gastrin as 2 peptides of 34 amino acid residues corresponding to the 2 heptadecapeptides extended at their N terminus. The heptadecapeptides are now generally called G17, and the big gastrins are called G34; the suffixes -s and -ns are used to define sulfation status if necessary. These peptides are all acid secretagogues. In addition, small amounts of related acid secretagogue peptides have been identified, notably G14 (the C-terminal tetradecapeptide of G17, also called minigastrin)

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 93 (12), G6 (the C-terminal hexapeptide of G17) (13), and an N-terminally extended peptide of 71 residues (G71, also called component 1) (14). The physiologic importance of these other gastrins remains uncertain.

Gene Structure and Expression The complementary DNA (cDNA) encoding gastrin was first cloned from pig antrum (15,16), and subsequently from human (17), rat (18), and other common species. The human gene is located on chromosome 17 and contains three exons (19). The sequence in the coding region predicts a precursor of 104 (porcine) or 101 (human) amino acid residues with a characteristic N-terminal signal peptide and a single sequence corresponding to the peptides isolated from tissue (Fig. 4-1). The different forms of gastrin described earlier therefore originate by alternative posttranslational processing from a single precursor generated from a single gene. There is conservation throughout the mammals of primary amino acid sequence in the region corresponding to the C-terminal portion of G17 and of the cleavage sites generating G34 and G17. The C-terminal sequence of gastrin is shared with the intestinal hormone CCK, reflecting a common evolutionary history of the two peptides (Fig. 4-2). Distinct genes encoding peptides more or less similar to mammalian gastrin and CCK have been cloned in chicken (20), turtle (21), bullfrog (22), and an elasmobranch fish (23). Although the C-terminal amide sequence (WMDFamide) is well conserved, there is variation across the major vertebrate groups in the immediately preceding sequence, which seems to be linked to specificity for stimulation of acid secretion. In adult mammals, the gastrin gene is expressed principally in the pyloric antral mucosa. In some species, particularly humans, there is also expression in the duodenal mucosa. In addition, there may be low-level expression in the pituitary. During development, the gastrin gene is expressed in pancreatic islet cells, and in the rat, the shift to an adult pattern of expression occurs around birth (24). Some work indicates that a number of epithelial tumors, including colorectal and lung carcinomas, may express the gastrin gene (25–27); however, the pattern of processing of the precursor peptide in these cells often differs from that in normal endocrine cells (see later). Expression of the gastrin gene in G cells is regulated by the gastric luminal contents. Amino acids that stimulate Preprogastrin gene exon 1

gastrin release also increase gastrin messenger RNA (mRNA), and fasting decreases gastrin mRNA (28). Moreover, intragastric acid decreases gastrin mRNA, and inhibition of acid secretion (e.g., with proton pump inhibitors) increases both mRNA abundance and rates of translation (29,30). The cellular signaling pathways remain uncertain, but activation of the mitogen-activated protein kinase (MAPK) pathway (e.g., by epidermal growth factor) stimulates gastrin gene expression via phosphorylation of Sp1 (31). There is also evidence for control of gene expression via the Wnt pathway and by transforming growth factor-β (32). The gastric pathogenic bacterium Helicobacter pylori is associated with increased plasma gastrin concentrations and increased gastrin mRNA abundance (33). This may be partially attributable to decreased production of somatostatin caused by locally increased concentrations of proinflammatory cytokines. But, in addition, there appears to be stimulation of nuclear factor-κB signaling in G cells of patients infected with Helicobacter pylori, which increases gastrin gene expression (34). In colorectal carcinoma, activation of Ras and the TCF-4/β-catenin signaling pathway has been implicated in gastrin gene expression (35,36).

Posttranslational Processing Studies using antibodies to peptides derived from different regions of progastrin, together with chromatographic separation, first indicated variation in the peptide products of preprogastrin between different cells expressing the gastrin gene. The kinetics of processing have since been studied using pulse-chase labeling methods (37,38). The initial product of translation, preprogastrin, is rapidly converted to progastrin by removal of the N-terminal signal peptide during sequestration into the lumen of the endoplasmic reticulum (Fig. 4-3). Cleavage of the signal peptide occurs at either Ala21 and Ser22, or Arg26 and Ser27 (39,40). Thereafter, the conversion of progastrin to its final secretory products involves endoproteolytic cleavage at pairs of basic residues, carboxypeptidase cleavage of C-terminal basic residues, Serphosphorylation, Tyr-sulfation, and C-terminal amidation. Phosphorylation of Ser-96 and Tyr-87 occurs in the trans-Golgi network (TGN). There is evidence that Serphosphorylation might determine subsequent cleavage at the adjacent pair of Arg residues (41). In endocrine cells, cleavage at pairs of basic residues (Arg-57,58, Lys-74,75, and

exon 2

exon 3

Preprogastrin mRNA RR

KK

RR

Preprogastrin

FIG. 4-1. Schematic representation of the relationship between the preprogastrin gene, preprogastrin messenger RNA (mRNA), and the peptide precursor preprogastrin.

94 / CHAPTER 4 CCK Caerulein Gastrin

SO-3 -Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 SO-3 pGlu-Gln-Asp-Tyr-Thr-Gly-Trp-Met-Asp-Phe-NH2 SO-3 -Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2

FIG. 4-2. Amino acid sequence of the C-terminal region of gastrin, cholecystokinin (CCK), and caerulein. Shared sequences are indicated by the box; note also the sulfated tyrosine residue at position 7 (CCK, caerulein) or 6 (gastrin) from the C-terminus; gastrin commonly exists in both Tyr-sulfated and unsulfated forms.

Arg-94,95) is mediated by subtilisin-like prohormone convertases (SPC), followed by carboxypeptidase E removal of C-terminal basic residues. Both SPC1/3 and SPC2 have been identified in G cells (42). Pulse-chase labeling studies indicate that in rat G cells, most progastrin is initially cleaved at the pairs of Arg residues to yield a 35-residue peptide with COOH-terminal glycine (G34-Gly). These cleavages appear to occur after sequestration into vesicles of the

regulated pathway of exocytosis and exhibit a t1/2 of about 12 minutes after exit from the TGN (37). In addition to G34-Gly, there are two other products: an N-terminal progastrin fragment and a COOH-terminal tryptic fragment that includes Ser-96, both of which are thought to be biologically inactive (see Fig. 4-3). Subsequently, G34-Gly is converted to the corresponding C-terminal amidated peptide (G34) via the enzyme peptidyl α-amidating monooxygenase (PAM).

SPC2, SPC3; Carboxypeptidase E Progastrin

G34-Gly

-Gly

G17-Gly

-NH2

G34 G17

A ER:

Preprogastrin

Golgi TGN:

Progastrin

G34-Gly

-NH2

Constitutive secretion G34-GlyOH

G34

Regulated secretion

Secretary vesicles: G17-Gly

B

PAM

-Gly

Non-classical gastrins

G17-GlyOH

G17

Classical gastrins

FIG. 4-3. Biosynthetic relations among different forms of gastrin. (A) Relations among progastrin, the Gly-extended gastrins (G34-Gly, G17Gly), and the amidated gastrins (G34, G17). The relevant posttranslational processing enzymes are shown. The stippled box corresponds to the signal sequence in preprogastrin. (B) Biosynthetic routes by which preprogastrin is converted to the secretory products, the subcellular compartments in which conversion occurs, and the secretory pathway taken by different products of preprogastrin. ER, endoplasmic reticulum; PAM, peptidyl α-amidating monooxygenase; TGN, trans-Golgi network.

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 95 This is a two-step reaction involving the generation of a hydroxy-glycine intermediate by the oxygenase domain of PAM, followed by conversion to the C-terminal amide by the lyase domain of the enzyme. The product, G34, may then be converted to G17 by cleavage at Lys-74,75 (38,42–44). There may also be some cleavage of G34-Gly to yield G17-Gly. The pH in secretory vesicles in endocrine cells is approximately 5.5, and prohormone convertases have acidic pH optima. Alkalinization of secretory vesicles has relatively little effect on cleavage at pairs of Arg residues, but it significantly inhibits the cleavage of G34 at Lys-74,75 (45). Some research has suggested that progastrin and the COOH-terminal Gly-gastrins might have biological activities that are distinct from those of the amidated peptides; for this reason, it is useful to refer to these peptides as the “nonclassical gastrins” (see Fig. 4-3). This reflects their new-found status as biologically active peptides, rather than their existence per se, which has been appreciated for many years (46,47). By analogy, the peptides G34 and G17 that were originally isolated and characterized by Gregory and Tracy (8,11,48), and that possess the defining biological property of the hormone, the stimulation of acid secretion, may be called the “classical gastrins” (see Fig. 4-3). There is cell-to-cell variation in posttranslational processing. For example, in human duodenum, G34 predominates over G17 (49), whereas the opposite is true in antral G cells. Moreover, in gastrinoma cells, there is considerable variation between tumors, which probably reflects variable expression of processing enzymes (46). In colorectal carcinoma cells, the main products of gastrin gene expression appear to be progastrin and the Gly-gastrins (25,26,50,51). Because endopeptidase cleavage and COOH-terminal amidation occur in secretory vesicles of the regulated secretory pathway (52) and because in nonendocrine cells these vesicles are scarce or absent, it is thought that the main products of secretion are largely unprocessed peptides that pass directly from the TGN to the cell surface via the constitutive route of secretion.

Cellular Origins In adults of most mammals, G cells identified by immunohistochemistry are found predominantly in the antral mucosa; in humans, there are also significant numbers in the duodenum. G cells are “open”-type endocrine cells; that is, their apical border projects into the gastric lumen and characteristically terminates there in a tuft of microvilli. There is considerable variation in the morphology of secretory vesicles in G cells. Although differences in fixation may account for some of the reported variation seen by electron microscopy, it also seems probable that vesicles recently budded from the TGN are smaller and more electron dense than older vesicles, and that internal pH influences morphology (53–55). The results of [3H]-thymidine incorporation experiments suggest a turnover time for hamster G cells of 10 to 15 days and a cellular derivation from neck precursor cells (56). There is likely to be physiologic control of G-cell numbers

because the labeling index can be increased by administration of bombesin (57), and there is G-cell hyperplasia in achlorhydria (58). Double labeling immunohistochemistry suggests that at an early stage in their differentiation, progenitor endocrine cells in the antrum express both gastrin and somatostatin genes (59). The basic helix-loop-helix (bHLH) transcription factor, neurogenin 3, is required for the development of these cells because both G and D cells are absent in mice lacking the gene, although endocrine cells in the gastric corpus such as ECL and X-cells are present (60,61). In contrast, the bHLH transcriptional repressor, Hes1, is a negative regulator of endocrine cell numbers, and in mice with deletion of the Hes1 gene, there is hyperplasia of pyloric antral and intestinal endocrine cell populations (62). There is also a requirement for the homeobox gene PDX-1 for the final differentiation of G cells, and there is loss of G cells and increased numbers of D cells in mice deficient in PDX-1 (63).

Assay For routine assay purposes, particularly for the determination of plasma gastrin concentrations, radioimmunoassay (RIA) is the method of choice (64). Assays based on determination of acid secretion, or other aspects of parietal cell function, are less sensitive but may be valuable where the distinction between biological and immunological activity is important. Most routine RIAs use antibodies binding at the C-terminus of the amidated gastrins. This sequence is shared with CCK, and thus care is needed to use antibodies with epitopes extending into the unshared sequence to avoid problems of cross-reactivity. Many different antibodies have been described, some showing remarkable capacities to distinguish between peptides differing in individual amino acid residues, in the presence or absence of the C-terminal amide or in Tyr-sulfation status. Even so, many antibodies show approximately similar affinity for the major C-terminally amidated gastrins, and these are usually the most useful for routine assays of plasma hormone concentrations. However, because there are multiple peptides derived from progastrin, the initial validation of a gastrin RIA should include studies of immunoreactive peptides after separation by chromatography. Assays using antibodies reacting at the C-terminus of progastrin (and not with amidated gastrin or Gly-gastrin) or at the C-terminus of Gly-gastrin (and not with progastrin or amidated gastrin) have been used by several groups and are of particular value in the study of nonclassical gastrins (25,26,46,47).

Release Mechanisms Plasma concentrations of the amidated gastrins in fasting humans are in the range 10 to 30 pmol/liter. After a mixed meal, plasma concentrations increase twofold to threefold with a peak at 30 to 60 minutes. The main circulating form

96 / CHAPTER 4 of gastrin in basal conditions is G34, and the main form at the peak of the postprandial response is G17 (65). The secretion of gastrin is regulated by nervous reflexes, by the direct action of luminal chemicals on G cells, and by indirect actions of the luminal contents mediated via other endocrine cells or neurons (Fig. 4-4). The mechanisms regulating gastrin release have been studied extensively in vivo (66), in the isolated perfused rat stomach (67,68), and in isolated G-cell preparations (69). The main luminal nutrients releasing gastrin are aromatic amino acids. The relevant transduction mechanisms remain uncertain. There is, however, evidence that G protein–coupled receptors may play a role as luminal sensors in enteroendocrine cells (70). In other systems, the aromatic amino acids that release gastrin have been shown to bind to and activate the external calcium-sensing receptor (CaR) (71), and there is evidence that CaR is expressed by G cells (72). The release of gastrin is inhibited by luminal acidification less than pH 3, and this provides a feedback-inhibition mechanism autoregulating gastrin release during a meal (73). Acid stimulates antral D cells to release somatostatin that then acts as a paracrine inhibitor of G-cell function (68). A number of neurohumoral factors (e.g., secretin, vasoactive intestinal peptide [VIP]) that inhibit G-cell function probably also work by release of somatostatin (67). Acute neutralization of the gastric contents does not alone stimulate gastrin release. However, prolonged inhibition of acid secretion, which occurs with administration of proton pump inhibitors (74,75) or in conditions where parietal cells are lost (e.g., pernicious anemia) (76), leads to hypergastrinemia. There is also increased gastrin release in conditions of antral inflammation, for example, with H. pylori infection (33,77), which is thought to be caused by direct stimulation of G cells by proinflammatory cytokines and indirect stimulation via cytokine suppression of D-cell function (see Fig. 4-4). In animal models, there is evidence that prolonged inhibition of acid secretion leads to bacterial

H+

H. pylori

Peptides/ aminoacids

+

+ NFκB/ Sp1 G-cell

D-cell −

SOM

TNFα, IL-1β

− +

overgrowth and depression of D-cell function, which could account for increased gastrin release (78). Distension of the stomach has little effect on gastrin release, but sham feeding or vagal stimulation increase gastrin release in humans and dogs. The effects of vagal efferent stimulation on the G cell are, however, complex with evidence for both cholinergic-excitatory and cholinergic-inhibitory pathways regulating gastrin release (66). In addition, there is clear evidence of a noncholinergic excitatory pathway, and the bombesin-like peptide, gastrin-releasing peptide (GRP), has been considered as a putative noncholinergic neurotransmitter controlling gastrin secretion. Thus, there are GRPcontaining nerve fibers in the vicinity of G cells, release of gastric GRP occurs with luminal or vagal stimulation, and administration of GRP antibodies blocks luminal stimulation of gastrin release (79–82). However, studies using a GRP receptor antagonist suggest that in humans, postprandial gastrin release is not mediated by GRP, although there is a role for the latter in control of acid secretion (83).

Metabolism There are important differences in the metabolic clearance rates of G17 and G34. In both humans and dogs, G17 is cleared from the peripheral circulation with a t1/2 that is about fivefold less than that of G34 (84,85). There is no evidence that G34 is converted to G17 in the peripheral circulation. There is, however, evidence that G17 may be cleaved to yield shorter C-terminal fragments such as G14 (86). No single organ has a dominant role in the metabolism of G17, and most capillary beds in the body remove similar proportions of G17 arriving in the arterial blood. Small C-terminal fragments of G17 may be transported by the liver into bile, but this does not apply to G17 or G34; nor do the latter appear in urine. Interestingly, however, N-terminal fragments of G34 (which are biologically inactive) have been described in urine (87). Enzymes responsible for cleavage of G17 include neutral endopeptidase 24.11 (also known as enkephalinase) and angiotensin-converting enzyme. Neutral endopeptidase 24.11 cleaves G17 on the C-terminal side of multiple residues including Trp4, Ala11, Gly13, and Asp16 (88–90).

Receptors, Transduction, and Structure–Activity Relations

Gastrin GRP EGF

FIG. 4-4. Control of G-cell function. G-cell function is stimulated by luminal peptides and amino acids, the neuropeptide transmitter GRP (gastrin-releasing peptide), and the growth factor EGF (epidermal growth factor). It is inhibited by luminal acid, via the paracrine mediator somatostatin from D cells. H. pylori infection depresses D-cell function via proinflammatory cytokines, which also enhance G-cell function. IL-1, interleukin-1; SOM, somatostatin; TNF, tumor necrosis factor.

Carboxy-terminal amidated peptides of the gastrin-CCK family act at the CCK-1 (also known as the CCK-A) and CCK-2 (also known as the CCK-B or gastrin-CCKB) receptors (Table 4-1). The former has low affinity for gastrin, but high affinity for CCK, and the latter has high affinity for both gastrin and CCK (91). The CCK-2 receptor is therefore the primary mediator of the physiologic effects of gastrin. It is normally expressed by parietal cells, ECL cells, some smooth muscle cells, neurons of the central and peripheral

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 97 TABLE 4-1. Characteristics of gastrin and cholecystokinin receptors Characteristic

CCK1

CCK2

Alternative names Agonist selectivity Distribution

CCK-A CCK>>>gastrin Pancreatic acinar cells, pylorus, vagus, enteric neurons, CNS neurons L-364,718 (devazepide), loxiglumide

Gastrin/CCK-B CCK = gastrin Parietal cells, ECL cells, CNS neurons

Selective antagonists Receptor coupling Primary GI functions

Gαq/11, Ca2+, PKC Pancreatic enzyme secretion, gall bladder contraction, inhibition of gastric empying, satiety

L-365,260, L-740,093 YM022, CI-988, YF476, spiroglumide, gastrozole Gαq/11, Ca2+, PKC Acid secretion, parietal cell maturation, ECL cell proliferation

CCK, cholecystokinin; CNS, central nervous system; ECL, enterochromaffin-like; GI, gastrointestinal; PKC, protein kinase C.

nervous systems, and (depending on the species) pancreatic acinar cells. The main endogenous ligand for CCK-2 receptors in the brain is likely to be CCK, because this is abundantly produced by central nervous system (CNS) neurons, whereas gastrin is not. In the periphery, however, receptors are exposed to both circulating hormones, and the main endogenous ligand in this case is gastrin because its concentrations in plasma are 5 to 10 times greater than those of CCK. The nonclassical gastrins—that is, progastrin and the Glygastrins—are not endogenous ligands of the CCK-2 receptor. However, these peptides are now known to have their own characteristic pattern of biological activities that are thought to be mediated by non–CCK-1/non–CCK-2 receptors (92,93). The original studies of Tracy and Gregory (94) established that the C-terminal tetrapeptide amide (WMDFamide) is the smallest fragment of G17 with full agonist activity at CCK2 receptors. Thus, the nonclassical gastrins (such as progastrin and the Gly-gastrins), which lack the COOH-terminal amide moiety, have low or no affinity for CCK-2 receptors. Several CCK-2 receptor antagonists have been described, including L-365,260, L-740,093, YM022, YF476, spiroglumide, and gastrozole (91,95,96). Results obtained using these compounds support the idea that gastrin regulates postprandial increases in gastric acid secretion (97). Both CCK-1 and -2 receptors belong to the seventransmembrane domain, G protein–coupled receptor superfamily (91). Homology studies suggest that they are closely related to each other. Signaling mechanisms downstream of the CCK-2 receptor have been studied in isolated parietal and ECL cells (98,99), in cancer cell lines expressing the receptor (e.g., AR4-2J cells) (100), and in transfected cells (101). The receptor is coupled to Gαq/11, and activation leads to an increase in intracellular Ca2+ and protein kinase C (PKC). In addition, there is activation of the MAPK and phosphatidyl inositol kinase (PI3K)/Akt signaling pathways, and these have been linked to a variety of outcomes including proliferation, migration, inhibition of apoptosis, and tubulogenesis (102–105). At least in part, it now seems possible that activation of these pathways may be secondary to autocrine and paracrine activation by growth factors released by gastrin (52). A CCK-2 receptor variant in which intron 4 of the gene is retained (CCK-2Ri4sv) has an extra 69 amino acid residues

in the third intracellular loop. This variant appears to be expressed in colorectal tumors but not adjacent normal colon, in Barrett’s esophagus but not normal esophagus, and in pancreatic cancer (106–108). It has somewhat increased constitutive activity compared with the wild-type receptor and slightly higher affinity for Gly-gastrin, although still relatively low (<0.001) compared with G17 (106). In HEK293 cells, which have low Src activity, expression of CCK2Ri4sv is reported to be associated with an agonist-independent increase in Src, and it has been suggested that expression of this form of the CCK-2 receptor could contribute to the increased Src activity in some tumors (109). Research suggests that induction of CCK-2 receptor expression occurs in some circumstances. Thus, in transgenic mice overexpressing gastrin, there is increased proliferation in the stomach and induction of the CCK-2 receptor on proliferating cells (110). There are also rapid increases in expression of the receptor at the margin of cryoulcers induced in rat stomach (111), and greater expression in the epithelium of Barrett's esophagus compared with esophageal epithelium from unaffected individuals (112). Together, these data indicate that the CCK-2 receptor gene is expressed in response to tissue injury or inflammation. In human colon cancer cells, increased transcription has been reported in response to overexpression of oncogenic Ras via an MEKdependent mechanism (113). Recently, signaling mechanisms activated by the nonclassical gastrins (progastrin and Gly-gastrin) that are independent of CCK-2 receptors have attracted attention (100,114,115). Although a full characterization of the relevant receptors is still required, there is some evidence that progastrin and Glygastrin may act at separate receptors (93,116). Activation of Src has been linked to both progastrin and Gly-gastrin (116,117). The actions of Gly-gastrin may be either MAPKdependent or -independent (114,115) and include stimulation of PI3K (115).

Biological and Physiologic Actions The amidated gastrins have a wide range of biological actions on gastrointestinal tissues. Proof of the physiologic actions of a circulating hormone have, in the past, been

98 / CHAPTER 4 based on experiments showing equivalence in the responses to similar plasma concentrations of hormone released endogenously and administered exogenously. This approach works well for acute effects but is less satisfactory for long-term effects (e.g., cell turnover or control of gene expression). In these cases, the use of receptor antagonists may be valuable, as may be studies of genetically modified animals in which the gene of interest has been deleted. The application of these approaches to gastrin has both confirmed traditional views of the physiology of the hormone in control of acid secretion and produced new insights into roles in the control of cell proliferation and differentiation in the gastric epithelium (3,4,52). Consult the previous edition of this book (118) for a full account of the earlier evidence for the physiologic role of gastrin in control of acid secretion. This edition focuses on more recent work. Parietal Cell Function and Control of Acid Secretion At a given concentration of endogenous plasma gastrin, the increase in gastric acid output after intragastric instillation of an amino acid test meal is comparable with that produced during infusion of exogenous G17 (119). Moreover, the postprandial increase in gastric acid secretion is inhibited by gastrin immunoneutralization (120) and by CCK-2 receptor antagonists (66). Pharmacologic studies using histamine H2 receptor antagonists indicate that gastrin stimulates acid secretion mainly via release of histamine from ECL cells that, in turn, acts as a paracrine mediator in the gastric mucosa–stimulating parietal cells (Fig. 4-5) (52,121,122). In support of this, gastrin releases histamine in isolated perfused rat stomachs (123); increases intracellular Ca2+ in ECL cells (124); and in mice in which the gene encoding histidine decarboxylase (HDC) has been deleted (thereby blocking histamine synthesis), there is little or no acid response to gastrin (125,126). In addition, gastrin regulates ECL cell numbers and the expression of genes in ECL cells that direct histamine synthesis and storage (see later). H+ +

Parietal cell

ECLcell

Histamine Gastrin ECL-cell responses to gastrin ECL-cell proliferation Histamine release Histamine synthesis, and storage, HDC, VMAT2, CgA gene expression

Proliferating cell

HB-EGF TGFα Amphiregulin Parietal-cell responses to gastrin Parietal cell numbers Parietal cell maturation Acid secretion (via histamine) Growth factor synthesis and secretion

FIG. 4-5. Actions of gastrin on histamine-secreting ECL (enterochromaffin-like) and acid-secreting parietal cells. EGF, epidermal growth factor; HDC, histidine decarboxylase; TGF, transforming growth factor; VMAT2, vesicular monoamine transporter type 2.

The question of whether gastrin acts directly on parietal cells has attracted considerable attention. Parietal cells express CCK-2 receptors, and gastrin directly stimulates purified preparations of isolated parietal cells (127,128). One idea is that relatively high plasma concentrations of gastrin are required to directly stimulate acid secretion from parietal cells. More recent data in mice in which the genes encoding either gastrin or the CCK-2 receptor have been deleted suggest there might be other roles for the direct action of gastrin on parietal cells including stimulation of parietal cell maturation and migration (129–131). Thus, in the absence of the gastrin gene (i.e., Gas-KO mice), parietal cells appear morphologically immature, and there is reduced basal acid secretion and insensitivity to stimulation by gastrin, histamine, or carbachol (129,130). However, infusion of gastrin in Gas-KO mice for more than 24 hours induces the capacity for secretagogue stimulation of acid secretion (129,132). With prolonged infusion of G17, acid secretion in Gas-KO mice increased initially, and then faded over a period of 2 weeks; interestingly, when Gas-KO mice were given G17 and G17-Gly, there was maintained secretion over many weeks, suggesting an interaction between the two peptides to maintain parietal cell function (132). Although gastrin is required for the normal regulation of gastric epithelial organization and function, prolonged hypergastrinemia accompanied by inflammation may also be associated with disruption of epithelial organization, at least in experimental animals. For example, in transgenic mice with lifelong increase of plasma amidated gastrin (InsGas mice), there is initially (up to about 3 months of age) increased acid secretion and increased parietal cell numbers, but with age there is a progressive loss of parietal cells and expansion of the mucous neck cell population (133). Infection of these mice with Helicobacter felis accelerates this progression, and by 18 months invasive gastric cancer develops in many (133). Control of Enterochromaffin-like Cell Function As noted earlier, gastrin stimulates histamine release from ECL cells, increases the production and storage of histamine by the ECL cell by stimulating the expression of key genes, and regulates ECL cell number (74,123,124,134–136). Histamine is synthesized from histidine by the cytosolic enzyme HDC (EC 4.1.1.22.), and then is sequestrated into secretory granules by vesicular monoamine transporter type 2 (VMAT2). The abundance of the mRNA encoding HDC is regulated by physiologic concentrations of circulating gastrin and may be increased within the time taken to digest a single meal (137). Hypergastrinemia in both humans and rodents is associated with increased abundance of mRNA encoding HDC and VMAT2, as well as the endocrine cell secretory protein, chromogranin A, which is thought to stabilize amine-containing secretory granules (134,138–142). The transcriptional mechanisms by which gastrin regulates these three genes have been investigated in human gastric epithelial cell lines and commonly involve transcription

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 99 factor binding to a variety of GC-rich regions and activation of Cre sites (143–148). In patients and in experimental animals, hypergastrinemia is associated with ECL cell hyperplasia (149). It appears that ECL cells have the capacity for proliferation; thus, this response may reflect a direct mitogenic action of gastrin (150). With prolonged hypergastrinemia there may be a progression to dysplasia and ECL cell carcinoid tumors (9,149,151,152). ECL cell carcinoid tumors are now well recognized to be produced in experimental animals with hypergastrinemia due to long-term administration of proton pump inhibitors (153,154); the ECL cell response is prevented by removal of the pyloric antrum, providing direct evidence for the role of an antral hormone (155). ECL cell carcinoid tumors also develop spontaneously in a sub-Saharan rodent Mastomys, and this has been attributed to amino acid substitutions in the CCK-2 receptor in this species that increase constitutive activity (156,157). In humans, increase of plasma gastrin alone, as occurs, for example, in sporadic gastrinomas, seems to be associated with a low incidence of ECL cell carcinoids (<1 %); the combination of increased plasma gastrin with either mutation of the multiple endocrine neoplasia type 1 (MEN-1) gene or with inflammation (e.g., chronic atrophic gastritis) increases the incidence of ECL cell carcinoids (151,152,158).

H+

TFF-1

Proliferation differentiation migration

HB-EGF, TGFα, amphiregulin, COX-2 products, Regs FGF-1 IL-8 Som

Histamine Gastrin

Control of Gastric Epithelial Cell Proliferation It has been recognized for many years that experimental administration of gastrin stimulates growth of the acidsecreting part of the stomach, leading to increased thickness of the mucosa and increased parietal cell mass (159). Moreover, gastric epithelial cell proliferation rates, determined by BrdU incorporation, increase with feeding partly because of increased gastrin secretion (160). Gastric epithelial progenitor cells are not thought to express CCK-2 receptors; instead, gastrin is considered to act indirectly via release of growth factors (see earlier) (52), presumably originating in parietal or ECL cells (Fig. 4-6). However, in Gas-KO mice, all the major gastric cell types are nevertheless represented, although the mucosa is relatively thin, indicating that development of the gastric mucosa does not exhibit an absolute requirement for gastrin. It appears likely that gastrin plays a role in determining the relative proportions of different cells and their maturation. Thus, in addition to effects on ECL cell number described earlier, there are initially increased numbers of parietal cells in transgenic mice overexpressing gastrin (133), and increased parietal cell mass is found in gastrinoma patients. Parietal cells are terminally differentiated so that gastrindependent increases in cell number may be secondary to either stimulation of progenitor cell proliferation or increased commitment of progenitor cells to the parietal cell lineage. Other Actions of Gastrin Gastrin contracts smooth muscle of the antrum and small intestine (161,162) and the lower esophageal sphincter (163).

FIG. 4-6. Putative paracrine mediators of the action of gastrin in the gastric epithelium relevant to cellular proliferation, differentiation, and cell migration. COX-2, cyclooxygenase-2; EGF, epidermal growth factor; FGF-1, fibroblast growth factor-1; TFF-1, trefoil factor-1; TGF, transforming growth factor.

Depending on the species and tissue, these may be either direct or indirect effects mediated by enteric neurons. Their physiologic significance in normal digestion remains uncertain. Studies using gene arrays and proteomic methods have started to reveal a number of previously unidentified target genes of gastrin. These include plasminogen activator inhibitor-2 (164), matrix metalloproteinase-9 (MMP-9) (165), and trefoil factor-1 (166). Therefore, it appears that in certain circumstances, for example, in cancer or tissue regeneration, gastrin may influence tissue organization, and this involves, at least in part, remodeling of the extracellular matrix (see Fig. 4-6).

Actions of Nonclassical Gastrins Several different roles for Gly-gastrin in the stomach have been described, but the physiologic significance of these effects remains rather uncertain. Synergistic interactions between amidated and Gly-gastrin for stimulation of acid secretion in the rat were reported several years ago (167). More recently, it has been found that administration of the two peptides in combination enhances and maintains

100 / CHAPTER 4 parietal cell function in mice in which the gastrin gene has been deleted (132). Moreover, Gly-gastrin appears to inhibit the loss of parietal cells and gastric atrophy that occur in mice overexpressing amidated gastrin and infected with H. felis (168). Several groups have reported that Gly-gastrin stimulates the proliferation of both transformed and nontransformed cells (169–172). In addition, transgenic mice overexpressing Gly-gastrin exhibit increased proliferation in the colon (173) and are prone to the development of lung adenocarcinomas. Moreover, the expression of Gly-gastrin (but not progastrin or amidated gastrin) in lung adenocarcinomas is associated with decreased patient survival (27). In addition to proliferative effects, the oncogenic properties of Gly-gastrin may also include stimulation of cell migration (115) and cell invasion, at least in part, by induction of MMP-1 and -3 (174,175). Like Gly-gastrin, progastrin stimulates cell proliferation both in vitro and in vivo. In a mouse model, the stimulation of colon proliferation by progastrin was shown to occur with fragments of the precursor that included the C-terminal 26-residues; Gly-gastrin or fragments of the extreme C terminus (e.g., the C-terminal tryptic peptide of progastrin) did not show activity in this assay (176). In transgenic mice overexpressing progastrin, there is increased epithelial cell proliferation in the colon and increased thickness of the colon mucosa (177). These mice also exhibit increased susceptibility to the damaging effects of the carcinogen azoxymethane (177–180). Moreover, susceptibility to the carcinogenic effects of azoxymethane is seen in mice expressing a mutant form of progastrin in which the dibasic amino acid residue cleavage sites have been deleted (180), indicating that the carcinogenic effects of progastrin are not dependent on processing to Gly-gastrin or amidated gastrin. Normally, in mice exposed to 8G γ-irradiation, there is complete inhibition of mitosis in the colon. In contrast, in irradiated mice that overexpress progastrin, colonic cells remain in the cell cycle. Therefore, it appears that progastrin allows cells to stay in the cell cycle even after DNA damage, which presumably contributes to its apparent effects as a cocarcinogen (181). In this irradiation model, there is no difference in rates of colonic epithelial cell apoptosis between control mice and mice overexpressing progastrin, although it has been reported in IEC6 cells that progastrin has inhibitory effects on apoptosis (182).

Conditions of Gastrin Excess Increased plasma gastrin concentrations in humans may be caused by a gastrin-secreting tumor (gastrinoma) or by increased secretion from antral G cells most commonly caused by a failure of acid inhibition. Gastrinomas occur in approximately one patient per million of population per annum and are typically located in either the pancreas or duodenum; they may be sporadic or on a background of MEN-1 (183). Plasma gastrin concentrations in gastrinoma patients are usually in the range 50 to 10,000 pmol/liter and

are accompanied by high basal acid output (>15 mEq/hr). Provocative tests that are useful for diagnosis in those patients with plasma gastrin and acid secretory rates overlapping or just greater than the reference range include rapid intravenous injection of secretin and intravenous calcium (184). As noted earlier, a clear example of the consequences of loss of acid inhibition of the G cell is pernicious anemia, where achlorhydria over many years leads to plasma gastrin concentrations up to 2000 pmol/liter. The long-term use of proton pump inhibitors similarly leads to disinhibition of the G cell and increased plasma gastrin concentration (75, 185,186). Hypergastrinemia also occurs in postvagotomy patients, where again increased gastrin release may be secondary to reduced acid output. In the relatively unusual condition of isolated retained antrum, surgical exclusion of the antrum from the rest of the stomach removes it from acid inhibition, thus increasing gastrin release, which stimulates acid secretion from the rest of the stomach, leading to a clinical picture resembling gastrinoma (183). Antral G-cell hyperfunction (increased gastrin released from antral G cells, hyperpepsinogenemia, but normal G-cell density) and antral G-cell hyperplasia were described many years ago (187,188), but remain poorly defined and may well reflect extreme responses to H. pylori infection.

CHOLECYSTOKININ Overview CCK was first described in 1928 as a stimulant of gall bladder contraction present in intestinal extracts (189). Independently, Harper and Raper (190) later showed that extracts of intestine were capable of stimulating pancreatic enzyme secretion, and that this property was distinct from the capacity of secretin to stimulate the flow of pancreatic juice; they called the active factor pancreozymin (PZ). For many years the relation between CCK and PZ was uncertain. However, the purification of the two activities to homogeneity by Jorpes and Mutt (191) made it clear that they were separate actions of a single hormone. For a while the term cholecystokinin-pancreozymin (CCK-PZ) was in use; the hormone now is commonly referred to as CCK regardless of the biological activity under consideration. CCK is produced by a wide variety of neurons in addition to gut endocrine cells and has multiple biological actions. Its primary gastrointestinal role is the regulation of protein and fat digestion in the upper small intestine (192). This is achieved by matching the delivery of digestive enzymes from the pancreas and bile salts from the gall bladder, with the delivery of nutrients by delaying gastric emptying and inhibiting food intake. Some actions of CCK released from the gut are hormonal and involve direct effects on target cells (e.g., pancreatic acinar cells, gall bladder smooth muscle), but others may be mediated by activation of vagal reflexes (193). In addition, CCK produced in central and peripheral neurons

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 101 acts locally as a peptide neurotransmitter. This chapter focuses on the role of CCK as a gut hormone.

Peptide Structure Like the structurally related hormone gastrin, CCK is a linear peptide existing in multiple molecular forms. It was originally isolated from porcine intestine by Mutt and Jorpes (194) as a 33-residue peptide. The C-terminal pentapeptide amide of CCK was shown to be identical to that in gastrin; in addition, it was shown to possess a sulfated tyrosine residue at position 7 counting from the C terminus (whereas in gastrin there is a tyrosine at position 6). At about the same time, the amphibian skin peptide caerulein was isolated and sequenced by Anastasi and colleagues (195); it was shown to possess a closely similar C-terminal sequence and to have CCK-like biological properties (see Fig. 4-2). Multiple molecular forms of CCK have since been isolated and characterized. By analogy with the system for naming the different gastrins, the various forms of CCK that include the C-terminal pentapeptide amide sequence are designated by the number of amino acids in the chain. The peptide first identified by Jorpes and Mutt is therefore CCK-33; subsequently the group identified N-terminal extended forms, namely CCK-39 and CCK-58 (196,197). In addition, both longer (CCK-83) and shorter forms (CCK-8 and CCK-22) have been isolated and sequenced from intestine (198–200). In most cases, these peptides appear to occur predominantly in the tyrosine-sulfated state, although unsulfated CCK-58 is known to exist (201). The stability of different forms of CCK during extraction can be a significant practical problem that requires careful attention in any comparative study of the tissue and species distribution of CCK variants (202).

Gene Structure and Expression A single gene encodes the different forms of CCK and is expressed in both gut endocrine cells and CNS neurons. The cDNA in rat predicts a 345-base mRNA transcript encoding a 115-amino acid-residue precursor with a single copy of the biologically active sequence (Fig. 4-7) (203). The amino acid sequence immediately downstream of the putative -Phe-amide in pro-CCK (i.e., -Gly-Arg-Arg-Ser-Ala-Glu-) is identical to the corresponding sequence in progastrin, indicating conservation of an important site for cleavage and posttranslational processing. Moreover, as in progastrin, species differences in precursor sequence are mainly located in the N-terminal region of the precursor. The CCK gene is located on chromosome 3 (204). The gene structure exhibits similarities in organization with the gastrin gene, including the presence of three exons, the first of which is untranslated (see Fig. 4-7). The similarity in gene organization, as well as in amino acid sequence in biologically important regions, strongly indicates a common evolutionary origin of the two hormones through gene

duplication. Peptides resembling CCK can be found in all the main chordate groups and appear to have been relatively well conserved during chordate evolution (205). Interestingly, in a primitive chordate, Ciona intestinalis, there is an unusual member of this peptide family that has sulfated tyrosine residues at both positions 6 and 7 from the C-terminus, therefore resembling both gastrin and CCK (206). The abundance of CCK mRNA in small intestine is increased by nutrients that also release CCK and is inhibited by fasting (207). The transcriptional control mechanisms have been studied in several cell lines including STC-1 cells. The latter are derived from an intestinal endocrine tumor in a mouse doubly transgenic for the polyoma small T antigen and SV40 early region antigen (208). These cells express CCK and other proximal small intestine hormones. The expression of CCK in STC-1 cells can be increased by peptone by mechanisms that involve increased intracellular cyclic 3′,5′-adenosine monophosphate (cAMP), increased calcium, and stimulation of the MAPK pathway (209,210). Mutational studies have identified a site −72 to −83 upstream of the transcriptional start site that overlaps a putative Cre/AP-1 site and seems to be required for responsiveness to peptone. In addition, CCK gene transcription in STC-1 cells is stimulated by the VIP-like neurotransmitter pituitary adenylyl cyclase-activating polypeptide via activation of the transcription factor cyclic adenosine 3′5′-monophosphate response element binding protein (CREB), which binds the putative AP-1/Cre site (211).

Posttranslational Processing The different forms of CCK described earlier arise through cleavage of pro-CCK in many cases at a single basic residue (Arg) (Fig. 4-8). Some of the N-terminal pro-CCK–derived peptides produced by endopeptidase cleavage have been identified (212). There are apparent differences in the patterns of pro-CCK–derived peptides in intestinal endocrine cells and CNS neurons, which have led to the view that there might be cell-specific patterns of posttranslational processing (212). However, an alternative view is that patterns of posttranslational processing in brain and gut are similar, and that differences in degradation during peptide extraction lead to variation in the identity of the recovered peptide (213). Studies in transfected cell lines suggest that SPC1/3 and SPC5 are capable of cleaving pro-CCK (214,215). There is a requirement for carboxypeptidase E to remove C-terminal basic residues because mice in which the gene encoding this enzyme is deleted are CCK deficient but have increased concentrations of Gly-Arg–extended variants of CCK (216,217).

Cellular Origins CCK in the small intestine is localized to I cells. These are predominantly located in the jejunum, which corresponds

102 / CHAPTER 4 preproCCK gene exon 1

exon 2

exon 3

preproCCK mRNA

preproCCK

FIG. 4-7. Schematic representation of the relationships between the preprocholecystokinin gene, preprocholecystokinin messenger RNA (mRNA), and the peptide precursor preprocholecystokinin. CCK, cholecystokinin.

with the relatively high concentrations of CCK in mucosal extracts of this region of intestine (218). The I cell is an open cell projecting to the intestinal lumen. It has a turnover time of a few days and appears to differentiate from an enteroendocrine cell lineage that includes the secretin cell. The bHLH transcription factor neurogenin 3, is required for the development of I cells and other intestinal enteroendocrine cells (61). Neurogenin 3 increases expression of the bHLH transcription factor, BETA2/NeuroD, which is required for the differentiation of intestinal secretin and CCK cells (219,220). In addition to the CCK produced in I cells, there are also small amounts of CCK elsewhere in the gut that reflect production in enteric neurons, including a subset of cholinergic secretory motor neurons of the submucosal plexus (221). The expression of CCK in CNS neurons is beyond the scope of this chapter; however, it is worth noting that the concentrations of CCK in many regions of the CNS may exceed those in the intestine (222,223). Signal

Neuronal populations expressing the CCK gene include GABAergic cerebral cortex neurons (224) and dopaminergic mesolimbic neurons (225).

Assay Plasma CCK is been measured by RIA (226), although a bioassay based on amylase release from rat pancreatic acinar cells has been useful in some studies (227). In RIAs it is important to take steps to ensure that the shared amino acid sequence between CCK and gastrin does not compromise specificity. Two approaches typically have been used. Antibodies that react equally with all peptides containing the common C-terminal pentapeptide amide may be used, providing steps are taken to separate gastrin and CCK (228). This can be achieved by high-pressure liquid chromatography or gel filtration chromatography, but inevitably such

R

R

K

R

R

preproCCK

RR *

CCK83

NH2

*

NH2 CCK58

*

CCK39

*

CCK33

NH2 NH2

*

NH2 CCK22

*

NH2 CCK8

FIG. 4-8. Relations among the main forms of cholecystokinin (CCK) isolated from small intestine or brain. Cleavage occurs at single Arg (R) or Lys (K) residues except for the sequence causing the C-terminal amide (−NH2).

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 103 assays make it difficult to achieve high throughput. Alternatively, antibodies that react selectively with CCK and exhibit little or no cross-reactivity with gastrin can be used (229). Care is needed to determine the relative immunoreactivities of different forms of CCK, and there is some evidence that CCK58 in particular may exhibit lower affinity than other forms of CCK for antibodies binding at the C terminus (230). Because plasma concentrations are relatively low (see later), it is often necessary to include a concentration step before assay.

Release Mechanisms The concentrations of CCK in the peripheral circulation during fasting are about 1 pmol/liter, and they increase up to about fivefold with feeding. In several species, CCK-58 is the predominant circulating form of the hormone (202,231). The main components of the diet responsible for CCK secretion are protein and fatty acids (227). Aromatic amino acids were suggested many years ago to stimulate CCK release based on pancreatic secretory responses in vivo (232); in the same model, intact protein failed to increase pancreatic enzyme secretion, but after digestion with pepsin became a strong stimulus (233). Factors other than CCK may, of course, account for a proportion of the pancreatic response. The question of whether intact protein releases CCK is also complicated by the existence of a number of CCK-releasing factors that are sensitive to protease degradation and are protected by ingestion of dietary protein. Several putative CCK-releasing peptides have been described in the gut lumen (234–236). The so-called luminal CCK-releasing factor has been isolated and sequenced from the luminal contents; it releases CCK when infused intraduodenally, and its effects are blocked by specific antibodies (235). It is produced in pancreas and small intestine (237) and is believed to stimulate CCK release by direct action on I cells to increase intracellular calcium (238). Degradation of CCK-releasing peptides by proteolytic enzymes in the intestinal lumen provides a mechanism to autoregulate CCK release. Consistent with this, it is well established that there is inhibition of CCK release by trypsin in the gut lumen (239). Moreover, prolonged administration of trypsin inhibitors (e.g., soybean trypsin inhibitors) eventually leads to pancreatic hypertrophy, which has been attributed to sustained increase of plasma CCK concentration (240). Stimulation of CCK release by dietary fat requires conversion of triglyceride to fatty acids. In humans, fatty acids with a chain length longer than C12 are effective releasers of CCK in vivo, and fatty acids with a chain length less than 10 are ineffective; similar observations have been made in STC-1 cells (241,242). In the latter, C12 increases intracellular calcium; there appears to be a transient increase in Ca2+ attributable to release from intracellular stores, and a sustained increase caused by Ca2+ entry. Of several cell lines tested (PC12, BON, Caco-2, IIC9), only STC-1 cells responded to C12 in the presence of external calcium, suggesting a cell-specific sensing mechanism for fatty acids

with chain length longer than C12 (243). The primary cellular sensing mechanisms in these two cases remain unclear, but it is notable that a family of at least three G protein– coupled receptors is now known that may function as fatty acid sensors (GRP-40, GPR41, GRP-43); however, whether these play a role in the gut remains uncertain.

Metabolism The half-life of CCK58 in the canine circulation is 4.4 minutes compared with 1.3 minutes for CCK8 (244). There is some evidence that CCK-8 is preferentially cleared by the liver, whereas longer forms are not (245,246). There is degradation of CCK8 by neutral endopeptidase 24.11; however, unlike G-17, where sulfation inhibits degradation by neutral endopeptidase 24.11, in the case of CCK-8, it is enhanced by sulfation (247). A novel CCK-inactivating peptidase has been purified from brain and characterized as a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). An inhibitor of this enzyme, butabindide, has been developed that protects endogenous CCK from degradation and enhances the effect of CCK in inducing satiety (248).

Receptors, Transduction, and Structure–Activity Relations CCK acts at both CCK-1 (also known as CCK-A) and CCK-2 (also known as CCK-B or gastrin-CCKB) receptors (see Table 4-1) (91). The latter are likely to be important in mediating the CNS actions of CCK because the alternative ligand, gastrin, is virtually absent from the brain. The CCK-1 receptor is expressed by pancreatic acinar cells, gall bladder and other smooth muscle, some peripheral and CNS neurons (particularly vagal afferent neurons), and some gastrointestinal tumors (149). The CCK-1 receptor was first cloned from rat pancreas, and the cDNA sequence predicted a protein with seven-transmembrane domains belonging to the G protein– coupled family of receptors (249). The receptor is linked to Gαq/11 and consequent stimulation of inositol phosphate turnover, mobilization of intracellular calcium, and activation of PKC (250). There is also activation of the MAPK cascade and tyrosine phosphorylation of p125 focal adhesion kinase and paxillin. The high affinity of naturally occurring peptides for CCK-1 receptors depends on the C-terminal pentapeptide amide shared with gastrin and a sulfated tyrosine residue at position 7 from the C-terminus. Unsulfated peptides, or peptides in which there is a sulfated tyrosine at position 6 from the C-terminus (as in gastrin), have low affinity for CCK-1 receptors. The CCK-1 receptor affinities for CCK-8 and CCK-33 appear to be similar (251). However, studies indicate that the N-terminal sequence of CCK-58 may influence the conformation of the C-terminal octapeptide region so that the biological properties of CCK-58 may differ from that of shorter fragments (252); in particular, CCK-58 appears

104 / CHAPTER 4 relatively more potent as a stimulant of fluid secretion from the pancreas (253,254). A number of useful antagonists of the CCK-1 receptor have been developed. The first to be described (e.g., dibutyryl cyclic guanosine monophosphate, asperlicin, proglumide, and benzotrypt) had relatively low affinity, and in the case of proglumide there was also low specificity. However, some of these compounds have proved to be useful starting points for drug development (255). Thus, glutamic acid derivatives developed from proglumide, such as loxiglumide, have high affinity and are suitable for administration to humans. Moreover, benzodiazepines based on asperlicin, such as L-364,718, have also been widely used in experimental studies. There have been extensive studies of the mechanisms by which CCK increases calcium in pancreatic acinar cells (250,256,257). In response to stimulation by CCK, there is an increase in intracellular calcium in the apical pole of the cell in the region (where secretory granules are located) that triggers exocytosis (256). At physiologic concentrations, CCK produces oscillations of intracellular calcium that are restricted to the apical pole. However, in response to high concentrations, there is a more global increase in intracellular calcium. The latter is also associated with intracellular trypsin activation and vacuolation, which may be relevant in the pathogenesis of acute pancreatitis (258). The spatial restriction of CCK-induced calcium oscillations is thought to be a property of the polarized distribution of mitochondria, which are able to buffer calcium (256). Inositol-1,4, 5-trisphosphate has been recognized as a mediator of CCKinduced increases in intracellular calcium for many years. More recently, cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate have also been identified as putative mediators of the increase in intracellular calcium induced by CCK, and it has been suggested that the characteristic pattern of intracellular calcium responses evoked by CCK are attributable to interactions between these signaling pathways (257).

Pancreas Postprandial pancreatic enzyme secretion is inhibited by CCK-1 receptor antagonists (263). Pancreatic acinar cells express CCK-1 receptors, and CCK stimulates enzyme secretion in vitro, supporting the idea that CCK acts directly on these cells. However, there is also cholinergic stimulation of pancreatic acinar cells by acetylcholine released from postganglionic vagal efferent fibers. Some evidence indicates that, in addition to direct effects on pancreatic acinar cells, CCK might also activate vagovagal reflexes to stimulate enzyme secretion; these would account, for example, for the rapid pancreatic secretory response to nutrient in the intestine compared with the slower response to injection of CCK (Fig. 4-9) (264,265). In addition to stimulation of secretion, prolonged administration of CCK also stimulates synthesis of pancreatic enzymes and cellular proliferation (266,267). Moreover, there is stimulation of pancreatic growth in rats by diets containing trypsin inhibitors and by protein-rich diets (70% casein); in both cases, the effects are reduced by administration of a CCK-1 receptor antagonist (240,268). However, in mice lacking the CCK gene, the pancreatic hypertrophy that occurs in response to a high-protein diet persists, indicating

+

−

Ach CCK

Biological and Physiologic Actions The main actions of circulating CCK are stimulation of pancreatic enzyme secretion, stimulation of gall bladder contraction, inhibition of gastric emptying, and inhibition of food intake. Mice in which either the CCK or the CCK-1 receptor genes are deleted have, in recent years, provided useful experimental models for studies of these functions (255,259–261). In addition, a strain of rat (the Otsuka Long Evans Tokushima fatty [OLETF] rat) in which the chromosomal locus of CCK-1 receptor is lost has proved useful (262). In addition to gastrointestinal effects of CCK, there are also a number of important CNS actions that may reflect the role of CCK as a transmitter in the brain; these include modulation of opioid analgesia and dopaminergic activity, anxiogenic effects, and inhibition of food intake. This chapter focuses on gastrointestinal effects of CCK.

+

CCK

Pancreatic exocrine secretion Ach

Ach

VIP Ach NO Inhibition of gastric emptying

FIG. 4-9. Activation of vagovagal reflexes by cholecystokinin (CCK). The latter acts on vagal afferent neurons serving the stomach and small intestine; stimulation of these pathways leads to inhibition of food intake, reflex relaxation of the gastric corpus with inhibition of gastric emptying, and reflex stimulation of pancreatic enzyme secretion. There are also direct effects of CCK on smooth muscle in the gastric antrum and on pancreatic acinar cells. Ach, acetylcholine; NO, nitric oxide; VIP, vasoactive intestinal peptide.

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 105 that diet-linked pancreatic hypertrophy is not associated with CCK in this case (261). Gall Bladder Contraction The delivery of bile to the duodenum requires contraction of the gall bladder and relaxation of the sphincter of Oddi, and both responses are stimulated by CCK. Gall bladder contraction occurred in response to similar plasma concentrations of endogenous CCK (released by a test meal) and exogenous CCK delivery by intravenous infection (269). Moreover, postprandial gall bladder contraction is blocked by CCK-1 receptor antagonists, which is compatible with the idea that this is a physiologic action of the hormone (270,271). In CCK-1 receptor KO mice, there is evidence of biliary stasis (259). Gall bladder smooth muscle expresses the CCK-1 receptor compatible with direct actions of CCK on the smooth muscle (272). However, in guinea pig gall bladder, there is evidence for a presynaptic site of action to facilitate gall bladder contraction (273,274). In the case of the sphincter of Oddi, the release of inhibitory neurotransmitters (nitric oxide and VIP) appears to be the primary mechanism producing smooth muscle relaxation (275,276). Gastric Emptying and Satiety The control of food delivery to the small intestine is mediated by CCK through inhibition of gastric emptying and inhibition of food intake. The two responses can be considered together because both are likely to involve activation of a vagal afferent pathway by CCK, and because there is evidence for functional links. The gastric emptying of test meals is reduced by administration of CCK-1 receptor antagonists, suggesting this is a physiologic effect of the hormone (263,277). Smooth muscle in the distal stomach may constitute one site of action of CCK in reducing nutrient flow across the pylorus (278), and in humans, ultrasound studies indicate that endogenous CCK plays a role in regulating contractions of the antrum (241). In addition, however, there is considerable evidence that CCK activates a vagovagal reflex leading to relaxation of the proximal stomach (see Fig. 4-9) (277,279,280). Vagal afferent nerve fibers express CCK-1 receptors, and CCK stimulates vagal afferent nerve discharge (281–284). There is evidence to suggest that vagal afferent neurons projecting to both stomach and jejunum are stimulated by CCK (283–285); moreover, brainstem neurons responding to the discharge of vagal afferent neurons activated by gastric mechanoreceptors also respond in the same way to CCK (286). In the case of vagal afferent neurons projecting to the stomach, it would appear that CCK is likely to act after delivery via the circulation; in the case of vagal afferent fibers serving the intestine, it is possible that CCK released by I cells acts locally. The effects of CCK on gastric emptying in the rat appear likely to be mediated by activation of this vagal afferent pathway because these effects are inhibited both by vagal nerve section and by application of the sensory neurotoxin capsaicin (277,279).

The most important behavioral response to peripheral administration of CCK is inhibition of food intake. This was first described in gastric fistula rats and has since been recognized in many different experimental circumstances (287,288). Similar to the effects of CCK on gastric emptying, the effect of peripherally administered CCK on food intake is inhibited by CCK-1 receptor antagonists and by lesion of vagal afferent nerve fibers, either surgically or by perivagal application of the sensory neurotoxin capsaicin. Studies using two CCK-1 receptor antagonists that differ in their capacity to cross the blood–brain barrier (devazepide, which penetrates the CNS, and A-70104, which does not) indicate that endogenous CCK released by nutrient inhibits food intake from a peripheral site, most likely CCK-1 receptors expressed by vagal afferent nerve fibers (289,290). Stimulation of these neurons by CCK increases c-fos expression in brainstem neurons, and research has identified prolactin-releasing peptide (PrRP) as a potential transmitter in these neurons. The latter inhibits food intake on CNS administration, thus the data are compatible with the idea that PrRP is a central peptide transmitter mediating the effects of CCK-vagal stimulation (291). Inhibition of gastric emptying and food intake may well be linked because gastric retention due to inhibition of emptying by endogenous CCK enhances sensations of fullness or satiety in healthy subjects (292). In addition, reduction of food intake in humans is increased by the combination of a low dose of exogenous CCK and moderate gastric distension, suggesting synergistic interactions between CCK and gastric mechanoreceptor stimulation (293). Several different types of interaction between CCK and other regulators of food intake have been identified at the level of the vagus nerve. Thus, potentiating interactions between CCK and leptin have been reported for stimulation of vagal afferent nerve discharge and inhibition of food intake (294–297). In contrast, in both rat and human nodose ganglia, there are neurons expressing CCK-1 and Orexin type-1 receptors, and the orexigenic peptide, Orexin A, inhibits the excitatory effect of CCK on rat vagal afferent nerve discharge (298). In addition, CCK decreases expression of CB1 receptors by vagal afferent neurons. CB1 receptors respond to endogenous cannabinoids such as anandamide that can stimulate appetite via a vagal mechanism (299). Interestingly, the expression of CB1 receptors in vagal afferent neurons is increased in fasted rats and decreased by refeeding through a mechanism sensitive to CCK-1 receptor antagonists (300). Because CB1 receptors are associated with stimulation of food intake, their down-regulation by CCK suggests an additional mechanism by which CCK modulates appetite signaling to the CNS. Gastric Secretory Effects There is expression of CCK-1 receptors by gastric somatostatin-expressing D cells and also by chief cells (301,302). In general, CCK is not a strong stimulant of acid secretion (even though its potency at CCK-2 receptors is comparable

106 / CHAPTER 4 with that of gastrin). However, after administration of CCK-1 receptor antagonists, the acid secretory response to CCK increases, indicating an inhibitory role for these receptors and revealing an acid-secretory response to CCK mediated by stimulation of CCK-2 receptors (303,304). It appears likely that CCK acts as an enterogastrone, that is, an intestinal hormone released by fat that inhibits gastric acid secretion. This effect is attributable to the presence of CCK-1 receptors on D cells and stimulation of somatostatin release by CCK, which, in turn, inhibits acid secretion and gastrin release (302–304). Studies comparing gastric function in mice in which either the gastrin gene or both gastrin and CCK genes have been deleted support the idea that CCK exerts an inhibitory effect via the D cell (305). In addition to CCK-1 receptors expressed by gastric D cells, there may also be expression by pepsinogen-producing chief cells, the physiologic significance of which remains uncertain (306).

Conditions of Excess and Deficiency Increased plasma CCK concentrations occur in vagotomized patients, after administration of CCK-1 receptor antagonists, and in infection of the small intestine (241,307). HyperCCKemia may also occur after prolonged ingestion of protease inhibitors, which accounts for the pancreatic hypertrophy seen in animals fed untreated soy flour (rich in soybean trypsin inhibitor). Conversely, it has been reported that decreased CCK secretion occurs in celiac disease (228). Whether increased plasma CCK concentrations contribute directly to human disease remains to be determined; moreover, the contributions of dysfunctional CCK signaling in the CNS to psychiatric disease remain far from clear.

SOMATOSTATIN Overview Somatostatin was first isolated from sheep hypothalamus on the basis of its ability to inhibit secretion of growth hormone (308). Subsequently, somatostatin-containing cells (D cells) were found to be widely distributed in the gut mucosa and in pancreatic islets (309). Somatostatin is also widely distributed in neurons of the autonomic and central nervous systems. There are two main naturally occurring somatostatins: peptides of 14 (Som-14) and 28 (Som-28) amino acid residues. They act at multiple receptors. Somatostatin released from enteroendocrine and pancreatic endocrine cells is mostly thought to act locally as a paracrine factor (310). This chapter focuses on the role of somatostatin in the gastrointestinal tract.

Peptide Structure The structure of the tetradecapeptide (Som-14) was determined after isolation from ovine hypothalamus; an alternative

name is somatotropin-release inhibiting factor (308,311). The higher molecular weight form (Som-28) was first isolated from porcine intestine and later hypothalamus (312). The sequence of Som-14 is contained within the C-terminal tetradecapeptide of Som-28 and is liberated by cleavage at a pair of basic residues (-Arg-Lys-) (Fig. 4-10). Other peptides of 25 and 20 amino acid residues also have been reported (313,314).

Gene Structure and Expression The human somatostatin gene is located on chromosome 3q38. The predicted precursor, human preprosomatostatin, contains 116 amino acids and has a single copy of the sequence corresponding to Som-28 located at its extreme C terminus (315,316) (see Fig. 4-10). The precursor of rat somatostatin exhibits a similar organization (317), and both rat and human genes contain a single intron (316,318). The somatostatin gene is well conserved in mammals; in some fish (e.g., catfish and anglerfish), there are two somatostatin genes and evidence that, although the sequence of Som-14 is conserved in one of them, there is divergence in the other (319,320). In keeping with the idea that somatostatin acts locally in several different systems, it seems likely that gene expression is differentially regulated in different populations of somatostatin-producing cells (321). For example, there is evidence for differential regulation of somatostatin gene expression in antral versus corpus D cells (322); in the rat gastric antrum, the abundance of Som mRNA is determined by luminal factors, notably gastric acid, whereas in the corpus, it is not. Thus, prolonged increases in gastric pH lead to a decrease in antral somatostatin mRNA, whereas fasting increases mRNA abundance (28,323,324). These changes may be partly because of the action of gastric acid in inhibiting bacterial overgrowth that leads to inflammation in the antral mucosa and suppression of D-cell function (325,326). In contrast, somatostatin gene expression in corpus D cells appears to be under the control of spinal primary afferent neurons and particularly

Preprosomatostatin

Prosomatostatin

RK

63

1

92

Som14

Som28

Som28 1-12

Som14

Som28 1-12

FIG. 4-10. Biosynthetic relations among different forms of somatostatin. Preprosomatostatin (stippled box indicates the signal peptide) is converted to prosomatostatin, which may be cleaved either to generate Som-28 and an N-terminal peptide, or to Som-14 and an N-terminal peptide that includes the 1-12 sequence of Som-28. Som, somatostatin.

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 107 calcitonin gene–related peptide released at the peripheral terminals of these nerve fibers (322). This system is thought to be part of the mechanisms by which neurons trigger protective mechanisms in response to mucosal damage by acid or noxious agents (327). The cis-acting mechanisms regulating somatostatin gene expression include stimulation by the cAMP-response element (CRE) and action of its transactivating factor CREB by cAMP or PKC (328–330).

Posttranslational Processing There have been extensive studies of preprosomatostatin posttranslational processing in isolated islets from teleost fish, where the aggregation of islet cells into a relatively compact body is particularly appropriate for biosynthetic studies (331,332). There is a characteristic N-terminal signal peptide in preprosomatostatin that is cleaved in the endoplasmic reticulum, and thereafter prosomatostatin is conveyed through the Golgi complex to secretory granules. Cleavage of the precursor is initiated in the TGN by a decrease in pH (333). The liberation of Som-28 and Som-14 occurs by cleavage at pairs of basic residues mediated by enzymes of the SPC family. In mice in which SPC-2 is deleted, there is impaired processing of prosomatostatin in the islets of Langerhans (and also of proglucagon and proinsulin), whereas in mice in which SPC1/3 is deleted, there is normal production of Som-14 (and normal processing of proglucagon, but impaired cleavage of proinsulin) (334,335). In stomach, duodenum, colon, and pancreas, Som-14 predominates, but in the small intestine, Som-28 accounts for more than 50% of total activity (310). Studies following the incorporation of [35S]-cysteine suggest that in gastric D cells, there is no substantial conversion of Som-28 to Som-14, and that Som-14 is generated by cleavage of prosomatostatin (336). Cleavage of the latter generates prosomatostatin 1-64, which has been isolated from both intestine and pancreas (337).

Cellular Origins Somatostatin-containing D cells are found throughout the gastrointestinal tract and pancreas (309). They are particularly abundant in the corpus and antral regions of the stomach. These cells may extend long cytoplasmic processes that approach nearby target cells (G cells in the antrum, parietal cells in the corpus), thereby facilitating paracrine signaling networks (338). In the gastric corpus, D cells do not project to the lumen, and thus are “closed” endocrine cells; however, in the antrum and throughout the intestine, they do extended into the lumen, and thus are “open” cells. There are also abundant D cells in the pancreatic islets that are often localized around the periphery. In addition, throughout the gastrointestinal tract, somatostatin is produced by some types of enteric neurons (339,340). These may occur in the submucosal plexus and project to the mucosa, or in the myenteric plexus and project distally to other myenteric

plexus neurons (221,341). In submucosal plexus neurons, somatostatin often is colocalized with neuropeptide Y, CCK, and choline acetyltransferase, and in myenteric neurons with choline acetyltransferase (221,341).

Assay RIAs of somatostatin typically make use of antibodies to Som-14 that react with both Som-14 and Som-28. In addition, there are antibodies to the N terminus of Som-14 or to the cleavage site in Som-28 that may provide the capacity for specific assay of different forms (342). Assay of somatostatin in plasma requires an extraction step, for example, by acetone, ethanol, or adsorption to ODS silica (343,344).

Release Mechanisms Concentrations of somatostatin in peripheral plasma are 10 to 30 pM and are thought to represent contributions from many different tissues. After protein- or fat-rich meals, there are increases in circulating peptides corresponding to both Som-14 and Som-28 (345,346). To study secretion from defined populations of somatostatin-producing cells, many authors have made use of assays of peptide in the venous effluent from organs in vivo or from isolated perfused organs, as well as secretion into the media from isolated cells incubated in vitro (67,68,347–351). The control of somatostatin release from gastric D cells has been studied intensively (310,352). Intraluminal acid is a stimulant for antral D-cell somatostatin release (353,354). In vivo, D-cell function also is modulated by a variety of neurohumoral factors and is impaired in infection with H. pylori. A number of hormonal and neuropeptides that inhibit acid secretion, including gastrin-releasing peptide, secretin, glucagon, vasoactive intestinal polypeptide, and CCK, are reported to stimulate gastric somatostatin release, which is consistent with the idea that paracrine release of somatostatin from D cells mediates inhibitory actions on acid secretion (67). In addition, the peptide neuromodulator GRP is reported to stimulate somatostatin release, whereas in the perfused rat stomach, there is cholinergic inhibition of secretion (355,356). The relevant control mechanisms therefore are relatively complex with both excitatory and inhibitory neural and endocrine influences on the D cell (Fig. 4-11). In addition to the hormonal stimuli described earlier, gastrin also is reported to stimulate somatostatin release independently of gastric acid, suggesting a negative feedback relation between G and D cells within the antrum (357). The physiologic mechanisms regulating D-cell function are subverted in antral inflammation. In particular, in H. pylori infection there is depressed D-cell function and depressed D-cell numbers identified by immunohistochemistry (which may simply reflect loss of cellular somatostatin stores). These changes are restored by eradication of the bacterium (358).

108 / CHAPTER 4 Parietal cell

ECL cell

+ Hist

−

Parietal cell

D-cell

− SOM

SOM + D-cell stimulation GRP CCK CGRP/amylin PACAP, VIP, Sec IL-4

− D-cell inhibition Ach GABA Opioids Nociceptin

FIG. 4-11. Control of gastric corpus D-cell function and action of somatostatin (SOM) in the corpus. There are both excitatory and inhibitory neurohormonal inputs to the D cell; paracrine inhibition of enterochromaffin-like (ECL) cell and parietal cell function leads to depression of acid secretion. Ach, acetylcholine; CCK, cholecystokinin; CGRP, calcitonin gene–related peptide; GABA, γ-aminobutyric acid; GRP, gastrin-releasing peptide; IL-4, interleukin-4; PACAP, pituitary adenylyl cyclase–activating polypeptide; VIP, vasoactive intestinal peptide.

Experimental infection of the mouse stomach with other bacteria also inhibits D-cell function (359). The D cell appears to be a target for Th-1 cytokines such as interferon-γ, which depresses function, whereas the Th-2 cytokine IL-4 stimulates D-cell function (360).

Metabolism In the dog, Som-28 has a longer half-life and slower clearance rate than Som-14 (t1/2 2.8 vs 0.6 minutes, respectively) (361,362). Studies of the molecular mechanisms of degradation during perfusion of the rat liver indicate that there are at least three distinct cleavages of Som-28. Tryptic-like cleavage of the Arg-Lys generates Som-14, and both Som-28 and Som14 are subject to endopeptidase cleavage through the cyclized (ring) segment and to aminopeptidase cleavage (342).

Receptors, Transduction, and Structure–Activity Relations There are five different somatostatin receptors, SSTR-1 to SSTR-5, all belonging to the G protein–coupled receptor superfamily (363). The different receptors are differentially expressed, with SSTR-2 and SSTR-5 being important in the gut. In the case of SSTR-2, there are two splice variants: SSTR-2a and SSTR-2b. Immunohistochemical studies indicate that SSTR-2a is expressed primarily by human G cells in the antrum, but also by intestinal G cells (364); interestingly, there was also localization to enteric neurons but not to epithelial cells in the gastric corpus. In contrast, in the rat, there is evidence for localization of SSTR-2 to ECL cells (365). Somatostatin receptors are coupled to pertussis toxin– sensitive G proteins. Multiple signal transduction pathways

have been described, including Gi/o-mediated inhibition of adenylate cyclase, activation of inwardly rectifying K+ channels, inhibition of voltage-dependent Ca2+ channels, and inhibition of intracellular Ca2+ mobilization (361). These effects are likely to account for inhibition of hormone release. In addition, activation of SSTR-2 may depress MAPK activity, and thus inhibit proliferation (361,366). Many somatostatin analogues have been developed (367). The SSTR-2 agonist octreotide is widely used clinically to treat endocrine tumors by suppression of hormone release and inhibition of tumour cell proliferation and metastasis. Other applications include management of intractable diarrhea and bleeding esophageal varices (368). More recently, a number of other agonists showing selectivity for different receptor subtypes have been developed including nonpeptide ligands and a number of antagonists (363). In addition, radioactively labeled somatostatin analogues, for example, [111In-DTPA-D-Phe1]-octreotide, have been used in diagnostic imaging of neuroendocrine and pituitary tumors (368).

Physiologic Actions Somatostatin has a wide spectrum of biological actions in many different parts of the body (363). The first clear role to be identified for somatostatin released by hypothalamic neurons was inhibition of the secretion of pituitary hormones such as growth hormone, prolactin, and thyroid-stimulating hormone. In addition, somatostatin inhibits release of a wide variety of peptide hormones from all regions of the gut and endocrine pancreas; it also has inhibitory effects on gastric acid secretion, pancreatic exocrine secretion, intestinal absorption, and proliferation, and it has complex actions on gastrointestinal motility. It inhibits cytokine secretion from epithelial cells and T lymphocytes, and deactivates macrophages (363,368). Within the gastrointestinal tract, the effects of somatostatin in the gastric epithelium have attracted sustained attention (see Fig. 4-11). In particular, D cells provide a point of convergence for a variety of factors that inhibit G-cell or parietal cell function, and thus represent a crucial link in the negative feedback control of acid secretion. The importance of the system is indicated by evidence that immunoneutralization of somatostatin prevents inhibition of gastric acid secretion caused by upper-intestinal fat, but not ileal or colonic fat (369). Mice in which the SSTR-2 gene has been deleted have high basal acid secretion (but normal plasma gastrin), and this was not changed by administration of Som-14; administration of gastrin antibody reduced basal acid output, indicating a major role for SSTR-2 in the control of acid secretion through inhibition of gastrin release (370).

Conditions of Somatostatin Excess or Deficiency Rare neuroendocrine tumors producing somatostatin have been described, about half of which have been associated with MEN-1. The associated clinical syndrome is

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 109 characterized by steatorrhea, cholelithiasis, hyperglycemia, and weight loss (371,372). Within the gastrointestinal tract a clear example of reduced production of somatostatin is provided by patients infected with H. pylori. Infection in the antrum leads to reduced tissue content of somatostatin and reduced D-cell number with a consequent increase in G-cell function. The depression of D-cell function is reversible on eradication of H. pylori infection (373).

GHRELIN Overview Ghrelin is a relatively recently discovered gastric peptide that stimulates food intake. It was identified as a naturally occurring ligand of the growth hormone secretagogue (GHS) receptor (2). It is widely recognized that growth hormone secretion is stimulated by growth hormone– releasing factor and inhibited by somatostatin. In addition, however, some synthetic peptides increase growth hormone secretion through receptors that are distinct from those for growth hormone–releasing factor (374–376). The naturally occurring ligand for these receptors was uncertain until Kojima and colleagues (2) identified a novel 28-residue gastric peptide, ghrelin, as fulfilling this role. Independently, Tomasetto and colleagues (377) identified a novel motilinlike peptide in stomach. It is now clear that ghrelin and motilin are structurally similar (Fig. 4-12), and that ghrelin comes from distinct endocrine cells, X- or A-like cells, of the gastric corpus; in addition to stimulation of growth hormone secretion, ghrelin is a potent stimulant of food intake.

differences at just two positions in the rat and human peptides.

Gene Structure and Expression The human ghrelin gene consists of four exons and three introns and is located on chromosome 3p25-26 (379). The rat ghrelin gene has a similar organization, but the mouse gene has five exons and four introns (380). Studies of the promoter region of the human ghrelin gene have identified an E-box motif that appears to regulate expression in a medullary thyroid carcinoma cell line (381). A number of polymorphisms have been reported. One, Arg51Gln, alters an endopeptidase cleavage site, and thus leads to the production of a proghrelin peptide in place of the 28-residue peptide (382). Another polymorphism, Leu72Met, occurs in the C-terminal cleavage product of proghrelin. There are suggestions that both polymorphisms may be associated with obesity, but their functional significance for human disease remains to be fully explored (383).

Posttranslational Processing Preproghrelin has 117 amino acid residues; the sequence of the biologically active 28-residue peptide follows immediately after the signal peptide and is liberated by cleavage at a pair of basic residues on the C-terminal side (2,377). A C-terminal fragment of the precursor is secreted into plasma (384). The n-octanoylation of Ser-3 is a posttranslational event (see Fig. 4-12); both acetylated and desacyl-ghrelin appear to occur naturally (385). In addition, decanoylated (C10:0) and possibly decanoylated (C10:1) peptides may occur naturally (386).

Peptide Structure Cellular Origins Ghrelin is a 28-amino-acid peptide that is modified by n-octanoylation of Ser in position 3 (2). In addition, alternative mRNA slicing yields a 27-amino-acid residue peptide corresponding to des(Gln-14) ghrelin (378). The structural similarities between human ghrelin and motilin amount to six residues in the N-terminal and midregion of the peptides. Unlike motilin, which is absent in the rat, ghrelin is present in all mammalian species examined so far, and its sequence is relatively well conserved; for example, there are

The X- or A-like cells of the gastric corpus (387) that produce ghrelin account for about 20% of the total endocrine cell population in this region of the stomach. Relatively smaller amounts of ghrelin are also produced in the intestine and colon. Ghrelin-producing cells of the gastric corpus are of the closed type, but in lower regions of the gut, they are the open type, suggesting that there may be different types of regulatory mechanisms in different populations of

CH3 (CH2)6 C=O O

Ghrelin Motilin

G-S-S-F-L-S-P-E-H-Q-R-V-Q-Q-R-K-E-S-K-K-P-P-A-K-L-Q-P-R G-S-S-F-L-S-P-E-H-R-V-Q-Q-R-K-E-S-K-K-P-P-A-K-L-Q-P-R F-V-P-I-F-T-Y-G-E-L-Q-R-M-Q-E-K-E-R-N-K-G-Q

FIG. 4-12. Amino acid sequences of human ghrelin and motilin showing common residues (boxes). Note the presence of an n-octanoyl side chain on Ser-3 of ghrelin.

110 / CHAPTER 4 ghrelin-producing cell (388). Gastrectomy reduces plasma ghrelin concentrations by about 65%, which is compatible with the idea that some circulating ghrelin is of extragastric origin (389). Other extragastrointestinal sites that might contribute to circulating ghrelin include the islets of Langerhans, arcuate nucleus of the hypothalamus, kidney, testis, and immune cells (376). During development, ghrelin appears in the rat stomach at E17; there is also ghrelin expression in the placenta. In pituitary, there are initially high concentrations of ghrelin at birth that decline thereafter (390).

Assay RIAs for ghrelin have made use of antibodies reacting at both the N and C termini (385). Antibodies directed at N terminus require acylation. Importantly, there are greater plasma concentrations of immunoreactive ghrelin measured by C-terminal–specific antibodies, which is consistent with the idea that there are greater circulating concentrations of desacyl ghrelin than the acetylated form (385,391).

Release Mechanisms In humans, circulating concentrations of ghrelin are greatest in the interdigestive period. The mechanisms regulating release of ghrelin currently are attracting considerable attention (376). Plasma concentrations of ghrelin increase before a meal and decrease with ingestion of food (392,393). Oral or intravenous glucose loads also decrease plasma ghrelin (394). There is a relation between ghrelin and body weight: concentrations are high in anorexic subjects (389) and reduced in obese subjects (395). In both cases, plasma concentrations adjust after changes in body weight. Concentrations are markedly reduced in patients after gastric restriction/bypass surgery (396). One study (397) reports eradication of H. pylori to be associated with increased plasma ghrelin, but another study (398) reports similar plasma concentrations of ghrelin in patients infected with H. pylori compared with control subjects. Most human studies of plasma ghrelin to date have not considered H. pylori status, and further work is needed to clarify the significance of infection as a possible confounding variable. In the rat, plasma ghrelin concentrations are greatest at the end of the dark and light periods (399); these periods correspond to maximum and minimum gastric volumes, suggesting that the control of ghrelin is complex. All the major nutrient groups are reported to depress ghrelin secretion when instilled into the rat stomach, and glucose and fat also reduced ghrelin on intravenous administration (400).

Metabolism In human subjects, exogenously administered acylated ghrelin disappeared from plasma more rapidly than total

ghrelin, with half lives of 9 to 13 and 27 to 31 minutes, respectively, suggesting that deacylation occurs after secretion (401). Studies in subjects with renal failure and nephrectomized mice suggest that the kidney is important for the clearance of desacyl ghrelin (391).

Receptors, Transduction, and Structure–Activity Relations Ghrelin acts at the GHS-1 receptor. Alternative mRNA splicing generates two forms of the receptor: type 1a is functional, but a truncated form, type 1b, is not (402). The receptor is coupled to Gαq/11, and activation leads to stimulation of phospholipase C, increased intracellular calcium, increased inositol polyphosphate turnover, and PKC activation (403). Interestingly, the ghrelin receptor has high constitutive, or ligand-independent, activity, which can be revealed using CREB activation as an assay and implying that receptor expression on its own (i.e., in the absence of ligand) may be associated with biological responses (404). GHSs that act via the GHS receptor have been known for many years, including the synthetic hexapeptide, His-D-TrpAla-Trp-D-Phe-Lys-amide (GHRP-6) (374,375). Moreover, a number of other GHS receptor ligands were identified through drug-discovery programs before the discovery of ghrelin as the endogenous ligand (376,405). The characteristic actions of ghrelin on food intake and growth hormone release are properties of the acylated peptide. A report suggests that desacyl ghrelin had actions in the opposite direction to the acylated peptide. These included inhibition of food intake and inhibition of gastric emptying (406); moreover, mice overexpressing desacyl ghrelin appear to be smaller than control animals (407). The mechanism of action of desacyl peptide and the relevant receptor currently are unknown.

Biological and Physiologic Actions The stimulation of growth hormone release by ghrelin occurs in vivo and in vitro and was the defining activity of the peptide (2). In addition, ghrelin exerts a number of other actions including effects on other hormones, the cardiovascular system, sleep, cell proliferation, and carbohydrate metabolism (379,405). Most strikingly, however, ghrelin stimulates food intake and adiposity, and plays a role in the regulation of digestion (408,409). Mice in which the GHS-1 receptor gene is deleted are insensitive to the effects of ghrelin administration, indicating that the orexigenic effects of ghrelin are mediated by GHS-R (410). However, both these mice and mice in which the ghrelin gene has been deleted exhibit normal body weight and food intake (411,412). These observations suggest ghrelin is not an essential regulator of food intake in the mouse. Studies in these mice do, however, suggest that ghrelin plays a role in determining fat versus carbohydrate utilization as energy substrates, particularly during high-fat intake (412).

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 111 The idea that ghrelin stimulates food intake by acting directly on the hypothalamus has subsequently been modified by the observation that the effects of circulating ghrelin on appetite require an intact vagus nerve (413). Thus, GHS-1a receptors are localized to vagal afferent nerve fibers that project to the stomach (414), ghrelin inhibits the resting discharge of vagal afferent fibers, and lesion of vagal afferent fibers with capsaicin inhibits the appetite-stimulating actions of ghrelin (413). Ghrelin exerts a number of effects on gastrointestinal function that, taken as a whole, point to a role in stimulating the delivery of nutrients to the upper small intestine. They include stimulation of gastric motility, gastric emptying, and gastric acid secretion (415,416). The stimulation of gastric motility and secretion in the rat after intravenous administration of ghrelin is blocked by vagotomy and atropine, indicating an indirect effect. In conscious rats, intravenous ghrelin and des(Gln-14)ghrelin were equally potent in stimulating gastric emptying; ghrelin also increased intestinal transit times and reversed postoperative delay in gastric emptying (417). On both intracerebroventricular and intravenous administration, ghrelin stimulated fasted-type gastrointestinal motility patterns in the rat (418). Notably, the effects of ghrelin in the rat (which lacks motilin) are reminiscent of those of motilin in other species. Central and peripheral ghrelin administration has potent gastroprotective effects against ethanol-induced ulcers. The mechanism appears to depend on nitric oxide release and primary afferent nerve fibers (419). It is known that activation of spinal afferent neurons releases CGRP, which is gastroprotective (327), and it seems possible that ghrelin activates this pathway.

Conditions of Excess and Deficiency There are increased plasma ghrelin concentrations in anorexia and decreased concentrations in obesity (see earlier). In the Prader-Willi syndrome (characterized by excessive appetite and obesity), there is also increased ghrelin, which it is thought might contribute to hyperphagia (420).

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404. 405. 406. 407. 408. 409.

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CHAPTER

5

Postpyloric Gastrointestinal Peptides Ella W. Englander and George H. Greeley Jr. Neuropeptide Y, 134 Structure, 134 Neuropeptide Y Receptors, 134 Gastrointestinal Localization of Neuropeptide Y, 134 Secretion, 137 Actions, 137 Motilin, 137 Structure, 137 Motilin Receptor, 137 Distribution, 137 Actions on the Gastrointestinal Tract, 138 Mechanism of Action, 139 Release, 139 Pathophysiology, 140 Erythromycin and Motilides, 140 Peptide YY, 140 Structure, 140 Gastrointestinal Localization of Peptide YY and Developmental Appearance of Intestinal Peptide YY, 140 Peptide YY Receptors, 142 Actions on the Gastrointestinal Tract, 143 Regulation of Peptide YY Secretion, 145 Neural Control of Peptide YY Release, 146 Trophic Actions of Peptide YY on the Gastrointestinal Tract, 146 Inhibitory Action of PYY (3-36) on Appetite and Food Intake, 147 Pathophysiology of Peptide YY, 147 References, 148

Secretin, 121 Structure, 122 Gastrointestinal Localization of Secretin, 122 Secretin Receptor, 122 Actions on the Gastrointestinal Tract, 123 Regulation of Secretin Secretion, 125 Pathobiology of Secretin, 126 Intestinal Somatostatin, 126 Distribution, 126 Receptors, 126 Secretion, 126 Actions, 126 Vasoactive Intestinal Polypeptide and Related Peptides, 126 Structure, 127 Gastrointestinal Localization of Vasoactive Intestinal Polypeptide, 127 Vasoactive Intestinal Polypeptide Receptors, 128 Actions on the Gastrointestinal Tract, 129 Regulation of Vasoactive Intestinal Polypeptide Secretion, 129 Vasoactive Intestinal Polypeptide Peptides, 130 Neurotensin, 130 Structure, 130 Gastrointestinal Localization of Neurotensin, 131 Neurotensin Receptors, 131 Actions on the Gastrointestinal Tract, 132 Regulation of Neurotensin Secretion, 133

SECRETIN The current science of endocrinology started with the discovery of intestinal secretin; secretin was the first hormone discovered. The science of endocrinology can be defined as the study of chemical messengers produced within endocrine organs that are secreted into the general circulation and act on specific target tissues some distance away to evoke a physiologic response. In 1902, Bayliss and Starling

E. W. Englander: Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550. G. H. Greeley Jr.: Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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122 / CHAPTER 5 (1) showed that infusion of hydrochloric acid into a denervated loop of the small intestine caused secretion of pancreatic juice in dogs. They reasoned that a chemical substance released by the acidified intestine and transported by the bloodstream to the pancreas triggered the stimulation of pancreatic secretion. They also showed that an acid extract of the intestinal mucosa given intravenously stimulated pancreatic secretion. They then proposed that the exocrine pancreas was stimulated by a chemical factor produced in the intestinal mucosa and released by luminal acid. They called this substance secretin. The endocrine nature of the underlying regulation of pancreatic secretion was also demonstrated by yet another experiment. In 1926, Farrell and Ivy (2) transplanted the tail of the pancreas into the mammary gland of a lactating dog. Feeding the dog resulted in an increase in pancreatic secretion. Because the transplanted pancreas was separated from its normal nidus and nerve supply, these investigators concluded that a bloodborne factor provoked pancreatic secretion after ingestion of a meal. Finally, in 1965, Mutt and coworkers (3) reported the amino acid sequence of secretin. Secretin is a 27-amino-acid peptide hormone produced in enteroendocrine “S” cells of the small intestine. Secretin is released into the general circulation in response to a decrease of luminal pH after a meal; its primary physiologic action is a stimulation of pancreatic bicarbonate secretion, although it also inhibits gastric acid secretion, gastric emptying, colonic contractions, lower esophageal sphincter tone, and motility. Secretin is used clinically in diagnosis of gastrinomas in patients with Zollinger–Ellison syndrome (ZES).

pancreatic exocrine secretion (4). The amidated, carboxy terminal of secretin is also essential for biological activity. Examination of the rat and pig secretin complementary DNA (cDNA) show that the secretin precursor consists of a signal peptide, a short amino-terminal peptide, the secretin sequence, an amidation sequence (glycine-lysine-arginine [GKR]), and a 72-amino-acid carboxyl-terminal peptide (Fig. 5-3) (5). The secretin gene consists of four exons spanning 813 base pairs, and like glucagon, VIP, and PACAP, the secretin sequence is encoded by a single exon (see Fig. 5-3) (6–12). The structure of secretin is highly conserved during mammalian evolution with amino acid substitutions limited to changes in positions 14, 15, and 16 (13–18).

Gastrointestinal Localization of Secretin The greatest tissue concentration of secretin peptide is found in enteroendocrine secretin or “S” cells of the mucosal epithelium, in the villi of the proximal small intestine (19). They are called S cells because of the small size of secretin granules. The density of secretin cells decreases progressively from the duodenum to the ileum. Interestingly, the highest secretin expression (messenger RNA [mRNA]) is seen in the ileum; secretin mRNA is also detected in the colon (5). Developmentally, intestinal secretin cells are first identified on day 17 of gestation in rats (20), and both secretin expression and peptide show their greatest levels 2 days before birth (6). Secretin expression is also detectable in β cells of the fetal rat pancreas (21). The greatest level of secretin expression in the pancreas occurs on day 19 of gestation, and then decreases to undetectable levels during adulthood.

Structure Secretin is a 27-amino-acid peptide (Fig. 5-1) structurally related to other regulatory peptides; together they are called the secretin family of peptides (Fig. 5-2). These peptides are glucagon, vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase–activating peptide (PACAP), gastric inhibitory polypeptide (GIP), growth hormone–releasing hormone (GHRH), peptide histidine isoleucine (PHI), and peptide histidine methionine (PHM). The entire 27-aminoacid sequence is essential for full activity; although an amino-truncated variant, secretin (5-27) will stimulate

Secretin Receptor The secretin receptor has been cloned (22) and is a G protein–coupled seven-transmembrane receptor linked to adenylate cyclase activation and stimulation of cyclic 3′,5′adenosine monophosphate (cAMP) production (22–24). The secretin receptor has a 427-amino-acid sequence and a molecular weight of 48,696 daltons. The secretin receptor is related structurally to the receptors for VIP, parathyroid hormone, and calcitonin (22).

Human Secretin His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2

Rat Secretin His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Gln-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2

FIG. 5-1. Comparison of amino acid sequences for human and rat secretin. The sequences differ in only a single residue that is underlined.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 123 Secretin

THR-PRO-ARG-HIS-SER-ASP-GLY-THR-PHE-THR-SER-GLU-LEU-SER-ARG-LEU-GLN-ASP-

Glucagon

SER-PHE-PRO-ALA-SER-GLN-THR-GLU-PRO-LEU-GLU-ASP-PRO-ASP-GLN-ILE-ASN-GLU-ASP-LYS-ARG-HIS-SER-GLN-GLY-THR-PHE-THR-SER-ASP-TYR-SER-LYS-TYR-LEU-ASP-

SER-ALA-ARG-LEU-GLN-ARG-LEU-LEU-GLN-GLY-LEU-VAL

SER-ARG-ARG-ALA-GLN-ASP-PHE-VAL-GLN-TRP-LEU-MET-ASN-THR-LYS-ARG VIP

SER-SER-ILE-SER-GLU-ASP-PRO-VAL-PRO-VAL-LYS-ARG-HIS-SER-ASP-ALA-VAL-PHE-THR-ASP-ASN-TYR-THR-ARG-LEU-ARG-LYSGLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ASN-SER-ILE-LEU-ASN-GLY-LYS-ARG

PACAP GIP

HIS-SER-ASP-GLY-ILE-PHE-THR-ASP-SER-TYR-SER-ARG-TYR-ARG-LYS-GLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ALA-ALA-VAL-LEU-ASP ALA-LEU-PRO-SER-LEU-PRO-VAL-GLY-SER-HIS-ALA-LYS-VAL-SER-SER-PRO-GLN-PRO-ARG-GLY-PRO-ARG-TYR-ALA-GLU-GLY-THR-PHE-ILE-SER-ASP-TYR-SER-ILE-ALA-MET-ASPLYS-ILE-HIS-GLU-GLU-ASP-PHE-VAL-ASN-TRP-LEU-LEU-ALA-GLU-LYS-GLY

GHRH

VAL-ARG-ARG-HIS-ALA-ASP-ALA-ILE-PHE-THR-SER-SER-TYR-ARG-ARG-ILE-LEU-GLY-

PHI

ASN-ALA-ARG-HIS-ALA-ASP-GLY-VAL-PHE-THR-SER-ASP-TYR-SER-ARG-LEU-LEU-GLY-

GLN-LEU-TYR-ALA-ARG-LYS-LEU-LEU-HIS-GLU-ILE-MET-ASN-ARG-GLN-GLN

GLN-ILE-SER-ALA-LYS-LYS-TYR-LEU-GLU-SER-LEU-ILE-

-GLY-LYS-ARG

FIG. 5-2. Alignment of the amino acid sequences for the rat secretin family of peptides. Nonidentity in sequences is indicated by underlining. All sequences shown are for the rat, except for human gastric inhibitory polypeptide (GIP). GHRH, growth hormone–releasing hormone; PACAP, pituitary adenylyl cyclase–activating polypeptide; PHI, peptide histidine isoleucine; VIP, vasoactive intestinal peptide.

Actions on the Gastrointestinal Tract The primary physiologic action of secretin is the stimulation of pancreatic bicarbonate secretion (25–28). Administration of secretin intravenously (0.03 CU/kg/hr [CU = 2.5 pmol]) will increase pancreatic secretion of water and bicarbonate in humans, dogs, and rats (29–33). That secretin is the physiologic effector of pancreatic bicarbonate secretion has been verified in dog experiments where infusion of physiologic amounts of exogenous secretin results in a stimulation of pancreatic bicarbonate secretion. In these experiments, physiologic amounts of secretin means that secretin was given at doses that reproduce circulating secretin levels measured in response to physiologic amounts of acid in the duodenal lumen of dogs. More importantly, the role of secretin is supported by the finding that bicarbonate secretion is reduced by systemic immunoneutralization of secretin in dogs. A significant increase in bicarbonate secretion is measured when plasma secretin levels increase by only 1 to 2 pM, and there is a linear dose–response relation between

plasma secretin levels and bicarbonate secretion up to 40 pM (34). In experimental settings, secretin given alone intravenously is a weak stimulant of pancreatic enzyme secretion. However, small amounts of secretin will potentiate the stimulatory effects of cholecystokinin (CCK) on enzyme and bicarbonate secretion in humans and in dogs (Fig. 5-4) (33). Secretin can also stimulate bile salt-independent bile flow (35–40); however, the physiologic relevance of this finding is unknown. Secretin, administered at physiologic doses, will inhibit pentagastrin-stimulated acid secretion and meal-induced acid secretion in dogs (41–43). Administration of secretin to humans also results in an inhibition of pentagastrin-stimulated acid secretion (44). It has been shown that immunoneutralization of systemic secretin increased plasma gastrin levels and acid secretion in response to a test meal in dogs, implying that secretin has a role as a physiologic inhibitor of acid secretion in the dog (41,43). In dogs, the inhibitory action of secretin on acid secretion has been shown to be mediated in part by prostaglandins (Fig. 5-5) (45). In addition, inhibition

Signal Peptide Rat

N-Terminal Peptide

M E P L L P T P P L L L L L L L L L S S S F V L P A P P R T P R Secretin

H S D G T F T S E L S R L Q D S A R L Q R L L Q G L V G K R S E C-Terminal Extension Peptide

E D T E N I P E N S V A R P K P L E D Q L C L L W S N T Q A L Q

D W L L P R L S L D G S L S L W L P P G P R P A V D H S E W T E

T T R Q P R

FIG. 5-3. Amino acid sequence for rat secretin precursor. The signal peptide, N-terminal peptide, secretin sequence, and C-terminal extension peptide are identified. (Modified from Kopin and colleagues [5], by permission.)

124 / CHAPTER 5 Actual value by Secretin (.03 Cu/kg/hr) and CCK-8 (.03-.25 µg/kg/hr)

∆ HCO3− Output (mEq/15 min)

.35

Sum of values by two individual hormones

.30 .25 .20 .15 .10 .05

SE C C .03 C K S .03 & EC C .0 C 3 K .0 3 SE C C .03 C K S .06 & EC C .0 C 3 K .0 6 SE C C . C 03 K . S 12 & EC 5 C C .0 K 3 .1 25 SE C C .03 C K SE .25 & C C .0 C 3 K .2 5

.00

FIG. 5-4. Mean increased pancreatic bicarbonate outputs greater than basal values in response to intravenous secretin alone (0.08 CU/kg/hr [CU = 2.5 pmol]), cholecystokinin (CCK)-8 alone at four different doses (0.03, 0.06, 0.125, and 0.25 µg/kg/hr), and a combination of secretin and CCK-8 in five dogs with pancreatic fistulas. Bars represent means ± standard error of the mean. (Modified from Chey and colleagues [33], by permission.)

of pentagastrin-stimulated acid secretion and stimulation of pancreatic bicarbonate secretion are not strictly parallel during duodenal acidification in dogs, implying that secretin is not the only factor released by the luminal acid that also exerts an inhibitory action on acid secretion (46). In rats, a low physiologic dose of secretin will inhibit gastric acid secretion that is mediated by prostaglandins (47). In dogs, Pentagastrin

administration of secretin in combination with serotonin (5-HT) results in a greater inhibition of acid secretion when compared with either agent alone (Fig. 5-6) (48). The relevance of the inhibitory action of 5-HT on acid secretion is strengthened by the finding that duodenal acidification causes secretion of intestinal 5-HT (49). Secretin causes a reduction in the serum gastrin response to food in both humans and dogs (50). Administration of secretin will reduce basal gastrin levels in dogs, healthy subjects, duodenal ulcer patients, and patients with pernicious anemia (51–53). The inhibitory actions of secretin on meal-induced gastrin secretion, acid secretion, and gastric emptying are similar in healthy subjects and patients with duodenal ulcer (51,54). In rat studies, secretin has been shown to inhibit gastrin release via its stimulation of stomach somatostatin (SRIF) secretion (55,56). Under a variety of experimental settings, administration of exogenous secretin affects secretion of insulin, glucagon, somatostatin, and pancreatic polypeptide (PP) (55,57–61). Abundant data indicate that secretin has an inhibitory role in regulation of gastric emptying in dogs and humans (41,62–64). Secretin also stimulates pepsin secretion in dogs, cats, and humans (65–68), and secretin stimulates pepsinogen secretion from isolated dog chief cells maintained in culture (69). In humans, the administration of secretin at doses as low as 0.03 U/kg significantly inhibits gastric emptying of a liquid meal (63). This inhibitory action of secretin is not potentiated by CCK. Infusion of secretin will stimulate gastrin secretion in patients with gastrinoma and ZES (53,70,71). In fact, intravenous (IV) infusions of secretin are used in locating gastrinomas during their surgical removal (72). Secretin appears to cause gastrin secretion by a direct action on stomach gastrin “G” cells, because in vitro experiments show that secretin will stimulate gastrin release from cultured gastrinoma cells (73).

Secretin

*

*

0 B1 B2 15

45

75 Minutes

105

135

165

FIG. 5-5. Gastric acid output after infusion of secretin (3.3 CU/kg/hr [CU = 2.5 pmol]) alone or in combination with indomethacin, an inhibitor of prostaglandin synthesis (2 mg/kg bolus + 0.5 mg/kg/hr) in six dogs. Each point represents the means ± standard error of the mean. Note that inhibition of gastric acid secretion was achieved only in the secretin infusion alone group. B, basal value. *p < 0.05 versus values before secretin administration. (Modified from MacLellan and colleagues [45], by permission.)

10

*

PG+5-HT

*

PG+5-HT+Secretin

Secretin * + Indomethacin

1

PG+Secretin

2

PG

3

20 Secretin alone

∆H+ (mEq/30 min)

Gastric acid output (mEq/15 min)

4

*

**

FIG. 5-6. Gastric acid outputs in conscious dogs during infusion of pentagastrin (PG) alone, PG with secretin, PG with serotonin (5-HT), or PG with 5-HT and secretin. *p < 0.05 versus PG alone. **p < 0.05 versus PG + secretin or PG + 5-HT. (Modified from Mate and colleagues [48], by permission.)

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 125

Acidification of the duodenal and jejunal lumen is the primary physiologic stimulus for release of secretin into the bloodstream and the subsequent stimulation of pancreatic exocrine secretion (25–28). In the dog, the pH threshold for stimulation of secretion release is 4.5 (26), and the amount of pancreatic bicarbonate that flows into the intestinal lumen is directly dependent on the length of small intestine that is exposed to the acid and the acid load (82). An increased basal acid secretion will cause an increased secretin release (83). Similar to another intestinal hormone, CCK, systemic levels of secretin are exceptionally low (30,84–86). Plasma secretin levels of approximately 10 pM will stimulate pancreatic bicarbonate secretion, and an increase in plasma secretin levels of only 1 to 2 pM will activate pancreatic bicarbonate secretion significantly (87,88). Measurement of an increase in plasma secretin levels after a normal meal is not agreed on, implying that secretin may not be released after a meal (89,90). However, acidified meals definitely cause secretin release (84). Some studies show that plasma secretin levels do not increase until several hours after a meal (91,92). As explained for the pancreatic bicarbonate response, the increased plasma secretin response is related to the amount of acid entering the duodenal lumen (84,91,93). In a study of humans, a liquid test meal increased plasma secretin levels by 2 to 3 pM when duodenal pH decreased to 4, and it was shown that administration of exogenous secretin at doses that mimicked this plasma concentration increase also stimulated bicarbonate secretion (27). In humans, administration of low amounts of acid into the duodenum, followed by neutralization, caused a short-lived increase in plasma secretin levels of approximately 1 pM (34). Exceptionally small bolus injections of secretin in humans (0.1–0.5 pmol/kg) will result in a stimulation of pancreatic bicarbonate secretion and increases in plasma secretin levels of 2 to 6 pM (34). Direct proof for involvement of secretin in the stimulation of pancreatic bicarbonate secretion has been shown by immunoneutralization of systemic secretin—the pancreatic bicarbonate response was decreased by approximately 80% (41). Together, these findings indicate that secretin acts as a physiologic-hormonal regulator of bicarbonate secretion; however, it is not exactly clear the extent to which this effect is present in humans after a meal (94). Fat in the duodenal lumen is a strong inhibitor of gastrin and gastric acid secretion, but the role of secretin in mediating

30 Plasma secretin (pg/ml)

Regulation of Secretin Secretion

this inhibitory action of fat is not settled (95). Pancreatic bicarbonate and secretin release are stimulated by duodenal infusion of oleate; the small increase in plasma secretin levels implies a role for secretin in the bicarbonate response (Fig. 5-7) (96). Alcohol can stimulate secretin release in humans most likely by a stimulation of gastric acid secretion (97). Intraduodenal administration of bile will also cause secretin release in humans (97). In contrast with other gut hormones, the vagal nerve does not influence secretin release in dogs and humans. In dogs, vagotomy and atropine treatment do not affect secretin release in response to intraduodenal administration of acid in dogs, and atropine treatment does not alter the secretin response to duodenal acidification in humans (95,98). Furthermore, vagotomy does not influence secretin release in response to acid in humans (99). In dogs, vagal stimulation does not affect secretin, and atropine administration did not modify secretin release to acid (98). In contrast with the lack of vagal influence over secretin release, there is a significant interaction between secretin and vagal cholinergic control of the exocrine pancreas. Vagal stimulation will potentiate the stimulatory effects of secretin on pancreatic bicarbonate secretion in dogs and cats (100,101). In addition, vagotomy or anticholinergic drugs will reduce pancreatic secretion of

*

*

20 * 10

0 *

*

0.3 Bicarbonate output (mEq/15 min)

Secretin also stimulates the pylorus or inhibits the lower esophageal sphincter at pharmacologic doses (74,75). Physiologic doses of secretin will relax the sphincter of Oddi in humans (76). Administration of secretin to cats and dogs will increase secretion from intestinal Brunner’s glands (77,78). Although the growth-promoting action of secretin on the pancreas is not completely accepted, exogenous secretin has been shown to exert a trophic action on the exocrine pancreas (79–81).

0.2 * 0.1

0 0

0.25 0.5 1.0 2.0 Sodium oleate (mmol/15 min)

FIG. 5-7. Effects of increasing doses of intraduodenal administration of sodium oleate (0.25–2.0 mmol/15 min) on plasma secretin levels and pancreatic bicarbonate outputs in dogs with modified Herrera cannulas. *p < 0.05 versus 0 minute values. (Modified from Faichney and colleagues [96], by permission.)

126 / CHAPTER 5 water and bicarbonate stimulated by intraduodenal administration of acid or food in humans (29,102–104) and in dogs. Somatostatin will inhibit secretin release (105,106), whereas bombesin has no effect. There is some evidence to suggest that a luminal secretinreleasing peptide mediates secretin release in rats; however, this peptide factor has never been isolated, nor its sequence identified (107).

muscle relaxation. SSTR3 is found in the smooth muscle cells of the rat and can mediate contractions of guinea pig stomach and intestinal smooth muscle cells (125,127,128). SSTR2 may have a role in the regulation of gut motility because SSTR2 is found on nitric oxide synthase-expressing myenteric neurons, which may be inhibitory motor neurons (129). Cortistatin will activate all SRIF receptor subtypes (112,121).

Secretion Pathobiology of Secretin Plasma levels of secretin are altered in various clinical conditions. Hypersecretinemia has been observed in patients with ZES and excessive gastric acid secretion (83). In patients with duodenal ulcers, secretin and pancreatic bicarbonate secretion are unaltered compared with healthy subjects (108). In patients with celiac sprue, the decreased release of secretin may have a role in their reduced pancreatic exocrine secretion (108).

INTESTINAL SOMATOSTATIN Distribution Intestinal somatostatin (SRIF) has been detected by immunoassay in intestinal extracts (Fig. 5-8) (109,110), and immunohistochemical studies show intestinal enteroendocrine somatostatin-containing “D” cells scattered throughout the small and large intestines (111,112). SRIF is also found in intestinal nerves and nerve plexuses; SRIF-containing nerve fibers are observed around ganglion cells of the myenteric plexus of the intestine (113–115). In the intestine, approximately 90% of the SRIF is found in the mucosa and 10% in the muscle layer (116). In the myenteric plexus, SRIF-containing neurons may function as interneurons and are involved in the descending inhibitory reflex of peristalsis (117). In the intestine, the primary molecular form is SRIF-28 (110,118). Interestingly, a novel peptide, called thrittene, was isolated from monkey ileum that is structurally identical to SRIF (119). The amino acid sequence of thrittene is identical to that of the N-terminal 13 amino acids of SRIF-28. Because the gene encoding this peptide has not been isolated, it may represent a processing product of the SRIF precursor and not the product of a novel gene. There is also cDNA of a novel 17-amino-acid SRIF-like peptide called cortistatin (112,120,121).

Receptors Five SRIF receptor subtypes have been identified (SSTR15) (122–124). SSTR1, SSTR2, and SSTR3 are found in the intestine (124–126). In the gastrointestinal (GI) tract, SRIF receptors appear to be involved in regulation of smooth

In humans, SRIF-28 is the primary form of SRIF that is found in the general circulation in response to ingestion of various nutrients (Fig. 5-9). This finding indicates that systemic SRIF is derived from intestinal “D” cells and not the stomach (130).

Actions SRIF exerts a general inhibitory action on release of intestinal hormones, secretion, and absorption. SRIF inhibits the cyclic interdigestive motor and electrical activity of the intestine (131,132). Infusion of physiologic amounts of SRIF will inhibit gastric motility and intestinal phase 2, a period of irregular contractions, and stimulation of phase 3, a period of rhythmic contractions. The interval between activity fronts is decreased by approximately 50% (133). SRIF can inhibit basal and stimulated bicarbonate secretion from rat duodenal Brunner’s glands (134). SRIF inhibits intestinal transport of ions, causing a lower intestinal secretion (135). SRIF inhibits secretion of almost every gut hormone tested including ghrelin, secretin, CCK, GIP, and motilin (126,136,137).

VASOACTIVE INTESTINAL POLYPEPTIDE AND RELATED PEPTIDES VIP is a 28-amino-acid peptide that was first discovered in porcine duodenal tissue extracts. VIP belongs to the secretin family of peptides that includes glucagon, secretin, GIP, PACAP, GHRH, PHI/PHM, and two reptilian peptides, helodermin and helospectin. VIP was named for its strong and persistent vasodilatory effects. VIP relaxes vascular and nonvascular smooth muscle and will stimulate electrolyte secretion from the exocrine pancreas and intestine. VIP and PHI/PHM are processed from the same precursor peptide. Unlike secretin, GIP, and glucagon, VIP is exclusively a neurotransmitter in the GI tract and is not normally secreted into the bloodstream unless it occurs as overflow from nerve endings or VIP-secreting tumors. In the GI tract and pancreas, VIP exerts a broad array of biological activities including regulation of relaxation of gastric and intestinal smooth muscle, stimulation of pancreatic exocrine and intestinal secretion, and an inhibition of gastric acid secretion and blood flow. Humans with a VIP-secreting tumor (VIPoma) will have the watery diarrhea syndrome and high plasma levels of VIP.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 127 Somatostatin concentration (pmoles/g wet weight) 0 200

1000

3000 Esophagus Cardia Fundus Antrum

Duodenum Jejunum Mid intestine Ileum Cecum

Colon Spleen Pancreas Gall bladder

Muscular layer

Mucosa or parenchyma

Liver

FIG. 5-8. Distribution of somatostatin in the gastrointestinal (GI) tract of the cat. Somatostatin levels are shown in the GI mucosa and parenchyma (right horizontal axis) and muscular layer (left horizontal axis). Maximal levels are seen in antral mucosa and in proximal duodenum. Substantial amounts of somatostatin are measured in the intestinal mucosa. Mean ± standard error of the mean is shown. (From Chayvialle and colleagues [109], by permission.)

Structure Said and Mutt (138–140) discovered VIP when screening extracts of the pig intestine for substances that stimulated vasodilation. VIP is a 28-amino-acid peptide that resembles several other regulatory peptides produced in the GI tract and pancreas including glucagon, GIP, GHRH, secretin, PACAP, PHI/PHM, and the reptilian peptides, helodermin, and helospectin (Fig. 5-10). The sequences for rat, pig, and human VIP are identical, indicating the conserved nature of VIP during evolution. The VIP gene consists of six introns and seven exons. The mRNA for VIP encodes for VIP and a second peptide belonging to the secretin family of peptides called PHM in humans and PHI in rodents. PHM and VIP are encoded by exons 4 and 5, respectively. Prepro-VIP is 170 amino acids; during processing, 5 different peptide fragments are formed, including prepro-VIP (residues 22–79) (also called N-terminal flanking peptide), PHI/PHM (residues 81–107), prepro-VIP (residues 111–122; bridging peptide),

the VIP sequence (residues 125–152), and prepro-VIP (residues 156–170; carboxy-terminal flanking peptide).

Gastrointestinal Localization of Vasoactive Intestinal Polypeptide Neuronal VIP/PHI is found throughout the entire length of the GI tract (109,141–143), the pancreas (144), and in salivary glands (145). In the GI tract, VIP is found in the superior and inferior mesenteric ganglia and in the submucous (Meissner’s) and myenteric (Auerbach’s) plexuses of the intestinal wall (146–149). The greatest concentrations of VIP/PHI are found in the colon and ileum, with lower amounts in the stomach fundus, duodenum, and jejunum (Fig. 5-11) (109,150). During development in rats, intestinal VIP first appears at 3 days of age, and levels increase progressively until 28 days of age, when they achieve adult VIP levels (151). VIP-containing autonomic nerve fibers are detected

128 / CHAPTER 5 Meal 120

*

*

Plasma somatostatin (pg/ml)

*

*

*

Portal

80 * Systemic 40 *

*

* *

0

−15

0

15

30

45

60

90

120

150

Minutes

FIG. 5-9. Somatostatin levels in portal and peripheral plasma after ingestion of a mixed meal in dogs. The test meal caused significant increases in portal and peripheral somatostatin levels lasting for 90 to 120 minutes after ingestion. *p < 0.05 versus basal somatostatin level. (From Chayvialle JA, Miyata M, Rayford PL, Thompson JC. Effects of test meal, intragastric nutrients, and intraduodenal bile on plasma concentrations of immunoreactive somatostatin and vasoactive intestinal peptide in dogs. Gastroenterology 1980;79:844–852, by permission.)

in all layers of the gut and around blood vessels (152). In immunohistochemical studies, VIP-containing fibers are readily evident in the lamina propria, where they are found with blood vessels and reach to the surface epithelium, and in the circular muscle layer. They are absent in the longitudinal muscle layer with the exception of the taenia coli (152). VIP-positive fibers appear to originate from intramural ganglion cells of the submucosal plexus (152,153) because extrinsic denervation will not reduce VIP staining or contents. In diseases that affect autonomic ganglionic cells such as Hirschsprung’s disease and Chagas disease, changes in VIP staining are found (154,155). A characteris-

tic feature of Crohn’s disease is neuronal hyperplasia with an increased VIP content in VIP-containing nerves (156). VIP staining is seen in autonomic nerve fibers of the exocrine and endocrine pancreas (144).

Vasoactive Intestinal Polypeptide Receptors Two VIP, high-affinity, G protein-coupled receptors have been characterized (157,158). They are called VIP receptors 1 and 2 (VPAC1, VPAC2). In the mouse, VPAC1 shows the greatest expression in the small intestine and colon, followed

VIP

HIS-SER-ASP-ALA-VAL-PHE-THR-ASP-ASN-TYR-THR-ARG-LEU-ARG-LYS-GLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ASN-SER-ILE-LEU-ASP-NH2

Secretin

HIS-SER-ASP-GLY-THR-PHE-THR-SER-GLU-LEU-SER-ARG-LEU-ARG-ASP-SER-ALA-ARG-LEU-GLN-ARG-LEU-LEU-GLN-GLY-LEU-VAL-NH2

PHI

HIS-ALA-ASP-GLY-VAL-PHE-THR-SER-ASP-PHE-SER-ARG-LEU-LEU-GLY-GLN-LEU-SER-ALA-LYS-LYS-TYR-LEU-GLU-SER-LEU-ILE-NH2

PACAP-27

HIS-SER-ASP-GLY-ILE-PHE-THR-ASP-SER-TYR-SER-ARG-TYR-ARG-LYS-GLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ALA-ALA-VAL-LEU-NH2

GHRH

TYR-ALA-ASP-ALA-ILE-PHE-THR-ASN-SER-TYR-ARG-LYS-VAL-LEU-GLY-GLN-LEU-SER-ALA-ARG-LYS-LEU-LEU-GLN-ASP-ILE-MET-SER ARG-GLN-GLN-GLY-GLU-SER-ASN-GLN-GLU-ARG-GLY-ALA-ARG-ALA-ARG-LEU-NH2

GIP

TYR-ALA-GLU-GLY-THR-PHE-ILE-SER-ASP-TYR-SER-ILE-ALA-MET-ASP-LYS-ILE-ARG-GLN-GLN-ASP-PHE-VAL-ASN-TRP-LEU-LEUALA-GLN-LYS-GLY-LYS-LYS-ASN-ASP-TRP-LYS-HIS-ASN-ILE-THR-GLN

Glucagon Helodermin

HIS-SER-GLN-GLY-THR-PHE-THR-SER-ASP-TYR-SER-LYS-TYR-LEU-ASP-SER-ARG-ARG-ALA-GLN-ASP-PHE-VAL-GLN-TRP-LEU-MET-ASN-THR HIS-SER-ASP-ALA-ILE-PHE-THR-GLU-GLU-TYR-SER-LYS-LEU-LEU-ALA-LYS-LEU-ALA-LEU-GLN-LYS-TYR-LEU-ALA-SER-ILE-LEUGLY-SER-ARG-THR-SER-PRO-PRO-PRO-NH2

Helospectin HIS-SER-ASP-ALA-THR-PHE-THR-ALA-GLU-TYR-SER-LYS-LEU-LEU-ALA-LYS-LEU-ALA-LEU-GLN-LYS-TYR-LEU-GLU-SER-ILE-LEUGLY-SER-SER-THR-SER-PRO-ARG-PRO-PRO-SER-SER

FIG. 5-10. Amino acid sequences of vasoactive intestinal peptide (VIP) and VIP structurally related peptides. Nonidentity in sequences is indicated by underlining. The species for each mammalian peptide are: VIP—human, rat, mouse, pig, sheep; secretin—cow, pig; peptide histidine isoleucine (PHI)—pig; pituitary adenylyl cyclase–activating polypeptide (PACAP)-27—human, rat, sheep; growth hormone–releasing hormone (GHRH)—human; glucagon—human, bovine, porcine; gastric inhibitory polypeptide (GIP)—human.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 129 by the liver and brain. The VPAC2 receptor is expressed in the stomach, pancreas, and intestine, as well as in the lung and brain. The VIP1 and VIP2 receptors have similar structural binding specificities for VIP, PACAP, and PHI (VIP = PACAP > PHI). VIP receptors are found in the exocrine pancreas, gastric and intestinal epithelial cells, vascular smooth muscle cells, and on pancreatic and biliary ductular epithelial cells. VIP is known to activate multiple intracellular pathways (159) including the activation of cAMP-dependent protein kinases A (PKA) and C (PKC) (160–162). In intestinal epithelial cells, VIP activates IL-8 production by mitogenactivated protein kinase (MAPK)–dependent pathways (163).

Actions on the Gastrointestinal Tract In the GI tract, VIP is considered a neurotransmitter of inhibitory motor neurons, and the primary activity of VIP is its smooth muscle relaxant activity (164–167). VIP mediates relaxation of the lower esophageal and internal anal sphincters and can antagonize the contractile effects of pentagastrin in several species (74,168–170). The role of VIP as a smooth muscle relaxant is supported by the intense VIPergic innervation of gut sphincters. Evidence also indicates that VIP mediates descending relaxation of the peristaltic reflex (171). VIP can inhibit the contractile activity of gastrin (168). The relaxing effect of VIP is most likely secondary to an increased intracellular cAMP level. VIP is released into the gastric venous effluent after either esophageal or fundic distension and electrical stimulation of the vagus (109,172). VIP exerts a dual effect on intestinal smooth muscle: VIP relaxes duodenal and ileal circular muscles in the rat and the colon in the guinea pig (173,174), and at high levels, VIP contracts the longitudinal muscles of the duodenum and ileum in the rat (174) and colon in the guinea pig (173), an effect mediated by intramural cholinergic neurons (174). In guinea pigs, VIP inhibits gall bladder resting tone and CCK-induced contractions (175,176). VIP is a strong stimulant of intestinal secretion, being more potent than secretin and glucagon (177). In fact, VIP is nearly 1000-fold more potent than secretin on intestinal secretion (178). In dog and human studies, VIP can reverse the normal absorptive process of the small intestine to a net secretion of chloride and bicarbonate against an electrochemical gradient (178,179). Patients with increased blood levels of VIP caused by a VIP-secreting tumor (VIPoma) will most likely have watery diarrhea. VIP receptors are found with intestinal enterocytes, suggesting that VIP acts directly to increase intestinal secretion (180). Other activities of VIP include an inhibition of food, histamine, and pentagastrinstimulated gastric acid secretion in dogs (181–183). The inhibitory action of VIP on acid secretion may be mediated by gastric SRIF (182). VIP can simulate pancreatic secretion in numerous species; however, it is less potent than secretin (184–187). VIP may be involved in biliary transport and secretion because VIP is a potent stimulant of fluid and bicarbonate secretion from cholangiocytes via cAMP-independent pathways (188). VIP may also play a role in regulation of

blood flow in the small intestine (189,190), and VIP can stimulate bile secretion in dogs and rats (191,192).

Regulation of Vasoactive Intestinal Polypeptide Secretion Because VIP is a neural peptide in the GI tract, systemic VIP levels do not increase in response to ingestion of a meal. Levels of VIP will increase, however, in the venous outflow of the gut in response to luminal infusion of fat or acid, electrical stimulation of extrinsic nerves, and mechanical distension of the gut mucosa (193,194). Release of VIP by the GI tract is blocked by hexamethonium but not by atropine (172,194). VIP secretion is induced by administration of acetylcholine or neostigmine (a cholinesterase inhibitor) that is blocked by atropine (194,195). These data indicate that

VIP concentration (pmoles/g wet weight) 0 600

400

200

200

400

600 Esophagus Cardia Fundus Antrum

Duodenum Jejunum Mid intestine Ileum Cecum

Colon Spleen Pancreas Gall bladder Liver Muscular layer

Mucosa or parenchyma

FIG. 5-11. Distribution of vasoactive intestinal peptide (VIP) in the gastrointestinal (GI) tract of the cat. VIP levels are shown in the GI mucosa and parenchyma (right horizontal axis) and muscular layer (left horizontal axis). Peaks are seen in both mucosa and muscle layers of duodenum and right colon. Substantial amounts of VIP are also measured in extracts of the gallbladder wall. Mean ± standard error of the mean is shown. (From Chayvialle and colleagues [109], by permission.)

130 / CHAPTER 5 intramural VIP-containing neurons are innervated by preganglionic cholinergic excitatory fibers. Plasma levels of VIP are increased in patients with the watery diarrhea syndrome, also called the Verner and Morrison syndrome that includes the triad of pancreatic islet tumor, severe watery diarrhea, and hypokalemia (196). VIP was shown to be the major effector of diarrhea because tumor removal resulted in the cessation of diarrhea together with the decrease in plasma VIP levels. VIP may also play a role in the increased intestinal fluid secretion induced by cholera toxin and Escherichia coli heat labile (LT) and heat stable (STa) toxins (197). Vasoactive Intestinal Polypeptide Peptides Pituitary Adenylate Cyclase–Activating Peptide PACAP is the most recently discovered peptide of the secretin family of peptides. PACAP is called PACAP because during its isolation from the hypothalamus, it was shown to simulate adenylate cyclase activity in rat anterior pituitary cells in culture (198). There are two PACAP variants, PACAP27 and -38 (160), and both forms are biologically active. The human PACAP gene has been cloned and is composed of five exons (199). The mouse PACAP gene is composed of six exons; the signal peptide, the PACAP-related peptide, and PACAP are encoded within exons 2, 4, and 5, respectively (199). The intron–exon organization and the production of alternate mRNA of the PACAP gene are similar to those of the GHRH gene, which supports the notion that the two genes originated by gene duplication. In fact, the close similarity of the structural organization for the genes of all members of the secretin family of peptides suggests that they originated from an identical ancestral gene by duplication. Although the greatest levels of PACAP peptide are found in the brain, PACAP is also found in nerve fibers and in myenteric and submucous ganglia of the GI tract (200–202). PACAP-27 is found in the intestine, whereas PACAP-38 is primarily nonenteric (203,204). PACAP containing enteric nerve fibers have been found in the stomach and are colocalized with type I PACAP receptors (PAC1) on enterochromaffin-like (ECL) cells. PACAP is localized in the enteric neural plexus of the GI tract, where it is believed to be a mediator of gastric acid secretion and intestinal motility. There are three receptors with a high affinity for PACAP; these are the classical VIP receptor (VPAC1), PAC1, and VPAC2. PAC1 has affinity for only PACAP-38 and PACAP-27, whereas the VPAC receptors show similar affinities for PACAP and VIP (205,206). The PAC1 receptor is found on gastric ECL cells, and the VPAC1 is found on gastric SRIFproducing D cells and chief cells (207). PACAP has been shown to stimulate ECL cell proliferation (208). Centrally administered PACAP also stimulates gastric acid and pepsin secretion in rats (209). Gut smooth muscle also contains VPAC1 and PAC1 receptors. These findings suggest that endogenous PACAP is involved in the neural regulation of gastric acid secretion, relaxation of colonic smooth muscle, and stimulation of pancreatic secretion (210,211). In humans,

PACAP has been shown to stimulate insulin and glucagon secretion (212), and IV infusion of PACAP will stimulate peptide YY (PYY) secretion in dogs (213). Peptide Histidine Isoleucine/Peptide Histidine Methionine PHI and PHM are cosynthesized with VIP from a common precursor, and they are colocalized with VIP in the GI tract (214). There is a carboxy-extended variant of PHI called peptide histidine valine (PHV) (215). The function of PHV is unknown. PHI exerts many of the effects of VIP; however, the potency of PHI can be one to three orders of magnitude less than those of VIP (216). PHI has been considered a weak agonist for VIP. In the GI tract, PHI, like VIP, enhances water and electrolyte transport in the jejunum (216,217), and PHI stimulates pancreatic glucagon secretion (218). A PHI/PHM-preferring receptor has been cloned from the goldfish (219).

NEUROTENSIN During the isolation of substance P from bovine hypothalamic extracts, Carraway and Leeman (220,221) discovered a peptide that potently stimulated vasodilation of exposed cutaneous tissue pieces in rats. This new peptide was sequenced and shown to consist of 13 amino acids. It was named neurotensin because it is found in the brain and exerts hypotensive activity. Neurotensin was later shown in intestinal enteroendocrine “N” cells and neural elements where it has endocrine, paracrine, and neural activity. The neurotensin gene codes for neurotensin and for another smaller peptide structurally related to neurotensin, called neuromedin N. In the GI tract, neurotensin influences gastric acid, pancreatic exocrine and biliary secretion, motility, and intestinal mucosal growth.

Structure Neurotensin is a tridecapeptide—the sequences for human, bovine, canine, porcine, and rat neurotensin are identical (Fig. 5-12) (222). The carboxy-terminal hexapeptide sequence is highly conserved in different species and is the locus of biological activity. Neuromedin N is a hexapeptide structurally related to neurotensin that is derived from the same precursor peptide (Figs. 5-12 and 5-13) (223). The precursor peptide for neurotensin (i.e., preproneurotensin) consists of 170 amino acids and contains both the neurotensin and neuromedin N sequences near the carboxy terminus (see Fig. 5-13). The neuromedin N and neurotensin sequences are flanked by pairs of basic residues that serve as cleavage sites for the endoproteases, prohormone convertases (PCs). In tissues that express pro–neurotensin/neuromedin N, the three carboxy-terminal Lys-Arg sites are differentially processed, whereas the dibasic site upstream of the neuromedin N sequence is not cleaved. In the GI tract,

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 131 Bovine/human/canine/porcine/rat NT

pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu

Guinea pig NT

pGlu-Leu-Tyr-Glu-Asn-Lys-Ser-Arg-Arg-Pro-Tyr-Ile-Leu

Chicken NT

pGlu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu

Opossum NT

pGlu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Val-Tyr-Ile-Leu

Amphibian xenopsin

pGlu-Gly-Lys-Arg-Pro-Trp-Ile-Leu

Porcine neuromedin N

Lys-Asn-Pro-Tyr-Ile-Leu

FIG. 5-12. Amino acid sequences of neurotensin (NT) from various species and related peptides. When compared with bovine NI, nonidentity in sequences is indicated by underlining.

Gastrointestinal Localization of Neurotensin In the GI tract, neurotensin is produced primarily in the ileal mucosa with smaller amounts in the jejunum, stomach, duodenum, and colon mucosa (Fig. 5-14) (229,230). Neurotensin is made in open-type enteroendocrine cells called “N” cells (230,231). Neurotensin has also been identified in the

1

20

140

Signal peptide

150 NmN

NmN–125

NmN

160

170

NT

T

NT

T

NT 1-8

myenteric plexus and is released from myenteric neurons in culture (232). Neurotensin found in myenteric neurons most likely functions as a neurotransmitter or neuromodulator. Developmental studies have shown that neurotensin/ neuromedin N expression is low in the fetal ileum, and then increases dramatically on postnatal day 3 in the rat (Fig. 5-15) (233,234). In the human fetal ileum, neurotensin/neuromedin N expression is low and increases dramatically in the adult (233). Interestingly, neurotensin/neuromedin N is transiently expressed in the human fetal colon at midgestation (weeks 16–22) and by 24 weeks of gestation; in newborn and adult specimens, neurotensin/neuromedin N expression is absent (234,235). The significance of these age-related changes in neurotensin/neuromedin N expression is unknown.

Neurotensin Receptors Three receptors for neurotensin have been isolated and characterized (236-239). These are the high-affinity NTS1 and the low-affinity nts2 and nts3. The nts1 and nts2 are G protein–coupled receptors, whereas the nts3 is not a G protein– linked receptor and has a gp95/sortilin structure. The NTS1 4000 Neurotensin concentration (ng/g)

processing of prepro–neurotensin/neuromedin N results primarily in the formation of neurotensin and a large peptide ending with the neuromedin N sequence at its carboxy terminal. It is called large neuromedin N. In the brain, processing of prepro–neurotensin/neuromedin N results in neurotensin and neuromedin N (224,225). Partial sequences of the carboxy terminal of neurotensin that contain five or more amino acids exhibit biological activity. The aminoterminal 10-amino-acid fragment is biologically inactive. There is a neurotensin-like hexapeptide, LANT-6, that is similar to neuromedin N, with an Ile residue substituted by an Asn in chicken LANT (226). Another peptide structurally related to neurotensin is called xenopsin, which is produced in the skin of the frog Xenopus laevis (227). The neurotensin gene has been cloned and is highly conserved among species (228). The rat neurotensin gene consists of 10.2 kb and includes four exons and three introns (228). The neuromedin N and neurotensin coding regions are tandemly arranged in exon 4.

5 dogs 3000

2000

1000 *

NT 9-13

0

FIG. 5-13. Schematic showing processing of the neurotensin/ neuromedin N precursor. Putative cleavage sites and products are shown. Tail peptide is a five-amino-acid peptide found in brain and intestinal extracts. NmN, neuromedin N; NT, neurotensin; T, tail peptide. (Modified from Shulkes and colleagues [252], by permission.)

Esophagus Fundus

Antrum Duodenum Jejunum

Ileum

Colon

Pancreas

Mucosa

FIG. 5-14. Distribution of neurotensin peptide in mucosal extracts of the dog gastrointestinal tract. *p < 0.05 versus esophagus, fundus, antrum, duodenum, ileum, colon, and pancreas. (From Doyle and colleagues [229], by permission.)

132 / CHAPTER 5 Fetal

Postnatal

Day 18 20

1

3

7

10 14 18 21 25 28 60 120

NT/N

Cyclophilin

A

NT/N mRNA/Cyclophilin mRNA (Relative densitometric units)

2 Ileum

1

0 18 20

1 3

60

7 10 14 18 21 25 28

Fetal

B

120

Postnatal Days

FIG. 5-15. Northern blot analysis of neurotensin/neuromedin N (NT/NM) mRNA levels in rat ileum during development. (A) Northern blot containing 10 µg poly (A)+ RNA per lane from the ileum. The Northern blot was probed sequentially with a rat NT/NM and a rat cyclophilin complementary RNA probe. (B) NT/NM mRNA abundance was quantitated in the ileum by scanning densitometric analysis of the autoradiogram. Values are expressed as relative densitometric units after correcting for differences in cyclophilin mRNA abundance in the same RNA preparations. (Modified from Evers and colleagues [233], by permission.)

receptor is expressed in the small and large intestine and in the liver. The nts2 is a low-affinity receptor that is structurally similar to NTS1 and is recognized by levocabastine; therefore, this site corresponds to what was called previously the neurotensin acceptor site. Amino acid substitutions in the second transmembrane domain may underlie the decreased affinity of this receptor when compared with NTS1. nts2 is not expressed in the GI tract. For intracellular signaling pathways, it is well established that neurotensin can stimulate MAPK phosphorylation and activation in different cell types including nontransformed and transformed human colonic epithelial cells (240,241) and pancreatic carcinoma cell lines (240,242) that express NTR1 (243,244). Neurotensin also stimulates the formation of inositol-1,4,5-trisphosphate (IP3), increases intracellular calcium (245,246), and activates extracellular signal-regulated kinase (ERK)1/2 in pancreatic MIA PaCa-2 cells (240), nontransformed human colonic epithelial NCM460 cells (241), and colonic adenocarcinoma HT29 cells (240). In colonic epithelial cells, neurotensin-stimulated MAPK activation depends on activation of small GTPases Ras (241). Binding of neurotensin to NTR1 in human colonic cells also transactivates epidermal

growth factor receptor, resulting in activation of MAPK (247). Studies in PC3 cells show that NTR1 is linked to Gq/11 because neurotensin stimulates phosphatidylinositol-specific phospholipase C (PLC)–mediated formation of inositol phosphate and release of intracellular Ca2+ (248,249).

Actions on the Gastrointestinal Tract Neurotensin exerts a large array of activities in the body (250–252). Systemic administration of neurotensin results in vasodilation, cyanosis, increased vascular permeability, hyperglycemia, hypoinsulinemia, hyperglucagonemia, and stimulation of pituitary hormone release (growth hormone, prolactin, adrenocorticotropic hormone, luteinized hormone, follicle-stimulating hormone) (253). Neurotensin causes a vasoactive stimulation of histamine release; it stimulates pancreatic, gastric, and intestinal secretion, and influences smooth muscle contraction. Administration of neurotensin intravenously to laboratory animals and humans inhibits gastric acid secretion (254,255). One report indicates that the inhibitory action of neurotensin

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 133 (Fig. 5-16) (261–266). Neurotensin alone is unlikely an endogenous effector of pancreatic secretion because intraduodenal administration of oleate increased neurotensin secretion much less than that required to produce an equal pancreatic secretion when neurotensin is given intravenously. As indicated for the inhibitory action of neurotensin in the regulation of acid secretion, neurotensin may act with other enteric factors to regulate pancreatic secretion. Neurotensin also influences esophageal and intestinal motility. In rats and humans, neurotensin can convert a fasting motor pattern to a fed pattern (267,268). Neurotensin appears to exert this effect via a neural mechanism because vagotomy, atropine, or hexamethonium treatment will block the effect of neurotensin on the activity front (269). Neurotensin also stimulates growth of normal of GI tissues including the pancreas, colon, and small intestine (Figs. 5-17, 5-18, and 5-19) (270–275).

on acid secretion is independent of the vagus but requires prostaglandin synthesis (255), whereas other laboratories indicate that neurotensin is ineffective on acid secretion in animals prepared with vagally denervated pouches or in humans with a prior vagotomy (254,256). The inhibitory action of neurotensin on acid secretion, together with the data that dietary fat will cause neurotensin secretion into the bloodstream (257), suggested that neurotensin is an enterogastrone. Although administration of medium doses of neurotensin (2.5–4 pmol/kg/min) will inhibit food-induced acid secretion in humans (258), the plasma neurotensin levels achieved with these infusions (~150 pM) are much greater than the levels measured after a meal. Furthermore, IV administration of neurotensin at doses that reproduced levels seen after a fatty meal or oleic acid failed to inhibit peptoneinduced acid secretion (259). Neurotensin must be given at much greater infusion rates to inhibit acid secretion. Together, these observations indicate that neurotensin is not an enterogastrone by itself, although it may act in concert with other enterogastrone candidates such as PYY, 5-HT, secretin, and GIP. Neurotensin does not inhibit histaminestimulated acid secretion, and neurotensin can also inhibit gastric motor activity (260). Neurotensin given alone has been shown to stimulate pancreatic bicarbonate and protein secretion in humans and dogs (261,262). Neurotensin can also enhance hormonal (i.e., CCK, secretin) and nutrient-induced pancreatic secretion

ID AA

1500

Protein

1000

100 Bicarbonate

500 0

0 ID AA + HCl

ID AA + HCl

10

IV NT (1 µg/kg/hr)

2000

150

1500

100

1000

50 0

Protein

500 0

Integrated protein output (mg/60 min)

Protein (mg/15 min)

Bicarbonate

NT

AA

HCI + HCl NT

AA + AA HCI + + HCl NT

6 4

Secretin

2 0

Bicarbonate (µEq/15 min)

200

8

AA + NT

(2 CU/kg/hr)

150

600

CCK-8

400

(500 ng/kg/hr)

Protein (mg/15 min)

2000

Bicarbonate (µEq/15 min)

IV NT (1 µg/kg/hr) 200

50

Dietary fat is the most potent secretagogue for release of neurotensin from the intestine (257,276). The levels of neurotensin increase from undetectable levels to ~20 to 50 pM when measuring extracted and concentrated plasma in a neurotensin immunoassay. Luminal glucose, amino acids, and saline are weak secretagogues for activation of neurotensin

Integrated bicarbonate output (mEq/60 min)

ID AA

Regulation of Neurotensin Secretion

200 0

FIG. 5-16. (Left) Pancreatic protein and bicarbonate outputs in response to intraduodenal (ID) infusion of amino acids (AA) alone or plus HCl (ID AA + HCl) with and without intravenous neurotensin (NT) in dogs prepared with Herrera-type pancreatic and gastric fistulas. (Right) Comparison of integrated (60 minutes) bicarbonate and protein responses with NT alone; AA versus AA + NT; HCl versus HCl + NT; and AA + HCl versus AA + HCl + NT. *p < 0.05 compared with basal levels. **p < 0.05, significance differences among groups. (Modified from Sakamoto and colleagues [262], by permission.)

134 / CHAPTER 5

Weight, g kg−1

25

*

*

12.5

NEUROPEPTIDE Y

0 150 DNA, mg kg−1

* *

*

75

0

Protein, g kg−1

3

* *

1.5

0

cells (283,284). A role for PKC-α and -δ is suggested because inhibition of PKC-α and -δ can block 4-phorbol12myristate-13-acetate (PMA)-stimulated neurotensin secretion (283). Furthermore, studies show that protein kinase D (PKD) activation plays a role in neurotensin secretion (285).

0

33 100 Neurotensin, µg kg−1

300

FIG. 5-17. Effect of chronic administration of neurotensin on weight, DNA, and protein contents of entire small intestine from same animals shown in Figure 5-18. There are doserelated increases in weight, DNA, and protein contents of entire small intestine after neurotensin treatment. *p < 0.01 compared with control groups. (Modified from Wood and colleagues [273], by permission.)

release (276). In rats, fatty acids and alcohols will cause neurotensin release (277). The minimal fatty acid chain length to cause neurotensin release is four carbons (277). Bombesin, an amphibian skin peptide that is structurally related to the mammalian gastrin-releasing peptide, will stimulate neurotensin secretion in the rat (278). In dogs, resection of the ileum abolishes nutrient-induced neurotensin release (279), indicating that the ileum is the primary source of circulating neurotensin after luminal nutrients. In humans, vagotomy did not affect neurotensin release (256). Release of neurotensin has also been studied in vitro using isolated-perfused organs and enriched intestinal enteroneuroendocrine N-cell populations. In the isolated perfused ileum, bombesin, carbachol, catecholamines, and substance P stimulated neurotensin release (280–282). In vitro cultures of ileal neurotensin cells showed that bombesin- and epinephrine-stimulated neurotensin release can be antagonized by SRIF and carbachol. Intracellular signaling mechanisms underlying regulation of neurotensin secretion have been examined using a human carcinoid cell line called BON that produces neurotensin and chromogranin A, a marker for endocrine/enteroendocrine

Neuropeptide Y (NPY)NPY belongs to the pancreatic polypeptide-peptide YY-neuropeptide Y (PP-PYY-NPY) family of peptides, also called the PP-fold family of peptides. NPY was first characterized from porcine brain extracts using a method that identified amidated peptides at the carboxy terminal (286). NPY is widely distributed in central and peripheral neurons, especially in the GI tract. NPY is one of the most potent orexigenic peptides isolated to date, and brain levels of NPY are changed in conditions where regulation of energy balance is altered including anorexia, bulimia nervosa, and diabetes. In the GI tract, NPY is found within intrinsic neurons of the submucosal and myenteric plexuses and in nerve fibers in the mucosa, submucosa, and muscle layers. NPY exerts primarily inhibitory effects on gut motility, blood flow, and intestinal secretion.

Structure Like PP and PYY, NPY is a carboxy-terminal amidated 36-amino-acid peptide (Fig. 5-20). The sequences for rat, rabbit, guinea pig, and human NPY are identical (287). The NPY gene has been cloned from several species including the rat and human (288–294). The NPY gene consists of four exons with the NPY sequence encoded in the second exon.

Neuropeptide Y Receptors The NPY receptor and the Y receptor subtypes are described later in this chapter (see Peptide YY).

Gastrointestinal Localization of Neuropeptide Y NPY has been localized in neurons of the central, peripheral, and enteric nervous systems of several species, including humans (295–300). The richest peripheral source of NPY is the GI tract (301). NPY and nitric oxide synthase are colocalized in all layers of the human gut wall (Fig. 5-21) (302). Studies on the distribution of NPY in the GI tract of several species show the greatest NPY concentration in the lower esophageal sphincters of the rat and pig and in the pylorus of the guinea pig (297). Immunocytochemical studies show NPY immunoreactivity within nerve cell bodies in the submucosal and myenteric plexuses, thus demonstrating the intrinsic origin of NPY. NPY is also found within nerve fibers in the mucosa, submucosa, and muscle layers of the

+40

First quarter

Second quarter

+40

Weight

**

DNA **

Protein

+20

% Change from control

**

0

+20

**

**

** *

0

−10

−10 Third quarter

* ** **

+20

0 −10

**

**

**

+40

**

**

Fourth quarter

+40

** **

+20

**

*

0 0

33

100

−10 300 0 Neurotensin, µg kg-1

33

100

300

FIG. 5-18. Effect of chronic administration of neurotensin on weight, DNA, and protein contents of proximal to distal quarters of rat small intestine. Saline or neurotensin in gelatin was injected subcutaneously in indicated doses every 8 hours for 5 days in groups of rats. Results are means ± standard error of the mean percentage change compared with control values for each corresponding quarter of small intestine obtained from rats given saline in gelatin. Values for weights of proximal to distal quarters of small intestine from control groups were 5.44 ± 0.16, 5.45 ± 0.15, 5.01 ± 0.09, and 4.83 ± 0.13 g/kg body weight. Values for DNA contents (proximal to distal quarters) from control rats were 27.6 ± 0.6, 24.1 ± 0.5, 22.6 ± 1.2, and 26.6 ± 0.8 mg/kg body weight. Values for protein content (proximal to distal quarters) from control groups were 579 ± 72, 544 ± 25, 499 ± 69, and 476 ± 39 mg/kg body weight. There were dose-related increases in weight, DNA, and protein contents of small intestine after neurotensin treatment. *p < 0.05; **p < 0.01 compared with control groups. (Modified from Wood and colleagues [273], by permission.)

3

*

(9)

4 2

0

(10)

(10)

(10)

(9)

0

25

NT 100

NT 300

NT 600

10

(10)

(10)

(10)

(9)

0

10 8 6 4 2

NT 600

15

12

NT 300

*

*

NT 100

*

*

Control

20

Protein content (mg/kg body weight)

14

Control

RNA content (mg/kg body weight)

6 NT 600

(10)

8

NT 300

NT 600

(10)

10

NT 100

NT 300

(10)

12

Control

NT 100

1

DNA content (mg/kg body weight)

2

Control

Mucosal weight (g/kg body weight)

*

5

*

14 *

(10)

(10)

(10)

(9)

0

FIG. 5-19. (Top) Colonic-mucosal weights and DNA contents, and (bottom) colonic-mucosal RNA and protein contents of rats given neurotensin (NT; 100, 300, 600 µg/kg) administered subcutaneously in gelatin every 8 hours for 10 days, compared with rats given saline (control) for 10 days. *p < 0.05 versus control. (Modified from Evers and colleagues [271], by permission.)

136 / CHAPTER 5 Porcine PYY Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-ArgHis-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2

Porcine PP Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asp-Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr-Ala-Ala-Glu-Leu-ArgArg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-NH2

Porcine NPY Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Gly-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-ArgHis-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2

FIG. 5-20. Amino acid sequences for porcine peptide YY (PYY), pancreatic polypeptide (PP), and neuropeptide Y (NPY). When compared with PYY, nonidentity in sequences is indicated by underlining.

Distribution of NPY in rat gastrointestinal tract

pmol/g

60

30

0

us

ag

h op

Es

A

h

S

LE

ac

m

o St

lor

Py

us

pe

Up

um leum un li ina m r Te

ej rj

lon

Co

Distribution of NPY in muscle layers of porcine gastrointestinal tract

pmol/g

140

70

0

us ES dus rum rus m I m II III um um um um um um ing rse ing oid tum L n nt ylo nu u um un un ile ile ile ec nd ve nd m c Fu A P de den en jej r jej per id nal Ca sce ans sce Sig Re o o d id M mi A Tr De r Du Du Duo M owe Up Te L

ag

h op

Es

B FIG. 5-21. (Top) Distribution of neuropeptide Y (NPY) immunoreactivity along the length of the rat and (bottom) pig gastrointestinal tracts. Values shown are in pmol/g wet weight and represent the mean of six rats and four pigs ± standard error of the mean, respectively. LES, lower esophageal sphincter. (From Allen and colleagues [297], by permission.)

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 137 GI tract (301,303-305). Within the submucosa, NPY is also found in cholinergic secretomotor neurons (306,307). A portion of the NPY immunoreactivity is extrinsic in origin because NPY disappears around blood vessels after either surgical or chemical sympathectomy (303,305,308). Colocalization experiments have shown that peripheral NPY is found in extrinsic, postganglionic, sympathetic-catecholaminergic neurons (300,301,305,309–311). In this case, NPY may function as a modulator of sympathetic transmission. In the rat and mouse, NPY is coexpressed with VIP in stomach and intestinal myenteric neurons (311,312). NPY is also produced in the rodent and human endocrine pancreas (313,314).

The mechanisms that regulate the cyclic pattern of motilin secretion and the exact stimulus for the release of motilin are unknown. The physiologic action of motilin is mediated by motilin receptors located on enteric neurons, ultimately leading to activation of muscarinic cholinergic neurons in the gastric antrum. The actions of motilin can be mimicked by the macrolide antibiotic, erythromycin, and a group of motilin agonists known as motilides. The motilides may be useful clinically as prokinetic agents for treatment of patients with delayed gastric emptying and impaired colonic motility.

Structure Secretion Because NPY is a neural peptide, its detection in the systemic circulation most likely will represent measurement of spillover from neural sources (e.g., nerve endings) into the general circulation. For example, stimulation of the vagal nerve in the isolated perfused pig pancreas causes a dramatic release of NPY into the venous effluent, most likely from postganglionic parasympathetic nerves (315).

Actions In the GI tract, NPY affects blood flow, motility, and intestinal secretion (315,316). NPY is a potent inhibitor of intestinal and pancreatic blood flow (317–319). Direct intraarterial administration of NPY to the cat colon causes a dosedependent and persistent vasoconstriction of the splanchnic circulation (317). NPY is approximately 25 times more potent than norepinephrine (317). NPY can also slow jejunal and colonic motility (317). NPY may inhibit colonic motility by an inhibition of excitatory enteric ganglia, as well as by a direct action on colonic smooth muscle in cats (320). NPY, like PYY, has been shown to inhibit fluid and electrolyte secretion from the small intestine (321–323). NPY may also act in the endocrine pancreas to regulate insulin secretion in a paracrine manner (313,314,324).

MOTILIN Motilin is a 22-amino-acid peptide that was characterized from extracts of the pig small intestine based on its ability to stimulate stomach myoelectric activity and pepsin secretion. It is called motilin because it stimulates gut motility. Motilin is produced in enteroendocrine “M” or “Mo” cells of the mucosal epithelium in the upper small intestine. Plasma motilin levels increase during the interdigestive period in a cyclic fashion, simultaneously with phase III contractions of the migrating myoelectric complex (MMC) in the stomach and upper small intestine. Abundant findings indicate that this increase in motilin secretion triggers initiation of phase III contractions of the migrating motor complex.

Motilin is a linear 22-amino-acid peptide (Fig. 5-22) (325,326). The prepromotilin sequence consists of 114 amino acids and includes a 25-amino-acid signal peptide, followed immediately by the motilin sequence, a dibasic cleavage sequence, and a variable (~65–70-amino-acid) carboxyl-terminal extension peptide. Like other GI peptides, prepromotilin is processed to the mature form—that is, the 22-amino-acid sequence, before it is released into the general circulation. Both the amino and carboxyl terminals of motilin are essential for full activity because neither amino- or carboxyl-terminal fragments stimulate gastric motor activity (327). Interestingly, substitution of the first residue in motilin will decrease activity approximately 300-fold (328), whereas substitution of residues 12, 13, 14, or 15 has marginal effects on motilin activity (329,330). The motilin gene consists of five exons separated by four introns and spans approximately 9 kb (331,332). Motilin is produced by exons 2 and 3.

Motilin Receptor The motilin receptor was identified and cloned from human stomach specimens (333). The motilin receptor is a heterotrimeric guanosine triphosphate–binding protein (G protein)– coupled receptor. Its amino acid sequence is 54% identical to the human receptor for the synthetic growth hormone secretagogues and the recently discovered stomach hormone, ghrelin. As expected, erythromycin, the macrolide antibiotic, binds to the motilin receptor. The motilin receptor is also found in enteric neurons of the duodenum, jejunum, and colon.

Distribution Motilin is primarily a duodenal hormone and is localized in enteroendocrine “M” or “Mo” cells. Motilin is also produced at lower levels along the entire length of the GI tract (334–338). In the cat GI tract, motilin mRNA levels are greatest in the duodenum and lowest in the colon; motilin is not detected in the stomach (339).

138 / CHAPTER 5 Human Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Monkey Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Ser-Lys-Gly-Gln

Porcine Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Horse Phe-Val-Pro-Ile-Phe-Thr-Tyr-Ser-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Arg-Gly-Gln

Sheep Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Val-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Tyr-Lys-Gly-Gln

Bovine Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Val-Arg-Arg-Met-Gln-Glu-Lys-Glu-Arg-Tyr-Lys-Gly-Gln

Canine Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Lys-Ile-Arg-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Feline Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Arg-Ile-Arg-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Rabbit Phe-Val-Pro-Ile-Phe-Thr-Tyr-Ser-Glu-Leu-Gln-Arg-Met-Gln-Glu-Arg-Glu-Arg-Asn-Arg-Gly-Gln

Guinea Pig Phe-Val-Pro-Ile-Phe-Thr-Tyr-Ser-Glu-Leu-Arg-Arg-Thr-Gln-Glu-Arg-Glu-Gln-Asn-Arg-Leu-Arg

Chicken Phe-Val-Pro-Phe-Phe-Thr-Gln-Ser-Asp-Ile-Gln-Lys-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

FIG. 5-22. Motilin amino acid sequences for several species. Nonidentity in sequences is identified by underlining.

Actions on the Gastrointestinal Tract The primary physiologic role of motilin is the stimulation of phase III contractions that originate during the interdigestive period in the antroduodenal region of the gut (340). Motilin exerts marginal effects on postprandial intestinal activity (341). During the interdigestive period, there is a cyclic increase of GI motility that consists of three phases and occurs at intervals of approximately 80 to 100 minutes (Fig. 5-23). Phase I is a period of quiescence lasting about 60 to 70 minutes; it is followed by phase II, which is a period of increased but irregular motor activity lasting approximately 20 to 30 minutes. Phase II is followed by phase III contractions and is characterized by intense and regular contractile activity that lasts approximately 5 to 10 minutes. This contractile activity starts in the stomach/lower esophageal sphincter and moves down the small intestine. As the phase III front reaches the distal small intestine, a new phase III is initiated in the stomach. The function of the MMC is thought to serve a housekeeping role by clearing the stomach and intestine of luminal secretions and contents during the interdigestive period.

Abundant evidence supports the idea that motilin is responsible for the initiation of phase III contractions. The increase in plasma motilin levels coincides with the increase in MMC activity. Other studies show that immunoneutralization of systemic motilin interrupts phase III contractile activity in the proximal gut but has no effect on the motility pattern of the distal small intestine (342,343). These findings support a role for motilin in initiation of phase III contractions in the proximal but not the distal gut. Although there is some argument regarding the role of motilin in humans, findings show that the onset of phase III contractions and motilin secretion are also tightly coordinated (344). A direct relation between motilin secretion and phase III activity, however, is more variable in humans when compared with dog findings (345, 346). IV administration of motilin will induce premature onset of activity fronts (344). Motilin can also stimulate gastric pepsinogen and pancreatic enzyme secretion, gallbladder contraction, and contraction of the lower esophageal sphincter (347–353). These stimulatory effects of motilin resemble those seen during normal phase III activity, suggesting that the observed changes in pancreatic secretion and pepsin secretion during phase III of

Plasma motilin concentration, pg/ml

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 139

500

Feeding 400

300

200

0

Gut motor pattern

Gastric antrum

Upper jejunum Mid intestine

Time intervals, 1 hr

FIG. 5-23. Changes in plasma motilin levels and gastrointestinal motor activity before and after feeding in a dog fasting 17 hours. This dog was prepared with three force transducers that were implanted on the gastric antrum 3 cm proximal to the gastroduodenal junction, on the upper jejunum 20 cm distal to the ligament of Treitz, and on the midintestine just midway between the ligament of Treitz and the terminal ileum. During the interdigestive state, plasma motilin levels increased at constant intervals in close association with the occurrence of episodes of strong contractions in the stomach. However, when bands of strong contractions (arrows) reached the midintestine, plasma motilin levels had decreased. (Modified from Itoh and colleagues [379], by permission.)

the MMC are caused by endogenous motilin. Evidence in dogs and humans shows that motilin can increase the gastric emptying rate (354). Exogenous motilin can be used clinically to treat diabetic gastroparesis or postvagotomy gastroparesis (355,356). Motilin also stimulates interdigestive blood flow in dogs that is apparently mediated by cholinergic and noncholinergic pathways (357). Interestingly, motilin and motilides will stimulate contractions of isolated colonic smooth muscle cells and can enhance propulsive motility of the colon (358–361). Although several studies have shown that motilin and macrolides stimulate gastric and small intestinal motility, there are contradictory findings on the effects of motilin on the colon. In the human, rabbit, and canine colon, motilin shows stimulatory actions (361–368), whereas other laboratories fail to show an effect in the human colon (369,370). The contractile activity of motilin is only partially inhibited by the muscarinic antagonist atropine in the rabbit, suggesting that motilin excites the rabbit colon by mechanisms independent of cholinergic motor nerves, as in the stomach (367). Motilin, at physiologic doses, can also stimulate insulin secretion via a vagal cholinergic muscarinic pathway in the dog (371).

Mechanism of Action The stimulatory action of motilin on phase III contractions is blocked by muscarinic-cholinergic antagonists, indicating that cholinergic neurons mediate motilin-stimulated phase III contractions (372–374). In addition, depletion of 5-hydroxytryptamine in nerve terminals or pharmacologic blockade of the type 3 5-hydroxytryptamine receptors hinders the stimulatory action of motilin on phase III contractions in the dog stomach (375), implying that 5-hydroxytryptamine neurons mediate the phase III effects of motilin.

Release In humans, plasma motilin levels increase after a meal, whereas in dogs, plasma motilin levels increase exclusively during the interdigestive period (376). Plasma motilin levels increase and decrease in a regular, cyclic pattern during the interdigestive period, that is, fasting (377–380). Motilin release occurs at regular intervals of 90 to 120 minutes and coincides with the onset of the phase III contractions of the

140 / CHAPTER 5 interdigestive myoelectric complex in the stomach and duodenum in dogs and humans (see Fig. 5-23) (379–383). Luminal administration of acid or alkali into the duodenum will increase plasma motilin levels in dogs (380,384), and dietary fat is a potent secretagogue for motilin release in humans but not dogs (382,383,385–387). Ingestion of a mixed meal prevents the regular cyclic increase in motilin levels (132, 379,388). The mechanism or the identity of the exact stimulus behind the cessation in the cyclic release of motilin and of the disruption of the MMC is unknown. A cholinergic neural mechanism is suggested for regulation of motilin secretion because the muscarinic antagonist, atropine, and the nicotinic antagonist, hexamethonium, prevent the cyclic increases of plasma motilin, and electrical vagal stimulation causes motilin secretion (372,389,390). Furthermore, carbachol-induced motilin secretion is blocked by atropine but not by hexamethonium, implying that muscarinic receptors mediate motilin secretion. Three to six weeks after truncal vagotomy, plasma motilin levels and alterations seen during the MMC resembled those before surgery, suggesting that enteric cholinergic neurons are not involved in motilin secretion in dogs (391,392). IV administration of secretin and PP inhibit motilin secretion, whereas bombesin stimulates motilin release (386,393,394). Interestingly, exogenous motilin can stimulate secretion of endogenous motilin via muscarinic receptors on motilin cells via preganglionic pathways involving 5-hydroxytryptamine 3 receptors in the dog (395).

Pathophysiology Plasma motilin levels have been measured in patients with a variety of GI conditions. In pregnant subjects or patients with constipation and idiopathic gastroparesis, decreased plasma motilin levels were measured (396–400). In surgical patients with normal gut motility and patients with irritable bowel syndrome or infantile colic, plasma motilin levels are increased (401–405). In addition, plasma motilin levels are increased in some diabetic subjects (406) and patients with ZES (407).

Erythromycin and Motilides The term motilide is derived from the words “motilin” and “macrolide,” indicating that the macrolide antibiotics, including erythromycin, are motilin agonists and bind to the motilin receptor. The macrolide antibiotic erythromycin given to either dogs or humans will cause contractions of the GI tract that resemble phase III contractions (408–410). Motilides exert all of the effects of motilin, causing smooth muscle contraction and activation of cholinergic muscarinic pathways. Erythromycin has been shown to bind to the cloned motilin receptor (333). Because motilides act at subantibiotic doses and are resistant to degradation, they are potentially useful clinically as prokinetic agents for the stomach and colon, especially for patients with diabetic gastroparesis.

PEPTIDE YY PYY is a 36-amino-acid peptide hormone that is structurally similar to two other GI peptides, PP and NPY. This family of peptides is called the PP-PYY-NPY family of peptides, or the PP-fold family of peptides. All 3 peptides are 36 amino acids in length with an amidated carboxy terminus. PYY is produced primarily in enteroendocrine “L” cells in the intestinal epithelium of the ileum-colon and is a candidate enterogastrone because PYY is secreted into the systemic circulation in response to dietary fat and is a potent inhibitor of gastric acid secretion. PYY can also inhibit pancreatic exocrine secretion, gastric emptying, and intestinal transit. Because of its potent antisecretory action, PYY may be an endogenous inhibitor of diarrhea. Based on this array of inhibitory actions in the GI tract, PYY is called an “ileal brake.” The primary role of an ileal brake substance is to enhance intestinal digestion and absorption of ingested nutrients. More recently, a natural variant of PYY, PYY (3-36), has been shown to be a potent inhibitor of food intake in experimental animals and humans.

Structure PYY is a 36-amino-acid peptide that is structurally related to 2 other pancreatic-gut peptides, PP and NPY (see Fig. 5-20) (286,411,412). Together, they compose the PP-fold family of peptides, and with the fish peptide Y (PY), they share a common hairpin-like three-dimensional structure called the PP-fold (412). It is called PYY because it has tyrosine residues at the amino and carboxy terminals. “Y” is the chemists’ shorthand for the tyrosine residue. Another endogenous PYY variant, PYY (3-36), occurs in the intestine and general circulation (413–415). The PYY gene has been cloned from several species including the rat and human (291–294,416–418). The conserved structural organization of the PYY, NPY, and PP genes suggests that each gene is derived from duplication of a common ancestral gene. Prepro-PYY consists of a signal peptide followed by the 36-residue active peptide, a cleavage sequence, Gly-Lys-Arg, and a flanking peptide at the carboxy terminal (290,419). During processing to the mature form, the precursor is cleaved at the carboxy terminal by a PC, and the Lys-Arg sequence is excised by a carboxypeptidase. The carboxy-terminal tyrosine is amidated and is essential for biological activity (420).

Gastrointestinal Localization of Peptide YY and Developmental Appearance of Intestinal Peptide YY PYY is found in “L” cells in the mucosal epithelium of the terminal ileum, colon, and rectum (Figs. 5-24 and 5-25) (421–430). PYY is not found in the muscle layer. Marginal amounts of PYY are found in the stomach antrum, proximal small intestine, and endocrine cells of the pancreas. In the lower intestine, PYY is coproduced with proglucagon (427);

800

Colon

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 141 Rat

†

Distal ileum

Mid-ileum

Mucosa Muscle

*†

Colon

*

Dog

1000

500

*†

Ileum-3

Ileum-2

1500

Jejunum-3

PYY (ng/g tissue)

2000

Fundus

Antrum

200

Jejunum

Duodenum

400

Ileum-1

PYY (ng/g tissue)

600

* †‡

*†

*

FIG. 5-24. Distribution of peptide YY (PYY) immunoreactivity in the digestive tract of the rat (full thickness) and dog (mucosal and muscle layers). In the dog, the duodenum, jejunum, and ileum were divided into three segments of equal length. The colon was divided into two segments. (Top) *p < 0.05 versus antrum, fundus, duodenum, jejunum, and midileum. †p < 0.05 versus distal ileum. (Bottom) *p < 0.05 versus antrum, fundus, duodenum, proximal, and distal jejunum; †p < 0.05 versus muscle layer of respective region; ‡p < 0.05 versus distal jejunum, proximal, and midileal mucosal layers. (Modified from Greeley and colleagues [423], by permission.)

in the pancreas, PYY can coreside with glucagon or PP (421,431,432). PYY is also detected in neuronal elements of the proximal GI digestive tract of the rat, cat, ferret, and pig (430). PYY is found in nerve cell bodies and nerve fibers of the canine stomach and intestinal myenteric plexus, and in the intestinal submucosal plexus (429). PYY is the first major gut peptide to appear during development in the mouse colon (433). On embryonic day 15.5, colonic PYY expression appears, followed by intestinal expression of glucagon, CCK, substance P, 5-HT, secretin, neurotensin, gastrin, and SRIF between embryonic days 16.5 and 18.5. In the rat, expression of PYY occurs on embryonic day 17 in the ileum and colon (417,434). Both PYY mRNA and peptide are detected initially at 17 days of gestation; their levels continue to increase during the postnatal period. The increase in intestinal PYY expression during the first weeks of life may be activated by ingestion of fat contained in the mothers’ milk. Dietary fat is a potent secretagogue for release of PYY (435), and fat is abundant

in rat milk, ranging from 22% in colostrum to 9% in late milk (436). After weaning, there is a coordinated decrease in levels of PYY mRNA and PYY peptide when pups switch from breast milk to an adult diet. Interestingly, the pattern of rat intestinal PYY gene expression agrees with the pattern of plasma PYY levels in human neonates (437). In newborn cord blood, plasma PYY levels are greater when compared with plasma PYY levels in healthy fasting adults, and plasma PYY levels increase during the first 12 days after birth in neonates fed with either breast milk or formula. These observations suggest that intestinal PYY gene expression is controlled by dietary nutrients, and that changes in the intestinal PYY gene expression have a role in the adaptation of the bowel to the changes in dietary intake. PYY has been detected in the fetal mouse pancreas at embryonic day 9.5, and it is detected in each of the four islet cell types (433). In the rat pancreas, PYY expression is first detected on day 15 of gestation and increases to its maximal on day 18 (417). At this time, pancreatic PYY mRNA levels

142 / CHAPTER 5

FIG. 5-25. Flask-shaped peptide YY cell of the open type in the rat colonic mucosa (original magnification × 360). (See Color Plate 1.) (Modified from Jeng and colleagues [495], by permission.)

are sevenfold greater than in the fetal and adult colon and 90-fold greater than those levels in the adult pancreas, in contrast with the intestine. Pancreatic PYY mRNA levels decrease dramatically after birth. Pancreatic PYY peptide levels have not been characterized during development. Although the functional significance of PYY in the pancreas during development is unknown, PYY may participate in the regulation of pancreatic and GI functions by endocrine and paracrine actions. PYY is measurable in the adult canine pancreas with the greatest levels in the right lobe and head of the pancreas, and PYY is released from the canine pancreas by vagal stimulation (438). The early development pattern of intestinal PYY gene expression suggests a role for PYY in the development of the intestine and in the regulation of epithelial cell replication or differentiation. The initiation of PYY gene expression comes before the appearance of mature morphologic characteristics in the colonic mucosa (i.e., formation of the crypts of Lieberkuhn). In addition, cells expressing PYY mRNA in the developing and mature colon are located at the base of the crypts adjacent to populations of immature cells and goblet cells undergoing differentiation (434). PYY may have a role in the regulation of the stomach and pancreas during the developmental period. Although gastric acid and pancreatic secretion are low during the postnatal period and do not achieve adult levels until weaning (439), PYY may have a role in their regulation because it is a potent inhibitor of gastric acid and pancreatic exocrine secretion in adult animals.

Peptide YY Receptors PYY, NPY, and PP bind to a family of G protein–linked receptors, called Y receptors, belonging to the rhodopsinlike superfamily (class 1) of receptors (412,440). There are five different Y receptor subtypes cloned from mammals (Y1, Y2, Y4, Y5, and y6; Table 5-1). The y6 receptor is given the lowercase designation because it encodes for a truncated receptor. All Y receptor subtypes are expressed in either the small or large intestine with specific distributions (440). Y1, Y2, and Y5 bind NPY and PYY preferentially, whereas Y4 binds PP preferentially. The pharmacologic profile of y6 is not agreed on (412,441). The characterization of the responses to PYY, NPY, and PP may reflect the result of the peptide’s activity on local neuronal receptors, as well as direct activity at the cell level on the targeted tissue. Y1 receptors are expressed only in nonepithelial colonic cells, whereas Y2 and Y4 receptors are found in epithelial and nonepithelial cells of either the small or large intestine. Expression of the Y5 receptor is restricted to epithelial crypts of the small intestine and nonepithelial cells of the colon. The Y1, Y2, Y4, Y5, and y6 receptors are coupled to inhibitory G proteins (Gi) and cause inhibition of cAMP synthesis (442–446). Activation of the Y1 receptor is coupled to MAPK in gut epithelial cells (IEC-6 [447]). Y1, Y2, Y4, and Y5 receptors also couple to PLC to cause release of Ca2+ from intracellular stores (442,443,445). The Y1 receptor is found in the gut and mediates an inhibition of fluid secretion in the intestine.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 143 TABLE 5-1. Cloned neuropeptide Y receptors with ligand-binding profiles, example locations, and example functions Receptor type Y1 Y2

Ligand-binding profile NPY ≈ PYY ≈ [Leu , Pro ] NPY > NPY2-36 > NPY3-36 ≥ PP > NPY13-36 NPY ≥ NPY2-36 ≈ NPY3-36 ≈ NPY13-36 >> [Leu31, Pro34] NPY 31

34

Y4

PP > PYY ≥ NPY > NPY2-36

Y5

NPY ≈ PYY ≈ NPY2-36 > hPP > [D-Trp32] NPY > NPY13-36 > rPP 1. NPY ≈ PYY ≈ [Leu31, Pro34] NPY >> PP 2. PP > [Leu31, Pro34] NPY > NPY ≈ PYY

y6

Example of locations

Example of functions

Gut, brain, heart, kidney

Vasoconstriction, anxiolysis; antisecretory in intestine

Brain, low levels in peripheral tissues

Presynaptic inhibition of neurotransmitter release, regulation of GI motility Inhibition of pancreatic secretion, GI motility Increased food intake

Small intestine, colon, prostate, and CNS Brain areas involved in food intake Small intestine and adrenal (mouse)

Unknown

CNS, central nervous system; GI, gastrointestinal tract; NPY, neuropeptide Y; PP, pancreatic polypeptide; PYY, peptide YY. Modified from Adrian TE. Peptide YY. In: Encyclopedia of hormones, Vol. 3. San Diego: Academic Press, 2003;161–170.

The Y4 receptor is found in the pancreas and mediates an inhibition of pancreatic exocrine secretion. On rabbit gastric circular smooth muscle cells (441), Y1, Y2, and Y4 receptors were shown to be negatively coupled to adenylyl cyclase by activation of Gi2, and Y2 and Y4 receptors were shown to be coupled to IP3-dependent (Ca2+)i release and activation of PLC-β by activation of Gq.

Actions on the Gastrointestinal Tract Studies in rats, dogs, and humans demonstrate that PYY is a general inhibitor of GI function. Based on its inhibitory actions on gastric acid secretion, gastric emptying, pancreatic secretion, and intestinal transit, PYY has been called an “ileal brake.” Infusion of PYY IV has been shown to inhibit gastric acid secretion in the dog, rat, rabbit, cat, and in humans (Fig. 5-26) (448–451). PYY can inhibit pentagastrin-stimulated, but not histamine and cholinergic-stimulated (i.e., bethanechol), gastric acid secretion in the dog (452). There is also evidence that PYY can decrease gastric acid secretion by blocking the cephalic phase of gastric acid secretion in the dog (453). In humans, PYY is a potent inhibitor of the cephalic phase of gastric acid secretion, indicating that part of the enterogastrone action of PYY is mediated via neural pathways (454). Furthermore, the inhibitory actions of PYY on acid secretion in humans are independent of SRIF and gastrin secretion (454). In the cat, PYY inhibits pentagastrin-stimulated acid secretion by stimulation of SRIF secretion. In the dog, PYY (1-36) is more potent than PYY (336) and PYY (4-36), indicating that the complete sequence of PYY is essential for full expression of its inhibitory action on acid secretion (420). A study using isolated rat gastric ECL cells showed that PYY may inhibit gastric acid secretion by blocking gastrin-induced histamine release and Ca2+ entry via activation of the Y1 receptor (455). In addition, there is evidence that PYY acts centrally in the brain to regulate gastric acid secretion (456). PYY, given peripherally, will inhibit thyrotropin-releasing hormone–activated vagal efferent

cholinergic projections to the stomach and acid secretion. Furthermore, PYY injected into the cisterna magna and dorsal vagal complex (DVC) results in an inhibition of gastric transit and antral motility (457,458). Interestingly, administration of PYY into the medullary brainstem area of the vagus (dorsal motor nucleus [DMN]) causes a dose- and vagal-dependent stimulation of gastric acid secretion in rats (459). The DMN is referred to as the DVC and includes the area postrema, the nucleus of the solitary tract, as well as the DMN. The medullary region contains vagovagal circuits that communicate between the periphery—that is, the GI tract and central nervous system (CNS). PYY receptors have been identified autoradiographically in the DVC of rats, and systemic radiolabeled PYY can penetrate into the DVC (460). These neural PYY receptors may be activated by PYY released from the GI tract or the brain because PYY is expressed in the brainstem (425,426) and GI tract (423). This centrally PYY-activated inhibition of gastric acid secretion may be part of the ileal brake. IV administration of PYY also inhibits pancreatic exocrine secretion. PYY can decrease the stimulatory effects of CCK, secretin, and neurotensin on the pancreas (Fig. 5-27) (461). PYY can also inhibit the stimulatory effects of endogenous secretagogues that are released in response to ingestion or luminal instillation of nutrients (461–463). PYY inhibits pancreatic secretion of juice, bicarbonate, and pancreatic enzymes. In addition, physiologic levels of PYY can inhibit meal-induced pancreatic secretion, and treatment with antiPYY serum enhances pancreatic secretion in the rat (464). PYY (1-36), as well as PYY (3-36) and PYY (4-36), exerts inhibitory actions with similar potencies on pancreatic secretion in the dog (420). With guinea pig acini, PYY cannot inhibit CCK bombesin-stimulated amylase secretion; however, PYY can inhibit VIP- and forskolin-induced amylase release (465). PYY may exert its inhibitory actions on pancreatic secretion by multiple pathways. PYY may act via adrenergic pathways to inhibit pancreatic secretion because phentolamine plus proprandolol administration will prevent the inhibitory effects of PYY on pancreatic secretion (466). The delay in

144 / CHAPTER 5 Pentagastrin (0.5 µg/kg-h) 6 4 2 0 PYY (100 pmol/kg-h)

Gastric acid output (mEq/15 min)

6 *

4

*

2 0 PYY (200 pmol/kg-h) 6 4

*

*†

* *

2 0 PYY (400 pmol/kg-h) 6 *

4

*†

2

* *†

*†

0 B1 B2

15 30 45 60 75 90 105 120 135 150 165 180 Minutes

FIG. 5-26. Inhibition of pentagastrin-stimulated gastric acid secretion by peptide YY (PYY) given intravenously in dogs prepared with gastric fistulas. *p < 0.05 versus pentagastrin alone; †p < 0.05 versus 100 pmol/kg/hr dose of PYY. (From Jeng and colleagues [495], by permission.)

intestinal transit of luminal nutrients by PYY is dependent on a β-adrenergic pathway that acts via serotonergic and opioid pathways (467). Evidence also supports the idea that PYY acts via intrapancreatic neural pathways (468) and centrally to inhibit neurally mediated pancreatic secretion (469). In the rat, PYY has been shown to exert its inhibitory action on the exocrine pancreas through the DVC of the CNS (469). The area postrema is the major site of PYYmediated inhibition of CCK and secretin-stimulated pancreatic secretion (470). PYY may also regulate pancreatic exocrine secretion by its inhibition of CCK secretion because IV administration of PYY blocks release of CCK in response to intraduodenal infusion of a fatty acid in dogs (463). Administration of PYY will also block CCK release in humans (471). PYY inhibits gastric emptying and intestinal transit. Unabsorbed nutrients, especially fats in the lumen of the distal small intestine (ileum), will inhibit upper gut motility (472–476). Ileal perfusion of carbohydrates alone or with an amylase inhibitor delays gastric emptying (476). This negative-feedback mechanism of the lower intestine represents a major and fundamental control mechanism of upper

gut motility and is part of the ileal brake. Because PYY is localized in the lower intestine and because it can inhibit gastric emptying and intestinal transit (319,477,478), PYY has been proposed as an ileal brake factor because it has all of the properties of the ileal brake. There is additional evidence to indicate that PYY is the ileal brake (479–481). In dogs, immunoneutralization of PYY increased intestinal transit of a test meal (479), and ileal perfusion of fat in dogs will increase the migrating motor (myoelectric) complex (MMC) cycle length, decrease the number of MMCs, and increase circulating levels of PYY (480). The fasting motor patterns of the upper gut, also called the interdigestive MMC, are characteristic patterns of intestinal electrical activity and are disrupted by ingested nutrients (480). The interdigestive MMC, cycle length, and blood levels of PYY are also parallel (481). Because of its potent antisecretory action, PYY may be an endogenous inhibitor of diarrhea (482). PYY or synthetic PYY analogues, given directly into the intestinal lumen are proabsorptive (483). PYY has been shown to enhance intestinal absorption of fluid and electrolytes in the jejunum and ileum (484,485). In agreement with the antisecretory action

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 145 IV Secretin (100 ng/kg-h) PYY 12.5 25

50

Secretin

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FIG. 5-27. (Top) Pancreatic bicarbonate output in response to a submaximal dose of secretin (100 ng/kg/hr, intravenously [IV]) alone (control) or in combination with graded doses of peptide YY (PYY; 12.5, 25, 50, 100, 200, 400 pmol/kg/hr) given each for 30 minutes in conscious dogs. PYY at 200 and 400 pmol/kg/hr inhibited secretin-stimulated pancreatic bicarbonate output significantly. (Bottom) Pancreatic protein output in response to a submaximal dose of cholecystokinin (CCK)-8 (50 ng/kg/hr, IV) alone or in combination with PYY (400 pmol/kg/hr, IV). PYY inhibited CCK-stimulated pancreatic protein secretion significantly. *p < 0.05 versus control; n = 6 dogs. (From Jeng and colleagues [495], by permission.)

of PYY, PYY receptors are found in the intestinal crypt cells where chloride secretion occurs (486). Occupation of the PYY receptor by PYY inhibits small bowel chloride ion secretion by decreasing basal and VIP or prostaglandinstimulated levels of cyclic AMP (321,442,444). PYY (3-36) has been shown to reduce appetite and food intake, both in normal and obese subjects (487). Interestingly, PYY was shown to reduce plasma ghrelin levels (ghrelin stimulates food intake), and endogenous PYY levels were reduced in obese subjects (488), suggesting that the PYY deficiency has a role in the cause of obesity. PYY may cause satiety by inhibition of gut motility and give a sense of fullness that is mediated by vagal afferents that originate in the GI tract and project to the hindbrain, terminating in the hypothalamus. In animal studies, PYY has been shown to inhibit hypothalamic NPY neurons and agouti-related protein neurons through inhibitory NPY-Y2 receptors that disinhibit adjacent proopiomelacortin-expressing neurons and decrease food intake. Systemic PYY can cross the blood–brain barrier, indicating that intestinal-derived PYY can influence central ingestive regulatory pathways (489).

Regulation of Peptide YY Secretion In humans, dietary fat is a potent stimulus for release of PYY from the intestine (490). In humans, PYY is released from the intestine in proportion to the calories ingested (490). In dogs, the ability of different nutrients given either into the duodenum or colon to stimulate PYY secretion was tested (435). Fatty acids (oleic acid) and glucose (2 g/kg) were shown to be the strongest PYY secretagogues when administered into the duodenum, whereas fatty acids and glucose are moderate stimulants of PYY release when given into the colon lumen. Amino acids and intact protein do not cause PYY release when given intraduodenally. However, a mixture of tryptophan and phenylalanine or intact protein administered into the colon of dogs will cause PYY secretion (435,438). Short-chain fatty acids (acetate, propionate, butyrate) given into the isolated perfused rabbit colon stimulate PYY release (491). Short-chain fatty acids are normal constituents of feces (492). In dogs, bile salts stimulate a dose-related release of PYY (493). Interestingly, plasma levels of PYY increase significantly within 15 to 30 minutes after ingestion of nutrients or in

146 / CHAPTER 5

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Neural pathways may have a role in the regulation of PYY secretion. In dogs, either atropine, hexamethonium, or atropine plus hexamethonium administration decreased food-induced secretion of PYY (499). Furthermore, β-adrenergic blockade with proprandolol or depletion of nerve terminal stores of catecholamines with reserpine did not influence nutrientinduced PYY secretion. IV administration of terbutaline, a β2-adrenergic agonist, causes PYY secretion (500). Truncal vagotomy increased resting-fasted and food-stimulated release of PYY. IV administration of bethanechol to dogs, a cholinergic agonist, also stimulated release of PYY and electrical stimulation of the vagus-stimulated PYY release. 350 Food Plasma PYY (pg/ml)

‡

14

PYY (pg/ml)

response to luminal administration of a fatty acid in humans, dogs (Fig. 5-28), and rats (464,490,494). PYY secretion in response to intraduodenal administration of fat causes PYY release even when the flow of chyme is prevented from reaching the distal intestine, and removal of the ileum-colon abolishes the PYY response to fat. This rapid release of PYY resulted in the speculation that another gut hormone that is released from the upper gut in response to a luminal nutrient triggered PYY release. It was also shown that IV administration of CCK caused a dose-dependent release of PYY (Fig. 5-29), and further experiments showed that a CCK-Atype receptor antagonist prevented the PYY response to a fatty meal or to exogenous CCK, supporting the role of intestinal CCK in causing PYY secretion (494,495). The influence of growth factors on PYY homeostasis has been studied (496,497). Both growth hormone and insulinlike growth factor-1 (IGF-1) stimulate intestinal PYY expression and peptide levels in rodents. Growth hormone and IGF-1 also stimulate PPY secretion. In terms of a molecular mechanism, the increase in intestinal PYY expression by IGF-1 involves Sp1 binding sites in the proximal promoter region of PYY (498).

300 250 200 150

30

60 90 Minutes

120

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FIG. 5-29. (Top) Integrated release of peptide YY (PYY) in response to graded doses of cholecystokinin (CCK)-8 given intravenously in six dogs. *p < 0.05 versus lower doses of CCK-8; †p < 0.05 versus 50 and 100 pmol/kg/hr CCK-8; ‡p < 0.05 versus lower CCK-8 doses; N = 5–7 dogs/dose). (Bottom) Plasma PYY levels in response to intravenous administration of CCK-8 (600 pmol/kg/hr) in conscious dogs. (From Jeng and colleagues [495], by permission.)

Together, these findings in the dog suggest that stimulated PYY levels are dependent on ganglionic transmission and an atropine-blockable postganglionic parasympathetic pathway. A vagal cholinergic mechanism may act in a tonic fashion to inhibit PYY secretion. Adrenergic mechanisms do not participate in nutrient-stimulated PYY release; however, electrical stimulation of the splanchnic nerves will increase PYY secretion, implying that the sympathetic nervous system has a role in PYY secretion. Cutting of intestinal intramural neural pathways does not interfere with food-induced release of PYY, suggesting that intramural neural mechanisms also do not participate in food-induced release of PYY (494). In the isolated, perfused rat ileum, bethanechol stimulated PYY release and the β-adrenergic agonist isoproterenol stimulated transient release of PYY (501,502).

0 B1

B2 15

30 45 60 Minutes

90

120

150

FIG. 5-28. Plasma peptide YY (PYY) levels in response to an oral mixed meal in conscious dogs. Notice that circulating PYY levels are increased 15 minutes after feeding the meal. B1 and B2 refer to basal plasma PYY levels. (From Jeng and colleagues [495], by permission.)

Trophic Actions of Peptide YY on the Gastrointestinal Tract The early developmental expression of PYY in the intestine suggests a regulatory role in the development of the alimentary

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 147 canal, as well as in mucosal cell replication or differentiation. PYY has been shown to stimulate intestinal growth in mice and rats (434). In adult female mice, administration of PYY increases intestinal growth in a dose-related manner (434). The stimulatory effects of PYY are greater on intestinal protein contents than on intestinal DNA contents. High doses of PYY (200 nmol/kg) increase DNA contents by 14% in the proximal small intestine, 18% in the distal small intestine, 20% in the proximal colon, and 28% in the distal colon. The same PYY dose increases protein contents by 47% in the proximal small intestine, 77% in the distal small intestine, 66% in the proximal colon, and 52% in the distal colon. Smaller doses (30 and 100 nmol/kg) of PYY did not affect bowel DNA content; however, they significantly increased the protein contents of the small intestine and colon to values that were significantly greater than those of control animals. High doses of PYY also increased the RNA contents of the various intestinal segments by 15% to 39%. In nursing-unweaned rats, the trophic effects of PYY are seen only in the proximal small intestine (434). PYY treatment increased DNA, RNA, and protein levels. In nursing rats, PYY increased the weight of the proximal small intestine by 12% to 15%, the total DNA contents by 43% to 50%, and the total protein contents by 22% to 20%. There were no stimulatory effects of PYY on the distal small intestine or colon in nursing rats. The absence of a trophic effect of PYY on the distal small intestine and colon in rats may be attributed to the low density of PYY receptors in the distal intestine (444). In rats, there are abundant high-affinity PYY/NPY binding sites in the duodenum and jejunum, a lower level in the ileum, and a complete absence in the colon (444). However, the intestinal pattern of PYY receptors cannot entirely explain the stimulatory actions of PYY. For example, PYY did not exert a stimulatory effect on the distal small intestine in rats, which has PYY binding sites (444), and PYY increased growth of the colon in mice, a place where PYY receptors are not expected. PYY may also act indirectly to stimulate intestinal growth through receptors located in stroma cells of the lamina propria or submucosa. In situ hybridization studies show the presence of PYY mRNA at several levels of the intestinal wall, including the lamina propria, submucosa, and muscle layer of the colon. PYY may act on neighboring cell targets that, in turn, influence gut cell proliferation. Other evidence supports an indirect mechanism of action for in vivo stimulation of intestinal cell growth by PYY. PYY cannot stimulate growth of a rat intestinal epithelial cell line (IEC-6, IEC-18 cells) (G. Gomez and G. H. Greeley Jr., unpublished data). PYY treatment, over a broad dose range and under different tissue culture conditions (e.g., serum-free or with serum), fails to increase cell numbers that are normally responsive to mitogens such as epidermal growth factor, platelet-derived growth factor, and insulin (503). Other studies have examined the trophic actions of PYY on the GI tract and reported that PYY administration failed to stimulate growth of the duodenum in rats (503). In this study, PYY was administered for 4 days only, and at a daily

dose that was approximately 15 to 60 times smaller than the dose range tested in rats or mice described earlier (434). Another earlier study also failed to show a trophic effect of a long-term PYY infusion on the intestine (504). This latter study showed that intestinal resection resulted in a dramatic increase in circulating PYY levels, as well as in the expected mucosal adaptive response. However, because exogenous PYY failed to stimulate bowel growth, the report concluded that PYY does not have a role in the trophic response after intestinal resection in the rat. Differences in experimental design may explain discrepancies among these various studies. Further evidence suggesting that PYY may be involved in the trophic adaptation of the intestine is provided by the finding that PYY content increases in the ileum that undergoes hyperplastic adaptation after subtotal colectomy in rats (G. Gomez and G. H. Greeley Jr., unpublished data). The study described earlier (434) did not address whether PYY exerts a trophic action on the intestine at physiologic doses. Evidence that the Y1 receptor is involved in intestinal cell growth is shown by a study where activation of the Y1 receptor in IEC-6 cells stably expressing this receptor caused cell growth and increased MAPK phosphorylation (505).

Inhibitory Action of PYY (3-36) on Appetite and Food Intake PYY (3-36) infusions in subjects of normal weight (Fig. 5-30) and in rodents will reduce appetite and food intake, and repeated administration of PYY will reduce weight gain in rodents (506). In mice, the inhibitory action of PYY (3-36) on food intake is not influenced by circulating ghrelin levels (507).

Pathophysiology of Peptide YY Plasma levels of PYY are altered in various clinical conditions (508–511). Plasma PYY levels are increased in patients with diarrhea associated with malabsorption of fat (510). In patients with gastric resection–induced dumping syndrome, due to rapid gastric emptying, plasma PYY levels are also increased (509). Systemic basal and postprandial levels of PYY are increased in patients with celiac and tropical sprue and in patients with massive small-bowel resection. The increase in PYY homeostasis is an expected adaptation because there is an increased load of unabsorbed nutrients in the distal intestine that stimulate PYY secretion via a luminal contact of PYY cells (485). In morbidly obese patients, both basal plasma PYY and postprandial PYY levels are reduced despite increased food intake (512,513). These observations are consistent with the idea that a decrease in PYY secretion is involved in the pathogenesis of obesity. Interestingly, in obese subjects, IV administration of PYY will reduce appetite and food intake with magnitudes similar to those seen in healthy subjects,

148 / CHAPTER 5 Obese subjects

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FIG. 5-30. Caloric intake by obese and lean subjects after infusion of peptide YY (PYY) (3-36) or saline. (A) Caloric intake by individual obese subjects. (B) Caloric intake by individual lean subjects during a buffet lunch 2 hours after the infusion of PYY or saline. (C) Mean ± standard error (SE) caloric intake by obese subjects. (D) Caloric intake by lean subjects during a buffet lunch 2 hours after infusion of saline or PYY. (E) Mean ± SE cumulative 24-hour caloric intake by obese subjects. (F) Caloric intake by lean subjects after infusion of saline or PYY. In all panels, the lean and obese groups each consisted of 12 subjects: 6 women and 6 men. (From Batterham and colleagues [513], by permission.)

indicating that obesity is not associated with an impaired response to PYY (see Fig. 5-30) (513).

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478. Savage AP, Adrian TE, Carolan G, Chatterjee VK, Bloom SR. Effects of peptide YY (PYY) on mouth to caecum intestinal transit time and on the rate of gastric emptying in healthy volunteers. Gut 1987;28:166–170. 479. Lin HC, Zhao XT, Wang L, Wong H. Fat-induced ileal brake in the dog depends on peptide YY. Gastroenterology 1996;110:1491–1495. 480. Dreznik Z, Meininger TA, Barteau JA, Brocksmith D, Soper NJ. Effect of ileal oleate on interdigestive intestinal motility of the dog. Dig Dis Sci 1994;39:1511–1518. 481. Wen J, Phillips SF, Sarr MG, Kost LJ, Holst JJ. PYY and GLP-1 contribute to feedback inhibition from the canine ileum and colon. Am J Physiol 1995;269:G945–G952. 482. Playford RJ, Domin J, Beacham J, Parmar KB, Tatemoto K, Bloom SR, Calam J. Preliminary report: role of peptide YY in defence against diarrhoea. Lancet 1990;335:1555–1557. 483. Liu CD, Hines OJ, Whang EE, Balasubramaniam A, Newton TR, Zinner MJ, Ashley SW, McFadden DW. A novel synthetic analog of peptide YY, BIM-43004, given intraluminally, is proabsorptive. J Surg Res 1995;59:80–84. 484. Bilchik AJ, Hines OJ, Zinner MJ, Adrian TE, Berger JJ, Ashley SW, McFadden DW. Peptide YY augments postprandial small intestinal absorption in the conscious dog. Am J Surg 1994;167:570–574. 485. Bilchik AJ, Hines OJ, Adrian TE, McFadden DW, Berger JJ, Zinner MJ, Ashley SW. Peptide YY is a physiological regulator of water and electrolyte absorption in the canine small bowel in vivo. Gastroenterology 1993;105:1441–1448. 486. Voisin T, Rouyer-Fessard C, Laburthe M. Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villus-crypt axis. Am J Physiol 1990;258:G753–G759. 487. Batterham RL, Bloom SR. The gut hormone peptide YY regulates appetite. Ann N Y Acad Sci 2003;994:162–168. 488. Korner J, Leibel RL. To eat or not to eat-how the gut talks to the brain. N Engl J Med 2003;349:926–928. 489. Nonaka N, Shioda S, Niehoff ML, Banks WA. Characterization of blood-brain barrier permeability to PYY3-36 in the mouse. J Pharmacol Exp Ther 2003;306:948–953. 490. Adrian TE, Ferri GL, Bacarese-Hamilton AJ, Fuessl HS, Polak JM, Bloom SR. Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 1985;89:1070–1077. 491. Longo WE, Ballantyne GH, Savoca PE, Adrian TE, Bilchik AJ, Modlin IM. Short-chain fatty acid release of peptide YY in the isolated rabbit distal colon. Scand J Gastroenterol 1991;26:442–448. 492. Rubinstein R, Howard AV, Wrong OM. In vivo dialysis of faeces as a method of stool analysis. IV. The organic anion component. Clin Sci 1969;37:549–564. 493. Izukura M, Hashimoto T, Gomez G, Uchida T, Greeley GH Jr, Thompson JC. Intracolonic infusion of bile salt stimulates release of peptide YY and inhibits cholecystokinin-stimulated pancreatic exocrine secretion in conscious dogs. Pancreas 1991;6:427–432. 494. Greeley GH Jr, Jeng YJ, Gomez G, Hashimoto T, Hill FL, Kern K, Kurosky T, Chuo HF, Thompson JC. Evidence for regulation of peptide-YY release by the proximal gut. Endocrinology 1989;124: 1438–1443. 495. Jeng Y-J, Hill FLC, Lluis F, Gomez G, Izukura M, Kern K, Chuo S, Ferrar S, Greeley GH Jr. Peptide YY release and actions. In: Thompson JC, Cooper CW, Greeley GH Jr, Rayford PL, Singh P, Townsend CM Jr, eds. Gastrointestinal endocrinology: receptors and post-receptor mechanisms. San Diego: Academic Press, 1990;371–386. 496. Gomez G, Udupi V, Qi X, Lluis F, Rajaraman S, Thompson JC, Greeley GH Jr. Growth hormone upregulates gastrin and peptide YY gene expression. Am J Physiol 1996;271:E582–E586. 497. Lee HM, Udupi V, Englander EW, Rajaraman S, Coffey RJ Jr, Greeley GH Jr. Stimulatory actions of insulin-like growth factor-I and transforming growth factor-alpha on intestinal neurotensin and peptide YY. Endocrinology 1999;140:4065–4069. 498. Wang G, Leiter AB, Englander EW, Greeley GH Jr. Insulin-like growth factor I increases rat peptide YY promoter activity through Sp1 binding sites. Endocrinology 2004;145:659–666. 499. Zhang T, Uchida T, Gomez G, Lluis F, Thompson JC, Greeley GH Jr. Neural regulation of peptide YY secretion. Regul Pept 1993;48:321–328. 500. Kogire M, Izukura M, Gomez G, Uchida T, Greeley GH Jr, Thompson JC. Terbutaline, a beta 2-adrenoreceptor agonist, inhibits gastric acid secretion and stimulates release of peptide YY and gastric inhibitory polypeptide in dogs. Dig Dis Sci 1990;35:453–457. 501. Plaisancie P, Bernard C, Chayvialle JA, Cuber JC. Release of peptide YY by neurotransmitters and gut hormones in the isolated, vascularly perfused rat colon. Scand J Gastroenterol 1995;30:568–574.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 159 502. Dumoulin V, Dakka T, Plaisancie P, Chayvialle JA, Cuber JC. Regulation of glucagon-like peptide-1-(7-36) amide, peptide YY, and neurotensin secretion by neurotransmitters and gut hormones in the isolated vascularly perfused rat ileum. Endocrinology 1995;136: 5182–5188. 503. Kurokowa M, Lynch K, Podolsky DK. Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation. Biochem Biophys Res Commun 1987;142:775–782. 504. Savage AP, Gornacz GE, Adrian TE, Ghatei MA, Goodlad RA, Wright NA, Bloom SR. Is raised plasma peptide YY after intestinal resection in the rat responsible for the trophic response? Gut 1985; 26:1353–1358. 505. Mannon PJ, Mele JM. Peptide YY Y1 receptor activates mitogenactivated protein kinase and proliferation in gut epithelial cells via the epidermal growth factor receptor. Biochem J 2000;350(pt 3): 655–661. 506. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR. Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 2002;418:650–654.

507. Adams SH, Won WB, Schonhoff SE, Leiter AB, Paterniti JR Jr. Effects of PYY[3-36] on short-term food intake in mice are not affected by prevailing plasma ghrelin levels. Endocrinology 2004; 145:4967–4975. 508. Wahab PJ, Hopman WP, Jansen JB. Basal and fat-stimulated plasma peptide YY levels in celiac disease. Dig Dis Sci 2001;46:2504–2509. 509. Adrian TE, Long RG, Fuessl HS, Bloom SR. Plasma peptide YY (PYY) in dumping syndrome. Dig Dis Sci 1985;30:1145–1148. 510. Adrian TE, Savage AP, Bacarese-Hamilton AJ, Wolfe K, Besterman HS, Bloom SR. Peptide YY abnormalities in gastrointestinal diseases. Gastroenterology 1986;90:379–384. 511. Adrian TE, Savage AP, Fuessl HS, Wolfe K, Besterman HS, Bloom SR. Release of peptide YY (PYY) after resection of small bowel, colon, or pancreas in man. Surgery 1987;101:715–719. 512. Alvarez Bartolome M, Borque M, Martinez-Sarmiento J, Aparicio E, Hernandez C, Cabrerizo L, Fernandez-Represa JA. Peptide YY secretion in morbidly obese patients before and after vertical banded gastroplasty. Obes Surg 2002;12:324–327. 513. Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR. Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med 2003;349:941–948.

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CHAPTER

6

Gastrointestinal Peptide Hormones Regulating Energy and Glucose Homeostasis Daniel J. Drucker Proglucagon Gene Structure and the ProglucagonDerived Peptides, 161 Regulation of Proglucagon Gene Expression, 162 Control of Proglucagon-Derived Peptide Secretion, 164 Proglucagon-Derived Peptide Metabolism and Clearance, 164 Glucagon Receptor Family, 165 Glucagon Receptor, 165 Glucagon-Like Peptide-1 Receptor, 165 Glucagon-Like Peptide-2 Receptor, 166 Glucose-Dependent Insulinotropic Polypeptide Receptor, 166 Biological Actions of Glucagon, 166 Glucagon Administration in Human Subjects, 167 Biological Actions of Glicentin, 167 Oxyntomodulin, 167 Biological Actions of Glucagon-Like Peptide-1, 167

Glucagon-Like Peptide-1 Receptor Agonists and the Treatment of Type 2 Diabetes, 169 Enhancing Incretin Action via Inhibition of Dipeptidyl Peptidase-IV, 170 Biological Actions of Glucagon-Like Peptide-2, 170 Glucagon-Like Peptide-2 Administration to Human Subjects, 171 Glucose-Dependent Insulinotropic Polypeptide, 171 Synthesis and Secretion, 171 Biological Actions of Glucose-Dependent Insulinotropic Polypeptide, 171 Glucose-Dependent Insulinotropic Polypeptide Administration in Human Subjects, 172 Pancreatic Polypeptide, 172 Orexins, 172 References, 173

Gastrointestinal hormones synthesized in functionally distinct populations of enteroendocrine cells and neurons play diverse roles in regulation of energy intake, nutrient absorption, and nutrient disposal. Most gut hormones are secreted at low basal levels in the fasting state, and plasma levels of most gut peptides increase rapidly but transiently after nutrient ingestion. The effects of gut hormones are increasingly complex and include regulation of food intake, exocrine secretion, gut motility, mucosal growth, nutrient absorption, and pancreatic endocrine function. Gut hormones also communicate with regulatory centers in the central nervous system (CNS) via afferent ascending nerves. This chapter

focuses on the biology of the proglucagon-derived peptides (PGDPs) and gut hormones with related actions on control of insulin secretion and energy balance.

PROGLUCAGON GENE STRUCTURE AND THE PROGLUCAGON-DERIVED PEPTIDES Before the molecular biology era, the best characterized member of the proglucagon peptide family was 29-aminoacid glucagon produced in pancreatic α cells. Antisera directed against pancreatic glucagon were shown to crossreact with immunoreactive peptides in gut extracts that were also detected in the circulation (1). These peptides, originally known collectively as “enteroglucagons,” were subsequently shown to consist of 2 principal forms: a larger protein of 69 amino acids designated glicentin, and a smaller 37-amino-acid peptide named oxyntomodulin (2). After cloning of complementary DNA (cDNA) and genes encoding mammalian

D. J. Drucker: Banting and Best Diabetes Centre, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

161

162 / CHAPTER 6 Proglucagon GRPP

Glucagon

IP-1

Glicentin Oxyntomodulin Pancreas Glucagon MPGF

GLP-1

IP-2

GLP-2

MPGF Intestine & Brain Glicentin Oxyntomodulin GLP-1 GLP-2 IP-2

FIG. 6-1. Structural organization of proglucagon gene and the proglucagon-derived peptides (PGDPs). Proglucagon undergoes tissue-specific posttranslational processing in the pancreas, intestine, and brain to liberate the indicated PGDPs. GRPP, glicentin-related pancreatic polypeptide; GLP-1 and GLP-2, glucagon-like peptide-1 and -2; IP-1 and IP-2, intervening peptides 1 and 2; MPGF, major proglucagon fragment.

preproglucagon, the structural relation among various PGDPs was clearly elucidated (Fig. 6-1). Two PGDPs related in sequence to glucagon, designated glucagon-like peptide-1 (GLP-1) and -2 (GLP-2) were found to be coencoded together with glucagon in a single proglucagon precursor (3–5). The human proglucagon gene is located on the long arm of chromosome 2 (6) and consists of six exons, with the sequences of glucagon and the GLPs encoded within separate exons (see Fig. 6-1). To date, no variants of the human proglucagon gene have been linked to heritability of specific human diseases. The mammalian genome encodes a single proglucagon gene that is transcribed to yield identical proglucagon messenger RNA (mRNA) transcripts predominantly in three cell types: pancreatic α cells, gut enteroendocrine L cells, and neurons in the caudal brainstem. The proglucagon mRNA transcript is structurally identical in all three tissues (7–9); hence, diversity in the generation of tissue-specific profiles of PGDPs is accomplished through tissue- and cell-specific expression of the prohormone convertases, proteases that differentially process proglucagon to yield either 29-aminoacid glucagon in the islet α cell or glicentin, oxyntomodulin, and both GLP-1 and GLP-2 in the gut L cell (see Figs. 6-1 and 6-2). Experimental studies have provided evidence supporting an essential role for PC2 in the cleavage of proglucagon to yield glucagon in pancreatic α cells, together with a larger, incompletely processed, secreted polypeptide designated major proglucagon fragment (10–13). The importance of PC2 for the generation of mature proglucagon is exemplified by the phenotype of mice with a targeted disruption of the PC2 gene. These mice exhibit mild hypoglycemia and markedly increased levels of incompletely processed proglucagon, together with deficiency of mature 29-amino-acid glucagon (14). In contrast with the importance of PC2 for generation of glucagon in islet α cells, PC1/3 appears to be required for processing of proglucagon in intestinal L cells (11,15). PC1 knockout mice exhibit multiple defects in prohormone processing including failure to generate significant amounts

of GLP-1 and GLP-2 (16). Although levels of mature GLP-1 are reduced in human subjects with an inactivating mutation of PC1, small amounts of bioactive GLP-1 can be generated in the absence of a functional PC1 enzyme (17). Intriguingly, PC1/3 has been localized to α cells in the embryonic pancreas, raising the possibility that GLP-1 may be liberated during development of the endocrine pancreas (18). PC1/3 expression, together with increased production of bioactive GLP-1, has also been localized to α cells in the setting of experimental diabetes; however, the biological implications of this finding remain uncertain (18,19).

Regulation of Proglucagon Gene Expression In the pancreas, proglucagon gene expression is stimulated by fasting and hypoglycemia, but is inhibited by insulin (20, 21), whereas in the intestine, proglucagon gene expression is up-regulated by nutrients (22). Gastrin-releasing peptide (GRP) and glucose-dependent insulinotropic polypeptide (GIP) (23) have been shown to increase levels of intestinal proglucagon mRNA transcripts in rodents. Fasting reduces and refeeding stimulates intestinal proglucagon gene expression (24), and a high-fiber diet (25) and short-chain fatty acids are potent inducers of proglucagon mRNA transcripts in enteroendocrine cells (26,27). Intestinal resection is associated with increased levels of proglucagon mRNA transcripts in the remnant intestine (28,29); however, the signals and mechanisms underlying this up-regulation remain unknown. Agents that activate the cAMP and protein kinase A (PKA) signaling pathways also increase proglucagon gene expression in the pancreas and intestine (8,30,31). Protein hydrolysates directly stimulate intestinal proglucagon gene expression through induction of gene transcription in part via DNA-promoter elements that mediate the response to cyclic 3′,5′-adenosine monophosphate (cAMP) (32). Intestinal proglucagon gene expression has been studied using primary cultures of intestinal cells, enteroendocrine cell lines, and transgenic mice. A primary determinant of intestinal proglucagon gene expression in studies of nontransformed intestinal cells appears to be the level of intracellular cAMP (8). Although agents such as phorbol esters, cholinergic agonists, calcium ionophores, and both GIP and GRP stimulate PGDP secretion in these cultures (33), only activators of cAMP generation enhanced proglucagon biosynthesis. Similarly, agents acting through the cAMP-dependent pathway appear to be the principal factors regulating proglucagon gene expression in mouse enteroendocrine glucagon SV40T antigen (GLUTag) (31,33) and secretin tumor cell line (STC-1) cells (34). Although GRP increases proglucagon gene transcription in STC-1 cells via the cAMP-response element (CRE) (23), GRP has not yet been shown to stimulate proglucagon gene expression in primary cell cultures or in the rodent intestine in vivo. Much less is known about the factors regulating the human proglucagon gene, largely because of the paucity of suitable models for studying human proglucagon gene expression.

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 163 RSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRQDFVQWLMNTKRNRNNIA Glicentin HSQGTFTSDYSKYLDSRRQDFVQWLMNTKRNRNNIA Oxyntomodulin Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG GLP-1(7-37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR GLP-1(7-36)amide HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPS Exendin-4 GLP-2 HADGSFSDEMNTILDNLAARDFINWLIQTKITD RNRNNIA IP-1 DFPEEVAIVEELG IP-2 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GIP APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY

PP

FIG. 6-2. Amino acid sequences of the proglucagon-derived peptides (PGDPs), exendin-4, glucosedependent insulinotropic polypeptide (GIP), and pancreatic polypeptide (PP). The lizard-derived exendin-4 shares 53% amino acid sequence identity with mammalian glucagon-like peptide-1 (GLP-1). Peptides that contain an alanine or proline at position 2 (indicated by underlining) are substrates for cleavage by the enzyme dipeptidyl peptidase-IV. IP-1 and IP-2, intervening peptides 1 and 2.

Transgenic mice expressing a human proglucagon gene promoter growth hormone transgene in the gut provide a potential model for studies of the human proglucagon gene promoter (35). Although nutrients up-regulated expression of the endogenous murine proglucagon gene, feeding had no effect on the human growth hormone (hGH) transgene, suggesting that important control elements for nutrientregulated control of human proglucagon promoter activity do not reside within the 1.6 kb of transgene human proglucagon gene promoter sequences (36). The control of human proglucagon gene expression has also been examined in NCIH716 cells, a cell line derived from a human adenocarcinoma exhibiting features of endocrine differentiation. Multiple factors, including palmitic acid, oleic acid, meat hydrolysate, carbachol, and GRP stimulate PGDP secretion from NCIH716 cells. In contrast with results of studies in rodent cells, cAMP enhanced GLP-1 secretion but failed to increase levels of proglucagon mRNA transcripts in NCI-H716 cells (37). Similarly, insulin, phorbol myristate acetate, or forskolin, all known regulators of rodent proglucagon gene expression, had no effect on proglucagon gene expression in NCI-H716 cells, and transfection studies using either the human or rodent proglucagon gene promoters demonstrate that NCIH716 cells do not support transcriptional activation of the isolated proglucagon gene promoter sequences (38). Hence, the available data suggest that NCI-H716 cells may express the human proglucagon in a constitutive, nonregulated manner. Cis-acting sequences within the rat proglucagon gene promoter have been identified as important control regions for islet cell–specific gene expression. Transcription factors important for islet cell–specific proglucagon gene expression include Brn4, Pax-6, Cdx-2/3, Isl-1, and members of the hepatocyte nuclear factor 3 (HNF-3 or Foxa) family (39–46). Pax6 and Cdx-2/3, in association with a coactivator protein, p300, interact synergistically to regulate proglucagon gene expression in islet cells (47). Pax-2 binds elements within the proglucagon gene promoter (48), but whether Pax-2 plays a role in the control of proglucagon gene expression remains uncertain (49). Similarly, although Brn4 is a potent activator of islet proglucagon gene expression, targeted disruption

of the Brn4 gene in mice does not produce abnormalities in α-cell development or changes in the levels of pancreatic glucagon mRNA transcripts (50). The proglucagon gene 5′-flanking sequences contain a CRE that confers cAMP responsivity to proglucagon gene transcription in pancreas and intestine (8,30,31,51). In contrast with our understanding of proglucagon expression in pancreatic islets, less is known about the factors that specify proglucagon gene expression in the intestine, partly because of the limitations of models for analysis of enteroendocrine gene transcription. Cell transfection studies using immortalized STC-1 or GLUTag cell lines indicate that DNA sequences between −1,252 and −2,292 in the rat proglucagon promoter are essential for specifying intestinal proglucagon expression (52). Several transcription factors thought to be important for control of islet proglucagon gene transcription may not be essential for enteroendocrine gene transcription. Genetic inactivation of the murine Foxa1 (HNF-3α) gene results in mice with mild hypoglycemia and inappropriately low levels of pancreatic proglucagon mRNA transcripts; however, levels of intestinal proglucagon mRNA transcripts are normal in Foxa1 mutant mice (46,53). Similarly, although members of the Foxa3 (HNF-3β ) family have been implicated in the control of proglucagon gene transcription in transfection studies, levels of pancreatic and intestinal proglucagon mRNA transcripts are normal in Foxa3−/− mice (54). The Pax-6 gene is important for cell lineage development in the gut and pancreas, as well as for control of pancreatic and intestinal proglucagon gene expression. Global inactivation of the murine Pax-6 gene results in major defects in formation of islet cell lineages (55), whereas mice homozygous for a dominant negative form of Pax-6 (SEYNeu) have significantly reduced levels of proglucagon mRNA transcripts in the pancreas (56) and both the small and large bowel, indicating that this transcription factor is essential for proglucagon gene expression in both islet and enteroendocrine cells (57). Conversely, increased expression of Pax-6 in primary intestinal cell cultures or in the rodent intestinal epithelium is associated with up-regulation of the levels of proglucagon gene expression (58).

164 / CHAPTER 6 Control of Proglucagon-Derived Peptide Secretion Glucagon, the principal secretory product from islet α cells, is secreted in response to hypoglycemia; however, the mechanism for sensing hypoglycemia and stimulating α-cell secretion likely involves the central and peripheral nervous system. In contrast, insulin and other factors secreted from the β cell, such as γ-aminobutyric acid (GABA) and zinc, appear to be the predominant factors inhibiting islet α-cell secretion. Glicentin, oxyntomodulin, GLP-1, and GLP-2 (Fig. 6-2) are cosecreted from enteroendocrine L cells, which are predominantly localized to the distal ileum and colon (59,60). GLP-1– immunopositive cells may also be located in more proximal regions of the small bowel, in cells that produce both GLP-1 and GIP (61). Multiple forms of GLP-1 are released in vivo, including GLP-1(1-37) and GLP-1(1-36)NH2, which appear to be biologically inactive (62–64), and GLP-1(7-37) and GLP-1(7-36)NH2, which are biologically active. A major proportion of GLP-1 is amidated at the C-terminal glycine residue [GLP-1(1-36)NH2 and GLP-1(7-36)NH2], likely via the activity of peptidylglycine α-amidating monooxygenase (65). C-terminal amidation may enhance the survival of GLP-1 in plasma (66). Both GLP-1(7-37) and GLP-1(7-36) NH2 appear equipotent (66–68); however, most circulating GLP-1 in humans is GLP-1(7-36)NH2 (69). In pigs and rats, approximately half of the GLP-1 is glycine extended, whereas in dogs, the glycine-extended forms of GLP-1 predominate. GLP-1 secretion from intestinal endocrine cells is stimulated by neural signals, endocrine factors, and direct nutrient contact with gut L cells (70). Intestinal PGDP secretion is regulated by several intracellular signals, including PKA, PKC, and calcium (33,71,72). A combination of studies using fetal rat intestinal cultures, the perfused rat ileum, and experiments performed in humans, rodents, and dogs have demonstrated that GLP-1 secretion is regulated directly by nutrients such as fatty acids, butyrate, peptones, or amino acids such as glutamine. Nevertheless, the rapid increase in plasma levels of the PGDPs within minutes of nutrient ingestion invokes a role for both neural and endocrine factors in control of L-cell secretion (70,73–75). Several neurotransmitters, including muscarinic agonists (76,77), regulate L-cell secretion both in vitro and in vivo. In humans, basal plasma levels of intact GLP-1 typically are within the 5- to 10-pM range in the fasting state, and increase to approximately 50 pM after meal ingestion (69,78). A small but detectable defect in meal-stimulated GLP-1 secretion has been observed in some subjects with obesity and type 2 diabetes (78). Mixed meals appear to be the most potent stimulus for GLP-1 secretion; however, individual nutrients including glucose, fatty acids, and dietary fiber also stimulate PGDP secretion (74,79–81). As most GLP-1–producing L cells, and hence stored GLP-1, is localized to the distal portion of the small intestine, a role for neural, endocrine mediators, or both in the rapid, nutrient-stimulated increase in plasma GLP-1 seems likely (70,82). Candidate mediators for indirect stimulators of GLP-1 include the more proximally located duodenal hormone GIP and the neurotransmitter acetylcholine (77,82–84). Nevertheless, species-specific

differences have been identified in studies of GLP-1 secretion because GIP does not stimulate GLP-1 secretion in humans (85,86), whereas the neuropeptide GRP stimulates GLP-1 secretion in both humans and rodents (87). Further evidence for the importance of GRP derives from studies of mice with inactivation of the GRP receptor that exhibit a reduced plasma GLP-1 response to gastric glucose (88). The neuropeptide calcitonin gene–related peptide may also regulate GLP-1 release (89). The vagus nerve plays a major role in controlling GLP-1 release from the distal L cells in response to ingested nutrients (90). Intestinal GLP-1 secretion appears to be inhibited by insulin and somatostatin-28 (33,91), as well as by the neuropeptide galanin (92).

PROGLUCAGON-DERIVED PEPTIDE METABOLISM AND CLEARANCE Little is known about the metabolism of glucagon, glicentin, and oxyntomodulin; however, the kidney plays a major role in the catabolism and clearance of all three peptides (93,94). Glucagon action is terminated via extracellular and intracellular degradation pathways. Glucagon-degrading activity within hepatic endosomes has been attributed to cathepsins B and D (95), and glucagon and GLP-1 are substrates for the widely expressed membrane-bound neutral ectopeptidase (NEP) 24.11 (96). The clearance and degradation of GLP-1 has received considerable attention because of the therapeutic potential of the peptide. The half-life of circulating native GLP-1 is less than 2 minutes (97,98), principally because of the protease activity of dipeptidyl peptidase-IV (DPP-IV), an aminopeptidase that specifically cleaves dipeptides from the amino terminus of proteins containing an alanine or proline at position 2. Oxyntomodulin and GLP-2 are also substrates for DPP-IV (99,100). DPP-IV is widely expressed in a large number of cell types in many tissues including the vascular endothelium of the small intestine, directly adjacent to the sites of GLP-1 release (97,101,102). Furthermore, in addition to a cell-associated membrane-bound form, a soluble DPP-IV molecule is also found in the circulation. DPP-IV catalyzes the cleavage of GLP-1 at the position 2 Ala residue to yield GLP-1(9-37) or GLP-1 (9-36)NH2. More than 50% of GLP-1 is metabolized to its N-terminal truncated form by DPP-IV within 2 minutes of peptide administration (103). Complementary evidence for the importance of DPP-IV in GLP-1 metabolism is derived from studies of rodents with mutations or inactivation of the DPP-IV gene. DPP-IV– deficient rats exhibit a prolonged half-life of GLP-1 (104,105), and mice with a targeted inactivation of DPP-IV exhibit increased levels of plasma GLP-1 (106). Moreover, rats and mice with mutant or inactivated DPP-IV genes exhibit improved glucose tolerance and increased levels of circulating intact GLP-1. DPP-IV is also critical for GLP-1 inactivation in humans, because intravenous or subcutaneous GLP-1 is rapidly degraded (within 30 minutes) to GLP-1(9-36)NH2 after administration of GLP-1 to healthy or diabetic human subjects, and this N-terminally shortened peptide accounts

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 165 for more than 75% of the immunodetectable circulating GLP-1 (98). Although GLP-1 (9-36)NH2 functions as a weak competitive pharmacologic antagonist of the GLP-1 receptor at the β cell and in the gastrointestinal system (107), experiments in anesthetized pigs treated with DPP-IV inhibitors revealed that GLP-1 (9-36)NH2 could paradoxically elicit modest insulin-independent antihyperglycemic effects (108). In contrast, infusion of GLP-1 (9-36)NH2 in healthy human subjects had no effects on glucose tolerance, insulin secretion or sensitivity, or GLP-1 action (109). Thus, the biological importance of N-terminally cleaved GLP-1 (9-36)NH2 remains uncertain. NEP 24.11 also exhibits endoproteolytic activity on GLP-1 and may also contribute to the metabolism of GLP-1 (96,110). Because GLP-2 is cosecreted with GLP-1, factors identified as important for GLP-1 secretion are similarly important for GLP-2 secretion, predominantly nutrients (111–113). GLP-2 also contains an alanine at position 2 and is degraded by DPP-IV (100). However, the half-life of exogenously administered GLP-2 is comparatively longer than that measured for GLP-1, at ~7 minutes (114). Although limited information is available about perturbations in levels of GLP-2 in human disease, patients with extensive resection of the small and large bowel exhibit significant reductions in circulating levels of GLP-2 after meal stimulation, whereas preservation of the colon appears important for maintaining levels of GLP-2 in adult human subjects (115,116). In contrast, the colon may be less important for preservation of plasma levels of GLP-2 in infants with nutrient malabsorption consequent to intestinal surgery (117). Patients with inflammatory bowel disease often exhibit increased circulating levels of GLP-2, in association with reductions in plasma levels of DPP-IV activity (118). The primary route of clearance for GLP-1 and GLP-2 appears to be through the kidney via mechanisms that include glomerular filtration and tubular catabolism (119–122). Patients with uremia have increased levels of circulating, immunoreactive GLP-1 (123), and bilateral nephrectomy or ureteral ligation in rats is associated with increases in the circulating half-life of GLP-1 (119). A role for tissues other than the kidney, such as the liver and lung, in GLP-1 clearance has not been clearly established (119).

intracellular cAMP and intracellular calcium (125). Oxyntomodulin is capable of binding to and activating both the glucagon and GLP-1 receptors; however, the anorectic actions of oxyntomodulin appear to require a functional GLP-1 receptor (126). The human Gcgr gene is localized to chromosome 17q25, and several reports have described an association between a Gly40Ser Gcgr mutation and an increased incidence of type 2 diabetes; however, this finding has not been confirmed in different populations with type 2 diabetes (127,128). Furthermore, cells expressing a transfected Gcgr containing the Gly40Ser mutation exhibit decreased affinity for glucagon in vitro, and human subjects with the Gly40Ser mutation exhibit a paradoxically decreased glycemic response to glucagon infusion in vivo (128,129). Hence, the biological significance of the Gly40Ser substitution remains uncertain. Gcgr mRNA transcripts have been detected in liver, islet β cells, brain, adipocytes, heart, and kidney. Rat Gcgr mRNA transcripts have also been detected in tissues such as spleen, thymus, adrenal gland, intestine, ovary, and testis where glucagon action remains poorly defined (130). Glucagon binding sites have been identified in multiple brain regions, and Gcgr transcripts have been detected in cortex, cerebellum, hypothalamus, and brainstem; however, the biological actions of glucagon in specific CNS regions remain unclear (131). Loss of glucagon action has been studied using antisense oligonucleotides directed against the Gcgr in diabetic mice (132,133), or in Gcgr−/− mice. Remarkably, overlapping phenotypes were seen in these experiments, including increased circulating levels of glucagon, mild fasting hypoglycemia together with improved glucose tolerance in Gcgr−/− mice, decreased body weight, and increased pancreatic mass associated with considerable hyperplasia of islet α cells (134,135). Furthermore, reduction of Gcgr expression in db/db mice was associated with lower levels of blood glucose, triglycerides, and free fatty acids (132,133). The improvement in glycemic control after loss of Gcgr function in these rodent studies appears to be attributable in part to increased circulating levels of bioactive GLP-1 derived from the pancreas. Gcgr expression is regulated in a tissue-specific manner with glucose and cAMP, important for Gcgr expression in hepatocytes (136,137), whereas glucose increased but cAMP and the glucocorticoid dexamethasone decreased levels of Gcgr mRNA transcripts in rat islets (138).

GLUCAGON RECEPTOR FAMILY GLUCAGON-LIKE PEPTIDE-1 RECEPTOR Separate receptors have been identified for glucagon, GLP-1, GLP-2, and GIP (124); however, the mechanisms underlying actions of PGDPs such as glicentin and oxyntomodulin remain poorly understood.

GLUCAGON RECEPTOR The glucagon receptor (Gcgr) is a member of the seventransmembrane–spanning G protein–coupled receptor superfamily, and it responds to glucagon with increases in both

The glucagon-like peptide-1 receptor (GLP-1R) was cloned from a rat pancreatic islet cDNA library (139). The human GLP-1R gene has been mapped to chromosome 6, band p21.1 (140); however, human GLP-1R mutations or polymorphisms have not been linked to the development of type 2 diabetes. The GLP-1R is expressed in lung; kidney; stomach; heart; intestine; α, β, and δ cells of the pancreatic islets; and multiple regions of the CNS (131,141–143). The GLP-1R couples to multiple G proteins, including Gαs, Gαq/11, and Gαi1,2, leading to activation of several intracellular

166 / CHAPTER 6 signaling pathways (144), increased adenylate cyclase and phospholipase C, and activation of PKA and PKC, respectively (139,145,146). GLP-1R activation also increases intracellular calcium (147–149), phosphatidylinositol-3 kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling pathways (144,150). Little is known about the factors that regulate levels of GLP-1R mRNA. GLP-1R expression is down-regulated in response to GLP-1, activation of PKC, high glucose, or dexamethasone, whereas treatment of diabetic rodents with DPP-IV inhibitors is associated with up-regulation of pancreatic GLP-1R expression (151). The GLP-1R undergoes homologous and heterologous desensitization and internalization in islet cell lines, in association with receptor phosphorylation (152–154). However, desensitization of the GLP-1R has not been observed after long-term administration of GLP-1R agonists in vivo. Exendin (9-39), an N-terminally truncated peptide derived from the lizard GLP-1R agonist exendin-4, functions as a relatively specific GLP-1 receptor antagonist (155,156) and is commonly used to examine the consequences of transient loss of GLP-1R action.

GLUCAGON-LIKE PEPTIDE-2 RECEPTOR The GLP-2 receptor was cloned from hypothalamic and intestinal cDNA libraries and is also coupled to cAMP generation when expressed in heterologous cell lines (157). GLP-2R expression is highly restricted, predominantly to the stomach, small and large bowels, and the CNS (158–160). Immunocytochemistry localized the GLP-2R to human enteroendocrine cells (158) and specific regions of the murine and rat CNS (159,160), whereas in situ hybridization has localized the receptor to neurons in the CNS and the murine enteric nervous system (161,162). The GLP-2R is highly specific for GLP-2 and is not activated by physiologically relevant concentrations of related members of the glucagon peptide superfamily (157,163). GLP-2 dose-dependently activates adenylyl cyclase, cAMP formation, and PKA in cells expressing a heterologous rat or human GLP-2R, as well as in primary cell cultures from the CNS and the intestinal mucosa (157,160,164,165). Furthermore, GLP-2R activation activates c-fos in cells transfected with the GLP-2R (164) and in the murine gastrointestinal tract (161). GLP-2 also activates constitutive nitric oxide synthase (NOS) activity and endothelial NOS protein abundance in the gut, which appears to regulate GLP-2– induced intestinal blood flow and glucose uptake (166). Activation of the GLP-2 receptor is also associated with enhanced cell survival. GLP-2R activation inhibits cycloheximide-induced apoptosis in a PKA-independent manner (167); in contrast, PKA regulates the antiapoptotic properties of GLP-2R signaling after inhibition of PI3K (168) in transfected fibroblasts or exposure to glutamate in hippocampal neurons (160). Although GLP-2 administration promotes rapid growth of the intestinal epithelium after administration in vivo (169), whether GLP-2R activation can

directly stimulate cellular proliferation requires further study. Incubation of colonic intestinal cells or rat astrocytes with GLP-2 results in increased cell proliferation (170–172); however, baby hamster kidney (BHK) fibroblasts transfected with the GLP-2 receptor did not exhibit a significant mitogenic effect in response to GLP-2 (164). Furthermore, GLP-2 treatment paradoxically inhibits cell proliferation in epithelial cells derived from the small intestine, yet stimulates cell proliferation in cell lines derived from the colon in a GLP-2R–independent manner (173). Hence, the molecular basis for GLP-2R activation promoting mitogenesis appears complex, likely indirect, and highly cell-type or tissue specific.

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE RECEPTOR Genetic mapping and linkage studies have localized the human glucose-dependent insulinotropic polypeptide receptor (GIPR) gene to chromosome 19 band q13.3 (174). The GIPR is a member of the seven-transmembrane domain, heterotrimeric, G protein–coupled Gcgr superfamily (124, 175), and it is expressed in the pancreas, stomach, small intestine, adipose tissue, adrenal cortex, lung, pituitary, heart, testis, vascular endothelium, bone, and brain (175,176). Activation of GIPR signaling leads to generation of cAMP and increases in intracellular calcium in pancreatic islet cells, adipocytes, osteoblasts, endothelial cells, and heterologous cell lines transfected with GIPR (176–179). The GIPR also signals via PI3K, MAPK, and phospholipase A2 pathways (180–182). The GIPR undergoes rapid and reversible homologous desensitization (73) in vitro. Studies using the βTC3 islet cell line implicate a role for regulator of G-protein signaling 2 (RGS-2), G-protein receptor kinase 2 (GRK-2), and β-arrestin-1 in GIP-induced receptor desensitization (183). GIP action is diminished in human subjects with type 2 diabetes (86), and defective GIP action in Zucker diabetic fatty rats has been correlated with reduced levels of GIPR mRNA transcripts in pancreatic islets (184). The absence of GIP action has been studied in mice with genetic disruption of the GIPR (185). GIPR knockout mice exhibit a mild defect in glucose tolerance (185), but they are resistant to the development of obesity and insulin resistance after high-fat feeding (186). Despite the roles of GIP and GLP-1 as the two dominant incretins regulating insulin secretion, combined disruption of both the GLP-1 and GIP receptors produces only modest impairment of glucose-dependent insulin secretion (187,188).

BIOLOGICAL ACTIONS OF GLUCAGON Glucagon exerts a number of important physiologic actions in tissues such as the liver, the endocrine pancreas, the vascular bed, and the gastrointestinal tract. Glucagon regulates hepatic glucose production via activation of glycogenolysis

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 167 and gluconeogenesis and by inhibition of glycolysis. Glucagon modifies the activity of enzymes important for glucose production and modulates expression of genes encoding enzymes in the glycolytic or gluconeogenic pathways (189). Glucagon also regulates fatty acid metabolism via reduction of malonyl coenzyme A (CoA) and stimulation of fatty acid oxidation. The cAMP-dependent transcription factor CREB is a downstream mediator for glucagon action. CREB, together with activation of the nuclear receptor coactivator peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 (PGC-1) and suppression of PPARγ activity, ultimately results in activation of hepatic gluconeogenesis (190). Glucagon increases cAMP and stimulates lipolysis in adipocytes, thereby providing free fatty acids as substrate for fat-burning tissues. Glucagon also inhibits insulin-stimulated glucose transport in adipocytes (191). In the peripheral vascular system, glucagon acts as a vasodilator via effects on local vascular tone, and glucagon increases cardiac output and heart rate, possibly via direct effects on the heart. Pharmacologic doses of glucagon increase renal blood flow, glomerular filtration rate (GFR), and urinary electrolyte excretion; however, lower concentrations of glucagon do not affect renal blood flow, GFR, or solute excretion (192). Although the kidney exhibits significant gluconeogenic capacity, the available evidence does not support a role for endogenous glucagon in the control of renal glucose output (193). The β cell expresses receptors for glucagon coupled to cAMP and stimulation of insulin secretion. The threshold for glucagon-stimulated cAMP accumulation in isolated β cells is ~1 nM glucagon, which is greater than the concentrations required for cAMP stimulation by GLP-1 or GIP (194). The physiologic importance of intraislet or circulating glucagon for β-cell physiology remains unclear, given the high concentrations of glucagon required to stimulate the β cell in vitro.

GLUCAGON ADMINISTRATION IN HUMAN SUBJECTS The most common use of glucagon therapeutically is in the acute management of severe hypoglycemia. Diabetic patients with hypoglycemia generally respond quickly, with a rapid increase in blood glucose, after glucagon administration (195,196). Glucagon is also used to inhibit gastrointestinal motility during radiologic or endoscopic investigations, and glucagon administration has been shown to benefit selected patients with refractory bronchospasm or symptomatic bradycardia (197,198).

BIOLOGICAL ACTIONS OF GLICENTIN Glicentin is a 69-amino-acid PGDP that contains the sequence of 29-amino-acid glucagon flanked by peptide extensions at both the amino and carboxy termini (see Fig. 6-2). Although cosecreted with GLP-1 and GLP-2 from

gut L cells, the specific biological actions of glicentin remain elusive. Glicentin has been shown to activate signal transduction pathways leading to stimulation of intracellular calcium and reduction of cAMP formation in smooth muscle cells derived from rabbit antrum (199). Furthermore, administration of glicentin to rodents induces small-bowel growth in some (169,200) but not all studies (201). To date, a distinct receptor responsible for transducing the biological actions of glicentin has not yet been identified.

OXYNTOMODULIN Oxyntomodulin contains the sequence of 29-amino-acid pancreatic glucagon together with an 8-amino-acid carboxyterminal extension (see Fig. 6-1). The original biological action described for oxyntomodulin was stimulation of acid secretion from the oxyntic glands in the stomach (202). Additional actions ascribed to oxyntomodulin include stimulation of intestinal glucose uptake (203), regulation of insulin secretion (204), reduction of gastric emptying (205), and inhibition of meal-stimulated gastric acid secretion (205–207). More recent studies have demonstrated an inhibitory effect of oxyntomodulin on food intake after both intracerebroventricular and peripheral administration in rats (208,209) and after intracerebroventricular administration in mice (126). Oxyntomodulin infusion also produces satiety and inhibits food intake after short-term intravenous infusion in human subjects (210). Although oxyntomodulin is a weak agonist at both the GLP-1 and glucagon receptors (211–214), the anorectic actions of oxyntomodulin are blocked by the GLP-1R antagonist exendin (9-39) (208) and are eliminated in the absence of a functional GLP-1R (126). Given the overlapping actions of oxyntomodulin with glucagon and GLP-1 and the lack of evidence for a distinct oxyntomodulin receptor, the available evidence suggests that many of the pharmacologic actions of oxyntomodulin represent heterologous activation of related PGDP receptor systems.

BIOLOGICAL ACTIONS OF GLUCAGONLIKE PEPTIDE-1 GLP-1 exerts multiple physiological actions leading to control of energy intake and nutrient assimilation (Table 6-1) (215). The original physiologic role described for GLP-1 was that of an incretin hormone. GLP-1 is secreted after nutrient ingestion and stimulates insulin secretion in a glucosedependent manner (85,216,217). GLP-1 also increases insulin gene transcription, mRNA stability, and biosynthesis by mechanisms that involve both cAMP/PKA-dependent and -independent pathways, as well as increases in the levels of intracellular Ca2+ (145,218,219). GLP-1 increases expression of the sulfonylurea receptor (SUR1) and inwardly rectifying potassium channel (Kir 6.2) in β cells, and it prevents glucose-dependent inhibition of KATP-channel activity (220).

168 / CHAPTER 6 TABLE 6-1. Summary of glucagon-like peptide-1 action Pancreas Stimulates glucose-dependent insulin secretion Increases insulin gene transcription, messenger RNA stability, and biosynthesis Inhibits glucagon secretion Stimulates somatostatin secretion Enhances β-cell responsivity and glucose competence Induces β-cell neogenesis and proliferation Inhibits β-cell apoptosis Gastrointestinal tract Inhibits gastric emptying Inhibits gastric acid secretion Cardiovascular system Increases heart rate and blood pressure in rodents Improves myocardial contractility Reduces cardiomyocyte apoptosis Central nervous system Inhibits food and water intake Stimulates luteinizing hormone–releasing hormone secretion Increases thyroid-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, and corticosterone/cortisol secretion in vivo Thyroid, lung, and kidney Stimulates calcitonin secretion from C cells in the thyroid gland Enhances mucous secretion, pulmonary muscle relaxation, and surfactant secretion in lung Promotes diuresis and natriuresis in kidney

The physiologic importance of GLP-1 has been demonstrated using GLP-1R antagonists, immunoneutralizing antisera, and GLP-1R knockout mice. Elimination of GLP-1 activity with GLP-1 immunoneutralizing antisera or the GLP-1R antagonist exendin (9-39) results in impaired glucose tolerance and diminished glucose-stimulated insulin levels in animals and humans (221–224). Similarly, mice with a targeted inactivation of the GLP-1R gene (GLP-1R−/−) are glucose intolerant and exhibit defective glucose-stimulated insulin secretion (225). GLP-1 confers glucose sensitivity to β cells, thereby improving the ability of the endocrine pancreas to sense and respond to glucose (226,227); however, GLP-1R signaling is not required for preservation of β-cell glucose sensitivity in the mouse (228). The demonstration that GLP-1 up-regulates the expression of components of the β-cell glucose sensing system (i.e., glucose transporters and glucokinase) may provide a partial mechanism for the effects of GLP-1 on β-cell glucose responsivity (229,230). GLP-1 also inhibits glucagon and stimulates somatostatin secretion (217). The increase in somatostatin secretion appears to be direct, via GLP-1Rs on somatostatin-secreting pancreatic δ cells (231), whereas the inhibitory effect of GLP-1 on glucagon secretion may be indirect, perhaps through stimulation of insulin and somatostatin, both of which inhibit glucagon secretion. However, GLP-1 may also inhibit glucagon secretion directly, via interaction with GLP-1Rs on α cells (142). Basal GLP-1 signaling in the fasting state is important for glucoregulation because administration of the antagonist exendin (9-39) to humans increases fasting glucose

and glucagon, suggesting that even the low basal levels of GLP-1 exert a tonic inhibitory effect on glucagon-secreting α cells (156). The insulinotropic and glucagonostatic effects of GLP-1 are glucose-dependent (232,233). Thus, when blood glucose levels decrease, GLP-1 no longer stimulates insulin secretion or inhibits glucagon secretion, thereby reducing the possibility of hypoglycemia. GLP-1 administration also leads to a number of changes in β-cell differentiation and function, including induction of proliferation and neogenesis of pancreatic β cells, reduced apoptosis, and increased differentiation of exocrine-like cells toward a more differentiated β-cell phenotype (234). GLP-1 activates the expression of immediate early genes which encode for transcription factors that regulate islet cell proliferation and differentiation. Furthermore, the differentiation of pancreatic exocrine cells toward a differentiated endocrine and β-cell–like phenotype is associated with induction of genes required for glucose-sensing and insulin gene expression (235–237). The molecular mechanisms mediating the GLP-1R–dependent activation of the endocrine differentiation pathway are poorly understood but may involve synergy with transforming growth factor-β and coordinate changes in Smad transcription factor activity (238). GLP-1R agonists also stimulate β-cell neogenesis and proliferation, and increase β-cell mass in both normal and diabetic rodents (235,239–242). Conversely, elimination of GLP-1R signaling in the mouse is associated with reduced numbers of large β-cell clusters and alterations in islet α-cell topography (243), and GLP-1R−/− mice exhibit a reduced adaptive response and greater hyperglycemia after partial pancreatectomy (244). The intracellular signal transduction pathways whereby GLP-1 mediates its proliferative effects have not been clearly defined, but they likely involve the induction of PI3K, epidermal growth factor receptor transactivation, and activation of p38 MAPK and PKCξ (245,246). GLP-1 may also regulate β-cell mass via increasing the resistance to apoptosis (246–250). GLP-1 inhibits apoptosis in β-cell lines and in nontransformed rodent β cells. Similarly, treatment of diabetic mice or rats with GLP-1R agonists reduces β-cell apoptosis (247,251,252). Furthermore, GLP-1R signaling appears to be essential for β-cell survival because GLP-1R−/− mice exhibit more severe hyperglycemia and enhanced sensitivity to the cytotoxic effects of streptozotocin on β cells in vivo (247). Importantly, the antiapoptotic actions of GLP-1 have been demonstrated in experiments using human islets (249,250). Incubation of freshly isolated human islets with GLP-1 for 72 hours reduced the expression of proapoptotic genes, improved cell viability, and enhanced glucose-stimulated insulin secretion (249). Complementary studies demonstrated that GLP-1 prevented apoptosis induced by high glucose or palmitate in human islets in vitro through mechanisms involving PKB and Akt (250). In the gastrointestinal tract, GLP-1 inhibits pentagastrinand meal-induced gastric acid secretion and gastric emptying (253,254). The reduction of gastric emptying attenuates meal-associated excursions in blood glucose, appears

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 169 dependent on the vagus nerve, and likely involves both central and peripheral GLP-1 actions (255,256). Vagal afferent denervation or GLP-1R antagonism with exendin (9-39) eliminates the effects of central and peripheral GLP-1 on gastric emptying. Furthermore, a recombinant GLP-1-albumin protein (Albugon) that does not appear to cross the blood– brain barrier retains the ability to inhibit gastric emptying (256), which is consistent with the importance of ascending GLP-1R–dependent pathways for control of gut motility. GLP-1 increases systolic, diastolic, and mean arterial blood pressure and heart rate in rats (257) and increases heart rate in calves (258). The effects of GLP-1 on the cardiovascular system are mediated through central and peripheral mechanisms (257,259,260). In experimental models of cardiovascular dysfunction such as pacing-induced ventricular failure or coronary occlusion and reperfusion in dogs, GLP-1 administration significantly improved ventricular function (261,262). GLP-1 may also exert a direct effect on cardiovascular parameters through interaction with GLP-1Rs in the heart (263). GLP-1 improved myocardial glucose uptake and cardiac contractility in dogs with pacing-induced heart failure (262). Furthermore, GLP-1 improved regional wall motion recovery after transient coronary artery occlusion and a 24-hour reperfusion period in dogs (261). Moreover, a 72-hour infusion of GLP-1 in patients after acute myocardial infarction and angioplasty resulted in markedly improved ventricular function (264). Conversely, GLP-1R signaling appears essential for normal cardiac structure and function because GLP-1R−/− mice exhibit ventricular hypertrophy and an impaired cardiovascular response to external stress (265). Intracerebroventricular administration of GLP-1 inhibits short-term food and water intake in rodents (225,266,267), and peripheral administration promotes satiety and suppresses energy intake in healthy, diabetic, and obese humans (268–270). Pharmacologic administration of GLP-1 may modify feeding behavior through direct interaction with satiety centers, or through mechanisms involving induction of interoceptive stress and visceral illness (271,272). The recombinant GLP-1-albumin protein Albugon rapidly inhibits food intake and activates neuronal c-fos expression without directly penetrating the CNS (256). Administration of either GLP-1 or noxious substances such as lithium chloride results in a similar pattern of neuronal c-fos activation in rats and elicits comparable aversive responses consistent with induction of visceral illness (272–275). Thus, the observation that excess GLP-1 induces nausea and reduction of food intake in human subjects may reflect activation of central aversive signaling pathways. Consistent with the cytoprotective actions of GLP-1 on β cells, activation of GLP-1R signaling promotes neuronal survival in diverse modes of cytotoxic injury (276,277). Furthermore, GLP-1R−/− mice exhibit a learning deficit that is improved after localized restoration of CNS GLP-1R signaling and display enhanced sensitivity to neuronal injury and increased seizure activity after kainate administration (278). These findings have engendered interest in the potential use of GLP-1R agonists as neuroprotective agents (279).

GLP-1 also modulates components of the hypothalamicpituitary axis. GLP-1 stimulates cAMP formation and thyroidstimulating hormone (TSH) release from cultured mouse pituitary thyrotrophs and isolated rat anterior pituitary cells (280) and enhances luteinizing hormone-releasing hormone secretion from rodent hypothalamic neuronal cell lines. Furthermore central GLP-1 administration stimulates TSH, luteinizing hormone, corticosterone, and vasopressin secretion in rats (281,282); however, GLP-1R−/− mice do not exhibit a major impairment of hypothalamic-pituitary function (283). Nevertheless, short-term infusions of GLP-1 transiently increase plasma levels of adrenocorticotropic hormone and cortisol in healthy human subjects (284). Notably, experimental evidence in rodents clearly indicates that systemic administration of GLP-1 or exendin-4 results in rapid activation of brainstem proglucagon neurons, indicating that peripheral GLP-1 is capable of activating the CNS circuits that, in turn, produce GLP-1 in the brain (285).

GLUCAGON-LIKE PEPTIDE-1 RECEPTOR AGONISTS AND THE TREATMENT OF TYPE 2 DIABETES GLP-1 reduces blood glucose levels through stimulatory effects on insulin secretion, reduction of glucagon secretion and gastric emptying, and indirectly through promotion of satiety leading to weight loss and improved insulin sensitivity. Short-term administration of native GLP-1 rapidly decreases plasma glucose in patients with type 2 diabetes (286–288). Furthermore, postprandial administration of native GLP-1 significantly attenuated meal-related glycemic excursion in diabetic patients, and a 3-week trial of preprandial subcutaneous GLP-1 injections improved postprandial glycemic control and reduced plasma glucagon in subjects with type 2 diabetes (289). The efficacy of native GLP-1 for the treatment of type 2 diabetes was illustrated in studies of continuous subcutaneous GLP-1 infusion for 6 weeks. Subjects treated with GLP-1 exhibited improved β-cell function, decreased fasting and postprandial glucose levels, a significant reduction in HbA1c, in association with modest weight loss and improved insulin sensitivity (290). Hence, there are considerable data from human clinical studies that administration of native GLP-1 exhibits therapeutic utility in the treatment of diabetic patients (291,292). Nevertheless, the therapeutic use of native GLP-1 would require multiple daily subcutaneous injections or continuous administration via subcutaneous infusion, because of the short circulating half-life of the native peptide in vivo (98,103,293,294). Accordingly, considerable effort has been devoted to development and characterization of GLP-1R agonists that are resistant to DPP-IV–mediated degradation, and that exhibit more prolonged durations of action in vivo. Exendin-4 is a naturally occurring GLP-1–related peptide isolated from the venom of the Heloderma suspectum lizard (295). Exendin-4 exhibits 53% identity to mammalian GLP-1, is a potent agonist at the GLP-1 receptor (155), and is

170 / CHAPTER 6 encoded by a separate gene distinct from lizard GLP-1 (296). Analysis of exendin-4 action in preclinical studies demonstrates that exendin-4 is a long-acting GLP-1R agonist that exhibits a full spectrum of GLP-1R–dependent actions in normal and diabetic rodents (297–299). Exendin-4, subsequently renamed Exenatide for clinical use, also has been shown to potently reduce blood glucose in human subjects with type 2 diabetes. Acute intravenous infusion of exendin-4 to healthy volunteers reduced fasting and postprandial glucose in association with inhibition of gastric emptying and reduced food intake (300). Repeated subcutaneous administration of exendin-4 for 4 weeks improved blood glucose and significantly decreased HbA1c (301). The clinical efficacy of Exenatide administration was examined in a series of phase 2 and 3 trials in patients with type 2 diabetes. Addition of Exenatide twice daily to treatment regimens previously encompassing metformin, a sulfonylurea, or both agents produced a significant reduction in HbA1c with prevention of weight gain in a 4-week study (302,303). Exenatide has completed phase 3 clinical trials as add-on therapy in diabetic patients with inadequate glycemic control who were treated previously with metformin or a sulfonylurea agent, or both, and is the first GLP-1R agonist approved for the treatment of type 2 diabetes. Additional GLP-1R–based agonists in clinical development include Liraglutide, a human DPP-IV–resistant analogue that exhibits noncovalent binding to albumin and a prolonged pharmacokinetic profile after once daily administration (304,305). Liraglutide has completed phase 2 clinical trials and significantly reduced HbA1c with no associated weight gain in a 12-week monotherapy study (306). CJC1131 is a human GLP-1 analogue that forms a covalent bond to human serum albumin and exhibits GLP-1R–dependent actions in preclinical studies (242). CJC-1131 also has been shown to effectively reduce glycemia in 12-week studies of subjects with type 2 diabetes. Taken together, the available evidence strongly suggests that one or more injectable GLP-1R agonists may be used for the treatment of type 2 diabetes.

stimulate insulin secretion, and increase the levels of intact GLP-1 and GIP in preclinical models of diabetes (307–309). Moreover, administration of DPP-IV inhibitors to human subjects with type 2 diabetes results in significant reduction of glycemia, together with decreased levels of circulating glucagon and an improvement in the insulin/glucose ratio (310,311). Several DPP-IV inhibitors currently are in latestage clinical development for the treatment of type 2 diabetes (291,292,312).

BIOLOGICAL ACTIONS OF GLUCAGONLIKE PEPTIDE-2 GLP-2 is a 33-amino-acid peptide cosecreted with GLP-1 from gut endocrine cells in a nutrient-dependent manner (112,113). The principal biological consequence of exogenous GLP-2 administration is the expansion of the mucosal epithelium in the small bowel (169), caused by enhanced crypt cell proliferation and a reduction in enterocyte apoptosis (313,314). The intestinotrophic actions of GLP-2, which are summarized in Figure 6-3, have been demonstrated in mice (169,315), rats (100,316,317), pigs (318,319), and human subjects (320). GLP-2 also acutely enhances hexose transport (321,322), reduces epithelial permeability (323), enhances gut barrier function (324), and reduces gastric motility and acid secretion (325,326) after exogenous administration in vivo (see Fig. 6-3). Whether GLP-2 action is essential for the growth and survival of the developing or adult mucosal epithelium remains uncertain. Although GLP-2(3-33) modestly attenuates exogenous GLP-2 action, it exhibits properties of both a partial antagonist and a weak agonist (327); hence, optimal tools for analysis of loss of GLP-2 action have not yet been identified.

Gut endocrine L cells Blood flow

ENHANCING INCRETIN ACTION VIA INHIBITION OF DIPEPTIDYL PEPTIDASE-IV The rapid degradation of native GLP-1 has fostered efforts directed at preventing GLP-1 degradation for the treatment of diabetes. DPP-IV, also known as CD26, is a widely expressed cell surface–associated peptidase that also circulates as a soluble form in the plasma. DPP-IV cleaves peptides at the position 2 alanine and appears to be essential for normal glucose homeostasis, because mice with genetic inactivation of CD26 exhibit increased levels of GLP-1 and reduced glycemic excursion after glucose challenge (106). Because DPP-IV is the dominant enzyme regulating GLP-1 degradation (98,103,106), considerable efforts have focused on the development of DPP-IV inhibitors for the treatment of type 2 diabetes. DPP-IV inhibitors reduce blood glucose,

Hexose transport

GLP-2R+ gut endocrine cells

GLP-2

Proliferation Apoptosis

Secondary mediators

Permeability Motility GLP-2R+ Enteric neurons

FIG. 6-3. The actions of glucagon-like peptide-2 (GLP-2) in the gastrointestinal epithelium. GLP-2 secreted from gut endocrine cells acts via gut endocrine cells, myofibroblasts, or enteric neurons to promote diverse actions in the gut epithelium. GLP-2R, glucagon-like peptide-2 receptor.

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 171 The trophic and cytoprotective actions of GLP-2 also have been examined in the setting of experimental intestinal injury. GLP-2 facilitates mucosal adaptation in rats after major small-bowel resection (317), and intravenous GLP-2 prevents mucosal hypoplasia in rats fed parenterally (328). The extent of mucosal damage is markedly attenuated by exogenous GLP-2 in mice with dextran-sulphate–induced colitis (329) or after indomethacin-induced enteritis (330). Similarly, GLP-2 administration improved intestinal disease activity scores and survival in mice with chemotherapyinduced enteritis (331), in rats with autoimmune enteritis (332), and in rats after occlusion of the superior mesenteric artery (333). Hence, pharmacologic GLP-2 administration promotes intestinal healing and prevents mucosal injury in a diverse number of experimental models of gut injury (234,334).

GLUCAGON-LIKE PEPTIDE-2 ADMINISTRATION TO HUMAN SUBJECTS Because exogenous GLP-2 administration significantly increases the surface area and absorptive capacity of the small-bowel epithelium, GLP-2 or structurally related GLP-2 analogues may exhibit therapeutic potential for the treatment of human subjects with short bowel syndrome (335). Administration of native GLP-2, 400 µg subcutaneously twice daily for 35 days, to human subjects without a terminal ileum and colon significantly improved energy absorption and body weight, and increased lean body mass. GLP-2– treated subjects exhibited reduced enteral fluid loss and reduced gastric emptying but no change in small-bowel transit time (320). The potential therapeutic efficacy of a more potent DPP-IV–resistant GLP-2 analogue currently is being examined in separate clinical trials of patients with Crohn’s disease or short bowel syndrome. GLP-2 also appears to regulate bone mass through direct effects on bone resorption. Administration of GLP-2 twice daily to human subjects with short bowel syndrome reduced markers of bone turnover in a 5-week study (336). Because food ingestion is known to regulate gut hormone release and bone resorption, the relation between gut peptides and bone resorption was studied after single-dose administration of various peptides in healthy postmenopausal women. Although administration of GIP and GLP-1 had no significant effect on bone turnover, GLP-2 reduced circulating levels of the C-terminal telopeptide region of type I collagen and decreased urinary excretion of dihydropyrimidine dehydrogenase (DPD)/ creatinine, both markers of bone resorption (337).

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE Synthesis and Secretion GIP, a 42-amino-acid peptide and the first incretin hormone to be identified (338,339), is synthesized in and released

from intestinal K cells in response to nutrient ingestion. GIP was originally identified on the basis of its ability to inhibit gastric acid secretion in dogs, and subsequently was shown to potentiate glucose-stimulated insulin secretion (338,339). The sequence of GIP is highly conserved across species, with more than 90% identity at the amino acid level for human, rat, mouse, porcine, and bovine GIP. The peptides encoded within the N- and C-terminal segments of pro-GIP have no known biological function. GIP is expressed predominantly in the stomach and in the K cells of the proximal small intestine. GIP mRNA also has been detected in the rat submandibular gland (73). There is little information about the factors that regulate GIP gene expression; however, nutrients up-regulate levels of GIP mRNA in the rat duodenum and submandibular salivary gland, whereas fasting significantly reduces levels of GIP mRNA (340,341). GIP secretion reflects the rate of nutrient absorption, rather than the simple presence of nutrients in the small intestine. Fat is a potent stimulus for GIP secretion in humans, whereas in rodents and pigs, carbohydrates are more effective secretagogues (342). GIP contains an alanine at position 2 and is a substrate for enzymatic inactivation by DPP-IV (293). The half-life of biologically active intact GIP(1-42) is estimated to be less than 2 minutes in rats (103, 343) and approximately 7 and 5 minutes in healthy subjects and patients with type 2 diabetes, respectively (343). After intravenous infusion of GIP in humans, intact bioactive GIP accounted for approximately 40% of the total detectable amount of immunoreactive GIP, whereas only about 20% of total immunoreactive GLP-1 remained intact in the same studies (343). GIP is cleared through the kidney, and levels of GIP are increased in patients with uremia or chronic renal failure.

Biological Actions of Glucose-Dependent Insulinotropic Polypeptide The increasingly diverse actions of GIP on various tissues are summarized in Table 6-2. The dominant actions of GIP on the islet β cell are the enhancement of glucose-dependent insulin secretion via increase of intracellular cAMP, inhibition of adenosine triphosphate–sensitive K+ channels, increase in intracellular Ca2+, and engagement of the β-cell secretory machinery (344). GIP also enhances insulin gene transcription and up-regulates the expression of glucose transporters and glucokinase, which are components of cellular glucose sensors (345). GIP functions as a β-cell growth factor for islet β cells in vitro via activation of cAMP/PKA, MAPK, and PI3K-dependent pathways (346,347). Whether GIP is also important for growth and survival of islet β cells in vivo remains uncertain. GIP also regulates adipocyte lipid metabolism (342) including stimulation of glucose uptake and increases the sensitivity of insulin-stimulated glucose transport. GIP enhances fatty acid production and insulin-stimulated incorporation of fatty acids into triglyceride, augments lipoprotein lipase

172 / CHAPTER 6 TABLE 6-2. Summary of glucose-dependent insulinotropic polypeptide action Pancreas Enhances glucose-stimulated insulin secretion Stimulates insulin gene expression Promotes β-cell proliferation and reduces β-cell apoptosis Adipose tissue Stimulates insulin-dependent glucose uptake Increases insulin receptor affinity Enhances fatty acid synthesis and incorporation into triglyceride Augments lipoprotein lipase synthesis and activity Reduces glucagon-stimulated lipolysis Bone Stimulates alkaline phosphatase activity and collagen type I messenger RNA Increases bone mineral density in rodents Other tissues Up-regulates intestinal hexose transport Stimulates glucocorticoid secretion in rodents Modulates vascular bed-type–dependent endothelial tone

synthesis and activity, and reduces glucagon-stimulated lipolysis in adipose tissue. GIP may also have lipolytic effects in adipocytes (348). The signaling mechanism(s) activated by GIP in adipocytes have not been fully elucidated (342). Intriguingly, GIPR null mice are resistant to diet-induced obesity and exhibit relative reductions in adipose tissue mass after high fat feeding, together with a reduction in expression of the key enzyme Acyl CoA:diacylglycerol transferase 1 (Dgat1) in adipose tissue (186). GIP may also regulate bone formation in osteoblast cells including increases in alkaline phosphatase activity and levels of collagen type I mRNA (178). GIP treatment increases bone mineral density in the ovariectomized rat (349), although acute administration of GIP to human subjects was not associated with changes in markers of bone turnover (337). GIP stimulates glucocorticoid secretion in rats via a cAMP/PKAdependent signaling pathway (350). Although GIP does not appear to regulate cortisol secretion in healthy human subjects (351), aberrant expression of the GIPR in adrenocortical adenomas is associated with the pathogenesis of meal-induced Cushing syndrome (351–353).

Glucose-Dependent Insulinotropic Polypeptide Administration in Human Subjects Because GIP stimulates glucose-dependent insulin secretion, several studies have examined the therapeutic potential of GIP for the treatment of type 2 diabetes. Remarkably, although GIP is a potent insulinotropic agent in healthy humans, GIP action is significantly diminished in human subjects with type 2 diabetes (86,354). Unlike GLP-1, GIP does not significantly inhibit glucagon secretion or gastric emptying in humans (355,356). Although GIP analogs engineered for resistance to DPP-IV action exhibit enhanced insulinotropic properties in preclinical studies (357), the

potential of these analogs to reduce blood glucose in human subjects with type 2 diabetes has not been carefully examined. Hence, the available evidence suggests that GIP is unlikely to be a therapeutic candidate for the treatment of diabetic human subjects (358).

Pancreatic Polypeptide Pancreatic polypeptide (PP) is a structurally related member of the peptide YY/neuropeptide Y (PYY/NPY) family. PP is a 36-amino-acid peptide predominantly expressed in the endocrine pancreas; however, rare PP-immunopositive enteroendocrine cells have been described in some but not all species (359). PP appears to preferentially recognize the NPY 4 receptor (360). PP secretion is stimulated by food ingestion and exercise, and vagal tone is an important determinant regulating PP secretion in rodents and human subjects. Studies in rodents have also demonstrated an anorectic role for either centrally or peripherally administered PP (361). Intriguingly, PP not only reduces food intake but also increases energy expenditure after intraperitoneal administration in genetically obese mice (362). Similarly, transgenic mice with PP overexpression in pancreatic islets exhibit reduced milk intake during the neonatal period, with decreased food intake together with reduced body weight a feature of older PP transgenic mice (362). Although the available evidence clearly implicates PP as a regulator of food intake, whether PP is essential for body weight homeostasis has not yet been determined. The biological actions of PP in human subjects remain somewhat obscure; however, PP, like PYY(3-36), exerts anorectic actions in vivo. Administration of PP reduces food intake in healthy human subjects (363) and in patients with Prader–Willi syndrome (364).

Orexins The orexin peptide families, also referred to as the hypocretins, are produced predominantly in the CNS. Original studies of orexin biology linked orexin action to the control of feeding behavior (365), whereas more recent data have implicated a role for orexins in the regulation of arousal and sleep physiology (366,367). Orexin-like immunoreactivity and orexin receptor mRNA transcripts have also been localized to the enteric nervous system in both the submucosal and myenteric ganglia (368). Intriguingly, fasting activates orexin plus neurons in the gut, and circulating levels of plasma orexin-A are increased after fasting in both rodents and humans (369). Intriguingly, orexin is also secreted from islet α and β cells in a glucose-dependent manner, and administration of orexin-A increases plasma levels of glucagon and glucose in fasted rats (370). Both orexin and the OX2 receptor have been localized to enteroendocrine cells; however, the precise function of the orexin system in gut endocrine cells remains poorly understood.

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 173 In summary, enteroendocrine peptides exert increasingly complex actions on the control of gut motility, epithelial integrity, cytoprotection, satiety, and pancreatic endocrine function. The actions of many of these peptides, as revealed through studies using antagonists and genetic loss of function mutants, are essential for control of glucose and energy homeostasis. Moreover, GLP-1 and GLP-2 agonists are being evaluated in clinical trials for the treatment of diabetes and short bowel syndrome. Hence, understanding the pleiotropic actions of these peptides has relevance for understanding the biology of gut hormone action and the potential treatment of specific human diseases.

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CHAPTER

7

Growth Factors in the Gastrointestinal Tract John A. Barnard and Kirk M. McHugh Introduction, 184 Growth Factors Defined, 184 Compartmentalization of Growth Factor Function in the Gastrointestinal Tract, 185 “Redundancy” in Growth Factor Families, 185 β Family of Peptides and Transforming Growth Factor-β Receptors, 186 Overview and Discovery of Transforming Growth Factor-β, 186 Transforming Growth Factor-β Genes, 186 Transforming Growth Factor-β Protein and Activation, 186 Transforming Growth Factor-β Receptors and Receptor Interacting Proteins, 187 Smad Signaling Is Induced by Transforming Growth Factor-β, 188 Biological Actions of Transforming Growth Factor-β in the Gastrointestinal Tract, 189 Lessons from Gene-Targeted and Transgenic Mice, 192 Epidermal Growth Factor Family of Peptides and Receptors, 193 Overview and Discovery of the Epidermal Growth Factor Family, 193 Epidermal Growth Factor, 194 Transforming Growth Factor-α, 195 Amphiregulin, 198 Heparin-Binding Epidermal Growth Factor–Like Growth Factor, 198 Betacellulin, 199 Epiregulin, 199 Epigen, 199 Regulated Shedding of Epidermal Growth Factor–Related Peptides by Metalloproteases, 200 Epidermal Growth Factor Receptor Family, 200 Insulin-Like Growth Factors, 205 Overview and Discovery of Insulin-Like Growth Factors, 205

Insulin-Like Growth Factor Genes and Proteins, 206 Insulin-Like Growth Factor Receptor Genes and Proteins, 206 Insulin-Like Growth Factor Binding Protein Gene and Protein Family, 206 Insulin-Like Growth Factor Family Distribution in the Gastrointestinal Tract, 207 Biology of the Insulin-Like Growth Factor Family in the Gastrointestinal Tract, 208 Elimination of Insulin-Like Growth Factor Function by Gene Targeting, 209 Trefoil Factor Family of Peptides, 209 Overview and Discovery of Trefoil Factor Family, 209 Trefoil Factor Family Genes, 210 Trefoil Factor Family Proteins, 210 Trefoil Factor Family Receptors and Binding Proteins, 211 Intracellular Signaling by Trefoil Factor Family, 211 Biological Actions of Trefoil Factor Family Proteins, 211 Hepatocyte Growth Factor, 213 Overview and Discovery of Hepatocyte Growth Factor, 213 Hepatocyte Growth Factor Gene, 213 Hepatocyte Growth Factor Protein, 213 Hepatocyte Growth Factor Receptors, 214 Intracellular Signaling by Hepatocyte Growth Factor, 214 Biological Actions of Hepatocyte Growth Factor, 214 Fibroblast Growth Factor Family, 216 Overview and Discovery of Fibroblast Growth Factor Family, 216 Fibroblast Growth Factor Genes, 216 Fibroblast Growth Factor Proteins, 216 Fibroblast Growth Factor Receptors, 217 Intracellular Signaling by Fibroblast Growth Factor, 218 Biological Actions of Fibroblast Growth Factor, 219 References, 221

J. A. Barnard and K. M. McHugh: Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, Columbus, Ohio 43205. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Acedemic Press, 2006.

183

184 / CHAPTER 7 INTRODUCTION

Historically, growth factors have been named based on their most prominent biological activity or the activity that led to their discovery. Subsequent work has, in most cases, resulted in the description of activities that are misrepresented by the original name. For example, EGF was discovered and so named because it stimulated epithelial cell growth in the newborn mouse eyelid, but further studies showed that EGF is also a powerful mitogen for fibroblasts. TGF-β was discovered and so named because it induced morphologic transformation of fibroblasts in culture, although, ironically, it ultimately proved to be one of the most important tumor suppressors ever discovered. Some growth factors also exhibit activities that are quite unrelated to cell growth; for example, EGF inhibits gastric acid secretion and hepatocyte growth factor (HGF) stimulates cell motility. Notably, the distinction between the term growth factor and related terms such as cytokine, gut peptide, and hormone is not distinct and other chapters in this volume cover peptides that may, in some classifications, be considered “growth factors.” Polypeptide growth factors modulate cellular functions by several potential pathways. Most do not regulate function by a classical endocrine mechanism, but rather by autocrine, paracrine, or both pathways (Fig. 7-1). A growth factor that regulates cellular function by an autocrine pathway is synthesized and secreted by a cell type that also has functional cellsurface membrane receptors and intact intracellular signaling pathways for that same factor. If adjacent but histogenetically

Growth Factors Defined Growth factors are polypeptides that regulate cell growth and modulate other critical cellular functions by binding to specific homodimeric or heterodimeric, high-affinity, cellsurface membrane receptors. Interaction between ligand and receptor activates intracellular signaling pathways, typically by phosphorylation of cytoplasmic proteins that translocate into the nucleus and specifically modify gene expression. Growth factors usually are secreted, but may also be found in transmembrane, cytosolic, and extracellular matrix (ECM)– associated compartments. Biological effects usually are apparent at extremely low concentrations, usually in the nanogram per milliliter range. Structurally and biologically related growth factors are classified into “families,” although this is not necessarily reflected in the confusing nomenclature of individual growth factors. For example, two of the most relevant and extensively studied growth factor families in gastrointestinal biology are the transforming growth factor-β (TGF-β) and the epidermal growth factor (EGF) families. TGF-β1, a member of the TGF-β family, is not related structurally or biologically to transforming growth factor-α (TGF-α), which is a member of the EGF-related peptide family, and is closely related to EGF structurally and functionally.

2

1

3

Epithelial cells

Mesenchymal cells

Splanchnic vasculature

FIG. 7-1. Paradigms for growth factor signaling in the gastrointestinal tract. Polarized epithelial cells are shown in relation to mesenchymal elements in the lamina propria and the splanchnic vasculature. Endocrine (1), paracrine (2), and autocrine (3) signaling pathways are shown; the pathways for each growth factor family are described in detail in the text.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 185 different cell types also possess functional receptors and signaling pathways, these cells are regulated by a paracrine route. Juxtacrine growth regulation occurs when a growth factor anchored in the cell-surface membrane stimulates a biological activity in an adjacent cell (1). Intracrine growth regulation occurs when a polypeptide growth factor binds to an intracellular receptor and is biologically active without exiting the cell (2). It is likely that most growth factors contribute to cellular and tissue homeostasis by simultaneous use of multiple pathways. The biological significance of autocrine growth factor pathways is considerable. Autocrine regulation of normal cellular homeostasis is an intricate servomechanism for fine regulation of biological function and cellular homeostasis. However, disruption of autocrine control may have devastating pathophysiologic consequences. This is best exemplified by the autocrine hypothesis that emerged in the early 1980s to explain the unchecked growth characteristic of malignant cells in human neoplastic disorders; that is, malignant cells secrete stimulatory polypeptide factors for which the cells themselves have functional receptors (3). Numerous examples now exist in support of amplified autocrine growth stimulatory pathways or interrupted autocrine growth inhibitory pathways leading to human cancers. This chapter discusses many examples of normal physiologic responses and pathophysiologic states that may result from a disturbance in this intricate balance of regulatory control.

Compartmentalization of Growth Factor Function in the Gastrointestinal Tract Perhaps more so than other organ systems, study of the biological activity of growth factors in the gastrointestinal tract requires consideration of multiple complex spatial variables. The apical domain of the gastrointestinal epithelium contacts a highly heterogeneous external environment, including dietary constituents and endogenous luminal factors such as proteases, extremes of pH, bacteria, and growth factor– laden secretions from more proximal organs. The basolateral domain of the epithelium is largely protected from these intraluminal influences by the tight junction, but this cell surface contacts extracellular fluids and is subject to physiologic and pathophysiologic alterations therein. A healthy tight junction maintains this spatial compartmentalization. However, in the context of epithelial injury and disruption of tight junctions, growth factors and their receptors that are ordinarily spatially segregated may contact and exert biological activity in cell or tissue compartments to which they are not usually exposed. Other compartmental variables to be considered are the horizontal (proximal-to-distal) and vertical (crypt-to-surface epithelium) axes in the gastrointestinal tract. Thus, a large number of complex variables contribute to growth factor biology in the gastrointestinal tract. With the exception of the EGF-related peptide family, little progress has occurred in the understanding of these spatial relations. Later, this chapter makes an effort to synthesize what is

known about these spatial relations into three dimensions for the EGF-related peptide family.

“Redundancy” in Growth Factor Families Virtually every growth factor family is composed of multiple structurally and biologically related proteins, implying a potential for redundancy of biological function. The concept of redundancy should be viewed with caution. True, in the artificial “one-dimensional” setting of cultured cells, studies clearly support functional redundancy in most growth factor families. However, the development of growth factor knockout mice in the early to mid-1990s emphasized that complex spatial, compartmental, and kinetic considerations must be invoked in the analysis of growth factor function. Under these terms, redundancy is much less commonly observed. Analysis of EGF and TGF-α, two of the best-studied members of the EGF-related peptide family, is illustrative of a potentially redundant system that, in reality, is much less redundant than might be predicted. In cell culture, these growth factors are usually equipotent in stimulation of cell proliferation and migration. However, differential activity potentially exists at the cell, tissue, and organ levels, and it may explain differences in biological activity between TGF-α and EGF in more complex models. At the cellular level, differences in receptor–ligand interaction are observed. For example, although EGF and TGF-α bind the mammalian EGF receptor (EGFR) with similar affinity, Winkler and colleagues (4) developed an anti-EGFR antibody that markedly inhibits TGF-α when compared with EGF binding, suggesting that the two peptides bind differently to the same receptor. This line of evidence is further supported by differential binding of mammalian TGF-α and EGF to NIH3T3 cells transfected with a chicken EGFR (5). Moreover, there are convincing data that intracellular routing and degradation of TGF-α receptor and EGFR complexes are different, partly because of differences in the isoelectric point of the two proteins (6). An integrated quantitative model of endocytic trafficking of EGF and TGF-α predicts whether signaling begins at the cell membrane in the case of TGF-α, or the endosomal compartment in the case of EGF (7). Compartmentalization of the polarized epithelial cell membrane into brush border and basolateral domains raises interesting questions regarding targeting of the transmembrane precursors of TGF-α and other EGF-related peptides to the cell surface, as well as the localization of EGFRs on these two domains. TGF-α is sorted to the basolateral membrane with approximately 95% efficiency in Madin– Darby canine kidney (MDCK) cells (8), an observation with important implications regarding biological activity in vivo. This observation is even more intriguing in light of subsequent observations that EGF is targeted to the apical domain of MDCK cells (9). Additional considerations occur at the tissue and organ levels. For example, that EGF is not synthesized by the normal small intestinal epithelium, whereas TGF-α is synthesized

186 / CHAPTER 7 by cells in the differentiated compartment of the epithelium (10,11) suggests that cell and tissue compartmentalization of EGF and TGF-α synthesis may result in major differences in activity. Thus, TGF-α is a more physiologic ligand for the small intestinal epithelial EGFR than is EGF. Similar paradigms of differential growth factor distribution have been observed in other organ systems (12). In pathophysiologic situations, alternate patterns of growth factor expression may be observed. For example, Wright and coworkers (13) described emergence of a novel EGF-producing cell lineage in chronically injured gastrointestinal mucosa. EGF is not normally produced in the small bowel, with the possible exception of Brunner’s glands of the duodenum, (14); thus, expression may depend, in part, on the coexistence of cellular injury and other pathophysiologic processes. Other examples of EGF-related peptides demonstrating differential biological activity despite structural similarity and affinity for the same receptor are observed in the complex milieu of the gastrointestinal luminal contents. Fasting human pancreatic juice destroys both TGF-α and EGF, whereas feeding the milk protein casein preserves EGF but not TGF-α bioactivity (15). EGF and TGF-α are also partially degraded by gastric juice, resulting in attenuation of bioactivity (16). These studies highlight the potential contribution of luminal and extracellular events on differential bioavailability of EGF-related peptides.

β TRANSFORMING GROWTH FACTOR-β FAMILY OF PEPTIDES AND RECEPTORS Overview and Discovery of Transforming β Growth Factor-β TGFs were first identified in 1978 as a reversible, transforming activity in cell culture medium conditioned by murine sarcoma virus–transformed mouse fibroblasts (17). Treatment of normal fibroblastic cells suspended in soft agar with a partially purified protein preparation from transformed mouse fibroblasts stimulated growth of colonies (anchorage-independent growth) and morphologic transformation; hence, the term transforming growth factors. Purification of the transforming activity identified two structurally unrelated peptides: TGF-α and TGF-β (18,19). Thereafter, the history of these two important growth regulatory proteins diverges rather sharply and has left in its wake a confusing nomenclature. Currently, three mammalian TGF-β “isoforms” are designated TGF-β1, TGF-β2, and TGF-β3. TGF-β is the prototype for a superfamily of structurally related, evolutionarily conserved peptides that share a cluster of conserved, disulfide-linked cysteine residues and exert a multitude of biological activities related to cell growth and differentiation, morphogenesis, and cell fate determination. The TGF-β superfamily includes activins and inhibins (20), bone morphogenetic peptides (21,22), nodal (23), growth and differentiation factor, Mullerian inhibiting substance (24),

Drosophila decapentaplegic peptide (25), and TGF-β, among others. TGF-β–related peptides are synthesized by a wide variety of cell types as large precursor molecules undergo regulated activation to release the biologically active protein. In some instances, activation occurs intracellularly, and mature peptide is secreted bound to a peptide antagonist. Other members of the family undergo regulated activation after secretion into the extracellular space. Cell-surface receptors and their primary intracellular signaling proteins also are related structurally and are conserved within the TGF-β superfamily. By far, the most extensively studied TGF-β superfamily member in gastrointestinal biology is TGF-β, which is the focus of this section. β Genes Transforming Growth Factor-β The three mammalian TGF-β growth factor family members have nearly identical biological activity in cell culture systems, but complex and distinctive phenotypes in gene-targeted and transgenic mouse models. The proteins are designated TGF-β1, -β2, and -β3 and are often referred to as TGF-β isoforms (26). The genes have been cloned in humans and many other species. In humans, TGF-β1, -β2, and -β3 are localized to chromosomes 19, 1, and 14, respectively (27,28). Major structural differences exist in the 5′-flanking regions of each gene, suggesting that observed differences in tissue distribution and differential regulation of TGF-β expression may be mediated by gene transcription. Transactivating and cis-acting factors in the promoters of all three isoforms have been extensively mapped and characterized (29–33). β Protein and Activation Transforming Growth Factor-β The three mammalian TGF-β isoforms share approximately 70% amino acid homology. All are first synthesized and secreted in biologically inactive, latent precursor complexes that are not structurally competent to bind to cell-surface TGF-β receptors. “Activation” of TGF-β occurs when the mature, biologically active form is liberated from the precursor complex by a process described later. Mature, activated TGF-β is a 25-kDa homodimer that functionally interacts with the receptor. The carboxy terminal 112 amino acids exhibit 70% to 75% conservation of sequence between isoforms and 100% conservation of the 9 cysteine residues. The amino-terminal precursor sequences are strikingly dissimilar (34). The overall amino acid sequence for mature isoforms is highly conserved in mammals. Expression of individual isoforms in cells and tissues in vivo is highly variable, especially during embryogenesis (35), perhaps accounting for the variable phenotype observed in gene-targeted mice. Activation of TGF-β under physiologic and pathophysiologic situations has been a relatively neglected area of TGF-β research, despite its critical importance in regulation of TGF-β activity. Latent TGF-β consists primarily of the N-terminal TGF-β precursor sequence, termed the

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 187 latency-associated peptide (LAP), noncovalently associated with the mature, C-terminal TGF-β sequence (36,37). The latent TGF-β complex also includes a second gene product, the latent TGF-β binding protein (LTBP). Four LTBPs have been cloned and fully sequenced (38). These molecules are structurally closely related to fibrillins, large extracellular glycoproteins with EGF-like and cysteine repeats. LTBPs are structurally unrelated to TGF-β and associate with the latent molecule by disulfide bridging. Mature, biologically active TGF-β is released from the latent complex by physicochemical phenomena such as extremes of pH, but the physiologic mechanism operative in vivo is less well understood. Proteolytic activation by proteases such as plasmin has been extensively characterized in vitro (39). Thrombospondin has been proposed as an attractive physiologic activator in vivo, based on the observation that plasminogen null animals do not replicate the abnormalities observed in TGF-β1–deficient mice, whereas thrombospondin null mice do (40). It has been hypothesized that thrombospondin confers a conformational change in the TGF-β latent complex by specifically binding to sequences in the LAP, releasing mature TGF-β for binding to TGF-β receptors. Other factors contributing to the activation of latent TGF-β include the presence of mannose-6 phosphate residues on the LAP (41a), transglutaminase-dependent cross-linking of latent TGF-β to ECM proteins (41b), and interaction with αvβ6 integrin (42). Finally, other extracellular proteins may bind TGF-β after activation, further modifying its activity. Some of these include decorin, biglycan, and α2-macroglobulin (43,44). β Receptors Transforming Growth Factor-β and Receptor Interacting Proteins The TGF-β family of receptors also has a complex nomenclature (45). Herein, this chapter focuses on receptors that bind the three mammalian TGF-β isoforms, fully recognizing that other members of the TGF-β superfamily such as the activins and bone morphogenetic proteins (BMPs) bind to structurally related TGF-β receptors and activate closely related signaling pathways that result in closely related biological activities. TGF-β receptors originally were described based on crosslinking patterns to 125I-TGF-β and designated receptor types I (~53 kDa), II (~70 kDa), and III (~300 kDa). Each receptor, now named TGF-βRI (or Alk5 in some systems), TGF-βRII, and TGF-βRIII, was cloned and extensively characterized in the early 1990s. The TGF-β receptors have an extracellular ligand-binding domain, a transmembrane sequence, and an intracellular sequence. Intracellular serine/threonine kinase domains are present in type I and II receptors, whereas the intracellular domain of the type III receptor has no known catalytic activity (46–48). All three isoforms of TGF-β bind to receptor types I, II, and III, but exceptions exist (see later in this chapter). Once the ligand is appropriately engaged, TGF-βRI and TGF-βRII heterodimerize to form the active

signaling complex. TGF-βRIII (betaglycan) is thought to function by binding TGF-β and increasing its affinity for TGF-βRII. TGF-β signaling is initiated from a heterotetrameric receptor complex involving TGF-βRI and TGF-βRII (49) (Fig. 7-2). In the basal state, TGF-βRII is constitutively homodimerized and constitutively autophosphorylated in a ligand-independent manner by its intrinsic serine/threonine kinase activity (50). Signaling from TGF-βRII does not occur in the absence of TGF-βRI, even when a ligand is engaged (51–53). TGF-βRI also forms ligand-independent homodimers, but it is unable to bind TGF-β in the absence of TGF-βRII. Ligand binding to TGF-βRII recruits TGF-βRI into a heterotetrameric receptor complex resulting in transphosphorylation and activation of TGF-βRI. Multiple TGF-βRII– mediated serine and threonine phosphorylation sites are present in the Gly/Ser (GS)-rich domain of TGF-βRI, which is located just C terminal to the juxtamembrane domain. A T204D (aspartic acid substitution for threonine) mutation in the GS region of TGF-βRI results in a constitutively active receptor, and this mutant receptor has become a valuable reagent for the study of TGF-β signaling in vitro (54). TGF-βRIII, or betaglycan, is an abundant, heavily glycosylated, high-molecular-weight proteoglycan that migrates diffusely in the range of 280 to 330 kDa in polyacrylamide gels (48). Endoglin, another TGF-βRIII, is found predominantly in endothelial cells (55). Mutations in an activin receptor-like kinase (Alk-1) and endoglin lead to altered TGF-β signaling and vascular arteriovenous malformations comprising the clinical entity termed hereditary hemorrhagic telangiectasia (56). The TGF-βRIII receptors are believed to function as “reservoirs,” acting as a sump for TGF-β and a presenting receptor for the active signaling TGF-βRI/ TGF-βRII complex. Like TGF-βRI and TGF-βRII, TGF-βRIII is constitutively homodimerized. Multiple lines of evidence support the hypothesis that betaglycan functions to facilitate high-affinity binding of all three TGF-β isoforms to TGF-βRII. This is especially true for TGF-β2, which has a low affinity for TGF-βRII in the absence of TGF-βRIII, but a 10-fold enhancement of activity when betaglycan is coexpressed (57). Data suggest that endoglin binds only TGF-β1 and TGF-β3 in the presence of TGF-βRII (58). Both endoglin and betaglycan can be coimmunoprecipitated with TGF-βRII (59,60). Many TGF-β receptor interacting proteins have been identified in cell culture experiments, but the exact importance of most of these in human biology needs further study. Examples of TGF-β receptor interacting proteins include the FK-506 binding protein FKBP12 (61), the α subunit of farnesyl transferase (62), serine threonine receptor-associated protein (STRAP) (63), SARA (Smad anchor for receptor activation) (64), Bα (a subunit of phospholipase A2) (65), Daxx (66), Disabled-2 (67), transforming growth factor-β receptor interacting protein (TRIP)-1 (68), tumor necrosis factor receptor-associated protein-1 (TRAP)-1 (69), TLP (TRAP-1 like protein) (70), SMIF (71), and mammalian light chain 7 (mLC7)-1 (72). All bind to cytoplasmic sequences in TGF-βRI or TGF-βRII and modulate TGF-β signaling.

188 / CHAPTER 7 Smad2

Smad4

Smad6/7 P P

P

P P

Smad3

P

P P Smad complex TGFβRI + TGFβRII P

P

P

P

Target gene -growth inhibition -differentiation

TGF TGFβ

FIG. 7-2. Transforming growth factor-β (TGF-β) signaling in the gastrointestinal epithelium. TGF-β is secreted from the basolateral surface of polarized intestinal epithelial cells and activates heterotetrameric TGF-β receptor (TGF-βR) complexes, also on the basolateral membrane domain. The primary growth inhibitory signaling pathway activated by TGF-β is the Smad pathway. Negative feedback from inhibitory Smad6 and Smad7 are also shown. The primary outcome of TGF-β signaling in the intestinal epithelium is growth inhibition.

Individual discussion of these proteins is beyond the scope of this chapter, but the role of FKBP12 on TGF-β signaling is illustrative of how these proteins potentially modulate TGF-β signaling. FKBP12 binds to the GS region of TGFβRI and is believed to help stabilize its inactive conformation. Phosphorylation of the GS region by TGF-βRII is predicted to dissociate FKBP12, creating a conformation for signaling. Thus, FKBP12 is a brake on TGF-β signaling.

Smad Signaling Is Induced by Transforming β Growth Factor-β TGF-β signaling occurs primarily by activation of Smad proteins, although a relatively substantial body of literature describes Smad-independent signaling by TGF-β (26,73). Herein, this chapter focuses on Smad signaling, a signaling cascade unique to the TGF-β pathway. Nine distinct Smad proteins have been identified in mammals. These are highly conserved signaling molecules related to “Mad” proteins in Drosophila and “Sma” proteins in Caenorhabditis elegans, hence the designation “Smads.” Smads are categorized as receptor-regulated R-Smads (Smad1, Smad2, Smad3, Smad5, Smad8, and Smad9), commonpartner Co-Smads (Smad4), and inhibitory Smads (Smad6 and Smad7) (74). For mammalian TGF-β signaling, Smad2 and Smad3 are the R-Smads, whereas activins and BMPs activate alternate R-Smads. Phosphorylation of Smad2 and Smad3 by TGF-βRI is necessary for TGF-β signaling. Phosphorylation by the TGF-βRI kinase occurs on serine residues in a highly

conserved SSXS motif in the MH-2 domain (75). Smad4, which does not contain this motif, is not phosphorylated. SARA facilitates interaction among Smad2, Smad3, and TGF-βRI (64). Phosphorylated Smad2 and Smad3 form heteromeric complexes with Smad4 (76). This configuration of Smads is believed to be required for translocation of the TGF-β signal into to the nucleus. However, variations on this “traditional” model of TGF-β signaling are increasingly being invoked to explain differences in the genomic and biological responses to TGF-β in models of diminished or deficient Smad2 or Smad3 function (see review by Flanders [77]). TGF-β signaling is negatively regulated at multiple points in the ligand/receptor/signaling cascade. Key proteins involved in negative regulation of mammalian Smad signaling are Smad6 and Smad7 (78,79). Although structurally related, Smad6 and Smad7 lack the SSXS phosphorylation site required for activation of Smad2 and Smad3. Both are induced by TGF-β treatment and exert negative feedback on TGF-β signaling (80). Smad7 binds to activated TGF-βRI and inhibits phosphorylation of Smad2, assembly of Smad complexes, and nuclear translocation of Smads (79). Smad6 and Smad7 also recruit E3-ubiquitin ligase activities known as Smurfs to the activated TGF-βRI, resulting in ubiquitination and degradation of the receptor (81). Other potent negative regulators of Smad function are the c-SnoN and c-Ski nuclear oncoproteins. C-SnoN and c-Ski interact with Smad2, Smad3, and Smad4 in the nucleus to repress transcription of TGF-β–responsive genes (82). After TGF-β exposure, c-SnoN is rapidly targeted for ubiquitin-dependent degradation by the proteasome (83), releasing TGF-β target genes from

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 189 transcriptional repression. Later, c-SnoN is markedly up-regulated, a presumed mechanism for negative feedback of TGF-β signaling (84). As the Smad complex accumulates in the nucleus in response to TGF-β signaling, transcription is modulated, both positively and negatively, by direct binding to DNA and interaction with a large number of potential DNA-binding proteins. Smad3 and Smad4 bind to the Smad-binding element (SBE) with low affinity, whereas Smad2 does not bind at all (85). The SBE consists of the 4-base-pair sequence 5′-AGAC-3′ that contacts a specific domain in the MH-1 region of Smad3 and Smad4. Because the SBE is short, interactions with Smad3 and Smad4 occur with low affinity. The SBE also occurs often in the mammalian genome. Thus, it is interesting that TGF-β does not signal promiscuously and nonspecifically. TGF-β also leads to both gene activation and gene repression in a given cell, depending on the target gene. These complexities are explained by the extensive repertoire of cell type–specific transcriptional corepressors and coactivators available to form a transcriptional complex with Smad proteins. In their review, Shi and Massague (26) point out “much work is needed for the delineation of cell type specific gene responses that mediate the action of the TGF-β family.” Although a great deal is known about the multifaceted biological response to TGF-β, the specific transcriptional programs induced by a growth inhibitory versus an angiogenic response, for example, is likely a function of the specific expression and stoichiometry of Smad coactivators and repressors within a given cell type. That is, the TGF-β response is read in different ways depending on the cellular context and the portfolio of available transcription factors. Consideration of all the potential DNA-binding partners is beyond the scope of the present application; however, many of these are catalogued in Table 7-1 and include well-characterized transcriptional modulators such as histone deacetylases and intermediate binding partners such as mSin3A and NcoR (86). Several merging concepts in TGF-β signaling deserve comment. These relate to progress in the understanding of how Smad proteins are partitioned between the cytoplasm and nucleus in the quiescent state and in response to receptor activation. First, Smad distribution within the cell is actively TABLE 7-1. Smad-associated transcriptional regulators Sequence-specific transcription factors Jun/Fos Sp1 Basic helix-loophelix (bHLH) CREBP E2F ATF-2 β-catenin Others

Coactivators

regulated by nucleocytoplasmic shuttling. It is clear that Smad2 and Smad3 are predominantly retained in the cytoplasmic compartment in the absence of TGF-β, but Smad4 is distributed in both the nuclear and cytoplasmic compartments. Evolving theories and pathways are being explored to more clearly understand how nuclear pore complexes and cytoplasmic and nuclear retention factors contribute to Smad localization and function (87). Second, it has become clear that TGF-β receptor signaling and Smad activation occur in distinct endocytic compartments, including caveolin-1 lipid raft–dependent and clathrin-dependent pathways. Segregation of receptor and Smads in these compartments may contribute to the intensity and duration of Smaddependent TGF-β signaling (88). Finally, it has been determined that Smad translocation into the nucleus is critically dependent on the scaffolding protein ELF, because ELF β-spectrin–deficient mice have a phenotype similar to Smad mutant mice (89). A variety of additional signaling pathways are activated in response to TGF-β. In some instances, these pathways directly influence Smad signaling, whereas in others, signaling may occur in parallel with or independent of Smad activation. A critical challenge in the field will be to integrate all TGF-β signaling into a clear comprehension of how the multitude of TGF-β biological responses occurs. In some cell types, TGF-β induces mitogen-activated protein kinase (MAPK) activity (90), whereas it does not in others (91). It has been suggested that increased MAPK activity in oncogenic Ras-transformed cells phosphorylates Smad2 and Smad3 in the linker domain, resulting in defective translocation of the Smad complex into the nucleus and TGF-β resistance (92). Conversely, others have found that EGF and HGF both appear to phosphorylate Smad2 and induce Smad2dependent transcriptional responses (93). These interactions clearly deserve further study. c-Jun N-terminal kinases (JNKs) phosphorylate Smad3 outside its -SSXS motif and facilitate TGF-β signaling (94). A TGF-β–stimulated MAPK kinase kinase (TGF-β activated kinase [TAK-1], TGF-β activated kinase 1) and downstream cascade has been identified (95), together with activating, interacting proteins TAK-1 binding protein (TAB)1 and TAB2 (96). TAK-1 activity simulates nuclear factor (NF)-κB activation (97). TGF-β signaling via protein kinase C (PKC) (98), Rho-like GTPases (99,100), p38 (101), and JNKs (94,99,102) have been reported and are reviewed extensively by Yue and Mulder (73).

Corepressors

p300/CBP ARC105 Smif

c-ski c-SnoN TGIF

Others

Histone deacetylase complex (HDAC) mSin3 N-CoR Others

CBP, CREB-binding protein, CREBP, cyclic AMP response element binding protein.

β Biological Actions of Transforming Growth Factor-β in the Gastrointestinal Tract TGF-β is described as a “multifunctional” or “pleiotrophic” peptide because of its diverse biological activities, many of which have important implications for gastrointestinal homeostasis. Alterations in TGF-β signaling have been extensively studied in the context of gastrointestinal neoplasia. These and other diverse activities of TGF-β are reviewed later.

190 / CHAPTER 7 Differentiation and Inhibition of Cell Growth The three mammalian TGF-β isoforms are potent and reversible growth inhibitors in epithelial cells, including those of the gastrointestinal tract (103,104). Although TGF-β is a differentiating factor in certain cellular contexts (105), a complete program of enterocyte differentiation is not induced by TGF-β. Under precise experimental conditions, TGF-β may induce limited characteristics of the differentiated phenotype in vitro (103,106). TGF-β arrests cells growth in the mid and late G1 phase of the cell cycle by inhibition of critical cellular events required for completion of G1 traverse and S-phase entry. Important early G1 events include inhibition of the growthpromoting transcription factor c-myc (107) and stimulation of c-jun expression (108). Late G1 events include inhibition of expression or activity of cyclin-dependent kinase activities (e.g., cyclin D, cyclin E, cyclin A, and their respective catalytic partners, the cyclin-dependent kinases: cdk2, cdk4, and cdk6) (26,109–114). TGF-β may also stimulate cyclin-dependent kinase inhibitor activities such as p21/waf1/cip1 (115), p27/kip (116), and p15/ink (117). To a large extent, the specific mechanism appears to be dependent on the cell type being studied. In cultured intestinal epithelial cells, down-regulation of cyclin D1 expression is a key step in G1 arrest induced by TGF-β (118). A result of depressed cyclin-dependent kinase activity is inhibition of phosphorylation of the retinoblastoma gene product, leading to a late G1 cell cycle arrest (111). Apoptosis, or programmed cell death, is critical for maintenance of the gastrointestinal epithelium. Although TGF-β induces or suppresses apoptosis depending on the cell type being studied, in the intestinal epithelium, TGF-β induces apoptosis in vitro (119,120) and in vivo (121). The mechanisms regulating TGF-β induction of apoptosis in the intestine are not well understood, but they appear to be Smad3 dependent (120) and mediated in part by down-regulation of Bcl-2 expression (122). In summary, TGF-β is a critical autocrine and paracrine growth inhibitor in epithelial cells, including those of the gastrointestinal tract. Consequently, it is not surprising that TGF-β, its receptors, and its signaling transduces all have tumor suppressor functions; this concept is explored later in this chapter. Although TGF-β is widely appreciated as a potent growth inhibitor in epithelia, it stimulates growth of fibroblastic cells. The kinetics of TGF-β–stimulated growth are delayed, suggesting involvement of a secondary mitogen. This is believed to occur by induction of the profibrotic cytokine, plateletderived growth factor (PDGF) (123). One reported study indicates that Smad-independent activation of c-abl kinase activity may be an important mediator of the proliferative effect of TGF-β on fibroblasts (124). Induction of Extracellular Matrix Formation and Fibrosis A major biological effect of TGF-β is modulation of cells and genes that contribute to ECM deposition. The result is a

prominent increase in ECM formation, recruitment of fibroblasts, and fibrosis. As a consequence of this potent activity, TGF-β has been implicated in fibrotic diseases of virtually every organ system, including the intestine, pancreas, and liver. TGF-β induction of fibrosis occurs by multiple pathways, including increased synthesis of ECM proteins (collagen, fibronectin) and proteinase inhibitors (plasminogen activator inhibitor), decreased synthesis of matrix metalloproteinases (MMPs; plasminogen activator, collagenase, elastase), and modulation of matrix receptors and binding proteins (integrins). Several comprehensive reviews highlight the effect of TGF-β on ECM (125–127). It has been of long-standing interest to determine whether signaling pathways responsible for the fibrogenic response to TGF-β are separate and distinct from the growth responses. This distinction remains relatively unclear but is an area of intense ongoing investigation, because it may ultimately have important therapeutic consequences. It appears increasingly clear that most of the fibrogenic responses to TGF-β are dependent on Smad3, because Smad3-deficient mice are resistant to fibrosis in several models (77). Although these studies are convincing, it remains difficult to reconcile these observations with other published work. For example, in a widely referenced study, Hocevar and coworkers (128) described fibronectin synthesis in a Smad4-deficient fibrosarcoma cell line, indicating independence from Smad4, and presumably Smad3, signaling. The effect of TGF-β on ECM synthesis appears to have clinical significance because a large number of fibrotic diseases are associated with increased TGF-β activity, and TGF-β increases wound healing in several models, including incisional wounds in the gastrointestinal tract (129). This may occur as a result of increased synthesis of collagen by human intestinal smooth muscle cells (130). Notably, however, unexpected observations have been made in models of wound healing in Smad3 gene–targeted mice. In these mice, wound healing is accelerated in the absence of Smad3. More analysis is needed to ascertain the role of TGF-β in the complicated milieu of a wound, which includes inflammatory cells, epithelial cells, fibroblasts, and myofibroblasts that respond in well-characterized stages of wound healing such as reepithelialization and wound contraction. Each of these stages may be differentially affected by TGF-β. Effect on Immune Function TGF-β is produced by many immune system cells, including T cells, B cells, macrophages, neurokinin cells, and others. The effects of TGF-β on the immune system are complex because both proinflammatory and immunosuppressive activities are modulated by TGF-β (131). The marked inflammation observed in the gastrointestinal mucosa of TGF-β1 gene–targeted mice (see later in this section) underscores the importance of TGF-β1 as an immune suppressor in the intestine (131–133). In mice, TGF-β promotes the expansion and suppressive function of CD4+/CD25+ T-regulatory cells (134), perhaps by induction of the transcription factor Foxp3.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 191 Transgenic mice with impaired TGF-β signaling in T cells are more susceptible to dextran sodium sulfate–induced colitis (134). Similarly, transgenic mice overexpressing a dominant negative TGF-βRII directed to the intestinal epithelium by an intestinal trefoil factor promoter or a liver fatty acid binding protein promoter are also markedly more sensitive to induction of colitis by dextran sodium sulfate (135,136). Interestingly, TGF-β levels are increased in the colonic mucosa of patients with inflammatory bowel disease, a disorder characterized by excessive immune response to mucosal antigens (137). This apparent paradoxical observation has been potentially explained by increased mucosal levels of inhibitory Smad7 and impaired TGF-β signaling in active inflammatory bowel disease (138). One of the most prominent effects of TGF-β on immune cell function is its activity as an IgA switch-inducing factor in naive B cells. TGF-β induces IgA− B cells to initiate IgA synthesis and secretion, which is a key observation because IgA is the dominant isotype in the gastrointestinal tract (139,140). The immunosuppressive effects of TGF-β may also have important implications for colorectal tumorigenesis. Early studies in fibrosarcoma cells suggested that TGF-β suppresses tumor immune surveillance, perhaps favoring establishment of metastasis (141). More recent studies show that TGF-β production by tumor-infiltrating T lymphocytes suppresses tumor growth (142). Stimulation of Cell Migration and Angiogenesis TGF-β promotes cell migration and chemotaxis in a broad variety of cell types, including fibroblasts, neutrophils, glial cells, and intestinal epithelial cells (126,143,144). The concentration of TGF-β required to stimulate migration is far less than that required for modulation of cell growth. In a model of intestinal epithelial wounding, TGF-β expression is upregulated, and restitution of the epithelial surface is accelerated (145). Conversely, loss of TGF-β signaling diminishes wound healing and renders mouse models more susceptible to chemically induced colitis (136). TGF-β appears to be much less potent than growth factors such as EGF or HGF in models of chemotaxis (directed migration) in intestinal epithelial cells (144). Some studies suggest migration in epithelial cells is stimulated by activation of Smad-dependent pathways, as well as p38 and JNK pathways (146). As a result of its pleiotrophic effects on cellular migration, cell proliferation, and ECM deposition, TGF-β is a major contributor to blood vessel growth and angiogenesis (147). Defects in vasculogenesis occur in TGF-β1 (148), TGF-βRI (147), and TGF-βRII gene−targeted mice (149). TGF-β stimulates expression of genes such as vascular endothelial growth factor (VEGF) (150,151), basic fibroblast growth factor (bFGF), and cyclooxygenase-2 that contribute to angiogenesis, as well as a large number of genes expressed in endothelial cells related to matrix deposition, a necessary precursor for angiogenesis (150,152). VEGF- and bFGFstimulated invasion of matrices in vitro is further supported

by low concentrations of TGF-β (pg/ml range) (153). The proangiogenic properties of TGF-β are believed to contribute to its tumor-promoting effects late in oncogenesis (154). Transforming Growth Factor-β Expression in the Normal Gastrointestinal Tract In the mouse intestinal epithelium, immunoreactivity for all three TGF-β isoforms is seen beginning midgestation (155). All three TGF-β isoforms are found in the adult normal gastrointestinal mucosa (10,104,156), but the distribution along the vertical axis of the crypt-villus unit and the epithelial and nonepithelial compartments of the intestine has been reported in various forms. The preponderance of data suggest TGF-β1 immunostaining in the colon is greatest in differentiated epithelial cells, although expression in lamina propria cells is also quite prominent (104,121,137,156–162). Expression of TGF-βRII protein in the murine and human small intestine and colonic epithelium occurs in a distribution nearly identical to ligand; that is, increased expression in the differentiated compartment and little or no expression in the proliferative compartment (156,163,164). Expression of TGF-βRI and TGF-βRIII in normal intestine has not been examined extensively, but it appears to mirror, in part, the distribution of ligand (165). Studies show that TGF-βRII is localized and activation of Smad phosphorylation occurs from the basolateral domain of polarized intestinal epithelial cells (166). Collectively, data on the distribution of TGF-β and its receptors in the gastrointestinal epithelium support the view that TGF-β is a major autocrine or paracrine inhibitory axis, or both, in the intestinal epithelium. A Complex Role for Transforming Growth Factor-β in Gastrointestinal Cancer Resistance to the tumor-suppressive actions of TGF-β is common and occurs by genetic and epigenetic mechanisms. Progress has delineated well-described genetic defects in the TGF-β tumor suppression pathway. Examples of genetic defects include: (1) inactivating mutations in noncoding polyadenine repeats of the TGF-βRII gene in individuals with microsatellite instability, including hereditary nonpolyposis colon cancer syndrome (167,168); (2) sporadic genomic mutations in TGF-βRII (169,170); (3) mutations in Smad proteins including sporadic, inactivating, missense mutations in Smad2 and Smad4 (171–173) and germline Smad4 mutations, primarily in exon 9, in juvenile polyposis (174). Epigenetic defects in TGF-β signaling are also increasingly recognized. Epigenetic defects in TGF-β signaling also occur in a significant fraction of colon cancer cells, such that in total, more than 90% of colorectal cancers are resistant to growth inhibition by TGF-β. An important epigenetic mechanism for resistance to growth suppression by TGF-β is oncogenic activation of Ras, a guanine nucleotide binding protein that plays a central role in organizing and transmitting cellular growth signals. Activating Ras mutations occur in more than 50% of

192 / CHAPTER 7 colorectal cancers (175), and Ras-transformed cells in culture are resistant to growth inhibition by TGF-β (92,176–182). Proposed mechanisms, among others, include decreased expression of TGF-βRII (177,182), ineffective Smad complex assembly and nuclear translocation (92), and degradation of Smad4 before nuclear translocation (183). For example, Smad2 and Smad3 do not accumulate in the nucleus of TGF-β–resistant mammary and lung epithelial cells overexpressing oncogenic Ras (92). This observation is explained by Ras-mediated phosphorylation of extracellular signal– regulated kinases 1 and 2 (ERK1/2) consensus sites in the linker region of Smad2 and Smad3, a site distinct from the TGF-β phosphorylation site in the SSXS domain. The result is inhibition of Smad accumulation in the nucleus. An analogous Ras-mediated phosphorylation event interferes with translocation of Smad1 in BMP-treated cells (184). Using an inducible Ras model, investigators have observed Smad4 degradation in the cytoplasm, prohibiting accumulation of a functional transcriptional complex in the nucleus (183). In contrast, studies in TGF-β–resistant, Ras/ErbB2-transformed, mammary epithelial cells show that Smad complexes are able to translocate into the nucleus and activate transcription from the Smad7 promoter (185). Others have also found that Smad complexes accumulate in the nucleus without hindrance in Ras-transformed intestinal cells, but their function is inhibited by interference with the transcriptional repressor TIEG (186). EGF and HGF both appear to induce Smad2dependent transcriptional responses, presumably by stimulation of ERK activity (93). In aggregate, these studies indicate that the effect of Ras on Smad signaling is dependent on the cell line being studied, specific technical aspects of the assay being used, or the stoichiometry of Smad complex formation in individual cell types. It has been established that tumor-suppressive TGF-β activity occurs by growth inhibitory Smad-dependent signaling. Until recently, however, the “double-edge” role of TGF-β as both a tumor suppressor and a tumor promoter could only be indirectly inferred (128,147,187). For example, it has been noted often that TGF-β levels are increased in metastatic tumors, including those of the gastrointestinal tract, potentially further driving prooncogenic signaling (188,189). In fact, increased expression of TGF-β is a risk factor for recurrence and reduced survival of colorectal cancer (190). Metastatic lesions express relatively more TGF-β1 than primary colorectal cancers, and the serum of patients with colorectal cancers has approximately fourfold more TGF-β than that of control subjects (191). Some colon carcinoma cell lines are growth stimulated by TGF-β (192). In colorectal cancers with mutant, inactivated TGF-βRII in which there is presumably no potential for prooncogenic signaling, the prognosis is better than in individuals with retention of one allele (193). Conversely, restoration of TGF-βRII in cells that carry a mutation in this gene increases cell invasiveness in vitro (187). Thus, once tumors escape growth regulation by TGF-β, if the potential remains for residual TGF-β signaling, the result is an enhanced potential for tumorigenesis.

More definitive evidence has emerged in support of tumor promotion by TGF-β using bitransgenic mice that overexpress the ErbB2 oncogene and a mutant-activated TGF-β receptor, each under the direction of a mouse mammary tumor virus (MMTV) mammary epithelial cell promoter (194). In these mice, induction of tumor formation by ErbB2 is delayed because of tumor-suppressive actions of TGF-β. However, an increased percentage of metastatic foci developed in the lungs, which is consistent with a prooncogenic effect for TGF-β later in the stages of tumor formation. Similar tumor-promoting actions of TGF-β were reported in cyclin D1/TGF-β1 bitransgenic mice (195). It is clear that events such as Ras transformation may have an additive adverse interaction with TGF-β signaling. In Ras-transformed cells, loss of autocrine growth inhibitory TGF-β signaling occurs coincident with a synergistic stimulation of angiogenic molecules (196), matrix molecules (197), and epithelial-to-mesenchymal transition (187), which is believed to be a key factor in tumor growth and metastasis in vivo. Ras overexpression also transcriptionally activates the TGF-β1 gene (198). Increased levels of TGF-β in this context further contribute to “undesirable” tumor-promoting signaling in growth-resistant cells.

Lessons from Gene-Targeted and Transgenic Mice Use of gene-targeted and transgenic mice has become a valuable methodology for study of TGF-β biology, especially relating to carcinogenesis. Illustrative examples have been cited throughout this chapter, and this section describes additional mouse models. Several investigators nearly simultaneously accomplished disruption of TGF-β1 by homologous recombination in the early 1990s (132,133,148). Embryonic lethality caused by defective yolk sac vasculogenesis is observed in approximately half of TGF-β1 null mice (148). In the remainder, no developmental defect is observed at birth. At 2 to 3 weeks of age, the deficient animals experience development of a rapidly fatal wasting syndrome characterized by massive infiltration of multiple organs with mixed inflammatory cells. Inflammatory lesions are most prominent in the heart, lungs, and gastric mucosa. The small bowel and colon are less involved. Peyer’s patches are small. These studies suggest an important role for TGF-β1 in immune homeostasis. Housing mice in a germ-free environment does not ameliorate inflammation, indicating independence of the inflammatory process from microorganisms (199). The predominant phenotype in TGF-β2 knockout mice is perinatal mortality caused by multisystem developmental abnormalities (200). TGF-β3 – deficient mice have defective palate formation (201). No overt abnormalities in gastrointestinal development are observed in TGF-β2 and TGF-β3 knockout mice. The rapidly lethal phenotype in TGF-β1 gene–targeted mice was cleverly circumvented by breeding TGF-β1–deficient mice with Rag2-/- mice that lack both B and T cells (202). Survival was prolonged, and carcinoma of the cecum and

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 193 the three knockout approaches, a lethal wasting syndrome develops early in life and is associated with immunodeficiency (208,209). In the remaining model, metastatic colon adenocarcinoma was found at 4 to 6 months of age in Smad3-deficient mice (210). Compound mutant Smad4 and adenomatous polyposis coli (APC) mice experience development of more aggressive tumors than APC mice alone, indicating cooperativity in these two tumor-suppressive pathways (211).

colon developed as early as 3 months of age, indicating loss of tumor suppression by TGF-β. Overexpression of TGF-β in the liver of transgenic mice has been reported to cause hepatic fibrosis and atrophy of the pancreas (203). Overexpression in the pancreas causes chronic pancreatitis and fibrosis (204). An attractive alternate approach for determination of the roles for TGF-β in development has been study of mice with a dominant-negative TGF-βRII transgene, thereby circumventing the “problem” of ligand redundancy. A dominant-negative TGF-βRII under control of a metallothionein (MT) promoter results in fibrosis of the pancreas together with macrophage inflammation, neoangiogenesis, and defective acinar cell differentiation (205). No discernible effects on the liver or other gastrointestinal structures were observed. Disruption of the gene encoding LTBP results in reduced deposition of TGF-β in ECM, decreased phosphorylation of Smad2, and development of colorectal cancer (206). This observation highlights the biological importance of the latent form of TGF-β. Targeted mutagenesis of various Smad genes has been accomplished by a number of investigators, and the characteristics of these mice have been summarized in an excellent review (207). Deletion of Smad2 and Smad4 results in profound defects in embryologic cellular differentiation and is lethal in the perigastrulation interval. Heterozygotes show no abnormalities. Targeted deletion of Smad3 has been accomplished by three groups of investigators. Gross defects in embryogenesis are not observed, although in two of

EGF TGFα AR EPIGEN

EPIDERMAL GROWTH FACTOR FAMILY OF PEPTIDES AND RECEPTORS Overview and Discovery of the Epidermal Growth Factor Family The EGF-related family of regulatory peptides shares sequence homology, affinity for the same receptors, and a profile of similar biological activities (212,213). All EGFrelated peptides are expressed in the gastrointestinal tract, although there are important differences in the distribution and circumstances of expression (214). These proteins are defined by high-affinity binding to the EGF receptor (Fig. 7-3) and conserved spacing of six cysteine residues and intervening amino acids in the mature peptide sequence according to the formula XnCX7CX4CX10CXCX5GX2CXn (212,213). Using this definition, the mammalian EGF family now includes the following seven members: EGF, TGF-α, amphiregulin (AR),

ErbB2: No ligand, preferred heterodimerization partner for all other ErbB receptors, ErbB2/ErbB3 is most active

BTC HB-EGF EPR

Heregulins Neuregulins

ErbB-1: Traditional EGF receptor, commonly overexpressed in GI neoplasia

Extracellular domain

Cysteine-rich domain TM domain

Transmembrane domain

Kinase domain

Cytoplasmic domain ErbB-1 HER1 EGFR

ErbB-2 HER2 Neu

ErbB-3 HER3

ErbB-4 HER4

ErbB4: Stimulates cellular migration when expressed alone

ErbB3: Lacks kinase activity; contains 6 putative PI3K binding sites

FIG. 7-3. Epidermal growth factor (EGF) receptor family. The four mammalian EGF receptors (EGFRs) are shown, together with their ligand specificities. The text boxes illustrate some of the distinctive features of each receptor. Note that ErbB2 and ErbB3 do not have specific high-affinity binding for members of the closely related EGF peptide family, but signaling may occur as a result of heterodimerization with ErbB1 or ErbB4. AR, amphiregulin; BTC, betacellulin; EPR, epiregulin; GI, gastrointestinal; HB-EGF, heparin-binding epidermal growth factor–like growth factor; HER1, human epidermal growth factor receptor 1; PI3K, phosphatidyl inositol kinase; TGF-α, transforming growth factor-α; TM, transmembrane.

194 / CHAPTER 7

TGFα

AR

HB-EGF

BTC

EPR

EPIGEN

EGF Ectodomain cleavage sites EGF-like domain Heparin-binding domain

FIG. 7-4. Epidermal growth factor (EGF)–related peptide family. Diagram showing each transmembrane precursor of the seven EGF-related peptides discussed in this chapter. Note that amphiregulin (AR) and heparin-binding epidermal growth factor–like growth factor (HB-EGF) are unique in the occurrence of heparin binding domains. All are processed proteolytically to yield the “mature” peptide sequence. BTC, betacellulin; EPR, epiregulin; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PI3K, phosphatidyl inositol kinase; TGF-α, transforming growth factor-α.

heparin-binding epidermal growth factor–like growth factor (HB-EGF), betacellulin (BTC), epiregulin (EPR), and epigen; these are shown in Figure 7-4. Each is synthesized as a glycosylated integral membrane propeptide with functionally distinct cytoplasmic, transmembrane, and extracellular domains. The extracellular domain includes a mature peptide sequence that is processed by regulated proteolytic cleavage by ADAM (a disintegrin and metalloprotease) sheddases (215). There are a large number of more distantly related EGF-like molecules and molecules that contain EGF-like repeats. These include trefoil peptides, cripto and various viral proteins, among others. Glial growth factor, acetylcholine receptor–inducing activity, heregulin, and neu differentiation factor (now collectively termed the neuregulins) are distantly related to EGF and bind to EGFR-like molecules, but they are not further referenced in this chapter. Many key regulatory proteins have single or multiple EGF-like repeats that uniquely contribute to their biological function.

Epidermal Growth Factor Epidermal Growth Factor Gene and Protein During the past half century, the study of EGF, its receptors, and signaling pathways has defined many important paradigms for understanding modern-day molecular and cell biology. As a consequence of career-long work in this field, Stanley Cohen and Rita Levi-Montalcini were awarded the Nobel Prize in 1987 for their seminal studies of EGF. As discussed throughout this chapter, more key emerging therapeutic modalities are relevant to EGF biology than to any other growth factor. The human EGF gene, located on chromosome 4q25-27, is 120 kb in length and contains 24 exons, 23 introns, and 9 EGF-like repeats (216–218). The 1217-amino-acid

transmembrane precursor is encoded by a 4.7- to 4.9-kb mature RNA species. Promoter sequences that regulate expression of EGF are not well understood, and factors that regulate EGF expression largely remain unknown (219). The mature, soluble peptide is 53 amino acids in length. The major sites of EGF synthesis under basal physiologic circumstances are the salivary glands and the kidney (220). Extremely low levels of EGF mRNA are detectable in the lactating mammary gland, the pancreas, and the duodenum. EGF immunoreactivity has been reported in a variety of gastrointestinal cells and tissues, including the mouse and human pancreas, the liver, Brunner’s glands of the duodenum, the normal stomach, and the normal esophagus. In aggregate, these observations of low-level EGF expression should be viewed with caution, because RNA levels are low or undetectable, and immunohistochemical findings have not been confirmed in other studies (10,11,221–232). These discrepant findings may be due to antibody cross-reactivity with other EGF-related peptides (especially in early years of study), developmental regulation of EGF expression, species variability, and other technical differences. A study using a semiquantitative polymerase chain reaction (PCR) assay detected EGF, TGF-α, AR, HB-EGF, BTC, and EPR transcripts throughout the gastrointestinal tract (233). Notwithstanding these discrepant observations, it is clear that gastrointestinal secretions have sufficient EGF concentrations, at least from the salivary glands, to exert a biological effect on the gastrointestinal mucosa (234,235). EGF expression may be induced in certain pathophysiologic states. For example, although undetectable in the normal liver, EGF expression is induced in the liver after partial hepatectomy and may be up-regulated in cirrhosis (224,225). In the context of chronic intestinal injury and ulceration, a novel EGF-producing epithelial cell lineage emerges in the intestine, perhaps contributing to epithelial restitution and

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 195 repair (13). EGF secretion by the salivary gland and pancreas can be stimulated by drugs and gastrointestinal hormones such as secretin, pentagastrin, theophylline, and sildenafil (234,236). Although aberrant EGF expression occasionally is detected in gastrointestinal neoplasia, including pancreatic (223,237,238), esophageal (239), gastric (230,240–242), hepatocellular (243), and colorectal carcinoma (244), far more attention has been given to overexpression of other EGFrelated peptides in these cancers (see later). The potential for EGF to contribute to gastrointestinal neoplasia certainly exists as suggested by the occurrence of hepatocellular carcinoma in transgenic mice overexpressing a secretable form of EGF (243). Biological Effects of Epidermal Growth Factor in the Gastrointestinal Tract Hundreds of studies have been published on EGF and gastrointestinal function. Multiple examples are given in the subsequent section on TGF-α, the rationale being that EGF and TGF-α bind the same receptor, and a nearly identical spectrum of biological activity has been reported. The basis for focusing on TGF-α is that it appears to be the more physiologic ligand, at least in the healthy adult gastrointestinal tract. Substantive differences in the mitogenic activities of EGF and TGF-α in gastrointestinal cell cultures have not been found (214,245). The nonmitogenic effects of EGF and TGF-α on the gastrointestinal tract, such as inhibition of gastric acid secretion, are also similar. Any proposed differences can potentially be related to varying loci of growth factor production, subtleties in receptor binding, targeting to domains of polarized cells, intracellular routing, and differential stability in gastrointestinal fluids. The result is, at most, a modest difference in bioactivity (246).

α Transforming Growth Factor-α Transforming Growth Factor-α Gene and Protein TGF-α was discovered as a secreted, EGF-like transforming activity in virally transformed cells. The human TGF-α gene is located on chromosome 2p11-13, contains 6 exons, and spans 70 to 100 kb of DNA (247,248). Its 4.5- to 4.8-kb TGF-α mRNA transcript encodes a 160-amino-acid integral transmembrane precursor protein. A signal peptide in the amino terminus is cleaved before insertion of the TGF-α precursor into the cell membrane. The extracellular domain includes a “mature” 50-amino-acid sequence that is 35% identical to EGF. The 23-amino-acid hydrophobic transmembrane domain is followed by a 39-amino-acid intracellular “cytoplasmic tail” that contains 7 cysteine residues, some of which are covalently linked to palmitate (249). The reported sizes of the mature TGF-α protein range from 5 to 30 kDa, a variation that may reflect differential glycosylation and proteolytic cleavage.

Mature TGF-α is cleaved from its membrane-bound precursor by a zinc-dependent metalloprotease disintegrin tumor necrosis factor–converting enzyme (TACE), also known as ADAM17 (250). This process is termed ectodomain shedding. ADAM-mediated proteolysis has emerged as a critical step in regulating the bioactivity of TGF-α and other EGF-related peptides. The ADAM proteases and shedding of EGF-related peptides are discussed separately later. Early studies performed before the discovery of ADAMs showed that the initial step in release of mature peptide is rapid cleavage at an extracellular N-terminal alanine/valine site (215,249). Release of mature peptide results from a slower proteolytic cleavage step at alanine/valine residues that flank the C terminus of the mature sequence (251). Factors that regulate cleavage include a carboxy-terminal valine in the cytoplasmic tail, adenosine triphosphate, PKC, and G-protein signaling pathways (249,251–253). TGF-α mRNA and protein are widely expressed in normal cells and tissues, including those of the gastrointestinal tract (254,255). The TGF-α promoter has been extensively characterized, is GC rich, does not have a TATA box, and is highly dependent on the Sp1 transcription factor (256). Additional promoter elements include binding sites for AP-2 (257), p53 (258), and TEF1 (259); sequences responsive to tumor necrosis factor-α (TNF-α) (260); estrogen (261); and a site responsive to EGF and TGF-α (262). The latter element is of interest because autoinduction and cross-induction between EGF-related peptide family members has been described in vivo and in vitro (245,263,264). This phenomenon underscores the complexity of interactions within the EGF-related peptide family. Transforming Growth Factor-α Expression and Regulation of Proliferation in the Liver, Pancreas, and Intestine Hepatic TGF-α mRNA appears early in gestation in the rat, peaks during embryogenesis, and subsequently declines approximately 90% from late gestation to adulthood (265,266). The normal adult liver is a quiescent organ, and TGF-α expression is low. However, expression increases in scenarios marked by robust hepatocyte proliferation, a finding that is compatible with TGF-α and EGF being potent autocrine mitogens for hepatocytes (267). For example, in the partial hepatectomy model, 70% resection of liver mass initiates a burst of hepatic DNA synthesis within 12 to 15 hours, resulting in a complete recovery of liver mass in 96 hours (268). Hepatic TGF-α mRNA begins to increase within 6 to 8 hours of a partial hepatectomy and peaks at about twice baseline levels at 24 hours, the time of peak DNA synthesis (269,270). TGF-α protein levels increase approximately 13 hours after partial hepatectomy, coincident with the onset of DNA synthesis (271), and peak about twofold greater than baseline levels (272). TGF-α levels also increase the blood of rats subjected to partial hepatectomy (273). Notwithstanding, TGF-α is not the primary stimulus for hepatocyte proliferation after partial resection, because a normal

196 / CHAPTER 7 proliferative response to hepatectomy is observed in TGF-α null mice, suggesting other EGF-related peptides may compensate for TGF-α deficiency (274). Overexpression of TGF-α in transgenic mice increases liver weight and DNA content twofold to threefold, and the fraction of cells engaged in DNA synthesis fivefold (275,276). Multifocal, well-differentiated hepatocellular carcinomas (277) develop in some strains of TGF-α transgenic mice, and coexpression of c-myc accelerates this process (278). In humans with hepatoblastoma and hepatocellular carcinoma, TGF-α is expressed at increased levels both in serum and tumors (279–281). A key role for EGF-related peptide signaling in a rat model of hepatocellular carcinoma is suggested by the prevention of tumor formation by administration of gefitinib, an EGFR tyrosine kinase inhibitor (282). In the pancreas, TGF-α mRNA and immunoreactivity are detected in fetal pancreatic acini, ductular epithelium, and in islet cells, whereas expression in adult pancreas is predominantly in the ductular epithelium (223,254,283,284). No abnormalities in pancreatic morphology or function are noted in TGF-α knockout mice (285,286), indicating the growth factor is dispensable for normal pancreatic development. Studies in a variety of in vivo and in vitro models indicate that both EGF and TGF-α stimulate cellular proliferation in the pancreas, predominantly in ductal cells (287–291). TGF-α is overexpressed in pancreatic cancers and is increased in the serum of patients with pancreatic carcinoma (237,238,292). Coexpression of the receptor and either EGF or TGF-α occurs in 38% of tumors and correlates with advanced disease. Overexpression of TGF-α in the pancreas of transgenic mice (275,276) results in interstitial pancreatic fibrosis, multifocal pseudoductular acinar metaplasia, and sometimes pancreatic carcinoma (293). Development of pancreatic carcinoma is accelerated in TGF-α /c-myc (294) and TGF-α /p53−/− bitransgenic mice (295). Several interesting observations suggest that TGF-α and other EGF-related peptides are important in regulation of progenitor cell populations in the pancreas. For example, the homeobox gene Pdx1, a marker for primitive pancreatic epithelium, is increased in MT-TGF-α transgenic mice (296a). Newly formed β-cell populations in a ligated pancreatic duct model are associated with increased expression of HB-EGF and BTC, as well as TGF-α (296b). Finally, evidence suggests that TGF-α activates expression of Notch signaling, which is important in the regulation of cell fate decisions, and together these proteins contribute to pancreatic carcinogenesis (297). Overexpression of EGF in pancreatic islets causes disorganization and enlargement of islets of Langerhans. Glucose homeostasis is not disturbed (298). TGF-α is readily detectable in gastrointestinal tissues in fetal and adult life (228,299–302). In humans, expression is found as early as the 10th week of gestation and persists throughout the gastrointestinal tract into adulthood. The preponderance of evidence suggests that TGF-α expression is greatest in differentiated epithelial cell compartments of the gastrointestinal tract, implying that the nonmitogenic actions of TGF-α may predominate under

normal physiologic circumstances. This assumes that physical access of this autocrine factor to the proliferative cell compartment may be limited; this is a speculation that remains to be proved. Because TGF-α is sorted, shed, and released from the basolateral surface of the intestinal epithelial cell, it is feasible that autocrine proliferative activity is able to access the proliferative compartment via the lamina propria (303). In the human esophagus, TGF-α mRNA and protein are found primarily in the differentiated epithelial cell population (226,304,305). In the human and murine stomach, expression is also found in the differentiated, nonproliferative cell population of the epithelium, especially in parietal cells (229,304,306–309). In most reported studies, TGF-α in the small intestine and colon is also principally found in differentiated, surface epithelial cells (10,11,303,304,310). Gastrointestinal biologists and physiologists have long been intrigued by the compartmentalization of the gastrointestinal epithelial functions into apical (brush border) and basolateral domains. This spatial compartmentalization is especially important for understanding how autocrine and juxtacrine growth factors function. The sorting of TGF-α and other EGF-related peptides therefore has been of intense interest. It is now clear that TGF-α is sorted to the basolateral domain of polarized epithelial cells with greater than 90% efficiency (8). Redundant motifs in the cytoplasmic tail, including a dileucine motif and eight amino acids in the juxtamembrane region of pro–TGF-α, are responsible for sorting (311). Myristoylation-dependent binding of Naked-2 to the cytoplasmic tail is believed to contribute to the escort of pro–TGF-α to the basolateral membrane (312), and evidence suggests that binding of membrane associated guanylate kinase inverted-3 (MAGI-3) to the PDZ domain also facilitates sorting (313). Once targeted to the basolateral membrane, the protein is rapidly cleaved by ADAM17 and quickly bound to EGF receptors, which are also located on the basolateral domain. EGF and TGF-α stimulate epithelial proliferation in normal gastrointestinal epithelium in vitro and in vivo. For purposes of this discussion, it is assumed that the mitogenic effects of TGF-α and EGF are equivalent, as this is the case in most assays. Studies are collated for this discussion. Neutralizing antibodies to EGF and TGF-α reduce the growth of cultured cells, including epithelial cells, exposed to human milk (314–316). TGF-α is a mitogen for gastric mucous cells, fundic epithelial cells, small intestinal epithelial cells, and colonic epithelial cells cultured in vitro (103,245,317– 320). TGF-α may also be an “indirect mitogen.” For example, some work shows that treatment of intestinal epithelial cells with substance P activates EGFR signaling by rapidly promoting metalloproteinase-induced cleavage of membraneanchored TGF-α (321). A number of studies have examined a role for EGF-related peptides on gastrointestinal epithelial cell proliferation in vivo. The methods used to deliver exogenous EGF-related peptide in these studies are of interest in light of the basolateral (serosal) distribution of EGFR in the gastrointestinal tract (see later in this section for a description of this observation). Parenteral administration of EGF or TGF-α stimulates

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 197 small intestinal and colonic epithelial growth (322–325). Overexpression of TGF-α in the small intestine of MT-TGF-α transgenic mice increases crypt cell proliferation, resulting in increased villus height (326). In contradistinction, transgenic mice overexpressing EGF under the direction of an β-actin promoter are growth retarded, but they show no substantive intestinal, pancreatic, or hepatic phenotypes (327). Limited evidence also suggests EGF stimulates proliferation in humans (328). In most reported studies enteral administration of EGF or TGF-α does not stimulate proliferation of normal adult gastrointestinal mucosa. These findings are congruous with the observation that the EGFR is localized to the basolateral domain of the intestinal epithelial cell. It should be recognized, however, that enteral administration of EGF might permit access to the basolateral receptor and stimulate growth under circumstances of developmental immaturity or pathologic injury to the epithelial barrier. It is well known that early in development, the intestinal epithelial barrier is more permeable to macromolecules. A significant body of evidence suggests luminal growth factors may actually gain access to and activate EGFRs on the basolateral membrane in immature animal models, although cellular proliferation is not necessarily the observed biological end point in these studies (329–331). EGF may also traverse the mucosa intact by transcytosis in the newborn interval (332). In experimental models of intestinal atrophy, parenteral administration of EGF or TGF-α promotes epithelial growth and reverses atrophy (333–337). In most, but not all, partial small intestinal resection models, the adaptive proliferative response observed in residual intestine is enhanced by administration of EGF, perhaps, in part, by increased expression of EGFR (338–340). Overexpression of EGF in the intestinal epithelium of transgenic mice also enhances the proliferative response to resection (341). However, postresection proliferation occurs in TGF-α–deficient transgenic mice, suggesting that sufficient overlap exists with other EGF-related peptides (342). The gastric mucosa of TGF-α overexpressing mice is also hyperplastic and shows lineage disturbances reminiscent of Ménétrièr’s disease (343,344). Clearly, the preponderance of experimental evidence cited in this chapter suggests that TGF-α and EGF stimulate proliferation in the intestinal mucosa. However, these observations do not establish an absolute requirement for EGF or TGF-α in normal intestinal epithelial proliferation. Genetargeting experiments that specifically address this issue clearly conclude that EGF and TGF-α are not required for normal intestinal proliferation (285,286). Although TGF-α gene–targeted mice have hair follicle abnormalities leading to wavy whiskers and occasional ocular abnormalities, no gastrointestinal phenotype is observed (285). A gastrointestinal phenotype is also not observed in EGF gene–targeted mice (345). In the context of disruption of the epithelial barrier, luminal EGF may also gain access to the basolateral domain, leading some experts to call EGF a “luminal surveillance factor” poised to contribute to proliferation, migration, and

repair of injured mucosa (346). In support of this, enteral EGF administration ameliorates mucosal injury in a rat model of neonatal enterocolitis (331), and improved outcomes were reported in a clinical trial of EGF enemas in patients with ulcerative colitis (347a). Interestingly, neither transgenic overexpression of EGF in the intestinal epithelium nor intravenous EGF ameliorates chemotherapeutic agent–induced mucosal injury induced by fluorouracil (347b). A different study found that EGF administered intraperitoneally protects the small intestinal mucosa against methotrexate-induced injury (348). EGF null mice are more sensitive to cysteamineinduced duodenal lesions, but they are no more sensitive to dextran sulfate sodium–induced colitis (233). Interestingly, TGF-α–deficient mice have increased susceptibility to dextran sulfate sodium–induced colitis (349). Triple knockout EGF, TGF-α, and AR mice are growth retarded, have reduced levels of goblet cells, develop spontaneous duodenal erosions, and show blunted ileal villi (233). Whether these findings are the result of reduced proliferation or a loss nonmitogenic activity contributing to cytoprotection is unclear. A number of factors may contribute to the variable results in experiments testing in vivo activities of EGF-related peptides. These include the route and dose of EGF-related peptide, the presence or absence of mucosal injury, the developmental stage of the model being studied, and the model of injury or read-out of EGF-related peptide biological activity. Even the feeding status of the animal may play a factor, because inhibition of luminal proteases by food may increase bioactivity of EGF in the intestinal lumen that is otherwise degraded soon after entry into the proximal bowel (350). Wong and Wright (351) provide an excellent review of the effects of EGF-related peptides on intestinal growth. Additional evidence for the mitogenic properties of TGF-α in the gastrointestinal epithelium is the substantial body of data showing it is overexpressed in gastrointestinal cancers. TGF-α may be overexpressed in neoplasms of the esophagus (352,353), stomach (230,240,241,354–356), and colon (357–362). Often, the level of overexpression correlates with tumor aggressiveness. Serum levels of TGF-α are also increased in patients with gastrointestinal tumors. Activation of the EGFR by excessive ligand production or other key mechanisms leads to an amplified autocrine axis that is amenable to therapeutic intervention. As discussed later in this chapter, this field has evolved to the development of inhibitors of EGFR as a therapeutic strategy for the treatment of gastrointestinal neoplasia. Nonmitogenic Activities of Epidermal Growth Factor and Transforming Growth Factor-α EGF-related peptides have key biological activities that are unrelated to cellular proliferation. These so-called nonmitogenic activities are outlined in a superb review by Uribe and Barrett (363). Nonmitogenic activities of EGF-related peptides include inhibition of gastric acid secretion; modulation of sodium, chloride, and glucose transport; stimulation of amylase and mucin secretion; stimulation of mesenteric

198 / CHAPTER 7 and mucosal blood flow; stimulation of smooth muscle contraction; and stimulation of cell motility, migration, and chemotaxis, among others. In aggregate, these activities work to promote adaptation to gastric, small intestinal, and colonic mucosal injury and ulceration (364), as discussed earlier.

Amphiregulin AR was first identified in MCF-7 breast carcinoma cells treated with the tumor promoter phorbol myristate acetate. The name amphiregulin comes from the ability of AR to either stimulate or inhibit cell growth, depending on the cell line being studied (365). The proteins originally described as colorectum-derived growth factor and Schwannoma cell– derived growth factor are now known to be AR (366,367). The AR gene is located on human chromosome 4q13-4q21 (368). Many proteins and compounds that stimulate cellular proliferation also induce AR gene expression. Examples include other members of the EGF-related peptide family (245,369), oncogenic Ras (370,371), phorbol esters (368), and prostaglandin E2 (372), likely through a cyclic 3′,5′adenosine monophosphate response element in the AR promoter. Structurally, AR is distinguished from EGF and TGF-α by an N-terminal heparin-binding domain in the mature peptide sequence. The N-terminal sequence of AR is extremely hydrophilic and contains a nuclear localization signal that is required for mitogenic activity of AR (366). Because of differential processing and variable glycosylation, multiple molecular weights of AR are noted on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranebound form has a molecular weight of 50 kDa, whereas the mature peptide ranges from 22 to 46 kDa (373,374). Shedding of AR occurs by the TACE/ADAM family of metalloproteinases. The metalloproteinase inhibitor batimastat inhibits shedding of AR, as it does other EGF-related peptides (374). Inhibition of tyrosine phosphatase activity promotes metalloproteinase-dependent cleavage of AR (375). AR binds exclusively to ErbB1. AR mRNA is expressed in normal and neoplastic cells of the gastrointestinal tract (368,376–378). It is detectable in the surface epithelium of the normal colon, whereas little or no expression is found in the proliferative zone (377,378). Most colon cancers express AR at a greater level than adjacent normal colon tissue (376–379). Polarizing Caco-2 and HCA-7 colon carcinoma target AR to the basolateral membrane, and secretion occurs into the basolateral cell culture medium (380). Cytoplasmic sequences in pro-AR are required for sorting (381). AR is a potent mitogen for most epithelial cells, and all the requisite elements of a growth stimulatory autocrine loop exist in colon carcinoma cells (378–380). In vitro, inclusion of heparin-like glycosaminoglycans in growth assays enhances proliferation induced by AR (382), whereas heparin antagonists block proliferation (383). Treatment of the GEO colon cancer cell line with antisense AR oligomers reduces cell growth (384).

AR is also an important mitogen in the liver and pancreas. In the normal human liver, AR gene expression is barely detectable; however, expression is increased in the context of cirrhosis (385). In the partial hepatectomy model, AR expression is induced to a maximum level 6 hours after resection, and the onset of DNA synthesis is delayed in AR gene–targeted mice (385). As expected, AR is mitogenic in isolated hepatocyte cultures. Transgenic overexpression of AR in the exocrine pancreas results in proliferation in small intralobular ducts (386), but the excessive fibrosis observed in TGF-α transgenic mice is not observed (275,276). AR-deficient mice are normal and have no obvious defects in the normal gastrointestinal system, although the proliferative response to partial hepatectomy is delayed and less robust (345,385).

Heparin-Binding Epidermal Growth Factor–Like Growth Factor HB-EGF was first identified in conditioned medium of a cultured macrophage cell line using heparin-affinity chromatography (387). The human gene locus for HB-EGF is 5q21 (388). Like AR, a host of proliferative stimuli and other perturbations rapidly induce HB-EGF gene expression, including TNF-α, oxidative stress, serum, other EGF-related peptides, insulin-like growth factor-1 (IGF-1), gastrin, v-jun, oncogenic Ras, phorbol esters, angiotensin II, and TGF-β (389–400). The mRNA is super induced by the protein synthesis inhibitor cycloheximide (245,391), thus defining HB-EGF as an “immediate-early gene.” This suggests HB-EGF plays a key role in mediating the earliest responses to proliferative stimuli and other cellular perturbations. Like other EGF-related peptides, HB-EGF is synthesized as a transmembrane propeptide that is processed by the ADAM family of metalloproteinases to release soluble mature peptide (401). The HB-EGF transmembrane precursor is apparently unique among EGF-related peptides in that it is capable of functioning as a receptor molecule by binding and internalizing the diphtheria toxin (402). The molecular weight of mature HB-EGF is 20 to 22 kDa, although multiple higher molecular weight forms have been noted (390,403). Mature HB-EGF contains an N-terminal hydrophilic heparinbinding domain similar to that found in AR and BTC. Interestingly, the C-terminal product of HB-EGF shedding is retained intracellularly, translocates to the nucleus, and triggers nuclear export of the promyelocytic leukemia zinc finger transcriptional repressor (404). Thus, one of the most prominent activities of HB-EGF, stimulation of cellular proliferation, may occur by two distinct mechanisms: activation of the EGFR by soluble HB-EGF and release of cells from transcriptional repression by intracellular C-terminal HB-EGF. HB-EGF is found in many human tissues and body fluids in the picogram per milliliter concentration range, including human amniotic fluid and breast milk (405,406). However, with the exception of prominent expression of HB-EGF mRNA and protein expression in normal gastric parietal cells (407), expression is low or undetectable in the normal

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 199 gastrointestinal epithelium and in the liver (408,409). In vitro, the Cdx-2 homeobox gene induces expression of HB-EGF, which contains a Cdx-2 binding element (410), suggesting it may play a role in intestinal development. HB-EGF is found in the developing pancreas and the homeodomaincontaining transcription factor PDX-1 binds to the HB-EGF promoter (411). The rapid induction of HB-EGF in response to a variety of experimental perturbations in vitro is also observed in vivo. For example, HB-EGF mRNA is rapidly induced after acute injury to the kidney (397), brain (412), and intestine (413). In the intestine, HB-EGF protects against hypoxic/ ischemic injury in vitro (413) and in vivo (414), perhaps by suppression of NF-κB activation (415). Substantial data support a role for HB-EGF in wound healing (390). HB-EGF binds to ErbB1 and ErbB4 and induces tyrosine phosphorylation of each (416–418). In cells expressing ErbB4 but no EGFR, HB-EGF signals a chemotactic, but not a proliferative, response, suggesting these two receptors mediate different biological activities (417). Receptor-binding affinities and biological activities of HB-EGF are accentuated by cell-surface heparan sulfate proteoglycans (HSPGs) (419–421). There are interesting, but limited, data suggesting that HB-EGF also specifically binds to N-arginine dibasic convertase to modulate cellular migration in an EGFRdependent manner (422). The dominant activities of HB-EGF are stimulation of cellular proliferation, cell survival, and cell motility (390). Most cell types proliferate in response to HB-EGF, including gastric cell, pancreatic cell, intestinal epithelial cells, and hepatocytes (245,420,421,423). Although liver-specific overexpression of HB-EGF in transgenic mice has no effect on basal measures of proliferation in the liver, in the context of partial hepatectomy, labeling indices are fivefold greater than in nontransgenic control animals (424). In renal epithelial cells, intestinal epithelial cells, and hepatocytes, enforced expression of a membrane-anchored form of HB-EGF or exogenous administration of HB-EGF causes resistance to apoptotic stimuli such as TNF-α (425–427). In this capacity, HB-EGF serves as a “survival” factor. Although only a limited number of studies have addressed HB-EGF expression in gastrointestinal neoplasms (428), overexpression has been observed in pancreatic, liver, and gastric neoplasia (429–432). Transgenic overexpression of HB-EGF in the pancreatic islet cells leads to fibrosis and metaplasia of ductular epithelium (433).

Betacellulin BTC was first isolated from a mouse insulinoma cell line (434). The gene is located on human chromosome 4q13-q21. The molecular weight of unprocessed BTC is 40 kDa (435). The mature peptide is released by an ectodomain-shedding mechanism that appears to be uniquely regulated in comparison with other EGF-related peptides (see later) (436). Mature, soluble BTC is approximately 32 kDa and consists of 80 amino acids. Like AR and HB-EGF, BTC contains

consensus glycosylation sequences and a heparin-binding domain (434). The pattern of BTC binding to ErbB receptors mirrors that of HB-EGF and EPR (437–439); however, BTC is unique in its ability to bind ErbB2 and ErbB3 heterodimers in the absence of ErbB1 and ErbB4 (437,438). Studies suggest that BTC induces distinctive patterns of EGFR tyrosine phosphorylation, leading to alterations in the profile of adapter protein recruitment and differences in signaling pathway activation (440,441). Again, these findings emphasize that redundancy in growth factor families should be viewed with some skepticism, because detailed scrutiny of the complexity of regulation of these proteins typically reveals unanticipated differences. BTC is found in bovine milk (442). The 3-kb BTC mRNA transcript is widely expressed in normal tissues, including the small intestine, liver, colon, and pancreas (434,443,444). BTC is increased in intestinal epithelial cells and in epidermal cells transformed by oncogenic Ras (371,445). Gastric cancer cell lines also express BTC mRNA, although the transcript is detectable in primary tumors only by PCR (429). In the pancreas, BTC is detectable in this normal fetus, in the adult by immunohistochemistry (446), and as early as embryonic day 13 by reverse transcriptase PCR (447). Studies of BTC function in the gastrointestinal tract are limited. Treatment of fetal pancreas explants with BTC favors development of a β-cell lineage (447). BTC is mitogenic for a number of fibroblastic and epithelial cell lines (434,448). BTC gene-targeted and transgenic mice have not yet been generated.

Epiregulin EPR was relatively recently discovered (449,450). The gene is on human chromosome 4q13.3. The molecular weight of the purified protein is 5.4 kDa, and the mature peptide is 46 amino acids in length. N-terminal sequences seen in AR, HB-EGF, and BTC are not present in EPR, and binding to heparin does not occur. The 4.8-kb EPR RNA transcript has been detected in a wide variety of cell lines and in normal stomach, small intestine, and colon (450,451). Similar to AR, EPR inhibits growth of some cell types and stimulates growth of others (449,452). It stimulates growth of gastric epithelial cells (453), and its expression is increased in models of partial hepatectomy (454) and in tumors of the colon and pancreas (455,456). Although the affinity of EPR for the ErbB1 is less than EGF, EPR is more potent in mitogenic assays (449,457–459). EPR gene-targeted mice show no overt developmental defects, a normal proliferative response to partial hepatectomy, but increased sensitivity to dextran sulfate sodium-induced colitis (451).

Epigen Epigen, the newest and presumably the last EGF-related peptide, has been identified as an expressed sequence tag in a mouse keratinocyte library (460). It is located on human

200 / CHAPTER 7 chromosome 4q13.3. The mRNA is found in the liver. Expression in other gastrointestinal organs has not been investigated. The protein is composed of 152 amino acids, and the biological function appears to be similar to EGF, although preliminary evidence suggests it may be far more potent (461).

Regulated Shedding of Epidermal Growth Factor–Related Peptides by Metalloproteases All EGF-related peptides are first synthesized as anchored transmembrane precursor proteins that are subsequently cleaved to release biologically active autocrine or paracrine peptide growth factor. This process is termed shedding. One of the most exciting areas of EGF-related peptide biology in recent years has been elucidation of the cellular molecular mechanisms by which shedding occurs. It has been learned that this is a complex, highly regulated process subject to therapeutic manipulation in diseases such as cancer in which EGF-related peptide signaling is often hyperactive. The significance of shedding as a key regulatory step in EGF-related peptide signaling was first highlighted by the observation that TACE gene–targeted mice develop a phenotype similar to TGF-α and EGFR null mice (250,285,462). TACE null fibroblasts isolated from these mice were dramatically impaired in their ability to shed mature TGF-α. This mouse model implied that cleavage of EGF-related peptides by TACE is critical for growth factor activity. Subsequent studies support this assertion. For example, it is clear that TACE is required for activation of the EGFR by TGF-α in tumors, a finding that also calls into question the importance of juxtacrine signaling by membrane-anchored growth factor (463). TACE belongs to a large family of zinc-dependent disintegrin-metalloproteinases designated ADAMs (464–466). These proteins are also variably referred to as sheddases or convertases. TACE, or ADAM17, is the major metalloproteinase involved in EGF-related peptide shedding, although multiple other members of this large family of 39 proteins may also participate. Adding to the complexity is a great deal of cell-type specificity of ADAM function. Notwithstanding, currently, it appears that ADAM17 is the major protease involved in TGF-α, AR, and HB-EGF shedding (467–469), whereas ADAM10 is the major protease involved in shedding of EGF and BTC (436,468). Cleavage of HB-EGF by other metalloproteinases such as MMP-3 may also contribute to shedding (470). Shedding can be activated by a variety of stimuli, including phorbol esters, calcium ionophores, Ras activation, calmodulin antagonists, tyrosine phosphatases inhibitors, GTP-γs, and EGFR autocrine signaling (374,471–477). Emerging evidence indicates that the well-known phenomenon of EGFR transactivation by ligands of G protein-coupled receptors (GPCRs) occurs by activation of ADAM activity, release of mature EGF-related peptide, and activation of the EGFR (478).

A potentially important dividend realized by expanding our understanding of ADAM and MMP-induced shedding is the potential for modulating EGFR activation using metalloproteinase inhibitors. The broad-spectrum metalloprotease inhibitor batimastat blocks shedding of EGF-related peptides in a manner that suggests an integral role for metalloproteases in the regulation of biological activity of these proteins (375,479). However, to date, clinical trials of metalloproteinase inhibitors have not been successful, perhaps because of a lack of specificity of currently available inhibitors (480).

Epidermal Growth Factor Receptor Family Overview The mammalian EGFR family is composed of four membrane-anchored receptor tyrosine kinases. Each is characterized by a single extracellular binding domain, a transmembrane domain, and a single intrinsic tyrosine kinase domain extending into the cytoplasm (481–484). The classical EGF receptor (ErbB) is also known as HER1 (human epidermal growth factor receptor 1), or ErbB1, because of homology with the v-erbB oncogene encoded by the avian erythroblastosis virus (485). The remaining three receptors are designated HER2/ErbB2, Her3/ErbB3, and Her4/ErbB4. The overall homology within the ErbB family is 40% to 50%, and all cysteine positions are conserved in the extracellular domain. The phenomenon of receptor dimerization is a key principle in understanding ligand-induced signaling by EGFRs (486), and it is a recurrent theme in the biology of most growth factor families. Within the EGFR family, ligand binding is able to transduce signals by two distinct pathways, receptor homodimerization and autophosphorylation and receptor heterodimerization and transphosphorylation. When one collectively considers the seven EGF-related peptides discussed in this chapter in the context of the extraordinary number of potential dimerizing partners in the ErbB family, the rich complexity and diversity of signal transduction within the EGF family quickly becomes apparent. In most contexts in this chapter, the classic EGF receptor, ErbB1, is referred to as the EGFR. When it is desirable to facilitate understanding of the pattern of homodimerization and heterodimerization with other members of the mammalian family, the designation ErbB1 will be used. The remaining three EGFR family members are designated by ErbB2, ErbB3, and ErbB4, respectively. Epidermal Growth Factor Receptor (or ErbB1) The EGFR gene is found on human chromosome 7p11 (487,488) and contains 110 kb DNA, including 26 exons and 25 introns (488). The molecular weight of the protein is 170 kDa. It is no doubt the most extensively characterized receptor protein in the field of growth factor biology. Both extracellular and intracellular domains have been extensively

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 201 characterized by point mutants, deletion mutants, insertional mutants, and chimeric receptors. The extracellular domain is characterized by an extensively N-glycosylated N-terminal domain and a cysteine content of about 10%. The hydrophobic transmembrane sequence traverses the cell membrane only once. Located in the cytoplasmic domain are a C-terminal tyrosine kinase and five tyrosine autophosphorylation residues. When phosphorylated, the latter serve as docking sites for intracellular signaling proteins that bind Src homology-2 (SH2) domains in intracellular adaptor molecules that link directly to signaling cascades. Studies suggest that EGFRs may exist in a ligandindependent, “predimerized” configuration (489), the existence of which is regulated by sequences in the cytoplasmic domain. When ligand is engaged, the EGFR homodimerizes or heterodimerizes with ErbB2, ErbB3, or ErbB4, depending on the ligand and cell type. Biophysical studies and X-ray crystallography indicate that receptor dimerization requires interaction between two molecules of ligand and two molecules of receptor (490–492). Engagement of a single ligand with a monomeric receptor results in a conformational change in the receptor that favors receptor-receptor interaction. Dimerization of EGFRs results in intermolecular autophosphorylation of key tyrosine kinases in the cytoplasmic domain. Congruous with this model is the dominant-negative effect observed by transfection of cells with a mutant EGFR containing an inactive tyrosine kinase moiety resulting in a reduction of high-affinity binding sites for EGF, a reduced rate of receptor endocytosis, and diminished receptor signaling (493). After binding any of its ligands, EGFRs are rapidly internalized by clathrin-dependent and clathrin-independent endocytic pathways. The extent to which signaling continues after endocytosis remains unclear, although it is generally believed that signaling continues and endocytic mechanisms are able to modulate the magnitude, specificity, and duration of signaling (494,495). Interestingly, endocytosis is inefficient for all other ErbB receptors, and signaling from the cell membrane is sustained (496). Once internalized, most EGFR is sorted to lysosomes for degradation, whereas approximately 25% is recycled to the cell membrane. The result is receptor down-regulation and attenuated signaling. Precisely how the various ErbB heterodimers undergo trafficking is not entirely clear. Data suggest that the EGFR is ubiquitinated by the ubiquitin protein ligase, c-Cbl, thus targeting the receptor/ligand complex for endocytosis (497,498). An old but intriguing observation that must be accommodated in models of EGFR signaling is that continuous exposure of EGFR to ligand for at least 8 hours is required for S-phase entry and DNA synthesis (499,500). Epidermal Growth Factor Receptor Signaling When ligand is engaged, specific residues in the cytoplasmic domain of dimerized or heterodimerized EGFR are tyrosine autophosphorylated. These phosphotyrosine sites create docking motifs for signaling proteins or adapter proteins with

SH2 or phosphotyrosine binding domains. SH2 domains are found in a large number of proteins and consist of approximately 100 amino acids that recognize phosphopeptide motifs (501). Recruitment of SH2 proteins to phosphotyrosine motifs in the activated EGFR may result in their direct phosphorylation by EGFR tyrosine kinase, activation of their intrinsic catalytic activities, or establishment of platforms for recruitment of additional signaling or adaptor proteins, as shown in Figure 7-5. Examples of catalytic activities recruited to the activated EGFR include Src kinases, Abl, protein tyrosine phosphatase (PTP), phospholipase Cγ (PLCγ), and phosphoinositol-3 kinase (PI3K) (502). Examples of adapter proteins include Shc, Grb2, Crk, Grb7, and Nck, among many others (503,504). Thus, the complexity of EGFR signaling is multiplied many fold by the diversity of potential catalytic activities and adaptor molecules recruited to the EGFR. A full discussion of all adaptor proteins, catalytic activities, and downstream signaling cascades activated by the EGFR is beyond the scope of this chapter, but there are several excellent reviews of this topic (502,503,505). A few classical and illustrative examples are provided below. The most extensively studied signaling pathway activated by the EGFR tyrosine kinase is the Ras/MAPK pathway (501,506). Central to activation of the Ras pathway is Grb2, a cytoplasmic adaptor protein with two SH2 motifs. Grb2 is constitutively bound to the Ras exchange factor Sos. Binding of Grb2 to the activated EGFR relocates Sos to the EGFR and the cell membrane, facilitating interaction and activation of Ras by guanine nucleotide exchange. Activation of Ras is a central event in a multiplicity of physiologic and pathophysiologic cellular events, the most prominent of which is activation of cellular proliferation and survival. This variety of cellular response results from Ras activation of a complex downstream network of interacting and intersecting Ras effectors (507–510). In addition to activation of the classical Raf/MEK/ERK signaling pathway, other Ras-effector cascades activated by Ras include PI3K, Ral GEF, and Rho-kinase pathways, among others (510). Mutational activation of the K-Ras gene occurs in about 50% of colorectal cancers (175) and more than 75% of pancreatic cancers (511). Activated Ras increases levels of EGF-related peptides and phosphorylation of the EGFR (512). Interestingly, this creates amplifying loops of autocrine EGF-related peptide production and activation of EGFR, as detailed in Figure 7-6. Termination of this autocrine loop by EGFR tyrosine kinase inhibitors reduces cell growth and indices of cellular transformation in Rastransformed cells (513). This in vitro phenomenon is also observed in vivo. The hypomorphic EGF tyrosine kinase activity characteristic of wav-2 mice decreases formation of epithelial tumors when these mice are mated with mice overexpressing the adapter protein Sos, a Ras activator (514). Study of cells isolated from these mice attributed the decreased number of tumors to loss of an EGFR-dependent “survival function,” which is a well-recognized consequence of EGFR activation (515).

202 / CHAPTER 7

PIP2

DAG IP3 JAK1 STATs

PI-3K PLCγ

EGFR/ErbB2

P P

Ca++

PKC

GPCR

AKT

Shc Grb2

Ras

Sos TGFα

MKK6

MEKK-1

Raf

p38

MKK -7

MEK

MAPKAP2

JNK

ERK

TACE/ADAM17

TGFα (pg/ml)

FIG. 7-5. Epidermal growth factor receptor (EGFR) signaling. In the gastrointestinal epithelium, EGFR activation typically occurs by ligand that has been targeted to and proteolytically cleaved from the basolateral domain of the polarized enterocyte. EGFRs, also located on the basolateral domain, are activated by ligand engagement, resulting in activation of intrinsic tyrosine kinase activity. Scaffolding proteins dock to the activated receptor and link to a complex variety of signaling cascades. ADAM, a disintegrin and metalloprotease; DAG, diacylglycerol; ERK, extracellular signalregulated kinases; GPCR, G protein–coupled receptor; IP3, inositol-1,4,5-triphosphate; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ; STAT, signal transducer and activator of transcription; TACE, tumor necrosis factor–converting enzyme; TGF-α, transforming growth factor-α.

60 50 40 30 20 10 0

PGE2 production

Ras activation

Pai, et al. Nature Med 8:289 Shao, et al. J Biol Chem 279:14287

RIE Ras Du et al. Mol Cancer Res 2:233

24 h

8h 12 h

4h

1h 2h

45′

0

30′

15′

Ligand auto- and cross-induction

COX-2 induction

EGFR family activation

EGFR ligand

TGFα AR HB-EGF

Bernard et al. J Biol Chem 269:22817

BTC 1B15

FIG. 7-6. Autoamplifying loops of epidermal growth factor (EGF)–related peptide activity that potentially contribute to unregulated growth in the intestinal epithelium. A large body of data now supports the “autoamplifying” nature of EGF-related peptide signaling. In the context of neoplastic disorders, multiple autoamplifying loops contribute to ongoing, unregulated cellular proliferation. Contributors to this loop are increased production of EGF-related peptides and induction of cyclooxygenase-2 (COX-2) in cells transformed by oncogenic Ras, extensive induction of other EGF-related peptides, induction of COX-2 expression, and increased prostaglandin production by EGF-related peptides. Recent evidence suggests that prostaglandin E2 (PGE2) transactivates the EGF receptor.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 203 PLCγ is also a key signaling molecule for receptor tyrosine kinases, including members of the EGF family (502,516). PLCγ hydrolyses phosphatidylinositol 4,5-biphosphate, releasing the second messenger molecules inositol-1,4, 5-triphosphate (IP3) and diacylglycerol (DAG). It may also function as a guanine nucleotide exchange factor (517a). PLCγ is distinguished among the PLC isoenzymes by two SH2 and one SH3 motifs that associate with the activated EGFR. Activated EGFR directly phosphorylates and activates PLCγ (517b). The result is stimulation of cellular proliferation and migration, including cells isolated from the gastrointestinal tract (518). PLCγ also appears to be an essential signaling molecule for maintenance of intestinal epithelial integrity by EGF (519). Involvement of members of the Src family of cytosolic, nonreceptor, tyrosine kinases in EGFR signaling has long been recognized, especially with regard to oncogenesis (520,521). Both SH2 and SH3 domains are found in Src family members. Downstream targets include PI3K and PLCγ, among others. Evidence suggests that the EGFR itself is phosphorylated by Src, leading to increased tyrosine kinase activity (521). Overexpression of Src proteins enhances EGF-mediated cell proliferation and transformation, whereas inhibition of Src activity blocks EGF-induced cellular proliferation. Thus, there is a bidirectional synergy between Src family members and the EGFR family (521). The phospholipid kinase PI3K phosphorylates the membrane lipid phosphoinositol to generate phosphoinositides (522). Phosphoinositides are second messenger molecules that recruit cytoplasmic proteins to the cell membrane. PI3K is activated by association with virtually all receptor tyrosine kinases, including the EGFR, or their associated adaptor proteins. PI3K contains two SH2 and one SH3 domain and is a critical downstream effector of Ras. The serine threonine kinase Akt, also known as protein kinase B (PKB), accumulates as sites of PI3K activation by binding directly to phosphoinositides. Akt catalytic activity results in phosphorylation of a host of other cellular proteins that are critical for cell proliferation and survival. Signal transducer and activator of transcription (STAT) proteins are a family of seven latent cytoplasmic transcription factors that are well-known signaling molecules for cytokines such as interferons (523). Less well studied is the phosphorylation and activation of STAT proteins by the activated EGFR. Activated STAT proteins dimerize and translocate to the nucleus where they bind to specific sequences of target genes. The activation of STAT proteins in response to EGFR activation appears to be Src-dependent in many cell types (524). Some work suggests that EGF treatment of primary hepatocytes results in activation of STAT5b in a Src-dependent manner, although activation of STAT5b is not required for stimulation of cellular proliferation (525). Migration of esophageal epithelial cells appears to be dependent on EGF activation of STAT proteins (526). The outcome of STAT activation in normal and transformed cells of gastrointestinal origin deserves further investigation.

Epidermal Growth Factor Receptor Expression in Normal Gastrointestinal Tissues and Gastrointestinal Neoplasia The EGFR is expressed in all cells at a level ranging from 20,000 to 200,000 receptors per cell. An exception is hematopoietic cells, which do not express receptor. Receptor levels can be much greater in neoplastic cells. Expression in the normal gastrointestinal tract begins during embryonic development and persists throughout adulthood. In the liver, multiple studies have shown that EGFR levels are quite high in the fetus, and then decrease after birth (527–529). Notwithstanding the high fetal levels of EGFR, the normal hepatic architecture observed in EGFR gene-targeted mice indicates hepatic organogenesis is not dependent on EGFR signaling (462,530). EGFR mRNA is detectable in normal human hepatocytes, most prominently at the periphery of the hepatic lobule (225). EGF is found in significant concentrations in bile, and it has been suggested that hepatic EGFR serves as a means to clear plasma of EGFR ligands by biliary excretion (531,532). After partial hepatectomy, the EGFR is initially up-regulated, and then down-regulated (529,533), perhaps because of increased ubiquitination and internalization of receptor (534). EGFR is also expressed in normal human pancreas, primarily in ductal and acinar epithelial cells (232). The EGFR is expressed in normal fetal and adult gastrointestinal tract. In the human fetus, EGFRs are present in the human gastric glands as early as the 12th week of gestation (535) and are detectable in fetal jejunum and colon between week 15 and 19 of gestation (536). The ontogeny of intestinal EGFR expression is reviewed comprehensively in an excellent article by Chailler and Menard (537). In the adult esophagus, EGFRs are localized to the basal and suprabasal layers, and little or no receptor is found in the surface epithelium (226,346,538–540). Expression in increased in the chronically inflamed esophagus (305). In the stomach and intestine, EGFR is found in parietal cells and has been variously reported in the proliferative regions of the crypt/villus axis or all along the horizontal axis of the epithelium in the small intestine and colon (346,541). An extensive body of literature concludes that the EGFR is located primarily on the basolateral membrane of polarized gastrointestinal epithelial cells and polarized epithelial cells in other tissues (329,380,542–548). A 22-amino-acid sequence in the EGFR juxtamembrane sequence contains information necessary for targeting the receptor to a basolateral localization (549). In polarized Caco-2 human colon carcinoma cells, levels of basolateral EGFR are 15-fold greater than apical receptor, and no bioactivity is detected when the apical compartment is treated with ligand (550). Similar observations have been made using polarized MDCK cells, rat proximal tubular cells, and other colon cancer lines. Despite these findings, it has been proposed that low levels of apical EGFR in the gastric epithelium protect against apical gastric acid (551) and a functional role for apical receptor should not be dismissed.

204 / CHAPTER 7 The EGFR is overexpressed in many types of cancers. In many instances, overexpression or amplification correlates with a poor prognosis. Overexpression of the EGFR and coexpression of its ligands are common in pancreatic carcinoma and also appear to portend a poor prognosis (238,287,291,552–555). In human hepatocellular carcinoma, overexpression of EGFR and its ligands may play a less important pathogenetic role (556–559). EGFR is overexpressed in Barrett’s epithelium and in adenocarcinoma of the esophagus and may correlate with a poor prognosis (353,558,560,561). In gastric cancer, a correlation has been established between overexpression of the EGFR and a poor prognosis, but overexpression is not particularly common (562–564) and not all studies corroborate this correlation (565–567). Expression of the EGFR and its ligands have been detected in human colon adenoma and carcinoma cell lines, in animal models of colon carcinoma, and in human disease (242,244,359–361,562,568,569). Metastatic colorectal carcinomas express more receptor than the primary tumors (570,571). Collectively, overexpression of EGFR and its ligands in gastrointestinal neoplastic disorders has led to a number of exciting and intensively studied therapeutic approaches that target the EGFR system.

distantly related neuregulin family are ligands for ErbB3 (585). When ErbB3 is expressed alone, binding with the EGF-related peptide family does not occur, but binding and signaling may occur when ErbB3 heterodimerizes with remaining EGFR family members. ErbB3 is considered a “kinase-impaired” receptor because its tyrosine kinase activity is significantly less than other ErbB receptors (586). ErbB3 is also unique among ErbB receptors in that multiple (six) YXXM consensus sites in the cytoplasmic tail permit binding of and signaling by PI3K (587). Distinct physiologic roles for ErbB3 in the gastrointestinal tract have not been identified. ErbB3 protein expression is detectable in normal human esophagus, stomach, small intestine, and colon (588). The predominant expression is in the differentiated, surface epithelium. Expression is also detectable in fetal and adult pancreas and liver (529,589). ErbB3 expression is upregulated by insulin and partial hepatectomy in the rat (529, 590). ErbB3 immunoreactivity is low or undetectable in normal human colonic epithelium, whereas expression is often detectable in most colon carcinomas (376,578). Increased expression is also found in gastric, hepatic, and pancreatic cancers (591–593).

ErbB2

ErbB4

c-ErbB2 is located on human chromosome 17q21, and the protein product is a 185-kDa glycoprotein with an intrinsic, cytoplasmic tyrosine kinase domain (572). ErbB2 is also referred to as the neu protooncogene, or HER2, and is the only member of the EGFR family with no identified ligand. ErbB2 appears to be the preferred heterodimeric partner with the other ErbBs, and heterodimer formation with ErbB2 increases affinity of ligand for the heterodimeric receptor complex (439,573). Formation of heterodimers with ErbB1, ErbB3, and ErbB4 induces tyrosine transphosphorylation of ErbB2. Overexpression of ErbB2 is associated with uncontrolled cell proliferation in animal models, as well as human neoplastic disorders (574,575). ErbB2 amplification and overexpression have been most extensively studied in mammary carcinoma, where it is overexpressed in about 20% of cases and is associated with a poor prognosis (575). Expression of immunoreactive ErbB2 in normal colonic epithelium ranges from undetectable to low and is found from the base of the crypt to the surface epithelium (576–579). ErbB2 overexpression in human colon cancer is infrequent (507–510,579,580). Increased expression of ErbB2 occurs in a small subset of tumors of the stomach, pancreas, and esophagus, and in tumors of the neuroendocrine system, but not the liver (488,511,512,567,581–583).

The ErbB4 gene is found on human chromosome 2q33 (594). The gene product is a 180-kb glycosylated, transmembrane protein with a tyrosine kinase domain in the cytoplasmic tail. Several distinctive features of ErbB4 have been described are reviewed in detail by Carpenter (595). Alternative spicing of the RNA occurs as evidenced by the absence of a PI3K domain in the cytoplasmic tail of one variant (596) and variation in the juxtamembrane domain of another (596). Interestingly, ErbB4 is the only EGFR that is susceptible to proteolytic cleavage by TACE (375,597,598). After cleavage, the intracellular domain translocates to the nucleus (599), but its function in the nucleus is uncertain. Cells that exclusively express ErbB4 undergo chemotaxis, but fail to proliferate (417). This differential activity of ErB4 may be the result of differential expression alternatively spliced isoforms with and without PI3K domains (600). The dominant ligands for ErbB4 are BTC, HB-EFG, EPR, and neuregulins. Expression has been identified in the fetal pancreas and the upper gastrointestinal tract, but not the liver (589,601). Expression of ErbB4 in gastrointestinal neoplasms has not been reported extensively, but misexpression of ErbB4 in pancreatic, gastric, and colon cancer has been observed (601–603).

ErbB3

Epidermal Growth Factor Receptor and ErbB Receptor Knockout Mice

The c-ErbB3 gene is located on human chromosome 12q13 and encodes a glycosylated, transmembrane protein with a molecular weight of 180 kDa (584). Members of the more

Multiple EGFR mutant and gene-targeted mice have been described and offer an interesting insight into the importance of this ligand in gastrointestinal physiology. A naturally

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 205 occurring recessive mouse mutation termed waved-2 has striking phenotypic similarities with the TGF-α knockout mouse described earlier in this chapter (285). Waved-2 mice have a single amino acid substitution in the tyrosine kinase domain of EGFR, the result of which is 80% to 90% reduction of EGFR tyrosine kinase activity, but normal levels of receptor mRNA and protein (604). Thus, the waved-2 mouse is an EGFR hypomorph or “partial” EGFR knockout. No gastrointestinal phenotype is apparent in normal waved-2 mice; however, under pathophysiologic circumstances, a phenotype emerges. For example, the normal proliferative response is blunted, and the accelerated enterocyte apoptosis is increased after partial small-bowel resection in waved-2 mice (605,606). The waved-2 allele reduces the number of small intestinal polyps in the APCmin mouse model by 90% (607). Waved-2 mice have an increased susceptibility to dextran sulfate sodium-induced colitis (608). These observations emphasize that EGFR signals critically important proliferative, survival, and cytoprotective functions in the gastrointestinal epithelium. The waved-5 mutation is an EGFR tyrosine kinase “dead” mutant with dominant negative activity (609). Three groups of investigators accomplished EGFR knockout nearly simultaneously in the mid-1990s (462, 530,604). The phenotype is quite variable, ranging from peri-implantation lethality to normal newborn pups that develop wasting and death in the first and second postnatal weeks. The predominant defects in EGFR null pups are observed in epithelial tissues, although a great deal of phenotypic heterogeneity is seen, likely because of the genetic background of the deficient mice. Null mice demonstrate delayed eyelid development, thin skin, disorganized hair follicles and limited hair growth, impaired central nervous system development, impaired pulmonary alveolar development, and renal structural and functional abnormalities. Miettinen and coworkers (462,610) described short, attenuated intestinal villi, reduced jejunal epithelial DNA synthesis, altered pancreatic islet morphology, and reduced pancreatic duct branching. Terminally, the intestine of EGFR null mice was hemorrhagic and dilated with complete loss of villus architecture. Threadgill and colleagues (530) found reduced esophageal bromodeoxyuridine incorporation, but normal intestinal morphology, differentiation, and proliferation in a CD1 background. Sibilia and Wagner (611) found no obvious gastrointestinal abnormalities. ErbB2 and ErbB4 null mice die on embryonic day 10.5 because of defective cardiac myocyte development and central nervous system development (612–614). The effect of deficiency of these receptors on gastrointestinal, liver, and pancreas development is uncertain, although a cell-specific knock out of ErbB2 in the enteric nervous system and colonic epithelial cells has been reported (615). The mutant mice were growth retarded and had distended colon, reminiscent of Hirschsprung’s disease. This phenotype was dependent on the loss of ErbB2 expression in the colonic epithelium, because mice with absent ErbB2 expression in

the enteric nervous system were normal. ErbB3 null mice survive until approximately embryonic day 13.5, but they have marked abnormalities in the central nervous system and the heart and show attenuation of the gastric epithelium and mesenchymal layers, decreased numbers of enteric ganglia, and abnormal pancreatic development (612,616). Transactivation of ErbB Signaling A major development in the understanding of EGFR biology in recent years has been the elucidation of various mechanisms of receptor transactivation. Transactivation is the term used to describe activation of the EGFR directly or indirectly by heterologous ligands. Transactivation has been reported extensively for the ErbB1 receptor tyrosine kinase and is now considered a well-established pathway for receptor activation. GPCR agonists, cytokine receptors, prostaglandins, growth hormone (GH) receptor, and various adhesion receptors such as integrins all cause tyrosine phosphorylation of ErbB1 (372,617–620). In many cases, GPCR-mediated transactivation of the EGFR occurs by activation of ADAMs that induce ectodomain shedding to produce EGF-related peptides (478,621). In addition, physical, chemical, and electrical stimuli also increase the level of tyrosine phosphate on ErbB1. Blockade of Epidermal Growth Factor Signaling: A Therapeutic Option in Clinical Medicine Inhibition of ErbB receptors and their signaling constituents has become a viable option for treatment of human tumors that overexpress EGF-related peptides, EGFR-related receptors, or both. In fact, the first approved cancer therapeutic agent based on growth factor biology was the anti-ErbB2 monoclonal antibody trastuzumab. Multiple potential therapeutic options to block EGFR signaling currently are in the drug development pipeline, ranging from antibody-mediated receptor blockade, antibodies that serve as carriers of cytotoxic compounds, highly specific EGFR tyrosine kinase small molecule inhibitors, ADAM inhibitors, specific signaling pathway inhibitors, and antisense oligonucleotide or ribozyme inhibitors. All these strategies are at various stages of testing in animal models and in human trials (465,622–624).

INSULIN-LIKE GROWTH FACTORS Overview and Discovery of Insulin-Like Growth Factors The IGF ligand/receptor system includes three structurally related ligands (insulin, IGF type I [IGF-I], and IGF-II) and two high-affinity cell-surface receptors (IGF-I and IGF-II receptors). An important physiologic factor regulating the interaction between the IGFs and their receptors is the IGF binding protein family (IGFBP1-6). GH is structurally unrelated to IGFs, but its actions are primarily mediated by regulation of IGF-I synthesis and secretion in hepatocytes;

206 / CHAPTER 7 hence, GH and its biology are referenced occasionally in this chapter. The IGF system functions as a leading endocrine, paracrine, and autocrine regulatory axis for cellular proliferation, survival, and apoptosis in many organ systems, including the gastrointestinal tract. In addition, it has general activities relating to energy metabolism, body size, carcinogenesis, and various organ specific functions. A number of excellent, comprehensive reviews are available for further study (625–633).

Insulin-Like Growth Factor Genes and Proteins IGFs are two closely related members of the insulin superfamily of peptide hormones. IGF-I and IGF-II are 67% identical, single-polypeptide chains that share ≈40% amino acid identity with insulin. Historically, IGF-I and IGF-II were referred to as somatomedin-C and somatomedin-A, respectively. Current nomenclature, and this chapter, uses the terms IGF-I and IGF-II. Unlike insulin, IGFs are not produced and stored in pancreatic islets, but they are synthesized and secreted by most cells in the body in a highly regulated manner. The IGF-I gene is located on human chromosome 12q22q23. The genomic sequence is large, spanning more than 80 kB DNA, including 6 exons (634). At least 4 transcriptional start sites have been identified, and IGF-I mRNA species range from about 1.0 to 8.0 kB. Complex, tissuespecific alternate slicing patterns have been observed, but the biological significance of these variants has not been clearly delineated for the gastrointestinal tissues (635). For human pro-IGF-I, two primary translation products exist, IGF-IA and IGF-IB. The most commonly recognized mature IGF-I sequence is a 70-amino-acid, secreted protein. Most IGF-I in the peripheral circulation is synthesized in the liver in response GH. In this context, IGF-I is an endocrine growth factor and it exerts feedback to the pituitary to down-regulate GH synthesis. Most growth-stimulating activities of GH can be mimicked by IGF-I. It is clear that GH regulates IGF production in other tissues as well, including the gastrointestinal tract (see later). In malnutrition, the stimulatory effect of GH on IGF-I is markedly reduced. IGF-I binds IGF receptor 1 with high affinity and IGF receptor 2 with low affinity. The human IGF-II gene is located on chromosome 11p15.5. Like the IGF-I precursor, the IGF-II precursor is large, and multiple splice variants have been described, many of which are developmentally regulated and contain gutspecific promoters (636). A pro-IGF-II–containing carboxyterminal precursor sequence is secreted from cells before processing to the mature 67-amino-acid protein (629). The IGF-II gene is highly unique in that it is “imprinted” in 90% of humans, meaning that normally one allele is silenced on the basis of parental origin. When IGF-II genomic imprinting is lost, IGF-II levels are increased and a substantially increased risk for colorectal cancer exists (637). IGF-II binds IGF receptors 1 and 2 with high affinity.

Insulin-Like Growth Factor Receptor Genes and Proteins The IGF-I receptor (IGF-1R) gene is located on human chromosome 15q26.3 and is 70% identical to the insulin receptor gene (638). The protein is synthesized as a single polypeptide chain that is cleaved by proteolysis at a tetrabasic amino acid site resulting in α and β subunits. These subunits associate by disulfide bridging into α and β heterodimers that further associate by disulfide bridging to the mature heterotetrameric α2β2 receptors. Structurally, this overall configuration is identical to the insulin receptor; in fact, hybrid IGF-1R and insulin receptors are well recognized (639). The α subunits form the extracellular ligand-binding domain, whereas the β subunits are transmembrane and intracellular sequences that carry the tyrosine kinase signaling domain (626). IGF-1Rs bind IGF-I and IGF-II with high affinity (KD is ≈ 1 × 10−8 to 1 × 10−9) and insulin with a 100-fold reduced affinity. Most biological activities of IGF-I and IGF-II are mediated by the IGF-IR. IGF-I binding results in tyrosine autophosphorylation of the receptor, activation of its intrinsic tyrosine kinase activity, and phosphorylation of intracellular substrates (Fig. 7-7). Foremost is the insulin receptor substrate 1 (IRS-1), a 185-kDa intracellular signaling protein with multiple phosphorylation sites and SH domains permitting docking of multiple intracellular signaling molecules (640). The result is activation of a variety of signaling cascades, the most prominent of which are the Ras and phosphoinosotide-3 kinase pathways. As expected from previous discussion on EGF signaling, activation of these pathways results in cellular proliferation and survival, the predominant activities of IGFs. The IGF-IIR is identical to the cation-independent mannose-6 phosphate receptor and has no structural homology with the insulin receptor or the IGF-IR (633). It is located on human chromosome 6q26. The IGF-IIR receptor is a single, transmembrane polypeptide that binds IGF-II with a 100-fold greater affinity than IGF-I. It has 15 cysteine-laden, contiguous, extracellular repeats and a short intracellular sequence with no recognizable signaling motifs (628). Its role in IGF-II signaling is controversial, and most experts believe it down-modulates IGF-II activity by regulating its endocytosis and intracellular degradation by targeting the proteins to the lysosome. This is consistent with a growing body of data suggesting that the IGF-IIR is a tumor-suppressor gene, acting as a “sink” or reservoir for IGF-II (630,641).

Insulin-Like Growth Factor Binding Protein Gene and Protein Family IGFBPs are a well-characterized family of six secreted proteins, designated IGFBP1-6, that bind IGFs with high affinity and display a broad spectrum of biological activity. Extensive information about the gene organization, protein structure, and molecular biology and physiology of IGFBPs can be found in the reviews referenced at the beginning of this section.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 207

PI-3K

IRS-1 β-chain

IGFIR α-chain

AKT

α-chain β-chain

IGF-1

IRS-1

Shc Grb2 Sos

Ras

Raf

MEK

Target gene

ERK

-growth stimulation -survival

FIG. 7-7. Insulin-like growth factor (IGF) signaling. Signaling is initiated from the basolateral membrane domain of the polarized enterocyte. Intrinsic tyrosine kinase activity in the cytoplasmic domain of the IGF-I type I receptor (IGF-IR) is activated by IGF-I or IGF-II. Insulin receptor substrate 1 (IRS-1) is a key adaptor protein in IGF receptor signaling, linking activated receptor to the canonical Ras signaling pathway, as well as activation of phosphatidylinositol-3 kinase (PI3K) signaling. The result is stimulation of cellular proliferation and cell survival.

The IGFBPs are potent modulators of IGF activity, in part because the affinity of IGF-I and IGF-II for their binding proteins is 10- to 100-fold greater than affinity for the IGF receptors. Both IGF-I and IGF-II bind to all six IGFBPs, albeit with some differences in affinity. The structure of all six IGFBPs can be divided into three domains, highly conserved C- and N-terminal domains and a central domain that is unique among IGFBP family members. The C- and N-terminal domains bind IGFs. It is the central domain that is modified by posttranslational modifications and confers functional diversity among IGFBPs (631). Depending on the experimental setting and IGF function being assessed, IGFBPs may potentiate or inhibit IGF activity. In most contexts, IGFBPs reduce IGF activity by reserving it in a biologically inaccessible pool. Regulated proteolysis of IGFBPs is a key mechanism for release of IGF, thus regulating its bioavailability and bioactivity (631). For example, addition of IGFBP-3 to colon cancer cell culture medium decreases bioactivity of IGF-I. Addition of MMP-7 cleaves IGFBP-3 into four fragments, releases IGF-I, and potentiates IGF-I biological activity (631). Other IGFBP functions include transport of IGFs in extracellular fluids. IGFBPs are detectable in plasma and extracellular fluids and are expressed during development, as well as during adult life. The primary circulating IGFBP is IGFBP-3, which carries approximately 75% of circulating IGF-I and IGF-II (631). Activities of IGFBPs that take place independent of IGF binding are recognized; for example, IGFBP-3 activates TGF-β receptor signaling in several cell culture models,

including intestinal smooth muscle cells (642). This may be an additional mechanism by which IGFBPs inhibit cellular proliferation.

Insulin-Like Growth Factor Family Distribution in the Gastrointestinal Tract A large number of studies have described the presence and distribution of IGFs and IGF receptors in the gastrointestinal tract (635,636,643–653). IGF-I, IGF-II, and IGFBPs are also found in human breast milk and gastrointestinal tract secretions (654–656). At least a portion of enterally administered 125I-IGF-I and -II can be recovered intact from gastrointestinal tissues of suckling rats, indicating that the peptide is stable in the milieu of the neonatal stomach and small intestine (657,658). Collectively, these studies unambiguously document expression of this ligand receptor system in the gastrointestinal tract; however, a consistent picture of distribution along the crypt villus axis and in the epithelial versus mesenchymal compartments has not emerged. This lack of clarity is a result of the variety of species studied and the variety of technical and molecular approaches used to study the distribution of IGF family members. Several general conclusions can be drawn, however. First, it is clear that both IGF-I and IGF-II bind to intestinal epithelial cells in vitro and in vivo (648,650,652,659,660). Second, similar to the EGF and TGF-β receptor systems, both IGF-IR and IGF-IIR are targeted to the basolateral membrane domain of the

208 / CHAPTER 7 enterocyte and other epithelial cells (661,662). Finally, multiple components of the IGF system, including IGFBPs, are expressed in the lamina propria of this intestine, implying an important role in regulation of epithelial-mesenchymal interactions (663,664). In support of this, transgenic mice in which IGF-I is overexpressed in the intestinal lamina propria under the direction of an α-smooth muscle actin promoter shows increased proliferation of the ileal epithelium (665). As pointed out earlier, most circulating IGF-I is synthesized in the hepatocytes in response to GH. Consequently, hepatocyte levels of IGF-I are very high and IGFBPs are also synthesized in the liver. Interestingly, however, the normal hepatocyte is not considered a major target for IGF action, because the receptor is expressed at extremely low levels (666). Hepatic stellate cells and myofibroblasts also express IGFBPs and IGF-I (667), and it is these cells that are believed to play a role in fibrogenic response to IGFs in the liver (668). IGF-II and IGF-IIR are highly developmentally regulated. Intestinal IGF-II RNA transcripts are readily detectable during late fetal gestation, but are much less apparent in adult rat samples (635,645). Similar observations have been made in human stomach and intestine (653). IGF-IIR levels also decrease during rat and human development, suggesting the IGF-II/IGF-IIR axis may play an important role in intestinal development (653,669). A clear pattern of developmental expression of IGF-I/IGFIR has not emerged. Fluctuations in expression are observed and the degree of change is less apparent than that described for IGF-II (635,652,653,670).

Biology of the Insulin-Like Growth Factor Family in the Gastrointestinal Tract The gastrointestinal tract is a major target organ of IGF action (671). One of the most prominent effects is stimulation of intestinal epithelial proliferation and maintenance of cell survival by reduction of apoptosis. Other activities relate to the diverse effects of this ligand/receptor family on somatic growth, energy balance, and metabolism of glucose, carbohydrate, and proteins. Insulin-like Growth Factor Stimulates Cellular Proliferation Multiple lines of evidence indicate that IGFs are mitogenic in intestinal epithelial and smooth muscle cells and on hepatic stellate cells in vitro and in vivo. In vitro, IGFs stimulate intestinal epithelial proliferation, but, in most instances, significantly less so than other growth factors such as EGF (103,660,672–675). When EGF is provided with IGF-I or insulin, a synergistic effect on intestinal epithelial proliferation often is observed. Isolated intestinal smooth muscle cells and isolated hepatic stellate cells in vitro also proliferate in response to IGF (676,677). The in vivo evidence for a proliferative effect of IGF on the intestine is dramatic and has been observed in multiple

experimental models using both enteral and parenteral IGF. Several illustrative examples are cited here. Oral feeding of IGF-I to neonatal pigs increases indices of small intestinal weight, DNA content, protein content, and villus height in the small intestine (678). These effects are most prominent in the proximal small intestine and are not seen in the pancreas or stomach. In a fetal sheep model, in utero ligation of the esophagus deprives the distal intestine from growth regulatory peptides in amniotic fluid. A 10-day infusion of IGF-I distal to the ligation results in increased small intestinal growth (679). Potten and coworkers (680) found a modest stimulation of small intestinal crypt labeling index after brief treatment of mice with an intraperitoneal injection of IGF-I. Similarly, a 14-day parenteral treatment of adult rats with IGF-I increased crypt depth and villus height 30% over control values (681). In this study and others, the proximal small intestine is the most responsive region. In multiple models, infusion of LR3IGF-I, an N-terminal–extended analogue of IGF-I that has a reduced affinity for IGFBPs, better stimulates proliferation in the epithelium and muscularis of the small intestine than the parent peptide (682,683). IGF-I also has prominent effects on intestinal growth under various pathophysiologic conditions. For example, IGF-I augments adaptive mucosal proliferation in the small intestine of rats that have undergone a partial small intestinal resection (684–686). The mucosal atrophy observed in the jejunal mucosa in parenterally fed rats is blunted by administration of inclusion of IGF-I in the parenteral nutrition solution (687). Similarly, the small intestinal atrophy that occurs in the context of chronic liver disease or sepsis is also reduced by administration of IGF-I (688,689). Studies in transgenic mice also support a proliferative effect of GH or IGFs on the intestinal mucosa. Transgenic mice overexpressing GH have increased bowel length and mass, but a normal intestinal crypt cell proliferation rate, suggesting that GH increases survival of intestinal epithelial cells (690). As expected, plasma and intestinal mucosal IGF-I concentrations were increased, suggesting that IGFs are “survival factors.” In transgenic mice overexpressing IGF-I under a MT-I promoter, circulating GH levels are undetectable because of negative feedback from IGF-I, thus permitting dissection of the specific effects of GH and IGF-I on the intestine. MT-IGF-I mice exhibit a significantly greater small intestinal length and mass, an increased villus height, greater crypt depth, and a higher crypt cell mitotic index compared with control littermates (691). Indices of differentiation are not altered. Overexpression of IGF-II in a transgenic mouse either does (692) or does not (693) increase the mass of the gastrointestinal tract, but detailed effects on the gastrointestinal tract were not reported in either case. Thus far, detailed attention to the development and function of the intestine in IGFBP overexpressing mice has not been reported. Collectively, the studies outlined previously suggest that IGF-I and GH, the latter of which is already used extensively in the clinical arena for other indications, may be useful therapeutic agents in patients with short bowel syndrome.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 209 Indeed, an increasing body of data suggests GH improves intestinal function in patients with short bowel syndrome (694), although not all studies and authors support this view (695). In animal models of short bowel syndrome or intestinal atrophy, it is clear that IGF-I is more potent in stimulating intestinal growth than GH. It has been hypothesized that this is because of induction of suppressor of cytokine signaling-2 (SOCS-2) by GH, but not IGF-I (696). In cell culture systems, SOCS-2 is growth inhibitory, apparently blunting the proliferative response to GH in the intestine. New clinical trials using IGF-I may offer more encouraging results in subjects with decreased gastrointestinal mucosal function. Insulin-like Growth Factor Is a “Survival Factor” Studies referenced earlier suggest that IGFs increase intestinal growth by inhibition of apoptosis. Indeed, MT-IGF-I transgenic mice exhibit a lower background level of spontaneous apoptosis in small intestine crypts and also a lower level of apoptosis in response to irradiation (697). This observation is congruent with reports that IGF-I inhibits apoptosis in cultured cell lines (698). Insulin-like Growth Factor Is Profibrogenic Several lines of evidence support the profibrogenic actions of IGFs in the intestinal tract. IGF-I stimulates proliferation and inhibits apoptosis of cultured fibroblasts, myofibroblasts, and smooth muscle cells and increases collagen synthesis in intestinal smooth muscle cells (699,700). Transgenic overexpression of IGF-I in the lamina propria of SMP8-IGF-I mice increases the mass of the muscularis propria and length of the intestine (665). The adaptive response to surgical resection of the small intestine in this same SMP8-IGF-I mouse is characterized by a marked lengthening of the residual bowel, suggesting IGF-I autocrine activity increases the mesenchymal elements in the intestine (701). In Crohn’s disease, a human disorder characterized by fibrosis and stricture formation, mucosal IGF-I and IGFIR levels are increased relative to normal intestine (702). Despite the weight of this evidence, it should be noted that not all models support a profibrogenic response to IGF-I. For example, carbon tetrachloride injury of the SMP8-IGF-I mouse liver results in reduced collagen synthesis and amelioration of the extent of liver injury (703). Insulin-like Growth Factor in Gastrointestinal Cancer The prominent effects of IGF ligands and receptors on cellular proliferation and survival suggest this growth factor axis may play an important role in neoplastic disorders. Multiple lines of evidence now support this view (630,632). Circulating IGF-I levels vary considerably among healthy individuals, and population studies suggest an overall trend toward increasing cancer risk, including cancer of the gastrointestinal tract, in persons at the high end of the normal range of IGF-I blood levels. Loss of IGF-II imprinting, resulting in

a modest increase in IGF-II levels, is now widely accepted as a marker for colorectal cancer risk (637). Increased expression of IGF-I, IGF-II, and IGF-IR occur in colorectal cancers (704).

Elimination of Insulin-Like Growth Factor Function by Gene Targeting Gene targeting has been used extensively to study the function of the IGF system (this topic has been reviewed by Dupont and Holzenberger [633], Wood [705], and Butler and LeRoith [706]). A prominent effect is fetal growth retardation, implying that the IGF system is a major regulator of in utero growth. For example, IGF-IR–deficient mice weigh approximately 45% of their normal littermates and die of respiratory failure within minutes of birth (707). Significant abnormalities occur in several organ systems and include hypoplasia of skeletal muscle, central nervous system anomalies, skin and hair follicle abnormalities, and delayed bone development. IGF-I–deficient mice are about 60% of normal birth weight. Depending on the genetic background, some mice die in the newborn period, whereas others survive into adulthood. Placental development is normal (708). No significant intestinal, liver, or pancreatic phenotype has been observed in IGF, IGF receptor, or IGFBP gene–targeted mice.

TREFOIL FACTOR FAMILY OF PEPTIDES Overview and Discovery of Trefoil Factor Family Trefoil factor family (TFF) represents a relatively new class of biologically active peptides that appear critical in maintaining gastrointestinal epithelial integrity under normal and pathologic conditions (709–713). TTF peptides are differentially synthesized wherever mucin secretion occurs, being prominently expressed within the gastrointestinal mucosa. TFF proteins are constituents of mucous gels and have been shown to rapidly modulate mucosal restitution through a combination of motogenic, cell-scattering, antiapoptotic, and anti-inflammatory activities. Several members of the trefoil family have also been implicated as gastrointestinal-specific tumor-suppressor genes, whereas the coordinate expression of TFF peptides within neural tissues suggests a potential neuromodulatory role for these proteins both within the brain and peripheral targets including the gastrointestinal tract. The trefoil, or P domain, is a conserved 38- to 39-aminoacid motif containing 6 cysteine residues held together by 3 pairs of disulfide bonds configured 1-5, 2-4, and 3-6 (714). This domain produces a compact “three-leafed clover” protein structure that has marked thermal and enzymatic stability, a property that is most likely critical to the function of trefoil proteins within the harsh environment of the gastrointestinal tract (715–719). The trefoil domain has been highly conserved through evolution and is found in numerous species from

210 / CHAPTER 7 Xenopus to humans (709). Three distinct trefoil peptides have been identified in mammals. TFF1, originally designated pS2, was isolated from an estrogen-sensitive breast cancer cell line (720). TFF2, or spasmolytic polypeptide, was identified in the early 1980s during purification of porcine insulin (715), whereas intestinal trefoil factor, or TFF3, was isolated from rat intestine in 1991 (721). TFF1 and TFF3 each contain a single-trefoil domain, whereas TFF2 contains two-trefoil motifs as a result of genomic duplication (721–723).

Trefoil Factor Family Genes The TFF proteins are the product of a multigene family organized in a 50-kb head-to-tail linear arrangement on human chromosome 21q22.3 (724–726). A similar clustered organization for the TFF genes is observed on a 40-kb syntenic region on mouse chromosome 17q (721,728). Genetic studies indicate that TFF genes share several common regulatory motifs including those binding hepatocyte nuclear factor 3 (HNF-3)/forkhead, GATA6, AP-1, upstream stimulatory factor (USF), and the Myc/Max/Mad network of proteins (729–734). In addition, the individual TFF genes each contain a subset of specific regulatory elements that modulate cell-specific and physiologic expression (729). The combinatorial use of these regulatory motifs therefore provides the ability for coordinate as well as independent expression of each TFF member. The 4.3-kb human TFF1 gene is composed of three exons with its single-trefoil domain encoded by exon 2 (735). The proximal promoter domain of TFF1 contains a series of standard regulatory elements that includes a TATA-box, a CAAT element, and a GC-rich element. Additional specific regulatory elements including an AP-1 binding site, estradiol responsive element (ERE), heat shock factor 2 (HSF-2) motif, GATA consensus sequence motif, and a tissue-specific enhancer that potentially binds EGF, TPA, c-Ha-ras, and c-jun have also been characterized in association with the TFF1 gene promoter (729,733–737). TFF1 gene expression has been shown to be responsive to insulin, IGF-1, bFGF, and retinoic acid (738,739). The presence of an ERE within the TFF1 promoter provides rationale for the original identification of this TFF member in estrogen-sensitive breast cancer cell lines. Interestingly, the ERE is missing from the mouse TFF1-promoter domain (740). The human TFF2 gene is 4.6 kb is length and is composed of 4 exons with its duplicated trefoil domains encoded by a single exon (734). TFF2 expression appears to be modulated by AP-1, USF, PEA3, Ets-like factor, HSF-2, and GATA6 and has been shown to be responsive to asparin (730,731,734,741,742). No ERE is observed within the TFF2 promoter, which explains its lack of responsiveness to estradiol. Stomach-specific expression of both TFF2 and TFF1 appears to be principally modulated by the HSF-2 and GATA consensus motifs found within their promoters (731). The 3.5-kb human TFF3 gene is composed of 3 exons with the trefoil domain encoded by exon 2 (743). The TFF3 gene contains potential binding sites for SP-1, AP-1, CF1,

and HRE (743,744). Unlike its gastric counterparts, TFF3 does not contain HSF-2 and GATA consensus motifs and is unresponsive to GATA6 (729,731). Goblet cell–specific transcription of TFF3 appears to be modulated by the presence of a 9-bp goblet cell response element (GCRE), the expression of which can be modified by adjacent positive and negative regulatory elements (745). In addition, a second goblet cell silencer inhibitor (GCSI) that interacts with a specific GCSI binding protein has been shown to modulate TFF3 expression in certain cancer cell lines (746–748). Interestingly, the mucin-2 (MUC2) promoter also contains two GCREs providing a common regulatory pathway for coordinate regulation of these genes (749). TFF3 expression has been shown to be responsive to neuropeptides, acetylcholine, hypoxia, and short-chain fatty acids (744,750–752). A variety of studies suggest that the epigenetic regulation of TFF transcription may play an important role in modulating their expression (751,752). Variations in histone acetylation have been postulated to modulate TFF gene transcription in cancer cell lines, although hard evidence for this mode of regulation remains to be identified (751). In contrast, good evidence exists to support the observation that CpG methylation of TFF genes plays an important role in their expression. Mouse studies indicate that all three TFF genes appear demethylated in gastrointestinal tissues and methylated in nongastrointestinal tissues, whereas TFF1 and TFF3 possess reciprocal methylation states in the stomach and intestine specifically limiting their expression patterns to these tissues (752).

Trefoil Factor Family Proteins TFF proteins are small 7- to 12-kDa, highly stable proteins that are produced by mucin-secreting epithelial cells found throughout the body (709–713,753–756). Synthesized as precursors containing an N-terminal signal sequence, TFF proteins are copackaged into mucous granules within the Golgi apparatus and eventually secreted onto the epithelial cell surface (713). Although trefoil peptides are principally expressed within the gastrointestinal tract, their expression has also been observed in a variety of other tissues including conjunctiva, uterus/endocervix, salivary glands, respiratory tract, nonlactating breast, gallbladder, pituitary, hypothalamus, hippocampus, frontal cortex, and cerebellum (757–769). Human serum contains approximately 150 pg/ml TFF1, whereas human urine has a TFF1 concentration of 14 ng/mg creatinine (770–772). The concentration of TFF2 in gastric juice has been reported to be between 1 and 20 µg/ml with dramatic diurnal variation (773). Within the gastrointestinal tract, trefoil proteins display cell-specific patterns of expression that are both temporally and spatially regulated (753). TFF1 and TFF2 are considered “gastric” peptides showing complementary expression within the pit mucous cells and mucous neck cells of the stomach, respectively (753,758,764,774–777). TFF2 expression is also observed within Brunner’s glands of the duodenum

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 211 (776,778,779). In contrast, expression of TFF3 appears “intestinal,” being primarily limited to goblet cells of the small intestine and colon (740,780–782). These cell-specific patterns of TFF expression result in a regional colocalization of specific TFF proteins and secretory mucins, thereby providing a potential molecular basis for the functional mucosa specificity. TFF expression occurs early in embryonic development, with all three TFF proteins coordinately expressed within the gastrointestinal tract (753,783,784). As development proceeds, the expression of the individual TFF peptides becomes progressively more restricted, resulting in the cell-specific patterns observed in the adult. Similar developmental regulation of TFF expression has also been observed in the brain (767). It is important to note that there is a large degree of species variation in the expression patterns of TFF proteins.

Trefoil Factor Family Receptors and Binding Proteins The direct receptors or binding proteins, or both, for the various TFF peptides have not yet been identified. TFF peptides are expressed at high levels at the apical surface of the normal gastrointestinal tract, suggesting that their association with high-affinity receptors may be problematic (709–710). It has been proposed that TFF–mucin interactions may mediate many of the biological properties of trefoil factors; however, TFF3 elicits a functional response only when applied to the basal surface of monolayers (785). In addition, findings that TFF proteins rapidly activate several intracellular signaling pathways strongly suggest that specific receptors/binding proteins exist. Although two TFF2-binding proteins have been identified, neither has been shown to mediate the cellular responses observed for TFF2 (786–789).

Intracellular Signaling by Trefoil Factor Family A variety of mechanisms have been postulated as modulating the intracellular responses of TFF proteins. These include activation of the EGFR and ras-dependent MAPK pathway (790–792). TFF3 induces phosphorylation of β-catenin and the EGFR, resulting in the intracellular redistribution of E-cadherin, which complexes with catenins (619,793,794). In addition, TFF proteins directly decrease the expression of catenins and the APC gene product, which completes with E-cadherin (794). The subsequent convergence of these two pathways therefore has a direct negative effect on the adhesive properties of cells, thereby promoting cell migration. TFF proteins inactivate apoptosis by a similar convergence of pathways including the phosphorylation of Akt, as well as the blockage of p53-independent and -dependent apoptosis through the EGFR and PI3K pathways (711). These pathways promote cell survivability in the absence of proliferation, which when combined with the motogenic pathways above result in a coordinated cellular response

supporting migration in the absence of programmed cell death and proliferation.

Biological Actions of Trefoil Factor Family Proteins In vitro studies, as well as recent transgenic knockout models, have helped to clarify the specific roles of TFF peptides within the gastrointestinal tract (795–800). From this work, it seems clear that the primary function of TFF proteins is the establishment and maintenance of mucous epithelial surface integrity (Fig. 7-8). TFF proteins function both in an autocrine and paracrine manner within the gastrointestinal tract, with optimal biological activity involving the interaction of two distinct trefoil domains (712,801). TFF2, which contains two trefoil domains, is eight times more efficient at promoting cell migration that monomeric TFF1 (799–800). The monomeric TFF proteins, TFF1 and TFF3, have been shown to form homodimers and heterodimers through disulfide linkage at a seventh conserved cysteine residue found within the C-terminal dimerization domain (775,802–806). Even so, monomeric TFF3 is capable of promoting cell migration, suggesting that dimerization may not be essential for TFF function (717,800,807). That TFF peptides are compact, highly stable proteins that are cytoprotective and promote healing when administered orally, subcutaneously, or intravenously makes them prime therapeutic targets for the prevention and resolution of gastrointestinal mucosal injury (808–810). Mucosal Defense TFF proteins have been shown to be coexpressed with mucins and enhance the protective characteristics of the mucosal defense barrier within the gastrointestinal tract (753,811,812). The addition of TFF2 to mucin preparations results in increased elasticity and viscosity of the mixture (755,780,813–816). It is postulated that TFF proteins are capable of cross-linking with mucin glycoproteins to form a physical barrier designed to protect the epithelial lining of the gastrointestinal tract (775,817). In addition, TFF peptides may directly affect the packaging of mucin glycoproteins within goblet cells, thereby regulating their secretion and subsequent function (711,712). Tissue Repair One of the fundamental actions of TFF proteins in the mature gastrointestinal tract is to promote epithelial cell restitution after mucosal injury (709–713,818). Loss of mucosal continuity within the gastrointestinal tract results in a coordinate increase in TFF expression in the regions immediately juxtaposed to the insult (712,819). This response is rapid, occurring as quickly as 30 minutes after injury, and is evident during inflammatory bowel disease, as well as a variety of ulcerative conditions (810). The coordinate expression of all three TFF members at the site of injury is similar to the pattern observed during early gastrointestinal

212 / CHAPTER 7 Mucosal defense

Cell proliferation

Cell-cell adhesion

Cell migration/ tissue repair

Differentiation Inflammatory response

Apoptosis

= Mucins = TFF

Transcriptional effects Enterocyte

Enterocyte Goblet cell Cell-substrate adhesion

Tumor suppression Potential neuropeptide

FIG. 7-8. Schematic representation of the biological action of trefoil factor family (TFF). TFFs are cosecreted with mucins from goblet cells and deposited on the apical surface of the gastrointestinal epithelium where they interact to increase mucosal defense. In addition, TFFs promote restitution by decreasing cell proliferation, cell–cell adhesion, cell–substrate adhesion, and apoptosis, thereby promoting cell migration in the absence of cell death. TFFs have also been proposed to be circulating neuropeptides, as well as play a role in tumor suppression.

development and may reflect a return to a more permissive microenvironment that is necessary for normal wound healing. It has also been suggested that the TFF expression supports the appearance of the ulcer-associated cell lineage, thereby directly stimulating gland regeneration in areas of ulceration (779,780,820,821). During restitution, TFF peptides act as motogens, supporting cell migration in the absence of cellular proliferation and apoptosis (709–713,717,754,755,783,790,799–800, 811,813,822–824). TFF proteins are believed to promote cell migration by the disruption of cell–cell and cell–substrate adhesion while promoting cell survivability. TFF-induced disruption of cell–cell adhesion appears to occur via rapid phosphorylation of β-catenin, causing the down-regulation of adhesion molecules associated with adherens junctions such as E-cadherin (619,712,793,794,825). In a similar manner, TFF treatment has been shown to induce focal adhesion kinase (FAK) phosphorylation, resulting in the downregulation of focal adhesion complexes and disruption of cell–substrate adhesion (712,822). Although the precise mechanism remains ambiguous, TFF proteins are believed to prevent cell death through the convergence of multiple pathways causing ERK/MAPK activation (712,717,790–791). TFF function is linked to the expression of other growth factors and signal transduction pathways that work in synergy to promote healing in the gastrointestinal tract. For example, although TFF proteins appear to work independent of TGF-β, they function synergistically with EGFR ligands such as TGF-α and erbB family members (619,709–713, 778,790,798,807,813,826–829). These complex interactions

generate a coordinate and sustained response to mucosal injury that is most likely critical to its eventual resolution. Although TFF3 knockout mice appear to develop normal gastrointestinal tissues, they show significantly impaired mucosal healing after injury and eventually die of extensive colitis (797). In a similar manner, TFF2 knockout mice display an increased susceptibility to injury by nonsteroidal antiinflammatory drugs (796). These observations highlight the important role that TFF plays in the mucosal defense and repair of the gastrointestinal tract. Development The coordinate expression of all three TFF isotypes in the developing gastrointestinal tract before the appearance of mature mucin-secreting cells suggests that TFF peptides may play a role in the differentiation and maturation of gastrointestinal epithelium. TFF1 promotes gastrointestinal differentiation by decreasing apoptosis and delaying the G1-S-phase transition in cells (830). This hypothesis is supported by several knockout studies, which demonstrate that TFF1 is essential for the normal differentiation of antral and pyloric mucosa, whereas TFF3 is critical in modulating intestinal cell proliferation rates (795,797). Inflammation Both TFF1 and TFF2 are expressed at high levels in rat lymphoid tissues and have been shown to participate in the immune response (831,832). In addition, the TFF proteins

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 213 have been shown to induce directed monocyte migration, block complement deposition in vitro, and increase interleukin-6 (IL-6) secretion (829,831–835). Transgenic studies suggest that the SHP2-ERK/MAPK pathways are capable of inducing TFF1 expression, whereas IL-6 appears capable of regulating TFF3 expression (836). These observations suggest that TFF proteins may have direct effects on the inflammatory response providing an additional mechanistic synergy designed to promote wound healing within the gastrointestinal tract. Tumor-Suppressor Genes TFF1 was originally isolated from an estrogen-responsive human breast carcinoma cell line. Changes in TFF gene and protein expression have been documented in a wide range of epithelial cancers including those of the gastrointestinal tract, breast, pancreas, lung, endometrium, ovary, prostate, bladder, and cervix (754,837–845). Both TFF1 and TFF3 knockout mice show increased rates of gastrointestinal tumor development, whereas loss of TFF1 expression has been reported in human gastric cancers as a result of somatic mutation (795,797,846). In addition, overexpression of TFF1 and TFF3 has an antiproliferative effect on a variety of cell lines (837–845). These observations support the contention that at least TFF1 and TFF3 may represent tumorsuppressor genes within the gastrointestinal tract. It is postulated that TFF peptides promote the metastatic capacity of tumor cells by promoting cell migration in the absence of apoptosis. Neuropeptide TFF proteins are coordinately expressed in the developing and mature brain independent of mucous production, suggesting a potential role for these factors in normal neural development (767). Expressed of TFF1 has been reported in the rodent hippocampus, frontal cortex, and cerebellum with staining principally occurring within astrocytes (767). TFF3 is expressed in the hypothalamus of both rats and humans, with TFF3 and oxytocin being stored in the same secretory vesicles (767–769,847). These observations suggest that TFF3 may be playing a neuromodulatory role within the brain, as well as in peripheral targets including the gastrointestinal tract.

HEPATOCYTE GROWTH FACTOR Overview and Discovery of Hepatocyte Growth Factor HGF, or scatter factor, was originally identified in the early 1980s as a component of serum that was highly mitogenic for hepatocytes (848–850). More recent studies indicate that HGF plays a key role in gastrointestinal development and repair (851–857). HGF is principally synthesized in cells of mesenchymal origin and represents a pleiotrophic cytokine that induces cell proliferation, motility, differentiation, and

morphogenesis via its high-affinity receptor c-met (857–861). HGF can stimulate the growth of gastrointestinal epithelial cells in vitro and enhance intestinal mucosal repair in vivo, and it plays a critical role in gastrointestinal morphogenesis during development (851–857).

Hepatocyte Growth Factor Gene The single-copy human HGF gene is composed of 18 exons and is located on chromosome 7q21.1 (862–864). IL-1, TNF-α, and TGF-β and a variety of growth factors have all been shown to regulate HGF expression at the level of gene transcription (865–868). A novel regulatory element that binds inducible transcription factors in response to extracellular stimuli has been identified within the basal promoter of the HGF gene (869). Both HGF mRNA and protein expression are up-regulated by EGF, TGF-α, and PDGF, whereas TGF-β down-regulates expression of HGF (870,871). Heparin is a strong inducer of HGF expression, whereas estrogen stimulation through the EGFR induces HGF transcription (872,873). HGF has been shown to regulate met transcription providing a direct regulatory feedback loop between ligand and receptor (874,875).

Hepatocyte Growth Factor Protein Principally a product of mesenchymal-derived cells, HGF is a heparin-binding glycoprotein that is synthesized as a 728-amino-acid precursor that contains a 29-amino-acid signal sequence and 25-amino-acid prosequence (876,877). HGF is synthesized and secreted as an inactive single-chain pro-HGF that is processed into a mature, biologically active heterodimer by extracellular cleavage (876–882). Mature HGF heterodimer is composed of a 60-kDa α-chain and a 30-kDa β-chain that is proteolytically cleaved from pro-HGF at 494 arg-495 val by the serine proteinase HGF activator (HGFA). The α-subunit of HGF possesses four kringle domains that are believed to mediate receptor-ligand binding and have been shown to be necessary for the correct biological activity of HGF, whereas the β-subunit of HGF has an inactive serine protease domain (876,883–886). HGFA is related to coagulation factor XII and has been shown to activate pro-HGF in response to liver, renal, and gastrointestinal injury, as well as during gastrointestinal development (870,887–891). Although HGFA is produced and secreted mainly in the liver and circulates in the plasma as an inactive precursor, HGFA is also expressed at high levels throughout the gastrointestinal tract (887,891,892). The developmental and physiologic expression patterns of HGFA provide a secondary mode of regulation for HGF biological activity by spatially and temporally delimiting the production of active HGF heterodimer formation. HGFA is synthesized as an inactive precursor that is activated by thrombin (890,893). HGFA activity has also been shown to be regulated by HGFA inhibitor type 1 (HAI-1) and HAI type 2 (HAI-2), both of which are Kunitz-type serine

214 / CHAPTER 7 proteinase inhibitors (894–896). In vitro studies indicate that HAI-1 is also a specific cell-surface binding protein for active HGFA (896).

Hepatocyte Growth Factor Receptors The high-affinity receptor for HGF is the transmembrane tyrosine kinase c-met (880). Originally identified as an activated oncogene in an N-methyl-N′-nitrosoguanidine-treated human osteogenic sarcoma cell line, c-met is a member of the receptor tyrosine kinase family that possesses significant homology to Ron and c-sea (897–901). The c-met receptor is expressed by a variety of epithelial/endothelial cells including those of the gastrointestinal tract, as well as gastrointestinal smooth muscle cells (902). The c-met receptor is a heterodimer composed of a 50-kDa α-chain that is disulfide linked to a 145-kDa β-chain forming a 190-kDa complex (903). The c-met receptor α-chain is exposed to the cell surface, whereas the β-chain spans the cellular membrane and contains an intracellular tyrosine kinase motif (880, 904–906). As with many growth factor receptors, c-met is found primarily on the basolateral domain of polarized epithelial cells supporting the proposed paracrine stimulation via HGF secreted from closely associated mesenchymal cells (903,907). However, studies indicate a second paracrine mode of action for HGF involving luminal expression of HGF in body fluids that is bound by a c-met/src-related gastrointestinal tyrosine kinase (gtk) complex found in the brush border membranes of enterocytes (907). Autocrine regulation of HGF action has also been described in some cancer cell lines (908,909). c-Met expression is regulated by a number of factors including HGF, PMA, ConA, EGF, IL-1, IL-6, TNF, and the steroidal hormones estrogen, progesterone, tamoxifen, and dexamethasone (874,903,910,911). HGF binding has been shown to result in c-met polyubiquitination leading to its degradation, thereby providing a potential regulatory feedback loop between HGF activation and receptor turnover (903). HGF also directly promotes expression of the met gene (875).

Intracellular Signaling by Hepatocyte Growth Factor Binding of HGF to the c-met receptor has been shown to initially induce tyrosine phosphorylation of the β-chain (912). The differential recruitment of substrate molecules to the phosphotyrosylated segments on the C-terminal cytoplasmic domain of the c-met receptor is proposed to activate distinct signaling pathways (901,913–915). For example, STAT has been shown to be critical in mediating tubular morphogenesis, whereas cell scattering appears to be regulated through the Ras-MAPK signaling pathway. In a similar manner, PLCγ activation appears critical for HGF-induced cell motility, with phosphorylation of PLCγ resulting in the

formation of DAG and IP3, thereby stimulating the PKC and Ca2+ mobilization signaling pathways, respectively (903,910). HGF-mediated ERK activation appears critical in signaling cell proliferation, whereas the nuclear translocation of ERK is believed to regulate transcription factors (903). HGF binding to c-met has also been shown to activate the guanine-nucleotide binding protein, ras, thereby inducing alterations in cell movement and cell shape by cytoskeletal remodeling (903).

Biological Actions of Hepatocyte Growth Factor HGF has been shown to be a mitogen, motogen, and morphogen (915a). HGF activity represents a classic example of epithelial/mesenchymal induction, with the most common mode of action for HGF being activation of the c-met receptor via paracrine mechanisms. It is generally proposed that cells of mesenchymal origin produce pro-HGF, releasing it into the local microenvironment where it is processed to biologically active mature HGF heterodimers (Fig. 7-9). As discussed earlier, active HGF associates with heparin and is bound to epithelial cells via the high-affinity c-met receptor, resulting in receptor phosphorylation and activation of multiple intracellular signaling pathways that ultimately lead to a variety cellular responses. The biological activity HGF is directly regulated within the local microenvironment by the complex interaction of both activator and inhibitor molecules. The precise interplay between these molecules appears essential in determining whether active HGF heterodimers are present in sufficient quantities to bind to the c-met receptor and elicit a biological response. Development The morphogenic properties ascribed to HGF are believed to occur by a unique combination of actions including alterations in cytoskeleton, focal contacts, and cellular junctions (903). HGF stimulates the growth of gastrointestinal epithelial cells in a dose-dependent and region-specific manner, with distinct gastrointestinal epithelial subpopulations displaying different mitogenic responses (851,854,857). In vitro studies indicate that subepithelial intestinal fibroblasts regulate intestinal epithelial cell proliferation through the paracrine action of HGF (916). Studies indicate that HGF is capable of cross-linking with gtk, a src-related kinase that is highly expressed in the brush border of the small intestine, thereby inducing gtk kinase activity and promoting modulation of the growth and differentiation of gastrointestinal epithelial cells (907). Developmental studies indicate both HGF and c-met are expressed in human fetal tissue by 6 weeks of development with expression observed in the placenta, lung, liver, pancreas, and throughout the developing gastrointestinal tract (917,918). During development, HGF has been shown to induce branching tuburogenesis in the kidney, lung, and mammary gland, whereas in the adult, HGF appears critical

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 215 HGFA

Thrombin

HGFA

IL1β HAI-1

Mucosal Injury

HGFA Liver & GI Tract

HS HS proHGF

Activation

proHGFA

HGF

Met

HGF Promotes proliferation migration differentiation

Epithelial cell

Epithelial cell

Epithelial cell

Promotes angiogenesis HS HGF Mesenchymal cell t Me

Endothelial cells

FIG. 7-9. Proposed model of hepatocyte growth factor (HGF) wound healing in the gastrointestinal (GI) tract. Circulating pro-HGF activator (proHGFA) is synthesized in the liver and gastrointestinal tract and becomes activated by thrombin on mucosal injury. In the presence of cytokines such as IL-1β, sequestered HGFA inhibitor type 1 (HAI-1)/HFGA complexes are released from the site of injury, thereby activating pro-HGF into active HGF, which interacts with heparin sulfate and binds to the c-met receptor promoting proliferation, differentiation, and migration in gastrointestinal epithelial cells and angiogenesis in vascular endothelial cells. HS, heparan sulfate.

in the regeneration of the same organs (859,919–926). Genetargeting studies indicate that HGF-met signaling is required for normal development of liver, skeletal muscle, placenta, and specific neural crest lineages (917,927). Consequently, HGF knockout mice are embryonic lethal as a result of impaired placental and liver development. Overexpression of HGF in transgenic mice results in an increase in renal failure and tumorigenesis (928,929). Interestingly, transgenic mice that overexpress HGF throughout the digestive tract experience development of ulcerative proctitis, rectal prolapse, and intestinal pseudoobstruction (857). These studies provide strong evidence that the HGF-met signaling is critical in the development and pathogenesis of numerous organ systems including the digestive tract (930). Tissue Repair HGF has been shown to induce the proliferation and migration of gastrointestinal epithelial cells in vitro, and gastrointestinal expression of HGF and its receptor c-met is greatly increased after acute injury and in regions of ulceration (856,896,931). In addition, HAI-1 expression has been shown to be up-regulated in regenerative epithelium (896). During epithelial injury, inflammatory cytokines such as IL-1β have been shown to promote the release of active HGFA/HAI-1 complexes from the cell surface (896). These studies suggest that HAI-1 may function as a reservoir of active HGFA in

regions of epithelial repair, thereby providing efficient localization of active HGF at the site of mucosal injury. Studies suggest that HGF-mediated repair occurs through a TGF-β–dependent pathway that involves the integration of both motogenic and mitogenic pathways (932). For example, HGF has been shown to inhibit the assembly of adherens junctions by directly modifying the stability and phosphorylation pattern of E-cadherin (933). In addition, HGF appears capable of directly regulating cell adhesion by decreasing expression of E- and P-cadherin at the protein level (934). HGF has also been shown to enhance the phosphorylation status of β-catenin, resulting in the dissociation of β-catenin and E-cadherin, thereby promoting the dismantling of cell– cell adhesion complexes (935,936). HGF is capable of modulating cell-matrix adhesion by altering the pattern of integrin expression, as well as modulating the recruitment of integrins, cytoskeletal proteins, pp125FAK, and paxillin into focal adhesion complexes (930,937). HGF stimulates matrix degradation by several mechanisms including increasing the production of plasminogen activator type 1, tissue factor, MMPs, collagenase-1, and stromelysin-1 (938,939). In addition, HGF binds directly to several matrix molecules such as fibronectin, heparin sulfate proteoglycan, and thrombospondin-1 (903). HGF has been shown to regulate cell motility by the selective up-regulation of motor-related proteins including α-tubulin, myosin II, actin, kinesin, and myosin 1α (940).

216 / CHAPTER 7 Tumorigenesis HGF and c-met are expressed in a wide range of tumors and have been implicated in regulating tumor cell motility, growth, proliferation, invasion, progression, and neovascularization (903,941–944). A variety of studies indicate that both HGF and c-met are highly expressed or overexpressed in gastrointestinal tumors including those of the esophagus, stomach, colon, and rectum (903,936,945–953). Distinct mutations and isoforms of c-met have been reported in both hereditary and sporadic forms of cancer (903,954). Both c-met and HGF have been shown to be valuable prognostic indicators, and HGF represents a viable target for cancer treatment, with inhibition of its function reducing many of its effects in cancer cells (947,951,955–963).

FIBROBLAST GROWTH FACTOR FAMILY Overview and Discovery of Fibroblast Growth Factor Family Fibroblast growth factors (FGFs) are a large family of structurally related proteins that are involved in diverse cellular processes including proliferation, differentiation, migration, and apoptosis (964). Originally identified in the early 1970s, FGF was characterized and named based on its ability to stimulate the proliferation of 3T3 fibroblasts (965). Since then, an additional 21 members of the FGF family have been identified in vertebrates (Table 7-2) (964,966). FGFs elicit their biological responses by binding to and activating FGF receptors (FGFRs) (967–969). Studies suggest that FGFs and their receptors are differentially expressed throughout the developing and mature gastrointestinal tract, where they

appear to play an important role in modulating gut development and epithelial repair mechanisms after injury (964, 970–973).

Fibroblast Growth Factor Genes Each FGF family member is the product of a single, unlinked gene ranging in size from 5 to 100 kb (966,974). Although most FGF genes are found scattered throughout the genome, some clusters have been observed in both humans and mice (966). Human FGF3, FGF4, and FGF19 are clustered on chromosome 11q13; FGF6 and FGF13 map to chromosome 12p13; and FGF17 and FGF20 are clustered on 8p21-p22. The most common genomic organization of the prototypic FGF gene is three coding exons, with exon 1 containing the initiation start site (966). Even so, alternative splicing, initiation sites, and amino termini have been reported for distinct FGF family members, making the precise organization and regulation of these genes best examined on an individual basis.

Fibroblast Growth Factor Proteins The FGF family currently consists of 22 members that have been subdivided into 7 distinct subclasses based on a combination of differential criteria including structure, biochemical properties, receptor affinities, and expression patterns (see Table 7-2) (966,975). Human FGF15 and mouse FGF19 have not been identified. However, evolutionary studies indicate that these two FGFs are most closely related to one another, suggesting that FGF19 may be the human ortholog

TABLE 7-2. Fibroblast growth factor family members and receptor affinity Ligand

Subfamily

Synonym

FGF-1

FGF1

Acidic FGF, aFGF

FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGF-11 to -14 FGF-16 FGF-17 FGF-18 FGF-19 FGF-20 FGF-21 FGF-22 FGF-23

FGF1 FGF7 FGF4 FGF4 FGF4 FGF7 FGF8 FGF9 FGF7 FGF11 FGF9 FGF8 FGF8 FGF19 FGF9 FGF19 FGF7 FGF19

Basic FGF, bFGF Int-2 kFGF, Kaposi FGF, hst-1 hst-2 KGF AIGF GAF KGF-2

High-affinity receptors FGFR1, IIIb and IIIc; FGFR2, IIIb and IIIc; FGFR3, IIIb and IIIc; FGFR4 FGFR1, IIIb and IIIc; FGFR2, IIIc; FGFR3, IIIc; FGFR4 FGFR1, IIIb; FGFR2, IIIb FGFR1, IIIc; FGFR2, IIIc; FGFR3, IIIc; FGFR4 FGFR1, IIIc; FGFR2, IIIc FGFR1, IIIc; FGFR2, IIIc, FGFR4 FGFR2, IIIb FGFR1, FGFR2, IIIc; FGFR3, IIIc; FGFR4 FGFR2, IIIc; FGFR3, IIIb and IIIc, FGFR4 FGFR1, IIIb; FGFR2, IIIb Function intracellularly independent of FGFRs FGFR4 FGFR1, IIIc; FGFR2, IIIc FGFR2, IIIc, FGFR3, IIIc FGFR4 FGFR1, IIIc unknown FGFR2, IIIb FGFR3, IIIc

AIGF, androgen-induced growth factor; GAF, glia-activating factor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; KGF, keratinocyte growth factor.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 217 of mouse FGF15 (966). Human FGFs range in size from 17 to 34 kDa and share several common features including a high affinity for heparin and the presence of a homologous 120-amino-acid central core domain that has been shown to be critical in FGFR binding (966). Structural studies have identified 12 antiparallel β strands within the central core domain including a β trefoil structure composed of four β sheets organized in a triangular array (967,968,976). The primary heparin-binding site is composed of several basic amino acids conserved on two β sheets and appears structurally distinct from the FGFR-binding site (969,976). Most FGFs contain an amino-terminus signal sequence and are consequently readily secreted from cells in which they are produced (966). However, a significant subset of FGFs including FGF1, 2, 9, 11 to 14, 16, and 20 lack an obvious signal peptide (977–986). Of this group, FGF9, 16, and 20 are nevertheless secreted from cells, and FGF9 has been shown to contain a noncleaved amino-terminal hydrophobic domain that is required for secretion. Although FGF1 and FGF2 are not secreted from cells, they can be found on the cell surface and within the ECM, suggesting that they are released from cells by a mechanism that is independent of the endoplasmic reticulum–Golgi pathway. In contrast, FGF11 to 14 do not appear to be secreted from cells, and it remains to be determined whether these FGFs function by interacting with FGFRs or in a receptor-independent manner. Interestingly, FGF2 and FGF3 have alternative high-molecular weight isoforms possessing an additional nuclear localization amino-terminus sequence that results in their transport to the nucleus. The precise biological function of these nuclear-localized FGFs remains unclear. Mammalian FGFs are differentially expressed in most tissues examined in both a developmental and tissue-specific manner (964). Although FGF subfamilies tend to display similar patterns of expression, individual FGFs are clearly expressed in unique sites as well (966). FGF3, 4, 8, 15, 17, and 19 appear to be expressed almost exclusively in the developing embryo, whereas the remaining FGFs are expressed both during embryonic development and in the adult (966). These patterns of expression strongly suggest that FGFs play an important role in embryonic development, as well as a potential physiologic role in adult tissues.

of FGFR and FGF ligand expression provide one mechanism whereby their biological functionality is delimited during development and in the adult. Structurally, FGFRs consist of an extracellular ligandbinding domain, a single-transmembrane domain, and a split intracellular kinase domain (Fig. 7-10) (964,992). The extracellular region contains two to three immunoglobulin (Ig)-like domains that are necessary for FGF binding. These Ig-like domains are capable of regulating both ligand affinity and specificity. The sites for heparin binding and ECM and cell adhesion association are located between Ig domains I and II. The intracellular region contains the functional domain responsible for FGFR tyrosine kinase activity, as well as additional sites that are necessary for protein binding and phosphorylation or autophosphorylation of the receptor molecule. Alternative splicing of FGFR transcripts is capable of generating FGFR isotypes that possess unique functionalities (988,991). For example, alternative splicing of the C-terminal portion of the third Ig domain generates distinct FGFR isotypes designated IIIb or IIIc that have been shown to possess unique binding specificities for FGFs (993–995). In addition, FGFR isotypes that lack the transmembrane domain, acid box, or signal peptide have also been reported (988,996–1000). Clearly, these changes in functional domain inclusion would have profound effects on the biological activity associated with receptor-ligand binding. Despite the complexity of receptors and receptor isoforms, most FGFRs Signal peptide targeted secretion I

Ig-like domains affects FGF binding affinity and selectivity II

III

Heparin-binding domain/ CAM homology domain proper interaction with extracellular components

Alternatively spliced Ig III domain

Transmembrane domain maintains receptor conformation for ligand activation

Fibroblast Growth Factor Receptors FGFRs are a gene family of receptor tyrosine kinases with at least four signaling members identified in vertebrates and designated FGFR1, FGFR2, FGFR3, and FGFR4 (987). FGFR genes generally contain 18 exons, possess similar exon-intron organization, and are randomly dispersed throughout the genome with no apparent linkages to FGF gene locations (987). FGFRs are differentially expressed in a tissue-specific manner throughout development and into adulthood, and they are capable of producing multiple receptor isoforms through alternative splicing of primary transcripts (964,966,974,987–991). The overlapping patterns

Acidic sequence optimal HSPG interaction

Juxtamembrane domain adaptor molecule binding

Kinase domains catalytic activity & adaptor molecule binding

FIG. 7-10. Schematic representation of a prototypic fibroblast growth factor receptor (FGFR) with functional domains identified. CAM, cell adhesion molecule; HSPG, heparan sulfate proteoglycan.

218 / CHAPTER 7 associate with similar adapter molecules to transmit intracellular signaling, and consequently have conservation of their intracellular adapter protein-binding sites (987).

Intracellular Signaling by Fibroblast Growth Factor FGFs bind to FGFRs, inducing their dimerization resulting in the autophosphorylation of specific cytoplasmic tyrosine residues and activating intracellular signal transduction pathways by controlling the protein tyrosine kinase activity and recruitment of signaling complexes (Fig. 7-11) (964, 1001). The binding of SH2 and phosphotyrosine binding signaling proteins to the phosphorylated FGFR provides a platform for the intrinsic catalytic activity of these molecules, as well as the binding of additional adapter proteins (964,1002,1003). In this manner, cell-specific signaling pathways can be modulated by ligand binding to the FGFR. Three primary signaling pathways have been associated with FGF binding to the FGFR including the Ras/MAPK, PLCγ/Ca2+, and PI3K/Akt pathways (964). Activation of the Ras/MAPK pathways involves the interaction of the lipidanchoring docking protein FRS2, which is bound by the adapter protein Grb2 and the PTP Shp2, resulting in complex formation with Son of sevenless and activation of ras by GTP exchange (1004–1009). This signal transduction

FGFR FGFR

pathway results in the phosphorylation of downstream transcription factors including c-myc, AP-1, and ETS family members through the MAPK signaling cascade (1010). Binding of PLCγ to FGFR results in the formation of two second messengers, IP3 and DAG, activating PKC and stimulating the release of Ca2+, respectively (964). This signaling cascade appears important in FGF-stimulated neural development including neurite outgrowth (1011–1013). FGFR is also capable of activating the PI3K/Akt pathway via three overlapping mechanisms involving Ras, Grb2/Gab1, and the FGFR alone (964,1014). Activation of this FGFR pathway has been shown to be critical in mesoderm induction (1014). Studies indicate that the activity of FGF signaling is regulated both within the cell, as well as within the extracellular space (964). Within the cell, many of the FGF signaling cofactors are regulated by FGF signaling directly, thereby creating regulatory feedback loops designed to modulate FGF activity (1015–1018). In addition, specific intracellular regulators of FGF have also been identified in several species. These include PTPs, which act as positive and negative regulators of FGF signaling, and Sprouty proteins, which interfere with FGF signaling via multiple mechanisms (1019–1028). A variety of transmembrane modifiers of FGF activity have also been identified including Sef, FLRT3, N-CAM, N-cadherin, and L1 (964,1011,1017,1029–1036). These interactions result in the formation of multiprotein

FLRT3

FGF HS FGF

P PLC

Ca2+

S2 FR P

Shp-2 DAG

FGF

FGFR

HS FGF

FGFR N-CAM

IP3

P13K

Gab1

Akt PKC

SOS

2. Differentiation

3. Proliferation & differentiation

Grb2

Raf

Ras

MEK

Transcriptional effects Nucleus

P

ERK

MKP3

1. Proliferation & differentiation

FIG. 7-11. Some of the intracellular signaling pathways activated by fibroblast growth factor (FGF) binding to the FGF receptor (FGFR). Fibroblast growth factor-heparan sulfate (FGF-HS) binding to the dimerized FGFR causes autophosphorylation of the receptor and activation of multiple intracellular signaling pathways including the Ras/mitogen-activated protein kinase (MAPK) pathway (1), phospholipase Cγ (PLCγ)/Ca2+ pathway (2), and the phosphatidylinositol-3 kinase (PI3K)/Akt pathway (3). DAG, diacylglycerol; ERK, extracellular signal–regulated kinase; FRS, FGF receptor substrate 2; MKP3, MAP kinase phosphatase 3; N-CAM, neural-cell adhesion molecule; PKC, protein kinase C; SOS, Son of sevenless.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 219 TABLE 7-3. Developmental phenotypes observed in fibroblast growth factor knockout mice Subfamily

Ligand

FGF1

FGF-1 FGF-2 FGF-4 FGF-5 FGF-6 FGF-3 FGF-7 FGF-10 FGF-22 FGF-9 FGF-16 FGF-20 FGF-8 FGF-17 FGF-18 FGF-11 FGF-12 FGF-13 FGF-14 FGF-19 FGF-21 FGF-23

FGF4 FGF7

FGF9 FGF8 FGF11

FGF19

FGF−/− phenotype Viable Viable Lethal, Viable Viable Viable Viable Lethal, ND Lethal, ND ND Lethal, Viable Lethal, ND Viable ND Viable Lethal, ND Viable

E4-5

P0 P0 E8 P0

E12.5

Description None identified Mild cardiovascular, skeletal, neural defects Inner-cell mass proliferation Long hair, angora mutation Subtle, muscle regeneration Mild inner ear, skeletal (tail), CNS defects Hair follicle growth, ureteric bud growth Multiple organ development, duodenal and colonic atresia ND Lung, heart, GI, skeletal, testes development ND ND Gastrulation defect; CNS, limb development Cerebellar development Skeletal and lung development ND Neuromuscular phenotype ND Neurologic phenotype Not clear ND Growth retardation, bone abnormalities

CNS, central nervous system; FGF, fibroblast growth factor; GI, gastrointestinal; ND, not determined.

complexes that appear critical in modulating FGFR interactions and its resultant signaling. Within the extracellular compartment, the interaction of FGF with heparin/HSPG has been shown to be an important feature of FGF functionality (990,1037–1040). HSPGs are critical in stabilizing FGF proteins, as well as modulating their binding and specificity FGFRs (990,1041–1044). In addition, these interactions appear important in modulating FGF release and diffusion within the extracellular compartment, thereby helping to delimit FGF activity within the local microenvironment (1045,1046).

Biological Actions of Fibroblast Growth Factor FGFs are a diverse family of growth factors that effect a wide range of biological functions including tissue morphogenesis, tissue repair, angiogenesis, and tumorigenesis via paracrine activation of FGFs (964). The precise temporal and spatial distribution of FGF ligands, FGFR isotypes, and cell-specific adaptor molecules provides a basis for functional diversity that is capable of establishing unique developmental and tissue-specific responses based on individual expression patterns alone. The critical, and yet

diverse, roles that FGFs and their receptors play in embryonic development are borne out by the wide range of anomalies observed in FGF and FGFR knockout mice (Tables 7-3 and 7-4) (964,966). In these studies, FGFs have been shown to be critical in implantation, mesoderm formation, gastrulation, chemotaxis, morphogenetic movements, neural induction, anterior-posterior axis formation, and endoderm formation, subsequently affecting a wide range of target tissues including those of the gastrointestinal tract. The physiologic recapitulation of these developmental programs in adult tissues, such as the gastrointestinal tract, appears critical in maintaining tissue homeostasis and promoting repair after injury. Finally, the role of FGFs in mediating cell growth and differentiation makes them and their receptors potential targets for gastrointestinal tumorigenesis. Development During embryonic development, FGFs can act as classic morphogens being both diffusible and capable of eliciting a biological response in a graded, dose-dependent manner (964). FGFs have been shown to be mitogenic for a wide range of cell types including fibroblasts, endothelial cells, and epithelial cells (964). A variety FGFs and FGFR subtypes are

TABLE 7-4. Developmental phenotypes observed in fibroblast growth factor receptor knockout mice Receptor FGFR1 FGFR2 FGFR3 FGFR4

FGFR−/− phenotype

Description

Embryonic lethal Embryonic lethal Viable Viable

Die around gastrulation with many defects Die either just after implantation or E10-11 Skeletal defects, long bone overgrowth, deafness No overt defects (lung defects/dwarfism with FGFR3−/−)

FGFR, fibroblast growth factor receptor.

220 / CHAPTER 7 differentially expressed within the gastrointestinal tract during development, suggesting that they play a key role in gut morphogenesis (964,1047). In general, FGF is expressed by mesenchymal cells and their derivatives found within the submucosal tissue, whereas expression of FGFR isotypes is observed in the epithelial cells of the overlying mucosa. Within the gastrointestinal tract, FGF-7, or keratinocyte growth factor (KGF) and its corresponding receptor, FGFR2-IIIb, have been extensively studied (1048–1051). KGF was originally isolated from the conditioned medium of human lung fibroblasts and characterized as a potent mitogen for keratinocytes (1052). Since then, KGF has been shown stimulate the proliferation of a variety of epithelial cell types (1053–1056). KGF and its receptor are expressed throughout the developing and mature gastrointestinal tract where they have been shown to enhance the proliferation, migration, and differentiation of gastrointestinal epithelial cells (1048,1054,1057,1058). KGF is principally produced by mesenchymal cells and acts in a paracrine manner by binding to the FGFR2-IIIb splice variant found in adjacent epithelial cells (1048). Recombinant KGF stimulates growth of the gastrointestinal tract, and unlike many other FGFs, its activity is principally limited to epithelial cells (1050, 1051,1054). KGF is believed to promote goblet cell differentiation through interactions with the GCSI binding protein, and KGF has also been shown to differentially regulate digestive function by specifically modulating enzyme activities within the gastrointestinal tract (747,1051,1059). Involvement of the FGF signaling pathway in early mesoderm and endoderm formation places the members of this growth factor family in a position to dramatically affect the early stages of embryonic development. Consequently, ablation of FGF or FGFR expression, or both, in mice results in a significant degree of embryonic lethality, making the observation of specific gastrointestinal defects difficult. Even so, alterations in gastrointestinal development have been observed in FGF and FGFR knockout mice. For example, FGF-10 (KGF-2) and FGFR2-IIIb knockout mice show a wide range of gastrointestinal defects with incomplete penetrance and variable phenotype (1060–1064). These defects include duodenal and colonic atresia, as well as defects in cecal and anorectal development. The duodenal and colonic atresia observed in FGF-10−/− and FGFR2-IIIb−/− mice occurs independent of mesenteric vascular occlusion and appears to represent a fundamental defect in the development of these gastrointestinal segments (1062–1064). The defects in cecal development observed in these animals occurs as a result of improper epithelial proliferation and invasion into the developing cecal bud permitting maintenance of this immature gastrointestinal segment throughout development (1060). The anorectal defects observed in FGF-10−/− mice represent a developmental failure of anorectal communication that becomes evident as early as embryonic day 13.5 (1061). Additional studies suggest that misexpression of FGF-1, -7, and -10 within the developing foregut may result in esophageal atresia and tracheoesophageal fistula formation (1065). Taken together, these observations highlight that

important role that the FGF signaling plays in mediating distinct epithelial–mesenchymal interactions within the gastrointestinal tract, thereby controlling normal gut development, maturation, and function. Tissue Repair and Angiogenesis A variety of studies indicate the FGFs play an important role in wound healing and tissue repair (970,971,973,1050, 1066,1067). Within the gastrointestinal tract, FGFs have been shown to restore connective tissue in injured mucosa by promoting epithelial cell migration and proliferation, fibroblast proliferation, and angiogenesis (1067,1068). FGF expression appears critical in the formation of granulation tissue, which is composed of fibroblasts, macrophages, and endothelial cells and represents the primary source for regeneration of the lamina propria and microvessels within the healing ulcer (1067,1068). Within this context, it has been suggested that the chronic overexpression of FGF may promote fibrosis (1069). Expression of FGF2 and its receptor increases along ulcer edges after acute gastrointestinal injury, and KGF, or FGF-7, overexpression has been observed in inflammatory bowel disease (1070,1071). FGF2 expression increases VEGF mRNA synthesis and protein secretion in a dosedependent manner promoting reconstruction of the microvasculature within the mucosal scar, thereby providing critical oxygen and nutrients to the region under repair (1067, 1068,1072). The administration of recombinant FGF has been shown to decrease the severity of gastrointestinal ulceration, reduce the susceptibility to experimental ulceration, and promote epithelial restitution (970,1073–1076). For example, KGF pretreatment ameliorates chemotherapy and radiationinduced mucositis by significantly reducing atrophy, accelerating regrowth, decreasing ulcer formation, and improving crypt and villus survival (1074,1077–1080). KGF has also been shown to prevent graft-versus-host disease by maintaining gastrointestinal integrity and preventing subsequent proinflammatory cytokine generation (1081). KGF appears to mediate some of these cytoprotective responses by up-regulating the expression of genes involved in mucosal protection such as antioxidant enzymes and intestinal trefoil protein (TFF3) (1080). As with most other growth factors, the biological actions of FGF during ulcer healing are tightly associated with the expression and activity of additional growth factors found within the gastrointestinal tract. These growth factors appear to cooperatively interact in the process of ulcer healing by differentially affecting nearly all the cell types required for epithelial restitution. These include epithelial cell proliferation and migration by EGF>FGF>PDGF, fibroblast proliferation by FGF>PDGF, and angiogenesis by VEGF>FGF>>PDGF>>EGF (973). The overlapping functions associated with these growth factors are most likely designed to guarantee a high success rate in wound healing after acute gastrointestinal injury.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 221 Tumorigenesis That FGFs have been characterized as potent mitogens for a variety of cell types makes them potential targets for tumor development within the gastrointestinal tract, as well as other tissue types. FGF expression has been reported in a variety of gastrointestinal tumors, with FGF1 overexpression observed in a high percentage of colorectal adenomas, colorectal cancers, and gastric cancers (1082–1084). In contrast, expression of FGF2 was reduced in many of the same tumor types, and this reduction in FGF2 expression was strongly correlated with gastric tumor grade. Expression of FGF2 and FGFR1 has been reported in human esophageal cancers, and expression of KGF and FGFR2-IIIb has been reported in neoplastic colonic mucosa, with overexpression of FGFR2-IIIb observed in more differentiated tumor samples (1047,1084). In addition, FGF-induced signaling through the pp57 mitogen-activated kinase has been shown to promote cell growth in undifferentiated colon carcinoma cell lines, whereas FGF1 expression has been correlated with the proliferation and activity of stromal fibroblasts found within endocrine tumors of the gut (1085,1086). FGFs may also play a secondary role in promoting tumor growth by mediating localized angiogenesis within the developing tumors (1087). These studies indicate that aberrant FGF and FGFR expression may be important in the development and progression of gastrointestinal tumors.

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246 / CHAPTER 7 1063. Fairbanks TJ, Kanard RC, Del Moral PM, Sala FG, De Langhe SP, Lopez CA, Veltmaat JM, Warburton D, Anderson KD, Bellusci S, Burns RC. Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway. J Pediatr Surg 2005;40:390–396. 1064. Kanard RC, Fairbanks TJ, De Langhe SP, Sala FG, Del Moral PM, Lopez CA, Warburton D, Anderson KD, Bellusci S, Burns RC. Fibroblast growth factor-10 serves a regulatory role in duodenal development. J Pediatr Surg 2005;40:313–316. 1065. Crisera CA, Maldonado TS, Longaker MT, Gittes GK. Defective fibroblast growth factor signaling allows for nonbranching growth of the respiratory-derived fistula tract in esophageal atresia with tracheoesophageal fistula. J Pediatr Surg 2000;35:1421–1425. 1066. Szabo S, Sandor Z. Basic fibroblast growth factor and PDGF in GI diseases. Baillieres Clin Gastroenterol 1996;10:97–112. 1067. Jones MK, Tomikawa M, Mohajer B, Tarnawski AS. Gastrointestinal mucosal regeneration: role of growth factors. Front Biosci 1999; 4:D303–D309. 1068. Tarnawski A, Szabo IL, Husain SS, Soreghan B. Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways. J Physiol Paris 2001;95:337–344. 1069. Okunieff P, Barrett AJ, Phang SE, Li A, Constine LS, Williams JP, Rubin P, Wang X, Wu T, Chen Y, Ding I. Circulating basic fibroblast growth factor declines during Cy/TBI bone marrow transplantation. Bone Marrow Transplant 1999;23:1117–1121. 1070. Bajaj-Elliott M, Breese E, Poulsom R, Fairclough PD, MacDonald TT. Keratinocyte growth factor in inflammatory bowel disease. Increased mRNA transcripts in ulcerative colitis compared with Crohn’s disease in biopsies and isolated mucosal myofibroblasts. Am J Pathol 1997;151:1469–1476. 1071. Finch PW, Cheng AL. Analysis of the cellular basis of keratinocyte growth factor overexpression in inflammatory bowel disease. Gut 1999;45:848–855. 1072. Sako A, Kitayama J, Yamaguchi H, Kaisaki S, Suzuki H, Fukatsu K, Fujii S, Nagawa H. Vascular endothelial growth factor synthesis by human omental mesothelial cells is augmented by fibroblast growth factor-2: possible role of mesothelial cell on the development of peritoneal metastasis. J Surg Res 2003;115:113–120. 1073. Zeeh JM, Procaccino F, Hoffmann P, Aukerman SL, McRoberts JA, Soltani S, Pierce GF, Lakshmanan J, Lacey D, Eysselein VE. Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats. Gastroenterology 1996;110: 1077–1083. 1074. Danilenko DM. Preclinical and early clinical development of keratinocyte growth factor, an epithelial-specific tissue growth factor. Toxicol Pathol 1999;27:64–71. 1075. Han DS, Li F, Holt L, Connolly K, Hubert M, Miceli R, Okoye Z, Santiago G, Windle K, Wong E, Sartor RB. Keratinocyte growth

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CHAPTER

8

Developmental Signaling Networks Wnt/β-Catenin Signaling in the Gastrointestinal Tract Guido T. Bommer and Eric R. Fearon History, 248 Discovery of the Wnt Signaling Pathway, 248 Adenomatous Polyposis Coli Tumor Suppressor Gene at Chromosome Region 5q21, 248 β-Catenin Pathway, 249 Wnt/β Overview, 249 Wnt/β-Catenin Pathway Ligands, Receptors, and Extracellular Inhibitors, 249 Wnt/β-Catenin Signaling from the Cell Surface to the Nucleus, 252 Nuclear Access of β-Catenin: Subcellular Distribution, 258 Nuclear Action of β-Catenin, 258 Wnt/β-Catenin/T-Cell Factor Target Genes, 259

Noncanonical Wnt Signaling Pathways, 260 β-Catenin Pathway in Gastrointestinal Physiology, 260 Wnt/β Wnt/β-Catenin in Gastrointestinal Development, 260 Wnt/β-Catenin Pathway in Gastrointestinal Homeostasis and Architecture, 260 β-Catenin Pathway Defects in Gastrointestinal Wnt/β Tumors, 261 Colon and Rectum, 261 Esophagus, 263 Stomach, 263 Liver, 264 Pancreas, 264 References, 264

A major objective for the gastrointestinal biology field is to obtain in-depth and comprehensive knowledge of specific signaling pathways, factors, and mechanisms that regulate crucial cell fate decisions in epithelial cells in the developing and adult gastrointestinal tract. Substantial progress toward a more complete understanding of pathways and proteins with key roles in regulating cell proliferation, survival, differentiation, and motility, among other processes, has been achieved, in part through the recognition that cancers in the

gastrointestinal tract often co-opt critical developmental pathways, with essentially constitutive (unregulated) activity of the pathway in cancer cells. As such, understanding how the deregulated pathways contribute to the altered phenotype of the cancer cells can provide important clues into the function of the pathway in normal cells, complementing other types of research investigations that might be performed to explore the function of the pathway in normal cell physiology. The Wnt proteins represent a conserved family of secreted proteins with pleiotropic and context-specific activities in embryogenesis and adult tissues, including the gastrointestinal tract. Defects in Wnt signaling play a major role in the pathogenesis of colorectal and subsets of various other gastrointestinal cancers. This chapter highlights how findings from a broad array of different models and approaches have revealed the significance of Wnt pathway function in development and normal adult tissues and how derangements in Wnt signaling underlie the genesis of tumors of the gastrointestinal tract.

G. T. Bommer: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan 48109. E. R. Fearon: Division of Molecular Medicine and Genetics, Departments of Internal Medicine, Human Genetics, and Pathology, University of Michigan School of Medicine, Ann Arbor, Michigan 48019. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

247

248 / CHAPTER 8 HISTORY Discovery of the Wnt Signaling Pathway In the early 1980s, Nusse and Varmus (1) determined that the proviral integrations of the mouse mammary tumor virus (MMTV) that were present in the genomic DNA of many independent MMTV-induced carcinomas were often associated with the same chromosomal region, and the cellular gene that was inappropriately activated by these integrations was hence termed Int-1, an abbreviation for “integration site 1.” After the introduction of the Int-1 gene into certain nontumorigenic epithelial cells, the cells acquired abnormal phenotypic traits associated with cancer cells, implicating Int-1 as a presumptive oncogene (2). Independent studies found that mutations of a Drosophila homologue of the Int-1 gene can lead to flies without wings, termed wingless (wg) mutants (3,4). After recognition of the relationship between Int-1 and Wg and further efforts revealing that the proteins encoded by Int-1 and Wg are members of a much larger collection of related signaling molecules, the family of genes and proteins was collectively termed WNTs (pronounced “wints”), for the combination of wingless and integration site 1 (5). A considerable fraction of current knowledge of key components in Wnt signaling has been obtained from genetic analysis of mutant phenotypes in Drosophila. As noted earlier, Wg mutants in Drosophila initially caught the attention of investigators because of the major defects seen in wing morphogenesis. However, it was subsequently found that embryos and larvae carrying Wg mutations also show other phenotypes, such as rather subtle changes in the distribution of specific mechanosensory hairs (so-called cuticular denticles) within body segments, as well as changes in the development of the eye. Of some interest is that a similar denticle phenotype akin to that seen in Wg mutants subsequently was also described for other Drosophila mutant flies, including those with mutations in the Dishevelled (dsh), Armadillo (arm), and Pangolin genes. In flies carrying inactivating mutations in a gene termed Zeste-white-3 (zw-3), essentially the opposite phenotype was observed, namely a naked cuticle phenotype. By examining flies for more pronounced phenotypes, or conversely more subtle phenotypes, in progeny generated by crossing a particular mutant fly with a defined phenotype to another mutant fly carrying a defect in some other gene, it is possible to order signaling pathways genetically, even in the absence of in-depth knowledge of the molecular changes leading to the observed phenotypes. These types of studies are referred to as epistasis experiments. Using this type of epistasis approach, it was possible to define the relationship and order of major components of the Wg pathway in Drosophila by the mid1990s (6–9). Wnt genes in certain vertebrate species, such as Xenopus laevis, were studied quite intensively in the late 1980s and early 1990s. In Xenopus, ectopic expression of certain Wnts was found to lead to a duplicated body axis (10,11).

However, it was not until the mid-1990s that a confluence of events led to major breakthroughs in understanding of the mechanisms through which Wnt proteins mediate signaling activity in invertebrate and mammalian cells. Arguably, one of the more significant events in elucidating Wnt pathway function in mammals was the discovery of the means by which the adenomatous polyposis coli (APC) protein functions to regulate β-catenin levels and localization.

Adenomatous Polyposis Coli Tumor Suppressor Gene at Chromosome Region 5q21 Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome affecting about 1 in 7000 to 1 in 10,000 people worldwide (12). In general, hundreds to thousands of benign adenomatous polyps (adenomas) arise in the colon and rectum of individuals with FAP by late in the second decade of life. Because of the large number of adenomas that arise and the risk that one or more lesions will progress to cancer, the lifetime risk for colorectal cancer approaches 100% if affected individuals are not screened and definitively managed by surgical intervention. Overall, FAP accounts for less than 1% of all colorectal cancer cases. The relative rarity of FAP germline mutations in colorectal cancer belies the importance of insights into Wnt pathway function gleaned from work on FAP. The tumor suppressor gene at chromosomal region 5q21, which when mutant underlies FAP, was first identified by linkage analysis and positional cloning, and subsequently was termed APC (13–15). Notwithstanding the central role of the APC gene in the development of adenomas and carcinomas in patients with FAP, interest in the APC gene was considerably heightened because of its prominent role in the development of sporadic colorectal tumors. Roughly 80% of sporadic colorectal adenomas and carcinomas have somatic mutations inactivating APC (16). Consistent with the “two-hit” hypothesis for the role of tumor suppressor gene mutations in cancer originally proposed by Knudson to explain the origins of sporadic and familial forms of retinoblastoma, somatic mutation or chromosomal loss of the remaining wild-type APC allele occurs in colorectal neoplasia arising in patients with FAP, as well as in sporadic lesions (17). Although the primary amino acid sequence of the 2843 amino acid APC protein was found to have some repeated sequence motifs when the APC gene was first identified, definitive insights into APC protein function were initially slow to emerge. The identification of the β-catenin protein as a major binding partner for APC was perhaps the major catalyst for uncovering what appears to be the most significant function of APC in normal and cancer cells. β-catenin is an abundant cellular protein that was first identified because of its role in linking the cytoplasmic domain of the E-cadherin cell–cell adhesion molecule to the cortical actin cytoskeleton, in part via binding of β-catenin to α-catenin, a vinculinlike molecule. However, the role of APC in binding to β-catenin appears to have little direct relation to the function

DEVELOPMENTAL SIGNALING NETWORKS / 249 of β-catenin in E-cadherin–mediated cell adhesion. Rather, APC appears to be a major regulator of the “free” pools of β-catenin in the cytosol and nucleus. By combining insights gained from Drosophila genetics, it was soon possible to identify glycogen synthase kinase-3β (GSK-3β), the mammalian homologue of the Drosophila zw-3 protein described earlier, and an apparent scaffolding protein termed Axin as key players that work together in a “destruction complex” targeting the free pool of β-catenin for proteasomal degradation. By mutational inactivation of APC, stabilizing β-catenin mutations, or in the presence of Wnt ligands, this destruction machinery is inhibited and allows β-catenin to translocate to the nucleus and act as a transcriptional coactivator for a family of HMG box proteins termed T-cell factor (TCF) or lymphoid enhancer factor (LEF) transcription factors (18–20). As described further later in this chapter, the role of β-catenin/TCF–mediated transcription in normal and neoplastic gastrointestinal cells has continued to be a major focus of attention for researchers in the Wnt and gastrointestinal biology fields.

β-CATENIN PATHWAY Wnt/β Overview The pathway initiated by the binding of a number of different Wnt proteins to their cell-surface receptors and which leads ultimately to the accumulation of β-catenin (the vertebrate homolog of Armadillo) in the nucleus has been termed the canonical pathway by many authors to distinguish it from other distinct intracellular signaling pathways, so-called noncanonical pathways, that appear to be activated in parallel by Wnt proteins binding to surface receptors (Fig. 8-1). These noncanonical Wnt pathways include activation of c-Jun N-terminal kinase (JNK) signaling or increases of intracellular calcium ion levels that lead to activation of protein kinase C (PKC). For simplicity, this chapter uses Wnt/β-catenin pathway to refer to the canonical Wnt pathway. In addition, this chapter emphasizes the terminology for the mammalian proteins, pointing out the relevant findings from other species where appropriate. In the absence of Wnt proteins, a “destruction complex” consisting of proteins such as APC, Axin, Casein kinase 1α (CK1α), and GSK-3β subjects β-catenin to a cascade of phosphorylation at several serine and threonine residues in the amino (N)-terminal sequences of β-catenin. These phosphorylation events cause β-catenin to be recognized and ubiquitinated by an E3 ubiquitin ligase complex containing the protein β-TrCP. After polyubiquitination, β-catenin is degraded by the proteasome. Binding of Wnt proteins to their receptors inhibits the phosphorylation cascade, allowing free β-catenin to accumulate in the cytoplasm and translocate to the nucleus where it can activate the transcription of target genes via interaction with TCF transcription factor proteins. The transcriptional program activated by β-catenin/TCF likely varies to some degree from one cell type or context to

another, reflecting that β-catenin/TCF may act with other transcription factors to modulate the relative levels of expression of target genes, rather than mediate all-or-none effects on the expression of target genes. Many β-catenin/TCF target genes likely play a role in physiologic cell fate decisions in embryonic development, adult tissue homeostasis, and the development of cancer, and the identities and functional activities of a number of presumptive β-catenin/TCF target genes are discussed later. β-Catenin Pathway Ligands, Receptors, Wnt/β and Extracellular Inhibitors Wnt proteins are small cysteine-rich glycoproteins. Currently, 19 human Wnt genes have been identified. Although secreted, Wnt proteins are not easily recovered from the supernatant of Wnt-expressing cells, which has been attributed to posttranslational modifications, particularly palmitoylation of the proteins, rendering them more hydrophobic than expected (21). Despite their apparent tight association with cells, Wnt ligands still achieve a significant spread from their site of production in vivo, allowing for the formation of a morphogen gradient. This observation has led to the concept that Wnt proteins might be transported by an extracellular vesicle-based transport system, termed the argosomes (22). Similar to other morphogens, the development of a morphogen gradient for Wnts seems to be regulated by the local composition of high-molecular-weight proteoglycans (23–26) and possibly by the presence of extracellular inhibitors such as secreted frizzled-related protein 1 (SFRP1) or Wnt inhibitory factor 1 (WIF1; see later; and Fig. 8-1). Wnt proteins interact with a heterooligomeric receptor complex consisting of a member of the so-called Frizzled (Fz) family and a low-density lipoprotein receptor–related (LRP) protein (27) (Figs. 8-2 and 8-3). Given the existence of 19 human Wnt genes, 12 human Frizzled genes, and 2 human LRP genes involved in Wnt signaling, there is a considerable potential for combinatorial diversity and functional redundancy in this signaling pathway, making it difficult to identify the physiologic role of specific receptor–ligand pairs by standard genetic approaches. Expression of Wnt ligands and receptors is tightly regulated spatially and temporally during development. This tight regulation of expression would seem to imply that, even though the activity of Wnt proteins might be redundant in certain model systems, the differing Wnt expression patterns seen in vivo lead to unique functional consequences. Importantly, Wnt proteins are defined by sequence similarity, rather than functional activity. As such, Wnt proteins can be subsetted according to their ability to induce β-catenin stabilization, a property that correlates well with the ability of the Wnt proteins to induce significant morphologic transformation of mammary epithelial cells (2,28,29). The Wnt proteins that stabilize β-catenin and induce morphologic transformation include Wnt-1 and Wnt-3, among others.

250 / CHAPTER 8

β–cat. β–cat.

AXIN

α–cat. β–cat.

β–cat. β–cat.

A FIG. 8-1. Two-state model of Wnt/β-catenin signaling pathway. (A) Absence of Wnt stimulation. In the absence of Wnt ligands, free β-catenin is continuously phosphorylated in its N-terminal region. This phosphorylation cascade takes place in a destruction complex consisting of adenomatous polyposis coli (APC) and Axin, as well as the enzymatic activities glycogen synthase kinase-3β (GSK-3β) and casein kinase 1α (CK1α). Phosphorylation of serine 45 by CK1α allows for the subsequent phosphorylation of threonine 41, serine 37, and serine 33. Phosphorylated serine 33 is then recognized by β-TrCP, which initiates ubiquitination and proteasomal destruction of β-catenin. This degradation is supported by the nuclear export of β-catenin by APC and Axin. In the absence of nuclear β-catenin, target gene promoters are silenced by recruitment of transcriptional repressors of the transducin-like enhancer of split/groucho-related gene (TLE/Grg) family via T-cell factor/lymphoid enhancer factor (TCF/LEF) DNA-binding factors. Several extracellular antagonists can prevent the effective stimulation of Wnt signaling: Secreted frizzled-related protein (SFRP) and Wnt inhibitory factor 1 (WIF1) directly bind to Wnts, thereby preventing binding to their receptor. Dickkopf (Dkk) binds to low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) via the third and fourth β-propeller domain. Although binding sites to not overlap with Wnt binding sites on these proteins, establishment of LRP5/6-Wnt-Frizzled receptor complexes is inhibited. Recruitment of Kremen proteins via Dickkopf leads to internalization of Wnt receptor complexes.

This group of Wnt ligands has sometimes been referred to as canonical Wnt proteins, whereas others have called them activating Wnts. Both terms essentially highlight the effects on β-catenin. Interestingly, although the Wnt-1 and Wnt-3 genes were initially discovered because they were activated by proviral insertions in a murine mammary carcinoma model, as yet, gene amplification, marked overexpression of the Wnt-1 or Wnt-3 genes, or both have not been revealed as common mechanisms for Wnt pathway dysregulation in human cancer. This issue is discussed further later.

Frizzled (Fz) genes encode proteins with seventransmembrane domains, and they have a high affinity for Wnt ligands, with a binding affinity (Kd) less than 10−8 M (30,31) (see Fig. 8-2). Binding of Wnt ligands to Fz proteins is assumed to occur via Wnt interactions with a cysteine-rich domain (CRD) in Fz, because this domain is conserved among all Fz proteins and also in some extracellular inhibitors of Wnts that bear considerable similarity to Fz proteins, namely the SFRP family. Different affinities of the CRDs of Fz proteins for different Wnt ligands have been proposed as an explanation

AXIN

β–cat. α–cat.

β–cat.

β–cat.

β–cat.

β–cat.

B FIG. 8-1. cont’d (B) Presence of Wnt stimulation. Wnt ligands bind to a receptor complex consisting of members of the Frizzled and the low-density lipoprotein receptor related protein (LRP5 and LRP6) families. Intracellular phosphorylation of LRP5/6 and phosphorylation of Dishevelled allow for the recruitment of Axin to the cell membrane, thereby disrupting the destruction complex that normally targets β-catenin for proteasomal degradation. Efficient action of the destruction complex is also inhibited by the recruitment of FRAT1/GBP (frequently rearranged in T-cell lymphoma/GSK-3β binding protein) that displaces GSK-3β from its binding to Axin. Stabilized β-catenin translocates to the nucleus and activates target gene transcription via recruitment of chromatin remodeling proteins (Brg1, CBP/p300), basal transcription apparatus (TIP49), and coactivators (Legless/BCL9 and Pygopus). HDAC, histone deacetylase complex; RYK, related to tyrosine kinase.

ii iii

Wnt

Wnt norrin

Dickkopf

EGF LDL-A LDL-A LDL-A

Propeller

EGF

Propeller

EGF

Propeller

EGF

Propeller

SXV

TM

TM

TM

TM TM

TM TM

CRD

PPP(S/T)P

LRP5/6

Axin

Dishevelled 6 JNK

FIG. 8-2. Schematic drawing of Frizzled protein structure. Frizzled receptors contain seven-transmembrane domains (TM). The N-terminal extracellular cysteine-rich domain (CRD) mediates binding to ligands such as Wnt of Norrin (30,36). Interaction with Dishevelled occurs immediately intracellular of the transmembrane domain (109). Several proteins involved in noncanonical signaling interact with the C terminus (265, 266). JNK, c-Jun N-terminal kinase. Binding regions for interacting proteins are shown later.

FIG. 8-3. Schematic drawing of low-density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/6). LRP5/6 have a single-transmembrane region. The extracellular N-termini contain four YWTD β-propellers (propeller), each of which is followed by an epidermal growth factor (EGF) repeat. β-Propellers 1 and 2 seem to mediate ligand binding, whereas propellers 3 and 4 mediate interaction with antagonists of the Dickkopf family (32,33,47,48). Three LDL receptor type A domains (LDL-A) allow for oligomerization. In the intracellular domain, PPPSP/PPPTP motifs (P) mediate the phosphorylation-dependent interaction with Axin (75,95). Binding regions for interacting proteins are shown later.

252 / CHAPTER 8 why certain Fz receptors may preferentially signal via the Wnt/β-catenin pathway, whereas other Wnts signal predominantly or exclusively through noncanonical pathways (31). As noted earlier, the LRP5 and LRP6 proteins are singletransmembrane domain proteins with significant similarity to the low-density lipoprotein receptor (32,33) (see Fig. 8-3). Although the presence of both classes of cell-surface receptors, Fz and LRP5/6, seems to be required for canonical signaling to occur in cells, there is evidence indicating that, in some instances, the two parts of the receptor complex may independently transmit Wnt signals (34,35). Evidence is also accumulating that LRP5/6 generally are not involved in β-catenin–independent Wnt signaling (i.e., noncanonical signaling), which seems mainly to rely on Fz receptors. An additional complexity is that additional interacting proteins (apart from the Wnts and LRP5/6) may exist for certain Fz receptors (36). For instance, it has been suggested that, in some settings, the protein encoded by the gene known as RYK (for “related to tyrosine kinase,” the mammalian homologue of the Drosophila derailed gene) might be an essential component of the canonical signaling pathway, functioning as a Wnt coreceptor in conjunction with Fz proteins (37). Several inhibitors of Wnt ligand–receptor interactions have been identified (see Fig. 8-1A). Whereas SFRPs and WIF1 seem to inhibit ligand–receptor interactions by binding to Wnt proteins directly, Dickkopf (Dkk) proteins do so by interacting with the LRP receptors. There are five human SFRP proteins (SFRP1-5), and they constitute a family of secreted proteins containing a CRD similar to that present in Fz receptors. Binding of SFRPs to Wnts via the CRD domain prevents their interaction with membrane-bound Fz and LRP5/6 receptors, and antagonizes Wnt signaling. In addition, as is discussed later, there seems to be evidence that SFRPs, via unknown mechanisms, may even be able to antagonize β-catenin signaling that has been activated by mutations in cancer cells downstream of the receptor–ligand interaction. The observation that epigenetic inactivation of several of the SFRP genes may be an early event in the pathogenesis of several cancer types (particularly colorectal cancer) is an intriguing one, given this apparent antagonistic activity of the SFRPs on Wnt/β-catenin signaling (38–40). Despite the apparent inhibitory effects of the SFRPs on Wnt/ β-catenin signaling, it has been suggested that in some settings SFRPs could function to increase Wnt β-catenin signaling, possibly by serving as a carrier for the highly hydrophobic acylated Wnt proteins that are not readily water soluble (41). These apparently conflicting activities for SFRPs might be reconciled, in part, because different reagents were used in the various studies (wingless vs mammalian Wnts). Nonetheless, currently, it seems reasonable to suggest that the physiologic role of SFRPs in vivo has not been fully established. WIF1, a secreted protein, was uncovered in a functional screen for inhibitors of vertebrate Wnt signaling (42). WIF1 interacts with Wnts via a poorly understood domain termed the WIF domain (42). In Drosophila, the transmembrane Wnt-interacting protein Derailed, which likely functions in

noncanonical Wnt signaling, appears to contain a WIF domain. Evidence suggests that, in contrast to the situation for the Drosophila Derailed protein, the human homologue of Derailed, termed RYK, might actually have an essential role in Wnt/β-catenin signaling (37). Notably, Shifted recently has been identified as a putative Drosophila homolog of WIF1. Whereas no modulation of Wnt signaling by Shifted could be demonstrated in the fly, it was observed that Shifted supports another important developmental pathway, namely hedgehog signaling by increasing the ligand’s extracellular half-life and diffusion (43,44). It is currently unclear whether a similar cross talk also exists in mammals. Dkk proteins were originally identified because of their ability to inhibit dorsalization of Xenopus embryos, a process that is promoted by activating Wnt ligands (45,46). Dkk proteins are secreted proteins that bind to the extracellular domain of LRP5/6 with high affinity (47–49). The interaction of Dkk1 with LRP5 or LRP6 can inhibit the formation of an Fz-LRP5/6 receptor complex, thereby inhibiting Wnt signaling (48). Based on sequence similarity, the Dkk family in vertebrates comprises four members, known as Dkk1-4. Whereas Dkk1 and Dkk4 have been shown to act as antagonists of Wnt signaling, Dkk3 does not interact with LRP5/6, and Dkk2, in some experimental settings, might actually play a positive role in Wnt/β-catenin Wnt signaling. Mechanistic insights into these differential functional activities of the Dkk family members are lacking. In addition to the simple disruption of the formation of Fz-LRP5/6 receptor complexes (48), another model for the action of Dkk proteins has been suggested. Specifically, Dkk1 has been shown to interact with the transmembrane proteins Kremen1 and Kremen2. The Kremen proteins, upon Dkk binding, appear to promote internalization of the LRP5/6 proteins (50,51). The net consequence of decreased cell-surface levels of LRP5/6 is reduced responsiveness of a cell toward Wnt ligands.

Wnt/β-Catenin Signaling from the Cell Surface to the Nucleus As noted earlier, stabilization of the free pool of β-catenin is believed to be a pivotal event in the cellular response to activating Wnt ligands. In the absence of activating Wnt ligands, the half-life of the “free” pool of β-catenin is tightly regulated by phosphorylation at its N-terminus, ubiquitination, and proteasomal degradation. To understand how the interaction of Wnt proteins with their respective receptors leads to the inhibition of the destruction machinery regulating β-catenin, it is important to understand first how the destruction of β-catenin takes place in the absence of activating Wnts. Thus, this section first focuses on the physiologic life cycle of β-catenin molecules. β-Catenin and the Destruction Complex β-Catenin, a protein of 781 amino acids encoded by the CTNNB1 gene, serves a dual role as a cell adhesion molecule

DEVELOPMENTAL SIGNALING NETWORKS / 253 and transcriptional regulator (Fig. 8-4). In normal epithelial cells, most β-catenin seems to be bound in a cytoskeletal complex that on one side (via E-cadherin) mediates contact to neighboring cells and on the other side (via binding to α-catenin) is tethered to the intracellular actin cytoskeleton. β-Catenin is therefore a major constituent of specialized cell–cell contacts termed adherens junctions. β-Catenin bound in this compartment seems to be long-lived and not subject to regulation via Wnt signaling. In contrast to the membraneassociated β-catenin pool, in cells not exposed to activating Wnt ligands, only a small fraction of the cellular β-catenin seems to be present in the cytoplasm and the nucleus; this is the so-called free β-catenin pool. This free pool is subject to rapid degradation via the proteasome, and consequently has a short half-life. Stabilization and nuclear translocation of β-catenin from this pool is essential for the ability of β-catenin to modulate transcription via interaction with TCFs and potentially other transcription factors. Given the large capacity of the cytoskeletal pool, it is not surprising that ectopic expression of E-cadherin can decrease the amount of the free β-catenin and inhibit neoplastic growth (52,53). In its N-terminus, β-catenin contains a stretch of evolutionary conserved serine and threonine residues that are each localized four amino acids apart (i.e., serine 33, serine 37, threonine 41, and serine 45). CK1α appears to phosphorylate serine 45 constitutively (54). This so-called priming phosphorylation event allows for the sequential phosphorylation of positions 41, 37, 33, and 29 mediated by GSK-3β (55–58). The phosphorylation of serines 33 and 37 ultimately

33

37

41

creates an epitope that is recognized by the F-box protein β-TrCP, which as a part of an SCF E3-ubiquitin ligase complex triggers the transfer of ubiquitin to β-catenin (59). Ubiquitinated β-catenin is then rapidly degraded via the proteasome (60,61). The sequential phosphorylation of β-catenin, as described earlier, takes place in a multiprotein complex that has been termed destruction complex. It assembles on two essential scaffolding proteins: APC and Axin. Both seem to be necessary for full activation, but both seem to play different roles (62–65). In addition to the catalytic components CK1α and GSK-3β, this complex also contains various regulatory components that are discussed later in this chapter. A series of phosphorylations of APC mediated in part by GSK-3β seems to be important for the full binding capacity of APC to β-catenin (66) and might support the release of phosphorylated β-catenin from the destruction complex, allowing unphosphorylated β-catenin to bind to Axin (67,68), thereby recharging the destruction complex. Axin binds not only to β-catenin, but also to APC (63,69,70). By this scaffolding function, it is believed to increase the effective concentration of enzyme (GSK-3β and CK1α) and substrate (β-catenin) of the destruction complex (71). Although conflicting evidence has been published, interaction of Axin with β-catenin, as well as the stability of Axin, might also be regulated by GSK-3β–dependent phosphorylations (72–75). Inhibition of the destruction complex leads to the accumulation of N-terminally unphosphorylated forms of β-catenin in the cytoplasm and the nucleus (76,77). Similarly, mutation

45

βTRCP1, Ebi GSK3β, CKIα, α-catenin

APC, E-Cadherin, Axin, TCF/LEF Legless, TIP49

TAdomain

ARM ARM ARM

ARM ARM ARM ARM ARM ARM ARM ARM ARM

SYLDSGIHSGATTTAPSLSGK

CBP/p300

Brg1

FIG. 8-4. Schematic drawing of β-catenin protein structure. β-Catenin plays a dual role as a transcription factor and cell adhesion molecule. Its stability is regulated by the sequential phosphorylation of several N-terminal serine and threonine residues (indicated by boldface) mediated by glycogen synthase kinase-3β (GSK-3β) and casein kinase 1α (CK1α), which allows for recognition by the F-Box protein β-TrCP and consecutive ubiquitin- dependent proteasomal degradation (55,59). Phosphorylation-independent recognition of the N terminus by the F-Box Ebi leads to SIAH-dependent proteasomal degradation (84). Twelve armadillo repeats (ARM) mediate the interaction with adenomatous polyposis coli (APC), E-cadherin, α-catenin, Axin, T-cell factor/lymphoid enhancer factor (TCF/LEF), Legless (267), and TIP49/Pontin52 (164). The C-terminal transactivation domain interacts with histone acetyltransferase CBP/p300 (160) and chromatin remodeling factor Brg1 (163). Interaction sites with other proteins are indicated below the schematic representation.

254 / CHAPTER 8 of any of the potential GSK-3β and CK1α phosphorylation sites, such as is often seen in certain cancer types, leads to the accumulation of β-catenin and subsequent activation of the downstream transcriptional program. Given its dual role in cytoskeletal organization and transcriptional activation, β-catenin is capable of a variety of protein–protein interactions. The central portion of βcatenin contains 12 repeats that are homologous to the Drosophila armadillo protein, and are therefore termed armadillo repeats. These repeats form a superhelix of α-helices that shows a prominent groove on one side (78). This groove mediates several of the most important protein–protein interactions for β-catenin, including those with APC, E-cadherin, Axin, and TCFs. In the cytoplasm, APC and E-cadherin compete for the same binding site on β-catenin (79), which is consistent with the notion that cytoskeletal pool is not amenable to the destruction complex. Assembly of the adherens junctions additionally requires the binding of α-catenin to the N-terminus. Interestingly, this interaction, as well as the interaction with E-cadherin, is regulated by phosphorylation of specific tyrosine residues of β-catenin (Y142 and Y654, respectively), suggesting that this could represent a major determinant of the distribution into the cytoskeletal versus the signalingcompetent pool (80–82). Whereas the fruit fly genome encodes only a single homolog of β-catenin called armadillo, vertebrate genomes also encode the protein γ-catenin/plakoglobin, which also serves a dual role in cytoskeletal and nuclear processes. Both proteins seem to mediate similar protein–protein interactions with their partners APC, Axin, and GSK-3β. It also has been shown that γ-catenin is capable of transforming cells. In addition to the classical destruction complex, there are other pathways of β-catenin destruction. One of these alternative pathways also seems to be dependent on APC function. Through an interaction with the C terminus of APC, a protein complex involving the SIAH1 and Ebi proteins can trigger degradation of β-catenin in a p53-dependent manner, independent of any requirement for GSK-3β (84–86). Most tumors with biallelic inactivation of APC express C-terminally truncated APC proteins that are unable to support the degradation of β-catenin via the classical destruction complex and the SIAH1-dependent pathway. Consistent with the view that SIAH1-mediated degradation of β-catenin is independent of GSK-3β, cancer-derived mutants of β-catenin with Nterminal mutations in the phosphorylation motif can still be regulated by SIAH1. Mutations of the SIAH1 gene in gastric cancers have been shown to be associated with increased nuclear localization of β-catenin (87). Given that mutations of SIAH1 should leave the activity of the classical destruction complex intact, this suggests that SIAH1-dependent β-catenin degradation might be the major determinant of β-catenin stabilization in certain tissues. In addition to the classical GSK-3β–dependent destruction mechanism for β-catenin and the SIAH1-dependent destruction, β-catenin degradation can be induced by retinoid X receptor (RXR). This has been shown to be independent of APC (88).

Regulation of the Destruction Complex in Wnt/ β-Catenin Signaling The end point of the Wnt/β-catenin pathway is the stabilization of the free β-catenin pool and its translocation to the nucleus where it can activate transcription of target genes. The function of the destruction complex has been discussed earlier, so this chapter now discusses the mechanisms by which binding of Wnt proteins to their receptors leads to the inhibition of the destruction complex and subsequent nuclear accumulation of β-catenin. In general, the Wnt receptor complexes seem to consist of both LRP5/6 and Fz proteins (27,89). Although these two signaling components seem to work together in a physiologic setting, the mechanism of cooperation is not entirely clear. There are also instances when only one of the two receptor components may be required for β-catenin stabilization. For example, genetic data from Drosophila suggest that Arrow, the Drosophila counterpart of LRP5/6, does not function in noncanonical Wnt signaling, though Fz proteins are clearly required for noncanonical Wnt signaling. Another example is that Fz proteins do not seem to be required for the stabilization of β-catenin resulting from ectopic overexpression of certain mutant “activated” forms of LRP5/6, such as membrane-anchored forms of LRP5/6 lacking the extracellular domain (32,34). However, the physiologic significance of these latter observations is unclear, and the larger body of data appears to support the view that Wnt-mediated stabilization of β-catenin requires both a Fz and an LRP5/6 component. To make the receptor functions easier to understand, this section describes the main effects of LRP5/6 and Fz separately, trying to point out the possibilities for cooperation as the discussion progresses. This section also introduces the function of Dishevelled, an additional signaling protein that genetically acts downstream of Fz and LRP5/6 receptors and upstream of β-catenin stabilization. The Role of Axin and LRP5/6 in Wnt/ β-Catenin Signaling Axin acts as a scaffold for both substrates and kinases of the degradation complex (Fig. 8-5). It can bind to APC (90), β-catenin, GSK-3β (91), and CK1α (55). The N-terminal RGS (regulator of G-protein signaling) domain of Axin seems to be required for its interaction with APC (90). Binding to β-catenin, GSK-3β, and CK1α takes place in the central portion of Axin. The C-terminal DIX (Dishevelled Axin) domain can interact with a corresponding DIX domain on the adaptor protein Dishevelled (92) and mediate homodimerization of Axin. By bringing both substrate (β-catenin) and enzymatic activities (GSK-3β and CK1α) in close vicinity, Axin can increase the rate of phosphorylation of β-catenin enormously, by some estimates more than 24,000-fold (63,71). In addition to this scaffolding function, Axin also seems toplay a role in supporting the nuclear export of β-catenin (see later). Experimental data and mathematical models have suggested that Axin protein seems to be the limiting factor in the

APC

DIX

RGS

DEVELOPMENTAL SIGNALING NETWORKS / 255

GSK3β

βcatenin

Dishevelled

LRP5/6

FIG. 8-5. Schematic drawing of Axin protein structure. Axin acts as a scaffold protein for the destruction complex. Binding to adenomatous polyposis coli (APC) is mediated by a N-terminal RGS domain (regulator of G-protein signaling domain) (70,90), whereas interaction with glycogen synthase kinase-3β (GSK-3β) and β-catenin occurs in the central domain (69,91). The C-terminal DIX (Dishevelled Axin) domain mediates homodimerization and binds to Dishevelled (268). On Wnt signaling, Axin is recruited to the phosphorylated intracellular domain of LRP5/6 (75,95), thereby disrupting the destruction complex for β-catenin. Binding regions for interacting proteins are shown below.

β-catenin destruction complex, being present in the cell at levels estimated 5000-fold less than those of APC (93). Thus, regulation of the levels and localization of the Axin protein would seem to be a highly effective way of regulating the overall activity of the destruction complex. In the absence of activating Wnt proteins, Axin is bound to APC, GSK-3β, and CK1α, markedly increasing the rate of β-catenin phosphorylation (71). Also, in the absence of extracellular Wnts, LRP5/6 proteins seem to form inactive receptor complexes, perhaps via homodimerization mediated by their extracellular epidermal growth factor repeats (94). On binding of Wnt proteins to the receptor complex, LRP5/6 is rapidly phosphorylated within one or more of five highly conserved PPP(S/T)P motifs that are present in the intracellular domain (95) (see Fig. 8-3). Once phosphorylated at the serine/threonine residue in the motif, one or more of the motifs can bind Axin, resulting in sequestration of Axin by LRP5/6 at the plasma membrane, thereby reducing Axin’s availability to the destruction complex. Interestingly, these phosphorylated PPP(S/T)P motifs in LRP5/6 seem not only to be essential, but also sufficient for Axin binding, as demonstrated through studies showing that transfer of a single PPP(S/T)P motif to the C terminus of the low-density lipoprotein receptor (which lacks any such motifs normally) will allow the chimeric mutant low-density lipoprotein receptor to stabilize β-catenin. Membrane-tethered mutant forms of LRP5/6, harboring a deletion of the extracellular domain, actually mediate Wnt ligand-independent stabilization of β-catenin, suggesting that the binding of Wnts must abolish a negative regulatory effect of the extracellular domain of LRP5/6 on the PPP(S/T)P motifs in the cytoplasmic domain (34,94). In addition to the ligand-induced change of subcellular distribution of Axin, it has also been reported that this process is accompanied by increased phosphorylationdependent proteasomal degradation of Axin. It is currently unclear which kinase triggers this process, although GSK3β has initially been implicated (72–75). It has been

suggested that recruitment of Axin to the plasma membrane also requires Dishevelled, another key player of intracellular Wnt signal transduction (34,106) that genetically acts downstream of LRP5/6 and Fz receptors. Both Axin and Dishevelled contain DIX domains that mediate their interaction. Dishevelled itself seems to be recruited to activated receptor complexes by direct binding to Fz receptors. The complex of Dishevelled and Axin could therefore mediate an indirect interaction of Fz and LRP5/6 receptors, and might be responsible for the cooperation of both receptor components in Wnt signal transduction. LRP5/6-mediated relocation of Axin most likely not only inhibits the assembly of the classical destruction complex, but also inhibits the role of Axin in the nuclear export of β-catenin (96). This might explain the observation that Wnts can regulate the activity of β-catenin to some degree even in the complete absence of GSK-3β activity, which is considered to be essential for the classical destruction complex (74). Obviously, in the case of cells lacking GSK-3β, Wnt proteins cannot regulate β-catenin levels via inhibition of a destruction complex that is already dysfunctional. In this situation, alternative constitutive degradation pathways (e.g., SIAH1) and Axin dependent nuclear export of β-catenin could possibly keep the concentration of nuclear β-catenin low in the absence of Wnt ligands. Wnt ligand binding could then abrogate the Axin dependent nuclear export of β-catenin and allow a Wnt signal transduction even in the absence of GSK-3β function (74, 96–98). Notably, mammalian genomes contain a second Axin-like gene called Axin2 (initially termed Axil in the rat and conductin in the mouse). The Axin and Axin2 proteins are roughly 45% identical in sequence. Similar to Axin, Axin2 can increase the degradation of β-catenin (91). In contrast to the apparently ubiquitous expression of Axin, Axin2 expression seems to be much more tightly regulated (91,99). Interestingly, the Axin2 gene has been identified as a target gene for Wnt signaling, presumably mediating a negative feedback function by targeting β-catenin for destruction (100–103). Mutations in the genes for Axin and Axin2 that prevent their function in the destruction complex have been found in hepatocellular carcinoma, colorectal carcinoma, and gastric cancers at varying frequencies (104). To date, germline mutations associated with colorectal cancer and congenital developmental defects of the teeth (oligodontia) have only been described for Axin2 and only in a single kindred from Finland (105). Information is lacking on whether the second allele is missing in the tumors arising in individuals carrying a germline mutation, though both Axin and Axin2 might be expected to function as a classical “Knudson-type” tumor suppressor genes with biallelic inactivation in tumor cells. The Role of Fz and Dishevelled Proteins in Wnt/ β-Catenin Signaling As described earlier, Fz proteins are high-affinity receptors for Wnts (see Fig. 8-2). Whereas LRP5/6 proteins seem to be mainly involved in the pathway leading to β-catenin

256 / CHAPTER 8

Axin, actin & vesicles

DEP

PDZ

DIX

stabilization, Fz receptors also play a role in the signaling network that leads to the correct orientation of cells within a plane, so-called planar cell polarity signaling, as well as Ca2+/PKC and JNK signaling (see review by Veeman and colleagues [107]). Although the intracellular domain of Fz receptors is the least conserved region of this protein family, it seems to be necessary for the Fz-dependent β-catenin stabilization. The precise mechanism of action of Fz in stabilizing β-catenin, however, is still mysterious but seems to involve the action of Dishevelled proteins. Based on genetic epistasis analyses, Dishevelled proteins act downstream of both Fz and LRP receptors and upstream of the stabilization of β-catenin (Fig. 8-6). On binding of Wnts to receptors, Dishevelled rapidly gets phosphorylated. Notably, this phosphorylation seems to occur independent of the subsequent downstream signaling events (35,108). Some part of the total cellular Dishevelled then becomes redistributed to the plasma membrane, possibly by interactions between the N-terminal PDZ domain of Dishevelled and the intracellular domain of Fz (109). As described earlier, Axin also gets redistributed to the plasma membrane when the intracellular domain of LRP5/6 becomes phosphorylated. Dishevelled seems to be required for the effective redistribution of Axin to the plasma membrane (106), possibly by interaction of DIX domains that are present in both proteins (92). The importance of this interaction is underlined by the observation that the disruption of the Dishevelled–Axin interaction by the protein IDAX (inhibitor of Dishevelled Axin complex) can inhibit Wnt signaling (110). In addition, Dishevelled can bind to the GSK-3β inhibitor FRAT1/GBP (i.e., frequently rearranged in T-cell lymphoma/ GSK-3β binding protein). This protein is thought to compete with Axin for the binding of GSK-3β. By this mechanism, Axin molecules that are bound to Dishevelled might be

Frizzled FRAT1/GBP, Naked, IDAX




JNK, Rho

FIG. 8-6. Schematic drawing of Dishevelled protein structure. The N-terminal DIX (Dishevelled Axin) domain mediates the binding to Axin (268) and to the actin cytoskeleton (269). Interaction with Frizzled seems to require the PDZ (PSD, Disc-large, and ZO1) and the DEP (Dishevelled, Egl-10, and Pleckstrin) domains (109,270). Several inhibitory proteins also bind to the central PDZ domain such as GBP/FRAT1 (GSK3β-binding protein/frequently rearranged in T-leukemia-1) (112), IDAX (inhibitor of Dishevelled-Axin complex) (110), and naked (271). The DEP domain seems to mediate noncanonical signaling such as activation of c-Jun N-terminal kinase (JNK) and Rho (272,273). Binding regions for interacting proteins are shown later.

prevented from binding to GSK-3β and are therefore unable to act in the classical degradation complex (111–113). Because Fz receptors contain seven-transmembrane domains similar to G protein–coupled receptors, it has been suggested that Fz action might also depend on G proteins. Although there is some evidence that β-catenin stabilization upon activation of rat Fz 1 can be mediated by G proteins (114), the physiologic contribution of G proteins to most downstream effects of Wnt signaling is not yet clear (see review by Malbon and colleagues [115]). The Role of Adenomatous Polyposis Coli in Wnt/ β-Catenin Signaling Activation of the Wnt/β-catenin signaling pathway occurs in most colorectal cancers. In about 80% of these cases, this is attributable to biallelic inactivation of the APC tumor suppressor gene. Stabilizing β-catenin mutations accounts for about 10% of the cases (116,117). The biallelic inactivation of APC has some curious aspects that suggest that APC fulfills move functions than simply its role in the destruction complex for β-catenin. First, biallelic inactivation requires two distinct genetics events leading to either two distinct mutations in the APC alleles, or a mutation in one allele and chromosomal loss involving the other allele. This seems rather inefficient in light of the fact that Wnt/β-catenin signaling can be efficiently activated by a single point mutation in one of the two alleles of the gene encoding for β-catenin. Indeed, most noncolonic tumors with activation of the Wnt/β-catenin signaling pathway carry stabilizing β-catenin mutations. It is unclear what factors are responsible for the preference to activate Wnt/β-catenin signaling by biallelic inactivation of APC in the colon. Second, in most cases at least one APC allele is retained that leads to the expression of a truncated protein. Complete loss of APC expression is exceedingly rare. The situation is especially interesting in the case of patients with familial adenomatous polyposis. These patients carry a germline mutation in the APC gene. The localization of the germline mutation (i.e., the first hit) seems to predetermine the way of inactivation of the second allele in the tumors, suggesting that some truncated APC proteins are not sufficient to support the transformation of colonic epithelium. Third, the amount of APC protein does not seem to be the rate limiting factor in the assembly of the classical destruction complex. In fact, Axin is present at a 5000× lower concentration in the cells (93). The vast majority of cellular APC protein is therefore localized outside the classical destruction complex. Taken together, those observations imply that APC might have other tumor-suppressor functions in colon epithelial cells in addition to its role in the regulation of β-catenin stability. They also suggest that mutant APC proteins might either retain some functions that are essential for colorectal carcinogenesis or might acquire new “gain-of-function” activity.

DEVELOPMENTAL SIGNALING NETWORKS / 257

APC

Asef



Rac

15aa 15aa

15aa

ARM ARM ARM ARM ARM ARM ARM

MCR

Kap3



microtubules

β-catenin (15 and 20aa repeats) GSK3β (20aa repeats)

SIAH,

 EB1  micro- tubules

Axin (SAMP)

FIG. 8-7. Schematic drawing of adenomatous polyposis coli (APC) protein structure. APC binds to β-catenin via four 15-amino-acid (aa) repeats and seven 20-aa repeats. The latter also mediate binding of glycogen synthase kinase-3β (GSK-3β). Axin binds to SAMP repeats (containing the sequence serine-alanine-methionine-proline), which are interspersed in the last five 20-aa repeats. Most inactivating somatic mutations occurs in the mutation cluster region leading to truncated APC proteins that cannot bind to Axin anymore. Effects of APC on the actin cytoskeleton seem to be mediated by binding of the guanosine triphosphate exchange factor Asef to the armadillo repeats (142). Modulation of microtubule function depends on direct interaction of the C terminus, as well as indirect interactions via EB1 and Kap3 (134,274,275). Binding regions for interacting proteins shown later. MCR, mutation cluster region.

Human APC is a large protein of roughly 300 kDa (Fig. 8-7). Multiple interactions between APC and other proteins have been described (see review by Nathke [118]). The N-terminus of APC is predicted to promote dimerization with another APC molecule via a coiled-coil structure (119), and the N-terminal region might be involved in intramolecular interaction with the C terminus of APC. The main interactions of APC with Wnt-signaling pathway components take place in its central region. Four 15-aminoacid repeats in the central region mediate constitutive binding to β-catenin. Seven additional 20-amino-acid repeats in the central region also mediate binding to β-catenin (120) and are regulated in their affinity for β-catenin binding by phosphorylation of APC: The affinity of APC for β-catenin increases greatly when APC is modified by a cascade of phosphorylation events involving GSK-3β, a scenario somewhat reminiscent of the phosphorylation of β-catenin and the resultant effect on the affinity of β-catenin for β-TrCP binding (66). Interspersed between the last five of the seven 20-aminoacid repeats are 3 serine-alanine-methionine-proline (SAMP) repeats. These repeats are characterized by the presence of the SAMP sequence and mediate the interaction of APC with Axin (70,121). Mutations in colorectal cancers tend to occur in a central 650-amino-acid region of APC, which is commonly referred to as the somatic mutation cluster region (MCR). Most mutations lead to the expression of a C-terminally truncated APC protein that lacks the Axin-interacting SAMP repeats and several of the β-catenin–interacting 20amino-acid repeats (122). These proteins are therefore not capable of participating in the destruction of β-catenin. As explained earlier, the nature of the first hit, that is, the localization of the first mutation, seems to dictate the localization

of the second mutation, suggesting a selective advantage in the cancer cells for combinations of certain truncated APC proteins (123,124). Mammalian genomes encode for another homologue of Drosophilia armadillo, termed γ-catenin or junctional plakoglobin. γ-Catenin binds to APC, and its levels and localization in cells appear to be regulated by APC function (83). When overexpressed, wild-type γ-catenin seems to be capable of transforming cells. Although it had been controversial for some time whether γ-catenin does indeed have a transactivating function of its own or whether its function is mainly exerted by freeing β-catenin from its cytoskeletal location (125,126), recent data confirm that γ-catenin can act as a transcriptional activator independent of β-catenin perhaps in a cell-context manner (127–129). In addition to the classical destruction complex, APC seems to be involved in an alternative pathway of destruction for β-catenin. This pathway is dependent on the E3-ubiquitin ligase complex containing SIAH1 and Ebi, and requires interactions with the C terminus of APC, which usually is lost in colorectal carcinoma (84,85). Interestingly, the SIAH1 gene seems to be a target for regulation by p53. Notably, p53 is activated by accumulation of excessive amounts of βcatenin, suggesting a negative feedback regulation mechanism (130,131). In addition to its role in the degradation of β-catenin, APC seems to play important roles in intracellular shuttling of proteins (e.g., β-catenin) and cytoskeletal organization, thereby possibly influencing the chromosomal segregation in mitosis (see review by Nathke [118]). Several nuclear export and localization sequences are localized throughout the sequence of APC. The role of the nucleocytoplasmic

258 / CHAPTER 8 shuttling of β-catenin in the regulation of the transcriptional activity of β-catenin is discussed later. The C terminus of APC can interact directly with microtubules (132,133). Via interaction with the protein Kap3, APC associates with microtubule-associated motor proteins of the kinesin family, allowing its directional transport to the microtubule’s “plus” end (134). This leads to the formation of APC clusters at the microtubule’s end and seems to be able to stabilize them (135). APC’s action on microtubule function seems to be important in the correct segregation of chromosomes during mitosis. Consequently, it has been observed that cells lacking functional APC show more chromosomal segregation defects, and that tumor-derived C-terminally deleted fragments of APC seem to have a dominant effect on spindle function (136–140). Taken together, these data suggest that defects in APC function might lead to a certain degree of chromosomal instability that could foster the further development of gastrointestinal cancers. The significance of this effect in vivo has not yet been clearly established (141). Via interaction with the GTP exchange factor Asef, which modulates the activity of small G proteins such as Rac, APC may also function in modulating the actin cytoskeleton. Tumor-derived mutations might thereby regulate cell migration (142,143). Nuclear Access of β-Catenin: Subcellular Distribution The dual role of β-catenin in cell adhesion and nuclear transcriptional regulation implies that effective ways must exist not only to regulate the cytoplasmic levels of free βcatenin, but also to regulate the subcellular localization of β-catenin. Even though most colorectal carcinoma contain mutations activating the APC/β-catenin pathway, β-catenin is only localized in the nucleus of a minority of the cells (144,145), preferentially in cells with mesenchymal phenotype and localized at the invasion front. This has been attributed to differences in the microenvironment and also to differences in differentiation status. Given the necessity for nuclear accumulation of β-catenin to perform its transcriptional activity, understanding the pathways that regulate the distribution of β-catenin is of obvious importance in the understanding of the molecular pathogenesis of colorectal carcinoma development and progression. Interestingly, in tissue culture systems, dramatic changes of the subcellular distribution of β-catenin within the cells can be observed when cells are grown at different cell densities. It has been postulated that, in addition to the membranebound pool, different free pools of β-catenin should exist that can preferentially bind to TCFs or α-catenin (146). Whether and what kind of structural differences account for these different pools currently is not well understood. To a certain degree, phosphorylation of specific tyrosine residues in β-catenin seems to govern the decision whether β-catenin acts as a cytoskeletal protein (via interaction with α-catenin and E-cadherin) or whether it is capable of fulfilling its transcriptional function (via interaction with BCL9-2) (80).

Much more is known about factors that regulate the nuclear access of β-catenin. Initially, it was suggested that because of the similarity of the armadillo repeats with the repeats of Importin-β, β-catenin could directly interact with the nuclear pore complex. Despite its size, β-catenin was therefore postulated to freely shuttle between nucleus and cytoplasm. In this model, the subcellular distribution of β-catenin would be mainly determined by the distribution of its interaction partners between the nucleus and the cytosol (97). However, evidence has been presented that nuclear export of β-catenin might actually be supported by APC (147,148) and Axin (96). Although nuclear export of β-catenin mediated by APC seems to play a role in terminating the transcriptional activity of nuclear β-catenin, it has been noted that overexpression of APC constructs that are targeted to the nucleus leads to a nuclear accumulation of β-catenin without an increase in target gene transcription (149). The molecular basis for this apparent paradox seems to lie in the capacity of APC to reduce the amount of functional β-catenin-TCF complexes by its interaction with the protein CtBP (C-terminal binding protein) (150). Taken together, in addition to its contribution to the destruction complex, APC seems to reduce the nuclear activity of β-catenin not only by increasing its nuclear export, but also by sequestering it away from TCF. It has been suggested that the nuclear export functions of APC and Axin are being antagonized by the nuclear import function of the proteins Legless/BCL9 and Pygopus, which also serve as essential coactivators of β-catenin’s transcriptional function. However, the physiological relevance of this effect is still subject to debate (151–154). Nuclear Action of β-Catenin Stabilization of β-catenin and nuclear accumulation allows for an increased interaction with proteins of the TCF/LEF DNA-binding protein family. These proteins bind to the consensus site CWTTGWW. Mammalian genomes have four distinct genes encoding for TCF-LEF transcription factors. The proteins contain a highly conserved high mobility group box (HMG box) that mediates sequence-specific DNA binding (155; see also review by van Noort and Clevers [156]) (Fig. 8-8). Binding to its recognition sequence induces a bend in the DNA that potentially can alter the binding and access of other DNA-binding proteins (157). β-Catenin interacts with the N terminus of TCF/LEF transcription factors (18). In the absence of high levels of β-catenin, they repress the transcription of target genes by virtue of their interaction with transcriptional repressor proteins of the transducin-like enhancer of split/groucho-related gene (TLE/Grg) family (related to the Drosophila groucho protein) and possibly the repressor CtBP (14,158,159). These repressors recruit histone deacetylases, and induce changes in the chromatin structure, making the promoter inaccessible for transcriptional activation. In addition, CtBP might cooperate with nuclear APC to prevent the formation of functional β-catenin-TCF complexes (150). β-Catenin,

DEVELOPMENTAL SIGNALING NETWORKS / 259

HMG

β-catenin

Grg/TLE

DNA

FIG. 8-8. Schematic drawing of Lymphoid enhancer factor (LEF)/T-cell factor (TCF) protein. β-Catenin binds to the N terminus of TCF/LEF proteins. Recruitment of transcriptional repressor complexes is mediated by the interaction with TLE/Grg proteins (transducin-like enhancer of split/grouchorelated genes). The high mobility group (HMG) domain mediates interaction with specific DNA target sequences. Binding regions for interacting proteins are shown later.

when present in the nucleus, appears to displace the transcriptional repressors from the TCF proteins. Association with histone acetyl transferases (such as CBP/p300), (160,161) ATPases (such as TIP49) (162) and components of the SWI/SNF chromatin remodeling complexes (such as Brg1) (163) leads to a modification of the chromatin in the promoter region, making the DNA more accessible for transcription factors. The basal transcriptional machinery gets recruited to responsive promoters (164), and target gene transcription can begin. The proteins Legless/BCL9 (i.e., B-cell lymphoma 9) and Pygopus seem to play a major role as essential coactivators of β-catenin–dependent transcriptional activation (152–154). Previously, BCL9 has been implicated in the development of non–Hodgkin’s lymphoma in the mouse. Legless/BCL9 has been shown to directly interact with β-catenin and recruit Pygopus to this complex. In addition to the transcriptional coactivator function of BCL9/Legless and Pygopus, a role of these proteins in nuclear import of β-catenin has been suggested (151). However, it is still subject to debate how much this contributes to the essential function of Legless/ BCL9 and Pygopus in the Wnt/β-catenin signaling pathway. The importance of the nuclear import function is supported by that expression of armadillo mutants that are artificially targeted to the nucleus are transcriptionally active even in the absence of either BCL9 or Pygopus (151). Mammalian genomes encode for two BCL9/Legless proteins, BCL9 and BCL9-2. BCL9 seems to exert its nuclear transport function of β-catenin mainly due to nuclear localization sequences on its interaction partner, Pygopus. In contrast, the other homolog, BCL9-2, does not seem to rely on Pygopus for nuclear localization (159) and can specifically interact with β-catenin only when β-catenin is tyrosine phosphorylated at residue 142. This modification in parallel prevents the interaction of β-catenin with α-catenin, thereby representing an intriguing model of a switch between the cytoskeletal and transcriptional functions of β-catenin (80).

Wnt/β-Catenin/T-Cell Factor Target Genes Accumulation of β-catenin leads to fundamental changes of gene expression of a large number of genes. Several

microarray-based studies have formed our current knowledge of this transcriptional program in different tissue culture systems, animal models, and tumor entities (165–169). Interestingly, only a few genes seem to be regulated consistently in all experimental systems, suggesting that this transcriptional is highly dependent on the context in which its activation occurs. Several target genes are discussed briefly here and later in this chapter (see Wnt/β-Catenin Pathway in Gastrointestinal Physiology). For further information on this rapidly expanding field, refer to several reviews (170–172) and the Wnt Home Page (access online at: http://www. stanford.edu/~rnusse/wntwindow.html). The protooncogene c-myc was among the first target genes of β-catenin to be identified, and it plays an important role in cell proliferation and growth (173). By interaction with MIZ1, it is capable of down-regulating the cell cycle inhibitor p21CIP1, thereby releasing a major brake from the cell cycle (165). The progression through the cell cycle is also supported by induction cyclin D1, which is also directly up-regulated by β-catenin (174). Proinvasive factors also seem to be subject to β-catenin– dependent transcriptional regulation. So far, good evidence suggests that matrix metalloproteinase-7 (MMP-7)/Matrilysin (175,176), membrane-type 1 MMP (MT1-MMP) (177,178), Laminin-5 γ2 chain (178), and CD44 (179) are target genes of this pathway. When analyzing the transcriptional program induced by stabilized β-catenin, a marked resemblance to the expression pattern of intestinal cell progenitor cells was noted (165). Inhibition of this pathway conversely led to a pattern resembling differentiated epithelial cells. Several of the previously identified target genes subsequently have been found to be up-regulated in the presumptive intestinal cell progenitor population in vivo. In addition, it was noted that β-catenin coordinates the mutually exclusive expression pattern of EphB2/EphB3 receptors and their ligand Ephrin B1, which are capable of mediating repulsive signaling during cell migration. It also was shown that this coordinated expression is responsible for the correct positioning of proliferative and differentiating intestinal cells along the crypt axis (180). Interestingly, several well-conserved feedback mechanisms have been described. TCF/LEF proteins seem to play a significant role in two of those feedback loops: Stabilized β-catenin seems to specifically activate the transcription of an isoform of TCF1 that does not contain the binding site for β-catenin and can therefore block the transcriptional activation of TCFresponsive promoters. Consistent with this notion, TCF1 knockout mice spontaneously form intestinal polyps consistent with a tumor-suppressor function of this protein in the intestine (181). In contrast, β-catenin specifically increases the transcription of the LEF1 transcripts that can interact with β-catenin, and therefore harbor the potential to increase the transcription of target genes in a positive feedback loop (182). Among the most robust transcriptional targets are the Axin2/Conductin gene and the NKD1 and NKD2 genes (related to the Drosophila naked cuticle gene). Similar to Axin, Axin2/Conductin can mediate a negative feedback

260 / CHAPTER 8 by supporting the degradation of β-catenin. Nkd1 and Nkd2 seem to mediate negative feedback inhibition at the level of Dishevelled (102,103). Both proteins could play an important role in the termination of signaling after physiologic stimulation of this pathway.

NONCANONICAL WNT SIGNALING PATHWAYS Early studies have led to the notion that the transforming activity of Wnts correlates with their ability to stabilize β-catenin (29). It also was realized that Wnt proteins play a role in several signaling pathways without the involvement of β-catenin. These pathways collectively are referred to as “noncanonical Wnt signaling pathways” in the literature (see review by Veeman and colleagues [107]). One of the classic examples of noncanonical Wnt signaling is the establishment of the polarity of cells within a plane in vivo, that is, the organization perpendicular to the apicobasal axis of the cells. Signaling pathways involved in the specification of these decisions involve the JNK pathway and intracellular calcium-dependent signaling pathways. Disruption of components of this so-called planar cell polarity pathway in mammals has been shown to lead to an inhibition of the development of the inner ear (183) by disturbing the orientation of the cilia. Interestingly, disruption of Fz 6 leads to a lack of orientation of the hair fur (184). So far, there is no evidence that planar cell polarity pathways are involved in gastrointestinal physiology or pathology. There is evidence that the Wnt proteins that preferentially serve in planar cell polarity and other noncanonical pathways, such as Wnt5a, might act antagonistic to activating Wnt proteins and induce degradation of β-catenin (185). Thus, they seem to be able to antagonize the function of β-catenin in the transformation of cell lines (186–188). Consistent with this possibility, data implicate Wnt5a as a tumor suppressor in the lymphoid system (186). Part of the Wnt signaling pathway is mediated by inhibition of GSK-3β, which is a central player in multiple signaling pathways (189). In addition to the stabilization of β-catenin, numerous other important pathways are therefore possibly modulated by Wnt signaling, for example, Snail (190) and Notch (191), which are both involved in epitheliomesenchymal transformation (192–194). Future studies need to address the relevance of the cross talk between these pathways to developmental processes in vivo.

WNT/β-CATENIN PATHWAY IN GASTROINTESTINAL PHYSIOLOGY Wnt/β-Catenin in Gastrointestinal Development The gastrointestinal tract develops as a derivative of the endoderm (see reviews by Gregorieff and Clevers [170] and Wells and Melton [195]). Using tissue-specific knockout

approaches, it was demonstrated that β-catenin is essential for endoderm formation around embryonic day 6 of mouse development (196). Experiments in Xenopus suggest that this may be, at least in part, mediated by binding to Sox17, a DNA-binding protein containing an HMG box similar to TCF/LEF proteins (197,198). The source, as well as the identity, of the activating Wnt protein is currently unknown. Given the lack of formation of a primitive streak (a prerequisite for endoderm formation) in the respective knockout embryos, Wnt3 could possibly serve this function (199). In the further development of the gut tube from the endoderm, Tcf1 and Tcf4 seem to be essential mediators as demonstrated by severe developmental defects of Tcf4−/−/ Tcf1−/− embryos. In addition to the truncation of the hindgut, these embryos show an expansion of anterior gut structures such as the stomach (200). Interestingly, it has been shown that overexpression of stabilized β-catenin mutants in the lung endoderm of transgenic animals leads to the expression of genes typically specific for the intestine (201). Taken together, these two observations suggest that Wnt/β-catenin signaling is not only essential for endoderm development, but also for the development of an intestinal fate.

Wnt/β-Catenin Pathway in Gastrointestinal Homeostasis and Architecture The intestinal mucosa is exposed to one of the most toxic, aggressive, and changing milieus of the whole body. In contrast with the outer surface of the body, the intestinal barrier consists only of a single layer of fragile epithelial cells. To maintain an active barrier and also fulfill the main tasks of digestion and absorption, the intestine relies on the high turnover of several specialized cell types that continuously form from intestinal stem cells (171,202). Mature cells can be mainly subdivided into a secretory and an absorptive lineage. The secretory lineage consists of goblet cells producing the intestinal mucus and a small number of enteroendocrine cells that are further subdivided based on the type of hormone that they produce. In the small intestine, a third secretory cell type, the so-called Paneth cells, localizes to the bottom of the crypts, producing a variety of antibacterial substances. The absorptive lineage is also called enterocyte and fulfills the task of absorbing the nutrients from the intestinal contents (170). All intestinal cell types derive from intestinal stem cells. These stem cells localize to the base of the crypt in the colon and above the Paneth cells in the lower part of the crypts in the small intestine (203). With the exception of the Paneth cells, the differentiating daughter cells migrate actively up the crypt in a coordinated way and populate the intestinal surface and villi. After a lifetime of less than a week, these cells undergo apoptosis and are shed in the intestinal lumen. Wnt/β-catenin signaling seems to play a major role in maintenance of a progenitor cell compartment, in the maintenance of the intestinal architecture, and in the differentiation of the secretory lineage.

DEVELOPMENTAL SIGNALING NETWORKS / 261 Several lines of evidence demonstrate an essential role for Wnt/β-catenin signaling in the maintenance of a functioning intestinal progenitor compartment. Knocking out the DNAbinding adaptor TCF4 leads to severe developmental defects with a total absence of the intestinal progenitor cell compartment (204). A similar phenotype is observed in transgenic animals overexpressing the Wnt antagonist Dkk1 in the gut and on induced deletion of β-catenin in the small intestine (205–207). This is consistent with the previous observation that inhibition of Wnt/β-catenin signaling in colorectal carcinoma cell lines leads to a shift in gene expression from a progenitor type to a more differentiated absorptive cell type (165). As expected, overexpression of stabilized β-catenin mutants or induced inactivation of APC in the gut leads to epithelial hyperproliferation and expansion of the proliferative compartment (169). Evidence for a role of Wnt/β-catenin signaling in maintaining the architecture of the intestinal crypts originated from the observation that the tyrosine kinase receptors EphB2 and EphB3 are direct target genes of this pathway (180). These receptors play a well-characterized function in the guidance of axonal outgrowth and cell migration during the development of the central nervous system (208). As expected for a Wnt/β-catenin target gene, a high expression in the proliferative compartment of the intestinal crypts was noted. Analysis of double-knockout mice for EphB2 and EphB3 showed that this expression pattern is essential for a coordinated movement of differentiating cells and the localized maintenance of a stem cell compartment. Consequently, in these mice, proliferative and differentiating cells intermingled, and Paneth cells no longer homed for the base of the crypt (180). A role of Wnt/β-catenin signaling in the commitment to a secretory fate was initially suggested by the observation that induced deletion of β-catenin in the intestine or overexpression of Wnt antagonists leads to a striking reduction of goblet cells, Paneth cells, and enteroendocrine cells (204,205,207). In reverse experiments, it was demonstrated that induced activation of the Wnt/β-catenin signaling leads to an increased production of Paneth cells, suggesting that Wnt signaling might play a specific role in mediating the differentiation of this specific cell type. In this context, several Paneth cell– specific proteins have been shown to be direct Wnt/β-catenin target genes (209,210). Interestingly, a phenotype of increased goblet cell differentiation also has been described in animals that were treated with γ-secretase inhibitors thereby preventing efficient Notch signal transduction (211). Similarly, an increase of secretory lineage cells has been observed in mice with a targeted disruption of the Notch target gene HES1 (212), suggesting that the balance between Notch signaling and Wnt/β-catenin signaling could govern the decision between absorptive and secretory lineage differentiation. Although much has been learned about the role of Wnt/βcatenin signaling in cellular differentiation and the maintenance of a progenitor cell compartment, very little is known about which specific Wnt proteins and receptor complexes

are mediating these effects in vivo. Given the vast potential for redundancy and combinatorial diversity, this poses a major challenge to the understanding of the physiologic role of this signaling pathway. An additional layer of complexity is added by the extensive cross talk of Wnt/β-catenin with other signaling pathways such as Notch signaling and the bone morphogenic protein pathway (202).

WNT/β-CATENIN PATHWAY DEFECTS IN GASTROINTESTINAL TUMORS This section summarizes some of the data that are available on the involvement of the Wnt/β-catenin signaling pathway in gastrointestinal malignancy. See Table 8-1 for more detailed information.

Colon and Rectum Sporadic Colorectal Cancer Activation of the Wnt signaling pathway seems to be one of the earliest changes in the development of colorectal neoplasia, occurring as early as in aberrant crypt foci (213). Inactivating mutations of APC seem to account for up to 80% of the cases (214,215). APC is a large gene coding for 2843 amino acids and spanning more than 90 kBp. Although a plethora of mutations have been described, they do not seem to be distributed evenly over the coding sequence, and 60% seem to be clustered in a small region of exon 15 between codon 1250 and 1450, which therefore has been termed mutation cluster region (MCR) (16,216). The capacity to destabilize β-catenin is highly dependent on the presence of the SAMP repeats, which, via binding to Axin, seem to recruit APC to the destruction complex. Biallelic inactivation of APC almost invariably leads to the expression of truncated APC proteins that do not contain any of the Axin-interacting SAMP repeats and are therefore not capable of efficiently stimulating degradation of β-catenin. Interestingly, complete deletion of both APC alleles with complete loss of APC protein expression is rarely observed, suggesting that the selective advantage of APC mutations not only relies on the inactivation of APC functions, but also possibly on the retention of certain APC functions or even the generation of new functions of the truncated proteins (123,124). Different explanations to account for this selection advantage such as increased genomic instability (139,140) and increased migratory potential (143) induced by truncated APC proteins have been proposed. In aggregate, less than 10% of colorectal carcinomas have been reported to carry stabilizing β-catenin mutations, and most colorectal carcinomas with functionally significant β-catenin mutations are mismatch repair deficient (215,217). In mismatch repair–deficient colorectal carcinomas, mutations in a microsatellite repeat tract in the coding region of the Axin2 gene also seem to be present in a

262 / CHAPTER 8 TABLE 8-1. Percentage of activation of Wnt/adenomatous polyposis coli/β-catenin pathway in selected tumor entities Tumor entity

APC

β-catenin

Colon Carcinoma

80%

10%

Stomach Fundic gland polyps (236) Gastric adenoma (240) Carcinoma (242,243)

43–64% 8–27%

Esophagus Barrett’s esophagus Adenocarcinoma Squamous carcinoma

Axin

Axin2

0%

<10%

4%

2%

10%

3%

64–94%

1%

Lung Adenocarcinoma

3%

Ovary Endometrioid carcinoma

2%

Rare, but frequent nuclear localization <2% Rare

14–50%

Uterus Endometrial carcinoma Cervical carcinoma

11–45% Rare

Thyroid Carcinoma

46%

Liver Hepatocellular carcinoma

Rare

Hepatoblastoma

≤69%

Pancreas Ductal carcinoma Acinar cell carcinoma Solid pseudopapillary tumor Pancreatoblastoma

6%

Skin Melanoma Pilomatricoma

3–7%

10–43% (correlated with hepatitis C virus) ≤89%

7%

Rare 18% 90% 55% 2–22% 26–100%

For references see corresponding chapters and Gilles and colleagues (172). APC, adenomatous polyposis coli.

considerable fraction of cases, though such mutations are not seen in colorectal cancers where mismatch repair activity is intact (218). In contrast with the situation in hepatocellular carcinoma, the pathogenetic relevance of the rarely observed Axin mutations in colorectal cancers is unclear (219,220). Familial Adenomatous Polyposis Coli FAP is an autosomal dominant condition caused by the germline mutation of one allele of the APC gene that leads to the development of thousands of colorectal polyps in the second decade of life. The disease almost invariably progresses to the development of colorectal cancer unless the colon is surgically removed before its occurrence. It is observed in 1 in 7000 to 1 in 10,000 people and is responsible for less than 1% of colorectal cancers (12). Most mutations are nonsense or frameshift mutations leading to the production of truncated APC proteins lacking APC protein synthesis lacking Axin-interacting regions but still retaining some capacity to interact with β-catenin. The majority is localized in the N-terminal half, and almost 35%

of the mutations occur in two mutational hotspots (codon 1061 and 1309) (116,123). Only a small subset of mutations are large deletions or mutations that completely abolish APC expression. Some analyses also suggest that the localization of the germline mutation predetermines the mechanism by which the second allele is lost. This is consistent with the notion that truncated APC proteins serve important functions in colorectal carcinogenesis and complete loss of APC expression is selected against. Greater severity of polyposis is associated with mutations between codons 1250 and 1464 (221). However, clinical presentation is not consistent even within kindreds carrying the same germline mutation. The same is also true for the presentation of extracolonic manifestations. The most common extracolonic symptom is a benign alteration of the eye’s pigment epithelium, called congenital hypertrophy of the retinal pigment epithelium (CHRPE). It occurs in up to 60% of patients and is associated with mutations between codons 457 and 1444 (222). In addition, other tumors occur in greater frequency in patients with FAP such as desmoid tumors (associated with mutations between codon

DEVELOPMENTAL SIGNALING NETWORKS / 263 1445 and 1578 [223]), osteoma, gastric adenocarcinoma, thyroid carcinoma, central nervous system tumors, hepatoblastoma, as well as duodenal ampullary carcinoma. Depending on the constellation of extracolonic manifestations, several distinct syndromes have been described. Among these, Gardner syndrome refers to FAP cases with extensive polyposis, epidermoid cysts, desmoid tumors, and osteomas. Turcot syndrome comprises cases with polyposis and brain tumors, such as medulloblastoma (12). Attenuated forms of FAP (termed AAPC for attenuated adenomatous polyposis coli or AFAP for attenuated FAP) have also been described. In AAPC kindreds, some affected individuals in the family have as little as 10 to 20 colonic adenomas and no cancers by 50 years old, though other affected AAPC family members can have colonic adenoma numbers essentially indistinguishable from those with classical FAP (224). Although the likelihood of development of colorectal cancer is much smaller for an individual with AAPC, in some populations, a high frequency of a predisposing allele might lead to a significant contribution to the incidence of colorectal carcinoma (225). For example, 6% to 10% of Ashkenazi Jews carry the APC variant I1307K, which increases the colorectal carcinoma risk only about twofold without leading to overt polyposis (226–228). However, because of its high prevalence in this population, this mutation can significantly influence colorectal carcinoma incidence in this population.

Esophagus Malignancies of the esophagus can be subdivided into squamous cell carcinoma and adenocarcinoma. The latter seems to develop via a premalignant glandular metaplasia termed Barrett’s esophagus. Squamous Cell Carcinoma Human Fz 2 was first cloned from an esophageal squamous cell carcinoma (229). The percentage of carcinomas overexpressing Fzd2 was reported to increase from 23% of differentiated carcinomas to 86% of undifferentiated carcinomas. However, nuclear localization of β-catenin was reported in only 18% of all cases (230). The significance of these findings is still unclear. Barrett’s Esophagus and Esophageal Adenocarcinoma Activating mutations of β-catenin have been described in less that 2% of the cases of esophageal adenocarcinoma (233–235). Although loss of heterozygosity (LOH) for APC was reported to occur in up to 40% of the cases, inactivating mutations in the remaining allele were only found in 1 of 38 tumors with LOH (235). Given the fact that only the mutation cluster region was screened in these studies, mutations in the remaining APC gene cannot be excluded. The role of genetic events leading to the activation of the Wnt/β-catenin signaling pathway is therefore unclear.

However, reduced membranous and increased nuclear localization of β-catenin seem to be common and early events in the pathogenesis of Barrett’s esophagus, and are correlated with the development of dysplasia (231). The cause of these changes and their relevance to the progression to cancer are unclear because they have been reported to be associated with both better and worse clinical outcomes in esophageal adenocarcinoma (231,232).

Stomach Fundic Gland Polyps Fundic gland polyps are benign nondysplastic hamartomatous or hyperplastic lesions that constitute most gastric polyps. Sporadic fundic gland polyps rarely, if at all, progress to adenocarcinoma. This is in contrast with the gastric fundic gland polyps that have been observed in patients with familial APC mutations. These usually display biallelic inactivation of the APC gene product and occasionally progress to adenocarcinoma. Surprisingly, up to 94% of the polyps predominantly benign sporadic fundic gland polyps show stabilizing mutations of the β-catenin gene (236,237). In the rare instances of fundic gland polyposis, polyps from single individuals often do not all show the same mutation, suggesting that they are of polyclonal origin (238). That gastric fundic gland polyps almost never progress to gastric adenocarcinoma (or even only to dysplasia) and the activation of the Wnt/βcatenin signaling pathway in most cases seems difficult to reconcile. However, Hassan and colleagues (239) reported that syndromic and sporadic cases with stabilizing β-catenin mutations rarely show abnormal β-catenin staining. Although these findings require further confirmation, they could suggest that so far unknown mechanisms are able to control the activation of the Wnt/β-catenin pathway in gastric epithelium. Gastric Adenoma Unlike benign gastric fundic gland polyps, about 25% of the much rarer gastric adenomas progress to gastric adenocarcinoma, marking these polyps as premalignant lesions. Depending on the histologic subtype, biallelic inactivation of APC has been described in 64% (intestinal type) and 43% (foveolar type) of cases (240). Interestingly, the presence of an APC inactivation was reported to be inversely correlated with the tendency to progress to adenocarcinoma (241). The small number of studies and rare occurrence of these lesions currently limits our understanding of these observations. Gastric Adenocarcinoma Although nuclear localization occurs in approximately one third of gastric adenocarcinomas, less than 25% of these

264 / CHAPTER 8 cases show activating mutations of β-catenin (242,243). It is unclear what causes the nuclear localization of β-catenin in the remaining carcinomas without mutations. In rare instances, biallelic inactivation of SIAH1 gene, encoding a component of a E3 ubiquitin ligase complex, have been reported to be associated with nuclear β-catenin accumulation (87). Given the fact that the regulation of β-catenin via the classical destruction complex is still functional in these cases, the exact molecular mechanism leading to the stabilization of β-catenin awaits further elucidation. Germline mutations in the gene encoding E-Cadherin are associated with familial gastric adenocarcinoma. Epigenetic inactivation of ECadherin also seems to occur in a high percentage of sporadic diffuse-type gastric adenocarcinoma. However, although these changes would be expected to free a large part of the cytoskeleton-bound β-catenin, they do not seem to lead to an increase in TCF/LEF-dependent transcription (243,244), suggesting that effects outside of the Wnt/β-catenin signaling pathway might be more relevant.

Liver Nuclear localization of β-catenin is observed in a significant proportion of hepatocellular carcinoma (HCC). The pathogenesis of this tumor shows marked differences between different geographic regions which can be attributed to the different prevalences of hepatitis B, hepatitis C, and the exposure to aflatoxin. The occurrence of nuclear β-catenin seems to be associated only with some of these causative agents, which might explain the marked differences of its frequency in the literature ranging from 12% to 41%. Stabilizing β-catenin mutations seem to occur in a distinct subset of these tumors that are characterized by their good prognosis and lack of involvement of the hepatitis B virus. This subset seems to be associated rather with hepatitis C virus and is devoid of high chromosomal instability (245–249). Distinct from this group is a rare childhood tumor called hepatoblastoma that shows activating β-catenin mutations in a high percentage (250–252). Axin and Axin2 mutations have been observed in HCC in 8% to 10% and 3%, respectively (104,245,253). APC mutations seem to be extremely rare in HCC. It has been reported that the prolyl-isomerase PIN1 can inhibit the interaction of β-catenin and APC. Overexpression of PIN1 has therefore been suggested to account for the stabilization of β-catenin in the fraction of tumors that do not show other genetic defects in the Wnt/β-catenin pathway (254). Overall, the importance of the Wnt/β-catenin signaling pathway in hepatic carcinogenesis has also been demonstrated in animal models. Biallelic inactivation of APC in the liver leads to the development of HCCs. Overexpression of stabilized β-catenin mutants only leads to a hypertrophy of the liver, and coexpression with H-Ras leads to the development of HCCs (255–257). In addition, β-catenin seems to play an important role during hepatic development (258).

Pancreas Ductal adenocarcinoma of the pancreas, the most common form of pancreatic cancer, usually does not show abnormal localization of β-catenin. Except for two mutations in pancreatic cancer cell lines, no mutations in β-catenin or APC currently have been described (243,259,260). However, several rare tumor entities display a high percentage of abnormalities in the Wnt/APC/β-catenin signaling pathway. Up to 90% of solid pseudopapillary tumors (261,262) and twothirds of pancreatoblastomas harbor activating β-catenin mutations (263). In contrast, activation of Wnt/APC/β-catenin signaling pathway in 25% of the acinar cell carcinoma is mediated by biallelic inactivation of APC in most cases (264).

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270 / CHAPTER 8 252. Wirths O, Waha A, Weggen S, Schirmacher P, Kuhne T, Goodyer CG, et al. Overexpression of human Dickkopf-1, an antagonist of wingless/WNT signaling, in human hepatoblastomas and Wilms’ tumors. Lab Invest 2003;83:429–434. 253. Ishizaki Y, Ikeda S, Fujimori M, Shimizu Y, Kurihara T, Itamoto T, et al. Immunohistochemical analysis and mutational analyses of betacatenin, Axin family and APC genes in hepatocellular carcinomas. Int J Oncol 2004;24:1077–1083. 254. Pang R, Yuen J, Yuen MF, Lai CL, Lee TK, Man K, et al. PIN1 overexpression and beta-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma. Oncogene 2004;23: 4182–4186. 255. Colnot S, Decaens T, Niwa-Kawakita M, Godard C, Hamard G, Kahn A, et al. Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci U S A 2004;101:17216–17221. 256. Harada N, Miyoshi H, Murai N, Oshima H, Tamai Y, Oshima M, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res 2002;62:1971–1977. 257. Harada N, Oshima H, Katoh M, Tamai Y, Oshima M, Taketo MM. Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations. Cancer Res 2004;64:48–54. 258. Micsenyi A, Tan X, Sneddon T, Luo JH, Michalopoulos GK, Monga SP. Beta-catenin is temporally regulated during normal liver development. Gastroenterology 2004;126:1134–1146. 259. Gerdes B, Ramaswamy A, Simon B, Pietsch T, Bastian D, Kersting M, et al. Analysis of beta-catenin gene mutations in pancreatic tumors. Digestion 1999;60:544–548. 260. Kubozoe T, Tsujiuchi T, Murata N, Sasaki Y, Tsunoda T, Konishi Y, et al. Absence of beta-catenin gene mutations in pancreatic duct lesions induced by N-nitrosobis(2-oxopropyl)amine in hamsters. Cancer Lett 2001;168:1–6. 261. Abraham SC, Klimstra DS, Wilentz RE, Yeo CJ, Conlon K, Brennan M, et al. Solid-pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations. Am J Pathol 2002;160:1361–1369. 262. Tanaka Y, Kato K, Notohara K, Hojo H, Ijiri R, Miyake T, et al. Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm. Cancer Res 2001;61: 8401–8404. 263. Abraham SC, Wu TT, Klimstra DS, Finn LS, Lee JH, Yeo CJ, et al. Distinctive molecular genetic alterations in sporadic and familial adenomatous polyposis-associated pancreatoblastomas: frequent alterations in the APC/beta-catenin pathway and chromosome 11p. Am J Pathol 2001;159:1619–1627.

264. Abraham SC, Wu TT, Hruban RH, Lee JH, Yeo CJ, Conlon K, et al. Genetic and immunohistochemical analysis of pancreatic acinar cell carcinoma: frequent allelic loss on chromosome 11p and alterations in the APC/beta-catenin pathway. Am J Pathol 2002;160:953–962. 265. Yao R, Natsume Y, Noda T. MAGI-3 is involved in the regulation of the JNK signaling pathway as a scaffold protein for frizzled and Ltap. Oncogene 2004;23:6023–6030. 266. Yao R, Maeda T, Takada S, Noda T. Identification of a PDZ domain containing Golgi protein, GOPC, as an interaction partner of frizzled. Biochem Biophys Res Commun 2001;286:771–778. 267. Kramps T, Peter O, Brunner E, Nellen D, Froesch B, Chatterjee S, et al. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 2002; 109:47–60. 268. Kishida S, Yamamoto H, Hino S, Ikeda S, Kishida M, Kikuchi A. DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability. Mol Cell Biol 1999;19: 4414–4422. 269. Capelluto DG, Kutateladze TG, Habas R, Finkielstein CV, He X, Overduin M. The DIX domain targets dishevelled to actin stress fibres and vesicular membranes. Nature 2002;419:726–729. 270. Axelrod JD, Miller JR, Shulman JM, Moon RT, Perrimon N. Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 1998;12:2610–2622. 271. Rousset R, Mack JA, Wharton KA Jr, Axelrod JD, Cadigan KM, Fish MP, et al. Naked cuticle targets dishevelled to antagonize Wnt signal transduction. Genes Dev 2001;15:658–671. 272. Li L, Yuan H, Xie W, Mao J, Caruso AM, McMahon A, et al. Dishevelled proteins lead to two signaling pathways. Regulation of LEF-1 and c-Jun N-terminal kinase in mammalian cells. J Biol Chem 1999;274:129–134. 273. Moriguchi T, Kawachi K, Kamakura S, Masuyama N, Yamanaka H, Matsumoto K, et al. Distinct domains of mouse dishevelled are responsible for the c-Jun N-terminal kinase/stress-activated protein kinase activation and the axis formation in vertebrates. J Biol Chem 1999; 274:30957–30962. 274. Berrueta L, Kraeft SK, Tirnauer JS, Schuyler SC, Chen LB, Hill DE, et al. The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules. Proc Natl Acad Sci U S A 1998;95:10596–10601. 275. Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, et al. APC binds to the novel protein EB1. Cancer Res 1995;55:2972–2977.

CHAPTER

9

Hedgehog Signaling in Gastrointestinal Morphogenesis and Morphostasis Gijs R. van den Brink, Maikel P. Peppelenbosch, and Drucilla J. Roberts Patterning, 271 The Hedgehog Pathway, 272 Hedgehog Genes, 272 The Unusual Posttranscriptional Fate of Hedgehog Proteins, 273 The Receptor Complex, 274 Signal Transduction and Target Genes, 274 Role of Hedgehog Signaling in the Development of the Gut, 275 Development of the Gastrointestinal Tube, 275 Hedgehog Signaling and Foregut Development, 275 Hedgehog Signaling and Development of the Midgut, 278 Hedgehog Signaling and Development of the Hindgut, 278

Hedgehog Signaling and the VACTERL Association in Humans, 279 Hedgehog Signaling in Homeostasis of the Adult Gastrointestinal Tract, 279 Morphostasis, 279 Role of Ihh and Shh in Adult Gut, 280 Shh and Gastric Epithelial Homeostasis, 281 Shh and the Small Intestinal Crypt, 281 Ihh and Colonic Epithelial Differentiation, 281 Hedgehog Signaling and Carcinogenesis of the Gastrointestinal Tract, 282 Future Developments, 283 References, 283

The mechanisms that orchestrate the development of a multicellular organism from a single common ancestor cell have fascinated people for centuries. We are still a long way from truly understanding the robust programs that allow building of our specialized organs that regulate digestion, respiration, and hemodynamics and that try to guard us from invasion by other unsolicited organisms. However, in the past three decades, many of the genes that play a role in patterning our bodies have been identified, and much has been learned about the mechanisms involved in development by characterizing the nature of these patterning genes and the way they work together. This chapter discusses

patterning mechanisms, introduces the Hedgehog pathway, and then summarizes our current understanding of the role of Hedgehog signaling in development and maintenance of the gastrointestinal (GI) tract.

PATTERNING The human body is built from hundreds of specialized cell types that make up its organs. The development and postnatal maintenance of these cell lineages is tightly controlled and incredibly stable in time. Cells are capable of going through major shifts in protein expression in response to physiologic and environmental cues without changing their cellular phenotype. Each cell type is defined by the expression of a unique combination of proteins transcribed and posttranscriptionally modified from the templates of the 20,000 to 25,000 genes in the body. One of the central questions in biology is how the information contained in these genes can translate into a spatially complex multicellular organism. The idea that such complexity is possible because cellular phenotype is coupled to cellular position goes back

G. R. van den Brink: Laboratory for Experimental Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands. M. P. Peppelenbosch: Department of Cell Biology, University of Groningen, Groningen, The Netherlands. D. J. Roberts: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

271

272 / CHAPTER 9 at least a century (1). Much has been learned about the proteins involved in the specification of cellular position from developmental biologists who have used models such as the regulation of segmentation of the Drosophila larval epidermis (2), development of the vertebrate limb bud (3), and neurogenesis (4). Most of the evolutionary conserved signaling pathways that act to couple cellular position to cellular phenotype have been identified in genetic screens in Drosophila, most notably the systematic screens performed by Nusslein-Volhard and Wieschaus (5). The type of molecules that have emerged from these genetic screens can be classified in two broad categories: intrinsic and extrinsic factors. Intrinsic signals such as kinases, phosphatases, and transcription factors function within the cell, and thus act in a cell-autonomous manner. Extrinsic signals are soluble or transmembrane factors that act among cells and regulate cell-nonautonomous processes. Extrinsic information is critical for a complex tissue composed of multiple different cell types to organize appropriately. The idea that tissues might contain gradients of soluble factors that act in a concentration-dependent manner to control cell fate was already postulated at the beginning of the twentieth century (1). Such factors have been termed morphogens. Signaling molecules from the Hedgehog, Wnt, and transforming growth factor-β have been shown to act as morphogens in several models of development and are used in the regulation of cell fate throughout evolution and in most developing tissues. This chapter summarizes our current understanding of the Hedgehog pathway and its role in the development and postnatal maintenance of the GI tract.

THE HEDGEHOG PATHWAY Hedgehog Genes The Hedgehog gene was identified in the first systematic screen for patterning genes in Drosophila. In larvae of wildtype Drosophila, a band of bristles called denticles is present across the anterior half of each segment, whereas the posterior half is smooth (the so-called naked cuticle). In screening for mutations that affect the segmental pattern of the Drosophila larva, Nüsslein-Volhard and Wieschaus (5) identified a group of mutants that affected the patterning within the segments, but at the same time left the number of segments unaltered. In one of these segment polarity mutants, the posterior half of each segment failed to develop, resulting in a larva that was entirely covered by denticles (Fig. 9-1). This phenotype gave the larva the aspect of a Hedgehog (Hemiechinus), hence the name. After much effort, the nucleotide sequence of the Hedgehog gene was finally obtained (6), and the mammalian Hedgehog homologues were identified. Echelard and colleagues (7) identified three Hedgehog genes in the mouse. They were named Sonic Hedgehog (Shh), after a popular Sega computer game character, Desert Hedgehog (Dhh), after an Egyptian species of Hedgehog (Hemiechinus auritus), and Indian Hedgehog (Ihh), which is a Hedgehog species endemic in Pakistan (Hemiechinus micropus). Riddle and coworkers (8) found that Shh protein mediates the action of the zone of polarizing activity (ZPA) in the chick limb bud (Fig. 9-2). The ZPA is a region at the posterior margin of the chick limb

A

B FIG. 9-1. The hedgehog mutant. An artistic impression of the Drosophila Hedgehog mutant. Each segment of the wild-type Drosophila larva (A) has bristles or denticles at the anterior end and a smooth posterior end, the so-called naked cuticle. Hedgehog signaling specifies the posterior end of each segment; as a result, the mutant lacks the naked cuticles (B).

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 273 Anterior

S

O

Posterior

Cys 258

Gly 257

Shh ZPA

Hh-C

N

Hh-N

A N

O

A Hh-N

Hh-C

S

Shh soaked bead

Cholesterol

B S O

Shh Hh-N

B FIG. 9-2. Shh and the zone of polarizing activity (ZPA). (A) A small group of cells in the ZPA at the posterior end of the developing limb produces Shh and specifies digit identity along the anteroposterior axis. (B) Transplantation of an extra ZPA at the anterior end of the limb bud leads to a mirrorimage duplication of the digit pattern. The same can be achieved by transplantation of a bead coated in Shh protein.

bud that is responsible for normal anteroposterior patterning. Shh was shown to colocalize with this previously described region and to alter limb patterning in a way similar to ZPA grafting. At the same time, a Hedgehog homologue was identified in zebra fish (Danio rerio) and shown to be expressed in tissues with organizing activity (9). In 1995, a new Hedgehog homolog was found in zebra fish and was named TiggyWinkle Hedgehog, after a character in a children’s book (10). Most phyla of the eukaryotic kingdom have now been shown to contain Hedgehog homologues. The expression of vertebrate Hedgehog genes has been detected in a variety of tissues during development. Sonic hedgehog is expressed in the limb buds and midline tissues such as the gut, notochord, floor plate, and ventral forebrain. The Indian hedgehog gene is expressed in the gut, kidney, liver, and developing bones (11–13). The mouse Desert hedgehog gene is expressed in the developing gonad, endothelium, and Schwann cells (12). Amphioxus, the closest living relative of vertebrates, has a single Hedgehog gene (14). Amphioxus Hedgehog is expressed in the notochord, the floor plate, and the endoderm (14); this pattern of expression is maintained by vertebrate Sonic hedgehog, suggesting that Indian and Desert hedgehogs have been able to diverge in their expression patterns and their role in embryonic development.

The Unusual Posttranscriptional Fate of Hedgehog Proteins For the Hedgehog protein to act as a potent morphogen, it needs to be processed in different ways. After removal of the signal sequence, the approximately 45-kDa Hedgehog

Signaling peptide

N

Hh-C Inactive processed peptide

C FIG. 9-3. SHH protein processing. (A) In the first step of SHH precursor protein processing the 257Gly-258Cys peptide bond is converted to an internal thioester via an N to S shift. (B) This processing intermediate is cleaved by cholesterol transferase activity of the C-terminal end of the precursor protein that mediates a nucleophilic attack by cholesterol on the internal thioester. (C) Processing results in a 19-kDa biologically active N-terminal fragment that is covalently attached to cholesterol and a biologically inactive C-terminal fragment.

proteins are cleaved autocatalytically, yielding an 19-kDa N-terminal fragment that contains all the signaling functions and a 26-kDa C-terminal fragment that has no other function than to catalyze the cleavage (Fig. 9-3) (15,16). The unusual posttranslational processing of Hedgehog proteins is not limited to this autocatalytic processing of the primary translated product. In the course of the cleavage, the SHH protein is covalently palmitoylated (17). This is atypical because palmitoylation usually is restricted to the cytoplasm, and thus affects only proteins without a signal peptide and not proteins entering the secretory vesicular pathway. Palmitoylated recombinant SHH has approximately 30-fold greater biological activity than unpalmitoylable mutants, suggesting that this acylation is important for enhancing biological activity. In accordance, in a Drosophila mutant in the acyl transferase mediating the Hedgehog palmitoylation (the so-called skinny hedgehog mutant [ski]), the morphogen is functionally inactive (18–20). The main function of the palmitoylation seems to be to increase the hydrophobicity of the molecule, which may serve to target the molecule to lipid rafts, which could, for example, facilitate efficient interaction with the Hedgehog receptor Patched (21,22). Even more atypical than the palmitoylation is the covalent attachment of a cholesterol moiety to the C-terminal part of Hh-N (23), making Hedgehog proteins the only established example of protein sterolation. A role for the cholesterol moiety in regulating diffusion or release of Hedgehog has been suggested (24–26): the sterol anchors hedgehog to

274 / CHAPTER 9 biological membranes, thus hampering diffusion. In limbs of embryos containing sterolation-deficient hedgehog, digits close to the ZPA (the source of hedgehog in limb bud) are normal, but the more distant digits are absent, suggesting that sterolation determines the spatial distribution of Hedgehog protein but is not required for biological activity.

The Receptor Complex Hedgehog signal transduction is highly unusual, containing many features unique to this signaling system (27). The Hedgehog binding receptor is a 12-transmembrane protein patched (Ptc). However, unlike a conventional receptor, Ptc does not convey the Hedgehog signal to the intracellular components of the pathway itself. Rather, binding of Hedgehog to Ptc alleviates the inhibitory effect of Ptc on another membrane receptor, the seven-transmembrane protein Smoothened (Smo). In the absence of Ptc, Smo will constitutively activate Hedgehog target gene transcription (28). The mechanism of Smo inhibition by Ptc is unknown, but it is presumably a catalytic effect, because Ptc and Smo do not need to bind or colocalize, and Ptc also inhibits Smo when present substoichiometrically (29). Some homology exists between Ptc and some prokaryotic transporter molecules, but also the Niemann–Pick C1 (NPC1) protein, a transport protein involved in cholesterol homeostasis (30). This homology gave rise to the conception that, in the absence of Hedgehog, Ptc might act as a pump that controls the containment of a yet unidentified compound that binds to and inhibits Smo (Fig. 9-4) (29).

PTCH

SMO

Signaling downstream of Smo is still only partially understood but involves a microtubule-bound complex of proteins that regulates the activity of the Hedgehog pathway. This complex consists of the proteins Costal2 (Cos2), Fused (Fu), Suppressor of Fused (Su(fu)), and a Hedgehog target gene– activating transcription factor. In Drosophila, the Hedgehog pathway acts through a single transcription factor, Cubitus interruptus (Ci). In vertebrates, the Hedgehog pathway regulates three Ci homologues that have been termed Gli-1 to Gli-3 (31–34). Because Hedgehog signal transduction is even more poorly understood in vertebrates than in Drosophila, this section focuses on Hedgehog signal transduction in Drosophila. Transcriptional regulation by the Ci protein has been hard to elucidate because of the presence of both an activating and a repressor form of the protein. In Drosophila, all Hedgehog-responsive cells contain a 75-kDa repressor form of Ci that is generated from the 155-kDa full-length protein by a proteolytic event that splices off the transcriptional activation domain and nuclear export signal (see Figs. 9-4 and 9-5). The resulting truncated protein retains a DNA-binding domain and an N-terminal transcriptional repression domain (Fig. 9-6) (35). Hedgehog signaling inhibits Ci proteolysis and results in nuclear accumulation of full-length Ci and activation of target genes. Ci proteolysis is regulated by Shaggy, an orthologue of the vertebrate glycogen synthase kinase-3β (GSK-3β) that can hyperphosphorylate Ci after a priming phosphorylation by protein kinase A (PKA) (36,37). This aspect of Hedgehog signaling is reminiscent of the mechanism of action of the Wnt pathway, where GSK-3β negatively regulates Wnt signaling through its control of the breakdown of the effector of the Wnt pathway, β-catenin (38). Interestingly, Smo is genetically closest related to the Wnt receptor Frizzled, and both receptors seem to work by inhibition of GSK-3β with resulting inhibition of the proteolytic breakdown of the effector protein of the pathway. Several studies have shown that the kinesin-related protein Cos2 provides a physical link between Smo and Ci processing. On activation of Smo, the stability of Smo is increased and Ci processing is inhibited potentially through

Hh-N

A

Signal Transduction and Target Genes

PTCH

SMO

B FIG. 9-4. The Hedgehog receptor complex. A model of patched in human (PTCH)-Smoothened (SMO) interaction. (A) In the absence of Hedgehog, PTCH functions as a transmembrane pump that pumps out an SMO inhibitory compound that is yet to be identified. (B) Binding of Hedgehog to PTCH shuts down its pump function; as a result, the inhibition of SMO is released.

FIG. 9-5. The Cubitus interruptus (Ci) protein. Ci is a 155-kDa protein that is proteolytically processed in the absence of Smoothened (SMO) signaling. Splicing generates a 75-kDa N-terminal protein that contains that DNA-binding zinc finger (ZnF) and the nuclear localization signal (NLS); because this protein lacks the transactivating C-terminal end of the protein, it acts as a repressor of gene transcription. Activity of the fulllength protein (that also contains a nuclear export sequence [NES]) can be inhibited by binding of Suppressor of Fused (Su(fu)) to the N-terminal Su(fu) binding site.

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 275

SMO

Su(fu) Fu Cos2 Ci Proteasome P

Ci-R

P

PKA, GSK3β, CK1

FIG. 9-6. Cubitus interruptus (Ci) processing. A schematic representation of the factors that influence Ci processing. Ci processing occurs after phosphorylation by protein kinase A (PKA), glycogen synthase kinase-3β (GSK-3β), and casein kinase 1 (CK1), and it requires the activity of the proteasome. Inhibition of Ci processing via activation of Smoothened (SMO) requires functional Cos2 and Fu kinase activity. The full-length Ci protein can be inhibited by Su(fu).

release of Ci from the Cos2 complex (39–42). Cos2 forms a close association with Fu, a kinase that phosphorylates Cos2 and is required for Hedgehog signaling, potentially influencing the interaction of the Ci/Cos2/Fu complex and microtubules (31,43). Su(fu) was identified in genetic screens in Drosophila as a suppressor of mutants in Fu (44). Su(fu) is a negative regulator of Hedgehog signaling and acts either by sequestration of Ci in the cytoplasm or by direct interaction with Gli proteins on DNA (43). After the release of transcriptional repression, Hedgehog target genes are transcribed. Hedgehog target genes include Gli-1, HNF3β, Hox genes, bone morphogenetic proteins 2 and 4 (BMP2 and BMP4), and Patched- and Hedgehoginteracting protein (HIP, an extracellular hedgehog antagonist). Of these genes, the up-regulation of Patched and HIP seem to be involved in a negative feedback response to Hedgehog signaling, whereas transcription factors such as HNF3β and Hox genes are implicated in translating the extracellular morphogenetic signal to intracellular phenotypic interpretation. Studies that used microarray techniques have now identified a large number of possible Hedgehog target genes (45–48). Hedgehog target genes found in microarrays include molecules involved in angiogenesis, cell cycle, cell adhesion, signal transduction, apoptosis, and nerve formation; transcriptional regulators; Wnt signaling antagonists; protease inhibitors; and a metal-binding protein. The actual in vivo relevance of these genes remains largely unclear and awaits functional studies for further validation.

Cells from the endoderm will form the epithelial layer; mesodermal cells differentiate into mesenchymal cell types such as myocytes and fibroblasts, whereas neural cells originate from the ectoderm. The gut tube acquires regional specialization along the longitudinal axis of the developing embryo. The fate of cells that form this tube is restricted to become part of one of the roughly four specialized tissue units of the esophagus, stomach, small intestine, and colon. Other small groups of cells bud from the original tube to form the thymus, lungs, liver, and pancreas. A second axis of specialization along which the different issue layers of muscle, submucosa, and epithelium develop is the crosssectional or vertical axis. The constant renewal of epithelial cells in the adult occurs along this vertical axis. Hedgehog signaling plays a role in the specification of cellular fate along both the longitudinal and the vertical axes, and its regulation is involved in budding of gut tube–derived organs. The endodermal cell layer expresses hedgehog family members SHH and IHH. This section discusses our current understanding of the role of Hedgehog signaling in GI development in three subsections that cover the three segments of the gut and its derivatives: foregut (esophagus, trachea, lungs, thymus, stomach, liver, and pancreas), midgut (small intestine), and hindgut (colon and anus).

Hedgehog Signaling and Foregut Development Esophagus and Trachea

ROLE OF HEDGEHOG SIGNALING IN THE DEVELOPMENT OF THE GUT Development of the Gastrointestinal Tube The GI tract develops from a simple tubular structure composed of cells derived from all three germ layers.

After formation of the foregut primordium, the trachea buds from the ventral wall of the foregut (49,50). In the mouse foregut, Shh mRNA expression is initiated at 9.5 days postcoitum (dpc) in the endoderm of the foregut primordium and will extend along the ventral wall of the developing foregut toward the midgut (7,51). Expression of Shh in the murine esophagus has been described until at least 15.5 dpc (12,51).

276 / CHAPTER 9 At some later point in development, expression of Shh is down-regulated in the esophagus, because no Shh expression is found at 18.5 dpc (52) or in the adult esophagus (53). In the elongating trachea, Shh expression initially is lost at 11.5 dpc, but then it reappears around 15.5 dpc (51). In contrast, Shh is not expressed by the cells of the thyroid, which form as a primordium at 9 dpc, is completely dissociated from the gut tube by 11.5 dpc, and descends to its final position until 15 dpc (54). Ihh is not expressed at any point in foregut development; this is confirmed by that Shh and Gli mutant mice show similar foregut phenotypes (see later). The role of Shh signaling in foregut development is revealed in Hedgehog pathway mutant mice. In Shh−/− mice, the formation of the tracheal bud is delayed and the lungs become hypoplastic. The normal elongation of the trachea fits well with that no Shh expression is found during this phase. The hypoplastic lungs suggest a role for Shh signaling in branching morphogenesis. The esophagus fails to develop normally; only a small esophageal remnant is found in Shh−/− mice at later stages of development. Because Shh has been shown to signal mainly to the mesenchyme during development and seems to act as a growth factor for mesenchymal cells (55), it is possible that the esophagus degenerates because of a lack of mesenchymal cell growth to support its epithelium. The stomach seems to connect to the respiratory tract in Shh null mice (51). The importance of Hedgehog signaling in foregut development was confirmed in Gli mutant mice (56). Although Gli3−/− mice have a normal trachea and esophagus, in the Gli2−/− mutant mouse, the esophagus has a small lumen, has little mesenchymal cells, and fails to develop a small muscle layer. The lungs in the Gli2−/− mutant mouse are hypoplastic and have only one right lobe instead of the four lobes that are normally found. Although the trachea elongates normally in the Gli2 mutant mouse, it does appear hypoplastic at 18.5 dpc, suggesting that the induction of Shh expression in the trachea at 15.5 dpc (51) is involved in its growth along the vertical axis. The Gli2−/−Gli3−/− mouse has a phenotype that is more severe than the Gli2−/− mouse and seems identical to the Shh null mutant. Therefore, although Gli3 is not necessary for normal foregut development, it can rescue part of the phenotype in Gli2−/− mice. Interestingly, the Gli2−/−Gli3−/− phenotype is more severe than the Shh null phenotype, demonstrating no development of lung, trachea, or esophagus at all. This suggests that Gli signaling in foregut development is not entirely Shh dependent (56). This discrepancy between the Shh and Gli2−/−Gli3−/− null phenotype can be explained by the finding that Fgf signaling may be capable of activating Gli transcription factors (57). Some effectors of Hedgehog signaling have been found by their similarity of their null phenotype to the Hedgehog pathway mutant phenotypes. The forkhead transcription factor Foxf1 seems to play a role as a mesenchymal effector downstream of Gli signaling. Foxf1 is expressed in the splanchnic mesoderm. Foxf1+/− mutant mice display a phenotype comparable to Shh mutant mice with esophageal atresia

and tracheoesophageal fistula. It was further shown that ectopic Shh expression activates Foxf1 expression whereas Foxf1 expression is lost in Shh−/− mice (58). Together these data imply that Foxf1 is a crucial mesodermal mediator of the endodermal Hedgehog signal in the normal tracheoesophageal bifurcation and elongation (59). The homeodomain transcription factor Nkx2.1 (thyroidspecific enhancer-binding protein [TTF1] or thyroid transcription factor-1 [T/EBP]) also plays a role in tracheoesophageal bifurcation. In the mouse, Nkx2.1 is expressed in the endoderm of the tracheal diverticulum and endoderm of the thyroid, trachea, and lung at later stages of development, whereas no Nkx2.1 is detected in the esophagus or the rest of the GI tract (59). In this mouse, however, the Nkx2.1−/− tracheoesophageal pathology seems different from that observed in the mice defective in Hedgehog signaling. The Nkx2.1−/− mouse has a single tube that connects the pharynx to both the lungs and the stomach. The pathology in this mouse seems to arise from a failure of the tracheal diverticulum to elongate (see Fig. 1B of Minoo and colleagues [59]). This is consistent with the selective expression of Nkx2.1 in the trachea and lung, but not with the formation of a fistula between airway structures and the esophagus or stomach as in the Shh−/−, Gli2−/−Gli3+/−, and Foxf1−/− mice. It is therefore unclear whether this mouse is truly a model of tracheoesophageal fistula as Minoo and colleagues suggested (59). In conclusion, Hedgehog signaling seems to play an essential role in the normal separation of trachea and esophagus, the maintenance and growth of the esophagus, and the growth of the lungs. It seems to play this role through regulation of mesenchymal growth through induction of Foxf1. Thyroid Although the thyroid primordium is correctly specified at 9 dpc in the absence of Hedgehog signaling (56), careful examination of the thyroid phenotype in the Shh null mouse does reveal a role for Shh signaling at later stages of thyroid development (54). The thyroid primordium buds in the foregut endoderm and becomes completely separated at 11.5 dpc. It descends between 10 and 15 dpc to its final position after which it separates into two lobes and organizes into a follicular structure, then genes involved in thyroid hormone production are induced (60). In Shh−/− mice, the thyroid primordium forms, but separation from the foregut is delayed, because it remains connected through a narrow stalk initially. The thyroid does not separate into two distinct lobes but becomes a single unilateral mass, the size of a single thyroid lobe in control mice (54). The single thyroid gland of Shh null mice formed normal follicles that contained apparently normally differentiating thyrocytes. The observed thyroid phenotype may be indirect and secondary to Hedgehog signaling in an adjacent tissue because no Shh or Ptc expression was detected in the thyroid at any point of development. Interestingly, small foci of

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 277 ectopic thyroid tissue were found in the tracheal tube at 15.5 to 17.5 dpc in the Shh−/− mouse (54), suggesting that the induction of Shh in the trachea not only plays a role in tracheal growth along the vertical axis (see later) but also acts to suppress thyroid cell fate in the trachea. Stomach During development of the stomach, the gastric epithelium forms tubular structures that invaginate into the underlying mesoderm. These tubules contain two cell populations. The superficial or pit cells are similar throughout the stomach and produce soluble mucins that contain a protein backbone encoded by the MUC5AC gene. The cells that form the bottom of the tubules form the glandular region and have both exocrine and endocrine functions. In the adult, both cell populations originate from a common stem cell compartment between the pit and the gland region from where daughter cells migrate in two directions. The gastric glands of the proximal (fundus) and distal (antrum) stomach are functionally distinct. Both Shh and Ihh are expressed in the developing stomach in mice (12). At 12.5 dpc, Shh is expressed in the proximal but not the distal stomach (61). High expression of both Shh and Ihh is found in glandular epithelium of the stomach at 18.5 dpc (52). The epithelium of Shh−/− mice (but not that of the Ihh mutant) is hyperplastic, and patches of gastric epithelium are positive for intestinal brush border markers alkaline phosphatase and wisteria floribunda agglutinin (WFA) lectin (52). These data suggest that Shh signaling is necessary to induce a gastric phenotype in endodermal cells in the stomach. The chick has a proximal glandular stomach (proventriculus) and adjacent muscular stomach (gizzard). In the chick, proventriculus Shh is initially expressed uniformly throughout the endoderm (62). During proventricular unit formation, the gastric units start to form a superficial pit region and a glandular region. No Shh is expressed in the glandular region at this point. When Shh is ectopically expressed in developing gastric units in a proventricular explant model, gastric units fail to develop (62). These data may suggest that gastric unit formation has some similarities with the development of endodermal appendages such as the thymus and pancreas. It is unclear, however, if this downregulation of Shh during gastric unit formation also occurs in the mouse or human. Both Shh and Ihh are uniformly expressed throughout the developing gastric units in mice at 18.5 dpc (52). However, maturation of gastric glands in mice occurs after birth, and restriction of Hedgehog signaling may therefore occur at a later point in gastric unit maturation. In the fully developed stomach, Shh is expressed exclusively in gastric glands in both mice and humans (see later) (53). In other experiments using a chick stomach explant model, it was shown that treatment inhibition of Hedgehog signaling with cyclopamine disturbed endodermal differentiation in the gizzard (63). In the absence of hedgehog signaling, the differentiating epithelium failed to stratify and contained

nonpolarized cells. The mesenchyme of cyclopamine-treated animals contained multiple vascular structures (something that was also observed in the thyroid of Shh−/− mice [54]). The endoderm of both in vitro and cyclopamine-treated chick stomachs expressed both glucagon and insulin, suggesting that the epithelium differentiates toward a pancreatic cell fate. Chick embryos treated with cyclopamine in ovo also displayed pancreatic differentiation of the gizzard endoderm (63). These data suggest that Shh is necessary to suppress a pancreatic cell fate in chick gizzard endoderm. In conclusion, Shh seems to act to suppress more distal cell fates in gastric endoderm (small intestinal in the mouse and pancreatic in the chick), and data in the chick suggest that a temporary loss of Shh expression in the developing gastric unit may play a role in budding of units from the endodermal layer. Pancreas The pancreas is an endodermal-derived gland with both endocrine and exocrine functions. The bulk of pancreatic tissue (approximately 95–99%) is composed of exocrine acinar cells that produce digestive enzymes and pancreatic ducts that are lined by duct cells. The rest of the pancreas consists of small clusters of endocrine cells or Islets of Langerhans. Each cluster of endocrine cells has a core of insulin-producing β cells surrounded by glucagon-producing a cells, somatostatin-producing D cells, and pancreaticpolypeptide cells. The development of the pancreas is probably the best studied aspect of endodermal differentiation, and many of the intrinsic and extrinsic determinants have been described (64,65). The pancreas develops from two primordia that originate at the ventral and dorsal side of the gut tube. In the mouse, the pancreatic anlage is specified around 8.5 dpc. Pancreatic morphogenesis initiates at 9.5 dpc with the formation of the dorsal pancreatic bud. At this time, the first endocrine cells are also detectable (64). At 10.5 dpc, Shh is expressed uniformly throughout the endoderm of the gut tube with the exception of pancreatic precursor cells that are distinguished by their Pdx-1 positivity (66). Although no Shh is expressed in the developing or adult pancreas, low levels of Ihh and Ptc mRNA are found with reverse transcriptase-polymerase chain reaction in the murine pancreas throughout development and in the adult (61). When a Pdx-1 promoter was used to drive ectopic Shh expression in the pancreatic buds, development of the pancreatic mesoderm was strongly disturbed. The mesenchyme contained α-actin–positive smooth muscle cells and c-kit–positive cells of Cajal. Both cell types are normally found in intestinal mesenchyme. The pancreatic buds in transgenic animals did contain endocrine and acinar cells, but these failed to cluster into islets and acinar structures and produced mucins that are typical for intestinal epithelial cells (66). These data suggest that during early pancreatic development, suppression of Shh signaling is especially important for normal development of the pancreatic mesoderm,

278 / CHAPTER 9 and that although the pancreas contains both endocrine and acinar cells, their differentiation is not entirely normal. Data from Xenopus further support this concept (67). Xenopus injected with a constitutive active form of the Hh receptor Smo completely lack a pancreas, a phenotype more severe than that of the Pdx-1-Shh mouse. This may be because their transgenic mouse expressed Shh behind a pancreas-specific promoter and the ectopic Shh transcription therefore starts after the initial specification of a pancreatic domain has already been completed. It has been suggested that Hedgehog signaling may act to restrict pancreas growth (61). Shh−/− mice have a body weight of approximately 30% of wild-type embryos at 18.5 dpc, but their pancreas weight is similar to that in wild-type mice. The Ihh−/− mouse has a body weight of approximately 65% of wild-type mice at this stage of development and its pancreas is similarly 65% of the weight of the wild-type pancreas. It is difficult to infer from these observations if, for example, Shh affects body size but not pancreas size or if Shh restricts pancreas growth as suggested (61). Almost half of Ihh−/− mice develop an annular pancreas in which the pancreas encircles the duodenum. Histologically, the pancreas appears intact and contains normal islets. Cellular differentiation seems to be more or less normal in both Shh−/− and Ihh−/− mice. Only if the number of endocrine cells is expressed relative to body weight would there appear to be a relative increase in the number of endocrine cells in the pancreas, especially in the Shh−/− mice (61). The number of endocrine cells/pancreas weight seems a more logical measure, however, and this seems only modestly increased in Shh−/− mice (22% increase), whereas the number of endocrine cells/pancreas weight is increased by 75% in Ihh−/− mice (61). Because Ihh and not Shh is expressed in the developing pancreas, it seems that Ihh may act as a negative regulator of endocrine cell fate in the developing pancreas.

Hedgehog Signaling and Development of the Midgut The midgut will form most of the intestine distal from the common bile duct that derives its blood supply from the superior mesenteric artery: the distal duodenum, the jejunum, ileum, cecum, and the ascending and proximal transverse colon. The intestinal endoderm initially is pseudostratified and is converted into an epithelial monolayer in a proximalto-distal morphologic wave that starts at 15 dpc. In contrast with human development, morphogenesis of the murine intestinal epithelium is not complete until 3 weeks after birth when crypts have developed from intervillous epithelial cells (68). Ihh and Shh are both expressed in the developing midgut, and therefore have partially overlapping functions. They regulate endodermal–mesodermal interactions in the developing small intestine because both are produced by endodermal cells and the Hedgehog target gene and receptor Ptc is strongly expressed in the surrounding mesoderm (52,69). Experiments in the chick gut have shown that during early gut development, Hedgehog signaling acts to recruit mesoderm.

This induction of mesodermal proliferation may be limited by that Shh induces BMP4 expression in the mesoderm (70,71). Ihh is expressed throughout the midgut at 11.5 dpc (12). Ihh expression is maintained along the length of the midgut until at least at 18.5 dpc when Ihh is expressed at the base of the villi in the small intestine (52). Initially, Shh is expressed uniformly throughout the endoderm; however, similar to the expression of Ihh, this expression is restricted to the base of the villi of the proximal midgut around 14.5 dpc (12,52). What underlies the down-regulation of Shh in the intestine at 14.5 dpc remains to be determined. It seems to follow a restrictive influence exerted by the mesoderm around 13.5 dpc with down-regulation of Foxa2 (HNF3β) and GATA factors in the endodermal layer (72). Interestingly, the intestinal endoderm is converted to a monolayer at 15 dpc, and this event seems to follow these molecular changes that are orchestrated by the underlying mesenchyme. Indeed, experiments in Xenopus with constitutively active Smo (SmoM2) suggest that down-regulation of Shh expression may not only be essential for the appropriate differentiation of pancreatic mesenchyme, but also important in the midgut. After injection of SmoM2 mRNA into one to eight cell-stage Xenopus embryos, intestinal epithelial differentiation was completely disrupted specifically in the midgut (67). The interpretation of these experiments is difficult, however, because they involved injection of SmoM2 mRNA that is not regulated by a cell type–specific promoter; this will also induce Hedgehog signaling in the endoderm where it does not normally occur in intestinal development. Shh and Ihh mutant mice displayed different phenotypes. Whereas the duodenum of Shh−/− mice was overgrown with villi, villus size was reduced in the Ihh−/− mouse and intestinal precursor cell proliferation also was reduced (52). Both Ihh and Shh null mice show a reduction in the thickness of the smooth muscle layer, indicating that Hedgehog signaling stimulates smooth muscle cell differentiation or proliferation (52).

Hedgehog Signaling and Development of the Hindgut The hindgut forms the distal transverse colon, descending colon, sigmoid, and anorectum that develop around the inferior mesentery artery. Low levels of Shh are expressed at the base of the crypts in the colon at 18.5 dpc (52), whereas Ihh mRNA is expressed throughout the epithelial layer from 11.5 dpc until birth (12,52). In Ihh−/− mice, the epithelial cells fail to organize into a polarized monolayer and the epithelium does not organize into cryptlike structures (73). The colon is partially dilated in Ihh−/− mice with an abnormally thin wall and lack of neurons in the dilated areas, a phenotype reminiscent of Hirschsprung’s disease (52). These data suggest that signaling by Ihh plays a role in the conversion of the pseudostratified endodermal layer to an epithelial monolayer in the hindgut and regulates the survival of neural crest cells that have migrated into the intestinal mesoderm.

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 279 Signaling by Shh is important in the distal hindgut because Shh null mice were shown to have anorectal malformations (52,74). The anorectum develops with a complex interplay of all three germ layers as it forms at the junction of the endoderm and ectoderm (71,75). Ramalho-Santos and colleagues (52) found an imperforate anus in their Shh−/− mice: the colon ends in a blind sack and does not form a connection with the perineum. Shh−/− mice maintained by Mo and colleagues (74) display a failure to separate the anorectum from the lower urinary tract, which drain in a common cavity or outlet. Gli2−/−, Gli3+/−, and Gli2+/− Gli3−/− compound mutant mice also have a persistent cloaca and a common outlet for the digestive and urogenital tract but a phenotype that is less severe than Shh−/− mice. Gli2−/− mice have an imperforate anus with a rectovesical fistula (between the distal intestine and the bladder) and a single urethral opening in the perineum (see also the study by Kim and coworkers [76]). The phenotype of Gli3−/− mice is mild with slight anal stenosis (74). These phenotypes indicate that, although Gli3 does play a role in the transduction of the Shh signal to the anorectal mesenchyme, the contribution of Gli2 is much more important and is similar to the role of Gli signaling in foregut development.

Hedgehog Signaling and the VACTERL Association in Humans All of the developmental abnormalities observed in the Hedgehog pathway mutant mice also are found in humans. In humans, the nonrandom association of these abnormalities is denoted by the acronym VACTERL. VACTERL stands for vertebral defects (V), anal atresia (A), cardiac defects (C), tracheoesophageal fistula, renal anomalies (R), and limb defects (L) (77–80). This association is not recognized as a specific syndrome, and its different components are found together in varying constellations (see Rittler and colleagues [80]) and have been assigned various acronyms (such as VATER, VATERL, among others). One study that examined 2,493,999 births reported only a single case with combined recognized deformities in all 6 organs (80). However, all of these defects have been found by many observers in one or more of the several known human and murine mutants of the Hedgehog pathway (51,52,56,64,74,81–83; see also the review by Kim and colleagues [76]). The atresia or stenosis of the esophagus with associated tracheoesophageal fistula that was found in hedgehog mutant mice is observed in approximately 1 in 2000 to 1 in 5000 live human births (77). Just like in the Hedgehog mutants, this anomaly can be found in association with other GI malformations in humans such as anal atresia, midgut malrotation, duodenal atresia, and annular pancreas (84), and with malformations of other organs as part of the VACTERL association mentioned above. Although the similarity between malformations found in the VACTERL association and Hedgehog mutant mice suggests a role for defective Hedgehog signaling, in patients

with the VACTERL association, such a role has not yet been established.

HEDGEHOG SIGNALING IN HOMEOSTASIS OF THE ADULT GASTROINTESTINAL TRACT Morphostasis The molecules that are involved in the regulation of position-dependent cell fate have been found in genetic screens that used developmental models. However, positional information is something that is equally important to many cell types in the adult organism. Although adult tissues seem to be static and stable, many tissues are not, because they have a high cellular turnover rate and their microarchitecture can be disturbed, for example, in chronic inflammation. The GI tract is a good example of the importance of positional information in cellular renewal in the adult. At each level of the gut a common precursor cell produces a module of specialized cell types appropriate for its position along the anterior-posterior axis. These modules display elaborate patterns of differentiation along the radial axis. At some parts of the GI tract precursor cells generate two functionally different compartments of cells that migrate in opposite directions (e.g., the stomach). Even among cells that migrate in the same direction, opposite gradients of maturation can be observed for different cell types. Although stability of the resulting complex epithelial microarchitecture is the norm, instability often is observed in humans. This instability leads, for example, to the formation of metaplasia, atrophy, or hyperplasia such as in polyposis. Often, these unstable epithelial states are at a high risk to progress to GI cancer. GI epithelial cells undergo a well-controlled maturation process. Precursor cells generate descendants that withdraw from the cell cycle and induce transcription of cell lineage– specific proteins. When the cell is fully differentiated it is shed into the gut lumen or undergoes apoptosis to maintain cellular census. This is a rapid process that is dependent on the cell’s position along the axis of renewal. It was shown in an important experiment by Hermiston and colleagues (85) that such processes are regulated in a cell nonautonomous manner, and thus are controlled by extrinsic regulators of cell fate. The authors performed an elegant experiment in chimeric mice in which some of the intestinal epithelial cells overexpressed the cell–cell adhesion protein E-cadherin (85). In these mice, the descendants of the transgenic precursor cells migrated considerably slower along the axis of renewal than the progeny of normal precursor cells present in the same chimeric animal. Despite this difference in the speed of migration, differentiation and cell death of the epithelial cells occurred at the appropriate moment along the vertical axis and homeostasis was maintained. This experiment demonstrated that differentiation of the epithelial cell is completely dependent on its position along the vertical axis of renewal, and therefore is extrinsically regulated.

280 / CHAPTER 9

Villus

Pit Precursor cell

Crypt

Shh

Parietal cell Gland

Precursor cell

Shh

FIG. 9-7. SHH signaling in the adult stomach and small intestine. (A) SHH is expressed in the fundic glands of the stomach where it may act as a polarizing factor. Gastric epithelial cells migrate in two directions from the precursor cell compartment. Cells that migrate up form the gastric pit, whereas those cells that migrate down form the gastric gland. In humans, Shh is produced by parietal cells in the gastric gland. Gland but not pit cells express PTCH, suggesting that Shh signaling acts on gland cells only. (B) SHH is produced by a few cells at the base of the small intestinal crypt and may be involved in the regulation of precursor cell proliferation and triglyceride absorption.

Evidence also exists that the proliferative compartment of precursor cells receives negative feedback information from cells in the differentiated compartment (86). Through such negative regulation the amount of differentiated cells can regulate the pace of their own replacement. If differentiated cells in the superficial epithelium are lost by damage, such a mechanism allows for increased proliferation to guard epithelial integrity. The mediator(s) of this negative feedback loop have not been identified to date but are clearly soluble molecules capable of long-range signaling. The identification of the extrinsic controls of cell fate is fundamental to the understanding of the regulation epithelial homeostasis and its deregulation. Work from several laboratories has shown that morphogens seem to play an important role as extrinsic regulators of cell fate in the adult, and this section reviews what is known about the role of Hedgehog signaling in the adult GI tract.

zymogenic cells in both mice and rats (53,87). IHH is expressed in the colonic epithelium where it is produced by terminally differentiated enterocytes (73). During development of the GI tract Hedgehog signaling occurs mostly from the endodermal layer to the mesodermal layer, with the exception of the stomach where Hedgehog receptor and target gene Ptc is also expressed in the differentiating epithelium at later stages of development in the mouse (12,52,69). In contrast, in the adult epithelium of both humans and rodents, Hedgehog signaling seems to be mostly autocrine within the epithelium because the greatest levels of PTCH/Ptc are found in the epithelium (73,87). Intercrypt table

Ihh

Role of Ihh and Shh in Adult Gut Crypt

The expression patterns of IHH, SHH, and Hedgehog receptor patched (PTCH) in humans suggest that Hedgehog signaling occurs throughout the human adult GI tract, with the exception of the squamous epithelium of the esophagus (53,87,88). Both SHH and IHH are expressed in the adult GI tract. The expression pattern of SHH has been examined most carefully; SHH mRNA is expressed at high levels in the glands of the fundic stomach. Low levels of SHH mRNA are found in epithelial cells at the base of the crypts of the small intestine and colon (Figs. 9-7 and 9-8). SHH protein can be detected in the fundic glands where it is expressed by parietal cells in humans and by mucous neck cells and

Precursor cell

Shh

FIG. 9-8. Hedgehog signaling and the colonic crypt. Similar to the crypts of the small intestine, low levels of Shh are expressed around the precursor cells at the base of the colonic crypt. High levels of Ihh, however, are produced by differentiated colonic enterocytes at the luminal end of the crypt and at the intercrypt tables. Hedgehog receptor and target gene PTCH is also expressed by the differentiated enterocytes, indicating that these cells are the major target for hedgehog signaling in the colon.

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 281 Shh and Gastric Epithelial Homeostasis Renewal of gastric epithelial cells is a bidirectional process. The gastric mucosa is a flat surface that contains multiple invaginations or gastric units. Gastric precursor cells are located somewhere approximately halfway up these units. Cells that migrate toward the luminal surface secrete the protective soluble mucin MUC5AC and form the gastric pit (the upper part of the unit) (89). Cells that migrate down form the gastric gland and produce a variety of exocrine and endocrine factors involved in defense against bacterial invasion, digestion of food, and regulation of metabolism. In this homeostatic system, factors have to control asymmetric cellular differentiation where the appropriate amount of cells adopts either a pit or gland cell fate. The presence of such factors that act as polarizing signals in the gastric units is clear from histopathologic observations in patients with hypertrophic gastropathies. These gastropathies include Ménétrier disease and hyperplastic hypersecretory gastropathy (HHG) (90). In Ménétrier disease, transforming growth factor-α, a ligand for the epidermal growth factor receptor, is overexpressed, leading to excess proliferation and differentiation of pit cells and loss of glandular epithelial cells (91–93). In contrast, the hypertrophy in patients with HHG is characterized by an increased mass of gland cells. We have shown that Shh expression is restricted to one side of the gastric precursor cell region. In humans, Shh expression forms a gradient in gastric glands as it is highest in parietal cells close to the precursor cell compartment and gradually diminishes to low levels as parietal cells migrate to the bottom of the fundic gland (87). We have therefore proposed that SHH may act as a polarizing factor in the adult gastric unit, in analogy with its role as a polarizing factor in the developing limb bud. We have, indeed, been able to show that Hedgehog signaling controls proliferating cell nuclear antigen (PCNA) expression in gland cells and not pit cells, and it regulates the expression of gland cell–specific transcription factors (87). The gastric epithelium of Shh null mice shows intestinal transformation (52), and loss of gastric gland cell differentiation in patients with intestinal metaplasia of the fundic epithelium is characterized by loss of SHH expression, indicating that SHH seems to be essential in the induction and maintenance of a fundic gland cell phenotype (53,94). In vitro experiments in a gastric cancer cell line that expresses SHH suggest that SHH expression may be pH dependent and is stimulated by low pH (94). Interestingly, treatment of newborn rabbits with retinol increases the number of parietal cells (95). Retinoids have been shown to act through their control of Shh expression in the developing limb bud (96), thus the control of parietal cell differentiation by retinoids may well be Shh dependent. A role for Shh as a fundic gland cell type–promoting factor is further supported by mice deficient for α-Inhibin (97). α-Inhibin−/− mice acquire gonadal sex-cord-stromal tumors producing large quantities of Activin A and B. The fundic glands of these mice show increased amounts of pit cells, loss of parietal cells, and absence of zymogenic cells. It has been

shown in developmental models that Activin acts to inhibit Shh signaling (98), and that loss of activin signaling in the foregut leads to expansion of both Shh expression and gastric epithelial differentiation (99). Together, these data suggest that SHH may act as a central regulator of gastric gland cell fate that is stimulated by retinoids and inhibited by Activins.

Shh and the Small Intestinal Crypt In the adult small intestine, SHH is expressed by a few cells at the base of the small intestinal crypt (53). Levels of Shh protein diminish in a gradient from the duodenum to the ileum in 2-week-old mice (100). As judged by the expression of Ptc, Hedgehog signaling targets both epithelial cells at the base of the small intestinal crypt and mesenchymal cells (88). A small reduction of precursor cell proliferation was observed in small intestinal crypts of cyclopaminetreated adult mice, suggesting that Shh stimulates precursor cell proliferation (87). Wang and colleagues (100) observed increased proliferation of small intestinal precursor cells in anti-Hh–treated postnatal mice. In their experiment, mice were treated from postnatal day 1 to 15, when morphogenesis of the small intestinal crypts occurs, polyclonal crypts are “purified” to monoclonality, and crypts multiply by a high rate of crypt fission (68). Hedgehog signaling thus seems to play different role during crypt morphogenesis and in the fully mature intestinal crypt. These authors also treated adult mice with an anti-Hh antibody and observed disturbed triglyceride absorption in Hedgehog-inhibited mice (101). In conclusion, the limited data available about the role of Shh signaling in the small intestinal crypt suggest a role in precursor cell proliferation and the regulation of triglyceride absorption.

Ihh and Colonic Epithelial Differentiation Epithelial renewal in the colon is a unidirectional process from a precursor cell compartment at the base of the crypts toward the lumen where cells reach the flat surface (intercrypt tables) and undergo a death program. The colonic crypt contains three different functional cell types. A gradient of differentiating absorptive enterocytes is found from the crypt base toward the lumen of the gut, whereas most mucusproducing goblet cells are found in the middle of the crypt and the endocrine cells form a gradient toward the base of the crypt (102). Both IHH mRNA and protein are expressed by terminally differentiated enterocytes in the adult colon (73). Low levels of SHH mRNA can be detected at the base of colonic crypts, and SHH protein is difficult to detect but is probably expressed by a few cells at the base of the colonic crypt (53,88). Most Hedgehog signaling, however, occurs in the differentiated enterocytes as they express the highest level of PTCH protein and mRNA (73,88). Oniscu and colleagues (103) found SHH

282 / CHAPTER 9 expression in differentiated enterocytes using immunohistochemistry; however, because these cells do not express SHH mRNA but do express IHH mRNA, these results are most likely explained by cross-reactivity of the SHH antibody with IHH protein. The role of Hedgehog signaling in colonic enterocytes was addressed by treating rats with the Hedgehog inhibitor cyclopamine, a veratrum alkaloid that binds and inhibits Smo (104–106). Histologic examination of the colonic epithelium of these rats showed a remarkable change in the appearance of the colonic enterocytes, with altered distribution of the cytoskeletal protein Villin that is specific for microvilli and loss of expression of the brush border enzyme carbonic anhydrase IV. Interestingly, the enterocytes gained the expression of intestinal trefoil factor, a marker of the goblet cell lineage. In vitro experiments in the HT-29 cell differentiation model showed that butyrate-dependent HT-29 cell differentiation is dependent on induction of IHH expression. These data suggest that Hedgehog signaling is essential to the appropriate specification of the enterocyte cell lineage and acts to suppress a goblet cell phenotype in these cells (73). A possible mode of action for Ihh was suggested by that the expression of Wnt target genes Engrailed-1 and BMP4 was increased in the colon of cyclopamine-treated rats, and that the expression of both proteins that is normally restricted to the base of the colonic crypt extended up toward the differentiated enterocytes. These results suggested that IHH might act to suppress WNT signaling as a differentiating enterocyte moves from the precursor cell compartment in the crypt toward the differentiated compartment at the luminal end of the axis of renewal. Additional evidence for a role for IHH as a suppressor of WNT signaling was obtained in DLD1 cells in vitro (73). The importance of inhibition of WNT signaling in enterocyte differentiation lies in that WNT signaling seems to act to specify a precursor cell phenotype in colonic enterocytes (107). We found that expression of IHH is in its turn suppressed by WNT signaling, and because WNT signaling is aberrantly activated in most colon cancer cells, these cells have lost their expression of IHH, and therefore have lost their autocrine Hedgehog signaling. These data correlate well with findings by Berman and colleagues (108), who showed that growth of most carcinoma cells derived from the esophageal, gastric, biliary tract, and pancreas was dependent on Hedgehog signaling, but that this was not the case for colon carcinoma cells. Colon carcinoma cells did not display any endogenous activity of the Hedgehog pathway, nor was their viability affected by cyclopamine, an observation that correlates well with our own unpublished observations. Qualtrough and colleagues (109) have found that cyclopamine treatment resulted in detachment of cultured colon cancer cells; this detachment was not observed in our own studies or those of Berman and colleagues (108), and further experiments are necessary to show if this is caused by methodologic differences. Oniscu and coworkers (103) found that recombinant Hedgehog protein stimulates the proliferation of isolated primary murine colonic enterocytes. Because differentiating enterocytes do not proliferate, this

study examined the effect of Hedgehog signaling on intestinal precursor cells. Because low levels of SHH are found in the precursor cell area, SHH may act to stimulate precursor cell proliferation. In conclusion, low levels of SHH in the precursor cell area of the colonic crypt may act to control precursor cell phenotype or proliferation. The major target for Hedgehog signaling, however, is the differentiating colonic enterocyte. These cells produce IHH that seems to act in an autocrine fashion to suppress WNT signaling and stimulate differentiation.

HEDGEHOG SIGNALING AND CARCINOGENESIS OF THE GASTROINTESTINAL TRACT It is becoming more and more evident that disturbance of cellular interpretation of extrinsic information is a major step in the initiation of GI malignancies. Several morphogenetic pathways have been implicated, for example, in the development of colorectal cancer. First, mutations in the WNT pathway were identified as the cause of the familial adenomatous polyposis (FAP) syndrome and most sporadic colonic adenomas (38). Then hamartomatous polyposis syndromes such as juvenile polyposis were linked to mutations in the BMP receptor and the related intracellular signal transducer SMAD4 (110). Disturbed Hedgehog signaling has already been shown to lead to basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (111). Two independent groups have found a role for Hedgehog signaling in GI tract– derived malignancies. One showed that most cell lines derived from esophageal, gastric, biliary, and pancreatic cancers express both SHH and IHH mRNA and PTCH mRNA, indicating that autocrine Hedgehog signaling occurs in those cells. This was indeed confirmed by transfection of Hedgehog reporter constructs, which showed endogenous Hedgehog signaling that was inhibited by cyclopamine. Treatment with cyclopamine also greatly reduced the viability of these cell types, suggesting that Hedgehog signaling maintains a malignant phenotype in most GI cancer types (108). Another group made similar observations in the development of pancreatic cancer; the group showed that SHH is not normally expressed in ductal epithelium in the pancreas, but that expression of SHH is induced in pancreatic intraepithelial neoplasia in humans and seems to increase with the severity of the lesions (112). Mice with a Shh transgene behind a pancreas-specific promoter not only developed mesenchymal transformation to an intestinal phenotype, but also displayed an epithelial histology that resembled pancreatic intraepithelial neoplasia in human patients. The viability of 6 of 12 SMO-positive pancreatic cancer cell lines was shown to be responsive to cyclopamine, suggesting that cyclopamine nonresponsive cells either have Hedgehog activating mutations downstream of SMO or alternative growth in a Hedgehog-independent manner (112). No such role for Hedgehog signaling was found in the colon (108). Our own data suggest that this may be caused

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 283 by the fact that the initiating mutation in most colonic adenomas is a mutation in the WNT pathway that would suppress the expression of IHH, and thus the activity of the Hedgehog pathway (73). Indeed, we found that expression of IHH is lost in HT-29 and DLD1 colon carcinoma cells in vitro and in sporadic adenomas and those in patients with FAP in vivo (73). Loss of IHH expression is an early event in patients with FAP and was found in histologically normal epithelium with an aberrant β-catenin expression pattern that is typical for APC mutant epithelial cells (73). Loss of IHH expression could be restored in DLD1 cells by blocking WNT signaling with an inducible dominant negative TCF4. The WNTdependent loss of IHH expression may play an important role in the loss of differentiation that is one of the defining hallmarks in the earliest stages of adenoma formation. Indeed, many of the phenotypic abnormalities that we observed in the colonic enterocytes of cyclopamine-treated rats such as redistribution of villin expression, loss of brush border enzyme expression, and induction of intestinal trefoil factor (ITF) expression are also observed during colorectal carcinogenesis (113,114). We have not examined what happens to Hedgehog signaling in the later stages of colorectal carcinogenesis when colon cancer cells progressively accumulate novel gene mutations and assume a more poorly differentiated phenotype. Data from Oniscu and colleagues (103) suggest that with the expansion of the precursor cell compartment in the later stages of colorectal cancer development, the zone of SHH-expressing cells increases as well. Thus, Hedgehog signaling seems to maintain a malignant phenotype in cancers that originate from foregutderived epithelial tissues such as the esophagus, stomach, bile duct, and pancreas. In the adenoma to carcinoma sequence that leads to colorectal cancer, secondary loss of IHH signaling caused by WNT pathway activating mutations seems to lead to loss of differentiation in maturing colonic enterocytes.

FUTURE DEVELOPMENTS The similarities between morphogenesis and morphostasis of GI epithelium will receive much more attention in the coming years. We hope that the further application of insights derived from a variety of developmental models will increase our understanding of the molecular mechanisms of homeostasis of GI epithelial tissues. In most cases, signaling modules that have been studied in developmental models will be found to be extremely similar in the adult GI tract. They have the same ligands, the same receptors, the same intracellular signal transduction molecules, and importantly, the same target genes. Therefore, a large body of experimental data from a variety of experimental developmental systems awaits application in the adult system. For Hedgehog signaling, the first steps have now been made. The finding that Hedgehog signaling may influence the survival of proximal GI tract–derived cancers and the availability of a variety of Hedgehog pathway inhibitors suggest

that the first experimental clinical data in oncologic patients will soon appear.

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CHAPTER

10

Developmental Signaling Networks: The Notch Pathway Guy R. Sander, Hanna Krysinska, and Barry C. Powell Overview of Notch Signaling, 287 Structure of the Notch Receptors, 288 Structure of the Notch Ligands, 289 The Delta Subfamily, 289 The Jagged Subfamily, 289 The Notch Signaling Pathway, 290 Functions of Different Notch Receptors and Ligands, 292 Modifiers of Notch Signaling, 293 Interactions of Notch with Other Signaling Networks, 294

Notch Signaling in the Gastrointestinal System, 295 Overview of Gastrointestinal Structure and Development, 295 Notch in the Developing Gut, 297 Notch in the Enteroendocrine System, 298 Notch in the Mucosal Immune System, 301 Notch in the Enteric Nervous System, 302 Future Directions, 302 References, 302

The Notch signaling pathway is fundamentally important in biology. In broad terms, it enables the formation of different cell types in a tissue. Notch is a receptor, and interaction of Notch with its ligands on adjacent cells initiates a cascade of biochemical actions, causing Notch to move from the membrane to the nucleus to alter the activity of gene expression networks through transcriptional regulation. Notch is expressed in all manner of cells in the gut, the epithelium, the enteric nervous system, the mucosal immune system, and the vascular network, yet with few exceptions, we know little about the specifics of Notch function in the gut. Much of what we know about Notch comes from its action in other tissues. This chapter describes the Notch signaling pathway and its role in development. Then what is known of Notch signaling in the gastrointestinal tract is discussed, focusing

on recent research that implicates a vital role for Notch in the development of the enteroendocrine system.

OVERVIEW OF NOTCH SIGNALING The name Notch originates from an observation made by Mohr (1), who noted the formation of notches at the wing margin of the fruit fly, Drosophila melanogaster, caused by partial loss of function of what was later named the Notch gene. The Notch locus was cloned in 1983 by Spyros Artavanis-Tsakonas (2), who has continued to pioneer the field. One of the distinguishing features of the Notch signaling pathway is that it is activated by cell–cell contact. The Notch receptors are large, cell-surface proteins that are activated by a group of cell-surface ligands often collectively referred to as the DSL ligands, after the founding members: Delta, Serrate, and Lag2. Both receptors and ligands are singlepass, transmembrane (TM) proteins. Activation of the receptor by ligand results in a proteolytic cleavage of the intracellular portion of the receptor, which is then transported to the nucleus, where it induces specific gene transcription. Notch signaling is important in a multitude of processes; that is, in growth, differentiation, proliferation, and patterning in a wide variety of tissues through its control of cell fate (3–6). From the earliest stage of development of an organism

G. R. Sander: Child Health Research Institute, North Adelaide, and Department of Pediatrics, University of Adelaide, South Australia 5006, Australia. H. Krysinska: Child Health Research Institute, North Adelaide, South Australia 5006, Australia. B. C. Powell: Child Health Research Institute, North Adelaide, and School of Pharmacy and Medical Sciences, University of South Australia; Department of Pediatrics, University of Adelaide, South Australia 5006, Australia. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

287

288 / CHAPTER 10 there is a recurring need to regulate the production of different cell types and at the same time maintain the pluripotential capacity of the progenitor cell. Notch signaling is important in the specification of cell fate as groups of undifferentiated cells segregate into different cell lineages. As such, Notch signaling is not deterministic of a particular fate, but is instead permissive, enabling the choice of different fates. Depending on the cellular context, Notch signaling may result in diametrically different outcomes—inhibition or promotion of differentiation, proliferation or apoptosis.

STRUCTURE OF THE NOTCH RECEPTORS Notch proteins have been identified in a number of species. In general, invertebrates possess one or two Notch genes, whereas vertebrates have four. Much of the pioneering work has been performed in the fruit fly, D. melanogaster, which has only one Notch receptor and two ligands, Delta and Serrate (4). The sea urchin, Lytechinus variegatus (7), the invertebrate chordate, Amphioxus (8), and the frog, Xenopus, also have only one Notch gene (9). The worm, Caenorhabditis elegans (10,11), and the chicken have two Notch receptors (12), whereas, intriguingly, zebra fish, Danio rerio, have four receptors (13). In humans and mice there are four Notch receptors (4). The Notch genes are highly conserved and are thought to have arisen by duplication of a single ancestral gene. Notch receptors are heterodimeric TM proteins of about 300 kDa (Fig. 10-1). Both the extracellular ligand binding domain and the Notch intracellular domain (NICD) contain a number of conserved regions. Unlike other receptors, Notch receptors have no enzymatic activity of their own, but instead rely on a ligand-induced proteolytic cleavage to induce activity through release of the NICD. The NICD is translocated to the nucleus where it interacts with the CSL protein (CBF1 or RBP-Jκ in vertebrates, Su(H) in Drosophila, and Lag1 in C. elegans). This interaction converts CSL from a transcriptional repressor to an activator, leading to the activation of other transcription factors and gene networks (3,4). All four Notch receptors signal through CSL, the one nonredundant member of the Notch signaling pathway. The Notch extracellular domain contains multiple, tandemly arranged epidermal growth factor-like repeats (EGFR). The number of EGFRs varies between different Notch receptors; Notch1 and Notch2 have 36 repeats, Notch3 has 34, and Notch4 has 29 (14,15). Comparison of the amino acid sequences of the individual EGFRs shows that the homology between similarly positioned repeats in Notch receptors in different species is greater than the homology between EGFRs within the same Notch molecule (16). This linear conservation suggests that the tertiary structure of the EGFRs might be important for function of the receptor, perhaps conferring some degree of ligand specificity, enhancing or suppressing the interaction with ligands, or affecting the stability of the Notch protein. In Drosophila, EGFRs 11 and 12 have been shown to be necessary for ligand binding (17), but the

functions of the remaining EGFRs are not well understood. Modification of adjacent EGFRs, referred to as the Abruptex domain in Drosophila Notch (18,19), suggest that they mediate the interaction of adjacent receptors and ligands in the same cell membrane, an interaction that results in a reduced ability of the Notch receptor to be stimulated by a ligand on adjacent cells (20). Adjacent to the EGFRs are three repeats of a cysteine-rich region termed LNR (after the Notch-related region in the C. elegans LIN-12 gene). This region is implicated in dampening receptor activity, because deletion or missense mutations in the LNR region result in a constitutively active receptor (15,16). Proteolytic cleavages of the Notch receptor occur during maturation of the receptor and in its activation (21). The first site of proteolytic cleavage (S1) is located between the LNR and TM domains, and cleavage there is required for formation of the mature Notch heterodimer (22). The receptors are synthesized as single-polypeptide precursors of approximately 2500 amino acids. The precursor contains a leader peptide that is removed during maturation. In the trans-Golgi apparatus, a furin-like convertase cleaves the precursor protein, and it is reassembled as a heterodimer of the extracellular and TM domains. The two subunits of the mature Notch receptor interact by a noncovalent calcium-dependent bond. Processed receptor is transported from the Golgi to the cell membrane. There are two other proteolytic cleavage sites in Notch. The second site (S2) is located near the TM region and is the site at which Notch is initially cleaved on ligand binding (23–26). The TM region contains the S3 and S4 sites at which cleavage occurs after ligand binding (27). The cytoplasmic domain of the Notch receptor contains several conserved regions that regulate its function. The subTM region known as RAM and the six adjacent ankyrin (ANK) repeats are involved in binding the CSL transcription factor (28). Ankyrin repeats may mediate homotypic interaction between Notch molecules expressed by a cell (29). The ANK region is also known to interact with Deltex, a widely expressed ubiquitin ligase (30,31) that can act either as a positive or negative regulator of Notch signaling (see Deltex section later in this chapter). Directly following the ANK repeats is a region identified in Notch1 and Notch2 as a Notch cytokine response region (32). In myeloid cells, this region regulates differentiation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Human Notch1 and Notch2 also have a transcriptional activation domain (TAD) located downstream of the ANK region that has been shown to interact with general transcriptional coactivators such as p300/CBP associated factor (PCAF) and p300 (33). Farther toward the C terminus is a polyglutamine tract (OPA) of unknown function (34). Glutamine-rich regions are found in some transcriptional regulators where they appear to mediate protein–protein interaction, but a direct role for the OPA repeats in Notch function currently is lacking. A PEST sequence (proline, glutamic acid, serine, and threonine-rich region), a characteristic of short-lived proteins and frequent site of ubiquitination (35), is located at the C terminus of the receptor (36).

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 289 A C terminus Intracellular

N terminus Extracellular

TM S2

NLS

NLS ANK

Ligand-binding site

TAD

c c EGFR

LNR

PEST OPA

S4 S3

B Drosophila

TM

Notch

Vertebrate Notch1 Notch2 Notch3 Notch4

FIG. 10-1. Structure of Notch receptors. (A) The mature Notch receptor is a heterodimer, formed after proteolytic cleavage and noncovalent reassembly of the newly synthesized protein. The receptors contain a variety of domains that are often tandemly repeated. The extracellular domain is characterized by between 29 and 36 tandem epidermal growth factor-like repeats (EGFRs). EGFRs 11 and 12 are important in mediating receptor–ligand interaction. These are followed by three cysteine-rich LNR repeats, the transmembrane region (TM), the RAM domain, and six ankyrin (ANK) repeats that mediate protein–protein interaction and bind the CSL (CBF1 or RBP-Jκ in vertebrates, Su(H) in Drosophila, and Lag1 in C. elegans) transcription factor through which all Notch receptors signal. The downstream Notch cytokine response region (NCR) and transcriptional activation domain (TAD) appear to be specific for Notch1 and Notch2. At the carboxy terminus lie a polyglutamine tract (OPA) of unknown function and the PEST region (proline, glutamic acid [amino acid symbol, E ], serine, threonine rich) that may mediate receptor stability. (B) Notch receptor structure (but not number) is highly conserved among species. Whereas the invertebrate Drosophila has one receptor, most vertebrates have four. Primary differences between vertebrate receptors are in the variation in the number of EGFRs and the length of the region between the ANK repeats and the carboxy terminus. (Modified from Weinmaster [15], by permission from Elsevier and the author.)

STRUCTURE OF THE NOTCH LIGANDS The Notch ligands are also known as the DSL family (after Drosophila Delta and Serrate and C. elegans Lag2), and, like Notch, they are single-pass, TM proteins (Fig. 10-2). The DSL family also has tandem, multiple EGFRs in its extracellular domain. However, the most N-terminal EGFR differs from the others and is referred to as the DSL repeat. It is essential for ligand function (37). As for the Notch receptors, invertebrates have a more simplified repertoire of Notch ligands. Drosophila possesses two ligands, Delta and Serrate, C. elegans has three (each containing remarkably few EGFRs), and vertebrates possess several ligands. Higher vertebrates appear to have three Delta genes and two Jagged genes (15), Jagged being the vertebrate equivalent of Drosophila and chicken Serrate. Zebra fish exhibit more variability, with four Delta genes and three Jagged genes (38,39). The distinctive structural difference between the Delta subfamily and the Jagged/Serrate subfamily of ligands is the presence of a cysteine-rich region in the extracellular domain of the latter. The intracellular domains of all the

ligands are quite short, and although they do not share any substantial homology, they are essential for normal function.

The Delta Subfamily Delta ligands contain an N-terminal domain and DSL and EGFR motifs, but they lack the cysteine-rich region of the Jagged subfamily (see Fig. 10-2). The three vertebrate Delta proteins have a variable number of EGF repeats, with Delta1 and Delta4 having eight repeats and Delta3 having six repeats (40–42). Delta3 appears to be the most divergent member of this family. Drosophila has one ligand, and vertebrates have three.

The Jagged Subfamily The proteins of the Jagged subfamily of Notch ligands, referred to as Serrate in Drosophila and chicken but Jagged in other vertebrates, are much larger than the Delta family

290 / CHAPTER 10 A Equipotent cells

Difference in receptor/ligand expression initiated

Feedback amplification

B

1° fate

2° fate

FIG. 10-3. Schematic of classical, lateral inhibition model of Notch function. A population of equipotent cells expressing Notch receptor and ligand equally receives a stimulus that initiates a change in the receptors and ligands. A fluctuation in Notch receptor expression is amplified through positive and negative feedback mechanisms such that adjacent cells eventually express receptors or ligands. This results in adjacent cells that adopt distinct fates driven by their receptor–ligand profile. In the schematic, receptors are identified as cup-shaped molecules, and ligands as hammer-like molecules.

FIG. 10-2. Structure of Notch ligands. (A) The notch ligands are single-pass transmembrane proteins that also contain a variety of conserved tandem repeats. There are two families of ligands, Delta and Jagged (also known as Serrate in some species), but the overall structure of their extracellular domains is remarkably similar to that of the Notch receptors. A variable N-terminal domain (NT) and a DSL motif (Delta/ Serrate/Lag) are followed by tandem epidermal growth factorlike repeats (EGFRs), typically from 6 to 16. The two families of ligands then differ substantially. The Jagged family (upper) is distinguished by a conserved cysteine-rich domain (CR) preceding the transmembrane (TM) region. All ligands have short intracellular regions lacking any conserved structure that typifies the Notch receptors. (B) Notch ligand complexity, similar to that of the receptors, increases from invertebrates to vertebrates. Drosophila has one Delta and one Serrate ligand, whereas humans have three Delta ligands and two Jagged ligands. Within each family the ligands are similar. (Modified from Weinmaster [15], by permission from Elsevier and the author.)

with, on average, twice the number of EGFRs. Drosophila has one ligand, and vertebrates have two. The extracellular portion of the Jagged ligands contain an N-terminal domain of 100 to 165 amino acids, followed by the highly conserved DSL motif (see Fig. 10-2). C terminal to the DSL motif is 16 EGF repeats and a cysteine-rich region that distinguishes

Jagged ligands from Delta. Following the TM region is an intracellular domain of variable length.

THE NOTCH SIGNALING PATHWAY Most of our knowledge about the function of Notch signaling comes from studies of hematopoiesis, neurogenesis, and embryonic development. The amount of information regarding Notch signaling of these events is extensive. The essential requirement for Notch signaling in early embryonic development has made it difficult to tease out the functions of Notch by traditional gene knockout studies because, generally, the genome-wide knockouts are embryonic lethal. However, sophisticated studies using conditional transgenic and gene knockout mice are beginning to reveal more about Notch function in the adult (43–45). Notch signaling can be regulated in three ways: by lateral inhibition, by inductive signaling, or in a cell autonomous manner. Notch is best known for its role in lateral inhibition, the process whereby positive and negative feedback loops that amplify or down-regulate Notch signaling are established to prevent neighboring cells from adopting the same fate at the same time (Fig. 10-3). Initially, during the process of lateral inhibition, there is an equipotent population of precursor cells that express both Notch receptors and ligands on their surface. By as yet unknown means, Notch expression decreases in some cells, and they up-regulate ligand expression.

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 291 Increased ligand expression stimulates Notch signaling on previously unaffected neighboring cells, which then reduce ligand expression by mechanisms such as those described later. The resulting distinct cell populations (receptor-low/ ligand-high and receptor-high/ligand-low) adopt different fates and become different cell types (5,46,47). An example of lateral inhibition is provided by the formation of neurons and glial cells during vertebrate neurogenesis (48). When neural stem cells encounter a neurogenic signal in their environment, only some of them respond to it by up-regulating the Notch ligand, Delta, on their cell surface. This, in turn, activates the Notch receptor expressed on the neighboring cells and stops them from responding to neurogenic signals in the environment. The outcome is that the Delta-high cells differentiate into neurons, whereas the cells that were initially prevented from differentiating by Notch signaling adopt an alternative fate and become glial cells. When Notch receptors and ligands are present on different cells, inductive signaling can occur. During this signaling, cells expressing the ligand activate Notch signaling in the cell expressing the receptor, thus influencing their developmental fates (5,46,47). When the Notch receptor and ligand are expressed in the same cell, there is a possibility of a cis interaction between them that could drive them into differentiation of one lineage. Coexpression can also result in an inhibition of Notch signaling (49), a process that is mediated, in part, by glycosylation (50). Notch receptors and ligands that are expressed by adjacent cells interact via their extracellular domains. Epidermal growth factor repeats 11 and 12 of the receptor and the DSL motif of the ligand mediate this interaction. Attachment of fucose to serine or threonine residues within the EGF domains by the enzyme O-fucosyltransferase is essential for interaction (51), but how this mediates interaction and initiates downstream events is unknown. Interaction may induce conformational changes in the receptor that expose proteasesensitive sites. After ligand interaction, the receptor is cleaved, initially at the extracellular S2 site, followed by concurrent cleavages at sites S3 and S4 in the TM region (see Fig. 10-1). On ligand binding to the extracellular domain, Notch receptors undergo several proteolytic cleavages, resulting in a release of the NICD (Fig. 10-4). The first ligand-induced cleavage occurs at the S2 site and is mediated by an ADAMtype proteinase (23,25). Cleavage at S2 results in shedding of the extracellular domain and is followed by intramembrane, presenilin-dependent γ-secretase cleavages at S3 and S4 (52,53). The S3 cleavage site is close to the cytosolic membrane border, and S4 is approximately 12 amino acids upstream from S3, near the middle of the TM. The presenilin-dependent cleavages at sites S3 and S4 result in the liberation of NICD, which translocates to the nucleus, where it interacts with the CSL repressor complex to convert it to an activation complex, thereby altering gene expression. CSL is the major effector of Notch signaling and is the one nonredundant member of the Notch signaling pathway through which all four Notch receptors signal. In the absence of Notch signaling, CSL represses the transcription of target

genes via a repressor complex that includes histone deacetylases and various corepressors, SMRT (silencing mediator of retinoid and thyroid hormone receptors), SAP30, CIR, and SHARP (54–56). Ligand activation and nuclear transport of the NICD enables it to interact with CSL via the Notch RAM and ANK regions. This interaction displaces SMRT, histone deacetylases, and the corepressors and in their place recruits histone acetyltransferases and other coactivators, creating a transcriptional activation complex. In association with coactivators, histone acetyltransferase mediates chromatin remodeling to activate expression of Notch target genes (57,58). The level of nuclear NICD is controlled by various modifications including phosphorylation and ubiquitination. Degradation of NICD down-regulates signaling and resets the cell for future instructive Notch signaling. Most research has focused on the fate of Notch after ligand interaction, but there is some evidence for a role for the ligands after Notch interaction (59,60). First, Drosophila Delta has an intrinsic nuclear localization capability; second, the C terminus of Jagged1 is capable of interacting with PDZ-containing proteins. These features may reflect the existence of signaling pathways initiated in the ligand-expressing cell in response to Notch–ligand interaction, invoking bidirectional signaling mechanisms. The primary Notch target genes include transcription factors of the basic helix-loop-helix (bHLH) superfamily (61,62). Original target genes identified in Drosophila, Hairy and Enhancer of Split, have given the name to the vertebrate homologs, Hes (63). Since discovery of the Hes family, another family of Hes-related bHLH genes has also been shown to respond to Notch signaling (64–66). This is now referred to as the Herp (Hes-related bHLH protein) family (61). These two families include approximately 10 Notch bHLH target genes. Basic HLH transcription factors function as dimers, with two activator bHLH proteins required to bind DNA and activate transcription. With one exception (67), the primary Notch target genes are all bHLH transcriptional repressors that inhibit transcriptional activity by forming nonproductive dimers. Because there are more than 200 bHLH genes with diverse biological roles, there is incredible potential for Notch signaling to influence many cellular processes through this family. What genes are regulated by the bHLH Notch target genes? Because bHLH proteins function as dimers, other families of bHLH genes are often immediate targets. Two well-studied families are involved in neural and endocrine development. Originally identified in Drosophila as the Achaete-Scute complex and the Atonal proneural genes, they encode bHLH proteins involved in neuronal and epidermal lineage formation (62,68,69). In neuronal formation in Drosophila, the AchaeteScute genes, in turn, regulate Delta expression (70,71). Because Notch signaling is also thought to up-regulate itself through Hes (72), the outcome of these activities is to create a feedback loop to polarize receptor and ligand expression and reinforce Notch signaling via different bHLH proteins. Vertebrate homologs of the Drosophila Atonal genes include BETA2, neurogenin3, and Math1. These genes are also

292 / CHAPTER 10 Notch

Delta jagged

HDAC CoR

NICD

CSL

HAT

X

Hes

CoA

CSL

P

Hes

Ub

Hes

X

Math1

FIG. 10-4. Mechanism of Notch signaling. Interaction of the Delta or Jagged ligand on one cell with the extracellular domain of a Notch receptor on an adjacent cell leads to proteolytic cleavage that releases the Notch intracellular domain (NICD). The NICD then translocates to the nucleus, where it binds to the CSL (CBF1 or RBP-Jκ in vertebrates, Su(H) in Drosophila, and Lag1 in C. elegans) transcription factor, displacing a complex of corepressors (corepressor R [CoR] and histone deacetylase [HDAC]) that have hitherto blocked transcription of CSL target genes. Binding of NICD to CSL occurs via the Notch ankyrin domains and leads to recruitment of transcriptional coactivators (CoA and histone acetyltransferase (HAT)) and transcription of basic helix-loop-helix (bHLH) target genes from the Hes and Herp families. These proteins typically act as transcriptional repressors and turn off downstream genes such as Math1 that regulate the selection of a secretory or absorptive cell fate in the gastrointestinal tract (see Notch in the Enteroendocrine System). Posttranslational modification of the NICD, involving phosphorylation and ubiquitination mechanisms, leads to its degradation and a downturn in Notch signaling, potentially resetting the Notch switch.

important in vertebrate neuronal differentiation; however, exciting research has revealed their involvement in the differentiation of enteroendocrine cell lineages in the gut (73–79) (see Notch in the Enteroendocrine System later in this chapter). The cell cycle regulators cyclin D1 and p21 may also be primary targets of Notch signaling (80,81). In keratinocytes, activated Notch induces p21 and promotes differentiation, whereas in other cells, Notch increases cyclin D1 expression to promote progression of cells through the G1 phase of the cell cycle. Both pathways are CSL dependent. CSL and the bHLH genes are the main effectors of Notch signaling, but there is accumulating evidence that Notch can signal through CSL independently of the bHLH genes and is also able to signal independently of CSL itself (82,83). Further identification of specific target genes in different cell types is clearly important to understand the functions of Notch signaling in proliferation, differentiation, and apoptosis. Notch signaling is tightly regulated at a number of levels. The receptor, ligand, and other components of the Notch signaling pathway all appear to be targets for ubiquitination and degradation via the proteasome. Ubiquitination of Notch and Delta is mediated by E3 ubiquitin ligases. The C terminus of Notch, including the PEST sequence, appears to be essential for ubiquitination by the E3 ligase mSel-10 (84),

and phosphorylation of specific Notch residues appears to be the signal for mSel-10 action (85). This rapid downregulation of Notch, coupled with its intrinsically low level of expression, provides an explanation for difficulty in detecting nuclear Notch. Delta has also been shown to be a target for another ubiquitin ligase (86).

Functions of Different Notch Receptors and Ligands In general, vertebrates have four Notch receptor genes and five ligand genes, and these are usually expressed in different patterns throughout development, potentially providing a diverse array of receptor–ligand combinations. In addition, different ligands can signal through the same receptor and produce different outcomes. In Drosophila, in which there is only one Notch receptor, mutations in Delta and Serrate produce different phenotypes, even though they act through the same receptor (87). In this model system, cell interactions mediated between Delta and Notch appear to be more stable than interactions between Serrate and Notch (17,88), and this may lead to stronger or more sustained Notch signaling. The distinctive cysteine-rich region of Serrate may mediate this effect. However, the glycosyltransferase Fringe

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 293 may also modify the responses to these two ligands (see Glycosylation section later in this chapter). Both Delta and Serrate are expressed during wing formation in Drosophila, where, at the wing margin, Notch activation is modified by Fringe (89). Fringe enhances Delta-activated Notch signaling and inhibits Serrate-induced signaling to establish a dorsal ventral boundary at the wing margin that is necessary for development of the wing. Given the large size of the Notch receptors and the presence of various conserved domains, it is likely that they will exhibit different signaling capacities. However, the data on this aspect of Notch signaling are only beginning to emerge. The primary bHLH Hes target genes appear to be differentially activated by Notch, but the precise effects are not clear. Whereas Notch1 is a potent activator of Hes1 and Hes5, an ability that appears to be mediated by the ANK repeat region and another adjacent region of the intracellular domain (90), the action of Notch3 is controversial (91,92). There is evidence that Notch3 is a much weaker activator of the Hes1 promoter than Notch1 (91), but there is also evidence to the contrary (92). Interestingly, Shimizu and colleagues (92) show that the level of coexpressed CSL affects the ability of Notch to transactivate Hes promoters, a finding that may explain the controversy regarding Notch3 transactivation. They also report that the NICD of Notch1 and Notch3 differentially transactivates Hes1 and Hes5, and that Notch2 is also a weak activator. Interestingly, Shimizu and colleagues (92) show that coexpression of Notch2 inhibited activity of Notch1 and Notch3, indicating that each receptor can not only activate different downstream genes, but is also able to competitively influence each other. An explanation for this may stem from the fact that Notch2 appears to have a weaker TAD compared with Notch1 (47).

the mammalian glycosyltransferases of the Fringe family: manic Fringe, radical Fringe, and lunatic Fringe (95,96). There are a number of O-fucose sites in the extracellular domain of the Notch receptor, particularly in EGFRs 11 and 12, that mediate receptor–ligand interaction (50,51). Modification of Notch by Fringe results in a different responsiveness of the receptor to ligand binding, because elongation of glycans by Fringe increases the ability of the receptor to bind Delta (97) but inhibits binding of Jagged (95). Glycosylation by Fringe has been shown to play an important role in the boundary formation in Drosophila wing, eye, and leg, in formation of the chick limb, and in vertebrate somitogenesis (98). Phosphorylation

A range of interactions with other molecules and posttranslational modifications of the receptor and ligand act to regulate Notch activity (93). Posttranslational modifications include glycosylation, phosphorylation and ubiquitination, and they affect each step in the signaling pathway, from ligand binding outside the cell to gene activation in the nucleus. This section briefly discusses these three processes and the function of three specific modifiers of Notch signaling: Numb and Deltex, which are involved in ubiquitination of Notch, and Mastermind, a nuclear coactivator that may also facilitate Notch phosphorylation.

Phosphorylation is a well-known and widespread mechanism for regulating protein function, and a role for phosphorylation in regulating the Notch signaling pathway is starting to emerge. After ligand activation, the NICD appears to be phosphorylated at several sites downstream of the ANK repeats by glycogen synthase kinase-3β (99–102). Glycogen synthase kinase-3β has been shown to bind to the Notch2 NICD near the ANK region and phosphorylate several serine and threonine residues, a process that inhibits activation of the Hes1 target gene and leads to down-regulation of Notch signaling (102). Notch1 has also been shown to be phosphorylated by glycogen synthase kinase-3β, but, in contrast with the effect on Notch2, this appeared to stabilize Notch1 signaling (101). The sites of phosphorylation need to be clarified in these studies, and whether they represent genuine distinctions in the response of different receptors to phosphorylation or inherent differences in the cells studied is awaited with interest. Notch signaling is integral to myeloid cell differentiation in response to cytokines (32). Research now shows that phosphorylation of specific amino acids in a 30-residue segment of the Notch cytokine response region has a key role in this process (103). Granulocyte colony-stimulating factor induces phosphorylation of the Notch2 NICD, including a critical site at Ser2078 (different to that phosphorylated by glycogen synthase kinase-3β), that inactivates the NICD and promotes differentiation. In contrast, when cells are stimulated with granulocyte-macrophage colony-stimulating factor, the Notch2 NICD is not phosphorylated at these residues, the NICD is able to initiate the Notch signaling cascade in the cells, and differentiation is inhibited. The direct effects of phosphorylation of the NICD in myeloid differentiation remain to be determined but may well signal ubiquitination and protein degradation (104).

Glycosylation

Ubiquitination

Glycosylation is the process of attaching carbohydrates to the side chains of amino acids. The EGFRs of Notch receptors are heavily glycosylated (94), a process that can potentiate or inhibit Notch signaling. The addition of O-fucose is particularly important, because it becomes the site of a further elongation by addition of N-acetylglucosamine by

Ubiquitination, the attachment of ubiquitin molecules by ubiquitin ligases, marks proteins for internalization from the plasma membrane and for proteasome- or lysosome-mediated degradation. Because activation of Notch occurs via an extracellular signal, and the signaling portion of the receptor is cleaved and released intracellularly and is therefore

Modifiers of Notch Signaling

294 / CHAPTER 10 dissociated from further extracellular events, internal mechanisms must operate to ensure tight control over Notch signaling. At least four ubiquitin ligases appear to regulate Notch signaling via ubiquitination (105). Two regulate rapid turnover of the NICD, a third regulates the Delta ligand, and a fourth regulates the Notch antagonist, Numb. Ubiquitination of the NICD is regulated by the ubiquitin ligases Sel-10 and Itch in a process coupled with Notch phosphorylation. The NICD is ubiquitinated by these ligases at sites located C terminal to the ANK region, leading to degradation of the NICD by the proteasome, thereby down-regulating the Notch signal (84,104,106). Although both Notch1 and Notch4 have been shown to interact with the Sel-10 ubiquitin ligase, they respond differently (104). Whereas Notch1 NICD appears to be rapidly degraded, the Notch4 NICD is not. How Notch4 evades proteasome degradation and what the consequences are for Notch4-mediated signaling are unclear. Sel10 also interacts with presenilin (107), the protease that cleaves Notch at the membrane in response to ligand interaction and may therefore regulate Notch signaling at a number of steps. Both the membrane-bound and the activated nuclear Notch can be ubiquitinated by the E3 ubiquitin ligase Itch and degraded by the proteasome (108,109). However, downregulation of the membrane-bound Notch in differentiating myoblasts is achieved by ubiquitination of tyrosine residues by cCbl ubiquitin ligase and degradation by the lysosomal pathway, rather than the proteasomal pathway (110). Thus, several mechanisms for ubiquitinating Notch appear to operate to maintain tight regulation of Notch signaling. Numb Numb was discovered as a key protein in the regulation of neuronal development. Knockout of Numb causes embryonic lethality before embryonic day (E) 11.5 with defects in many neuronal lineages (111). In the Drosophila peripheral nervous system, a sensory organ precursor cell undergoes rounds of asymmetric divisions in development of the sensory organ. Numb is a membrane-associated protein that segregates into one daughter cell during precursor cell division and acts as an inherited determinant of cell fate (112). Notch acts as a binary switch in this system to specify cell fate, and Numb acts to negatively regulate Notch in the process. Numb is a cytoplasmic, membrane-associated protein that binds to the RAM and C-terminal sequences of the NICD to suppress Notch activity (93). It does this by recruiting the E3 ubiquitin ligase, Itch, enhancing ubiquitination of membrane-tethered and nuclear Notch (109). Deltex Deltex was identified in Drosophila in a genetic screen for molecules that interact with Notch to specify cell fate (113). Deltex binds to the ANK region of the NICD and can modulate Notch signaling either positively or negatively (30,114,115). There are several vertebrate Deltex homologs (115) that are widely expressed with Notch (116). Deltex has

been identified as an E3 ubiquitin ligase that transfers ubiquitin to itself and to heterologous substrates (117). This finding, together with the elucidation of Numb’s involvement in Notch ubiquitination, further emphasizes the importance of ubiquitination in regulating Notch signaling. Mastermind Like Numb and Deltex, Mastermind was identified in a screen for Notch modifiers in Drosophila. Mastermind is a nuclear protein that binds to the ANK region to coactivate Notch signaling, amplifying Notch induction of the target gene Hes1 (58). Mastermind has a key role, recruiting the general coactivators CBP/p300 to Notch target gene enhancers to promote nucleosome acetylation and gene activation in response to Notch signaling (118). Mastermind also appears to enhance phosphorylation and proteolytic turnover of the NICD. Fryer and colleagues (118) speculate that Mastermind may function as a timer, coupling transcriptional activation with disassembly of the Notch enhancer complex, in effect coordinating the turning on and off of transcription. The “off switch” may be completed by Notch ubiquitination via the ubiquitin ligase Sel-10, followed by degradation of the NICD by the proteasome (84,104,106).

INTERACTIONS OF NOTCH WITH OTHER SIGNALING NETWORKS The Notch signaling pathway is important in the specification of cell fate in a wide variety of cell types and is perhaps instructive for the generation of all cells in invertebrates and vertebrates (4). Depending on the context, Notch signaling may result in diametrically different outcomes, from inhibition or promotion of differentiation to proliferation or apoptosis. Not surprisingly therefore, the Notch pathway appears to interact with a variety of other important signaling networks. Key pathways where interactions with Notch have emerged are the fibroblast growth factor pathway, the transforming growth factor-β and bone morphogenetic protein (BMP) pathway, and the nuclear factor-κΒ, Wnt, Ras, and signal transducer and activation of transcription (STAT) signaling pathways (119–130). All these pathways are involved at some stage in gut function and, with the widespread expression of Notch in the gut (see Notch in the Developing Gut later in this chapter), potentially herald its extensive involvement in diverse cellular processes (76). In foregut development in Drosophila, STAT signaling is required for expression of Notch target genes that regulate epithelial morphogenesis (124). Development of the proventriculus, an organ derived from the foregut, depends on Notch signaling in two rows of boundary cells in the proventriculus primordium. This results in invagination of the foregut epithelium into the endodermal midgut layer, a process driven by a cytoskeletal protein of the spectraplakin family that responds to Notch signaling (131). Drosophila Notch and Janus-activated kinase (JAK) STAT mutants exhibit similar

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 295 defects in cell movement and proventriculus development. Although the exact nature of the link between STAT and Notch remains to be determined in this system, Kamakura and colleagues (130) have shown that in the central nervous system, the Notch target gene Hes interacts with JAK and STAT to promote STAT phosphorylation and activation. Other signaling pathways appear to modulate Notch signaling via the Hes and Herp Notch target genes (120,122,128), although interaction with NICD and CSL can also occur (121,127). In contrast, the Wnt pathway, which operates in diverse cell types during development and acts through the lymphoid enhancer factor 1/T-cell factor (LEF1/TCF) transcription factors (132), appears to regulate Notch signaling through direct control of Delta transcription (126). There are several LEF1 binding sites in the Delta1 promoter, and mutagenesis studies have shown that these are necessary for Delta1 expression, at least in the presomitic mesoderm. Wnt signaling is involved in the proliferation of intestinal epithelial progenitor cells, and mutation of TCF4 has dramatic consequences for intestinal morphogenesis (133). TCF4 knockout mice die soon after birth. At a time when the gut epithelium is normally in a highly proliferative phase, these mice lacked any proliferative epithelial compartments, and the neonatal epithelium was composed exclusively of differentiated, nondividing villus-type cells. TCF4 thus appears to have a critical role in maintaining intestinal crypt stem cells. Notch1, Notch2, and Delta1 are expressed in the intervillus epithelium at E18.5, where the stem cells and proliferative cells are found (134), and it is possible that Wnt acts through TCF4 to up-regulate Delta1 expression in gut epithelial stem cells, in turn activating Notch signaling in adjacent cells. In this scenario, in normal gut epithelium, the TCF4/Delta cells would retain their stemlike capability, and the Notch expressing cells would have a high proliferative capacity that fuels gut growth. This work offers tantalizing glimpses of how diverse signaling pathways may interact with Notch, and it appears certain that such interactions will prove relevant to gut development and function.

NOTCH SIGNALING IN THE GASTROINTESTINAL SYSTEM Overview of Gastrointestinal Structure and Development Much about the adult gastrointestinal tract is large. In humans, it is several meters in length with a surface area of more than 200 square meters! Furthermore, the gut epithelium is in a state of continual renewal, replenishing its epithelial surface every few days. In the small intestine, this amounts to replacement of a staggering 109 cells in mice and more than 1011 cells in humans every 2 to 4 days (135). Endocrine cells in the epithelium of the gastrointestinal tract represent the largest population of hormone-secreting cells in the body (136). The mucosal immune system and enteric nervous system, which are intimately linked with gut function, form

substantial and complex networks in their own right. The mucosal immune system, which is involved in the development of oral tolerance and defense against intestinal pathogens and toxins, is estimated to contain about half the total number of T cells in the body (137) and about 80% of all B cells (138). The enteric nervous system, which contains more than 14 different neuronal cell types, has a key role in intestinal motility, absorption, and secretion (139,140). This section discusses the emerging role for Notch signaling in the development and maintenance of these tissues and networks that underpin gastrointestinal function. The adult gut derives from all three germ layers in the embryo, namely the ectoderm, mesoderm, and endoderm. The gut epithelium and its associated glands derive from embryonic endoderm, whereas the nervous system derives from embryonic ectoderm. The mucosal immune system derives from the endoderm and mesoderm. The early stages of gut development—that is, the formation of the primitive gut tube—are similar between Drosophila and vertebrates (141,142), and much of our knowledge of Notch signaling in gut development is informed by studies in Drosophila. Essentially, in Drosophila and in mice, the endodermal layer invaginates in the anterior and posterior positions, forming foregut and hindgut primordial tubes, which then extend toward each other, fuse, and give rise to the primitive gut tube (142). The alimentary canal is divided into foregut, midgut and hindgut sections, each with distinct physiologic functions. In mice, the foregut gives rise to the proximal part of the duodenum, the midgut gives rise to the remaining parts of the small intestine, distal duodenum, jejunum and ileum, and the hindgut gives rise to the colon, caecum, and rectum (143). Development from the primitive gut tube to the established organ sees many changes. In mice, formation of the primitive gut tube commences at E8; by E13.5, the intestinal epithelium is beginning to undergo morphogenesis and cytodifferentiation, and at E15.5 appears as a pseudostratified epithelium. The epithelium then starts to fold, forming an undulating layer of epithelium folded upward into villi separated by proliferating intervillus epithelium. After birth, the intervillus epithelium is folded downward into pocket-like crypts. The crypts increase in number through fission in the next few weeks such that each villus is “fed” by cells emanating from several crypts. Looking down onto the epithelium from the lumen of the gut, a regular array of villi and crypts with several crypts surrounding each villus and a number of villi about each crypt can be seen. Apoptosis of cells at the apex of the villi occurs at birth, but the villi continue to lengthen for a few weeks as cell production exceeds the rate of apoptosis and cell loss. In humans, formation of mature crypts and villi occur earlier in gestation, and both are evident well before birth (144). The epithelium of the small intestine produces five types of differentiated cells: absorptive enterocytes, mucinproducing goblet cells, antimicrobial peptide-secreting Paneth cells, peptide-secreting enteroendocrine cells and antigen sampling M cells; the latter are specifically found in the

296 / CHAPTER 10 follicle-associated epithelium that overlies Peyer’s patches. All cell types are formed in crypts, which are bottle-shaped invaginations of the small intestine interspersed between finger-like villi (Fig. 10-5). The stem cells reside near the base of the crypt; four to six are predicted for small intestinal crypts (145–147). As the gut epithelium is replenished every few days, the cycle of production of new cells continues throughout life. The overwhelming majority of these cells become absorptive enterocytes, which, together with secretory goblet cells and enteroendocrine cells, move upward as they differentiate. A small number of cells, about 10 per crypt, behave differently. Known as Paneth cells, they migrate downward from the stem cells and complete their differentiation at the bottom of the crypt. Paneth cells are designed for antimicrobial defense, containing many secretory granules, and are relatively long lived with a lifespan of 20 days. The upwardly mobile cells are shed from the top of the villus in a continuous

stream, taking between 2 and 4 days to make the trip from newly minted cell to an apoptotic body. How this prolific and carefully orchestrated production of four different cell lineages is maintained is now coming to light (see Notch in the Enteroendocrine System later in this chapter). In the formation of the colon, the epithelium also becomes folded and forms villi and crypts, although this a transitory arrangement. In humans, the villi have flattened by birth, and the colonic epithelium has matured into an array of regularly spaced crypt pockets (Fig. 10-6). The stem cells are thought to reside at different crypt locations in the ascending and descending colon (145–147). In the ascending colon, they are thought to be located in the midcrypt, whereas in the descending colon, they are believed to be located at the crypt base. As for the small intestine, there are several stem cells per crypt, and they produce enterocyte, goblet, and enteroendocrine lineages. There are no Paneth cells in the adult intestine, perhaps because the stem cells have different potential or because an initiating signal is lacking.

Shedding cells

Villus

Epithelium Enterocytes Goblet cells Enteroendocrine cells

Epithelium Differentiation

Enterocytes Goblet cells Enteroendocrine cells

Differentiation

Proliferation

Proliferation Crypt Stem cells

Migration Crypts Migration

Paneth cells Stem cells

FIG. 10-5. Schematic of the crypt-villus structure of the small intestine. The epithelium is massively folded into finger-like villi and pocket-like crypts. The epithelial stem cells reside near the base of each crypt (white cells). Proliferative daughter cells variously migrate down to the crypt base and differentiate into Paneth cells or up toward the villus tip and differentiate into enterocytes, goblet cells, and enteroendocrine cells. Each crypt feeds a number of surrounding villi. (Modified from Sancho and colleagues [76], with permission, from the Annual Review of Cell and Developmental Biology, Volume 20 © 2004 by Annual Reviews www.annualreviews.org and from the author.)

FIG. 10-6. Schematic structure of the large intestine. The epithelial surface is packed with crypts but otherwise lacks the villus structure of the small intestine. The epithelial stem cells reside at or near the crypt base and give rise to enterocytes, goblet cells, and enteroendocrine cells. Paneth cells are not found in the large intestine. (Modified from Sancho and colleagues [76], with permission, from the Annual Review of Cell and Developmental Biology, Volume 20 © 2004 by Annual Reviews www.annualreviews.org and from the author.)

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 297 In the stomach, the epithelium forms deep tubular glands in the stomach wall, each gland with several different zones: the foveolus, isthmus, neck, and base (146). The cells lining the epithelium comprise different types of secretory cells; gastric foveolar cells of the mucosal surface and foveolae secret mucous, whereas the chief or peptic cells of the neck and isthmus secrete pepsinogen. Acid-secreting or parietal cells are found in the body compartment of the stomach at the base of the glands, endocrine cells are located in the fundus and body, and gastrin-producing cells are predominantly found in the antral mucosa. The stem cells are believed to reside in the middle of the glands in the vicinity of the neck and isthmus. Thus, the type of differentiation is connected with the direction of migration from the progenitor pool.

Notch in the Developing Gut The development of the primitive gut tube along the anteriorposterior axis is known to be regulated by Hedgehog and BMP signals (141,148), and the involvement of Notch at this stage has not been studied. Until recently, the mechanisms that regulate the development of the gut tube in the dorsoventral axis have not been well defined. It is now known through studies of cell fate specification in the Drosophila hindgut that Notch signaling plays an important instructive role in this patterning (142). A single row of boundary cells separates the dorsal and ventral cells in the hindgut. Notch is exclusively expressed in presumptive boundary cells (which lack Delta), and Notch signaling is activated on interaction with Delta expressed by adjacent ventral cells. This results in expression of steroid receptor genes and Crumbs, an apical polarity organizer, in the boundary cells that determines their fate. How the Delta expression domain is established in the ventral cells is unknown. A number of survey studies show that genes involved in Notch signaling are widely expressed throughout embryonic and adult gut, with varying spatial and temporal patterns, which is indicative of a variety of roles in intestinal function (77,134,149,150). The earliest stage in mouse embryonic gut development investigated for the expression of Notch receptors, ligands, and downstream signaling molecules is E13.5 (134). Schroder and Gossler (134) undertook a comprehensive survey of expression for Notch1-4; the ligands Jagged1, Jagged2, and Delta1-3; the three modifying glycosyltransferases of the Fringe family; and the downstream targets Hes1, Hes5, Hes6, and Hes7 at E13.5, E18.5, and postnatal day 25 by nonradioactive in situ hybridization. These experiments were the first to provide important spatial information on the expression of Notch genes in the gut, although the low sensitivity of the technique compromised the landscape view and is probably an oversimplification. The genes were variously expressed in the gut epithelium and in the mesenchyme, sometimes throughout each compartment and sometimes in discrete cells, pointing to complex and perhaps redundant interactions between the various receptors and ligands. At E13.5, when the gut is a relatively simple tube, most Notch pathway genes appeared to be expressed more

abundantly in the mesenchyme compared with the epithelium. Delta1 was expressed in mesenchymal cells adjacent to the epithelium and in scattered cells around the periphery of the mesenchyme. A knock in of LacZ into the Delta1 locus confirmed high Delta1 expression in the circular and longitudinal muscle layers in the mesenchyme and revealed a similar pattern in the fetal stomach (151). Delta3 and Delta4 were expressed more discretely in the E13.5 gut tube; Delta3 was weakly expressed in rare, scattered cells at the periphery of the mesenchyme, whereas Delta4 appeared to be more abundant in cells that overlapped the Delta1 and Jagged expression domains. Jagged1 and Jagged2 were primarily expressed in a mesenchymal layer beneath Delta1. All Notch receptors appeared to be expressed in low amounts; Notch1-3 were expressed in both the epithelium and mesenchyme, whereas Notch4 was restricted to the mesenchyme. Notch1 and Notch4 were expressed in a similar pattern to Delta4. The expression domains of the Fringe modifier genes and the Hes target genes generally overlapped with receptor or ligand expression. These expression domains indicated involvement of Notch signaling in diverse developmental processes in the epithelium and mesenchyme from an early age. At E13.5, the gut tube mesenchyme contains a variety of different cell types that are developing into muscle, angiogenic, and neuronal structures. Although the spatial distribution of the Notch components was consistent with a role for Delta1 in muscle development, and a role for Delta4 in angiogenesis was inferred based on expression in other tissues (42), it is important to test these assumptions by examining coexpression with diagnostic markers. At E18.5, the gut tube is undergoing dramatic changes in organization as the epithelium folds into villi-like extensions that project into the lumen. The intervillus epithelium, which is reshaped over the next few days into the crypts characteristic of the adult intestine, contains proliferating cells and in all probability the intestinal epithelial stem cells. There is a clear demarcation in Notch receptor and ligand expression at this time. Notch1 and Notch2 are expressed in a few cells in the intervillus epithelium, where they are partnered by Delta1 (134). That they are involved in the process of cell renewal seems certain, but at what stage this occurs is unclear. The other Notch receptors and ligands are expressed in the lamina propria or submucosa. Expression of the Notch target gene, Hes1, is largely confined to the intervillus epithelium, whereas Hes5 and Hes6 are mainly observed in the villus epithelium. Immunohistochemical detection of Hes1 protein shows that is restricted to the villi at E18 (73,77) and is not in the proliferative intervillus cells where Hes1 messenger RNA (mRNA) has been detected (134). These studies suggest the Hes genes and proteins may mediate the effects of Notch signaling that commenced in the intervillus region. In their study of Hes1 knockout mice, Jensen and colleagues (77) recorded the expression of several Notch signaling genes in the developing intestine of E18 mice. Where genes were expressed, these authors saw little change in their expression in intestinal compartments ranging from the stomach to the colon. Hes2 and Hes3 were absent from embryonic gut, and in contrast with the in situ localization studies of Schroder

298 / CHAPTER 10 and Gossler (134), Jensen and colleagues (77) detected Delta3 but not Hes5. Different findings in these studies may be attributable to the relative strengths of the different detection techniques used. Jensen and colleagues (77) used reverse transcriptase-polymerase chain reaction to detect mRNA expression in segments of gut, whereas Schroder and Gossler (134) used in situ hybridization. The Notch signaling genes and proteins are invariably expressed at low levels, and the detection techniques often need to succeed at their highest level of sensitivity. Thus, probe optimization, “macroscopic” versus “microscopic” tissue analysis, and regional variation can all contribute to apparent discrepancies in expression data. The use of knockin mice to express a readily detectable and long-lived reporter protein from the endogenous promoter can be particularly effective, as demonstrated for studies of Delta1, Math1, and neurogenin3 expression (73,74,151). At postnatal day 25, the gut resembles its adult form, with typical villus and crypt architecture. At this time, Notch receptor expression in the epithelium is restricted to the crypts where Notch1 appears to be the major active receptor. Notch2 is weakly and diffusely expressed in the crypts, whereas Notch3 and Notch4 are not expressed there. However, Notch3 and Notch4 are expressed in the vasculature of the mesenchyme, a pattern that has continued throughout gut development. Of the ligands, Delta1, Delta4, and Jagged1 are expressed in individual cells in the crypt epithelium, and Delta4 and Jagged1, together with Jagged2, are also expressed in the vasculature beneath the crypts. Hes1, Hes5, Hes6, and Hes7 are all expressed in the crypt epithelium, and as at E18.5, Hes5 expression extends up the villi. Hes5 is also expressed in the mesenchyme. Hes1 protein expression is also confined to the crypts in adult mice and is in direct contrast with what is seen in late embryonic (E17) gut, where it is present in the differentiating villus cells (73,77). This transition may occur just before birth, as Kayahara and colleagues (152) report that Hes1 protein is present in the intervillus region at postnatal day 1. Hes1 expression in nonproliferating villus cells at E17 suggests that it does not control the choice between proliferation and differentiation at this stage, whereas its expression in adult crypts implies it might have a role in what is considered to be the more classical aspect of Notch function. Work in our laboratory confirmed the presence of transcripts for Notch1-3, Jagged1, Jagged2, and Delta1 in adult rat gut from the esophagus through all the distinctive compartments of the stomach and small intestine to the large intestine (149). In situ hybridization techniques located Notch1 (Fig. 10-7), Notch2, Jagged1, and Jagged2 in a variety of cells in these tissue compartments. In the small intestine, they were abundantly expressed in the lower parts of the crypts, with the exception of Notch2, which was present in only a few crypt cells. The nature of these cells was not clear, and in the colon, Notch2 was not detected at all. The expression of Notch receptors and ligands in cells of the lamina propria and enteric nervous system (150) point to the much wider involvement of Notch signaling in gut function, extending beyond its role in epithelial function.

Notch in the Enteroendocrine System Endocrine cells in the gut epithelium are the largest population of hormone-producing cells in the body (136), and unlike other organs in which endocrine cells are found, enteroendocrine cells are scattered among other differentiating cell types. This necessitates an additional degree of control over differentiation of enteroendocrine cells from progenitor cells. It has been known for many years that enteroendocrine cells derive from common progenitor cells in the intestinal crypts, yet the mechanisms have remained elusive. Exciting research using several different strains of knockout mice now implicate the Notch signaling pathway as a critical player in the differentiation of enteroendocrine cell lineages in the gut (73–79). More than 10 different enteroendocrine cell types are found in the gastrointestinal tract of mammals based on their hormone profile and their ultrastructural appearance (153). Their distribution can vary markedly. Some occur throughout the gastrointestinal tract, some are found mainly in the stomach and small intestine, and yet other enteroendocrine cell types preferentially occur in the large intestine. Early hints of a role for Notch signaling in forming these lineages came from mice in which a bHLH transcription factor, BETA2, had been ablated (78). BETA2, also known as NeuroD, is a member of the proneural class of bHLH genes related to the Achaete-Scute genes of Drosophila, which are regulated by Notch (62,154). The BETA2-deficient mice acquired severe diabetes and died perinatally. Development of pancreatic islets appeared to be arrested between E14.5 and E17.5, a stage characterized by expansion of the insulinproducing β cell population. They lacked enteroendocrine cells expressing secretin or cholecystokinin, although they did make endocrine cells expressing other hormones. BETA2 interacts with the p300 transcriptional coactivator to regulate transcription of the secretin gene and the gene encoding the cyclin-dependent kinase inhibitor, p21 (79). By examining the effect of overexpression of BETA2 on secretin production and apoptosis in cell lines, Mutoh and coworkers (79) proposed that BETA2 coordinates transcriptional activation of the secretin gene and cell cycle arrest in the intestinal epithelium. In a different approach, Rindi and colleagues (75) used other sophisticated gene technology that specifically ablated three types of enteroendocrine cells in the mouse gut. Using the promoter of the secretin gene that is expressed by a subset of enteroendocrine cells, they made transgenic mice in which secretin-producing cells could be ablated by addition of the antiviral agent gangciclovir. Identification of endocrine cells in these mice showed loss of secretin and cholecystokinin- and peptide YY/glucagon-expressing cells. Reduced numbers of enteroendocrine cells producing gastric inhibitory peptide, substance P, somatostatin, and serotonin were also observed. They showed that BETA2 was expressed in enteroendocrine cell but not nonendocrine cells. Thus, BETA2 is a marker for enteroendocrine cells and is essential for secretin and cholecystokinin expression, but it is not

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 299

A

B

C

FIG. 10-7. Expression of Notch1 in adult duodenum. Specific radioactive riboprobes for Notch1 were hybridized to adult rat duodenal sections and developed; sections were counterstained with hematoxylin. (A) Bright field images; (B, C) dark field images. Notch expression is indicated by white grains in the dark field images. (C) This panel has been manipulated in Photoshop to remove the gray background cast by the stained tissue in B, which makes it difficult to distinguish the expression. Notch is expressed in the crypt region (bracketed) and in the lamina propria (arrow) and epithelium of the villus (arrowhead).

required for development of all enteroendocrine cells, indicating that there are at least two types of multipotent enteroendocrine progenitor cells. Direct involvement of Notch signaling in the development of endocrine cells has been provided by knockout of Delta1 and CSL, the transcription factor that mediates NICD function in the nucleus (155). Knockout of either of these genes resulted in accelerated differentiation of pancreatic endocrine cells. Knockout of Hes1, one of the immediate Notch target genes, provides further evidence for the role of Notch signaling in enteroendocrine development in the gut (77). A deficiency in Hes1 produces abnormal endocrine differentiation in the gastrointestinal tract and pancreatic hypoplasia. Normal enteroendocrine cell differentiation begins in the antral region of the stomach at E14.5, followed in the intestine at E16.5. Hes1 deficiency produced an increase in the number of enteroendocrine cells producing glucagon, cholecystokinin, gastrin, and somatostatin. The proportion of goblet cells was increased, whereas there were reduced numbers of enterocytes; these effects ultimately distorted villus morphology. Interestingly, although mitotic activity of E17 epithelium appeared unchanged, there was increased apoptosis at E19, which, surprisingly, was apparent in the crypts. In the knockout mice, there was an increase in expression of two Notch ligands, Delta1 and Delta3, and several bHLH transcriptional activators, principally Mash1, NeuroD/BETA2, and neurogenin3, in the gastrointestinal tract. These are consistent

with the notion that Hes1 inhibits Notch signaling via two mechanisms, feedback inhibition of the activating ligands and direct inhibition of Notch bHLH transcriptional activators, a hallmark of the lateral inhibition hypothesis for Notch signaling. Thus, Hes1 may act as a general repressor of bHLHcontrolled enteroendocrine development. Work by Lee and colleagues (74) extends our knowledge of enteroendocrine cell development, shedding light on their development in the stomach and the potential involvement of Notch in that process via the bHLH transcriptional activator, neurogenin3. Neurogenin3 lies downstream of Notch and is a target for repression by the Notch target gene, Hes1. Hes1 negatively regulates neurogenin3 through repression of its promoter (156). Knockout of neurogenin3 causes severe diabetes and death at postnatal day 3 (157). Neurogenin3 mice lack glucagon-secreting cells, somatostatin-secreting cells and gastrin-secreting cells in the glandular stomach. Serotonin-expressing enterochromaffin cells are not absent but are dramatically decreased. Similar effects occur in the pancreas. In addition to enteroendocrine cells, two other types of secretory cells are found in the gut epithelium, mucinproducing goblet cells and antimicrobial, peptide-secreting Paneth cells. Paneth cells differ in two notable respects from all other differentiated cells of the gut epithelium in that they migrate to the base of the crypt as they mature, where they reside for approximately 20 days before being shed.

300 / CHAPTER 10 They are thus much longer lived and migrate in a different direction to all other differentiating gut epithelial cells. A molecular explanation for the origin of these lineages has remained elusive. Knockout of another bHLH gene, Math1, a downstream target for Notch signaling, now provides insight into how differentiation of secretory lineages in the gut is coordinated (73). Math1 was previously known to be involved in cell fate determination in the nervous system (158,159). In using a LacZ knockin at the Math1 locus, Yang and colleagues (73) were able to carefully delineate where Math1 was expressed. In the gastrointestinal tract, Math1 expression is turned on specifically in the epithelium of the small and large intestine from E16.5 (73). Interestingly, it is not expressed in the stomach, which also has a number of resident secretory cell types. Math1 is abundantly expressed in the proliferative compartment of the gut epithelium of embryonic and adult mice, with scattered positive cells in the villi. Because LacZ staining is cytoplasmic, they were able to readily double stain for a nuclear proliferative marker, Ki-67, to precisely correlate Math1 expression with proliferation. This revealed that Math1 was expressed in most but not all proliferative cells, from the 4th to the 13th cell position in the crypts of the small intestine and from the 2nd to 4th position in the colon. Using a battery of morphologic, histologic, and immunohistochemical tests, Yang and colleagues (73) confirmed that Math1 is expressed in goblet, Paneth, and enteroendocrine cells, and that all these cells are absent in the knockout mice. Because enterocyte differentiation appears unaffected, Math1expressing progenitor cells appear to give rise to secretory cell lineages, and Math1-negative proliferative cells give rise to enterocytes. As with previous knockout studies of this family of bHLH genes, loss of Math1 decreased expression of one of the Notch ligand genes, Delta3, and another bHLH gene, NeuroD/BETA2. In contrast, expression of Notch1, Notch2, Notch3, Notch4, Delta1, or Hes1 was not affected. These effects are consistent with a model in which Math1 is upstream of NeuroD/BETA2 and provides positive feedback to down-regulate Delta3 and Notch signaling. Figure 10-8 outlines a simple model for specification of the four major types of differentiated cells in the intestine, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. It is suggested that they arise through a series of binary decisions mediated by the Notch signaling pathway. Simply, a stem cell produces two cells, one of which retains its “stemness” and a daughter cell that adopts a transit-amplifying phenotype and gives rise to all other lineages. There is circumstantial evidence for Notch involvement in the earliest binary decision. An RNA-binding protein known as Musashi1 has been found to have a central role in the maintenance of neural stem cells (160–163) and perhaps in breast epithelial stem cells (164). Musashi1 is expressed in a few select cells in the intestinal crypt at positions defined by labeling studies as likely sites for intestinal epithelial stem cells (152,165,166). Notch and Hes1 are abundantly expressed in the crypt (134,149), and both expression domains overlap with Musashi1. Musashi1 has also been shown to down-regulate

Secretory lineages Paneth

Goblet

Absorptive lineage

Enteroendocrine (10)

Notch?

Enterocyte

NeuroD/BETA2

Notch? Ngn-3

Notch? Math-1+ Math-1−

Math-1− Math-1+ Stem Notch? Stem

FIG. 10-8. Potential role of Notch signaling in intestinal epithelial differentiation. This model proposes that the four major cell lineages of the intestine arise from common pluripotent stem cells through binary cell decisions potentially mediated by Notch signaling. It is likely that Notch signaling activates the basic helix-loop-helix (bHLH) transcription factor Hes1, which antagonizes Math1, another member of the same superfamily. Expression of Math1 commits progenitor cells to a secretory cell fate, whereas Math1−ve cells become enterocytes. Subsequent expression of neuorogenin3 and neuroD/BETA2 in Math1+ve cells is necessary for specification of various enteroendocrine cell types. (Modified from Sancho and colleagues [76], with permission from Elsevier and the author.)

Numb, a Notch antagonist, thereby promoting Notch signaling (93,167). In the context of the intestine, the stem cells are predicted to express Notch, a Notch ligand, Musashi1, and Hes1, promoting autonomous Notch activity and inhibiting downstream transcriptional activators, such as Math1 and NeuroD/BETA2. The transit-amplifying cells are predicted to express Notch and Hes but not Musashi. The net result of this is that Hes continues to inhibit Math1 and NeuroD/BETA2 but results in negative feedback regulation of the Notch ligand. This stops autonomous Notch signaling and instead promotes polarization of Notch receptor and ligand expression on adjacent cells, effectively establishing lateral Notch signaling between adjacent cells. Cells expressing Notch and Hes continue to block Math1 and NeuroD/BETA2 induction and adopt an enterocyte fate. Adjacent cells expressing ligand down-regulate Notch and Hes, Math1 is derepressed, and its expression commits these cells to the secretory lineage. Subsequent activation of NeuroD/BETA2 in this lineage provides further specification in the development of enteroendocrine cell fates, principally the secretin, cholecystokinin, and L-cell lineage. Conceivably, other transcriptional activators from the large bHLH family may be regulated by Notch signaling to dictate the individual enteroendocrine cell types.

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 301 Notch in the Mucosal Immune System The intestine is a major T- and B-cell organ, estimated to contain about half the T cells and about 80% of all B cells in the body (137,138). These cells reside in the gut epithelium, the underlying lamina propria, and specific gut-associated lymphoid structures, such as Peyer’s patches, cryptopatches, and lymphocyte-filled villi. Much work has shown that Notch signaling is essential in the development of T and B cells (168), but its involvement in the mucosal immune system is relatively unexplored. In their in situ hybridization study of Notch receptors and ligands in the adult rat gastrointestinal tract, Sander and Powell (149) noted their expression in the lamina propria and Peyer’s patches. Notch1 was expressed at a low level in Peyer’s patches, in the coronal zone just beneath the follicle-associated epithelium, which typically contains T cells. Notch2 was more abundant and expansive in the corona and germinal center, the latter predominantly containing B cells. Neither of the two ligands investigated, Jagged1 or Jagged2 appeared to be expressed in Peyer’s patches; however, Jagged1 was expressed in scattered cells in associated high endothelial venules. Notch1 is also expressed in the lamina propria of the small and large intestines, whereas Notch2 is present only in the lamina propria of the large intestine. A downstream Notch regulator, Deltex (see Deltex section earlier in this chapter), has also been discovered in sheep Peyer’s patch B cells (169). These studies provide tantalizing evidence for a role for Notch in the adult mucosal immune system and warrant further investigation. In lymphocyte development, the decision to adopt a T- or B-cell fate involves Notch signaling. This requirement has been shown in a series of elegant experiments based on conditional inactivation of Notch signaling in mice expressing Cre recombinase under the control of the interferon-αregulated Mx promoter. Inducible inactivation of the Notch1 receptor or the CSL gene that mediates signaling by all Notch receptors results in a block in the development of T cells and ectopic development of immature B cells in the thymus (168,170–173). Wilson and colleagues (170) specifically investigated the effect of the inducible inactivation of Notch1 on the development of extrathymic T cells in the gut using a bone marrow reconstitution assay. Because inactivation of Notch1 was nearly completely effective in the bone marrow but only about 60% in the mice, they used a competitive, mixed bone marrow chimera assay to investigate the effect on T-cell production. They found a 10- to 15-fold decrease in T-cell receptor αβ and γδ T cells in the gut as a result of Notch1 knockout, confirming that Notch1 is necessary for development of extrathymically derived gut T cells. Because CD117+ lineage–negative T cells located in gut cryptopatches have been described as potential intraepithelial T-cell precursors (174), they then examined their relative abundance in the gut and thymus of reconstituted chimeras. In the gut of Notch1-deficient mice, they were reduced twofold, compared with ninefold in the thymus. Thus, Notch signaling is essential for the development of T cells in the thymus and extrathymic T cells in the gut. One model put

forward proposes that bipotential T-/B-cell progenitors migrate from the bone marrow to the thymus or the gut, at which locales Notch signaling is initiated and T-cell development continues. In addition to the classic functions of Notch in regulating development and cell fate selection, new insights into an ongoing role for Notch in lymphocyte function are emerging that are relevant to the gut. These studies reveal that Notch signaling can regulate T-cell receptor–mediated T-cell proliferation and apoptosis, development of Th1/Th2 immune responses, and the decision between immunity and tolerance (175–180). Jehn and colleagues (175) showed that Notch1 has an antiapoptotic capacity in T cells, where it is able to interact with a member of the orphan nuclear receptor family that normally orchestrates T-cell receptor–mediated apoptosis. In other work, the same group showed that Notch is required for T-cell receptor–mediated activation of peripheral CD4 and CD8 T cells (176). Engagement of the T-cell receptor resulted in Notch synthesis and activation and appeared to have a direct effect on interferon-γ production, specifically in CD8 T cells. Hoyne and colleagues (177) provide data that Notch signaling regulates the decision between immunity and tolerance. They showed that Notch1 and Jagged1 were expressed on peripheral CD4 and CD8 T cells and dendritic cells in the spleen of naive mice. Using retroviral gene transfer to constitutively express Jagged1 on antigen-presenting cells, they then showed that peripheral naive CD4 T cells differentiated into regulatory T cells, which were able to transfer tolerance to naive recipient mice. If blockade of Notch signaling in the receiving T cells could be shown to prevent development of regulatory T cells, that would add further weight to these provocative findings. Current work now also highlights a role for Notch signaling in mediating Th1 and Th2 responses to infection (179,180). Th1 and Th2 T cells are derived from CD4 T cells. Th1 cells mediate immunity to bacteria and viruses, characteristically secreting interferon-γ, whereas Th2 cells mediate immunity to multicellular pathogens, characteristically via secretion of interleukin-4 (IL-4), a key cytokine that is also involved in their differentiation (181,182). T cells do not directly recognize pathogens, but instead depend on dendritic cells to pass on this “information,” in the process initiating development of a Th1 or Th2 response. Amsen and coworkers (179) showed that dendritic cells engineered to express Delta induced a Th1 response by interacting T cells, whereas Jagged induced a Th2 response. They further showed that the IL-4 promoter contains several binding sites for the Notch effector, CSL, and thus provide a mechanism through which Notch signaling could initiate Th2 differentiation. Taken together, these studies indicate that Notch signaling can have profound effects on lymphocyte function, controlling the balance of differentiation of CD4 T cells into Th1 or Th2 lineages and regulating their proliferation and survival. Although these capabilities are yet to be specifically linked to functioning of the mucosal immune system, they have obvious implications for this major lymphocyte reservoir in its response to enteric infection.

302 / CHAPTER 10 Notch in the Enteric Nervous System Notch signaling has been well studied in the central nervous system, where it is known to have key roles in the maintenance and differentiation of neural precursor cells (183), whereas its role in the enteric nervous system is largely unexplored. The enteric nervous system is an independent and complex nervous system that controls gastrointestinal motility reflex and has a major role in regulating water and electrolyte transport and intestinal blood supply. The enteric nervous system is derived from the neural crest, and its precursors migrate along defined pathways to the gut (184). These precursors remain pluripotent even after colonizing the gut (185). There are more than 14 different neuronal cell types in the enteric nervous system (139,140) that appear to be derived from at least 2 precursor lineages: a sympathoenteric lineage that expresses the bHLH transcription factor and Notch target gene, Mash1, and a sympathoadrenal lineage that does not express Mash1 (186,187). Mash1 is the mammalian homolog of the Achaete-Scute complex of Drosophila, which participates in neurogenesis in the central and peripheral nervous systems. In Drosophila, Mash1 and Notch have been shown to interact. The vertebrate homologs show similar expression patterns in the developing brain and retina, suggesting that both genes participate in the development of vertebrate neural lineages (188). Mash1 knockout mice lack sympathetic and enteric serotenergic neurons and enteric neurons in the esophagus and die soon after birth (186). Enteric neurons of the stomach and small intestine are only partly affected. During development in Mash1 knockout mice, neural crest cells migrate to their correct positions but do not differentiate into neurons (189). These studies implicate Notch signaling in the development of enteric neurons. Notch signaling is traditionally associated with development and developmental decisions that lead to diversification of cell fates; however, it also has a role in postmitotic cells. Berezovska and colleagues (190) were the first to show that Notch is expressed in mouse and human adult brain, and subsequently, Sestan and coworkers (191) showed that Notch is involved in regulating the capability of neurons to elaborate and extend neurites. They found that formation of neuronal contacts activated Notch signaling, leading to restriction of neuronal growth and an arrest in maturation. Increased Notch activity was concomitant with an increase in the number of interneuronal contacts and cessation of neurite growth, whereas inhibition of Notch signaling promoted neurite extension. With the report by Sander and colleagues (150), we now know that Notch is involved in functioning of the adult enteric nervous system. They documented differential expression of Notch receptors and ligands in adult rat gut and used immunohistochemical markers to correlate their expression with specific neuronal subtypes. Notch1 and Jagged2 but not Notch2 or Jagged1 were detected in ganglia of the submucosal and myenteric plexus. Notch1 was expressed by cholinergic neurons lacking calretinin or nitric oxide synthase, and

therefore may be restricted to neurons that elaborate polarized projections to the intestinal muscle layers (192,193). In contrast, Jagged2 was more widely expressed than Notch1, prompting speculation that other Notch receptors may have roles in the enteric nervous system.

FUTURE DIRECTIONS In the years since the first Notch gene was cloned, we have come to appreciate how important the Notch signaling pathway is in biology. Our increasing knowledge of its interaction with other signaling networks heralds a better understanding not only for how cell fate is determined by this pathway, but also for the impact of Notch signaling on other cellular functions. There is still much to learn, and that is no more evident than when one considers the complexity of the gastrointestinal tract and the diversity of cells that express Notch. We are really only at the beginning of understanding Notch function in the gastrointestinal tract, but the few studies that have been done reveal the broad scope of Notch involvement in the gut. Current evidence implicates Notch in the development of intestinal epithelial cell lineages, but its role remains to be proven formally by direct manipulation of receptor or ligand activity. To better understand the extent of Notch signaling in the gut, we need to better define the cells in which the Notch receptors and its ligands are expressed. Given the low level of endogenous Notch expression, expanded application of knockin technology to monitor expression of Notch gene promoters with sensitive and stable reporters is expected to provide more precise data on expression. Is Notch expressed in intestinal epithelial stem cells? What are the cells in the lamina propria, in the Peyer’s patch, or in the enteric nervous system that express Notch, and what role does Notch play in them? With this knowledge in hand, studies of Notch function in gut cell lines and targeting of inducible gene knockouts to specific gut cells are needed to help map the mechanisms and consequences of Notch action in the gut.

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306 / CHAPTER 10 154. Sriuranpong V, Borges MW, Strock CL, Nakakura EK, Watkins DN, Blaumueller CM, Nelkin BD, Ball DW. Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol 2002;22: 3129–3139. 155. Apelqvist A, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, Hrabe de Angelis M, Lendahl U, Edlund H. Notch signalling controls pancreatic cell differentiation. Nature 1999;400:877–881. 156. Lee JC, Smith SB, Watada H, Lin J, Scheel D, Wang J, Mirmira RG, German MS. Regulation of the pancreatic pro-endocrine gene neurogenin3. Diabetes 2001;50:928–936. 157. Gradwohl G, Dierich A, LeMeur M, Guillemot F. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A 2000;97:1607–1611. 158. Bermingham NA, Hassan BA, Wang VY, Fernandez M, Banfi S, Bellen HJ, Fritzsch B, Zoghbi HY. Proprioceptor pathway development is dependent on Math1. Neuron 2001;30:411–422. 159. Ben-Arie N, Hassan BA, Bermingham NA, Malicki DM, Armstrong D, Matzuk M, Bellen HJ, Zoghbi HY. Functional conservation of atonal and Math1 in the CNS and PNS. Development 2000;127:1039–1048. 160. Okano H, Imai T, Okabe M. Musashi: a translational regulator of cell fate. J Cell Sci 2002;115:1355–1359. 161. Sakakibara S, Okano H. Expression of neural RNA-binding proteins in the postnatal CNS: implications of their roles in neuronal and glial cell development. J Neurosci 1997;17:8300–8312. 162. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H. Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 2000;22:139–153. 163. Sakakibara S, Nakamura Y, Yoshida T, Shibata S, Koike M, Takano H, Ueda S, Uchiyama Y, Noda T, Okano H. RNA-binding protein Musashi family: roles for CNS stem cells and a subpopulation of ependymal cells revealed by targeted disruption and antisense ablation. Proc Natl Acad Sci U S A 2002;99:15194–15199. 164. Clarke RB, Anderson E, Howell A, Potten CS. Regulation of human breast epithelial stem cells. Cell Prolif 2003;36(suppl 1):45–58. 165. Nishimura S, Wakabayashi N, Toyoda K, Kashima K, Mitsufuji S. Expression of Musashi-1 in human normal colon crypt cells: a possible stem cell marker of human colon epithelium. Dig Dis Sci 2003;48: 1523–1529. 166. Potten CS, Booth C, Tudor GL, Booth D, Brady G, Hurley P, Ashton G, Clarke R, Sakakibara S, Okano H. Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation 2003;71:28–41. 167. Imai T, Tokunaga A, Yoshida T, Hashimoto M, Mikoshiba K, Weinmaster G, Nakafuku M, Okano H. The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA. Mol Cell Biol 2001;21: 3888–3900. 168. Radtke F, Wilson A, MacDonald HR. Notch signaling in T- and B-cell development. Curr Opin Immunol 2004;16:174–179. 169. Gupta-Rossi N, Storck S, Griebel PJ, Reynaud CA, Weill JC, Dahan A. Specific over-expression of deltex and a new Kelch-like protein in human germinal center B cells. Mol Immunol 2003;39:791–799. 170. Wilson A, Ferrero I, MacDonald HR, Radtke F. Cutting edge: an essential role for Notch-1 in the development of both thymusindependent and -dependent T cells in the gut. J Immunol 2000;165: 5397–5400. 171. Wilson A, MacDonald HR, Radtke F. Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J Exp Med 2001;194:1003–1012. 172. Radtke F, Wilson A, Stark G, Bauer M, van Meerwijk J, MacDonald HR, Aguet M. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 1999;10:547–558.

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CHAPTER

11

Physiology of Gastrointestinal Stem Cells Alda Vidrich, Jenny M. Buzan, Sarah A. De La Rue, and Steven M. Cohn What Is a Stem Cell? 308 Organization of Structural/Proliferative Units in the Gastrointestinal Epithelium, 308 Small Intestine, 308 Colon, 308 Gastric Epithelium, 308 Morphogenesis of the Gastrointestinal Proliferative Units, 309 Clonality of the Structural/Proliferative Unit, 310 Studies of Chimeric Organisms, 310 X-Chromosome Inactivation, 312 In Vivo Stem Cell Mutagenesis Assays, 312 Stem Cell Number and Hierarchy, 313

Stem Cell Number, 313 Stem Cell Hierarchy, 314 Regulation of Stem Cell Number, 314 Putative Stem Cell Markers, 315 The Stem Cell Niche, 315 Regulation of Stem Cell Function, 316 Signaling Pathways, 316 Growth Factors and Peptides, 327 Inflammation: Cytokines and Other Mediators, 332 Stem Cell Plasticity, 335 Conclusion, 336 Acknowledgments, 336 References, 336

Epithelia, such as those lining the gastrointestinal tract, are able to undergo continuous self-renewal and repair because they contain a population of undifferentiated stem cells and long-lived progenitor cells that have a large capacity for selfrenewal and can give rise to daughter cell lineages that differentiate into the mature epithelial cell types needed for normal function of the epithelial layer. The “Unitarian hypothesis,” which advanced the concept that all of the differentiated cell types found in the small intestinal and colonic epithelia arise from a common multipotent progenitor or “stem” cell located near the base of each intestinal crypt of Lieberkühn was first proposed by Cheng and Leblond in 1974 (1–5). In the ensuing decades since this seminal work, investigators have further refined our knowledge of the role that epithelial stem cells play in normal gut development, in epithelial regeneration associated with mucosal injury or inflammation, and in intestinal neoplasia. A number of functional and kinetic properties of intestinal stem cells have also been defined from studies of the topographic arrangement of differentiated stem cell descendents in aggregation

chimeras (tetraparental mice), from in vivo mutagenesis studies, and from the radiobiologic response of intestinal crypts after γ-irradiation (6–16). However, the most dramatic progress in deciphering many of the molecular pathways involved in regulating these processes has been made through advances in our ability to experimentally manipulate the expression of genes in transgenic and knockout mice. The goal of using stem cells as a therapeutic modality for the restoration and repair of diseased organs has captured the imagination of both scientists and the lay public. However, the possibility of isolating and manipulating embryonic stem cells first became a reality more than 20 years ago, with the successful growth of murine embryonic stem cells in culture (17,18). The derivation of embryonic stem cell lines from human blastocysts was realized more recently (19,20). The potential therapeutic uses of human embryonic stem cells have met with significant opposition based on ethical considerations. Nevertheless, a number of human embryonic stem cell lines have been produced. Opposition to the generation of human embryonic stem cells has fueled the investigation of stem cells from adult tissues. Although obviously promising, these studies are hampered by the lack of identifiable stem cell markers and limited knowledge of the requirements for growth and maintenance of these cells in culture. Nevertheless, in vivo studies using knockout mice and mice expressing a variety of

A. Vidrich, J. M. Buzan, S. A. De La Rue, and S. M. Cohn: Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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308 / CHAPTER 11 mutated genes have begun the process of identifying and characterizing the genes, signals, and pathways that regulate epithelial stem cells in their native tissue environment or niche. These have been supplemented by studies of human diseases with underlying gene mutations that alter stem cell fate or function. Understanding the biology of stem cells, both embryonic and adult, is of fundamental importance to the studies of tissue and organ development, the homeostatic maintenance of renewing tissues, tissue repair, and intestinal neoplasia, as well as advancing developments in tissue engineering. This chapter reviews the current knowledge pertaining to the regulation of epithelial stem cells of the gastrointestinal tract during both gut morphogenesis and maintenance of intestinal homeostasis.

WHAT IS A STEM CELL? Certain tissues of the body display continual turnover because cells lost through injury or terminal differentiation must be replaced to maintain the functionality of the tissue. The rate of cell replacement changes to match the rate of cell loss so that the tissue can maintain homeostasis under either normal or perturbed conditions. The gastrointestinal tract is a prime example of a continuously and rapidly renewing tissue. Terminally differentiated cells of the intestine are shed from the villus tip into the gut lumen. These cells are replaced by enterocytes arising from the proliferation of rapidly amplifying transit cells, which are the progeny of stem cells residing in the bottom half of the crypts. Currently, there are no specific morphologic or molecular criteria for identifying epithelial stem cells in the gut. Although different characteristics have been attributed to the stem cell population in the literature, the definition of a stem cell advanced by Potten and Loeffler (16) is used for purposes of discussion in this chapter. They proposed that a number of characteristics should be used to identify stem cells, or the properties of “stemness” that specify the cells’ functional capability under a variety of circumstances (16). The properties defining intestinal epithelial stem cells include: (1) an undifferentiated cell with a relatively unlimited potential for proliferation, (2) the capacity for self-maintenance, (3) the ability to give rise to a variety of cell lineages (pluripotency), and (4) the ability to regenerate the entire epithelium in response to an injurious event.

ORGANIZATION OF STRUCTURAL/ PROLIFERATIVE UNITS IN THE GASTROINTESTINAL EPITHELIUM

and colonic epithelia arise from a common multipotent progenitor cell located in the lower region of the crypt or replicative unit of the intestine as first proposed by Cheng and Leblond (1–5). In this view of the organization of the crypt, stem cells are thought to reside in a restricted zone just above the Paneth cells at the crypt base. These stem cells have a high potential for self-renewal and a large potential for cell division. A small fraction of epithelial cells at this location (cell positions 4–5 from the crypt base) appear to replicate slowly (Tc > 30 hours) and are thought to represent the stem cell and long-lived progenitor cell populations (Fig. 11-1) (14,21,22). Transit-amplifying (TA) cells are more rapidly proliferating (Tc ~12–13 hours in the mouse) daughters of stem cells that undergo 4 to 6 divisions within a zone of proliferation located in the mid-crypt. Differentiation occurs as the progeny of the TA cell population migrate in vertically coherent and topologically constrained bands either onto the villus epithelium or toward the base of the crypt (1,16,23). Goblet cells and enterocytes undergo terminal differentiation as they are rapidly translocated onto the villus. Paneth cells arise during downward migration to the crypt base, while enteroendocrine cells differentiate during migration from the zone of proliferation in either direction. Despite the high turnover of cells in the adult small intestine, the sizes of the crypts and the villi remain constant. Thus, epithelial cell differentiation appears to be tightly coupled to cell migration, and the rates of cell renewal, migration, and cell loss must be tightly regulated and interrelated processes.

Colon The organization of the adult colonic epithelium is similar to the small intestine except that there are no villi in the colon. A pleuripotent stem cell located at or near the base of each colonic crypt gives rise to each of the three principal epithelial cell lineages present in the colon (Paneth cells are not found in the mouse colon under normal circumstances) (6,10). Rapidly replicating TA cells are found in the lower half of the crypt, and cell-cycle times in this region of the crypt have been estimated to be approximately 30 to 33 hours (24–27). As in the small intestine, the pleuripotent epithelial stem cell is thought to have a much longer cell-cycle time than the TA cell population (24). Cells differentiate during migration from the proliferative zone, which lies in the lower third of the colonic crypt, to a polygonal-shaped surface epithelial cuff that surrounds the orifice of each gland (9).

Gastric Epithelium Small Intestine In the small intestine, the progeny of replicating undifferentiated epithelial cells located in the crypts rapidly and continuously replace the differentiated epithelial cell population. All of the differentiated cell types found in the small intestinal

Patterns of gastric epithelial cell renewal in the mucous and zymogenic units of adult CD1 mice have also been defined by Lee and Leblond (28,29). Their studies suggest that granule-free stem cells located in the isthmus region give rise to daughter cells. These daughter cells differentiate

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 309

3 days Differentiated cells– enterocytes, goblet cells, enteroendocrine cells

Villus Crypt

Transitamplifying cells Tc~12_13 hrs

Pericryptal fibroblast

~150 cells

"Anchored" stem cell Tc > 30 hrs

Paneth cell

FIG. 11-1. Epithelial renewal and differentiation in the adult intestine. Each crypt contains one or more slowly cycling “anchored” clonogenic stem cells located at approximately cell positions 4 to 5 from the crypt base. One of the daughters of this stem cell eventually gives rise to long-lived progenitor cells and subsequently to the rapidly proliferating, undifferentiated transit-amplifying cell population. Enterocytes, goblet cells, and enteroendocrine cells differentiate during upward migration of epithelial cells onto the villi, whereas Paneth cells differentiate as they migrate to the crypt base.

as they migrate away from the isthmus in a bipolar fashion. In the peripyloric mucous gland, two types of exocrine cell precursors are produced. One type contains homogeneous dense mucous granules. These cells replicate several times, move upward into the pit region, and then migrate to the surface. The lifespan of these surface mucous cells is approximately 3 days. The other type of daughter cell possesses apical granules with dense cores. These latter cells migrate downward and subsequently differentiate into relatively longlived glandular mucous cells (28,29). Rapidly proliferating undifferentiated progenitor cells of zymogenic glands are also found in the isthmus. These undifferentiated cells are thought to give rise to immature forms of several cell types that subsequently undergo a complex bipolar migration. Surface mucous cells migrate upward to the luminal surface as their name implies. Parietal (acidsecreting) cells migrate downward to the neck and base of

the glands. Mucous neck cells also migrate downward and are believed to undergo successive transformation into mucozymogenic and then long-lived zymogenic cells. Enteroendocrine cells of the gastric epithelium arise from stem cells in the isthmus and join the downward migration into the glands (30).

MORPHOGENESIS OF THE GASTROINTESTINAL PROLIFERATIVE UNITS The molecular mechanisms that regulate intestinal development have been difficult to study in humans because many of these maturational and morphogenic events occur during the first trimester of prenatal development and are not accessible to easy experimental manipulation. In contrast to humans, rodents are altricial animals, thus many of

310 / CHAPTER 11 these same developmental events occur shortly before birth or during the suckling period and the suckling/weaning transition. This difference in the timing of developmental events has made the rodent an attractive model system for studying intestinal ontogeny. The rodent gastrointestinal epithelium undergoes a remarkable morphologic reorganization beginning in late gestation, changing from a poorly differentiated pseudostratified cuboidal epithelium to a simple stratified columnar epithelium (31–33). Nascent villi form by the upward growth of mesenchymal tissue into this monolayer of columnar epithelial cells. At birth, the proliferative unit of the mature intestinal epithelium, the crypt, has not yet formed (Fig. 11-2). The fetal and neonatal homolog of the crypt, the intervillus epithelium, is populated by the descendants of several pluripotent stem cells (7,10). Formation of the small intestinal crypts begins during early postnatal life by invagination of the intervillus epithelium into the underlying mesenchyme. This poorly understood process is completed by the suckling/weaning transition (postnatal day 14 in the mouse). Evidence suggests that, during the process of nascent crypt formation, the newly forming polyclonal crypt epithelium is converted to monoclonality (i.e., each crypt contains an epithelial cell population derived from a single multipotent stem cell; see Clonality of the Structural/Proliferative Unit later) (7). Morphogenesis of crypts in the colon resembles that of the small intestine but begins in late gestation (34–36). The number of glands in the stomach, small intestine, and colon expands greatly by a process of gland or crypt division (fission) beginning during the suckling period and extending through early (post-weaning) adulthood to reach the number of crypts present in the adult small and large intestine or glands present in the stomach (37). Since each crypt or gland is ultimately derived from at least one pluripotent stem cell, it Late suckling intestine

Newborn intestine

is likely that a marked expansion of the number of resident clonogenic stem cells within the gastrointestinal epithelium also occurs during this developmental period. During late fetal and early postnatal life, regional gradients of differentiation are also established along both the duodenal-colonic and the crypt-villus axes of the gut (6,38–43). Several studies have suggested that pericryptal fibroblasts in the mesenchymal tissue underlying the epithelium and secreted extracellular matrix components are important in regulating the establishment of region-specific differentiation programs (44–48).

CLONALITY OF THE STRUCTURAL/ PROLIFERATIVE UNIT The direct identification of stem cells in the gastrointestinal epithelium has been difficult due to the lack of specific morphologic or immunohistochemical markers that identify this unique and rare cell population within each intestinal crypt or gastric gland. Rather, the existence and functional properties of the parental stem or progenitor cell have been inferred by experimental characterization of the daughter lineages they produce. A number of studies have suggested that each adult crypt is monoclonal, that is, genetically derived from a single stem cell. These data come from a variety of experimental systems in which histochemical markers were used to identify the genetic origin(s) or tag lineages of cells within the crypt epithelium. The model systems used include allophenic tetraparental mice (7–9), use of X chromosome– linked enzyme markers (10,49–51), in vivo mutagenesis in mice heterozygous for cell-surface lectin markers (11,12), and studies tracing age-dependent extinction of transgene expression in the colon (6). In each of these systems (see later), the crypt behaves as if all of its cells are derived from a single multipotent stem cell. Pulse-chase labeling studies to investigate cell migration suggest that these stem cells reside at about the fourth cell position from the base of the crypt (14).

Studies of Chimeric Organisms Villi

Inter-villus epithelium

Replicating cells

Crypt

Stem cell

FIG. 11-2. Morphologic remodeling of the intestinal epithelium occurs in the suckling mouse. Arrows indicate cell migration pathways. Enterocytes, goblet cells, and enteroendocrine cells differentiate during migration onto the villus epithelium from the zone of replicating cells located midcrypt. Paneth cells differentiate during migration of progenitors to the crypt base.

Mouse embryo aggregation chimeras are formed from the fusion of two zygotes that differ in the expression of a marker for the tissue of interest. Adult mice derived from these embryonic chimeras are composed of tissues that are a mosaic of cells derived from two parental genotypes (7–9,52,53). This technique has provided many important insights into the clonal origins of epithelial cells in various tissues including the intestine. C57BL/6J Lac (B6) mice express binding sites for the lectin Dolichos biflorus agglutinin (DBA), whereas SWR mice do not. The intestines of C57BL/6J Lac (B6),SWR aggregation chimeras are mosaics of cells derived from the two parent strains. However, when binding of labeled DBA to crypt epithelial cells was examined histologically in these chimeric mice, crypts were composed of cells that were wholly positive or wholly negative for DBA staining, with no mixed

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 311 A

B

Villi

Crypt Pericryptal fibroblast Goblet cells Enterocytes

Paneth cells Enteroendocrine cells

CO

MO

−?

GLP-2

Stem cell

GLP-2R

Sign

al

Endothelial cell

Enteric neuron

Crypts

FIG. 11-3. The intestinal stem cell niche. (A) Illustration of the stem cell hierarchy of the crypt. (See Color Plate 2.) Chimeric mice can be generated by injecting pluripotent embryonic stem cells from one genetic background into blastocysts representing another genetic background. The small intestine of the resulting mouse contains cells representing both genetic backgrounds. The image on the left is a section prepared from the proximal small intestine of an adult chimeric mouse. The finger-shaped villus is supplied with epithelial cells from two adjacent crypts. One crypt is populated entirely by blue epithelial cells, representing one genetic background. The other crypt is populated entirely by nonblue cells, representing the other genetic background. Crypts do not contain a mixture of cells from both genetic backgrounds. Thus, they are monoclonal, that is, composed of epithelial cells ultimately derived from a single progenitor. This progenitor occupies the highest position in the stem cell hierarchy. The precise number of progenitor-derived multipotent stem cells that are active in each crypt is uncertain. Note that cells from each crypt migrate up the villus in an orderly fashion, forming distinct columns with discrete borders. Migration is completed in 3 to 5 days. The highly organized migration is illustrated further in the inset that shows a whole-mount preparation of intestine from a chimeric mouse. Striped villi contain differentiating epithelial cells derived from monoclonal blue crypts and nonblue (white) crypts. (B) Diagrammatic representation of the stem cell niche. An active multipotent stem cell (SC) gives rise to a C0 daughter that produces the enterocytic lineage and to descendants that generate secretory lineages (goblet, Paneth, and enteroendocrine cells; note that it is uncertain whether all are derived from M0). Glucagon-like peptide-2 (GLP-2) produced by a subset of enteroendocrine cells is able to stimulate proliferation of the Co daughter via interaction with enteric nervous system neurons that express the GLP-2 receptor (GLP-2R). The nature of the neuronal signal that affects Co is unknown. (Reproduced from Mills JC, Gordon JI. The intestinal stem cell niche: there grows the neighborhood. Proc Natl Acad Sci U S A 2001;98:12334–12336, by permission.)

crypts being observed (Fig. 11-3A). This strongly implies that each crypt is composed of a single clonal population of epithelial cells (7,8). The data from these mice were informative for the Paneth, mucous, and columnar cells, but a result could not be determined for the endocrine cells (8). Interestingly, nascent crypts were polyclonal during the first 2 weeks after birth—that is, composed of a mixture of cells from C57BL/6 and SWR parents (7). However, after this point, crypts were composed of cells derived from either C57BL/6 parents or the SWR parents, but not a mixture of cells from both parents. This subsequent “cleansing,” or purification, of the crypt is proposed to occur either by overgrowth and extrusion of

one of the stem cells over the other(s), or by fission of the crypts, with individual stem cells segregating into separate crypts. Another approach for identifying the clonal origins of cells within a crypt is by detecting the presence of the Y chromosome in crypt epithelial cells of mouse aggregation chimeras produced from fusion of a female and a male blastocyst. These chimeras were constructed in a similar manner to the C57BL/6J Lac (B6),SWR mice aggregation chimeras described earlier (30). Experiments conducted in the resulting XX/XY mice confirmed the observation that crypts (glands) were monoclonal (i.e., crypts containing a mixture of female cells and

312 / CHAPTER 11 male cells were not observed) from 2 weeks after birth. In addition, these experiments were extended to confirm the origin of the neuroendocrine cells within the crypt epithelium. A combination of in situ hybridization for the Y chromosome and immunohistochemistry for the enteroendocrine cell-specific marker gastrin was used. This technique demonstrated that enteroendocrine cells in the female areas of the chimeric stomach were Y chromosome negative, while those in the male areas were almost exclusively Y chromosome positive (30). Human chimerism can also result spontaneously from the occurrence of nondisjunction within a cell of the early developing embryo. An example of this is loss of the Y chromosome, which results in an XO/XY chimeric individual (54). Tissue was obtained from a single XO/XY individual during a prophylactic colectomy for familial adenomatous polyposis (FAP). The tissue was then probed using Y-chromosome– specific probes in a non-isotopic in situ hybridization assay. These probes revealed that the intestinal crypts in normal areas of the tissue were almost entirely composed of either Y chromosome positive or negative cells, with only 4 of the 12,614 crypts scored containing a combination of both. This observation is consistent with the Unitarian hypothesis, as proposed from the observations made during the aforementioned murine studies. Interestingly, in regions of the colonic tissue that contained tubular adenomas, approximately 76% of the crypts were polyclonal. This observation suggests that crypt monoclonality is actively maintained in the normal mucosa.

X-Chromosome Inactivation X-chromosome inactivation has also been used as a tag to trace the origins of epithelial cell lineages in the mouse intestinal tract. In gut epithelium, X-chromosome inactivation is completed by embryonic day 11.5 (E11.5), well before the commencement of stomach gland morphogenesis (49). A transgenic mouse line that contains a tandem insertion of copies of the lacZ reporter gene on the X chromosome (H253 mice) was used to examine the origins of cell lineages in the gastric epithelium (50). Hemizygous female H253 mice show mosaic expression of the transgene as a result of X-chromosome inactivation that normally occurs during development. The clonality of the gastric glands was examined in the fundic and pyloric regions of female H253 hemizygotes at different developmental time points. Most of the glands were found to be polyclonal at early developmental time points, with an estimated three or four stem cells per gland. However, by 6 weeks of age, the glands had become monoclonal, being either entirely positive or negative for β-galactosidase activity. Two alternative mechanisms were proposed as possible explanations for the appearance of this monoclonality: either emergence of a dominant stem cell clone, or purification through segregation of stem cells to daughter glands as gastric glands undergo fission (50).

In another study, the colonic crypts of H253 mice were also shown to be monoclonal (51). The additional observation was made that 5% to 10% of the glands retain their polyclonal nature into adulthood. The authors proposed that a possible explanation for this could be that these glands have not undergone fission, are doing so at a reduced rate, or had a greater original number of stem cells. None of these alternative explanations has been confirmed. The clonality of the fundic and pyloric glands in the human stomach has been studied by investigating polymorphisms in two X chromosome–linked genes: the phosphoglycerate kinase (PGK) gene and the androgen receptor (HUMARA) gene (50). These were carried out in a similar manner to the X-inactivation studies in mice; however, a somewhat different result was obtained. Although the pyloric glands appeared to be homotypic for both the PGK and HUMARA loci (i.e., they were monoclonal), approximately half of the fundic glands were observed to be heterotypic for both loci. This indicates that approximately half of the fundic glands are polyclonal and demonstrates that the situation in humans is more complex than in mice, with regional differences apparent in the gastric mucosa (50).

In Vivo Stem Cell Mutagenesis Assays Characterization of animals with induced somatic mutations in the intestine has provided further information regarding cell lineage relationships, and properties of stem cells and progenitor cells within the crypt epithelium. For example, mutations at the Dlb-1 locus, which encodes the DBA lectin-binding site, have been studied in the intestine in situ after in vivo mutagenesis with ethylnitrosourea. The F1 progeny of C57BL/6J (DBA receptor–positive) crossed with SWR (DBA receptor–negative) mice are heterozygous for expression of the DBA receptor. When this locus is abolished, either spontaneously or using ethylnitrosourea, cells that are negative for DBA binding occur. Examination of the crypts in the small intestine of these mice showed a subset of wholly DBA-negative mutated crypts that persisted indefinitely. This observation is consistent with a Dlb-1 gene mutation in a single stem cell that is able to give rise to the entire epithelial cell population in a given crypt (11,12). In addition to wholly DBA-negative crypts, partially unstained crypts were also seen. However, the partially negative crypts were extinguished over time, suggesting that they resulted from a mutation in a progenitor or TA cell rather than an anchored stem cell within the crypt. More recently, Bjerknes and Cheng (13) found that the mutagen, N-nitroso-N-ethylurea, can induce clones of cells in SWR mice (Dlb-1−/−) that are able to bind DBA. This results in a clearer signal, where clones of lectin-positive cells can be observed on an otherwise unstained background. Various progenitor cell populations were characterized within the crypt by the DBA-positive staining of differentiated progeny that they produce. Bjerknes and Cheng have used this specific

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 313 progenitor assay by somatic mutation (SPASM) technique to demonstrate the presence of long-lived progenitor cells committed to columnar cell differentiation (C0), or committed to mucous/secretory cell differentiation (M0). A third mixed progenitor cell type was also identified that could give rise to either mucous or columnar cell lineages. Data obtained in these studies allowed the researchers to estimate that mucous cell clones (Mo) persist for an average of 100 days. Such long-lived progenitors appear to be intermediate in the hierarchy between the stem cell and the rapidly replicating transit cell population (13). Short-lived committed progenitor cells for both columnar (C1) and mucous (M1) cells were also identified, in addition to pluripotent stem cells that were able to give rise to all epithelial cell types. Somatic mutagenesis of genes on the X chromosome has also been useful in studying clonal relations within the crypt. As one allele of an X-linked gene is inactivated in the adult intestine due to X-chromosome inactivation, a single mutation in the remaining active allele is sufficient to induce a mutant phenotype that can be topologically examined by immunohistochemistry or histochemical staining for enzyme activity. For example, Griffiths and colleagues (10) studied intestinal mutations induced by dimethyl hydrazine (DMH) in the X-linked gene, glucose-6-phosphate dehydrogenase (G6PD). When female mice were administered a single dose of DMH, either partially or wholly G6PD-negative crypts resulted. The presence of partially negative crypts was found to be transient, with a decrease in frequency occurring relative to the time after administration of DMH. In parallel to this decrease in the number of partially negative crypts, there was an increase in the frequency of wholly negative crypts. The initial explanation proposed for this phenomenon was based on the presence of multiple stem cells in a crypt, with random loss occurring after time. However, the possibility that “cleansing” could occur by crypt fission, with a concomitant increase in wholly negative crypts, has gained credence more recently. This would involve the segregation of stem cells from partially negative crypts into separate, monoclonal crypts that were wholly negative or wholly positive (55). Insights into the clonality of human colonic crypts have been gained from studies of spontaneous mutations within the gene locus encoding the enzyme O-acetyl transferase (OAT). The mucus secreted from the goblet cells in the colon of most of the human population contains O-acetylated sialic acid residues. However, 9% of the population are homozygous for a mutation in the gene that encodes the enzyme OAT, and instead secrete mucus containing non–O-acetylated sialic acid (56,57). O-acetylated mucus is not stained by the mild periodic acid-Schiff (mPAS) technique, whereas the non–Oacetylated mucus is. An individual who is heterozygous expresses active OAT, and therefore stains negative for the mPAS technique. However, when this individual loses the remaining active gene, the genotype is converted to OAT −/−, which can result in occasional positively mPAS-stained crypts. When this occurs, the goblet cells are observed to stain uniformly PAS positive, from the base to the luminal surface

of the crypt (58). This could be caused either by a spontaneous somatic mutation within a crypt stem cell or by loss of a functional OAT allele through mitotic nondisjunction within an individual crypt stem cell, which subsequently colonizes the crypt.

STEM CELL NUMBER AND HIERARCHY Stem Cell Number Although the many genetic studies cited in the previous section suggest that the adult crypt is ultimately derived from a single genetically determined stem cell, other systems for estimating stem cell numbers have yielded differing results. For example, estimates of stem cell number based on mathematical modeling from cell proliferation data obtained by either 3H-thymidine labeling or by quantifying mitoses have suggested that there are approximately four to six ultimate clonogenic stem cells per crypt in the small intestine (23,24). Although the rates of epithelial renewal in the colon can be accounted for by the presence of only a single stem cell per colonic crypt, the models are also consistent with a greater number of stem cells per colonic crypt. Another powerful experimental approach that has been used to assess the number and functional potential of progenitor cells is based on the capacity of these cells to regenerate individual crypts after injury. Originally described by Withers and Elkind, this approach has been used by a number of investigators to estimate the stem cell number in the intestinal and colonic epithelium after γ-irradiation or cytotoxic therapy (14,15,24,59–63). The microcolony formation assay is based on the capacity of surviving stem cells (clonogens) to regenerate crypt-like foci of replicating epithelial cells, which can be scored histologically 3 to 4 days after radiation injury (15,62). Within 3 to 6 hours after irradiation, many of the cells within the lower third of the crypt undergo p53-mediated apoptosis. The remaining transit cells within the crypt undergo cell-cycle arrest and cease replication, although they continue to migrate out of the crypt and onto the villus. Thus, the crypt is rapidly depleted of cells and disappears as a recognizable structure 24 to 36 hours after irradiation. If one or more clonogenic stem cells within a crypt survive irradiation, they will proliferate, ultimately giving rise to regenerative crypts that first appear in the small intestine approximately 72 to 96 hours after irradiation. These crypts, or “microcolonies,” can easily be counted, and the fractional crypt survival in response to varying radiation doses can be analyzed. An estimate of the number of stem cells (clonogens) in each crypt that retain the capacity to regenerate the epithelium can be derived from experiments measuring fractional survival of crypts after one or two dose fractions of irradiation (63–66). When extrapolated to zero dose, data from these experiments suggest that there may be four to six stem cells per crypt under unperturbed conditions in the small intestine.

314 / CHAPTER 11 Stem Cell Hierarchy The functional studies detailed earlier which indicate that crypts contain several stem cells with clonogenic potential is, on first appearance, at odds with the previously cited genetic analyses of crypt clonality that imply that crypts are monoclonal (i.e., derived from a single ultimate stem cell) in the adult intestine. However, the concept of crypt hierarchy proposed by Potten and colleagues (24,63,66) provides a potential resolution for this paradox. This concept arises from the observation that the number of clonogenic stem cells determined using the split-dose radiation injury model described earlier is highly dependent on the radiation dose used (63). When low doses of γ-irradiation are used to make this determination, there are approximately four to six clonogenic stem cells per crypt. However, as the dose of radiation is increased, the estimate of the number of clonogenic stem cells increases, reaching as high as 36 stem cells per crypt (24,63,66). Observations such as these coupled with studies examining the distribution of apoptosis as a function of cell position in the crypt, have led to the proposition of a threetiered hierarchy of stem cell potential within the lower crypt. In this model, the ancestral stem cell occupies the first tier, while a second tier of potential stem cells (the asymmetric daughters of the ultimate stem cell) is located above this level. This second tier of undifferentiated cells normally enter the TA population; however, they retain the ability to function as stem cells if the ancestral stem cells are lost or rendered nonfunctional. At even greater doses of irradiation, the immediate daughters of the second tier stem cells may also be recruited to function as clonogens to reconstitute crypts after injury. Thus, estimates of stem cell number obtained from functional assays such as radiation injury may depend on the nature of the experimental perturbations produced in the stem cell microenvironment.

Regulation of Stem Cell Number At steady state in the normal intestine, there is a continuous flow of cells from the crypt stem cell/TA cell compartment onto the villus tips. The number of stem cells in the crypt is tightly regulated with production of stem cells being balanced by cell loss. It is widely believed that, under unperturbed conditions, stem cell proliferation is asymmetric; that is, division of the parental stem cell gives rise to a daughter cell in addition to a new stem cell (self-renewal), thus maintaining a balance between crypt stem cell number and the production of differentiating cells. The precise mechanisms that regulate the proliferation of stem cells and whether they divide symmetrically or asymmetrically are unknown. In early studies, Potten and colleagues (67) postulated that selection of stem cells may occur through the expression of specific genes after division that enable autonomous stem cell function of one daughter cell. Alternatively, the expression of certain genes during mitosis results in a polarized cell division committing one of the

daughter cells to the differentiation pathway and the other to functioning as an autonomous stem cell (67). A third alternative is that local environmental signals may dictate stem cell behavior of progeny within the stem cell niche (68,69). Programmed cell death may also be an important mechanism for regulating stem cell number in the intestinal epithelium. In the adult small intestine, under unperturbed conditions, a persistent low frequency of apoptosis is seen in the crypts of animals and humans (14,70,71). This process, referred to as “spontaneous apoptosis,” occurs predominately in the lower regions of the crypt and appears to be restricted to the stem cell region, averaging four to five cell positions above the base of the crypt. Tight control of stem cell number is required to maintain a steady level of cellular output from the crypt onto the villus, thereby maintaining a stable crypt size. Thus, spontaneously occurring apoptosis is likely an inherent part of the regulatory mechanism that determines stem cell numbers and the size of the replicating transit cell population in the normal adult tissue (14,71). Various gene families have been found to be important in the regulation of apoptosis including p53, which is involved in the response of cells to DNA damage, and the Bcl-2 family of genes, whose products are capable of both suppressing and promoting apoptosis. In contrast with p53, Bcl-2 family members may be involved in regulating both spontaneous apoptosis and apoptosis that occurs in response to cellular injury. There are regional differences in the expression of both p53 and Bcl-2 family proteins between the large and small intestine. These differences directly relate to the frequency of spontaneous apoptosis and to tumor incidence in these regions of the gastrointestinal tract (72). Forced alterations in β-catenin/lef1 signaling can also induce apoptosis in the intervillus epithelium and alter the process of crypt stem cell purification to monoclonality described earlier, reinforcing the suggestion that apoptosis is a critical part of this process (73). The regional expression of the Bcl-2 gene family along the crypt-villus axis of the normal adult intestine has also been addressed. The family can be split into two groups: those proteins that are involved in suppression of apoptosis, which include Bcl-2 and Bcl-XL, and those that promote apoptosis, which includes Bax, Bad, Bcl-Xs, and Bak. Bak and Bad are expressed in the crypt, while Bcl-2 and Bcl-XL are expressed along the length of the villus (74). The proapoptotic protein Bax is strongly expressed in the stem cell region of the small intestine, that is, the lower region of the crypt. However, the distribution of Bax is quite different in the colon with little or no Bax found at the base of the crypts and moderate to high levels in cells of the surface epithelium (70). The precise cell-specific and regional expression of these proteins during normal intestinal development has not been fully characterized. Overall, these observations fit with the hypothesis that the ratio of deathpromoting and death-suppressing members of the Bcl-2 family in a given cell plays an important role in controlling its sensitivity to apoptosis.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 315 Bcl-2–related proteins and p53 also play a role in preserving the genomic integrity of the stem cell population in response to genotoxic damage. Low doses of radiation or genotoxic compounds can induce p53-dependent apoptosis in stem cells in the lower crypt (14,75–77). These studies suggest that altruistic cell death may be one mechanism for ensuring the genetic integrity of crypt stem cells faced with an onslaught of environmental agents with genotoxic or mutagenic potential. Stem cells in the small intestine are more prone to initiate this process of altruistic apoptosis than stem cells in the colon. These regional differences in damage-induced apoptosis may partially explain the propensity of stem cells within the colonic epithelium to accumulate mutations, leading to a greater incidence of carcinoma in this tissue. Induction of cell cycle arrest is another potential mechanism for enhancing the genomic integrity of the stem cell population. Together with the expression of p53 that occurs rapidly after radiation injury, the cell cycle regulatory gene p21waf1/cip1 is also induced (24,72,78). Cell-cycle arrest induced by p21waf1/cip1, p53, or other regulatory proteins at G1/S or G2 checkpoints likely plays a role in providing sufficient time for repair of DNA damage before S phase or mitosis. A third mechanism has been proposed by Potten and colleagues (79) for protecting the genomic integrity of intestinal stem cells. In this model, the stem cell retains the template DNA, while newly synthesized DNA strands are selectively segregated to the committed daughter cells after the second round of stem cell division. Although this proposal remains controversial, it offers another potential mechanism for protecting stem cells from mutations that inevitably occur during DNA replication.

Putative Stem Cell Markers Although no definitive markers of intestinal stem cells have as yet been established, Potten and colleagues have identified Musashi-1 (Msi-1) as a potential candidate marker for the intestinal epithelial stem cell (80). Msi-1 encodes a neural RNA-binding protein originally identified in Drosophila that appears to be required for the asymmetric division of both Drosophila and mammalian sensory neural precursor cells (81–83). The expression of the Msi-1 gene was examined in murine intestine after irradiation, as well as at several different developmental stages (80). In the neonatal mouse intestine, Msi-1 is expressed in the cytoplasm of undifferentiated epithelial cells in nascent crypts, while in the adult intestine, it is expressed by a small number of cells just above the Paneth cell zone and in cells intercalated between Paneth cells. In addition, not all proliferating cells (i.e., Ki67-positive cells) express Msi-1. The distribution of Msi-1 immunoreactivity is consistent with the location of the stem cell population within the crypt epithelium. Radiation injury increases the expression of Msi-1, which is localized in the lower half to one third of the crypt, although it is only present in a subset of proliferating cells.

The wave of Msi-1–expressing cells correlates with the restoration of the clonogenic stem cell compartment after injury. These data suggest that Msi-1 is a marker of stem cells and early lineage specific progenitor cells. Further examination of both Msi-1 expression in the developing and adult intestine (84) and Hes-1, a transcription factor under the control of Msi-1, showed that both Msi-1 and Hes-1 are expressed in the intervillus epithelium. However, Msi-1 is found in only a few cells located above Paneth cells, whereas Hes-1 is more broadly distributed in epithelial cells in the lower and mid crypt. This finding that Hes-1–expressing cells have a broader distribution supports the argument that they may represent rapidly replicating TA cells of the crypt. This is consistent with the demonstrated need for Hes-1 expression for the differentiation of the absorptive enterocyte (see later).

THE STEM CELL NICHE The existence of a specialized physical and structural microenvironment, or niche, in which stem cells reside was first proposed by Schofield (85) and is now widely accepted. The stem cell niche is understood to encompass both cellular and molecular components that surround, support, and interact with stem cells. It provides a stable, protective environment in which the function of stem cells and their daughters, the TA cells, is maintained and regulated by factors and signals produced by the cells forming the niche. Stem cells also interact with the niche components as the needs of the tissue, organism, or both change during development or in response to injury or other physiologic changes. In the intestine, the crypt is the niche that houses epithelial stem cells and their TA daughters (see Fig. 11-3). This structure is formed early in intestinal development by the invagination of the intervillus epithelium into the surrounding mesenchyme. A number of cell types comprise the mesenchymal component of the intestinal stem cell niche including intraepithelial lymphocytes, blood vessels, enteric neurons, and pericryptal fibroblasts. Subepithelial myofibroblasts surround the intestinal crypts, while a basement membrane lies between the epithelial cells lining the crypt and the myofibroblasts. The intestinal subepithelial myofibroblasts (ISEMFs) form a syncytium that merges with the pericytes of the blood vessels, as well as extending throughout the entire lamina propria. These cells form a fenestrated sheath that is tightly apposed to the epithelial layer, and they have been indicated to play an essential role in interactions between the epithelium and the mesenchyme (86). In addition, secretion of growth factors such as hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), various fibroblast growth factors (FGFs), and perhaps other growth factors by the ISEMFs plays an essential role in the differentiation of the epithelial cells that express receptors for these factors (86). The interstitial cells of Cajal are another type of myofibroblast found in the intestine. These cells localize proximal to mural neurons, where they play

316 / CHAPTER 11 various roles, including as pacemakers for smooth muscle in the intestines, propagating electrical events, and modulating neurotransmission (87). Although both types of fibroblasts exist as a syncytium, it is unclear whether the two networks connect (86). Bjerknes and Cheng suggest that enteric neurons may promote proliferation of stem cell clones and long-lived columnar progenitor cell clones that differentiate into absorptive cells when the neuronal circuit is activated by glucagon-like peptide-2 (GLP-2), an enteroendocrine cell product (88). The existence of a niche is demonstrated by its independent existence in the absence of stem cells. In the gut, proof of the existence of a niche came from targeted disruption of the T-cell factor 4 (Tcf-4) gene (89). In the absence of Tcf-4 intestinal crypts were found to be devoid of a proliferating cell population, while the pericryptal mesenchymal components of the niche remained. Similarly, a dose of lethal radiation will deplete the epithelial component of the intestinal niche. If a single stem cell survives, the remaining mesenchyme is sufficient to support restoration of the stem cell compartment within the regenerating crypt.

REGULATION OF STEM CELL FUNCTION Signaling Pathways TCF-4 and the Wnt Signaling Pathway Wnt proteins are a family of secreted, small signaling molecules (90) that are known to regulate embryonic development, tissue morphogenesis, and maintenance of adult tissue homeostasis, as well as to play a key role in cancer development. Furthermore, studies have demonstrated the central role of Wnt signaling in regulating crypt epithelial stem cell proliferation during normal development and in tissue homeostasis. This is illustrated by the observation that mice lacking Tcf-4, a downstream component of Wnt signaling, do not have replicative cells, and presumably stem cells, in the intervillus epithelium from E16.5 (89). These mice are unable to form nascent crypts and die shortly after birth. The intracellular signaling cascade that links Wnt signaling to activation of β-catenin/Tcf-4 has been largely defined and is summarized in Figure 11-4 (90). The primary receptors for Wnts are the Frizzled family of proteins (91,92). However, Wnt signaling also requires a transmembrane molecule of the low-density lipoprotein (LDL) receptor–related protein (LRP) family, with which it forms a trimeric receptor complex that transduces the Wnt signal to intracellular proteins (91,92). Signaling via Wnt receptors is complex and thought to involve Wnt binding to the Fz/LRP receptor, which activates the complex and causes Axin to bind to LRP with subsequent stabilization of β-catenin (a transcriptional regulator) (90,93–97). Stabilized, cytoplasmic β-catenin translocates to the nucleus, where it complexes with the TCF/LEF DNA-binding

proteins, converting the TCF complex from a transcriptional repressor to an activator of gene transcription (98–102). In the absence of Wnt signaling, cytoplasmic β-catenin complexes with Axin and adenomatous polyposis coli (APC), at which point it is phosphorylated by the serine/ threonine kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1α (103–106). Axin and APC are thought to provide scaffolding for the interaction between the kinases and β-catenin (107,108). Once phosphorylated, β-catenin is ubiquitinated and targeted for degradation by the proteosome (109–111). Support for the role of the β-catenin/Tcf family in the regulation of epithelial morphogenesis comes from a report by van Noort and colleagues (112). They demonstrated the presence of unphosphorylated nuclear β-catenin in the undifferentiated intervillus epithelium, confirming that activation of the β-catenin/Tcf signaling pathway occurs as early as E16.5 in the fetal homolog of the adult crypt. Studies by Wong and colleagues (73,113) suggest that members of the β-catenin/Tcf family also regulate the selection of a single, genetically dominant pleuripotent stem cell in each crypt. This selection occurs during postnatal intestinal development in the mouse as described earlier (see Morphogenesis of Structural/Proliferative Units and Clonality of the Structural/ Proliferative Unit). This group engineered the in vivo expression of a stabilized β-catenin mutant only in the 129/Sv cells of B6-ROSA26,129/Sv chimeric mice (73,113). In these mice, artificially sustained expression of a Lef-1/β-catenin fusion protein during the time when signaling by this complex would normally be declining resulted in a dramatically greater frequency of apoptosis in the population of cells expressing the transgene. The 129/Sv stem cells, which expressed the stabilized β-catenin mutant, were selectively eliminated from the chimeric intestine after weaning, by which time crypt morphogenesis was completed. These experiments suggest that early extinction of Lef-1/β-catenin signaling in a pleuripotent crypt stem cell results in its selection as the genetically dominant stem cell within the crypt. However, other regulatory mechanisms affecting this selection process cannot be excluded. It has been proposed that the Wnt family of ligands provides the extracellular signals that induce activation of β-catenin/Tcf-4 intermediate signaling pathways, although the precise identity of these extracellular signals remains to be elucidated. Thus, other extracellular signals may contribute to the activation of the β-catenin/Tcf-4 signaling cascade. For example, FGF2 can regulate Tcf/β-catenin– mediated transcriptional activity in endothelial cells (114). This may occur in intestinal epithelial cells, as has been indicated by our studies of FGF receptor-3 (FGFR-3)–mediated signaling events in intestinal mucosal development. During normal morphogenesis of the small intestine, robust expression of FGFR-3 was noted in the intervillus epithelium and the stem cell region of newly forming crypts (115). A marked reduction in both the number of crypts and the size of the epithelial stem cell population was observed in the intestine of suckling FGFR-3 null mice.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 317

FIG. 11-4. Diagrammatic representation of Wnt signaling. Cytoplasmic components of the Wnt signaling pathway. In naive cells (left), β-catenin forms a complex with Axin and adenomatous polyposis coli (APC). Axin acts as a scaffold for the protein kinases casein kinase 1α (CK1α) and glycogen synthase kinase-3 (GSK-3), and the PP2A protein phosphatase. PP2A may act on Axin, as well as other substrates in the Axin/APC complex. After phosphorylation by GSK-3 and CK1α, β-catenin is degraded by ubiquitination involving interactions with Slimb/-TrCP. After binding of the Wnt ligand (right), the Fz and low-density lipoprotein receptor–related protein (LRP) receptors recruit Dsh and Axin to the membrane, where they may interact with each other (dashed lines). Wnt signaling leads to inhibition of β-catenin degradation and its accumulation. As a result, β-catenin is stabilized in the cytoplasm and is no longer degraded. Negative regulators are outlined in black. Positively acting components are outlined in color. (Reproduced from Logan and Nusse [90], by permission.)

Furthermore, stimulation of the CaCo2 intestinal cell line by FGFR-3 ligands or transfection with a constitutively active form of FGFR-3 induced up-regulation of Tcf-4–mediated transcriptional activity in these cells (116). Conversely, inhibition of Tcf-4–mediated signaling in preconfluent Caco-2 cells by transfection of a dominant negative Tcf-4 mutant triggers precocious differentiation of these cells (117). Decreased activation of Tcf-4/β-catenin signaling is therefore a possible mechanism for the decreased size of the stem cell pool in FGFR-3 null mice, suggesting that regulation of intestinal stem cell dynamics by the Tcf-4/β-catenin family involves diverse extracellular signaling events that may be context dependent. Genes Downstream of β -Catenin/Tcf-4 Activation A number of target genes that respond to β-catenin/ Tcf-4 transcriptional regulation have been identified as mediators of stem cell proliferation that maintain the topologic organization of stem cells and progenitors within the crypt.

Eph Receptors and Ephrin Ligands The organization and hierarchy of cells within the intestinal crypt are highly ordered and topologically constrained, as discussed earlier. Studies have provided insights into possible mechanisms that may be involved in maintaining the relative positions of cells, including stem and progenitor cells, along the crypt–villus unit. Not surprisingly, the β-catenin/ Tcf transcription complex is involved in regulating this aspect of cellular organization along the crypt-villus axis. van de Wetering and colleagues (118) have identified a number of Tcf-4–regulated genes in colon cancer cells by expression of a dominant negative TCF (dnTCF) mutant. Among the genes down-regulated by expression of the dnTCF mutant were the EphB2 and EphB3 receptors. Conversely, ephrin ligands were up-regulated in these cells (118). Eph receptors and ephrin ligands play essential roles in cell–cell interactions, providing repulsive signaling in a wide range of developmental processes (119). In addition, the observation that the Tcf4–deficient E18 mouse embryos lacked intestinal EphB2 and EphB3 expression, while proliferating cells of normal intervillus epithelium express both Tcf-4 and EphB receptors,

318 / CHAPTER 11 Villus Crypt

Ephrin B1 Ephrin B2 EphB2

Stem cell

EphB3 Paneth cells

FIG. 11-5. Schematic representation of the expression gradients of EphB2, EphB3, and their ephrin ligands in crypts of the adult small intestine. Arrows show the direction of the gradient of Eph receptor and the respective ephrin ligand expression.

corroborated the regulation of EphB/ephrinB expression by signaling through Tcf-4 (118). The receptors for ephrins constitute a large subfamily of receptor tyrosine kinases. These receptors are grouped into EphA receptors that bind type A ephrin ligands and EphB receptors that bind type B ephrins (119). Both Eph receptors and ephrin ligands are membrane-bound proteins; thus, the interaction between the receptor and ligand requires direct cell–cell contacts. The most common type of interaction is generally one of repulsion of a cell neighbor, although examples of increased adhesion/attraction have been documented (119). Studies of EphB2 and EphB3 null mice demonstrate that expression of EphB receptors is necessary for the correct positioning of epithelial cells along the crypt-villus axis (120). EphB2 is expressed in a gradient along the crypt-villus axis with the greatest levels in cells at the bottom of the crypt (Fig. 11-5). Conversely, ephrinB ligands are expressed in an opposing gradient, with the highest levels observed in cells at the crypt-villus junction in adult intestine and at the periphery of the intervillus pockets in the neonatal gut, whereas no expression is observed at the crypt base. Only EphB3 is necessary for the downward migration of Paneth cells to the crypt base as Paneth cells distribute randomly throughout the crypt-villus axis in EphB3 null mice. Paneth cells do not express any of the ephrinB ligands, and therefore differ from the surrounding epithelial cells at the base of the crypt. Thus, the lack of ligand expression coupled with high expression of the EphB3 receptor provides Paneth cells with a positional address, or “zip code,” that directs their migration downward to the crypt base. Presumably, the lack of ephrinB ligands eliminates the “repulsive” response such expression would elicit in an area of EphB receptor–positive (crypt base) cells (120).

In the neonatal intestine, the interaction of EphB receptors on proliferating cells with their ephrin B1 ligand on adjacent differentiating cells gives rise to an organized topologic localization of these cells. This restricted topologic positioning is lost in the EphB2/EphB3 double-mutant intestine, where proliferating and differentiated cells intermingle. In addition, some intervillus regions of the double-mutant mice contain differentiated cells without an alteration in the position of proliferating cells in this region. This result suggests that in wild-type mice, expression of EphB receptors on proliferating cells actively restricts the migration of the ephrinBexpressing differentiated cells (120). Expression of the EphB receptor and the ephrinB ligand is more complex in the adult intestinal crypt, with different levels of expression along the length of the crypt (120). Nonetheless, EphB receptors still restrict the positions occupied by cells with different levels of ephrinB expression, suggesting that expression of EphB defines a location in the crypt that is restricted to stem cells and their rapidly proliferating daughters. Thus, in EphB2/EphB3 double-mutant mice, proliferating cells that normally localize toward the bottom of the crypt can be found at the crypt-villus junction, while differentiated cells with high levels of ephrinB that are generally found at the crypt-villus junction may relocate to the bottom of the crypt, where they are intermixed with islands of undifferentiated replicating cells. C-Myc The myelocytomatosis oncogene (c-Myc) is a member of the MYC family of transcription factors that are characterized by a basic helix-loop-helix leucine zipper (bHLH-LZ) motif. It binds E-box sequences as a heterodimer with MAX, another (bHLH-LZ) protein (121,122). The activated β-catenin/Tcf-4 complex has opposing effects on the expression of c-Myc and p21cip1/waf1, a key cell-cycle inhibitor and marker of differentiated cells (118). Transfection of colorectal carcinoma cell lines with dnTCF mutants showed that activation of c-Myc through this signaling pathway inhibits differentiation and promotes proliferation of these cells. More importantly, the stable transfection of only c-Myc into the dnTCF-expressing colorectal cell lines abrogated the inhibition of cell growth, and the induction of p21cip1/waf1 imposed by the expression of dnTCF. Finally, c-Myc regulation of p21cip1/waf1 expression is suggested by the demonstration that c-Myc binds to the p21cip1/waf1 promoter (118). These findings strongly suggest that c-Myc mediated the proliferative effects of the β-catenin/Tcf-4 complex in colorectal carcinoma cells through transcriptional inhibition of the cell cycle inhibitor p21cip1/waf1. This regulatory pathway also appears to be operative during mucosal morphogenesis that occurs in the normal fetal small intestine. Genes that are transcriptionally upregulated by β-catenin/Tcf-4, such as c-Myc, Enc1, and CD44, were either not expressed or expression was decreased in the intestinal intervillus epithelium of late fetal Tcf-4 null mice, which lack stem cells in the intervillus epithelium. In contrast, gene products that are normally down-regulated

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 319 by activation of Wnt signaling, such as p21cip1/waf1 and liver fatty acid–binding protein, were abundantly expressed in this region (118). Cohen and colleagues (123) transiently decreased c-Myc expression in mid-to-late gestation embryos by in utero adenoviral-directed introduction of either antisense c-Myc messenger RNA (mRNA) or the engineered F-TrCP-MAX ubiquitin-protein ligase, which targets the degradation of c-Myc. Both means of diminishing c-Myc expression resulted in highly decreased complexity of the intestinal villus structure and the lung alveoli, suggesting a failure of adequate expansion of the stem cells found in these epithelial structures at this developmental time point. Finally, gene expression analysis of mRNA expressed by epithelial cells at the crypt base, isolated by laser capture microdissection from Paneth cell–ablated CR2-tox176 mice, lends further (circumstantial) support for the hypothesis that c-Myc expression is necessary for maintaining a proliferative stem cell population (124). Many of the genes expressed by the epithelial cells occupying the lower crypt were identified as those involved in regulating c-Myc transcription and c-Myc protein stability, as well as transactivation of c-Myc target genes such as the recombinant RNA–binding proteins nucleolin and nucleophosmin. Caudal-Related Homeobox Genes As the name implies, the caudal gene of the fruit fly Drosophila melanogaster determines anterior-posterior patterning during development (125–127). Cdx1 and Cdx2 are members of the caudal-related homeobox gene family sharing sequence homology to the caudal gene and encode transcription factors that regulate key aspects of normal mammalian development (128,129). These transcription factors have important roles in the early morphogenesis of the intestine when the visceral endoderm is converted to a columnar epithelium with nascent villi (125,126,130). At this developmental stage, Cdx1 expression is restricted to the intervillus epithelium, which contains stem progenitor cells, suggesting a role for this transcription factor in crypt morphogenesis (125). Cdx1 and Cdx2 proteins are expressed in gradients along both the duodenal-colonic axis and the crypt-villus axis of murine intestine from early embryonic development through adulthood (131). Expression of both Cdx1 and Cdx2 is greatest in the distal small intestine with expression decreasing proximally. Cdx1 is expressed throughout the entire colon, while Cdx2 is only present in the proximal colon. Finally, there is a crypt-villus gradient of expression for both Cdx1 and Cdx2. In the adult intestine, Cdx1 is localized primarily in the nuclei of crypt epithelial cells, while Cdx2 is found mostly in the villi with some crypt expression (131). A number of studies suggest that Cdx1 and Cdx2 regulate region-specific morphogenic programs in the adult intestine (127,131–137). Thus, heterozygous Cdx2 null mice develop colonic polyp-like lesions that do not express Cdx2, presumably resulting from mutations that result in biallelic Cdx2 inactivation but do not include loss

of heterozygosity (138,139). These polyps display a mucosal heterogeneity with areas of keratinized stratified squamous epithelium, as well as areas of gastric and small intestinal– type mucosa (138–140). In contrast, Cdx1 expression is seen in gastric or esophageal mucosa that has undergone intestinal metaplasia (141). In addition, a role for Cdx1 and Cdx2 in intestinal differentiation is suggested by the observation that colorectal carcinoma has low or no expression of these transcription factors (141–143). Both Cdx1 and Cdx2 are involved in the regulation of intestinal epithelial cell differentiation and expression of intestine-specific gene products. For example, IEC6 cells, an intestinal epithelial cell line that does not differentiate in culture, can acquire a differentiated phenotype when induced to express transfected Cdx1 and Cdx2 (132,135). Cdx1 and Cdx2 also regulate the transcription of intestine-specific enzymes such as colonic carbonic anhydrase 1, sucrase isomaltase, and intestinal phospholipase A/lysophospholipase (IPAL) (127,133,144). In the case of Cdx2, this transcriptional regulation is achieved through the binding of Cdx2 to cis elements present in the promoters of sucrase isomaltase and carbonic anhydrase 1 genes, as well as several other intestine-specific genes (127,145–149). More recently, Yamamoto and colleagues (136) identified CDX consensusbinding sites in the MUC2 promoter. They determined that CDX2, but not CDX1, can bind to these sites and up-regulate the transcription of MUC2 mRNA, suggesting that CDX2 plays a role in the differentiation of intestinal goblet cells. Expression of Cdx1 by epithelial cells residing in the lower portion of crypts in the adult murine intestine raises the possibility that it plays a role in regulating some aspect of epithelial stem or progenitor cell fate, or both. Consistent with this hypothesis, the Cdx1 promoter contains four Tcf/ Lef1 binding motifs; Cdx1 expression is up-regulated by activation of Wnt/β-catenin signaling, which occurs in the intestinal stem and progenitor cell population; and Tcf-4 null mice at E17 specifically lack Cdx1 expression in the small intestine. These findings suggest a role for Cdx1 in mediating the β-catenin/Tcf-4 regulation of crypt morphogenesis, and possibly proliferation of some as yet unidentified epithelial cells in the stem cell niche (150). In vitro studies have yielded conflicting data regarding the role of Cdx1 in regulating intestinal epithelial cell proliferation. Some studies led to the conclusion that Cdx1 stimulated proliferation of cells in culture, while other studies reported inhibition of their proliferation (135,151,152). Similar conflicting data were obtained in studies examining the effect of Cdx1 expression in colon cancer cell lines (137,142). In contrast, Cdx2 inhibited cellular proliferation when expressed in both a cell line derived from normal intestine (IEC6 cells) and in a colon carcinoma–derived cell line (Caco-2 and HT29 cells) displaying tumor-suppressor activity (132,137,142,153,154). More recent investigations have clarified the role of Cdx1 in regulating intestinal epithelial cell proliferation, especially with respect to colon cancer. In addition, these studies point to differences in in vitro methodologies as the source of some of the prior conflicting results (155,156). The colon cancer

320 / CHAPTER 11 cell lines, DLD1 and HCT116, which transiently express CDX1, were found to undergo cell-cycle arrest in G0/G1 with an accompanying reduction in G1 kinase activity, cdk6 and cdk2 [156]. In addition, cyclin D1 mRNA and protein were diminished due to a reduction in the transcriptional activity of the cyclin D1 promoter. Lynch and colleagues (156) therefore postulated that Cdx1 may play a role in coordinating the exit of a crypt epithelial (TA) cell from the cell cycle, along with the induction of differentiation as it migrates out of the crypt. The observed loss of CDX1 in human colon cancer may be a contributing factor to the attainment of uncontrolled proliferation due to the inability to exit the cell cycle. Guo and colleagues (155) presented compelling evidence that both Cdx1 and Cdx2 can inhibit β-catenin/Tcf transcriptional activity. These data raise the possibility that expression of Cdx may mediate the transition of crypt epithelial cells from the progenitor compartment to the TA cell compartment. However, the current data do not allow for a definitive identification of the Cdx-responsive crypt epithelial cell population. Cell lines displayed variable sensitivity to the Cdx1/Cdx2-mediated inhibition of β-catenin/Tcf transcriptional activity and subsequent inhibition of cellular proliferation. The authors speculate that a gradient of sensitivity to the inhibition of proliferation by Cdx factors may exist among cycling crypt epithelial cells (155). Indeed, it could be envisioned that stem and progenitor cells might be relatively unresponsive to the antiproliferative effects of Cdx, while TA cells may be a more sensitive population. A model is proposed that reconciles the presence of both Cdx1 and Cdx2 in proliferating epithelial cells of the crypt with the findings of an antiproliferative role for these transcription factors (Fig. 11-6) (155). In epithelial stem and cycling progenitor cells of the crypt, the activated β-catenin/ Tcf signaling pathway promotes the expression of key target

FIG. 11-6. Model of Cdx1 and Cdx2 interaction with the Wnt/ β-catenin signaling pathway. TCF, T-cell factor. (Reproduced from Guo and colleagues [155], by permission.)

genes, such as cyclin D1, c-Myc, and Cdx1. Cdx1 enhances the expression of Cdx2, and together they function to slow cellular proliferation. However, this activity is dampened or inhibited in Cdx-expressing epithelial cells of the crypt by as yet unknown mechanisms. As cells migrate out of the crypt base, inhibition of Cdx1 and Cdx2 antiproliferative activities ceases and expression of Cdx gene targets, such as p21cip1/waf1 and KLF4, is induced. This regulates the withdrawal of differentiation-committed cells from the cell cycle. Active β-catenin/Tcf-4 signaling inhibits the expression of both p21cip1/waf1 and KLF4. Thus, the demonstrated ability of Cdx to inhibit β-catenin/Tcf-4 may be necessary for the subsequent induction of p21cip1/waf1 and KLF4 (118,155,157). SOX9 SOX proteins are a family of transcription factors that share a common high-mobility group DNA-binding domain with the TCF/LEF transcription factors. While some Sox proteins play a role in inhibiting cellular differentiation and maintaining stem cell multipotency, others are involved in the regulation of cell fate decisions and cellular differentiation (158–163). SOX9 expression is found throughout the developing and adult intestine (164). It localizes to the nuclei of epithelial cells located in the lower portion of crypts and also colocalizes with Ki-67, a marker of proliferating cells. Studies of LS174T cells (human colon carcinoma cells) transfected with an inducible dominant negative TCF4 construct demonstrated that the Wnt signaling pathway regulates SOX9 expression. Direct interference with the formation of the β-catenin/Tcf-4 complex in these cells also resulted in the immediate loss of SOX9 expression. The in vivo link between SOX9 expression and Wnt signaling is illustrated in Tcf-4 null mice that have decreased numbers of replicating cells within the intervillus region of the developing small intestine, do not form crypts, and also fail to express SOX9 in the intervillus epithelium (89,164). These findings suggest that SOX9 may be a downstream effector of Wnt/β-catenin/Tcf-4 signaling. More direct proof for this hypothesis comes from the identification of Wnt/β-catenin/ Tcf-4 target genes that are directly regulated by SOX9 (164). Thus, the expression of Cdx2 and Muc2, genes that are normally expressed in differentiated villus epithelial cells, was repressed when SOX9 was overexpressed in LS174T cells. Therefore, SOX9 likely mediates some aspects of the maintenance of the undifferentiated, proliferating stem cell compartment in the intestine by the Wnt/β-catenin/Tcf-4 pathway. Interestingly, other important regulators of this compartment (i.e., Cdx1 and c-Myc) that are direct downstream target genes of the Wnt/β-catenin/Tcf-4 pathway were not affected by SOX9 expression (164). It has therefore been speculated that SOX9 may represent a branching point of the Wnt signaling pathway (164). Finally, Sox9 is also found in the nucleus of Paneth cells in the small intestine, that is, in differentiated cells that do not cycle. This observation led Blache and colleagues (164) to suggest that SOX9 expression is not so much cell specific,

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 321 as specific to a cellular location within the tissue, in this case the intestinal stem cell niche (164). CD44 In rapidly renewing tissues such as the intestine, the rate of proliferation of epithelial cells in the intestinal crypts must be tightly matched to the rate of the cell loss. CD44 had been indicated to play a role in the maintenance of intestinal homeostasis under normal conditions. This family of cell-surface–expressed glycoproteins mediates both cell– cell and cell–matrix interactions (165,166). A single gene encodes all of the family members, while alternative splicing and differential glycosylation generate different isoforms (167–169). CD44 is thought to function as a cell-adhesion receptor linking extracellular hyaluronate to the cell and cytoskeleton in tumor progression and metastasis, as well as other processes (167,168,170–173). In addition, CD44 variants carrying the v3 exon contain heparan sulfate side chains that bind growth factors and facilitate growth factor receptor–mediated signaling (174–176). This finding is of significance for intestinal stem cell maintenance and function since heparan-sulfated CD44 binds to mesenchymal FGF2, heparin-binding epidermal growth factor, and HGF (174,176). This ability to bind growth factors is especially important for cells occupying the intestinal stem cell niche since the surrounding mesenchyme is known to secrete FGF2, which is produced during epithelial repair after injury and increases stem cell survival in radiation injury (60). In the intestinal epithelium, CD44 expression is confined to the basolateral membrane of a subset of proliferating epithelial (proliferating cell nuclear antigen–positive) cells that reside in the lower portion of the crypt and include the stem and progenitor cells (177). Tcf-4 null mice do not develop small intestinal crypts and show a loss of cycling cells in the intervillus epithelium, and also lack expression of CD44 (177). However, cells of the small bowel lamina propria, which are not affected by the disruption of Tcf-4 expression, expressed CD44 (177). These data suggest that the β-catenin/Tcf-4 signaling pathway controls the expression of CD44 by small intestinal epithelial cells, but that other regulatory pathways exist in nonepithelial cell types. Interestingly, the epithelia of colon and stomach that were not affected by Tcf-4 disruption also expressed CD44. Such epithelial compartments may be spared the effects of Tcf-4 disruption due to the expression of Tcf-3, which can also complex with β-catenin (89,178). Several investigators have raised the possibility that CD44 may regulate stem cell or progenitor cell fate as opposed to proliferation. Currently, there is no direct evidence that CD44 plays a role in regulating the proliferation of crypt epithelial cells, while its presence on only a subset of PCNA-positive epithelial cells would suggest that it does not (179). Nevertheless, CD44 expression is up-regulated in colorectal cancer as early as the adenoma stage, suggesting that it may play a role in adenoma formation through effects on nonproliferative mechanisms (173,180,181).

Indeed, studies by Lakshman and colleagues (182) demonstrate that CD44 is a negative regulator of apoptosis. Ultrastructural changes in the colons of CD44 null mice were consistent with events in the mitochondria associated with apoptosis including enlargement of the organelles and disrupted cristae. In addition, these mice displayed an altered balance in the expression of antiapoptotic and proapoptotic members of the Bcl-2 family in favor of increased apoptosis (183). Thus, expression of antiapoptotic Bcl-xl was decreased relative to wild-type mice, while the expression of Bax was unchanged (182). In addition, activation of both caspase-9 and -3 was evident in CD44 null mouse colon. Levels of the cell-cycle proteins cyclin A, p21, and pRb were found to be low, or the proteins were absent in the colon of CD44 null mice. These key regulators of the cell cycle have also been implicated in apoptosis in a variety of tissues (184–189). Although the small intestine of CD44 null mice was not examined, these data suggest that crypt epithelial cell expression of CD44 may act to limit apoptosis of these cells. Indeed, the frequency of apoptosis is low in small intestinal crypts of healthy animals, suggesting that this process is highly regulated and probably plays an important role in maintaining crypt stem cell numbers (70).

Genes That Modify Wnt Signals Dickkopf1 Dickkopf (Dkk) is a vertebrate mesenchymal multigene family that encodes a group of secreted proteins that inhibit Wnt signaling (190,191). Dkk1 is the prototype family member and is known to be critically important in head formation during vertebrate development, a process that requires the inhibition of Wnt signaling (192–194). Dkk1 binds to the Wnt coreceptors LRP5/6, and to the transmembrane molecule Kremen. The resulting complex affects the internalization of LRP, thereby abrogating Wnt signaling by eliminating the formation of the Wnt/Frizzled/LRP complex (195–197). Dkk1 is believed to be a pan inhibitor of the canonical Wnt signaling pathway (198,199). Although it is currently unknown whether Dkk1 is operative in the adult intestine under normal conditions, experimental expression of this inhibitor in mice has provided compelling and direct evidence that Wnt signaling is critical for regulation of the function of intestinal stem cells. Two studies have examined the impact that expressing Dkk1 has on intestinal physiology, either in plasma through an adenovirus shuttle mechanism or by transgenic expression in the intestine using villin to target Dkk1 to that tissue (198,199). Both studies demonstrated that expression of Dkk1 resulted in disorganization of the intestinal epithelium and distortion of crypt and villus architecture. Kuhnert and colleagues (198) report down-regulation of the Wnt target genes CD44 and EphB2 in small intestine and colon together with inhibition of epithelial proliferation. Pinto and colleagues (199) report a reduction in crypt size and number in villinDkk1 transgenic mice, together with decreased proliferation

322 / CHAPTER 11 and an absence of nuclear β-catenin expression. In addition, the epithelium that replaces the crypts in Dkk1 transgenic mice displays a differentiated enterocyte phenotype, indicating that inhibition of the canonical Wnt signaling pathway in the intestine leads to terminal differentiation. Of significance was the observation that secretory cells were absent in the Dkk1-expressing intestine, which suggests that Wnt signaling is also necessary for secretory cell lineage allocation, possibly through the regulation of the proliferation of a secretory lineage progenitor (199). Prior studies have demonstrated that secretory lineage progenitors express Math1 (see Notch Signaling Pathway later in this chapter). Consistent with this observation, Dkk1 transgenic intestines were devoid of Math 1 expression, indicating that Wnt/ β-catenin/Tcf-4 signaling may be necessary for the maintenance of epithelial cell progenitors committed to the secretory cell lineage (199,200). As detailed earlier, maintenance of the intestinal crypt stem and TA cell population is dependent on activation of the canonical Wnt signaling pathway, via the expression of c-Myc and repression of p21cip1/waf1 (118,199). As expected, Dkk1 transgenic intestines did not express c-Myc. Rather, p21cip1/waf1 was expressed by the epithelium replacing the crypts. These data further support opposing roles of c-Myc and p21cip1/waf1 in regulating proliferation and differentiation of the intestinal epithelium (118). Lastly, although there is no question that Dkk1 is a potent inhibitor of Wnt signaling, it is currently unknown whether Dkk1 is operative in the adult intestine under normal conditions. However, a study by Niida and colleagues (201) demonstrated that the Dkk1 promoter contains four TCF-binding elements, which suggests that Dkk1 is a downstream target of the Wnt signaling pathway that may participate in a negative feedback loop in Wnt signaling. There are two possible ways in which Dkk1 may act. First, it may have a localized “braking action” of Wnt signaling, thereby ensuring that crypt epithelial cell proliferation does not outstrip the ability of cells to transit out of the crypt and onto the villus, thus maintaining homeostasis in the crypt-villus axis. Second, it may also act as a facilitator of epithelial differentiation by blocking proliferation through the inhibition of Wnt signaling and its downstream proproliferation genes (c-Myc), with subsequent induction of p21cip1/waf1. Foxl1 (Fkh6) Foxl1 is a winged helix transcription factor that is expressed by mesenchymal cells (202–204). In the small intestine, Foxl1 is expressed by fibroblasts surrounding the crypts (203,205). Both the cellular localization of Foxl1 and the finding that mice with a targeted inactivation of Foxl1 display abnormal intestinal architecture suggest that this transcription factor mediates cross talk between the intestinal epithelium and the surrounding mesenchyme (206). Some research has provided a glimpse of the mechanism through which Foxl1 might affect intestinal architecture (207). Foxl1 null mice were found to have thickened gastric mucosa with branching or fission of the

gastric glands after weaning, while small intestinal villi were lengthened and there was a significant expansion of the crypt compartment compared with wild-type mice. At 4 and 12 weeks of age, Foxl1−/− mice had 40% and 22% more crypts than their wild-type littermates, suggesting an expansion of the crypt stem cell population (205). Increased epithelial cell proliferation was seen in Foxl1 null mice, which correlated with an increase in the nuclear levels of β-catenin in these cells, suggesting an activation of the Wnt/β-catenin pathway (207). Additional experiments revealed increased expression of the heparan sulfate proteoglycans (HSPGs) Syndecan-1 and Perlecan. Extracellular proteoglycans can modify Wnt signaling by acting as coreceptors to facilitate/enhance the interaction of Wnts with their high-affinity receptors, the frizzled proteins (207). Thus, it is postulated that Foxl1 modulates Wnt signaling indirectly by limiting the expression of HSPGs. Indeed, a mutation in the mammalian HSPG Glypican-3 results in a tumor-susceptibility syndrome (208). Notch Signaling Pathway A number of studies emphasize the role of the Notch signaling pathway in directing cell lineage decisions of epithelial cell progenitors in the intestinal crypts (200,209,210). Notch genes encode transmembrane receptors that interact with membrane-bound ligands on adjacent cells (211). This signaling pathway is operative in a variety of diverse tissues and cells including T and B cells (reviewed by Sander and Powell [211]). In the vertebrate intestine, four receptors (Notch1-4) and five ligands have been identified (Delta1, Delta3, Delta4, Jagged1, and Jagged2) (212). Activation of the Notch receptor results in cleavage of the intracellular domain of this protein and translocation of the fragment to the nucleus. Once it reaches the nucleus, truncated Notch activates the transcription factor Su(H) (Suppressor of Hairless), which then binds to regulatory sequences in downstream target genes such as hairy/enhancer of split (Hes). Hes protein inhibits the activity of a variety of bHLH transcriptional activators (213). The downstream effects of Notch signaling are context specific and can result in modulation of proliferation, either inhibition or induction of differentiation, or inhibition of apoptosis (213). Math1, a bHLH transcription factor, is a downstream effector of Notch signaling that is required for secretory cell fate specification (200). Normal architecture of the crypt-villus unit was present in Math1 null mice; however, the villi were populated only by enterocytes, and all secretory cell lineages were absent. In contrast, expression of Hes-1 likely supports the differentiation of the absorptive enterocyte since Hes1 null mice display increased expression of enteroendocrine cell markers and increased numbers of goblet cells (210). It is postulated that Hes1 exerts a suppressive influence on Math1 to ensure that the correct number of secretory cells and enterocytes is generated. In parallel with Math1 expression, Hes-1 is found in the embryonic intestinal precursor and persists in the adult intestine. Interestingly, Hes-1 is localized

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 323 to the nonproliferative compartment in the developing (E17) proximal intestine, while in the adult intestine, it is found only in crypts, suggesting multiple roles for Hes1 in enterocyte differentiation. As noted earlier, in Drosophila development, Notch signaling induces Drosophila E(spl) (enhancer of split, the Drosophila homolog of mammalian Hes 1) and regulates cell fate decisions by lateral inhibition, whereby a cell that is beginning to differentiate prevents its neighbor from differentiating (209,210). The reciprocal interaction of Notch and Delta on adjacent cells results in a cascade of intracellular signals in the Notch-bearing cell that suppresses the expression of differentiation-specific genes (Fig. 11-7). The observation that Delta1 and Delta3 are up-regulated within

the intervillus epithelium of the Hes1 null mouse supports the concept that Notch–Delta interactions regulate whether progenitor cells become committed to enterocyte-specific or secretory cell lineages. Other genes known to regulate differentiation of various secretory cell types, including Math1, NeuroD, and Neurogenin3 are prematurely expressed in the developing gut of Hes1−/− mice and provide further evidence that Hes1 functions as a suppressor of secretory cell differentiation (210). Other bHLH transcription factors are involved in the commitment of secretory cell lineage progenitors to differentiate into various secretory cell subtypes. For example, a study by Jenny and colleagues (214) demonstrates that

FIG. 11-7. Notch signaling pathway. (A) Low-power 4-mm section of adult murine small intestine. Precursor cells (brown) are stained for cyclin proliferating cell nuclear antigen (PCNA); enterocytes (red) express intestinal alkaline phosphatase (IAP); goblet cells (blue) secrete mucins. Inset shows high-power image of small intestine enterocytes and goblet cells. (B) Math1, a component of the Notch signaling pathway, influences intestinal epithelial cell fate decisions. In crypt progenitor stem cells that express high levels of Notch, the Hes1 transcription factor is switched on, and the expression of Math1 and other “prosecretory” genes is blocked. The result is that the precursor cells become enterocytes. In cells expressing low amounts of Notch, levels of Delta are high, production of Hes1 is blocked, and Math1 expression is induced. Production of the Math1 helix-loop-helix transcription factor allows precursor cells to make a choice whether to become goblet cells, Paneth cells, or enteroendocrine cells. Cr, crypt; Vi, villus. (See Color Plate 3.) (Reproduced from van den Brink GR, de Santa Barbara P, Roberts DJ. Development. Epithelial cell differentiation—a Mather of choice. Science 2001;294:2115–2116, by permission.)

324 / CHAPTER 11 neurogenin3 (ngn3) functions downstream from Math1 to direct enteroendocrine cell fate specification during development, as well as in the adult intestine. Neurogenin3 was expressed in a decreasing gradient along the cephalocaudal axis of the developing mouse intestine and in cells located in the proliferative compartment of the adult crypt. Expression of Ngn3 protein was also restricted to the crypt in adult mice. The role of ngn3 in regulating differentiation of the enteroendocrine cell populations was underscored by the observation that ngn3 null mice did not express any of the markers characteristic of enteroendocrine differentiation beginning at E15.5, a time when such markers are first detectable in the wild-type mouse. Other aspects of intestinal development appeared normal, suggesting that the effects of ngn3 disruption were specific to the development of the enteroendocrine cell lineages. NeuroD regulates the final stage of enteroendocrine cell differentiation by inducing cell-cycle arrest and up-regulating expression of genes involved in the synthesis of the neuroendocrine mediators made by these cells. Although Math1 was expressed in the intestine of ngn3 null mice, NeuroD was not expressed in these mice, suggesting that ngn3 functions downstream of Math1 but upstream of NeuroD, regulating the commitment of secretory cell progenitors to enteroendocrine cell lineage development before their exit from the cell cycle. Klf4, a zinc-finger transcription factor, is required for differentiation of the mature goblet cell phenotype in the colon (215). A marked decrease in the number of goblet cells in the colon was observed in Klf4−/− mice on postnatal day 1. However, all other epithelial subtypes differentiated normally, and colonic cellular proliferation was not altered compared to wild-type mice. Staining for acidic and neutral mucins confirmed the presence of some immature cell or goblet cell precursors, raising the possibility that Klf4 may not be involved in the initiation of the goblet cell lineage, but rather plays a role in its terminal differentiation. The small bowel did not show similar goblet cell alterations in response to Klf4 ablation. Therefore, it is likely that the differentiation programs for goblet cell specification in the colon are different from that of the small bowel. The Klf4 promoter contains binding sites for the caudalrelated homeobox protein Cdx2 (216). Transfected Cdx2 has been shown to effect the in vitro differentiation of rat small intestinal cells (IEC6) into goblet cells (132). These observations raise the possibility that Cdx2 functions via activation of Klf4 to effect terminal colonic goblet cell differentiation. However, as pointed out earlier, goblet cell differentiation in the colon may proceed by a different mechanism, since Cdx2 is primarily expressed in the proximal gut during embryonic development, as well as in the adult tissue (131). Whether Klf4 is involved in goblet cell lineage progenitor commitment or is operative in the terminal differentiation of the goblet cell phenotype remains to be determined. Hedgehog Signaling Pathway The hedgehog (Hh) pathway is involved in the developmental patterning of a variety of tissues including the

gastrointestinal tract (217,218). In the intestine, the Hh pathway regulates anterior-posterior and axial patterning during development. More recent work shows that Hh regulates intestinal stem cell proliferation and commitment of epithelial progenitors to differentiate (219–222). The vertebrate Hh family is composed of three members: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh), each of which encodes secreted protein products (223). Two of these, Shh and Ihh, are expressed in the intestine. The receptor for Shh is composed of two proteins: Patched (Ptch), a tumor suppressor and repressor of Hh target genes, and Smoothened (Smo), a seven-transmembrane domain protein that is the signaling component of the receptor complex (Fig. 11-8) (224). In the absence of Shh, Ptch represses Smo, while Shh binding of Ptch abrogates Smo inhibition. This leads to the activation of the transcription factors Gli1 and HRK4 (human Kruppel-related gene 4), which affects downstream gene expression in response to Shh binding (224,225). The induction of Ptch by Shh may be Hh

Ptch

Smo

Gli complex − Hh

Gli repressor

+ Hh

Gli activator Ptch Gli Bmp4

FIG. 11-8. Hedgehog (Hh) signaling pathway. The cellular response to Hh is controlled by two transmembrane proteins, Patched (Ptch) and Smoothened (Smo). The secreted ligand Hh binds to Ptch and relieves the repression of Smo signaling. Smo undergoes a conformational change, leading to the activation of its intracellular target(s), and a cascade of intracellular events that result in the translocation of the active form of the transcription factor Gli to the nucleus. Nuclear Gli activates target gene expression, including Ptch and Gli itself, as well as genes involved in controlling cell proliferation. In the absence of Hh signaling, Ptch represses Smo signaling and Gli becomes a transcriptional repressor of Hh target genes.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 325 a way to limit the response of Hh signaling in a given tissue (223,224,226). The importance of limiting Hh pathway signaling is underlined by data from human studies indicating that unrestrained Hh signaling in adults results in the development of various cancers (225,227). Shh and Ihh are expressed in the gut epithelium at sites of epithelial–mesenchymal interactions. Secretion of Hh induces the mesenchymal production of Ptch and of bone morphogenic protein 4 (BMP-4), a member of the TGF-β superfamily (218). Shh and Ihh display overlapping expression in the mouse gut endoderm; but at later developmental ages, more regionalized intestinal expression is established. At E14.5, Shh is expressed primarily in the fore stomach and duodenum, while Ihh is found from the hind stomach to the anus (218). Expression of mesenchymal Bmp in the small intestine parallels the epithelial expression of Hhs (218,219). For example, in the small intestine of E18.5 mice, both Shh and Ihh are found in epithelial cells at the base of villi where crypts will eventually form, while expression of Bmp4, Ptch, and Gli1 parallels this in the underlying mesenchyme (219). Similarly, Bmp4, Ptch, and Gli1 expression is found throughout the mesenchyme of the colon at E18.5. Whereas Ihh is expressed throughout the colonic epithelium at this developmental time point, expression of Shh is restricted to colonic crypt epithelial cells (219). The expression of Hh ligands and downstream target genes in late intestinal development and in the postnatal gut suggests a role for this gene family in regulating the size and location of the proliferative compartment, the patterning of the crypt-villus unit, or the maintenance of adult intestinal homeostasis (219,220,222). The potential role of Hh signaling in regulating small intestinal epithelial (stem) cell proliferation has been suggested by a number of studies. In one study, Ihh−/− mice displayed intestinal villi of reduced size compared with those of wildtype mice; this was accompanied by a 54% reduction in intervillus epithelial cell proliferation (219). The intestinal phenotype of Ihh−/− mice resembled that of Tcf-4 null mice, which lack a stem cell compartment. However, the intestinal localization and expression of Tcf-4 in Ihh−/− mice was not abnormal. These results suggest that normal Ihh pathway signaling may either induce additional Wnts or stimulate other pathway intermediates leading to the stabilization of β-catenin with subsequent activation of β-catenin/Tcf-4 signaling. This activation of β-catenin/Tcf-4 signaling is needed to maintain epithelial stem or progenitor cell proliferation in the intestinal crypts (118,199). However, when both Shh and Ihh signaling is inhibited, a different effect on stem or progenitor cell proliferation is obtained. Blockade of total Hh signaling in the intestine through the expression of an intestine-specific pan-Hh inhibitor, the hedgehog-interacting protein (Hhip), resulted in increased epithelial stem or progenitor cell proliferation, or both (220). This effect was mediated by mesenchymal cells that expressed: (1) Ptch1 and Ptch2, Hh receptors; (2) Gli1, a downstream transcription factor; and (3) BMP4, a target gene of the Hh signaling pathway (220). The strictly mesenchymal expression of Ptch1 and Gli1 suggests that Hh signaling in the small intestine is paracrine. Mice expressing

a villin-Hhip transgene in intestinal epithelial cells exhibited varied levels of transgene expression that correlated with the severity of the observed phenotype. The highest transgene expression resulted in dramatic histologic alterations in the architecture of the neonatal intestinal crypt-villus unit. This was exemplified by a flattened epithelium with regions that were pseudostratified, and the epithelium was hyperproliferative as assessed by Ki67 staining. These histologic alterations suggest that inhibition of Hh signaling interferes with the normal process of epithelial remodeling and villus formation. Lower transgene expression resulted in milder intestinal phenotypes showing disorganized villus architecture with crypt-like structures seen on the sides of thickened villi and villus branching (220). Again, proliferating epithelial cells were evident along the villi. Proteins expressed by differentiated villus epithelial cells were absent or greatly reduced from large areas of the intestine of villin-Hhip mice, underscoring the perturbation of epithelial differentiation along the crypt-villus axis. Hyperproliferation of crypt epithelial cells and disorganization of the villi was also reported in a study that inhibited Hh pathway signaling by the administration of a pan-Hh–specific antibody to mice late in gestation and after birth (228). The above studies with villin-Hhip mice indicate an interrelation between Wnt signaling and the Hh pathway. Gene targets of the β-catenin/Tcf-4 signaling pathway, Cdx1, Cd44, EphB2, and c-myc, were up-regulated and found in the ectopic cryptlike structures that formed in villin-Hhip mice (220). In addition, there was increased cytoplasmic and nuclear β-catenin localization in epithelial cells of the villus. Finally, both ISEMFs (which express Hh receptors) and the muscularis propria displayed abnormalities in the intestines of villin-Hhip mice. ISEMFs were mislocalized to the villus tips in close association with the ectopic proliferating epithelial cells. Taken together, these data suggest that epithelial Hh signaling to the surrounding mesenchyme defines the location of the proliferative compartment through reciprocal interaction between mesenchymal cells and the overlying epithelium. Hh signaling also establishes the polarity of the villus axis before crypt formation. Hh signaling might restrict epithelial stem or progenitor cell proliferation, or both, by interacting with the Wnt/β-catenin/Tcf-4 pathway, possibly through the (down) regulation of Wnt expression that is provided by the ISEMFs (86,118,229). Hh signaling seems to play a different role in the colon from that of the small intestine. Both in vivo and in vitro studies suggest a role for Hh signaling in regulating colonic crypt morphogenesis and commitment of progenitor cells to colonocyte specific differentiation. Differentiated colonic epithelial cells express Ihh; the colonic epithelium of wildtype mice at E16.5 consists of a monolayer of polarized epithelial cells with organized crypts and proliferating cells located at the crypt base. In addition, cells that have ceased to proliferate are located in the cuff region (221). By contrast, the colon of Ihh−/− mice is a multilayered epithelium composed of proliferating cells. However, no cryptlike structures were observed (221). The absence of cryptlike structures in Ihh null mice suggests that Ihh provides the requisite signals

326 / CHAPTER 11 for initiating crypt morphogenesis and subsequent epithelial cell differentiation in the fetal colon. Confirmation of this hypothesis was obtained by examining the consequence of inhibiting Hh signaling in vivo (221). The colon of adult rats treated with cyclopamine, an inhibitor of Hh signaling that binds the Smo receptor, displayed marked histologic changes, as well as loss of markers of mature differentiated colonocytes such as villin (specific for microvilli) and carbonic anhydrase IV (a brush border enzyme). In the colon, Ihh signaling also negatively regulates the Wnt pathway and restricts expression of downstream Wnt target genes to the crypt compartment. Cyclopamine-treated animals showed up-regulation of the Wnt pathway gene targets cyclin D1, BMP4, and Engrailed-1, with a concomitant increase in crypt epithelial cell proliferation (221). In the normal adult colon, two Wnt-regulated genes, Engrailed-1 and BMP4, display a gradient of expression along the crypt length, with greatest expression seen at the crypt base. Cyclopamine treatment abolished this gradient of expression, suggesting that normal Ihh signaling acts to restrict the effects of Wnt pathway activation, that is, expression of downstream target genes, to cells localized to the crypt base. Studies have confirmed that Ihh can regulate β-catenin/ Tcf-4 signaling in vitro (221). Finally, colonic adenomas from patients with FAP, where β-catenin/Tcf-4 signaling is increased as a result of mutations in the APC gene, do not express Ihh, suggesting that constitutive activation of the canonical Wnt pathway abrogates Ihh signaling (221). These data point to reciprocal regulation between the Wnt and Ihh signaling pathways that results in the restriction of epithelial cell proliferation to the crypt compartment. Bone Morphogenic Protein Signaling Pathway BMPs comprise a family of mesenchymal-derived, secreted, signaling molecules that are members of the TGF-β superfamily (230). Previous investigations show that the BMP pathway is involved in gut development and in the maintenance of adult intestinal homeostasis (230–232). More recent work has also begun to elucidate some of the mechanistic details of the role that BMP signaling plays in shaping the intestinal stem cell niche. BMPs bind to receptors (BMPR) with serine-threonine kinase activity to transduce a signal to the nucleus via (multiple) nuclear binding proteins, the Shads (230). BMP activity is antagonized by two soluble inhibitors, Noggin (gene product of Nog) and Gremlin (product of mouse gene Cktsf1b1) (233–235). Compelling evidence that Bmp4 plays a direct role in shaping the stem cell niche by restricting the topographic location of newly forming crypts comes from the in vivo inhibition of Bmp function (231,236). Intestinal expression of the Bmp inhibitor Noggin, under control of the mouse villin promoter resulted in ectopic formation of crypts perpendicular to the villus axis in the mouse small intestine (231). These crypts appeared normal in that there was a proliferative compartment at the base. Expression of crypt-specific genes was observed, most notably c-myc and

EphB3, genes regulated by the activated β-catenin/Tcf-4 signaling pathway. Finally, there was proper positioning of Paneth cells to the crypt base, and all the epithelial lineages appeared to be expressed. Gastric and duodenal juvenile polyps also develop in Smad4 null mice due to their inability to respond to a BMP signal (237,238). Smad4 forms part of the intracellular complex that transduces the Bmp signal from the cell surface to the nucleus to effect gene transcription in response to Bmp signaling (239). Nonsense mutations in human BMPR1A, a receptor for BMP4, underlie the formation of intestinal polyps in a subset of juvenile polyposis patients, further implicating BMP4 in the proper architectural arrangement of cells to form the crypt-villus unit (240). The BMP signaling pathway serves as an interface between two important signaling pathways: the canonical Wnt signaling pathway and the Hh pathway. Wnt/β-catenin/Tcf-4 signaling is known to be critical in maintaining the stem cell compartment and is a regulator of stem and progenitor cell proliferation in the crypt (118). BMP4 is a well known downstream target gene of this pathway. Hh proteins secreted by intestinal epithelial cells, known to be regulators of intestinal patterning during embryogenesis, are also emerging as key regulators of epithelial differentiation in the postnatal intestine through the repression of Wnt signaling (220,221). BMPs are also gene targets of the Hh pathway, especially BMP4 (220). BMP4, as well as other mediators responsive to Hh signaling, are expressed by mesenchymal cells surrounding Hh-secreting epithelial cells and constitute part of the communication network between the epithelium and the mesenchyme in the stem cell niche. Current data support the hypothesis that the spatial restriction of crypt formation to the intervillus epithelium at the base of villi is achieved by the cooperative interaction of the Bmp and Hh pathways during late intestinal development. Furthermore, this cooperation also serves to limit Wnt/β-catenin/Tcf-4 signaling to the stem cell niche (Fig. 11-9) (220,221,236). More direct evidence that BMP4 may restrict crypt epithelial stem and progenitor cell replication through the direct perturbation of downstream components of the Wnt signaling pathway has been reported (236). These authors show that mice in which the BMP receptor Bmpr1a had been conditionally inactivated develop small intestinal polyps. These polyps display an increase in the number of crypts in polypoid regions compared with the number of crypts found in normal intestinal segments of control mice (236). Wnt signaling, as measured by phosphorylation of LRP6, a component of the Wnt receptor complex, was active in both wild-type and Bmpr1a mutant mice (236). Thus, the observed expansion of the crypt proliferative compartment was not due to activation of the Wnt receptor in the Bmpr1a mutant mice. Rather, BMP regulates a downstream component of Wnt receptor interaction, the nuclear levels and activity of β-catenin (236). Based on their observations, He and colleagues (236) propose that activation of stem cell self-renewal depends on two signals, the Wnt signal as previously proposed by other groups and a

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 327 and epithelial cells to regulate a number of differing functions, including mucosal morphogenesis, normal cellular proliferation and epithelial turnover, differentiation, mucosal regeneration after injury, and tumorigenesis. Many different growth factor families have been found to affect mucosal function in the gastrointestinal tract including FGFs, the epidermal growth factor family, TGF-β, growth hormone and insulinlike growth factors (IGFs), hepatocyte growth factors, platelet-derived growth factors, and GLP-2 (241–251). In the following sections we discuss several growth factor families for which substantial evidence exists to imply a direct effect in regulating epithelial stem and/or progenitor cell fate or proliferation in the gastrointestinal tract.

Ihh

BMP4

Ihh BMP4

Fibroblast Growth Factors Notch

Stimulatory

Wn

t

Inhibitory

W

nt

nt

W

Wnt

Wnt

FIG. 11-9. Model of the signaling network that defines the intestinal stem cell niche and regulates the gradient of proliferation to differentiation. The stem cell niche is defined by epithelial-mesenchymal signaling, which restricts Wntexpressing cells to the base of the crypt. Mesenchymal expression of BMP4 is maintained by two signals, Wnt-expressing cells that surround the crypt epithelium and Indian hedgehog (Ihh)–expressing villus epithelium. Bone morphogenic protein 4 (BMP4), in turn, has an inhibitory effect on Wnt expression, restricting Wnt-expressing mesenchymal cells to around the crypt base. The Ihh expression domain is also regulated by an unknown Wnt-responsive factor, restricting the domain to differentiated villus epithelium. A gradient of factors sets up a microenvironment within the stem cell niche to promote adhesiveness of the stem cell, proliferation of the transit-amplifying population, and differentiation of epithelium. Wnt signaling is a likely candidate for creating morphogendependent differential responses within the microenvironment. Low levels of Wnt signaling near the mid and upper regions of the crypt may permit Notch signaling to affect cellular differentiation of the intestinal lineages. Notch, in turn, acts to suppress the Wnt signal. (Reproduced from Rizvi AZ, Wong MH. Epithelial stem cells and their niche: there’s no place like home. Stem Cells 2005;23:150–165, by permission.)

transient suppression of BMP signaling, possibly through the expression of the BMP inhibitor Noggin.

Growth Factors and Peptides Growth factors are a large heterogeneous group of peptides that mediate interactions between cells in the mesenchyme

The FGF family consists of a series of heparin-binding proteins encoded by at least 22 distinct genes that have been identified in many organisms, including human and mouse (252). FGFs regulate pleiotropic responses, during normal development, tissue homeostasis, and in injury repair. FGFs are biologically active for a wide variety of cell types, including cells of epithelial, mesodermal, and neuroectodermal origin. FGF1 and FGF2, the prototypical members of this family, lack a consensus signal sequence required for the classical secretory pathway. The lack of a signal sequence suggests that a major mode of release of FGF1, FGF2, or both may be through cell death or injury, and that these growth factors may have a prominent role in wound repair. However, the mechanism by which these two prototypical FGFs are released from cells has yet to be fully elucidated. FGF3-8, 10, 17-19, and 21-23 are readily secreted from cells due to the presence of appropriate amino-terminal signal peptides. FGF9, FGF16, and FGF20 lack a signal peptide but are nevertheless secreted (252). FGF11-14 also lack signal sequences and are thought to remain intracellular (253). Expression of a number of FGFs has been described in the gastrointestinal tract (60,115,243,244,254–260). However, the levels and patterns of individual FGF genes being expressed depend on the particular developmental stage or physiologic context. For instance, FGF2 is expressed during embryogenesis in the developing foregut and regulates patterning of the foregut endoderm for the formation of hepatic and pancreatic buds (241). Similarly, FGF10 can be detected in the developing mouse gut as early as E10.5 and is expressed in the mesenchyme surrounding the cecal bud at E11.5 (243,244). FGF10 regulates proliferation and infiltration of the colonic endoderm into the surrounding mesenchyme. Mice with a targeted knockout of FGF10 or its receptor, FGFR-2, have decreased endodermal replicative activity and do not develop a cecum (244,261). In the suckling mouse intestine when crypt morphogenesis is occurring, expression of FGF1, FGF9, and to a lesser extent FGF2 predominates (260). Expression of FGF10 is difficult to detect at this developmental stage but is induced in the adult intestine after experimental injury (262).

328 / CHAPTER 11 In contrast to the patterns of FGF ligand expression associated with early development of the gut and with nascent crypt formation in the suckling mouse intestine, basal expression of FGFs in the adult gastrointestinal tract is low. However, expression of several of the FGF genes can be induced in response to injury or inflammation. Levels of FGF7 (keratinocyte growth factor) are increased in the intestinal mucosa of patients with inflammatory bowel disease (IBD). Vidrich and colleagues (260) found that increased expression of FGF2, FGF7, and FGF10 in the ileum of SAMP1/YitFc mice, a mouse model that develops chronic ileal inflammation, paralleled the increase in the number of potential clonogenic stem cells per crypt that occurs in this model. The response to radiation injury is also characterized by marked increases in the expression of FGF2, FGF4, and FGF10. This coincides temporally with the appearance of regenerative crypts derived from surviving clonogenic stem cells and the subsequent expansion of crypt numbers through branching or fission (60,259). Proliferation of the surviving crypt stem cell population must occur to support the expansion in crypt numbers that replenishes the complement of proliferative units in the epithelium. Several FGFs have been shown to regulate proliferation, differentiation, or migration of intestinal epithelial cells in culture (259,263,264). Dignass and Podolsky (263) have demonstrated that FGF1, FGF2, and FGF7 have mitogenic activity for the intestinal epithelial cell lines IEC6, Caco-2, and HT-29 in culture. In an in vitro wound-healing model, these researchers also demonstrated that FGF1 and FGF2 promote cell migration and epithelial restitution through a TGF-β-dependent mechanism. More recently, the mitogenic activity of FGF4 and FGF10 on cultured intestinal epithelial cells has been described (259,264). FGFs may also play a role in regulating lineage commitment of undifferentiated progenitors. For example, a study by Iwakiri and colleagues (265) demonstrated that FGF7 could mediate goblet cell differentiation in the H2 subclone of the human colonic epithelial cell line HT-29 through regulation of the goblet cell silencer inhibitor element. The ability of FGFs to regulate the proliferation or fate of intestinal stem cells and progenitor cells in vivo has also been described (60,254–256,264,266,267). Pharmacologic studies of FGF2, FGF7, and FGF10 have demonstrated that these ligands can each enhance clonogenic crypt stem cell survival in the microcolony assay when they are administered before irradiation. Interestingly, these growth factors must be administered for several days before irradiation to achieve a dramatic effect on crypt stem cell survival after irradiation. Potten and colleagues (268) have suggested that a possible explanation for this effect is that FGF7 (and potentially other FGFs) increases stem cell number or induces changes in the cell cycle dynamics of the clonogenic crypt stem cell before radiation injury. The biological effects of FGFs on target cells are mediated by binding to high-affinity, cell-surface receptor tyrosine kinases encoded by at least four separate genes (269–277). Alternative splicing of the primary RNA transcripts results

in two distinct isoforms of each receptor (IIIb and IIIc) that differ in their ligand-binding affinities for various members of the FGF family. Tissue-specific and cell-specific differences in the pattern of receptor RNA splicing and expression of various isoforms have been found (252). The IIIb isoforms of FGFR-1, -2, and -3 are expressed predominantly in epithelial cells and have a more restricted ligand-binding profile than the corresponding IIIc isoforms. Our understanding of the role of FGFRs in regulating the fate or proliferation of stem and progenitor cells in the gastrointestinal tract is complicated by the existence of multiple isoforms of each receptor. In addition, individual FGFR isoforms may have significant binding affinity for multiple FGF ligands that overlap with the ligand-binding characteristics of the other FGFRs (252,278). Thus, multiple FGFRs may play overlapping or distinct roles in regulating the physiology of intestinal stem cells and progenitor cells under a variety of physiologic or developmental circumstances. For example, studies using laser capture microdissection and immunohistochemistry have shown that both FGFR-2 and -3 are expressed in intestinal epithelial cells. FGFR-2 mRNA was detected throughout the gastrointestinal epithelium during early gut development (243,244,261) and in both crypt and villus epithelial cells in the adult murine small intestine. FGFR-2 IIIb has been implicated in the regulation of epithelial regeneration after intestinal injury or associated with intestinal inflammation (254–256,264,266,267). The FGFR-2 IIIb ligands, FGF7 and FGF10, are both able to increase the number of surviving crypt epithelial stem cells when they are administered before irradiation (267,268,279). FGF7 can induce a human colonic epithelial cell line, HT29 subclone H2, to differentiate into goblet cells in vitro (265). Given the specificity of this ligand for FGFR-2 IIIb, these findings suggest that signaling via FGFR-2 may mediate aspects of lineage allocation decisions within the progenitor cell population, in addition to its effects on the clonogenic stem cell population. Expression of FGFR-3 in the small intestine and colon is developmentally regulated. Increased expression of FGFR-3 was observed as early as E15 and E16. FGFR-3 is also expressed at high levels in the suckling mouse intestine (postnatal days 4–21), a developmental time period characterized by formation of small intestinal crypts and expansion of crypt numbers through crypt fission (115). In contrast to the relatively uniform distribution of FGFR-2 along the crypt-villus axis, we found that cell-surface FGFR-3 was expressed only in crypt epithelial cells (Fig. 11-10) (115). Expression of FGFR-3 was not observed in the entire TA cell population, but rather was restricted to a smaller subset of replicating cells in the lower half of the crypt, where epithelial stem cells and undifferentiated progenitor cells are found (Fig. 11-11) (115). FGFR-3 IIIb was the dominant isoform expressed in both murine small intestinal and colonic crypts. In humans, FGFR-3 IIIb is expressed in the normal colon and in multiple intestinal epithelial cell lines (280). FGFR-3 is also highly expressed within regenerating crypts after radiation injury (115). The developmental stage-specific

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 329 A

B

C

D

BG

FIG. 11-10. Fibroblast growth factor receptor-3 (FGFR-3) expression in the suckling mouse intestine. Sections were incubated with rabbit anti–FGFR-3. (A) Immunoreactive FGFR-3 in the proximal jejunal crypts from a 14-day-old mouse (original magnification ×100). Open arrowheads point to Paneth cells, while the black arrowheads indicate FGFR-3–positive ganglion cells. The inset shows a higher magnification view of a crypt from the section in A (original magnification ×400). Staining is localized to the basolateral membranes of undifferentiated epithelial cells in the lower half of the crypt. Black arrow indicates the crypt-villus junction. Arrowheads denote Paneth cells showing barely detectable staining. (B) The specificity of the anti–FGFR-3 immunohistochemistry is demonstrated in a section from the proximal jejunum incubated with normal rabbit IgG in place of the primary anti–FGFR-3 antibody. (C) In the duodenum (original magnification ×100), staining is confined to the crypt epithelium with no staining in the adjacent Brunner’s glands (BG). (D) FGFR-3 immunoreactivity also is detectable in the lower third portion of colonic crypt epithelium (original magnification ×200). (See Color Plate 4.) (Reproduced from Vidrich and colleagues [115], by permission.)

changes in FGFR-3 expression, coupled with the observation that FGFR-3 is highly expressed within adult regenerating crypts after radiation injury, strongly suggest that one major function of this receptor is regulation of stem cell fate during crypt morphogenesis (115). Activation of FGFR-3 IIIb can induce proliferation of Caco-2 intestinal epithelial cells in vitro (280). Expression of FGFR-3 IIIb was down-regulated as these cells differentiated and correlated with the loss of proliferative responses to FGFR-3 IIIb–specific ligands. Induction of FGFR-3 IIIb expression in differentiated cells restored the proliferative response only to those FGF ligands that bind to FGFR-3 IIIb.

These data, as well as the observation that cell-surface expression of this receptor is restricted to undifferentiated cells in the lower crypt, imply that FGFR-3 has a restricted role in crypt morphogenesis. This likely involves mediating the proliferation or fate of stem cells or their immediate descendents, rather than regulating the proliferative response of the entire replicating crypt cell pool. The downstream signaling pathways through which FGFRs mediate their effects on epithelial stem and progenitor cells have only been partially defined. FGFRs can activate a number of downstream signaling pathways, including signal transducer and activator of transcription 1 and 3 (STAT1/3),

330 / CHAPTER 11

A

B

C

FIG. 11-11. Cell-specific patterns of fibroblast growth factor receptor-3 (FGFR-3) expression in mouse proximal jejunum. Proximal jejunum from 21-day-old FVB/n mice (A–C) received BrdUrd 2 hours before tissue harvest. (A) S-phase cells incorporating BrdUrd (red fluorescence) are found primarily in the lower two-thirds crypt. Solid white arrows indicate the location of the crypt-villus junction. (B) FGFR-3 is expressed along the basolateral surfaces of epithelial cells in the lower crypt (green fluorescence). Open arrows indicate the basilar surface of a crypt. (C) Colocalization of BrdUrd and FGFR-3. The S-phase cells in the upper portions of the crypt epithelium do not stain with anti–FGFR-3 (e.g., white arrowhead ). (See Color Plate 5.) (Modified from Vidrich and colleagues [115], by permission.)

and the phosphatidylinositol-3 kinase-dependent, phospholipase Cγ–dependent, and Ras/Raf/mitogen-activated protein kinase–dependent signaling cascades (280–282). An intriguing report demonstrates that FGF2, a ligand of FGFR-2 and -3, can stimulate Tcf-4/β-catenin signaling in some cell types (114). Activation of Tcf-4/β-catenin–mediated signaling is required for proliferation of the crypt stem cell population and normal morphogenesis of the intestinal crypt epithelium (see earlier in this section) (89,178). Although much attention has been focused on Wnt(s) as the candidate upstream mediator(s) of Tcf/β-catenin signaling pathway, the finding that other growth factors such as FGF2 can also regulate Tcf/β-catenin activity suggests the possibility that other upstream signaling pathways may be involved (114). The cellspecific pattern of FGFR-3 expression reported by Vidrich and colleagues (115) encompasses the stem cell and progenitor cell populations, which are regulated via Tcf-4 (89). In addition, the timing of FGFR-3 expression in the intestinal epithelium relative to Tcf-4 mRNA expression would support such a speculation (115,178). Insulin-like Growth Factors The IGFs, IGF-I and IGF-II, are small polypeptide growth factors (~7 kDa) that are expressed in many different tissues, including the gastrointestinal tract. A number of reports have emphasized the potential role of the IGF family in enhancing intestinal growth (245–247). IGFs also promote epithelial regeneration in a number of injury models including methotrexate-induced enteropathy (283), dextran sulfate–induced colitis (284,285), radiation injury (286–288), and intestinal adaptation after small-bowel resection (289–291). IGF-I also suppresses apoptosis of colon cancer cells (292,293) and promotes restitution of epithelial cell monolayers in culture after experimental wounding (294). IGF-I and -II bind to both type I and II IGF receptors in the gastrointestinal tract and may mediate muscular hypertrophy and fibrosis

in Crohn’s disease (295,296). Several IGF-binding proteins modulate the biological activity of IGFs. Evidence that IGFs may regulate intestinal stem cell or progenitor cell function, in addition to their effects on mesenchymally derived cells in the intestine, has emerged from recent studies. Several groups have reported that administration of IGF or overexpression of IGF-I in the intestine is protective against radiation enteritis (286–288,293). In IGF transgenic mice, this effect is mediated in part by suppression of radiation-induced apoptosis in the lower crypt (286). Maximal suppression of apoptosis was noted in the stem cell zone, at cell positions 3 to 5 above the crypt base. In the IGF transgenic mice, induction of the proapoptotic gene Bax by irradiation was also suppressed in the lower third of the crypt epithelium. The authors concluded that IGF-I enhanced mucosal regeneration in this model through direct effects on crypt cell replication or apoptosis within the stem cell compartment. Studies of intestinal development in transgenic mice that overexpress IGF-II in the intestine and in IGF-II knockout mice point to a role for this growth factor in regulating epithelial stem cell proliferation or crypt morphogenesis (51). The surface area and the number of crypts (structural/proliferative units) in the colon of IGF-II–deficient mice were reduced during the suckling–weaning transition and early postweaning period. Conversely, mice overexpressing an IGF-II transgene under the control of a keratin promoter (K:IGF-II transgenic mice) manifested a significant increase in the surface area of the colonic mucosa. This increase in luminal surface area was attributable to increased numbers of colonic crypts, while changes in the size of the crypts or the rates of spontaneous crypt epithelial apoptosis were not observed. Rather, the authors conclude that the increase in the number of crypts in the K:IGF-II transgenic mice was due to an accelerated rate of crypt fission. Since crypt fission results in the segregation of clonogenic crypt stem cells to daughter crypts, an increased rate of symmetric clonogenic stem cell division must accompany this process.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 331

Transforming Growth Factor-β Members of the TGF-β family are thought to be important regulators of epithelial homeostasis in the normal gastrointestinal tract. TGF-β mediates its

KGF

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Glucagon-like peptide-2 is a 33-amino-acid peptide produced by enteroendocrine cells (L cells) in the distal small intestine and colon (297). Studies have identified GLP-2 as a potent intestinal growth factor in rodents that acts by expanding the mucosa through stimulation of crypt epithelial cell replication (248,298,299). The trophic effects of GLP-2 are greater in the small intestine than in the colon (297,298). In addition to its trophic effects in the uninjured intestine, GLP-2 promotes epithelial regeneration in a number of models of epithelial injury and inflammation (300–303). GLP-2 may also regulate crypt epithelial apoptosis in response to experimental injury. Administration of GLP-2 in the setting of nonsteroidal anti-inflammatory drugs or chemotherapy-associated injury to the intestinal mucosa significantly reduced crypt epithelial apoptosis induced by these agents (301,304). The elegant studies reported by Bjerknes and Cheng (88) have provided important new insights into the mechanism of the effects of GLP-2 on epithelial stem cell and progenitor cells in the gut. An in vivo assay was developed that could distinguish effects of growth factors and other mediators on specific stem cell or progenitor cell subsets defined by the cell lineage(s) tagged by induced mutations in the Dlb-1 locus. Dlb-1 encodes a cell-surface binding domain for the lectin, Dolichos bisflorus, and the progeny of Dlb-1+ stem cells and progenitor cells can be identified histologically by their ability to bind this lectin. Thus, the lineages represented in the Dlb-1+ clone of cells along a crypt-villus unit define the functional capacity of the specific mutant progenitor or stem cell that gave rise to the clone (Fig. 11-12). Stem cell clones and columnar-specific progenitor cells specifically responded to GLP-2 administered to mice and resulted in expansion of columnar cell lineages in these mutant clones. Mucous cell progenitors were unaffected. These data point to a rather specific effect of GLP-2 on stem cells and only a subset of long-lived lineage committed progenitors within the lower crypt epithelium. The biological activity of GLP-2 is mediated by binding to a G protein–coupled receptor, GLP2R. However, expression of this receptor was found only in enteric neurons. GLP2R was not detected in crypt epithelial cells in the rodent gut in this study (88). The GLP2R+ neurons were shown to respond to GLP-2 stimulation, and the direct effect of these neurons on the crypt progenitor cell population was demonstrated by local inhibition of neuronal signaling. This intriguing finding implies that GLP-2 mediates its effects on epithelial stem cells and progenitor cells through an intervening local neural circuit that impinges directly on the function of the crypt epithelium and emphasizes the potential importance of the enteric nervous system in integrating the response of the stem cell population to physiologic stimuli.

GLP-2

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1 0 Stem Clones Epithelium

FIG. 11-12. Progenitor behavior detected by specific progenitor assay by somatic mutation (SPASM) after glucagon-like peptide-2 (GLP-2) or keratinocyte growth factor (KGF) administration. (A) Photomicrographs of typical NEU-induced Dlb-1+ clones derived from mucous progenitors, columnar progenitors, or stem cells. Dlb-1+ cells, labeled with horseradish peroxidase–conjugated lectin are stained brown. The tissue is counterstained with Alcian blue to aid identification of mucous cells (which are blue [arrow], unless Dlb-1+ [arrowhead] ). Columnar cells are pale blue (arrow), unless Dlb-1+ (arrowhead). Dlb-1+ mucous (arrowhead) and columnar (arrow) cells are both seen in stem cell clones. Original magnification ×320. (B) After GLP-2 treatment, columnar cell and stem cell clones are larger in comparison with those in control samples. Mucous cell clones were larger after KGF treatment. Control clones had 129.2 ± 28.3, 3.4 ± 0.3, 12.0 ± 3.5 (mean ± standard error of the mean) cells for stem cell, mucous cell, and columnar cell clones, respectively. (C) GLP-2 treatment decreases, but KGF treatment increases mucous cell density in the epithelium and stem cell clones. Control mucous cell density was 0.039 ± 0.001 in the epithelium and 0.032 ± 0.003 in stem cell clones. (See Color Plate 6.) (Reproduced from Bjerknes and Cheng [88], by permission.)

332 / CHAPTER 11 effects through binding to and forming a multimeric complex with type 1 and 2 TGF-β receptors on the cell surface (305). Binding of ligand results in phosphorylation of Smad2 and Smad3, which form a heterotrimeric complex with cytoplasmic Smad4. On translocation to the nucleus, this complex modulates the transcriptional activity of TGF-β target genes. Expression of several TGF-β isoforms (TGF-β1, TGF-β2, TGF-β3) has been reported in the adult intestine. TGF-βs are expressed in multiple epithelial and nonepithelial cell types within the intestinal tract (306). However, the epithelium is thought to be the predominant source of TGF-β synthesis under basal conditions (306,307). A number of observations suggest that TGF-βs may have a direct role in regulating epithelial homeostasis along the crypt-villus axis of the intestinal epithelium. Administration of exogenous TGF-β3 resulted in suppression of proliferation at cell positions 3 to 4 and at cell positions 13 to 27 from the base of the crypt, suggesting that TGF-β can regulate proliferation of both the stem cell and the TA cell populations in the small intestine and colon (242). A direct effect on the stem cell population is also suggested by the observation that prolonged administration of TGF-β results in significant protective effect on crypt stem cells using the microcolony formation assay after radiation injury (242,308). Booth and colleagues (242) suggest that this protective effect is mediated by cell-cycle inhibition of the stem cell population, rendering fewer stem cells sensitive to the cytotoxic effects of radiation injury during the sensitive phase of the cell cycle. A similar protective effect of TGF-β2 was observed using a methotrexate intestinal injury model and was attributed to alterations in the cell-cycle dynamics of the epithelial stem cell population (309). TGF-β also has a role in regulating intestinal inflammation, injury repair, and tumorigenesis. Mice homozygous for a targeted disruption of the TGF-β1 gene develop a multifocal inflammatory disease and die of organ failure at about 3 weeks old. Increased expression of TGF-β has also been noted in experimental models of intestinal injury in vivo and in IBD and may provide a mechanism for cross talk between the epithelial stem cell niche and the mucosal immune system (310,311). The inflammatory phenotype and early mortality observed in Tgf β1 −/− mice can be largely abrogated by introducing this targeted mutation into a Rag2−/− background. The Tgf β1−/− Rag2−/− mice develop multiple carcinomas in the cecum and colon by 5 months old, suggesting that TGF-β1 acts as a potent tumor suppressor gene altering some aspect of stem cell physiology in these regions of the gut. Surprisingly, the epithelial cell proliferation was not grossly altered in the hyperplastic epithelium, suggesting that other compensatory mechanisms may regulate the response of the TA cell population in the absence of TGF-β1. Mice heterozygous for targeted disruption of Smad4, a signaling molecule downstream of the TGF-β receptor, and an Apc gene mutation develop increased numbers of polyps due to somatic loss of the wild-type Apc and Smad4 alleles (237). The lesions formed in these compound mutant mice progress to form malignant

adenocarcinomas to a far greater degree than mice that are heterozygous for the mutant Apc allele alone (237). However, polyps found in compound heterozygotes for a target mutation of Smad2 and the same Apc mutation were not different from mice heterozygous for the Apc mutation alone, suggesting that there may be a much greater degree of complexity to this signaling pathway than originally thought (312).

Inflammation: Cytokines and Other Mediators The adult gastrointestinal tract is characterized not only by the rapid and continuous replacement of differentiated epithelial cells, but also by a remarkable capacity to quickly repair damage that disrupts mucosal integrity. Mucosal injury and ulceration are common features found in a variety of gastrointestinal diseases that can be induced by numerous causative agents including infectious agents, toxins, acute or chronic radiation injury, ischemia, and immune activation as seen in IBD. For example, IBD is characterized by cycles of clinically apparent disease activity marked by the presence of mucosal injury and inflammation, followed by periods of disease quiescence during which the injured mucosa is repaired and normal gut function is restored. Loss of mucosal integrity from any cause leads to a common set of histopathologic features that constitute the host response, which include inflammation and mucosal regeneration (313). Mucosal regeneration in the intestine after intestinal injury or inflammation is a multistep process that must ultimately involve changes in the dynamics of the proliferating crypt epithelial cells and the epithelial stem cell population (22, 229,314,315). Initially, a continuous simple epithelial cell monolayer has to be restored through migration of adjacent epithelial cells over the wound. Subsequently, intestinal crypts (the morphologic unit of cell renewal) must form within the reconstituted epithelium. A single surviving clonogenic crypt stem cell is sufficient to give rise to a regenerating crypt. Crypts may also undergo branching or fission as part of the regenerative process to replenish the epithelium with sufficient numbers of proliferative units (crypts) in areas where mucosal disruption is extensive and severe. Since the epithelial stem cells within a crypt segregate to daughter crypts when crypt fission occurs, an expansion of the stem cell number must accompany this reparative response. Finally, the normal morphologic relationship of the epithelium to the underlying mesenchymal tissue and vasculature must be reestablished, and normal region-specific epithelial differentiation and function must be reconstituted. Inflammation may have both localized and systemic effects on the epithelial stem cell and progenitor cell populations. An illustration of this phenomenon is provided by studies of the SAMP1/YitFc mouse. These mice are a recombinant inbred strain in which many features of human Crohn’s disease develop including segmental small intestinal inflammation that is confined to the ileum (316). These mice develop localized perturbations in lineage-specific epithelial differentiation in the ileum at an early age, when

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 333 histologic evidence of acute or chronic inflammation is first beginning to occur (260). Marked expansion of multiple secretory cell lineages, including increased numbers of Paneth, goblet, and intermediate cells, occur in regions of ileal inflammation (Fig. 11-13). Accompanying this localized expansion of secretory cell lineages is a loss of absorptive enterocytes. Changes in epithelial differentiation within the ileal mucosa persist into adulthood and are further amplified with the development of a local inflammatory response, reciprocal interactions between inflammation and epithelial differentiation. These observations favor a model in which the local inflammatory response in the SAMP1/YitFc ileum is associated with an increase in the number of the common secretory cell lineage progenitors at the expense of the columnar (absorptive) progenitor cell population. The number of stem cells per crypt also increases markedly at the onset of inflammation in the SAMP1/YitFc mouse (Fig. 11-14). However, the increase in crypt stem cell number occurs globally, both in inflamed regions of the

small intestine (ileum) and in the proximal jejunum, a region of the small intestine where inflammation does not develop in this model. The global increase in crypt stem cell number follows a time course that parallels the severity of inflammation. Thus, the mechanisms that regulate the global effects of inflammation on the crypt stem cell population are distinct from those that mediate local perturbations in progenitor cell replication, differentiation, or both, suggesting that different components of the inflammatory response are involved. A number of cytokines and other mediators associated with intestinal inflammation or injury can induce alterations in the crypt stem cell or progenitor cell populations when exogenously administered. These include tumor necrosis factor (TNF)-α, TGF-β3, trefoil family factor 3 (TFF3), interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-11, prostaglandins, cyclooxygenase-1 (COX-1), COX-2, FGF2, FGF7, and FGF10 (59,60,242,264,308,317–325). Expression of FGF2, FGF7, FGF10, and IL-11 were each increased

A

B

C

D

FIG. 11-13. Altered lineage allocation and crypt morphology accompany ileitis in SAMP1/YitFc mice. Ileitis in SAMP1/YitFc mice is characterized by the presence of a marked transmural acute and chronic inflammatory infiltrate and crypt abscesses. Loss of villi, marked crypt elongation, and increased numbers of Paneth cells and intermediate cells containing eosinophilic granules (A, open arrows ) are evident throughout the crypt of SAMP1/YitFc mice at 20 weeks of age. Note the eosinophilic granules in numerous cells located from the base of the crypt to the crypt-villus junction. In control AKR or C57BL/6 mice, Paneth cells are normally restricted to cell positions 1 to 4 at the crypt base. Periodic acid-Schiff-Alcian blue staining demonstrates increased numbers of goblet cells in areas of inflammation (B). Electron microscopic analysis of crypt epithelium of 10-week-old SAMP1/YitFc mice shows cells in the mid and upper crypt containing immature Paneth cell granules characteristic of intermediate cells (C). Electron micrograph of Paneth cells with mature granules from 10-week-old AKR mice is shown for comparison (D). (See Color Plate 7.) (Reproduced from Vidrich and colleagues [260], by permission.)

334 / CHAPTER 11 A

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FIG. 11-14. Regeneration of crypts in SAMP1/YitFc mice after radiation injury. The appearance of regenerating crypts is shown in hematoxylin and eosin–stained sections of ileum from 40-week-old SAMP1/YitFc mice (A) and age-matched AKR (B) mice 3.5 days after 14 Gy γ-irradiation. In adjacent sections, S-phase cells were detected by immunohistochemistry with anti-BrdUrd (C, D). A regenerative crypt (microcolony) is observed if one or more clonogenic stem cells within the crypt survive radiation injury and are able to subsequently replicate. Note that the number of regenerating crypts is markedly increased in the SAMP1/YitFc (C) mice compared with the age-matched AKR control mice (D). Crypt stem cell survival was globally increased in both inflamed (ileum, E) and noninflamed (proximal jejunum, F) regions of the small intestine in SAMP1/YitFc mice compared with AKR or C57BL/6 control mice (*, p < 0.01). Crypt stem cell survival after 14 Gy γ-irradiation (G) increases in parallel to the severity of the inflammatory response (H). (See Color Plate 8.) (Reproduced from Vidrich and colleagues [260], by permission.)

significantly coincident with the onset of inflammation and the increase in stem cell number in SAMP1/YitFc mice (260). Prior studies have suggested that FGF2, FGF7, and FGF10 may each play a role in regulating repair of injury to the intestinal epithelium (60,254–256,258,264,266,267, 326). Serum levels of FGF2 are significantly increased in patients with active IBD (258,326) and in the mouse small intestine after radiation injury. As discussed in the preceding sections, FGF2 enhances crypt stem cell survival after irradiation (60). FGF7 and its receptor are also expressed at greater levels in the colonic mucosa of patients with IBD (254–256). Given the effects of FGF7 or FGF10, or both, on crypt epithelial progenitors and mucous cell differentiation reported in other models of injury repair (264,279), it is tempting to speculate that the increase in FGF7 or FGF10 that occurs with inflammation in the SAMP1/YitFc ileum is responsible for the local perturbations in epithelial differentiation observed in this model, although this speculation has yet to be confirmed.

IL-11 is produced by intestinal myofibroblasts and possibly by other intestinal cells in response to various cytokines that regulate the inflammatory response (327). Some studies have shown that administration of IL-11 reduced the severity of colitis in HLA-B27 rats with established colonic disease (328,329). In other studies, IL-11 increased crypt stem cell survival in a model of severe cytoablative chemotherapy (324,328) or after γ-irradiation (325). This occurred in conjunction with increased villus length, preserved crypt/villus ratio, and reduced hepatic bacterial foci, as well as decreased apoptosis of intestinal epithelial crypt cells (324). Increased IL-11 levels are also associated with the development of inflammation in the ileum of SAMP1/Yit mice and may mediate the global increase in crypt epithelial stem cell number observed in this model (260). These data have led to the hypothesis that IL-11 may exert its beneficial effects by simultaneously affecting stem cell production or survival and immune system modulation including reduction of proinflammatory cytokines (324,325,329–332).

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 335 The effect of a particular cytokine or inflammatory mediator may also depend on the particular physiologic context present in the stem cell/progenitor cell niche. For instance, in mice without inflammation, blocking IFN-γ has little effect on proliferation of stem cells or progenitors in the lower crypt (323). However, in mice infected with the parasite, Trichuris muris increased proliferation was observed at cell positions 1 to 4, the region of the crypt containing the stem cell population, and at cell positions 11 to 15, corresponding to the TA cell zone. The increased proliferation in both the stem cell zone and the TA cell zone was abrogated by treatment with neutralizing anti–IFN-γ antibody but not by isotype-matched control immunoglobulin, suggesting that IFN-γ mediates stem cell proliferation in this physiologic context. The source of a particular mediator that regulates stem cell function may also depend on which cellular or enzymatic pathway(s) are activated in a particular inflammatory context. For example, in the absence of inflammation, prostaglandins produced by Cox-1 but not by Cox-2 were found to enhance crypt stem cell survival in the microcolony assay and to suppress radiation-induced apoptosis in the lower crypt of normal mice (59,61). However, when mice were exposed to a proinflammatory stimulus such as bacterial lipopolysaccharide before irradiation, crypt stem cell survival and radiation-induced apoptosis became highly dependent on prostaglandins produced by COX-2 (319). Protection of the stem cell population and inhibition of apoptosis in the lower crypt by lipopolysaccharide are dependent on TNF receptor I, suggesting that complex signaling networks are involved in mediating this effect. Distinct subsets of cells within the adaptive mucosal immune system may mediate the response of the stem cell/ progenitor niche to different immunologic or inflammatory stimuli. Kamal and colleagues (333) show that a distinct epithelial response occurs after infection with another parasitic worm, Trichinella spiralis. The predominant response in the small intestinal crypt epithelium to infection with T. spiralis was an increase in Paneth and intermediate cell numbers. The increase in Paneth and intermediate cells occurred in infected euthymic and nude mice but not in infected TCR(β/δ)−/− mice. Adoptive transfer of normal mesenteric lymph node cells to infected TCR(β/δ)−/− mice restored the increase in Paneth/intermediate cells observed after T. spiralis infection, suggesting a unique T-cell population mediates this effect on epithelial lineage allocation as part of the immune response to this parasite. This phenotype is reminiscent of the local perturbation in secretory cell lineage allocation observed in SAMP1/YitFc mice as described earlier (260). In contrast to T. spiralis infection, increases in crypt progenitor cell replication in colitis induced by dextran sodium sulfate (DSS) depended on the presence of luminal flora and activated macrophages (334). Intact Toll-like receptor signaling was also required since the increased proliferation of colonic epithelial progenitors did not occur after DSS treatment in Myd88−/− mice, which are defective in signaling via Toll-like receptors that recognize various components of the microbiota. Experiments using Csf1op/op

(lacking macrophages) Rag1−/− mice (depleted of lymphocytes) and mice depleted of neutrophils by treatment with a neutrophil-specific monoclonal antibody indicated that the increase in crypt progenitor cell proliferation in DSS colitis required macrophages but not T cells or neutrophils. Pericryptal macrophages isolated by laser capture microdissection from DSS-treated animals displayed an activated phenotype. These studies illustrate that a complex network of signals generated by both the luminal environment and the mucosal immune system are integrated within the stem cell niche to regulate the response of epithelial stem cells and progenitors to a broad range of differing physiologic or environmental circumstances.

STEM CELL PLASTICITY The traditional view that organ-specific stem cells have irreversibly lost the capacity to differentiate into cell types of other tissues has been long standing. However, this dogma has been challenged in the past few years by studies demonstrating that adult stem cells isolated from one organ or tissue may be able to differentiate into cells of other tissues under certain circumstances (reviewed by Anderson and colleagues [335]). An example of this phenomenon is illustrated by studies showing that transplantation of bone marrow cells into humans or lethally irradiated animals can give rise to the expected hematopoietic cell lineages, as well as to nonhematopoietic cells such as cardiomyocytes, hepatocytes, skeletal muscle fibers, and others (315,336). These findings raise the possibility of using bone marrow–derived cells for repopulating a failing organ with autologous functional cells. One demonstration of this concept is the rescue from liver failure of mice that are deficient in fumarylacetoacetate hydrolase (FAH) enzyme by the transplantation of purified hematopoietic stem cells (337). The transplanted cells differentiated into functionally normal, FAH-expressing hepatocytes and restored organ function. Several studies have begun to address the question of whether hematopoietic stem cells can also repopulate the gastrointestinal tract and give rise to the relevant intestinal cell lineages. Bone marrow cells from male donors transplanted into syngeneic female mice engrafted into the intestine and differentiated into ISEMF cells. The bone marrow origin of the ISEMF cells was verified by the presence of the Y chromosome. The expression of smooth muscle α-actin and absence of desmin, the macrophage marker F4/80, and the hematopoietic precursor marker CD34 was consistent with a myofibroblast phenotype (338). The persistence of these cells in the lamina propria for a period of at least 6 weeks suggested that the donor stem cells at this site were capable of sustained turnover and differentiation. Donorderived ISEMFs have also been observed in intestinal biopsies from human female patients with graft-versus-host disease who received bone marrow from a male donor (338). Another study reported the long-term persistence of bone marrow–derived epithelial cells in lung, intestine, and skin from the transplantation of a single purified hematopoietic

336 / CHAPTER 11 stem cell (339). However, these investigators did not find any evidence of mesenchymal ISEMF being derived from the single hematopoietic stem cell transplantation. This result suggests that the prior studies demonstrating the differentiation of bone marrow cells into intestinal mesenchymal ISEMF may have been due to the inclusion of fibroblast precursors in the transplanted bone marrow cell preparation. However, stromal cells do not generally survive transplantation, thus the discrepancy in some of the results of the two studies cited remains to be resolved (315). Nevertheless, both studies demonstrate that the transplanted bone marrow– derived cells were able to populate and persist in the recipient intestine, a heterotypic tissue. This demonstration is considered a prerequisite to proving that tissue-specific stem cells transplanted into another tissue have functionally crossed lineage boundaries and differentiated into cells of the recipient tissue (335). Before it can be accepted that organ-specific adult stem cells can differentiate into a cell of another organ, more rigorous criteria must be applied to the isolation of pure stem cells, as well as to the identification of the resulting lineages, which must include morphologic, molecular, and functional parameters (335).

CONCLUSION Significant progress has been made in elucidating the mechanisms and pathways involved in the establishment and maintenance of the intestinal stem cell compartment and crypt-villus unit. It is becoming increasingly clear that crypt morphogenesis, maintenance of the proliferative epithelial stem cell compartment, and crypt-villus homeostasis require the cooperation and convergence of multiple signaling pathways, some of which were hitherto believed to be active primarily during intestinal specification and development. The orchestration of these processes is achieved by sustained cross talk between the epithelial compartment and those cells that comprise the stem cell niche. Studies examining the signaling pathways involved in maintaining intestinal homeostasis have also yielded important insights into the mechanisms that underlie some gastrointestinal cancers, as well as clues to important control points for these pathways that may be targeted for therapeutic interventions. Although current research has addressed many questions, many more have been raised. What are the extracellular signals involved in the establishment of the crypt stem cell compartment during normal gut development, as well as in the maintenance of crypt stem cell homeostasis in the adult, and how are they integrated to determine the fate of individual stem cells or progenitor cells? What is the nature of the molecular signals that regulate stem cell selection within the crypt compartment that occurs during epithelial morphogenesis? The issue of whether crypts become genetically monoclonal by an active selection process involving apoptosis of unwanted cells or through a process of stem cell “competition” is open to debate and research. Indeed, the molecular definition of the intestinal stem cell is only beginning to emerge.

What are the molecular mechanisms for tightly regulating stem cell number within a narrow range in the normal adult crypt under steady-state conditions, and what is the nature of signals that regulate changes in the size of the stem cell population that occur in pathologic processes, such as mucosal injury repair, inflammation, and neoplasia? Finally, although a type of molecular addressing system has been identified that specifies cell positioning along the cryptvillus axis, characterization of the molecular signals controlling its expression has only just begun. It is clear that the many recent advances described earlier have added a significant new molecular dimension to the elegant and insightful studies of Cheng and Leblond that first outlined more than a quarter of a century ago the hierarchical organization of epithelial cells within the intestine. Although many questions remain, the ongoing lively research in intestinal stem cell biology is certain to provide intriguing and perhaps unanticipated answers and, in the process, to raise many new questions as well.

ACKNOWLEDGMENTS This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK06475, to S.M.C.) and through the University of Virginia Digestive Diseases Research Center (NIH P30 DK56703).

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331. Trepicchio WL, Bozza M, Pedneault G, Dorner AJ. Recombinant human IL-11 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production. J Immunol 1996;157:3627–3634. 332. Trepicchio WL, Wang L, Bozza M, Dorner AJ. IL-11 regulates macrophage effector function through the inhibition of nuclear factorkappaB. J Immunol 1997;159:5661–5670. 333. Kamal M, Wakelin D, Ouellette AJ, et al. Mucosal T cells regulate Paneth and intermediate cell numbers in the small intestine of T. spiralis-infected mice. Clin Exp Immunol 2001;126:117–125. 334. Pull SL, Doherty JM, Mills JC, et al. Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury. Proc Natl Acad Sci U S A 2005; 102:99–104. 335. Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–395. 336. Bhatia M, Wang JC, Kapp U, et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94:5320–5325. 337. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6:1229–1234. 338. Brittan M, Hunt T, Jeffery R, et al. Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut 2002;50:752–757. 339. Krause DS, Theise ND, Collector MI, et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001; 105:369–377.

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CHAPTER

12

Apoptosis in the Gastrointestinal Tract Leonard R. Johnson Cell Death, 345 Apoptosis and Necrosis, 345 Proteins Involved in Apoptosis, 346 Apoptotic Pathways, 347 Intestinal Epithelia: In Vivo Studies, 349 Growth and Differentiation, 349 Spontaneous Apoptosis, 350 Induced Apoptosis, 351 Intestinal Cells: In Vitro Studies, 358 Apoptotic Pathway in Intestinal Cells, 358

Polyamines and Apoptosis, 359 Additional Studies, 362 Anoikis, 363 Gastric Mucosa, 364 Helicobacter Pylori, 364 Additional Studies, 365 Conclusion, 366 References, 366

CELL DEATH

Apoptosis and Necrosis

Cell death is a common event in multicellular organisms and is responsible for tissue modeling during embryonic development, as well as homeostasis of cell number in various proliferative adult tissues. Examples of the former include the deletion of the tail in human embryos, the resorption of the tadpole tail as it turns into a frog, and the loss of tissue between developing digits. An example of the latter is the death of extra stem cells in gut crypts so that the number of proliferating cells is kept in balance with those being lost as they are shed into the lumen. Another example of cell death in the maintenance of tissues is the killing of targeted or infected cells by elements of the immune system. In addition to these types of cell death, which are obviously regulated, cells can be killed by outright injury. This often results in an inflammatory response and tissue damage. This type of cell death is usually considered to be accidental, whereas the former is usually referred to as programmed.

Programmed cell death occurs through a process known as apoptosis and is the result of triggering a signal cascade that activates specific enzymes that dismantle the cell. The word apoptosis originates from Greek and literally means “falling off ” as leaves fall from a tree. Although apoptosis had been recognized as a form of cell death for many years, and was described as pyknosis, the term itself was proposed by Kerr and colleagues in 1972 (1). The apoptotic process can be initiated by a variety of stimuli. These include cell damage, such as caused by radiation, chemotherapy, or reactive oxygen species. Withdrawal of growth factors or trophic substances also results in apoptosis of some cells. The binding of ligands to cell membrane receptors of the tumor necrosis factor (TNF) family is responsible for cytokine-induced apoptosis. The full course of apoptosis, from its initiation to the final fragmenting of the cell, usually requires several hours. At the start of apoptosis, dense chromosome condensation occurs at the periphery of the nucleus. The chromatin is compacted and segregated. At this time, the cell also begins to shrink as the cytoplasm condenses. The nucleus becomes fragmented, and the cell breaks up into membrane-bound apoptotic bodies, which are phagocytosed by surrounding cells. Morphologically, the defining feature of apoptosis is that the contents of the cell are never released but remain membrane bound.

L. R. Johnson: Department of Physiology, University of Tennessee College of Medicine, Memphis, Tennessee 38163.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

345

346 / CHAPTER 12 Regulator

Adapter

Effector

C. elegans

Ced-9

Ced-4

Ced-3

Apoptosis

Vertebrates

Bcl-2

APAF-1

Caspase-9

Caspase-3

Apoptosis

FIG. 12-1. Three types of proteins involved in apoptosis and first identified in Caenorhabditis elegans (C. elegans) are shown with their vertebrate counterparts. (Modified from Vaux and Korsmeyer [5], by permission.)

In contrast, in cells undergoing necrosis in response to tissue damage, the plasma membrane becomes permeable early in the process. The cells typically swell and burst, releasing their contents and often triggering an inflammatory response. Detection of cells undergoing necrosis is based on the early increase in membrane permeability. Small charged molecules, such as propidium iodide and various nucleic acid dyes, which would normally be excluded from the cell, enter and can be detected by their fluorescence.

Proteins Involved in Apoptosis The general steps involved in apoptosis are remarkably conserved during the evolutionary process. This suggests that the proteins involved are also similar. Identification of these proteins and the molecular mechanisms involved was largely the result of genetic studies in the roundworm Caenorhabditis elegans (2,3). Three C. elegans gene products (CEDs) are involved in apoptosis. CED-3 and CED-4 promote apoptosis, whereas CED-9 prevents it. CED-3 is an effector, a cysteine protease that cleaves its target proteins following aspartic acid residues. It exists as a zymogen and is activated by self-cleavage. CED-4 can bind to CED-3 and activate it. CED-9 can bind to CED-4 and prevent its activation of CED-3. Under normal conditions, apoptosis is prevented because CED-4 is bound to CED-9. Thus, CED-9 may be considered a regulator and CED-4 an adaptor protein. Mammals have evolved similar groups of proteins that fill the roles of effector, adaptor, and regulator (4). These are shown in Figure 12-1, together with their C. elegans counterparts (5). Effectors In vertebrates, apoptosis results from a complex set of biochemical events catalyzed by a family of cysteine proteases called caspases (6,7). These effectors exist in the cytoplasm as presynthesized proenzymes and are activated in response to physiologic, pathogenic, or cytotoxic stimuli (8). There are two basic types of caspases (Fig. 12-2): executioner caspases are responsible for the actual dismantling of the cell, and initiator caspases are capable of activating executioner caspases. The morphologic hallmarks of apoptosis in the nucleus, chromatin condensation and nuclear fragmentation, are initiated by caspase cleavage of specific proteins. Caspase-3 and -6 cleave lamin and various nuclear proteins responsible for the structural integrity of the

nucleus (9). The fragmentation of DNA is the result of the activation of a number of caspase substrates, such as caspase-activated DNAase (CAD) and DNA fragmentation factor (DFF). Both of these enzymes are constitutively present bound to inhibitory proteins and are released by the action of the caspases (10). Additional proteins cleaved and inactivated during apoptosis are responsible for the repair of DNA damage. These include DNA-dependent protein kinase and poly adenosine diphosphate ribose polymerase (11). DNA fragmentation results in a “laddering” that can be detected by enzyme-linked immunosorbent assay (ELISA) after staining with appropriate DNA antibodies linked to peroxidase and various dyes. Various membrane and cytoplasmic proteins are also targets of caspases. Many of these are involved in the maintenance of the cytoskeleton and include actin, β-catenin, gelsolin, fodrin, and spectrin. Actin itself is the major cytoskeletal protein responsible for the shape of the cell and cell motility. Early during the apoptotic process, phosphatidyl serine is externalized to the plasma membrane, providing a phagocytotic signal to other cells. Annexin V binds to phosphatidyl serine and, conjugated to fluorochromes, provides another means to detect apoptotic cells via fluorescent microscopy. The activation of caspase-3 itself also serves as an excellent assay of apoptosis. Colorimetric substrates are available for all the caspases, or Western blotting can be used to determine the shift in the amount of procaspase to the active form (12). Accessory Molecules Adaptor or accessory molecules are involved in the activation or inhibition of caspases (Fig. 12-3). Perhaps the most familiar of these is apoptotic protease activating factor-1 (APAF-1), which is analogous to CED-4 (see Fig. 12-1). Another such molecule is cytochrome c, which is released from mitochondria during the apoptotic process. Cytochrome c binds to APAF-1 monomers in the cytosol, resulting in the formation of the heptameric apoptosome. Caspases Initiator Caspases

Executioner Caspases

Caspase-8

Caspase-3

Caspase-9

Caspase-6 Caspase-7

FIG. 12-2. The major vertebrate caspases.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 347 Adapter molecules APAF-1

cIAP1

Cytochrome c

cIAP2

Smac / Diablo

BIRC3

Htr A2

Survivin

XIAP FIG. 12-3. Accessory or adaptor molecules that regulate caspase activity. Htr A2, high-temperature requirement service protease A2; XIAP, X-linked inhibitor of apoptosis protein.

The apoptosome then recruits and binds procaspase-9, which is proteolytically cleaved to form active caspase-9 (8,13). Caspase-9 then activates the executioner caspase-3, -6, and -7 (8,13). Inhibitors of apoptosis proteins (IAPs) are capable of directly inhibiting activated caspases and probably ensure that cells are not killed by spontaneous activation of caspases. Synthesis of IAPs is also stimulated by transcription factors such as nuclear factor-kappa B (NF-κB) and the extracellular signal–regulated kinases (ERKs) (14,15). Strong anticaspase activity has been demonstrated for a subset of IAPs, which includes XIAP (X-linked IAP), cIAP1, cIAP2, and ML-IAP (16,17). The most potent of these is probably XIAP, which has been targeted for anticancer therapy (18,19). IAP activity can be inhibited by additional regulators released from mitochondria. These include SMAC (second mitochondrial activation of caspase) and high-temperature requirement service protease A2. These will bind to XIAP in the cytoplasm, interfering with the inhibition of caspases (19,20). Regulators The final group of molecules involved in apoptosis is composed of the members of the Bcl-2 family of proteins

that regulate the release of cytochrome c from mitochondria and alter the ability of APAF-1 to activate caspases. Bcl-2 was originally discovered as an oncogene in B-cell lymphoma (21). Subsequent studies showed that Bcl-2 is capable of inhibiting apoptosis induced by a variety of signals (22,23). A large number of Bc1-2 proteins have been identified using a variety of techniques. All of these possess at least one of four conserved motifs known as Bcl-2 homology (BH) domains. Based on their structures and function, the Bcl-2 proteins can be divided into three groups (Fig. 12-4). The first consists of the antiapoptotic channel-forming proteins having four BH domains and a transmembrane anchor sequence. The second group consists of proapoptotic channelforming proteins having domains BH1, BH2, and BH3 plus the transmembrane region. The third group contains proapoptotic proteins having only the BH3 domain. They are often referred to as BH only proteins and dimerize with the membrane-anchored, channel-forming Bcl-2 receptors (24). Proapoptotic and antiapoptotic members can form homodimers and heterodimers. Whether a cell lives or dies may depend on the relative amounts of the two forms (25). Thus, a preponderance of proapoptotic Bcl-2 proteins will result in heterodimers with all available antiapoptotic members and an excess number of proapoptotic proteins to form homodimers. An excess of prosurvival proteins prevents the release of cytochrome c from mitochondria and may also prevent the activation of procaspase-9 by APAF-1 (26).

Apoptotic Pathways Activation of two major pathways initiates the process of apoptosis in most cells. An extrinsic pathway is initiated by ligands such as TNF-α, a pleiotypic cytokine that, depending on cell type and surrounding circumstances, can result in growth arrest, apoptosis, or proliferation (27). The second major pathway is the intrinsic or mitochondrial pathway,

Antiapoptotic channel-forming BH4

BH3

BH1

BH2

TM

Bcl-2, Bcl-w Bcl-XL, Mcl-1 Bcl-w

BH2

TM

Bax, Bok Bak

TM

Bik, Blk

Proapoptotic channel-forming BH3

BH1

Proapoptotic ligands BH3

BH3

Bad, Bid, Bim

FIG. 12-4. Regulators of apoptosis. Subgroups of the Bcl-2 family of proteins, showing their Bcl-2 homology (BH) domains and presence or absence of a transmembrane (TM) region, which is involved in mitochondrial anchoring. Examples of each are listed.

348 / CHAPTER 12 which is activated by DNA damage and often depends on p53 (28). Extrinsic Pathway: Death Receptors Death receptors of the TNF receptor (TNFR) family, such as Fas, TNF-α–related apoptosis-inducing ligand (TRAIL), and TNFR1, recruit, on binding of ligand, molecules that make up a death-inducing complex that includes procaspase-8 (13) (Fig. 12-5). TNF trimerizes TNFR1 on binding, inducing the association of the receptor’s death domains, which, in turn, bind to adapter proteins that recruit and activate a number of other molecules to the receptor complex (4). This results not only in the activation of caspase-8, which, in turn, activates caspase-3, but also the activation of pathways mediated by NF-κB, mitogen-activated protein kinases (MAPKs), and c-Jun NH2-terminal kinases (JNKs), which regulate apoptosis (29–31). The activation of these additional pathways explains why TNF-α usually does not cause apoptosis unless an inhibitor of protein synthesis is present. Nuclear factor-κB protects most cells from apoptosis, whereas its inhibition enhances cell death (29,32). Normally, NF-κB is bound in the cytoplasm to inhibitory κB (IκB) and remains inactive. Activation results from phosphorylation of IκB on serine residues, which involves phosphatidylinositol-3 kinase (PI3K) and the Akt serine-threonine kinase (33). Phosphorylation of IκB releases NF-κB to go to the nucleus and regulate gene transcription. Among its protein products are

Bcl-2, Bcl-XL, and various IAPs (34). Sustained activation of JNK induces apoptosis in a variety of cell lines (35,36). In some cell lines including intestinal epithelial cell-6 (IEC-6), JNK is inhibited by the sustained activation of MAP kinase kinase (MEK1), ERK1/2, or both (37,38). Intrinsic Pathway: The Mitochondrion The second major pathway is the intrinsic or mitochondrial pathway, which results in the release of cytochrome c from mitochondria (Fig. 12-6). As mentioned earlier, cytochrome c binds to APAF-1 in the cytoplasm and in the presence of adenosine triphosphate forms the apoptosome, which recruits and activates procaspase-9. Additional proteins such as SMAC are also released from the mitochondria. SMAC (or Diablo) binds to and inhibits cytosolic IAPs. The mitochondrial pathway is regulated by the Bcl-2 family proteins. DNA damage is the usual trigger for the release of cytochrome c. Agents such as ultraviolet irradiation, γ-irradiation, chemotherapeutic drugs, and some instances of cellular damage, such as hypoxia, often activate p53 (39). Activated p53 promotes the expression of a number of apoptotic genes including members of the Bcl-2 family such as Bax (39). Much of the tumor-suppressive activity of p53 depends on its ability to induce apoptosis, and many cancers

DNA damage

p53 TNF-α

FasL Bax, BH3-only proteins Fas

TNFR1

Bcl-XL, Bcl-2

Extrinsic pathway F ADD

TRADD

t-Bid

Procaspase 8

NF-κB

Caspase-8

Bcl2, Bcl-XL

Bid

Bad

ERK1/2 Cyto C IAPs

Procaspase-3

t-Bid SMAC

Nucleus

Caspase-3

APAF 1

Mitochondrial pathway

Procaspase 9

Caspase 9 IAPs Procaspase 3

Bcl-2

Apoptosis

FIG. 12-5. Extrinsic or death receptor pathway. ERK, extracellular signal–regulated kinase; FADD, Fas-associated death domain; IAP, inhibitor of apoptosis protein; NF-κB, nuclear factor-κB; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRADD, TNFR-associated death domain.

Caspase 3

Apoptosis

FIG. 12-6. Intrinsic or mitochondrial pathway, also sometimes referred to as the damage pathway. BH, Bcl-2 homology; IAP, inhibitor of apoptosis protein; SMAC, second mitochondrial activation of caspase.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 349 contain deletions or mutations that inactivate p53. Although most of the effects of p53 are believed to be due to its actions as a transcription factor, Chipuk and colleagues (40) show that p53 directly activated Bax to permeabilize mitochondria and initiate apoptosis. Depolarization and permeabilization of the mitochondrial membrane and the subsequent release of cytochrome c are determined by the relative amounts of antiapoptotic channelforming Bcl-2 family members such as Bcl-2 and Bcl-XL and proapoptotic channel-forming members such as Bax and Bak (see Fig. 12-4). However, BH3 only family members play an important role in this process by inactivating Bcl-2 in the mitochondrial membrane. Some BH3 only proteins such as Noxa and PUMA are transcriptional targets of p53 (41,42). Other members of this group are inactivated by posttranslational modifications that prevent them from interacting with prosurvival Bcl-2 family members (43). In some cells, the withdrawal of a growth factor initiates apoptosis. In these cells, Bad is sequestered by a cytosolic protein known as protein 14-3-3. That sequestration (binding) depends on the serine phosphorylation of Bad, which is dependent on Akt activation by growth factors (44). Removal of growth factor frees Bad to move to the mitochondria and bind Bcl-2 or Bcl-XL, resulting in their inactivation and apoptosis. The BH3 only protein Bid is cleaved by caspase-8 in response to the activation of the intrinsic pathway through TNF, Fas, or TRAIL receptors. Cleavage converts Bid to the active, truncated form, t-Bid, which is capable of binding to Bcl-2 and causing apoptosis (45). This process results in cross talk between the extrinsic pathway and the mitochondrial pathway (43). p53 has also been shown to release proapoptotic multidomain proteins and BH3 only proteins that were sequestered by Bcl-XL (40).

INTESTINAL EPITHELIA: IN VIVO STUDIES This section covers studies done in both the small and large intestines. Although there are certainly differences involving apoptosis, the primary one being the high incidence of colon cancer compared with cancer of the small bowel, there are also many similarities. The comparison and contrast between the two tissues avoid redundancy and often provide obvious explanations of differences in physiology and pathology. There is an abundance of studies examining the place of apoptosis in the development of colon cancer and other gastrointestinal tract malignancies. This is a subject in itself and is covered in Chapter 17. The intestinal epithelium provides an interesting yet complicated model in which to study apoptosis. This tissue has one of the most rapid turnover rates known, as cells are lost into the lumen and replaced by the progeny of stem cells. Apoptosis plays a role both in the loss of cells and in regulating the number of stem cells. Both processes, proliferation and apoptosis, are affected by hormones, growth factors, and influences that regulate the growth of all tissues in the body. In addition, however, the lining of the gut is

subjected to luminal contents in the form of nutrients, digestion products, bacteria, and growth factors. All of these have been shown to affect the growth of the tissue (46). The intestinal epithelium of the mouse has served as the primary model for studying gut apoptosis. For the most part, these studies have focused on describing spontaneous apoptosis and damage- or stress-induced apoptosis. Many of these have identified the roles of members of the Bcl-2 family of proteins and p53. However, few studies have examined the overall regulatory process of apoptosis in the intestine. There has been little progress in developing appropriate in vitro models of normal intestinal cells. The most widely used intestinal cell lines have been developed from tumors and are abnormal. Some such as the popular CaCo-2 line contain a mutated p53 gene. Although all cell lines by definition have altered rates of proliferation and death, the IEC-6 line has proved useful to elucidate the mechanisms regulating apoptosis in untransformed crypt cells that have not differentiated.

Growth and Differentiation The small intestinal epithelium of the adult mouse, like most vertebrates, is composed of protruding villi interspaced with numerous cavities called crypts (Fig. 12-7). The crypt-to-villus ratio varies from approximately 14 crypts per villus in the duodenum to about 6 crypts per ileal villus (47). This difference is the crypt-to-villus ratio probably accounts for the difference in the sizes of villi in the two locations. The longer duodenal villi each contain approximately 7800 cells, whereas those in the ileum contain about 2100 (47). The approximately 1.1 million crypts of Lieberkühn of the adult mouse (48) are flask-shaped structures and are the proliferative units of the intestine. Numerous studies in normal, transgenic, and chimeric mice have shown that crypts are monoclonal and are populated by the descendents of a single stem cell anchored near the base of the crypt (48–53; see review by Hermiston and colleagues [54]). After division of the stem cell, one daughter cell remains anchored as the new stem cell, whereas the other undergoes several divisions in the lower and middle thirds of the crypt. The total number of dividing and nondividing cells in a crypt varies according to its location just as the number of cells per villus varies. Duodenal crypts contain approximately 530 cells, whereas the average number of cells per ileal crypt is 360 (47). Each crypt produces 13 to 16 cells per hour (47). Most of these cells migrate out of the crypt and onto a villus, where they continue to migrate toward the villous tip. During migration the cells mature into three of the four terminally differentiated cell types of the adult small intestinal epithelium: the absorptive enterocyte, the enteroendocrine cell, and the mucus-secreting goblet cell. Some cells migrate to the base of the crypt where they differentiate into Paneth cells that produce lysozyme. Thus, each of the four main cell types arise from progenitors of a single multipotent stem cell (51,54). The cells that migrate onto a villus are subsequently shed

350 / CHAPTER 12

3 days

Villus Enterocytes Goblet cells Enteroendocrine cells

As in the small intestine, proliferation takes place in crypts. Cells migrate from the colonic crypts to a group of surface epithelial cells surrounding the crypt opening. Together with cells from additional crypts, the surface cells make up the so-called intracryptal tables, the flat areas between the crypts. Colonic crypts, like the crypts in the small intestine, are monoclonal, that is, populated by the progeny of a single stem cell (52,54,59). Crypts in the proximal colon produce approximately 6 cells per hour, whereas those in the distal colon have greater proliferative rates of about 21 cells per hour. The average cell turnover rate in the colon is increased to approximately 8 days compared with the 2 to 3 days found in the small intestine (60).

Spontaneous Apoptosis

Crypt

Proliferative transit cells

Stem cell(s)

In both animals and humans, a low rate of apoptosis occurs in the intestinal crypts unrelated to a known stimulus. The greatest frequency of apoptosis coincides with positions 4 and 5 from the base of the crypt, which is the presumed location of the stem cells (61). Apoptosis may therefore be responsible for eliminating extra stem cells. Apoptosis may also be responsible for removing excess cells from the villous tip. Although few apoptotic cells have been observed in sections from the lower and mid regions of the villi, this incidence increases with increasing distance from the crypt. This process of cell removal is certainly regulated to maintain the number of villous cells. However, it is uncertain whether apoptosis itself is regulated, or whether apoptosis occurs in response to detachment from the basement membrane.

Paneth cells

Crypt FIG. 12-7. Schematic of the crypt-villus unit of the small intestine, showing progress of an enterocyte from stem cell to transit cell to mature cell located on the villus to its exfoliation. Migration from the crypt-villus junction to the zone of extrusion at or near the villus tip takes approximately 3 days.

into the intestinal lumen by a process that is incompletely understood (55,56). Shedding involves apoptosis, and the question is whether the apoptosis occurs on the villus, or whether it is triggered by detachment from the substrate, a process known as anoikis. The whole process from proliferation to differentiation to shedding occurs in a few days. It takes only 2 to 3 days for a cell to move from the crypt to the apex of the villus where it is exfoliated (47,57). Cells are shed from the villous tip at the rate of 1400 cells per villus during a 24-hour period (58), making the epithelium of the small intestine one of the most rapid turning over tissues in the body. From the previous discussion, it is obvious that cell death, apoptosis, plays a significant role in maintaining the normal cell population of the small bowel epithelium. Despite the absence of villi, proliferation and migration are also involved in cellular differentiation in the colon.

Potten (62,63) has estimated that one apoptotic cell is found in every fifth longitudinal histologic section through the crypt of the mouse small intestine. These cells, for the most part, occupy positions 4 and 5 from the base of the crypt (the stem cell positions). Spontaneous apoptosis in the small intestinal crypt occurs at the same rate in p53-deficient mice as in wild-type animals (64). Thus, p53 does not appear to play a role in regulating the number of stem cells in the small intestine. Bcl-2 is not expressed in small-bowel epithelial cells (65), and as one would surmise, Bcl-2−/− and normal mice have the same rate of apoptosis of small intestinal epithelial cells (66). The apoptotic protein Bax, however, is strongly expressed in the stem cell region of the small intestine and in enterocytes at the villous tips (61). Thus, Bax may stimulate the removal of extra stem cells and play a role in the shedding of effete villous cells. Little or no Bax is found in the crypts of the large intestine (61). Thus, the localizations of the antiapoptotic and proapoptotic Bcl-2 family members may provide the explanation for the relative resistance of the small intestine to the development of cancer compared with the colon (61,67). These findings indicate that apoptosis plays an important role in regulating the number

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 351 of stem cells in the epithelium of the small intestine, and therefore the number of cells leaving the crypts and migrating onto intestinal villi. Potten and colleagues (61) have estimated that an extra stem cell will cause 60 to 120 additional cells per crypt. Thus, by virtue of the several divisions undertaken by daughter cells before they exit the crypt, an extra stem cell would seriously alter the equilibrium of cell number normally maintained in the crypt-villus unit. Apoptosis in the colonic epithelium occurs at a much lower rate than it does in the small intestine and is not confined to the crypts. Instead, cells are spontaneously eliminated at low rates regardless of their positioning. Knockout studies have shown that, as in the case of the small intestinal epithelium, spontaneous apoptosis in the colonic epithelium occurs independently of both p53 and Bax (65,68). In contrast, Bcl-2 is expressed in colonic epithelium, and knocking out this gene results in an increased rate of spontaneous apoptosis, which occurs primarily in the crypt at positions 1 and 2, the location of the stem cells (66). Bcl-2 is believed to play an important role in the regulation of cell number in the colonic epithelium, and its presence is an explanation for why the colonic mucosa is susceptible to cancer. Villus There is no doubt that detachment from the underlying matrix or substrate induces epithelial cell apoptosis. Detachment-induced cell death, or anoikis, has been shown to occur in isolated human intestinal cells (69) and in mammary, renal, and bronchial epithelial cells (70–72). In normal human IECs, activation of caspase-6 occurred within 15 minutes of detachment (73). The major questions being debated are whether apoptosis begins while cells are still attached to the villi, and, if so, how it is initiated. The first question arose out of the relatively infrequent observations of obvious apoptotic cells on the villus (61). Hall and colleagues (74) used sensitive microscopic and histologic techniques to determine that, on average, one apoptotic body was present per section of villous epithelium. Each section was 3 µm in thickness, and because the villi of the mice were 120 to 150 µm, these authors calculated that at any time 40 to 50 apoptotic cells were present on each villus. The distribution of apoptotic cells was not uniform, and they increased in number with position toward the villus tip. Apoptotic bodies are rapidly cleared, usually in less than 1 hour. Thus, during a 24-hour period, each villus may shed 960 to 1200 apoptotic cells (74). This calculation fits with that of Potten and Hendry (75) who report that 1200 cells migrate onto a villus each day. The finding that apoptosis occurred while cells were still attached to the villus was confirmed by Grossman and colleagues (76), who used an antibody directed against activated caspase-3 to detect dying cells. These authors report that epithelial cells of the small bowel activated caspase-3 as they approached the area of shedding and before they displayed the morphologic features of apoptosis. Because caspase-3 is the major executioner caspase, and its activation

is the culmination of the signaling pathway leading to apoptosis, we can conclude that the apoptotic process is well under way before effete cells appear apoptotic. In conclusion, these studies (74,76) provide strong evidence that intestinal cell number is regulated by apoptosis, as well as by proliferation, and that the apoptotic rate is linked to the proliferative rate. Do cells detach from the extracellular matrix (ECM) before being shed into the lumen? Using scanning electron microscopy, Shibahara and colleagues (77) observed protruding enterocytes at the tips of villi. These cells showed evidence of apoptosis such as vacuoles and chromatin condensation. Frequently, the intercellular spaces beneath these cells were occupied by large lymphocytes. Taken together, these data suggest, although they certainly do not prove, that mature enterocytes may detach from the ECM and undergo apoptosis before being shed into the lumen. The mechanisms involved in this process and anoikis are discussed later in this chapter in the Intestinal Cells section. Regardless of the mechanisms involved, the processes of cell proliferation and loss are linked. Additional support for this relation comes from a study in elderly adults. Ciccocioppo and colleagues (78) compared the rates of enterocyte apoptosis and proliferation in the duodenal mucosa of 12 humans with a mean age of 77.6 years with 12 others with a mean age of 37.7 years. The TUNEL technique was used to detect apoptosis, whereas proliferation was measured immunohistochemically by M1B-1 detection in endoscopically obtained biopsy specimens. Apoptosis had increased in the elderly adults to 15.3% of the cells examined from 2.1% in the younger age group. This was matched by an increased proliferative rate to 37.7% versus 15.8%. What is not known is whether the increased proliferation compensates for increased cell loss or whether the opposite situation exists.

Induced Apoptosis Many damaging agents have been shown to induce apoptosis of intestinal epithelia. These include ionizing radiation, chemical mutagens, and chemotherapeutic drugs and food products. In addition, activation of the extrinsic pathway via the so-called death receptors results in a more physiologic apoptotic response. Apoptosis is part of the result of procedures such as small-bowel resection and ischemia–reperfusion and has been investigated to determine its contribution to the overall response. A variety of agents has been tested for their protective effect on the gut mucosa and their ability to prevent apoptosis during exposure to radiation or chemotherapeutic agents. The goal of these studies has been to discover a means to protect the lining of the intestine during cancer therapy. A variety of cytotoxic drugs, mutagens, and ionizing radiation result in apoptosis of intestinal epithelia (79,80). In the whole animal, the most widely studied of these and the one most used as a model to study the effects of other agents on

352 / CHAPTER 12 apoptosis is radiation. Dose-dependent increases in apoptosis occur within the intestinal crypt after 3 to 6 hours of exposure to γ-irradiation (81,82). The cells most susceptible to radiation-induced apoptosis were at position 4 of the crypt—again, the stem cell location. These were the cells that died within the first 3 to 6 hours. Ijiri and Potten (80) found that all cells in the crypt were capable of apoptosis, but their susceptibility was dependent on the type of damaging agent. The early- and low-dose apoptotic response associated with stem cells of the small intestine is dependent on p53. Studies using p53−/− mice showed the absence of apoptosis that normally occurred 3 to 6 hours after γ-irradiation of wild-type animals (64,84). Knockout studies using Bcl-2−/− mice showed similar levels of apoptosis in small intestinal crypts 3 to 4 hours after γ-irradiation regardless of the presence of Bcl-2 protein (84). This result is entirely predictable, because the small intestinal epithelium does not express Bcl-2. As would also be expected from the distribution of the protein, increased numbers of apoptotic cells were found in colonic crypts of the Bcl-2−/− animals. Results using the chemotherapeutic agent 5-fluorouracil are similar to those with γ-irradiation, because p53 null mice are resistant to apoptosis after administration of this drug compared with wild-type animals (85). Apoptosis initiated by 5-fluorouracil could be attenuated by the administration of uridine, but not thymidine. The authors suggest that the p53-dependent cell death initiated by 5-fluorouracil was caused by the ability of the drug to damage RNA rather than DNA (85). In Bcl-2 null mice, 5-fluorouracil also had effects similar to those of γ-irradiation, because there was a significant increase in apoptosis at the base of colonic crypts 4.5 hours after administration of the agent (86). This same group has also examined the role of the antiapoptotic protein Bcl-w using the identical experimental design (87). Immunoblotting showed that Bcl-w was expressed in both the small intestine and the colon. Interestingly, spontaneous apoptosis was not altered by the absence of Bcl-w in crypts from either tissue. Both γ-irradiation and 5-fluorouracil, however, caused significantly greater levels of apoptosis in Bcl-w−/− mice than in their wild-type counterparts. Bcl-w, therefore, seems to play a significant role in damage-induced apoptosis in both tissues, whereas Bcl-2 regulates apoptosis only in the colon. Whereas p53, Bcl-2, and Bcl-w have important roles in the regulation of damage-induced apoptosis in the gut epithelia, Bax expression was without effect (86). Neither γ-irradiation nor 5-fluorouracil altered the incidence or location of apoptosis in intestinal epithelia of Bax null mice compared with their normal counterparts. In all of the studies discussed previously, apoptosis was quantitated by identifying apoptotic cells. Marshman and colleagues (88) characterized the activation of caspase-3 along the crypt-villus axis, comparing it with the existing histologic techniques for detecting apoptosis. In both large and small intestines, caspase activation was rare in control,

untreated mice. Exposure to 8 Gy radiation increased caspase-3 activity in the crypts of both tissues, where it was confined to apoptotic bodies. Thus, the determination of caspase-3 activity produced results identical to histologic techniques and provides an additional method for examining cell death in vivo. It is obvious from the experiments discussed earlier that knockout studies are extremely useful in determining whether a specific protein is involved in apoptosis. In fact, these data could not have been obtained in any other way. The primary disadvantages to these studies are: (1) the gene is absent from the entire animal, and it is unknown what compensatory changes have occurred; (2) the development of null mice is expensive and time consuming; and (3) most importantly, these studies tell nothing about the regulatory pathways that actually influence the levels of the proteins being studied. A study by Egan and colleagues (89) uses selective gene ablation to provide important data on the regulation of apoptosis in vivo. These authors examined the role of the transcription factor NF-κB (see Fig. 12-5) in radiation-induced apoptosis in small intestinal epithelium, in which the gene coding IκB kinase had been selectively ablated (thereby preventing the activation of NF-κB) using the Cre/loxP system of somatic DNA recombination. Using morphologic techniques and caspase-3 activation, the authors report the number of apoptotic cells to be approximately twofold greater in the mice in which IκB kinase had been ablated compared with control mice after treatment with 8 Gy γ-radiation. There were no significant effects of gene ablation on spontaneous apoptosis. Whole-cell extracts of isolated IECs from the conditional IκB kinase knockout mice contained greater levels of p53 than the control mice. The conditional knockouts also expressed significantly lower levels of the antiapoptotic proteins Bcl-2 and Bcl-XL. The authors concluded that IκB kinase–dependent NF-κB activation is a key signaling event leading to protection against radiation-induced apoptosis in intestinal epithelia (89). It is likely that one of the mechanisms involved is the stimulation of the transcription of Bcl-2 and Bcl-XL by NF-κB. To this point in the chapter, nothing has been stated about the role of the extrinsic, or “death receptor,” pathway in damage-induced apoptosis. Few experiments have been conducted in vivo regarding the TNFR family, although many have been done using cell lines (see discussion later in this chapter). However, the role of TNFR1 has been examined during the response to radiation-induced damage of the intestinal epithelium. These studies were prompted by observations indicating that p53 mediates apoptosis partly through its ability to transactivate members of the TNFR family (90). Mice in which TNFR1 had been knocked out (TNFR1−/−) and those injected with TNFR1-fusion chimeric (a competitive inhibitor of TNFR1) were partially (30–40%) protected from p53-dependent apoptosis induced by γ-irradiation. The authors also found that radiation induced a p53-dependent increase in the intestinal level of TNF, and that injection of a neutralizing anti-TNF antibody

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 353 decreased p53-dependent intestinal cell apoptosis by 60%. These data indicate that p53-dependent, radiation-induced apoptosis of IECs is mediated, in part, by the upregulation of TNF by p53 and its ability to subsequently induce cell death through TNFR1 (90). A large body of literature exists that deals with the death receptor and ligand system known as the Fas/Fas ligand (FasL) system (see Fig. 12-5). Pinkoski and colleagues (91) have reviewed this topic as it pertains to the liver and the gastrointestinal tract. The best characterized system of apoptosis involving the Fas/FasL system is that found in lymphoid cells. A major role in these cells of the Fas/FasL system is controlling the immune response by regulating the number of lymphocytes. Fas is also expressed in nonlymphoid tissues at sites of immune privilege (ovaries, uterus, testes) and at sites commonly infiltrated by lymphocytes (small and large intestines, liver, and lung). The role of the Fas/FasL system in controlling lymphocytes in these tissues as it pertains to immune privilege, graft-versus-host disease, inflammatory bowel disease, hepatitis B and C, alcohol liver disease, and Wilson’s disease is covered in Pinkoski and colleagues’ review (91) and is not discussed here. Small-Bowel Resection The most widely used model to study intestinal adaptation and mucosal proliferation is partial intestinal resection (92,93). After resection of a significant portion of the small bowel in many species, including humans, the remaining intestinal mucosa undergoes both structural and functional changes including small-bowel dilation, villus enlargement, increased crypt depth, epithelial cell hyperplasia, increased cell migration rate, and increased absorptive capacity per length of intestine (92–95). There have been a plethora of studies attempting to elucidate the mechanisms involved in the growth of the mucosa, but until fairly recently these have all targeted proliferation. Growth results from the equilibrium between cell renewal and cell loss. Cell loss is especially important in tissues that proliferate as rapidly as the intestinal epithelium. Interestingly, Helmrath and colleagues (96) found increased numbers of apoptotic bodies in both crypts and villi of mice after a 50% resection of the small bowel. Several groups have explored the significance of apoptosis in the response to small-bowel resection by examining the effects of various gene products involved in apoptosis on the adaptive response. Shin and colleagues (97) performed 50% intestinal resections on wild-type and homozygous p53-null mice to examine the role of p53 during increased enterocyte apoptosis and the overall adaptive response of the gut mucosa. In general, these investigators found approximately equal values for DNA content, protein content, villus height, crypt depth, and the proliferative index in the transected control and p53 null animals. Seven days after resection, there were significant increases in all these parameters in both groups and no significant differences in the increases themselves; that is, p53 had no significant effect on the

adaptive response. The apoptotic index was determined by counting apoptotic cells in the crypts, and the results indicated that apoptosis was not altered in the p53 null mice after small-bowel resection. In both groups, apoptosis increased approximately 25%, which matched the 23% increase in proliferation. Although this correlation supports a role for apoptosis in maintaining the new equilibrium in cell growth, its significance has not been established. The major conclusion from this study was that the increase in apoptosis after small-bowel resection occurs via a p53-independent mechanism (97). The Bcl-2 family of proteins also has been targeted to explain the increased rates of apoptosis and proliferation after resection. Welters and colleagues (98) examined the effects of 75% small-bowel resection in wild-type control mice and those overexpressing Bcl-2 in the small intestinal epithelium. They concluded that intestinal hyperplasia after resection was significantly increased in the mice overexpressing Bcl-2. Small-bowel resection produced the expected increases in jejunal mucosal weight and protein and DNA content compared with the sham-operated animals. These increases were significantly greater in the mice overexpressing Bcl-2 than in the wild-type resected group. The investigators did not quantitate apoptosis, thus the actual role of cell death in the results can only be surmised. Studies involving the genetic manipulation of the Bax gene, however, have shed considerable light on the role of apoptosis in the adaptive response to small-bowel resection. Stern and colleagues (99) compare adaptation to resection in Bax null mice with that of wild-type counterparts. They observed the usual increases in ileal wet weight, crypt depth, and proliferative rates in both groups. Resection significantly increased the rate of apoptosis in the control group; however, the apoptotic index in the Bax null mice was unchanged. These data support the conclusion that increased apoptosis after small-bowel resection is Bax dependent, and that enterocyte proliferation and apoptosis are regulated via different mechanisms during intestinal adaptation (99). Tang and colleagues (100) have approached the same question but have also examined the pathways involved. One week after resection, they observed significant increases in messenger RNA (mRNA) for caspase-8, Fas, FADD (Fas-associated death domain), and TRADD (TNFR-associated death domain) in Bax+/+ mice, but not in Bax−/− animals. They also found significant increased levels of caspase-8 and Fas protein after resection of Bax+/+ mice. Fas protein was increased in the control Bax+/+ animals as well. In response to resection, apoptosis occurred only in the Bax+/+ mice, prompting the conclusion that adaptation-induced apoptosis is mediated through the extrinsic or FAS pathway but requires the mitochondrial pathway as evidenced by the requirement for Bax. In the adapting Bax+/+ intestine, increased apoptosis was confined to the crypts and was accompanied by increased cell proliferation. Bax−/− mice exhibited an adaptive response similar in magnitude to the Bax+/+ animals; however, it occurred without an increase in cell proliferation and with

354 / CHAPTER 12 an actual decrease in apoptosis. The authors concluded from these data that adaptation-induced, Bax-dependent apoptosis may be the limiting factor determining the magnitude of the adaptive response (100). Knott and colleagues (101) examined the involvement of the extrinsic pathway directly by comparing the adaptive responses to resection in TNFR1 null mice and Fas null mice with wild-type control animals. They found that apoptotic and adaptive responses occurred normally in both null models and concluded that increased apoptosis in response to smallbowel resection does not appear to involve the extrinsic or death receptor pathway. Thus, these data and conclusions disagree with those described by Tang and colleagues (100). In support of their data, Knott and colleagues (101) also examined the expression of caspase-8, TNFR1, FasL, Fas, and TNF in the gut remnant at various postoperative times. They reported only modest changes in these parameters that did not correlate with the histologic appearance of apoptosis. Based on observations that inhibition of the epidermal growth factor receptor (EGFR) decreases adaptation and increases apoptosis and that intestinal levels of Bax correlated with EGF signaling, Jarboe and colleagues (102) examined whether Bax was required for the exaggerated apoptosis after resection in mice with deficient EGFR signaling. They crossed waved-2 mice (impaired EGFR signaling) with Bax null animals. After small bowel resection, enterocyte apoptosis increased in the EGFR-impaired mice and was prevented in the Bax−/− mice. Crossing the animals prevented the increased apoptosis observed in the waved-2 mice. The authors conclude that Bax is required for the increased apoptosis observed after resection, and that it is also necessary for the enhanced apoptotic response to defective EGFR signaling. To summarize the studies, apoptosis after massive smallbowel resection appears to be independent of p53 and dependent on Bax. There are differing results as to the involvement of the extrinsic or death receptor pathway, although the study in which this pathway was inactivated genetically (101) found no involvement of either TNFR1 or Fas. Ischemia–Reperfusion Ischemia–reperfusion induces an apoptotic response in the adult rat intestinal epithelium (103). These experiments demonstrate that apoptosis was induced by ischemia and exacerbated by reperfusion. Coopersmith and colleagues (104) examined this response in mice by quantitating the effects of ischemia–reperfusion on both immature crypt cells and differentiated villous cells. After transient occlusion of the superior mesenteric artery, apoptosis occurred in the undifferentiated epithelial cells of the proliferative compartment, as well as in the differentiated cells of the villus. The response in the villous cells was sevenfold over baseline compared with twofold in the crypt cells. The greatest increase in apoptosis occurred at the villous tip, perhaps reflecting the possibility that hypoxia was greatest in this region. Comparisons of p53+/+ and p53−/− mice also indicated that apoptosis after

ischemia–reperfusion is p53 independent (104). These investigators used a fatty acid–binding protein promoter to produce transgenic mice that overexposed Bcl-2 fivefold in both crypt and villous cells. Forced expression of Bcl-2 reduced apoptosis by approximately 50% in both crypts and villi compared with normal littermates. The authors suggest that these data indicate that both mature and immature cells undergo a commitment phase that is sensitive to Bcl-2. They also found that forced expression of Bcl-2 caused a similar reduction of p53-dependent apoptosis of crypt epithelial cells in response to γ-irradiation. Thus, the ability of Bcl-2 to inhibit apoptosis occurs independently of whether p53 is involved in the response. Factors affecting apoptosis of the intestinal epithelium after ischemia–reperfusion have been studied in detail by Fujimoto and his group of investigators (103,105–107). These studies demonstrate that injury and apoptosis were mediated by reactive oxygen species, inflammatory leukocytes, mitochondrial disfunction, and the release of cytochrome c. Another study from this group examined whether reactive oxygen species induced preoxidative mitochondrial damage by decreasing the mitochondrial membrane potential after ischemia–reperfusion, and whether ischemic preconditioning attenuated apoptosis by affecting the mitochondrial pathway (108). Ischemic preconditioning refers to the fact that a tissue can be made resistant to the effects of long-term ischemia–reperfusion by a prior brief period of occlusion. In this study, 60 minutes of ischemia followed by 60 minutes of reperfusion was used to generate damage. In some instances, the 60-minute occlusion of the superior mesenteric artery was preceded by a process of preconditioning, which consisted of 20 minutes of occlusion followed by 5 minutes of reperfusion. These experiments were conducted in rats, and the major stimulus caused an approximate sevenfold increase in apoptosis, which was decreased 30% by preconditioning. The investigators found that ischemia–reperfusion by itself caused mitochondrial respiratory disfunction, which resulted in a significant increase in the production of reactive oxygen species. Increased production of reactive oxygen species enhanced mitochondrial glutathione oxidation and lipid peroxidation. Peroxidation of lipids reduced the mitochondrial membrane potential, which led to the release of cytochrome c, caspase-9 activation, and finally, the activation of caspase-6-in the small-bowel mucosa. Each step in this scheme was inhibited significantly by ischemia preconditioning, which resulted in the reduction of apoptosis (108). This study therefore shed light on the mechanism by which ischemia–reperfusion damages the intestinal mucosa and the role of the mitochondria in that damage. Prevention of Apoptosis Radiation and chemotherapy for human tumors target the rapidly dividing cancer cells but also affect normal cells with high rates of proliferation. Foremost among these are the cells of the bone marrow and intestinal epithelia.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 355 Acute injury to the intestinal mucosa is the major complication of this therapy. The toxicity derived from the loss of mucosal cells consists of diarrhea, dehydration, and bacterial infection, which can lead to sepsis and shock. This sequelae often limits the dose of therapeutic agent, and hence the effectiveness of the therapy itself. In addition, the quality of the lives of patients can be compromised severely. For these reasons, considerable time and effort have been invested in attempting to discover an agent or procedure that would decrease the severity of the side effects on the mucosa of the gastrointestinal tract. There are threshold levels of radiation above which the bone marrow and intestinal mucosa are unable to repopulate themselves. In mice, exposure to 7 to 15 Gy total body radiation kills most proliferating cells of the bone marrow, but sufficient stem cells survive to repopulate the tissue (109). Greater doses result in an insufficient number of surviving stem cells, and the animal dies. After 8 to 14 Gy radiation, many proliferating IECs are killed, but a sufficient number of stem cells survive to repopulate the mucosa (62,81,110). The greater the dose of radiation, the smaller the number of surviving stem cells and the fewer the number of regenerating crypts. Three to four days after the radiation insult, the number of active crypts can be counted histologically as an assay of stem cell survival and the overall damage (110,111). This assay is quite useful for evaluating “protection” and has been termed a clonogenic assay, because a single surviving stem cell can generate a new crypt. Numerous studies have identified several different classes of compounds with radioprotective effects on the small-bowel mucosa. Growth Factors Although several growth factors have been shown to protect enterocytes from apoptosis, there are few systematic studies or series of studies involving these compounds. Epidermal growth factor (EGF) was shown to protect against mucositis induced by 5-fluorouracil (112). However, there have also been conflicting results, and a more recent report examined the role of EGF using both transgenic and classical approaches. Huang and colleagues (113) found that mice overexpressing EGF specifically in the small intestine showed no differences in weight, apoptosis, crypt cell proliferation, and mucosal architecture when compared with control animals after a course of fluorouracil. Treatment of normal mice with exogenous EGF also did not protect, despite a variety of doses, schedules, and routes of administration. The investigators also induced mucositis in mutant mice with specific defects in EGF signaling. Compared with control animals, clinical and histologic parameters after fluorouracil were not different in mice with functionally defective EGFRs or those lacking transforming growth factor-α (TGF-α), a major ligand for the EGFR (113). Granulocyte-macrophage colony-stimulating factor has been shown to reduce chemotherapy-induced intestinal damage in a rat model by decreasing glutathione generation and lipid peroxidation (114). Glucagon-like

peptide-2 also decreases injury of the gut caused by chemotherapeutical agents by preventing some of the apoptotic response (115,116). A similar claim has been made for keratinocyte growth factor in mice subjected to both radiation- and chemotherapy-induced injury. The authors found increased stem cell survival, and they suggest that it acts by stimulating cell proliferation (117). Interestingly, the protective effects of TGF-β3 have been attributed to its ability to induce cycle arrest before exposure to either radiation or chemical agents (118–120). Dunker and colleagues (121) found decreased rates of apoptosis in mice heterozygous for TGF-β2 and TGF-β3 with concomitant increases in Bcl-2 and Bcl-XL. Because it is unlikely that one peptide, keratinocyte growth factor, prevents apoptosis by stimulating cell proliferation, whereas another, TGF-β3 protects stem cells by inducing cell cycle arrest, it is safe to conclude that the mechanism(s) of action of these compounds remains to be elucidated. Podolsky and coworkers have undertaken a series of studies involving intestinal trefoil factor (ITF). Intestinal trefoil factor is produced and secreted by goblet cells of both the small and large bowels, enhances cell migration, and protects enterocytes from damage (122). Intestinal trefoil factor has been shown to decrease apoptosis of colonic epithelial cells in response to ceramide, serum starvation, and etoposide (123,124). Because apoptosis of goblet cells is characteristic of the intestinal response to radiation and chemotherapy, Beck and colleagues (125) hypothesize that these procedures decrease ITF expression, and that the loss of its effects on migration and cellular function impair healing. Three and a half days after exposure to 14 Gy total-body radiation, there was a marked reduction in goblet cell number and ITF mRNA. Intestinal trefoil factor enhanced migration of IEC-6 cells and wound repair in vitro after methotrexate. intestinal trefoil factor–negative (ITF−/−) mice were significantly more susceptible to 5-fluorouracil–induced weight loss, diarrhea, and increased intestinal permeability compared with wild-type control animals. Survival of the ITF−/− mice was also decreased after chemotherapy. Exogenous ITF given orally was able to improve crypt survival significantly after radiation and chemotherapy. Interestingly, ITF did not alter the expression of a host of molecules involved in apoptosis, including Fas, FasL, TNF, TNFR1, TNFADD, and numerous members of the Bcl-2 family of proteins, as assessed by microarray analysis. The authors suggest that ITF is involved in the protection against, and recovery from, radiation and chemotherapy (125). Several early studies demonstrated that basic fibroblast growth factor (bFGF) could protect endothelial cells from radiation-induced injury (126–128). Dignass and colleagues (129) found that bFGF is expressed in intestinal mucosa, and that it may regulate epithelial cell growth and migration. Houchen and colleagues (130) examined the effect of radiation injury on the expression of bFGF and the survival of crypt stem cells in the mouse small intestine. Fibroblast growth factor mRNA increased significantly 48 hours after exposure to 13 Gy and reached a peak at 96 hours.

356 / CHAPTER 12 COX-2) and by a specific COX-2 blocker. Lipopolysaccharide was protective in COX-1−/− mice, but its effects were eliminated in COX-2 knockout animals. Thus, the radioprotective effect of LPS on the mouse intestine is mediated through prostaglandin synthesized by COX-2 (137). Interestingly, the same group had previously obtained evidence that crypt cell survival in the mouse small intestine after irradiation was dependent on prostaglandin synthesized via COX-1 (138). This conclusion was based on the findings that COX-1 localized to the crypt cells, and 3 days after irradiation, prostaglandin E2 (PGE2), COX-1 mRNA, and COX-1 protein levels were increased. Although these two studies implicate PGE2 in an important role in the response to radiation damage, they also imply different mechanisms. A follow-up study identified the EP2 receptor as being responsible for mediating at least some of the survival effects of PGE2 on the murine intestinal mucosa (139). Riehl and colleagues (140) have obtained evidence that TNFR1 mediates the radioprotective effects of LPS. Crypt cell apoptosis and the number of surviving crypts were measured in wild-type, TNFR1−/−, and TNFR2−/− mice after 12 Gy radiation with and without pretreatment with LPS. Lipopolysaccharide reduced radiation-induced apoptosis in wild-type and TNFR2−/− mice but had no effect in the TNFR1 knockouts. Exogenous PGE2 protected the TNFR1−/− animals. TNFR1 and COX-2 expression were found in subepithelial fibroblasts and villous cells, but not crypt cells. The authors suggest that the subepithelial fibroblasts could be the source of the protective prostaglandins in vivo. Thus, as in the case of bFGF, the protective actions of LPS on crypt stem cells may be mediated by a second cell type.

The expression of FGF protein in the small intestine followed a similar time course. Treatment with FGF before, but not after, irradiation enhanced crypt cell survival threefold. Additional studies involving bFGF provided evidence that intestinal injury after irradiation in mice is secondary to apoptosis of endothelial cells (131). Receptors of bFGF were found in intestinal endothelial cells, but not in crypt or villous cells, with the exception of Paneth cells. Intravenous administration of bFGF prevented radiation-induced endothelial apoptosis and crypt damage, organ failure, and death from the gastrointestinal syndrome. To investigate whether endothelial apoptosis was causally responsible for the damaged intestinal epithelium, these investigators examined the responses of mice in which the acid sphingomyelinase (asmase−/−) gene had been knocked out. These animals did not synthesize the proapoptotic lipid, ceramide, in endothelia after irradiation. Apoptosis of the intestinal epithelia, damage to the gut, and death were prevented after irradiation in the asmase−/− mice. The authors conclude that endothelial apoptosis is the primary lesion initiating intestinal radiation damage in mice (131). They suggest that these data provide a new approach to prevent radiation damage to the bowel. Prostaglandins The E-type prostaglandins protect both the intestine and bone marrow from radiation damage (132–134). Lipopolysaccharide (LPS) protects the bone marrow from radiation injury (135) and stimulates prostaglandin synthesis by inducing cyclooxygenase-2 (COX-2) (136). Stenson’s group demonstrated that LPS administered at least 2 hours before irradiation led to a threefold increase in the number of surviving crypts 3.5 days later (137). Parenteral administration of LPS stimulated prostaglandin production in the intestine and induced COX-2 expression in subepithelial cells and villous but not crypt cells. The protective action of LPS was prevented by indomethacin (inhibits COX-1 and

Polyamines The polyamines, spermidine and spermine, and their precursor, putrescine, are found in virtually all cells of higher eukaryotes (141); they are intimately involved in, and

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FIG. 12-8. Apoptotic cells in mice subjected to 15 Gy γ-irradiation and killed 4 hours later. Mice received vehicle (control) or 2% DL-α-difluoromethylornithine (DFMO) in their drinking water for 4 days before irradiation. (A) Apoptotic cells per crypt-villus unit. (B) Position of apoptotic cells along crypt-villus axis. (Reproduced from Deng and Johnson [148], by permission.)

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 357

Lysophosphatidic Acid The phospholipid, lysophosphatidic acid (LPA), a normal constituent of serum, mimics the actions of polypeptide growth factors. Lysophosphatidic acid acts on a G-protein– coupled family of receptors to influence cell-cycle progression, the actin cytoskeleton, cellular differentiation, Ca+2 homeostasis, and cell survival (149–151). Lysophosphatidic acid has been shown to enhance the survival of Schwann cells and lymphocytes (152,153) and to suppress apoptosis induced by ischemia–reperfusion in cardiomyocytes (154) and by serum withdrawal in fibroblasts and renal epithelial cells (155,156). Deng and colleagues (157) examined the ability of LPA to prevent apoptosis in IEC-6 cells and the intestinal epithelia of mice. Injection of LPA 2 hours before exposure to 15 or 18.75 Gy γ-radiation significantly reduced the number of apoptotic bodies per crypt-villus unit compared with intestines from mice injected with vehicle alone. In IEC-6 cells, LPA rescued cells from apoptosis when applied up to 1 hour after camptothecin treatment or 2 hours after exposure to radiation. Lysophosphatidic acid also inhibited the activation of caspase-3. The ability of LPA to prevent apoptosis

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1 Control 2 Irradiation

1.00 Caspase-3 activity pmol/mg protein/min

required for, cell growth and proliferation (142,143). Intracellular polyamine levels are highly regulated and are dependent primarily on the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting step in polyamine biosynthesis, the decarboxylation of ornithine to form the diamine putrescine (144,145). DL-αdifluoromethylornithine (DFMO) blocks ODC and depletes cells of polyamines. DL-α-difluoromethylornithine is a specific and irreversible inhibitor of ODC, having no effects except those caused by the inhibition of ODC and the subsequent depletion of polyamines (145,146). Polyamines are involved in apoptosis, and whether they increase or inhibit it depends on the cell type or tissue involved and the stimulus for apoptosis (147). We have found, however, that DFMO and subsequent polyamine depletion prevent apoptosis in IEC-6 cells. (These studies are discussed in detail in the next section.) We have also shown that polyamine depletion inhibits irradiation-induced apoptosis in the epithelium of the mouse small intestine (148). Four hours after exposure to 15 Gy radiation, apoptotic cells increased from 0.6 to 2.7 per cryptvillus unit. In polyamine-depleted animals, the increase was reduced to 1.6 cells (Fig. 12-8A). Most apoptotic cells occurred at positions 4 through 7 within the crypts (see Fig. 12-8B). Polyamine depletion also prevented most of the increases in caspase-3 activity (Figs. 12-9A and B) and the proapoptotic protein Bax (see Fig. 12-9C) after irradiation. Four days after irradiation, the crypt survival rate was increased more than twofold in the mice treated with DFMO compared with control animals. Because DFMO is already approved for use in patients to retard tumor growth and because it can be administered orally, polyamine depletion may be useful therapeutically to counteract gastrointestinal toxicity caused by radiation and chemotherapy (148).

3 Irradiation DFMO

0.75

*

0.50

0.25

0

1

A

3

2

kDa Pro-caspase-3

−32

Active Caspase-3 unit

−19

Beta-actin

−42

B Irradiation

1

2

3

−

+

+

1

2

3

Bax

Beta-actin

C

FIG. 12-9. Polyamine depletion inhibits caspase-3 activation and Bas after irradiation of the mouse small intestine. Animals were treated as in Figure 12-8. (A) Caspase-3 activity determined by enzyme-linked immunosorbent assay. (B) Pro-caspase-3 and caspase-3 Western blots. (C) Bax levels assayed by Western blotting. DFMO, DL-α-difluoromethylornithine. (Reproduced from Deng and Johnson [148], by permission.)

even when administered after the apoptotic stimulus is interesting. It remains to be seen whether this effect can be reproduced in whole animals. Others Several cytokines have been reported to protect intestinal epithelia from programmed cell death. Interleukin-11 (IL-11) protects clonogenic stem cells in the intestinal crypts of mice after radiation (158), stimulates the recovery of small-bowel epithelial cells after cytoablative therapy (159), and reduces

358 / CHAPTER 12 increased both caspase-3 activity and DNA fragmentation. Thus, NO appears to exert an antiapoptotic effect on the rat gastrointestinal tract.

the severity of chemotherapy-induced mucositis in hamsters (160). Interleukin-15 has been shown to protect the intestinal mucosa from several chemotherapeutic agents including 5-fluorouracil (161). Leukotriene D4 increases cell survival by preventing apoptosis in IECs (162). The mechanism of this effect has been explored Wikström and colleagues (163). These investigators found that leukotriene D4 blocked the release of cytochrome c caused by the inhibition of COX-2. They found that COX-2 inhibition resulted in the activation of caspase-8 and the subsequent production of t-Bid, indicating that this pathway to apoptosis is blocked by prostaglandins. Leukotriene D4 also reduced the basal level of caspase-8 activation and the t-Bid generation. The effect of nitric oxide (NO) on intestinal apoptosis has been examined by Bersimbaev and colleagues (164) in the rat gastrointestinal tract. They exposed mucosal cells to NO delivered from the NO donor, S-nitroso-N-acetylpenicillamine, and examined the effect of intravenous LPS in the presence and absence of the inhibition of NO synthesis. Nitric oxide inhibited caspase-3 activity and apoptosis as measured by DNA fragmentation. Lipopolysaccharide increased both caspase-3 activity and apoptosis in gastric, ileal, and colonic mucosa 5 and 24 hours after LPS. Administration of an inhibitor of inducible nitric oxide synthase (iNOS) immediately after LPS injection significantly

INTESTINAL CELLS: IN VITRO STUDIES This section covers in vitro studies involving cultured cell lines and isolated enterocytes. Cell lines have been used to elucidate apoptotic pathways and to identify points at which these pathways can be controlled. Isolated enterocytes have been used primarily to examine the onset and events involved in apoptosis induced by detachment from substrate or ECM, the process referred to as anoikis.

Apoptotic Pathway in Intestinal Cells The extrinsic and intrinsic apoptotic pathways shown in Figures 12-5 and 12-6 are the consensus of many experiments conducted with a variety of different cell types. Figure 12-10 illustrates the responses to activation of the extrinsic pathway by TNF-α and cycloheximide (CHX), which is given to block the synthesis of antiapoptotic proteins (165), and activation of the intrinsic or mitochondrial pathway by either the

TNF-α

(167)

(167) IκBa

PI3K Akt

p53

Procaspase-8 IκBa = NF-κB (167)

ERK1/2 (38)

(166) Bax Caspase-8

NF-κB (167)

MEKK

Damage (12,148)

(167)

Bcl-XL Bcl-2 (12) MEK4/7

Bid (12)

IAPs

Procaspase-3 (12,38,120)

(38)

t-Bid

JNK (166)

MEK1 (38)

Nucleus Bcl-XL (12)

Caspase-3

Bad

Erk1/2

Bcl-2 CHX (166)

(12,38,166) Cyto c

APAF-1

Caspase-9 Apoptosis (12,38,165,166,167,168)

Procaspase-9 (12,166)

FIG. 12-10. Apoptotic pathways activated in intestinal epithelial cell-6 (IEC-6) cells by tumor necrosis factor-α (TNF-α) and damage induced by γ-irradiation or camptothecin. CHX, cycloheximide; Cyto c, cytochrome c; ERK, extracellular signal–regulated kinase; IAP, inhibitor of apoptosis protein; IκBα, inhibitory κBα; JNK, c-Jun NH2-terminal kinase; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol-3 kinase; TNF, tumor necrosis factor.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 359 topoisomerase I inhibitor, camptothecin (12), or γ-irradiation (148) in the IEC-6 cell line. This line was originally described by Quaroni and colleagues (166) and was derived from adult rat intestinal crypt cells. It remains a nontransformed, undifferentiated cell line. The pathways shown in Figure 12-10 are the first comprehensive portrayal of events after an apoptotic stimulus in IECs. As shown, the primary apoptotic effect of TNF-α is the activation of procaspase-8 to caspase-8 to initiate the extrinsic pathway. The mitochondrial pathway is also activated in response to TNF-α because caspase-8 cleaves Bid to produce t-bid (12). Tumor necrosis factor-α also initiates two survival pathways by activating PI3K, which leads to activation of NF-κB (167) and MEK, which leads to the activation of ERK1/2 (38). These transcription factors produce IAPs and Bcl-2, which would block the apoptotic response in the absence of CHX (165). Damage to IEC-6 cells causes the activation of Bax and the mitochondrial pathway with the subsequent release of cytochrome c (12).

Polyamines and Apoptosis Depletion of polyamines by the inhibition of ODC with DFMO leads to cell-cycle arrest without apoptosis in IEC-6 cells (168). Cell-cycle arrest was the result of the induction of p53 and the cell cycle inhibitors p21 and p27. The absence of apoptosis under these conditions prompted Ray and colleagues (169) to examine whether DFMO and polyamine depletion altered the response of IEC-6 cells to apoptotic stimuli. Both camptothecin and TNF-α/CHX significantly increased apoptosis as measured by DNA fragmentation and caspase-3 activity. Polyamine depletion prevented most of both responses. Cells that were cultured with exogenous putrescine together with DFMO had apoptotic responses indistinguishable from control samples, indicating that the protective effect was caused by polyamine depletion and not DFMO itself. Increasing the levels of putrescine by blocking its conversion to spermidine induced apoptosis in the absence of other stimuli (169). This result was in keeping with data from other cell types indicating that excess polyamines could stimulate apoptosis (170,171). An additional interesting report by Li and colleagues (172) indicates that polyamine depletion can either sensitize IEC-6 cells to apoptosis or prevent it, depending on the stimulus. These authors found that DFMO increased apoptosis in response to staurosporine and decreased it after TNF-α/CHX. Both responses appeared to depend on NF-κB, because they were prevented by the inhibition of NF-κB binding activity. To my knowledge, this is the only report where polyamine depletion sensitized IECs to an apoptotic stimulus. This observation has not been pursued, thus the mechanism remains unknown. Effects of Polyamine Depletion Yuan and colleagues (12) have shown that polyamine depletion prevented the release of cytochrome c from

mitochondria after camptothecin. This was due, in part, to increased levels of Bcl-2 and Bcl-XL and decreased levels of Bax and t-Bid in the cells incubated with DFMO. In addition to inhibiting the activation of caspase-3, the depletion of polyamines significantly reduces the activation of caspase-6, -8, and -9 in response to TNF/CHX (165). The remaining proapoptotic step shown in Figure 12-10 involves JNK, which has been shown to be activated by TNF-α/CHX through TNFR1 and TNF receptor-associated factor (TRAF2) (31,173). Tumor necrosis factor-α/CHX also activates JNK in IEC-6 cells, and that activation is dependent on the presence of polyamines (165). Polyamine depletion prevented JNK activation, caspase-9 activation, DNA fragmentation, and caspase-3 activation. Inhibition of JNK with the specific inhibitor SP-600125 prevented apoptosis, caspase-9 activation, and the release of cytochrome c. Thus, part of the antiapoptotic effect of polyamine depletion appears to be caused by the inhibition of JNK activity (165). The mechanism by which JNK activates apoptosis is not clear; however, these results indicate that JNK regulates a step(s) upstream of caspase-9 and modulates the mitochondrial pathway by influencing cytochrome c release or Bcl-2. Two other pathways shown in Figure 12-10 involve ERK and NF-κB. Both of these are activated by TNF/CHX and enhanced by polyamine depletion. A large number of reports point to the role of ERKs in mediating a strong antiapoptotic response (37,174,175). Polyamine depletion causes the sustained activation of ERK in response to TNF-α/CHX (38). Pretreatment of polyamine-depleted cells with a membranepermeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α–induced JNK activation and were significantly protected from apoptosis. Conversely, a dominant negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant negative MEK-1 cells did not prevent JNK activation or cytochrome c release and failed to protect cells from both TNF-α/CHXand camptothecin-induced apoptosis (38). The stable expression of a dominant negative mutant of JNK significantly protected IEC-6 cells from apoptosis. Thus, polyamine depletion in IEC-6 cells activates ERK, which inhibits JNK activation, leading to the protection of cells from apoptosis (38). Polyamine depletion rapidly activates NF-κB (176). Within 1 hour after administering DFMO, p65 had redistributed from the cytoplasm to the nucleus of IEC-6 cells. The degradation of IκBα also began within 1 hour and was complete by 5 hours. These times correlated with a 50% decrease in putrescine levels at 1 hour and a total absence of putrescine by 3 hours. The activation of NF-κB was prevented by the addition of exogenous putrescine to the DFMOtreated cultures (176). Li and colleagues (172) also reported that polyamine depletion led to activation of NF-κB from 4 to 8 days after constant exposure to DFMO. They found that inhibition of NF-κB binding activity by sulfasalazine or

360 / CHAPTER 12 MG-132 prevented the increased apoptotic response to staurosporine and the decreased apoptotic response to TNF/ CHX normally found in polyamine-depleted cells. Wang and his coworkers (177) have followed this study by showing that activation of NF-κB by polyamine depletion led to the synthesis of cIAP2 and XIAP, members of the inhibitors of apoptosis family, and the inhibition of caspase-3 activity. They did not try to reconcile these findings with their previous data indicating that NF-κB activation enhanced staurosporine-induced apoptosis (172). TNF-α–induced activation of NF-κB has been shown to require Akt (178,179). Akt, also known as protein kinase B1, is a serine/threonine protein kinase, originally identified as the oncogene transduced by the acute transforming retrovirus Akt (180,181). In quiescent cells, Akt resides in the cytosol in an inactive state. After cytokine stimulation, it translocates to the inner surface of the plasma membrane where PI3K-generated 3′-phosphoinositides reside, and Akt is phosphorylated at threonine 308 and serine 473 by pyruvate dehydrogenase kinase-1 (PDK1) for its full activation (see Fig. 12-10) (182). Akt has been shown to inhibit apoptosis by directly phosphorylating and inactivating Bad (183). Akt protects cells from apoptosis by inactivating caspase-9 and by activating NF-κB (184,185). Although Akt inhibits apoptosis by regulating the release of mitochondrial cytochrome c, the mechanism is not clear (186,187). Glycogen synthase kinase-3β (GSK-3β), an important downstream target of Akt, is inhibited by Akt phosphorylation (188). Increased activity of GSK-3β has been shown to induce apoptosis of several cell types (189). Zhang and colleagues (190) found that inhibition of Akt increases apoptosis in polyamine-depleted IEC-6 cells, and they suggest that Akt activation is responsible for suppression of caspase-3 activity through a process involving the phosphorylation of GSK-3β. Bhattacharya and colleagues (167) found that inhibition of GSK-3β had no effect on apoptosis induced by TNF-α in IEC-6 cells. They went on to show that inhibition of PI3K and overexpression of dominant-negative Akt (DN-Akt) significantly increased apoptosis in polyaminedepleted cells. Constitutive activation of Akt in response to polyamine depletion was prevented in DN-Akt cells, reversing the protective effect of polyamine depletion. Expression of DN-Akt, as well as PI3K inhibitors, prevented Akt activation and subsequent translocation of NF-κB to the nucleus. Constitutively active Akt (CA-Akt) expression increased the resistance to TNF-α–induced apoptosis and increased the activation of NF-κB. Polyamine depletion of cells transfected with DN-Akt prevented basal and TNF-α–induced IκBα phosphorylation. Prevention of NF-κB activation in cells transfected with DN-IκBα increased apoptosis basally and restored it in polyamine-depleted cells. Thus, protection from apoptosis by activated Akt in polyamine-deficient cells is dependent on the nuclear translocation and activation of the transcription factor NF-κB and is independent of GSK-3β activity. Akt regulates the mitochondrial pathway, preventing activation of caspase-9, and thereby caspase-3, via the actions of NF-κB (167).

Signal transducers and activators of transcription (STATs) proteins are a family of latent transcription factors that undergo ligand-dependent tyrosine phosphorylation and activation (191). STAT3 is the transcription factor responsible for activation of the acute phase-response genes and binds to the sis-inducible element (SIE) found in the c-fos promoter regulated by platelet-derived growth factor (PDGF) (192). Pfeffer and colleagues (193) found that inhibition of ODC with DFMO rapidly induced STAT3 activation as determined by its tyrosine phosphorylation, translocation from the cytoplasm to the nucleus, and presence in SIE-dependent DNA-protein complexes. Activation of STAT3 was accompanied by the activation of a STAT3-dependent reporter construct. Additional phosphorylation of STAT3 at serine 727 is thought to increase its transcriptional activity and is mediated by the MAPK pathway (194,195). Constitutive activation of STAT3 has been described in a wide variety of primary tumors and has been reported to induce cell survival in association with surviving expression in gastric cancer cells (196,197). STAT3 signaling appears to mediate the survival of oncogenic Ras-transfected IECs (198). Bhattacharya and colleagues (199) found that polyamine depletion induced STAT3 phosphorylation at both Tyr-705 and Ser-727, resulting in localization to the cell periphery and nucleus, respectively. Sustained phosphorylation of STAT3 at both residues occurred after polyamine-depleted cells were exposed to TNF-α. Inhibition of STAT3 increased the sensitivity of polyamine-depleted cells to apoptosis, and the protective effect of polyamine depletion was totally eliminated in cells expressing DN-STAT3. DFMO increased mRNA and protein levels of Bcl-2, Mcl-1, and BIRC3. These levels were significantly decreased in DN-STAT3– expressing cells. Inhibition of STAT3 in polyamine-depleted cells induced the activation of caspase-3 and apoptosis. Thus, STAT3 appears to play a prominent role in protecting polyamine-depleted cells from apoptosis. This protection may be mediated by the expression of antiapoptotic proteins, Bcl-2, Mcl-1, and BIRC3 (199). Regulation of Apoptotic Pathways It is naive to believe that polyamines affect each of the steps or even several steps in Figure 12-10. In eukaryotic cells, reversible protein phosphorylation by protein kinases and phosphatases regulates numerous processes, including apoptosis (200). The kinases themselves are dephosphorylated and inactivated by a type 2A protein phosphatase (PP2A). Previous studies have implicated serine/threonine PP2A in a wide variety of cellular functions including metabolism, transcription, translation, ion transport development, proliferation, differentiation, and apoptosis (200–202). Figure 12-10 depicts two major regulatory pathways—the ERK/JNK pathway and the PI3K/Akt/NF-κB pathway— that are altered by polyamine depletion. In addition, each of these pathways controls the levels or activities of Bcl-2 family proteins that are responsible for determining whether cytochrome c is released from mitochondria. The activities

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 361 Protein

Activity of Protein

Apoptosis

ERKs Akt IκBα NF-κB Bcl-2 Bad FIG. 12-11. The effects of serine-threonine phosphorylation on the activities of proteins regulating apoptosis. The consequences of the change in activity on apoptosis also is shown. ERK, extracellular signal–regulated kinase; IκBα, inhibitory κBα; NF-κB, nuclear factor-κB.

of many components of these pathways and the Bcl-2 proteins themselves are regulated by serine/threonine phosphorylation. Bcl-2, for example, is inactivated by PP2A, whereas Bad is activated by PP2A (201). An analysis of the effects of serine/threonine phosphorylation on the activities of proteins in these signaling pathways is shown in Figure 12-11. In each case, this effect is in the same direction as the change in activity reported for polyamine depletion. Also in each case, the change in activity, whether an increase or a decrease, acts to prevent apoptosis. In addition, Akt and the ERKs are activated within minutes of exposing IEC-6 cells to TNF-α. These activities peak around 3 to 4 hours, and then decrease to basal levels by 9 hours (38, 167). In polyamine-depleted cells, however, the activities of

300 DFMO 250

Activity

200 150 100 Control

50 0 0

3

6

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Time (h) TNF/CHX

FIG. 12-12. Generalized time course of the activities of Akt and extracellular signal–regulated kinase (ERK) after exposure of control and polyamine-depleted DL-α-difluoromethylornithine (DFMO) cells to tumor necrosis factor-α/ cycloheximide (TNF-α/CHX).

these enzymes increase to greater levels, and then plateau. Figure 12-12 is a generalized representation of these findings. Thus, there appears to be no inactivation of Akt and ERK in DFMO-treated cells so that polyamine depletion renders these enzymes constitutively active (38,167). The findings depicted in Figures 12-11 and 12-12 could be explained if PP2A were inactive in polyamine-depleted cells and its inactivation protected those cells from apoptosis. In support of this explanation are earlier reports indicating that polyamines can activate PP2A specifically compared with PP1 (203,204). Ray and colleagues (205) have shown that the inhibition of PP2A with either okadaic acid or fostriecin protected IEC-6 cells from apoptosis induced by TNF-α/CHX. Cells transfected with the siRNA for PP2A were also protected from apoptosis. Polyamine-depleted cells had only about one-third the PP2A activity of control cells or those incubated with putrescine in addition to DFMO. Inactivation of Bad involves the phosphorylation of serine residues 112 and 136, which decreases its affinity for Bcl-XL at the mitochondrial membrane (200,206). This frees Bcl-XL to prevent apoptosis. PP1 activates Bad by serine dephosphorylation. Current data suggest that PP1 itself is dephosphorylated and activated by PP2A, which is also responsible for dephosphorylating and inactivating Bcl-2 (200). Thus, decreased PP2A activity in polyamine-depleted cells may not only prevent the inactivation of Bc1-2, but also cause Bad to remain phosphorylated and inactive. Polyamine-depleted cells have been shown to have significantly greater levels of pSer112-Bad than control cells (205). Furthermore, the level of active pSer Bcl-2 decreased after TNF-α/CHX in control cells but increased significantly in cells treated with DFMO. In addition, okadaic acid increased the phosphorylation of these proteins similar to the effects of polyamine depletion. The inhibition of PP2A with okadaic acid also mimicked other effects of polyamine depletion, namely the prevention of cytochrome c release, inhibition of JNK activity, and the inhibition of caspase-9 and -3 in response to TNF-α/CHX (205). Type 2A protein phosphatase, therefore, appears to be a crucial point for controlling apoptotic pathways in IECs. Type 2A protein phosphatase itself is regulated by tyrosine kinases such as Src kinase and the EGFR, which phosphorylate it, inhibiting the enzyme (207,208). Signals transduced through a single receptor, such as the TNF-α receptor, can promote either cell proliferation or apoptosis, depending on how the cell is programmed to respond to receptor proximal events. Expression of v-Src rescues several cell types from apoptosis induced by cytokine withdrawal, irradiation, chemotherapeutic drugs, or detachment from the ECM (209–213). These findings suggest that constitutively activated Src can mimic the effects of cytokine receptors, integrins, and other receptor pathways that protect cells from apoptosis. The mechanism by which Src inhibits apoptosis is not well defined because of the divergent nature of Src signaling. However, current studies suggest the Src may regulate apoptosis by altering the strength and balance

362 / CHAPTER 12 of signals activating the various MAPK and PI3K/Akt pathways (214–217). We have shown that Src is constitutively activated in polyamine-depleted IEC-6 cells (218). Phosphorylation of Tyr 416 in the kinase domain completely activates Src and provides a binding site for SH2 domains of other proteins (219). Incubation of IEC-6 cells with DFMO for 4 days significantly increased the amount of pY 416-Src without affecting the level of Src protein. Pharmacologic inhibition of Src increased basal apoptosis and apoptosis in response to TNF/CHX in control cells. In polyamine-depleted cells, the inhibition of Src restored apoptosis to control levels. The expression of DN-Src in IEC-6 cells increased apoptosis significantly compared with vector-transfected cells and eliminated all protection by DFMO. Conversely, the expression of CA-Src produced cells resistant to apoptosis (218). Figure 12-13 depicts a model showing how the activation of Src might account for the effect of polyamine depletion on apoptosis. As noted earlier, the activation of STAT3 contributes to the resistance of polyamine-depleted cells to apoptosis. Interestingly, Src activation may also account for the activation of STAT3. The activation of STAT3 has been associated with Src activity in cell lines transformed with v-Src (220,221). Src is known to activate STAT3 in PDGF-stimulated fibroblasts (222). Zhang and colleagues (223) have proposed a mechanism for STAT3 activation in which Src, as part of a membrane-anchored scaffold, phosphorylates and activates Janus kinase (JAK), which, in turn, phosphorylates the cytoplasmic tail of a membrane receptor, which acts as the docking site for STAT3, which is then phosphorylated by Src. In conclusion, polyamine depletion has not only allowed investigators to define the effects of polyamines on apoptosis, but it has contributed to our understanding of the apoptotic process and how it is regulated in IECs. In DFMO cells:

+Src

+ Bcl-2-pSer

− PP2A-PTyr

+ Akt-pSer

+ ERKs-pTyr/Thr

− IKB-α-pSer

+ NFκB

− JNK

− Bad-pSer112

FIG. 12-13. Summary of the changes (positive [+] and negative [−]) in activity occurring as a result of polyamine depletion and how they can be accounted for by a Src-induced inhibition of type 2A protein phosphatase (PP2A). DFMO, DL-αdifluoromethylornithine; ERK, extracellular signal–regulated kinase; IκB-α, inhibitory κB-α; JNK, c-Jun NH2-terminal kinase; NF-κB, nuclear factor-κB.

Additional Studies Numerous studies using cultured or isolated intestinal cells have examined the regulation of various steps in the apoptotic pathway. For the most part these experiments agree with those involving polyamine depletion in cultured cells and those examining the same compounds in vivo. The importance of the Ras/MAPK/ERK pathway was emphasized in a study that showed dying enterocytes downregulate pathways converging on Ras (224). Ras-convergent proteins such as the EGFR, Erb-B2, and the Son of sevenless guanine nucleotide exchangers were rapidly down-regulated. Caspase inhibitors, however, prevented both down-regulation and apoptosis. Yan and colleagues (225) found that expression of the DN kinase inactive kinase suppressor of Ras (KSR), which regulates the TNF activation of the Raf/MAPK/ERK pathway, decreased survival and increased apoptosis of TNFα–treated IECs. Antiapoptotic pathways including NF-κB, cIAP2 gene expression, and ERK/MAPK activation were all inhibited in the DN-KSR cells. In the T84 cell line, Rudolph and colleagues (226) found that cyclic adenosine monophosphate (cAMP) inhibits apoptosis by up-regulating ERK1/2 through a protein kinase A (PKA)–independent mechanism. It is obvious from these studies and those discussed previously that a number of upstream regulators converge on the MAPK signaling pathway to prevent cell death in IECs. Denning and colleagues (227) obtained evidence that in response to oxidative stress, enterocytes are primed for apoptosis via Fas-mediated pathways. After exposure to H2O2, mRNA and protein levels increased for both Fas and FasL, and IECs underwent apoptosis. Apoptosis was associated with the activation of caspase-8. The susceptibility of normal enterocytes to Fas-induced apoptosis depended on levels of cIAP1 and cIAP2 (228). Thus, these results are basically identical to those showing that TNF-α will not cause apoptosis without concurrently inhibiting protein synthesis. The antral hormone gastrin is an important trophic agent for gastric and colonic mucosa. These trophic effects, as opposed to the acid secretion stimulatory action of the hormone, are primarily produced by progastrin (see Chapter 15). Although it is well established that gastrin increases growth by stimulating DNA synthesis and increasing cell proliferation, it has also been shown to inhibit apoptosis (229,230). Gastrin has been shown to prevent apoptosis in a pancreatic acinar cell line by activating the Akt (229) and MAPK (231) pathways. In IECs, pentagastrin decreased the release of cytochrome c from mitochondria and inhibited the activation of caspase-9 and -3 (230). Most studies involving effects of gastrin on apoptosis have dealt with cancer progression and are covered in Chapter 17. As frequently pointed out, COX-2 is overexpressed in colorectal adenomas and adenocarcinomas, and its inhibition by nonsteroidal anti-inflammatory drugs (NSAIDs) protects against colorectal cancer (232–234). One study produced some insight into the mechanisms involved in the prostaglandinmediated protection of cells from apoptosis. Nishihara and colleagues (235) show that cAMP agonists, such as PGE2,

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 363 cholera-toxin, and a cAMP analog that was membrane permeable, protected both normal and transformed intestinal cells (RIE-1 and T84) from apoptosis induced by anti-Fas antibody and staurosporine. Protection was associated with the rapid induction of cIAP2, but not six other members of the IAP family. Inhibition of COX-2 in cells overexpressing the enzyme decreased cIAP2 expression and increased apoptosis. Both of these were reversed by exogenous PGE2 and its subsequent cAMP-dependent induction of cIAP2. These results suggest an explanation for the anticancer effects of NSAIDs by identifying a mechanism whereby cAMP promotes cancerogenesis by inhibiting the normal apoptotic process (235). Two recent studies have examined the cytokines, plateletactivating factor (PAF) and IL-11, which may be involved in mediating the pathogenesis of ulcerative colitis and protecting against it, respectively, for their effects on the apoptotic pathway (236,237). Lu and colleagues (236) stably transfected IEC-6 cells to overproduce Bcl-2 under the control of a lactose-inducible promoter. In the absence of Bcl-2 overexpression, PAF caused Bax to translocate to the mitochondria, resulting in the collapse of the mitochondrial membrane potential and activation of caspase-3 and apoptosis. This was the first demonstration that PAF can activate apoptosis in enterocytes by a Bax-dependent mechanism, and that the entire process could be controlled by Bcl-2 expression. An earlier study had shown that Bax is down-regulated in the inflamed mucosa involved in ulcerative colitis compared with normal colon and inactive mucosa from a patient with ulcerative colitis (238). Even though IL-11 is being evaluated in patients for its effects on ulcerative colitis, its mechanism of protecting colonic cells has not been determined. Kiessling and colleagues (237) found that IL-11 induced signaling in HT-29 cells and colonic epithelial cells by activating the JAK/STAT pathway. IL-11 resulted in a dosedependent phosphorylation of Akt and decreased activation of caspase-9 and the inhibition of apoptosis.

Anoikis In 1994, Frisch and Francis (239) showed that detachment of epithelial cells from the ECM induced apoptosis, which they termed anoikis for the Greek word meaning “homelessness.” Cells undergoing anoikis have all the characteristics of apoptosis that have been discussed earlier in this chapter. The ECM controls cells through signals via a family of cellsurface receptors called integrins (240,241). Integrins are composed of α- and β-subunits. At least 15 α- and 8 βsubunits have been described, and the combination of particular α- and β-subunits accounts for binding and signaling specificity (241). As integrins bind to the ECM, they aggregate and direct the formation of a cytoskeletal and signaling complex that results in the assembly of actin filaments. The reorganization of actin filaments into stress fibers produces a positive feedback, resulting in additional integrin clustering. This causes the formation of a transmembrane complex

of ECM proteins, integrins, and cytoskeletal proteins such as talin, paxillin, vinculin, and actin. These aggregates are known as focal adhesion complexes. Focal adhesion kinase (FAK) is one of a variety of protein tyrosine kinases activated by integrins and targeted to the focal adhesion complex (242). Focal adhesion kinase is further phosphorylated within the focal adhesion complex, which then provides binding sites for other proteins such as paxillin. The formation of focal adhesions is necessary for cell migration, spreading, growth, and differentiation (239,243–245). Epithelial cells that have lost attachment undergo anoikis, and are thus unable to move and attach elsewhere. Anoikis is often lost in cancer cells because of the mutation and inactivation of genes necessary for apoptosis. These malignant cells are able to proliferate free of attachment to the ECM and to metastasize (246). Focal adhesion kinase is the primary enzyme linking detachment from the ECM to apoptosis. Focal adhesion kinase is overexpressed in tumor cells, and the inhibition of FAK results in apoptosis in breast cancer cells by activating a caspase-8–dependent mechanism (247). In IECs, activation of caspase-6 occurs within 15 minutes of detachment and 30 to 45 minutes before the activation of caspase-3 (248). In a second report, the same group of investigators found that FAK is cleaved in an orderly fashion by two different caspases. The first cleavage was mediated by caspase-3, resulting in a 94- to 92-kDa fragment, which was further cleaved by caspase-6 to an 84-kDa fragment (249). Thus, the targeting of FAK during anoikis not only dismantles the cytoskeleton, but also alters the cell’s signaling mechanisms. During the first 15 minutes after detachment, two initiator caspases, caspase-2 and -9, were activated followed by the activation of caspase-6 and -3. The release of cytochrome c was not detected before 60 minutes (250). These investigators concluded, therefore, that cytochrome c release did not play a role in the initiation of apoptosis. These results imply that caspase-9 activation proceeded by a mechanism different from the formation of the apoptosome with APAF and cytochrome c. The activation of EGFR signaling inhibits anoikis (251), and one well-established mediator of EGFR signaling is the c-Src kinase. Src is recruited to the focal adhesion complex, and the ability of FAK to transduce downstream signals is dependent on this protein tyrosine kinase (252). Rosen and colleagues (251) used TGF-α to activate the EGFR in IEC-18 and rat intestinal epithelial (RIE) cells. Transforming growth factor-α strongly inhibited anoikis by preventing the inhibition of Src activity that normally follows detachment. Detachment caused the down-regulation of Bcl-XL, which was necessary for anoikis and was prevented by Src activation. The authors conclude that anoikis was, at least in part, mediated by the loss of Src activity and the subsequent decreased expression of Bcl-XL (251). A follow-up article from the same group showed that v-Src induced resistance to anoikis was mediated by increased Bcl-XL (253). Transfection of IEC-18 cells with v-Src made them resistant to anoikis and up-regulated Bcl-XL. Both of these findings depended on the induction of the MEK/MAPK pathway.

364 / CHAPTER 12 From these studies it appears that detachment disrupts Src signaling from the focal adhesion complex, leading to the down-regulation of the MAPK pathway and decreased expression of Bcl-XL. Although Filmus and coworkers (251,253) have documented the importance of Bcl-XL in attenuating anoikis, they have also shown that exogenous Bcl-XL, no matter how highly expressed, cannot completely prevent it (254). A second mechanism was identified, in which anoikis was stimulated by detachment-induced up-regulation of FasL. They also demonstrated that the increase in the expression of FasL was dependent on a detachment-induced increase in stress-activated protein kinase 38. Vachon and colleagues (255) induced anoikis in enterocytes by pharmacologically inhibiting FAK or by expressing DN-FAK. They found that p38 activation increased transiently after the induction of anoikis in both differentiated and undifferentiated enterocytes. The activation of p38 was associated with the downregulation of the FAK/PI3/Akt pathway. Only p38β and p38δ were required for anoikis, specifically in undifferentiated and differentiated cells, respectively. The studies discussed previously have established that Bcl-XL levels play an important role in anoikis, and that Src can regulate Bcl-XL expression through a MAPK-dependent mechanism. Additional studies have examined the role of Ras during anoikis and found that it regulates Bcl-XL through a mechanism that is independent of both the MAPK and the PI3/Akt pathways (256). Using nonmalignant rat and human IECs, Rosen and coworkers (256) found that activated H- or K-ras oncogenes completely prevented the down-regulation of Bcl-XL triggered by detachment from the ECM. Inhibition of Bcl-XL in the Ras-transformed cells stimulated anoikis and inhibited tumorigenicity. Ras also induced the downregulation of the proapoptotic Bak protein, which could be reversed by pharmacologic inhibition of PI3K. Thus, the authors concluded that Ras-induced resistance to anoikis was mediated by at least two separate mechanisms: one that results in the down-regulation of Bak, and one that stabilizes the expression of Bcl-XL in the absence of ECM (256). These findings were supported by recent data showing that FTS (farnesylthiosalicylic acid), a direct inhibitor of Ras, restored the sensitivity to anoikis in Ras-transformed RIE cells (257). Ras inhibition of anoikis did not depend on Akt or MAPK. Several pathways have therefore been identified that appear to mediate anoikis in IECs. One involves Src, another the FasL, and apparently two separate pathways mediated by Ras.

GASTRIC MUCOSA The stem cells for the gastric mucosa are located in the neck area between the gastric pits and glands. Cells migrate toward the surface, differentiating into surface mucous cells and downward into the pits differentiating into parietal, chief, and endocrine cells. The surface cells are shed continuously with a 3- to 5-day rate of renewal under normal

conditions (258). Cell loss is enhanced by the physical presence of food in the lumen of the stomach and the friction between it and the surface cells produced by gastric motility. The turnover rates of the cells present in the gastric glands are considerably longer (259).

Helicobacter Pylori Apoptosis is involved in gastric ulceration, and the mediators are basically identical to those regulating apoptosis in IECs. Slomiany and colleagues (260) induced chronic gastric ulcers in rats with acetic acid and observed a 33-fold increase in caspase-3 activity at the onset of ulceration 2 days after damage. Caspase-3 activity then decreased as the ulcers healed. Konturek and colleagues (261) used water immersion and restraint for 3.5 hours to induce acute gastric stress ulcers in rats. Apoptosis was maximal 4 hours after the period of stress and declined to near basal levels 24 hours after injury. Bax levels and mRNA expression were high immediately after damage and peaked at 4 hours. They then decreased approximately 50% by 24 hours but were still significantly increased. Bcl-2 expression, in contrast, was low after injury, and then increased fourfold between 4 and 6 hours. It remained near this level throughout the remainder of the 24-hour period. Gastric infection by Helicobacter pylori is associated with gastritis, ulceration, and carcinoma (262). Additional studies have shown that incubation of cultured gastric epithelial cells with H. pylori inhibits cell proliferation (263,264). In addition to the inhibition of proliferation, the induction of apoptosis contributes to the decreased growth rate after H. pylori infection. Moss and colleagues (265) found low rates (2.9% of epithelial cells) of apoptosis in gastric biopsies from uninfected patients. The apoptotic cells were confined to the most superficial regions of the gastric glands. In H. pylori infection they were more numerous, comprising 16.8% of the epithelial cells, and were located throughout the depth of the glands. These investigators suggested that increased apoptosis could provide the stimulus for a compensatory increase in proliferation, a response associated with chronic H. pylori infection (266,267). It is unknown whether the rate of apoptosis in individuals infected with H. pylori depends on the presence of metaplasia. Eradication of H. pylori did not alter cell proliferation in patients with chronic gastritis, and H. pylori increased apoptosis only in the absence of metaplasia (268). A second study with similar conclusions found that after eradication of H. pylori a significant decrease in apoptosis occurred in nonmetaplastic but not in metaplastic tissue from the same individual (269). In general, H. pylori induces apoptosis in gastric epithelial cells in cell culture experiments (263,264,270–272). In addition, in gastric biopsy samples, H. pylori has been associated with twofold to fivefold increases in epithelial cell apoptosis that return to normal levels after eradication of the bacteria (265–267,270,273–275). Epidemiologic studies have implicated the cag island in the pathogenesis of

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 365 H. pylori (276), possibly resulting from the insertion of the CagA protein into host epithelial cells after bacterial attachment (277). Some studies have shown that cag-positive strains increase proliferation but not apoptosis (274,275), whereas others have found that apoptosis is increased significantly in patients with duodenal ulcer disease who, for the most part, are infected with cag-positive bacteria (278–280). Moss and colleagues (281) examined the effect of H. pylori on gastric epithelial proliferation and apoptosis in a large prospective study of 60 patients with nonulcer dyspepsia. They found that proliferation was increased twofold in both antral and corpus epithelial cells and was not related to cagA status but was positively correlated with the presence of gastritis. Apoptosis was increased in the antrum and body only in those with cagA-positive strains of H. pylori. Thus, these results do not support the hypothesis that a deficiency of apoptosis in the presence of cagA strains may lead to neoplasia. Additional evidence implicates another strain-specific H. pylori locus, vacA, in apoptosis. VacA encodes a secreted bacterial toxin, VacA, which induces apoptosis of gastric epithelial cells (282,283). Although a vacA gene is present in virtually all strains of H. pylori, the strains vary greatly in the production of vacuolating cytotoxin activity (284). The variation is due to a difference in the vacA gene structure and the resulting toxins produced. Cover and colleagues (284) have shown that VacA purified from a potent strain induced apoptosis in a dose-dependent manner that required acid activation of the toxin and the presence of ammonium. A mutant inactive toxin neutralized the effect of the toxin from the potent strain. The authors conclude that differences in levels of gastric epithelial cell apoptosis among individuals infected with H. pylori may depend on strain variations in VacA structure. A number of other mechanisms related to the bacteria have been suggested to account for the apoptotic activity of H. pylori (259). Of particular relevance is that H. pylori infection stimulates the release of cytokines such as IL-1, -2, -6, and -8, TNF-α, and interferon-γ (285–287). Their effects may be mediated by the TNF receptor family, and H. pylori has been shown to induce the expression of Fas and FasL (288,289). The induction of apoptosis by H. pylori in vitro is related to the ability of the bacteria to induce and activate the Fas/FasL pathway (288–290). Wu and colleagues (291) found that H. pylori sensitized human gastric epithelial cells and enhanced their susceptibility to TRAIL-mediated apoptosis. Urease has also been shown to contribute to apoptosis. Fan and colleagues (292) found that H. pylori urease binds to class II major histocompatibility complex molecules and induces apoptosis in N87 and Kato III gastric epithelial cells. There is additional evidence that LPS contributes to apoptosis (293). H. pylori can activate NF-κB via the IKKα signaling pathway (294). Suppression of NF-κB activation has been shown to sensitize cells to H. pylori–induced apoptosis, suggesting that NF-κB inhibits apoptosis after infection by the bacteria (295,296). In contrast, other studies indicated that NF-κB

activation enhanced H. pylori–induced apoptosis (297,298). Gupta and colleagues (297), for example, showed that direct inhibition of NF-κB abolished H. pylori–induced apoptosis in gastric epithelial cells. Kim and colleagues (299) found that suppression of NF-κB significantly increased caspase-3 activity and apoptosis in H. pylori–infected MKN-45 and Hs 746T gastric epithelial cell lines, as well as in primary gastric epithelial cells. Inhibition was accompanied by a reduction in iNOS mRNA and protein levels. These investigators conclude that NF-κB activation by H. pylori protects cells from apoptosis by up-regulating iNOS. As far as NF-κB is concerned, all results seem possible because Chu and colleagues (300) found that apoptosis in H. pylori–infected cells was not correlated with NF-κB but was related to a reduction in Bcl-2. The variable results in these studies may well be due to the use of different cell types, the measurement of protein levels at different times after infection, and the use of different strains of H. pylori. Nagasako and colleagues (301) found that Smad5, a protein that is involved in differentiation and known to inhibit growth, is induced by H. pylori infection, but only by strains positive for the CagA pathogenicity island. Suppression of Smad5 by interference RNA totally inhibited the induction of apoptosis by H. pylori. Thus, from this study it appears that the up-regulation of Smad5 may be responsible for the induction of apoptosis by H. pylori under some circumstances.

Additional Studies Unlike the case for the small intestine, there have been relatively few studies of apoptosis of normal gastric epithelial cells, other than those involving H. pylori. The only real exception involves NSAIDs. Because of the interest in gastric ulcer and the effects of aspirin, indomethacin, and other NSAIDs on the gastric mucosal barrier, there are a plethora of studies involving damage to the gastric mucosa. Beginning in the late 1990s, investigators started to examine the effect of these substances on apoptosis of gastric epithelial cells. Nonsteroidal anti-inflammatory drugs induce apoptosis in isolated rat gastric mucosal cells, as well as in gastric epithelial cell lines (302–305). Kusuhara and colleagues (302) induced apoptosis with both indomethacin and sodium diclofenac (nonselective inhibitors of COX). A selective COX-2 inhibitor had a weak effect. Apoptosis caused by the NSAIDs could be inhibited by caspase inhibitors, but not by exogenous PGE2. The authors suggest that the induction of apoptosis did not involve the inhibition of prostaglandin synthesis. This conclusion is in disagreement with a host of studies showing that gastric damage caused by a variety of agents can be prevented by exogenous prostaglandin. Zhu and colleagues (303) found evidence that indomethacin-induced apoptosis involved the up-regulation of the c-myc oncogene. Further investigation by the same group focused on protein kinase C (PKC). Indomethacin decreased the abundance of PKC-β1, and overexpression of

366 / CHAPTER 12 this isoform attenuated the apoptotic response of AGS cells (306). They obtained additional evidence that PKC-β1 protected against indomethacin-induced apoptosis through the up-regulation of the cell-cycle inhibitor p21 (waf1/ cip1). Fujii and colleagues (305) examined the effect of indomethacin on the mitochondrial pathway. Indomethacin induced apoptosis and caspase-3 activity in a time- and dosedependent manner. Cytochrome c release was also related to the dose of indomethacin and occurred immediately before and at the same time as caspase-3 activation. Z-VAD-FMK, a caspase inhibitor, inhibited both the increase in caspase-3 activity and apoptosis without altering cytochrome c release. This is one of the few studies that have examined the steps involved in the apoptotic pathway in gastric epithelial cells. A study by Hoshino and colleagues (307) examined the effects of PGE2 on apoptosis caused by the gastric irritant ethanol. This study followed a series of reports by Mizushima and coworkers (308) showing, in 48-hour primary cultures of guinea pig gastric epithelial cells, that gastric irritants induced apoptosis. They also determined that these irritants— ethanol, acid, NSAIDs, and hydrogen peroxide—induced apoptosis through a common pathway that involved mitochondrial disfunction (309). In each case, exogenous PEG2 inhibited irritant-induced apoptosis (310). In the Hoshino study (307), subtype-specific agonists were used to show that EP2 and EP4 receptors were involved in the PGE2-mediated protection from ethanol-induced apoptosis. Activation of these receptors was coupled to an increase in cAMP, and a cAMP analogue inhibited the ethanol-induced apoptosis. The mechanism of inhibition was shown to involve the activation of PKA, but not PI3K. This article also reported experiments showing that PGE2 blocked cytochrome c release and the activation of caspase-9, -8, and -3, which were stimulated by ethanol exposure. This thorough study provides the best description of the apoptotic process in gastric epithelial cells to date. In addition, it is relevant to the large volume of studies documenting gastric mucosal damage after exposure to various irritants. These data are also in agreement with all of the experiments indicating that prostaglandins can prevent gastric mucosal damage.

CONCLUSION The field of apoptosis is relatively new. Although the process of apoptosis or programmed cell death has been known for many years, its importance was only realized beginning in the early 1990s. Since that time the number of manuscripts being published in this field has increased at a logarithmic rate. To my knowledge, there are at least two journals devoted entirely to this field. Many of the published reports involve cancers or the development of neoplasia. I have not included these because they are dealt with elsewhere in these volumes as separate chapters. I have tried to review and summarize the literature pertaining to normal apoptotic processes in gastrointestinal epithelial cells. A great deal has been learned about programmed

cell death in the small and large intestines including the identification of susceptible cell types, the role of apoptosis in maintaining the equilibrium of cell numbers, apoptotic pathways, and the roles of Bcl-2 family proteins and IAPs. For the most part, similar information is sparse or lacking for the gastric mucosa. Studies are underway to identify agents that might prove useful in blocking unwanted intestinal cell apoptosis that occurs during radiation and chemotherapeutic treatment of cancer. Although we have reasonable evidence indicating how apoptosis is induced through the TNF receptor family or by DNA damage, information is lacking indicating the mechanism of protection from apoptosis initiated by other compounds. Additional studies of various signaling pathways that regulate apoptosis such as the PI3K, MAPK, and STAT cascades are necessary to determine their utility in clinical medicine and whether they can provide therapeutic regimens for conditions such as inflammatory bowel disease and cancer. The influence of phosphatases, which often determine the activity of many additional enzymes, remains largely unidentified in apoptotic pathways. Given the general interest in apoptosis, research will continue to expand at a rapid pace. Hopefully, a significant proportion of those studies will be directed at the gastrointestinal tract.

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Molecular Aspects and Regulation of Gastrointestinal Function during Postnatal Development James F. Collins, Liqun Bai, Hua Xu, and Fayez K. Ghishan Ontogeny of Secretory Function, 376 Ontogeny of Digestive Function, 377 Ontogeny of Intestinal Transport, 379 Transport of Macromolecules during the Neonatal and Suckling Periods, 379 Intestinal Transport of Hormones and Growth Factors during the Neonatal Period, 380 Postnatal Development of Transport Function, 381

Ontogeny of Transport Function along the Vertical and Horizontal Gut Axes, 391 Developmental Regulation of Gastrointestinal Function, 392 Acknowledgments, 394 References, 394

The mammalian intestinal tract undergoes dramatic maturational changes during the first few weeks of postnatal life. These changes in digestive and transport function occur under the influence of genetic and neurohormonal regulators that mediate development of the gut. In precocial species such as rats and mice, gut maturation occurs predominantly after birth during the suckling and weaning periods. Conversely, in altricial species such as humans and pigs, significant gut maturation occurs during the late fetal period with limited absorptive and secretory function being present at birth. In all mammals, dramatic genetic and functional changes occurring around the suckling/weaning transition. The neonatal intestine differs from that of adults in that digestive and secretory function is not fully developed, and thus nutrient absorption occurs throughout the small and large intestines and along the entire length of the cryptvillus axis. Many milk-derived factors are now known to have effects on intestinal function and to regulate the expression of genes involved in nutrient transport and immune function. These factors include immunoglobulins, growth

factors and peptide hormones, and antigens such as bacteria. On weaning, the gut epithelium is under the influence of a different set of physiologic regulators and dietary factors; thus, alterations in function rapidly ensue. This chapter reviews the key discoveries that have led to current knowledge and understanding of gastrointestinal function during ontogeny. This chapter discusses secretory, digestive, absorptive, and regulatory aspects of gut maturation and, whenever possible, provides insight into the molecular physiology of experimental findings. Ontogeny is development of a single individual, or a system within the individual, from the fertilized egg to death (1). As observed in several mammalian species, ontogenetic development of the intestine is an organized process driven by genetic and environmental factors that results in an adaptive, functional intestinal tract. Advances in cellular and molecular biology have allowed a better understanding of the mechanisms responsible for the ontogenetic changes of the mammalian intestine. The development of the gastrointestinal tract in the early stage of life is characterized by morphologic and functional changes of the intestinal epithelium. However, there are significant variations in the timing and rate of maturation that depend on the gestational period of mammals. For example, the mouse and rat, which have short gestational periods, show maturation of their gastrointestinal function at weaning. In contrast, intestinal development

J. F. Collins, L. Bai, H. Xu, and F. K. Ghishan: Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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376 / CHAPTER 13 occurs earlier in utero in species with long gestational periods, such as the sheep, pig, and human (2,3). The complete development of gastrointestinal function includes fully developed secretive, digestive, and absorptive functions as guided by genetic factors and influenced by neurohormonal regulation. The maturation of digestive and absorptive functions of carbohydrates, proteins, fats, minerals, and vitamins in the newborn have been determined in relation to the availability of hydrolytic enzymes, such as amylase, glycosidases, lactase, protease, and lipase. The anatomic differentiation of the human fetal gastrointestinal tract occurs by 20 weeks of gestation and resembles that of a newborn. However, secretive and absorptive functions develop at different stages; the gastric and pancreatic secretive processes are only minimal and can only be partially stimulated in the full-term newborn, whereas the intestinal absorptive function is partially developed by 26 weeks gestation. The gastrointestinal epithelium consists of multiple functional cell types, including enterocytes, enteroendocrine cells, goblet cells, and Paneth cells in the intestine, as well as parietal cells, chief cells, and mucous neck cells in the stomach. All four epithelial cell types of the intestine differentiate from common undifferentiated stem cells in the crypt compartment (4). These crypts contain only a small group of proliferating, undifferentiated stem cells that give rise to several cell phenotypes that migrate onto several adjacent villi (5). Enterocytes absorb water, solutes, vitamins, and other nutrients. Enteroendocrine cells secrete peptide hormones. Goblet cells produce a variety of mucins. Paneth cells secrete digestive enzymes, growth factors, and antimicrobial cryptdins. The enterocytes, goblet cells, and enteroendocrine cells differentiate as they migrate up the crypt-villus axis turning over every 5 days, whereas Paneth cells migrate downward turning over more slowly (4). As mature enteroendocrine cells and enterocytes migrate to the tips of the villi, they presumably undergo apoptosis and are extruded into the lumen (6). How stem cells are allocated to differentiate into different cell types at different stages of development is not fully understood. It has been proposed that the development of the gastrointestinal function is dependent on “hardwired” genetic programming coupled with dietary influences as triggered by neurohormonal factors (7). In the past decade, molecular biology techniques have been widely applied to investigate the development of gastrointestinal function. This chapter summarizes the most recent updates in the ontogeny of gastrointestinal function, especially in the developmental regulation of overall gastrointestinal and absorptive function.

ONTOGENY OF SECRETORY FUNCTION Secretion occurs in different segments of the gastrointestinal tract and is an important process of normal gastrointestinal function. Gastrointestinal secretion occurs throughout the gut from salivary glands, stomach, pancreas, small intestine, and colon. Acid secretion, through gastric H+,

K+-ATPase activity, results in acidification of the gastric lumen and creates an environment that promotes protein and bacterial degradation. Bicarbonate secreted by the pancreas neutralizes gastric acid, ensuring proper pH conditions for digestive enzyme function in the intestine. Chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) provides active intestinal secretion. Secretory processes thus are critical for normal gastrointestinal functions, including digestion and absorption. Developmental changes occur in secretory function of the various components of the gastrointestinal tract and are determined by genetic, environmental, and dietary factors. Furthermore, alterations in secretory function, as seen in patients with cystic fibrosis, result in chronic diarrhea and malnutrition, which is commonly observed in children. Normal growth of the human infant is dependent on a well-adapted gastrointestinal system that ensures adequate secretion, digestion, and absorption, as well as protection from infection. The development of gastrointestinal secretory function, particularly in response to dietary and hormonal stimulation, has been studied extensively over the last decade. Gastric acid secretion is a critical step for food digestion. Alterations in gastric acid secretion are seen in neonates in intensive care units (8,9). Parietal cells of the gastric mucosa contain a secretory membrane protein, H+, K+-ATPase, that is primarily responsible for gastric acid production (10). Gastric acid is produced in premature and term infants (11–15). Parietal cells and gastric H+, K+-ATPase are reported to be present at 13 weeks gestation (16) and increased significantly with gestational age (17). Using H+, K+-ATPase β subunit–deficient mice, researchers showed that gastric H+, K+-ATPase is required not only for the normal digestive function, but also for normal development of the gastric mucosa and attainment of the normal structure and composition of secretory membranes (18). Regulation of gastric H+, K+-ATPase during development has been studied predominantly with respect to gastrin. Human newborns have high circulating levels of gastrin, yet they have low acid secretion. Furthermore, exogenous gastrin is ineffective in stimulating acid secretion, despite the presence of anatomically developed parietal cells in newborns. Thus, it is unclear the precise role that gastrin plays in regulation of acid secretion during early postnatal life. Secretion of water and electrolytes by the gastrointestinal tract is central to its physiologic functions. Secretion of bicarbonate and potassium occurs along the entire length of the intestine, with the predominant electrolyte driving fluid secretion being chloride. Chloride is secreted into intestinal lumen via the coordinated action of the basolateral Na+-2Cl−-K+ cotransporter (NKCC1) and the apical CFTR (19). Chloride secretion is accompanied by paracellular and transcellular movement of sodium, and the resulting luminal accumulation of sodium chloride provides the osmotic basis for water movement. Potassium is secreted through apical K+ channels following the electrochemical gradient (20,21). A portion of bicarbonate secretion is also secondary to active secretion of chloride, which recycles into the cell through

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 377 apical anion exchangers in exchange for bicarbonate (22). The secretion of HCO3− by ductal epithelium of the pancreas has several functions. First, it neutralizes the gastric acid and protects the intestinal mucosa; second, it protects the pancreatic-biliary tract from ascending infection. Overall, the mechanism of bicarbonate secretion has been implicated as a complex regulatory process including neural, hormonal, and intracellular signal transduction in ductules and acinar pancreatic cells (23). Developmental change of intestinal secretion of electrolytes has been observed, especially for K+, Cl−, and HCO3−. Suckling rats have high colonic K+ secretion, and this has been shown to be a result of regulation by aldosterone (21). Intestinal Cl− secretion was also found in suckling animals from different mammalian species (24–28). The activity of cyclic adenosine monophosphate (cAMP)– dependent chloride channels and the CFTR transcript has been detected in fetal small and large intestines, with similar expression levels seen throughout prenatal and early postnatal development (29–32). Several studies have suggested that adults are more resistant to bacterial enterotoxin-induced secretory diarrhea. This phenomenon reflects that the development of secretive function is age dependent. In animal studies, the immature intestine is more susceptible than the mature intestine to bacterial enterotoxins (24,33,34). The jejunum of 2- and 3-week-old rats have higher sensitivity for stable toxin (ST) of Escherichia coli than that of the adult (34). Furthermore, the small intestine of 14-day-old pigs was more sensitive to cholera toxin (CT) challenge than that of 14-week-old animals (24). This reduced response to CT with age was found to be partially due to the presence of an antisecretory factor, which naturally inhibits fluid losses in enterotoxigenic diarrhea. CT and ST are members of a larger family of A-B toxins of bacterial (and plant) origin that are composed of structurally and functionally distinct, enzymatically active A and receptor-binding B subunits or domains. The mechanism of CT- and ST-induced diarrhea involves B pentamer–mediated receptor binding of the toxins, entry into a vesicular pathway, and translocation of the enzymatic A1 domain of the A subunit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase (cAMP). The activation of cAMP opens Cl− channels and inhibits Na+/H+ and Cl−/HCO3− exchangers, subsequently causing a sequence of events culminating in life-threatening diarrhea (35). It has been suggested that differential expression of G proteins and ion transporters (e.g., Na+, K+-ATPase and Cl− channels) during development may also alter the neonatal host’s responsiveness. Therefore, the developmental control of microvillous membrane receptors, signal transduction mechanisms, and ion transport systems in the gastrointestinal tract may, in part, contribute to the altered host sensitivity to toxigenic diarrhea of infancy (35). The developmental changes in responsiveness of intestinal secretory function by enterotoxins may include affinity or number of receptors on enterocyte membranes, receptor–effector transduction pathways, and neurohormonal

regulation (36). It has been shown that the number of ST receptors was increased in the brush-border membranes of porcine, murine, rat, and human intestine during development (34,37–39). In a pig model, the increase of the susceptibility during the first week of life and at weaning to ST-mediated diarrheal disease is partially caused by an increase of ST receptor numbers (38). In addition, ST receptor messenger RNA (mRNA) expression was found to be greatest in the perinatal period in the jejunum and ileum, suggesting that the age-dependent changes in ST receptors are transcriptionally or posttranscriptionally controlled, or both (40). However, the immature gut exhibited increased host sensitivity to CT stimulation that was not correlated with initial receptor binding, but was related to decreased Na+-K+-ATPase activity, suggesting that an underexpressed sodium pump may be partially responsible for the high incidence of secretory diarrhea in neonates (33). The signal transduction pathways of receptor–effector also show developmental changes. One study found that the interaction of adenylate cyclase and it receptors on stimulation of CT is significantly greater in preweaned rats than that in weaned rats (41). In addition, the CT-induced fluid secretion was closely correlated with increased adenylate cyclase activities. Thus, increases in adenylate cyclase responsiveness to CT are, at least in part, responsible for the increased incidence of toxigenic diarrhea in neonates. Furthermore, high levels of antisecretory proteins were found in 28-weekold pigs compared with 35-week-old pigs that are capable of inhibiting enterotoxin-induced secretion in older animals (42). These observations suggest that multiple factors are involved in the developmental regulation of intestinal secretion. Moreover, the enteric nervous system appears to modulate intestinal secretion. There are at least two major, final, common neuronal pathways influencing the secretory epithelium of the crypts, cholinergic and noncholinergic. The direct stimulatory effect of CT binding to enterocytes is estimated to cover only one third of the secretory response, and the remainder is induced indirectly through activation of the enteric nervous system. Cholera toxin–induced secretion in the small intestine is accompanied by the release of mucin from goblet cells (43). However, exposing cultured goblet cells in vitro to CT does not evoke mucous secretion (44). These observations indirectly suggest that the CT-induced mucous secretion is mediated by the enteric nervous system. Furthermore, CT-induced secretion can be attenuated by 5-HT, or serotonin (45–47). These findings suggest that the release of toxins activates the enteric nervous system and inflammatory mediators, thus evoking fluid secretion or influencing enterocytes directly, or both (48,49).

ONTOGENY OF DIGESTIVE FUNCTION Expression levels of digestive enzymes in the small intestinal brush-border membrane change dramatically from the neonatal period into adulthood because of dietary adaptation.

378 / CHAPTER 13 This topic has been investigated intensively since the 1970s. Disaccharidases are located in the brush-border membrane of mature enterocytes of the small intestine. The major disaccharidases found in humans are β-galactosidase (lactase) and α-glucosidases (sucrase, isomaltase, and glucoamylase). Lactase hydrolyzes lactose to glucose and galactose. Sucrase hydrolyzes sucrose to fructose and glucose. Isomaltase hydrolyzes the α-1, six bonds of α-limit dextrins to two glucose units. Maltase hydrolyzes maltose to two glucose units. Disaccharidase activities are greatest in the proximal and midjejunum. Glucoamylase activity is distributed evenly throughout the small intestine. Lactase levels continue to increase for 30 cm beyond the ligament of Treitz, and then decrease. This distribution is established in fetal life and continues into adulthood. Milk is enriched with lipids, but it contains relatively low carbohydrate levels. Lactose is the major carbohydrate in the human milk. Lactase activity, which peaks soon after birth, is low before the 12th fetal week and increases to only 30% of perinatal expression levels by the 26th to 34th week of gestation (50). The rapid ontogenic change in lactase activity in the intestine during the early stage of human life clearly demonstrates physiologically relevant dietary adaptation after birth. The lactase levels remain relatively high through 6 months of age, and then they decline at 5 years of age (51). In adults, two phenotypes exist in regard to lactose digestion. Most of the world population is characterized by a loss of lactase activity before adulthood, resulting in adult-type hypolactasia. Therefore, the ingestion of lactosecontaining products will result in osmotic-type diarrhea. The lactase-persistent phenotype is characterized by continued high lactase activity throughout adulthood. The adult-type hypolactasia is inherited in a recessive manner, whereas the persistence of lactase activity is inherited in a dominant manner. Several nucleotide polymorphisms have been described in the promoter region of the lactase gene. Two particular single-nucleotide polymorphisms (SNPs) are tightly associated with adult-type hypolactasia. A C at position− 13910 upstream of the lactase gene is 100% associated, and a G at position −22018 is more than 95% associated with lactase nonpersistence in the Finnish population (52). The functional role of these SNPs has been confirmed in two articles (53,54) demonstrating that the genomic region containing these two polymorphisms possesses enhancer activity. Currently, it is believed that lactase expression in both humans and animals is regulated via a complex spatial and development pattern in the small intestine. A conserved 150-base pair (bp) proximal promoter is important for regulation of lactase expression. This promoter region binds transcription factors such as CDX-2, hepatocyte nuclear factor 1α (HNF-1α), and GATA that are important for regulation of the lactase expression. In humans, all glucosidases are present by the third intrauterine month. An increase in maltase, glucoamylase, and sucrase-isomaltase occurs at the 12th week of gestation, and 70% of adult levels are reached between 26th and 34th

weeks gestation (50,55). Although these developmental changes have been well documented over the past several decades, the molecular mechanisms of how those genes are precisely regulated remain largely unknown, but transcriptional mechanisms have been suggested (56). Indeed, in the mouse and the rat intestine, sucrase-isomaltase is increased by weaning, and a combinatory role of HNF-1α, Cdx-2, and GATA-4 was shown to be involved in the timing and position of the sucrase-isomaltase expression along the small intestine (56). Similar to our advanced understanding of the molecular physiology of digestive enzymes on the intestinal brushborder membrane, there has been a remarkable advance in our knowledge of the events of the ontogeny of islets of Langerhans in the mammalian pancreas. In particular, there has been a dramatic shift from the previously held concept of one secreted hormone per cell type during ontogeny to a wider acceptance of a precursor cell for each of the four types of principal endocrine cell types, which ultimately produce insulin, glucagon, somatostatin, and pancreatic polypeptide (57). The current understanding of ontogenic change of the endocrine pancreas is that there is a series of stages in the differentiation of islet cells, and in one of these stages, they often can secrete more than one hormone (58). Early in development, glucagon and insulin secretion are usually mediated by the same cell (59). Somatostatin and pancreatic polypeptide are secreted together by cells much later in development, but at one point in mouse development, polypeptide YY is present in all four principal islet cell types (60). More recently, developmental biologists have demonstrated that pancreatic development requires complex combinations of transcription factors to control organogenesis and cell differentiation (61–63). In particular, the Pax family of transcription factors is involved in the formation of the pancreas and many other organs. Two of its members, Pax4 and Pax6, play important roles in islet differentiation (64,65). Pax genes encode key regulators, which are involved in the embryonic development of many organs including the eyes, brain, kidney, thyroid gland, immune system, and pancreas. A number of murine and human genetic disorders are linked to mutations in specific Pax genes. Pax genes 4 and 6 are required for development of the mouse pancreas with Pax6 directing the differentiation of A cells (65). Pax4 null mice lack serotonin and somatostatin-secreting cells in the gastric antrum, as well as most endocrine cell types in the proximal small intestine (66). Deletion of Pax6 eliminates glucagon-like peptide-1 (GLP-1)– and GLP-2–expressing cells in the distal intestine (67), gastrin and somatostatin cells in the antrum, and gastric-inhibitory-peptide (GIP) cells in the duodenum (66). In addition, reduced numbers of all four pancreatic islet cell types was seen in Pax6 null mice (62), and pancreatic endocrine cells were absent in Pax4 and Pax6 double knockout mice (65). A new member of the protein tyrosine phosphatase (PTP) family, called PTP-NP (for neural and pancreatic), is a receptor-type transmembrane molecule and an early marker of development (68). Overall, the absence of these endocrine

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 379 cell types in Pax4 and Pax6 null mice indicates that Pax4 and Pax6 play crucial roles in normal pancreatic development. Another regulatory peptide, adrenomedullin, has been found to modulate insulin secretion (69). Adrenomedullin is an almost ubiquitously expressed peptide that is involved in growth, development, and endocrine signaling. It is expressed by islet cells during development (70). This hormone appears first within cells containing glucagon, then eventually within all principal islet cell types, and ultimately only within those cells expressing pancreatic polypeptide (71). Thus, early expression of adrenomedullin is critical for normal islet development. The pancreatic duodenal homeobox gene-1 (Pdx-1) is a master regulator of both pancreatic development and the differentiation of progenitor cells into the β-cell phenotype (72,73). Pdx1 regulates insulin and somatostatin gene expression in the endocrine pancreas (74). The stable expression of Pdx-1 in a glucagon-secreting α cell line is sufficient to induce expression of the β-cell–specific genes insulin, glucokinase, and islet amyloid polypeptide (75,76). The patterns of expression of Pdx-1 in the developing pancreas are maintained throughout development and provide both spatial and temporal contributions to the commitment of endoderm to a pancreatic phenotype (77). Targeted disruption of the Pdx-1 gene in mice results in agenesis of the pancreas (78,79). Notably, a child born without a pancreas was shown to be homozygous for an inactivating mutation in Pdx-1 (80), underscoring the importance of the Pdx-1 transcription factor in pancreatic development.

ONTOGENY OF INTESTINAL TRANSPORT Two important developmental periods must be taken into account when one considers developmental aspects of intestinal transport function: the neonatal/suckling and the weaning/postweaning periods (36). These developmental stages are important because milk-derived factors, hormones, and macromolecules affect physiology of the gut, and there are distinct hormonal changes associated with weaning, when new dietary factors influence gut physiology. The degree of intestinal maturation also plays a major role in differences in absorptive function during ontogeny. Thus, mechanisms and levels of intestinal transport of nutrients and electrolytes vary throughout postnatal development. Furthermore, nutrient transport in the neonatal intestine occurs along the entire crypt-villus axis, whereas only enterocytes along the upper villus have an absorptive phenotype after weaning (81). Changes in transport function during postnatal development can be related to alterations in transporter turnover rates, density of transporters in the plasma membrane of intestinal epithelial cells, or changes in affinity constants and/or expression levels of different transporter isoforms. Changes in transport activity can also be related to several nonspecific factors, such as alterations in mucosal surface area, proliferation and differentiation of epithelial cells, membrane fluidity, and paracellular permeability (36). Plasma membrane fluidity

of intestinal cells changes during ontogeny due to altered cholesterol-to-phospholipid and protein-to-phospholipid ratios and also to changes in the lipid composition (82–85). Furthermore, these changes in the biophysical properties of the membrane can modify the activity of various transporters (86,87). Also during intestinal development in the postnatal period, there is a transient appearance of villus-like structures in the proximal colon. Enterocyte amplification also occurs, in which the absorptive surface area of microvilli increases (88,89), a phenomenon that may be caused by decreased cell turnover rates.

Transport of Macromolecules during the Neonatal and Suckling Periods In general, the neonatal intestinal epithelium is much more permeable than the adult intestine. During early postnatal life, growth factors, immunoglobulins, food antigens, other macromolecules, and bacteria may adhere to the gut epithelium and be absorbed from colostrum and milk. This transport occurs through enterocytes and specialized “M” cells (90), which appear early in fetal development (91,92). This process may be mediated by interaction of milk-derived factors with specific receptors on epithelial cells. Whole proteins derived from dietary sources may also be transported by M cells. The high permeability of the neonatal intestine for macromolecules decreases after birth, but the precise timing of this event is species dependent. In some rodent species and in pigs, transport capacity decreases a few days after birth, whereas in rats and rabbits, transport ceases completely around weaning (93–96). The process of “gut closure,” the dramatic decrease in epithelial permeability that occurs around the suckling/weaning transition, is dependent on epithelial-derived factors and increased pancreatic secretions into the gut lumen (97–99). Transport of macromolecules during the neonatal period may also occur by a nonspecific process, whereby adherent or soluble molecules are moved across the epithelium by vesicular transport. Only molecules adhered to the microvillus membrane are transported across the cells, whereas soluble molecules in the fluid phase are mostly degraded (100–102). This ability of the neonatal gut is due to an apical, tubular system and numerous supranuclear vacuoles, of which distribution and developmental pattern vary dramatically between mammalian species (36). That macromolecules are more likely to adhere to intestinal epithelial cells during the neonatal period (103) cannot be fully explained, although differences in membrane composition are one possible factor. Furthermore, the relative contribution of enterocytes versus M cells in nonspecific transport of macromolecules is unknown, although selective adherence of milk IgGs to M cells has been reported (104). Immunoglobulin transport in the neonatal period is enhanced by protease inhibitors found in colostrum and milk (105,106), which ensure that IgGs escape degradation and bind to their cognate receptors in the intestinal epithelium.

380 / CHAPTER 13 This process is important for the development of passive immunity, a process that is known to be involved in regulating the expression of development-specific genes in the developing intestine (107). Rodents express a specific IgG receptor that selectively binds to the Fc portion of immunoglobulin (the Fc receptor of the neonate [FcRN]), which mediates endocytosis and transport across epithelial cells (Fig. 13-1) (108,109). Fc receptor of the neonate is maximally expressed in the proximal small bowel, with decreasing expression levels in more distal gut segments (97). The FcRN/IgG interaction occurs at the low pH of the duodenum and uptake is mediated by endocytosis (109,110). Transported IgGs are then trafficked within vesicles that are protected from fusion with lysosomes to the basolateral membrane for extrusion into the interstitial space (111). Humans and other precocious mammals are born in a hypoglobulinemic state, and thus absorption of IgGs from the maternal milk is critical for development of passive immunity (112). Studies suggest that IgG may also be transferred by the placenta during late fetal development in humans. However, the intestinal epithelium also expresses IgG receptors in the fetal and neonatal periods, and macromolecular transport continues until after birth, when it gradually diminishes (113–115). Studies suggest that FcRN expression may be transcriptionally mediated by Sp family member transcription factors (116), which are known to regulate many genes during postnatal development (117,118). Furthermore, the neonatal intestine also actively A. Receptor-mediated absorption

secretes IgAs via the activity of the polymeric IgA receptor (PIgR) (119,120) (see Fig. 13-1), and interestingly, PIgR gene expression may be modulated at the transcriptional level by glucocorticoid hormones (121).

Intestinal Transport of Hormones and Growth Factors during the Neonatal Period Several lines of experimental evidence suggest that milkbased hormones and growth factors can survive digestion and are absorbed by epithelial cells of the neonatal, mammalian intestine (Table 13-1). Because gastric acid secretion and pancreatic secretion of proteases is decreased during the neonatal and suckling periods, peptide hormones and other proteins are able to reach the absorptive enterocytes of the proximal small bowel. The principal target of these milk-borne substances seems to be the intestinal epithelium, where they influence proliferation and differentiation of enterocytes (122–126). A small fraction of these absorbed molecules may survive intracellular proteolytic processes and become absorbed into the neonatal circulation; however, the physiologic importance of this observation is currently unknown. In suckling animals, degradation of proteins is low, but it increases dramatically coincident with weaning (126–129). This low degradative capacity facilitates the transport of biologically active substances by the neonatal intestine, as intestinal epithelial cells express B. Transcytotic absorption

C. Secretory component

IgG FcRN IgA PIgR

FIG. 13-1. Immunoglobulin absorption and secretion during neonatal life. (A, B) Two different mechanisms used during the suckling period to transport IgGs across the intestinal epithelium. The receptormediated pathway involves the Fc receptor of the neonate (FcRN) and endocytosis, followed by vesicular trafficking and release across the basolateral membrane. The transcytotic pathway can be observed in some mammals, and it involves nonselective endocytosis, where endocytosed molecules are either partially destroyed or partially transported into the interstitial fluids. (C) IgA secretion is shown, which also occurs at this developmental stage and involves the polymeric IgA receptor (PIgR). IgA dimers bind to the receptor on the basolateral membranes and are endocytosed and transported across the cellular cytoplasm, where the complexes are secreted into the gut lumen. (Modified from Pacha [36], by permission.)

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 381 TABLE 13-1. Transport of milk-derived hormones and growth factors across the gastrointestinal epithelium Milk-derived factor

Physiologic effects

Corticosterone Epidermal growth factor (EGF) Insulin

Hormone shown to be absorbed from formula-fed, glucocorticoid-deficient rats (435). Transport occurs in suckling and weanling rats. Some EGF is degraded during transport, whereas only a small amount is actually transported into the circulation (133,443,444). Orally ingested insulin decreases blood glucose levels in suckling rats (445). Neonatal calves receiving greater amounts of dietary insulin have higher circulating levels of insulin (colostrum vs milk) (446). Insulin-like growth factor may be absorbed in an undegraded form in pigs. Plasma concentration of IGF is greater in pigs fed a diet containing higher IGF levels (colostrum vs milk) (122,447). Orally ingested IGF-I modulates intestinal growth in rats (125) and is absorbed intact (448). Insulin-like growth factor type I is absorbed by a nonsaturable, nonreceptor-dependent mechanism (449). Prolactin is transferred from mother to rat pups and can be detected in the serum of the offspring; it can also be detected in the serum of suckling rats given an oral dose of prolactin (450). Some prostaglandins are absorbed intact and can be detected in the liver of neonatal rats (451,452). Oral administration to rats results in a small amount being absorbed in an undegraded form (453,454). Thyroid-releasing hormone treatment of maternal rats leads to an increase of serum levels in the pups (455), and TSH was shown to be absorbed in a biologically active form (456). Injection (subcutaneous) of neonatal rats with TSH increased serum T4 in both sucklings and weanlings, whereas oral administration had the same effect only in sucklings (457). Mouse pups born to TGF-β knockout mouse heterozygotes have detectable tissue levels of TGF-β, whereas those born to null females do not. Transforming growth factor-β given orally can cross the intestinal epithelium and can be detected in various tissues (458).

Insulin-like growth factor (IGF)

Prolactin Prostaglandins Somatostatin Thyroid-releasing hormone Thyroid-stimulating hormone (TSH) Transforming growth factor-β (TGF-β)

Modified from Pacha (36), by permission.

receptors that mediate transepithelial transport of these molecules across intestinal mucosa (Table 13-2) (130–132). Furthermore, in rat ileum, it has been demonstrated that epidermal growth factor (EGF) and nerve growth factor may be transported by fluid-phase and receptor-mediated mechanisms, but that only small fraction of both peptides actually reach the basolateral membrane for extrusion into the circulation (133,134).

Postnatal Development of Transport Function Most nutrient transporters are first expressed during gestational development and are particularly important during the third trimester when the fetus actively absorbs nutrients from the amniotic fluid. The postnatal ontogeny of nutrient transporters reflects the need to absorb increasing quantities of nutrients that are required for rapid growth and high metabolic activity. The developmental changes in transport capacities are due to several factors: (1) changes in intestinal mass, (2) physicochemical properties of the cell membranes, (3) and types of transporters being expressed and their kinetic properties. Furthermore, colonic absorption of some nutrients occurs in sucklings, a process that may compensate for decreased transport capacity in the small bowel and less developed colonic bacterial fermentation. Total intestinal transport capacity increases with age, predominantly because of increased intestinal mass, but surprisingly, the absorptive capacity for many carbohydrates and amino acids decreases in proportion to intestinal mass (135–138). In some mammalian species, this decline may

be up to 50% of initial values (137,139) and is not attributable to decrease of mucosal surface area (135). The phenomenon is not, however, fully understood, but three possible mechanisms could be hypothesized: (1) changes in transporter expression levels, (2) induction of other isoforms or related transport systems, and (3) alterations in transporter turnover rates. Decreased absorptive capacity could also be related to the smaller proportion of enterocytes that express various active transporter processes during early ontogeny. Ontogenic Regulation of Intestinal Monosaccharide Absorption Sugars are a major source of caloric intake at all life stages. The dietary sources of sugars vary from lactose in milk to complex carbohydrates found in other foods. All these carbohydrates are digested as mainly glucose, galactose, and fructose, before being absorbed in the small intestine (140). The existence of multiple intestinal carbohydrate transporters has been proposed on the basis of kinetic analysis (141,142), and two families of monosaccharide transporters have been identified: the facilitated-diffusion glucose transporter (GLUT) family and the Na+-dependent glucose transporter (SGLT) family. These transporters play a pivotal role in the transfer of monosaccharide across epithelial cell layers in the gut; and overall, several transporters contribute to the absorption of glucose, galactose, and fructose. Lactose in milk is hydrolyzed by lactase into glucose and galactose, and these sugars are absorbed by a Na+-dependent mechanism (143–146). This pathway is mediated by the sodium-dependent glucose transporter 1 (SGLT1), a member of SLC5 gene

382 / CHAPTER 13 TABLE 13-2. Developmental regulation of mucosal receptors of the small intestine Receptor

Developmental stage

Experimental observations

Epidermal growth factor (EGF)

F, S, W, Ad

Glucocorticoid

N, S, W, PW

Insulin

S, PW

Insulin-like growth factor type I (IGF-I)

N, S, W

Epidermal growth factor binding capacity decreases in the perinatal period, and then increases to adult levels by postnatal day 14. A factor present in rat milk decreases EGF binding (459). Epidermal growth factor receptor expression is greater during the gestational period than in suckling and adult rats (425). A higher binding capacity and lower receptor affinity is present after weaning in newborn pigs. Virtually no receptors were observed in suckling pigs (131). Binding capacity increases from fetal life through the suckling period, and then decreases until adulthood in rats (412). Binding capacity reaches a maximum during gestation, followed by a decrease to adult levels within 3 days of parturition of rabbits (460). Decreased binding capacity occurs during development in high- and low-affinity receptors, with no changes in binding affinity. Binding capacity is much greater in crypt cells than in villus cells in rats (130). Binding capacity rapidly declines after birth and remains low during the suckling period, and then increases by postnatal day 21 in pigs. Receptor affinity remains unchanged during ontogenic development (461). Age-dependent increase of binding capacity during suckling and weaning periods in rats, with decreased capacity noted thereafter (462). Receptor messenger RNA expression is significantly higher during fetal development of the rat intestine (463). Age-dependent decrease of binding capacity from early suckling period until adulthood in rats (462). An age-dependent increase in binding capacity has been noted in rats (464). Binding capacity decreases immediately after birth, and then increases within 2 weeks to adult levels, in the absence of alterations in receptor affinity. In the perinatal period, only high-affinity receptors are expressed, whereas later in postnatal development, high- and low-affinity receptors are expressed in rats (465). Receptor messenger RNA expression is not altered during postnatal development in rats (466). An age-dependent increase in binding capacity and expression of both highand low-affinity receptors has been noted in the absence of changes in affinity in rats (467). High- and low-affinity receptors have been described; the binding capacity of both receptors has been noted during ontogeny (468). Receptor binding capacity dramatically increases on weaning in rats, although receptor affinity does not change during postnatal development (469).

S, W, PW, Ad IGF-II

F, W S, W, PW, Ad

Prostaglandin E1 Somatostatin

W, PW, Ad N, S, W, PW, Ad

Thyroid hormone

N, S, W, PW

Vasoactive intestinal polypeptide

S, W, PW F, N, W

Vitamin D

S, W, PW, Ad

Ad, adult; F, fetal; N, neonatal; PW, postweaning; S, suckling; W, weanling. Modified from Pacha (36), by permission.

Sodium-Dependent Glucose Transporter 1 family (147), whose mRNA expression varies during ontogeny (148–151). Furthermore, SGLT1-mediated sugar absorption shows a gradient of activity along the horizontal axis of the small intestine, with greater activity in more proximal sections and decreasing activity distally. SGLT1 expression is high during neonatal life, and it decreases on weaning (138,152); also, because SGLT1 also transports galactose, the developmental pattern of galactose transport is thus identical to that of glucose (135,138). In contrast, fructose absorption is mediated by a Na+-independent, facilitated diffusion pathway involving the glucose transporter GLUT5. The monosaccharide exits enterocytes by facilitated diffusion via the glucose transporter GLUT2. The absorption process mediated by these transporters is regulated by many developmental and dietary factors. The following sections focus only on development-related regulation of these transporters.

Intestinal SGLT1 was cloned and characterized many years ago from rabbits and humans (153,154). SGLT1 is located at the brush-border membrane of the intestinal epithelial cells and is responsible for transporting glucose and galactose across the intestinal brush border (155). Mutations in this transporter gene in humans results in glucose-galactose malabsorption (GGM) syndrome (140,156), a disease characterized by neonatal onset of severe, osmotic diarrhea. Na+dependent glucose uptake in the intestine is modulated during postnatal development. A high glucose absorption rate is observed in the perinatal period, but absorption decreases thereafter (157–159). A similar pattern is also observed in intestinal SGLT1 transporter expression. In human intestine, SGLT1 mRNA expression first occurs in midgestation (~17 weeks) and the expression level increases in adults (148). In rat intestine, SGLT1 expression is higher in both

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 383 weanlings and adults compared with sucklings (138,159). These observations suggest that the functional regulation of glucose absorption during ontogeny may involve transcriptional regulation of SGLT1 gene expression. Some evidence suggests that another Na+-dependent glucose transporter may also be present in the intestinal epithelium. Kinetic studies of sugar absorption in intact animals, and in in vitro preparations and brush-border membrane vesicles, indicate that a low-affinity glucose transporter is also present in the apical membrane of the epithelial cells (160). The presence of a second glucose transporter in the brushborder membrane of enterocytes may explain why less severe symptoms are observed in some GGM patients with deletion mutations in the SGLT1 gene, and why symptoms may be observed in some GGM patients with no mutations in the SGLT1 gene (140). Facilitated Glucose Transporter 5 Fructose absorption is mediated by GLUT5 (140,161,162), which is an apically expressed, transmembrane protein that facilitates fructose movement down a chemical gradient. GLUT5 transporter complementary DNA (cDNA) has been cloned from the human, rabbit, rat, and mouse intestine (148,163–168). Fructose absorption increases dramatically in the small intestine during weaning in rats and rabbits, and to a lesser extent in pigs (135,137,138), and a similar pattern is also observed in intestinal GLUT5 transporter expression. In human and rat intestines, GLUT5 gene expression is very low before weaning and sharply increased at weaning (148,167,169,170). This stimulation in rats and in mouse (163,168,171–174) is linked to an increase of GLUT5 mRNA abundance. These observations then suggest that the functional regulation of fructose absorption during development involves transcriptional regulation of GLUT5 gene expression (175). Furthermore, GLUT5 expression may be precociously induced just before weaning by exogenous fructose administration (171). Fructose uptake in the intestine is also modulated by dietary factors including fructose content of the diet. Facilitated Glucose Transporter 2 As discussed earlier, glucose, galactose, and fructose are transported across the apical membranes of enterocytes by two different transport systems; however, they all share a common transport pathway for extrusion across the basolateral membrane, mediated by GLUT2. This transporter is involved in transporting these sugars out of enterocytes along the existing electrochemical gradients (161,174). GLUT2 is thus a facilitated diffusion glucose transporter expressed at the basolateral membrane of intestinal epithelial cells (174, 176). GLUT2 cDNA has been cloned from human, rat, and mouse (177–179). Similar to GLUT5, the expression of GLUT2 protein is also modulated during ontogeny and regulated by dietary factors. GLUT2 expression decreases during the late suckling period. The expression of GLUT2

protein increases with age; in the perinatal period, expression level of GLUT2 is low, and it reaches plateau after weaning (148,162,180–183). Diets containing high fructose or glucose levels can enhance intestinal GLUT2 expression in neonatal (suckling) rats through increasing GLUT2 gene transcription (184). Ontogenic Regulation of Intestinal Amino Acid Transport The adult mammalian intestine transports amino acids via Na+-dependent and -independent mechanisms, and single amino acids may be transported by multiple transport systems. In the neonatal intestine, Na+-independent lysine and alanine absorption, and Na+-dependent alanine, taurine, and glutamine absorption have been demonstrated (143,145, 185,186).The intestinal transport capacity for various amino acids varies throughout postnatal development and, in general, is greater in the neonatal period than later in life (135,138). These developmental changes may be due to changes in expression levels of individual amino acid transport systems expressed in the intestinal epithelium; this has been directly demonstrated in primary enterocytes and brushborder membrane vesicles (186,187). Na+-dependent amino acid and carbohydrate transport systems also are expressed in the colon during the neonatal period but rapidly decline toward weaning (188–191). The unusual transport ability of the colon for sugars and amino acids may compensate for lower transport capacity in the small intestine of neonates. In support of this supposition, lactose absorption has been detected in the proximal colon of newborns (192,193). However, despite advances in the understanding of intestinal amino acid transport during postnatal development, information about the relative contribution of individual amino acid transporter systems is lacking. Ontogenic Regulation of Intestinal Lipid Absorption Lipid absorption is particularly important in the neonatal period, because lipids constitute a major portion of consumed calories. In addition to absorbing lipids by pinocytosis (194), the neonatal intestine can also absorb fatty acids and cholesterol from dietary sources (195,196). Triglycerides are digested by gastric and lingual lipases (197) in the absence of bile acids, which are low in the neonatal period. Resulting catabolic products, fatty acids and 2-monoacylglycerols, are transported at greater rates in the neonatal intestine compared with adults (195,196,198). The brush-border fatty acid binding protein may also enhance lipid absorption by both diffusional and specific carrier-mediated processes (199), although a passive mechanism(s), independent of fatty acid binding protein, likely also exists (195). Furthermore, intestinal absorption of carnitine, which is an important player in lipid metabolism, is enhanced in the neonatal small intestine (200). It is then a logical assumption that absorbed fatty acids and monoacylglycerides are efficiently resynthesized into triglycerides, phospholipids, and cholesteryl esters, because the activity of reesterification enzymes in the neonatal gut is

384 / CHAPTER 13 comparable with adult levels (201). Expression of apolipoproteins, which are involved in the formation of macromolecular complexes of lipoproteins, is also regulated during ontogeny, with moderate expression levels during gestation (202,203). Postnatal Development of Water Transport Water is absorbed across the small intestinal epithelium in the absence of external driving forces and is secondary to active sodium-chloride transport. In proximal small bowel, sodium and water absorption are glucose dependent. In vivo experiments in suckling rats demonstrated a fourfold decrease in water transport in the small intestine compared with the colon (204), and a decrease in colonic water absorption during early postnatal development (205). Greater water absorption detected in these perfusion experiments may be mediated by paracellular pathways, as supported by the observation that an artificially created osmotic gradient results in water secretion. Furthermore, the connection between active sodium transport and glucose is sodium-glucose cotransport across the apical membrane of enterocytes by the Na+-glucose symporter, SGLT1. Na+ that enters cells with glucose is pumped out across the basolateral membrane by the Na+, K+-ATPase pumps, and glucose passes out across the basolateral membrane passively via GLUT2, with the net result being that glucose and sodium are transported across the epithelium. However, the coupling among Na+, glucose, and water transport is less well understood. It is commonly thought that Na+ transport increases the local osmotic pressure in the lateral intercellular spaces, and that this, in turn, generates osmotic water flow across the epithelium. Some work, however, suggests that water is cotransported together with Na+ and sugar through SGLT1 (155), but the physiologic importance of this observation is unknown. The gastrointestinal tract, like many other cells and tissues, also expresses water channels called aquaporins, with at least six aquaporin isoforms being expressed in the digestive system (206). Aquaporin-1 (AQP1) is widely distributed in endothelial cells of capillaries and small blood vessels, as well as in the central lacteals in the small intestine. Aquaporin-1 is also present in the duct system in the pancreas, liver, and bile duct and in colonic crypts (207). Aquaporin-3 is mainly expressed in the epithelia of the upper digestive tract, from the oral cavity to the stomach, and in the lower digestive tract, from the distal colon to the anus. Aquaporin-4 has been shown to be expressed in the basal membrane crypt cells and in gastric parietal cells (206), but its function in these tissues is currently unknown. Aquaporin-5 is expressed in acinar cells of the salivary, pyloric, and duodenal glands. In addition, the recently cloned AQP8 has been found to be strongly expressed in enterocytes (208,209), although evidence demonstrates that AQP8 null mice do not have an intestinal water transport defect (210). Aquaporin-8 is also expressed in salivary glands, pancreas, and liver, whereas AQP9 has been detected in the liver and intestinal goblet cells. Aquaporins have

important roles in the digestive system, such as the participation of AQP5 in saliva secretion, as demonstrated by the studies on AQP5 null mice (206). Currently, little is known regarding the developmental pattern of AQP expression in the intestine, even though remarkable changes in AQP expression have been reported in the developing kidney (211). In addition, water transfer across the digestive epithelia seems to occur not only via aquaporins, but also via other transporter or channel systems. Ontogenic Development of Sodium Chloride Absorption Ion and fluid transport processes undergo ontogenic changes in both the small and large intestines. In neonatal mammals, the colon appears to have a significant role in absorption of water and ions and in maintenance of normal fluid and electrolyte homeostasis. This role may be extremely important during early postnatal growth, when the small intestinal transport functions are not fully developed. Overall, the gastrointestinal tract processes about 9 liters water and 800 mEq Na+ daily in adulthood. Thus, a major function of the gastrointestinal tract is to absorb virtually all water and Na+ to maintain overall body water and Na+ homeostasis. Increased water and NaCl transport occurs early in postnatal development; high Na+ absorption occurs in the neonatal period and decreases later in postnatal life in the rat, rabbit, and human colon (205,212,213) and in the rat small intestine (36). The Na+ absorptive pathway that predominates in the colon is mediated by amiloride-sensitive Na+ channels (213–215), which gradually increase in abundance from fetal life through the suckling period (95,216,217) after which time they decline in abundance. Colonic Na+ transport persists until adulthood in rabbits (213), but in rats, it ceases after weaning (214,215) and is replaced by Na+-H+ exchange (36). In pigs, Na+ absorption changes from an electroneutral mechanism in the perinatal period to an electrogenic process later in postnatal development (218). Na+-H+ exchange activity increases during development and reaches maximal values after weaning (219–221). Three Na+-H+ exchanger (NHE) isoforms, NHE2, NHE3, and NHE8, are expressed in the mammalian small intestine, and the expression NHE2 and NHE3 is developmentally regulated (219,220,222). A functional coupling exists between Na+-H+ and Cl-HCO3 exchangers in the small intestinal epithelium, but the ontogenic expression pattern of the two transport systems are opposite to one another (223). As has been hypothesized for NHE activity in the developing gut, ontogenic changes in Cl-HCO3 exchanger expression may be transcriptionally mediated (224). Another colonic Cl transport system, which is unresponsive to luminal Na+, also has been reported (189,213). Chloride may also be transported by Cl channels and the NKCC1 in the neonatal intestine (32). In addition, the mucosal electrical gradient generated by active Na+ ion transport promotes diffusion of Cl across the epithelium via alternative mechanisms that may include transcellular- and paracellular-mediated pathways.

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 385 Sodium-Hydrogen Exchangers The sodium absorptive process in the small intestine involves carrier-mediated pathways. The NHEs play an integral role in electroneutral sodium absorption in the mammalian intestine. To date, nine NHE isoforms (NHE1-9) have been identified, and NHE isoforms 1, 2, 3, and 8 are known to be expressed in the mammalian intestinal epithelium. These NHEs have different tissue distribution, membrane localization, inhibitor sensitivity, and physiologic regulation (225), and they show 25% to 70% amino acid sequence identity between isoforms (226). NHE1 is expressed on the basolateral membrane of intestinal epithelial cells throughout the gastrointestinal tract and plays an important role in cell volume and intracellular pH (pHi) regulation (225). Loss of NHE1 function in knockout mice results in growth retardation, ataxia, seizures, and altered intracellular pH in pancreatic acinar cells (227,228). Because NHE1 does not contribute to the intestinal Na+ absorption process, it is not discussed here in detail. NHE2 protein is expressed on the apical membrane of enterocytes and is involved in transepithelial Na+ absorption in the mammalian gastrointestinal tract (225). Knockout of NHE2 in mice leads to abnormal parietal cell viability and enhanced, compensatory NHE3 expression in the colon (229,230). Intestinal NHE2 expression is modulated during postnatal development. NHE2-mediated Na+ uptake in rat jejunum is lowest in suckling rats, higher in weanling and adult rats, and still higher in adolescent rats (220) (Fig. 13-2). A similar pattern is also seen in the NHE2 protein and mRNA expression; NHE2 protein levels are lowest in sucklings and higher in weanling and adult rats (fourfold to sixfold increase), whereas NHE2 mRNA is lowest in suckling rats and higher after weaning (threefold to fivefold increase) (219). The mechanism of this ontogenic regulation on NHE2 function is thought to be at the transcriptional level; an approximately twofold greater transcription rate was observed in adolescent rats compared with suckling rats by nuclear run-on analyses (219).

Epidermal growth factor has shown to enhance Na+ absorption in gastrointestinal tract. In isolated rat hepatocytes, EGF treatment also significantly increases the pHi recovery rate (recovery from an intracellular acid load; a measurement of NHE activity) by 2.4-fold (231). In isolated rat enterocytes, administrating EGF (200 ng/ml) stimulates Na+-H+ exchange activity by 1.8-fold (232). In in vivo studies, EGF treatment increases jejunal NHE2 activity by almost twofold in suckling rats. The increase of NHE2 activity correlates with the increase of NHE2 mRNA abundance. Epidermal growth factor administration has no effect on NHE3 gene expression in these young rats. In vitro studies show that EGF stimulates pHi recovery in cultured rat intestinal epithelial cells by twofold, and a similar induction is observed in NHE2 gene promoter activity (233). These observations suggested that the effect of EGF on NHE gene expression in suckling rat jejuna is isoform specific, only for NHE2 but not NHE3, and this regulation is mediated by transcriptional activation of NHE2 gene expression. The NHE3 protein is also apically expressed NHE in intestinal epithelial cells. It is involved in transepithelial Na+ absorption in the mammalian gastrointestinal tract (225). Knockout NHE3 mice exhibit a moderate to severe absorptive defect in the intestine and mild diarrhea (229,234,235). As a key transporter involved in electroneutral Na+ absorption in the small intestine, NHE3 gene expression is regulated by many factors, and like intestinal NHE2, NHE3 function, and gene expression, it is also subject to developmental regulation. At the functional level, NHE3-mediated Na+ uptake in rat jejuna changes as follows: suckling = weanling < adult << adolescent rats (220) (see Fig. 13-2). At the protein level, NHE3 expression is lowest in suckling rats and increased up to sevenfold after weaning, whereas NHE3 mRNA expression is also lowest in suckling rats and increased approximately twofold after weaning (219). The ontogenic regulation of NHE3 expression likely involves both transcriptional and posttranscriptional mechanisms. The increase in rat small intestinal NHE3 expression after weaning coincides with a surge in levels of endogenous

2w

A

NHE activity in rat ileum Na+ nMol/mg protein/10 sec.

Na+ nMol/mg protein/10 sec.

NHE activity in rat jejunum 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 3w

6w

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 2w

3-4 mo.

3w

6w

B

FIG. 13-2. Sodium-hydrogen exchange (NHE) activity in rat small intestine. Brush-border membrane vesicles were purified by the Mg++ precipitation method from rats and pH-dependent uptake of radiolabeled sodium was assayed. (A) Sodium-hydrogen exchange activity in jejunum increases with maturation, and then declines into adulthood. (B) Sodium-hydrogen exchange (NHE) activity in ileum increases with maturation. The lowest activity is seen in suckling rats, and the highest activity is seen in adolescent rats. 3-4 mo., 3 to 4 months old; 2w, 2 weeks old; 3w, 3 weeks old; 6w, 6 weeks old. (A: Modified from Collins and colleagues [220], by permission. B: Modified from Kikuchi and colleagues [221], by permission.)

386 / CHAPTER 13 glucocorticoid hormones (236). In fact, studies have demonstrated glucocorticoid regulation of intestinal NHE3 gene expression in an age- and region-specific manner. In suckling rats, the responsiveness to methylprednisone treatment is observed only in the proximal, but not in the distal, small intestine. NHE3-mediated Na+ absorption is also increased by threefold, together with a twofold to threefold increase in NHE3 protein and a twofold increase in NHE3 mRNA expression (220,237). Conversely, in adult rats, the stimulatory effect of methylprednisone is observed only in the distal, but not in the proximal, small intestine, where NHE3mediated Na+ absorption is increased together with NHE3 protein and mRNA levels (237,238). These observations suggest that the effect of glucocorticoids on NHE3 expression is most likely due to genomic effects mediated by the glucocorticoid receptor. NHE8 protein has also been shown to be expressed on the brush-border membrane of intestinal epithelial cells. The function of NHE8 has not been fully defined, although it may contribute to intestinal Na+ absorption early in life, based on its protein localization and gene expression pattern (239). In the proximal small intestine, NHE8 expression is also regulated by development, but opposite to the expression pattern of NHE2 and NHE3. NHE8 protein expression is highest in weanling rats and ~56% lower in adults, whereas NHE8 mRNA abundance is similar in suckling and weanling rats, but twofold less in adult rats (239). Potassium Absorption K+ homeostasis is an important physiologic function, and it is maintained by K+ transport across the renal and intestinal epithelia and via regulation by hormonal factors and movement between extracellular and intracellular compartments. Na+ is extruded and potassium transported into enterocytes across the basolateral membrane of intestinal epithelial cells by the Na+-K+-ATPase, which is more strongly expressed in sucklings and weanlings than in adults (215, 240–242). The developmental increase in Na+-K+-ATPase activity is paralleled by mRNA expression for α- and β subunits of this protein, with expression levels decreasing toward adult levels around weaning (242,243). Although Na+-K+-ATPase activity and the number of ATPase molecules increase during development, total functional protein activity surprisingly is not altered, suggesting that additional regulatory mechanisms are involved (244). This ATPase plays a major role in intestinal potassium homeostasis as described later. The intestinal epithelium actively absorbs potassium from the body fluids, followed by secretion into the gut lumen; thus, the intestine plays a major role in overall body potassium homeostasis. K+ secretion has been documented in rat jejunum and ileum (36) and in pig and human colon (212,245), whereas absorption was detected in other experimental situations (246,247). These contradictory results are most likely related to methodologic differences used in the different studies, but it is likely that both processes do occur and are of physiologic importance. Additional studies

demonstrated active K+ absorption via H+-K+-ATPase in both the small (248) and large intestines of neonates (249,250). Furthermore, it was demonstrated that active K+ absorption and H+-K+-ATPase activity are higher during the neonatal period (249,250). Calcium Absorption Intestinal Ca2+ absorption in mammals involves two parallel processes: paracellular, nonsaturable movement through tight junctions between enterocytes, and transcellular transport, which is saturable and 1,25(OH)2 vitamin D3 dependent. In adult mammals, the paracellular pathway predominates under conditions of high calcium intake, whereas the transcellular process is more important under conditions of low calcium intake. In newborn rats, calcium absorption seems to be dependent on both pathways, but a paracellular, 1,25(OH)2 vitamin D3–independent pathway is predominant (251,252). The nonsaturable diffusional pathway also functions as the only absorptive pathway for Ca2+ in preterm, low-birth-weight infants (253). The appearance of saturable active Ca2+ absorption is associated with a substantial increase in the capacity for other steps involved in 1,25(OH)2 vitamin D3–dependent, transcellular Ca2+ absorption, including movement across the apical membrane of enterocytes along a steep electrochemical Ca2+ gradient (mediated by transient receptor potential channel isoform 6 [TRPV6]) (254–256), translocation through the cell (mediated by the intracellular calcium binding protein calbindin-D9k) (257–259), and extrusion at the basolateral membrane against an electrochemical gradient by plasma membrane Ca2+-ATPase (259–262). Many of the proteins responsible for intestinal calcium transport have been identified (as described earlier) (257); however, to date, expression patterns of these genes during postnatal development have not been determined. Phosphate Absorption Phosphate is one of the most important minerals in the body; it is a constituent of bone and teeth, and it plays an important physiologic role in the regulation of acid–base balance. Phosphate is found primarily in bone matrix (85%), whereas the remaining phosphate is divided between intracellular (14.5%) and extracellular fluids (<0.5%). In intracellular fluids, phosphate is a component of nucleotides (DNA and RNA), high-energy molecules (e.g., ATP), and metabolic intermediates. In extracellular fluids, phosphate is present in its inorganic form and serves as a buffer for H+. About 90% of the phosphate in plasma is free, whereas the remaining 10% is bound to serum proteins. The phosphorus level in blood in humans is between 3.0 and 4.5 mg/dl. Phosphate absorption in the small intestine involves transcellular and paracellular pathways. The paracellular pathway appears to be dependent on the magnitude of the electrochemical gradients across the intestinal epithelium (263) and on dietary inorganic phosphate (Pi) intake levels; it predominates under conditions of high Pi intake. Meanwhile, the transcellular pathway involves a carrier-mediated process and

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 387 is most important when dietary Pi intake levels are low. Early studies showed that active transport of Pi through the apical membrane of small intestinal epithelial cells is coupled with sodium in human, rat, and rabbit (263–268). Other studies showed that Pi is absorbed predominantly by secondary active Na+-dependent transport in a stoichiometry of 2Na+:1Pi, and it is now known that this process is mediated by the type IIb sodium-phosphate (NaPi-IIb) cotransporter (as discussed in detail later) (269–271). In agreement with the increased requirements of neonatal mammals for phosphate, the capacity of intestinal phosphate transport decreases during development (Fig. 13-3). Furthermore, sodiumphosphate cotransport activity is restricted to the duodenum, jejunum, and proximal ileum in suckling rats, and only to the duodenum and proximal jejunum in adult animals (271). For decades, the molecular identity of proteins involved in intestinal phosphate absorption was not known. However, beginning in 1998, the intestinal sodium-coupled phosphate transporter (NaPi-IIb) gene was cloned from mouse and human (270,272,273). The NaPi-IIb protein has moderate homology with the renal sodium-phosphate cotransporter (NaPi-IIa). The NaPi-IIb cotransporter is expressed at the apical membrane of intestinal epithelial cells (as well as in numerous other tissues) and is responsible for phosphate absorption in the intestine. Pi absorption across the epithelial cells (transcellular pathway) in the small intestine is modulated by many factors including age, dietary Pi content, hormones (vitamin D, estrogen, glucocorticoids, EGF). Here, the focus is on developmental regulation of this key intestinal transporter. Phosphate absorption in the small intestine decreases with age. The highest Pi uptake rate is observed in the suckling period, with the lowest uptake rate observed in the adult. This pattern has been reported in several species including rat, mouse, pig, and rabbit. In pigs, sodium-dependent phosphate uptake in intestinal brush-border membrane vesicles is greater in newborn piglets and decreased ~66% after weaning (274). In mouse, Na-dependent Pi uptake is greatest

in sucklings, then decreased ~85% at weaning, and further reduced ~94% at 8 weeks and 8 to 9 months of age (275). In rat, Na-dependent Pi uptake in suckling rats is significantly greater (threefold) than in adult rats (276). And finally, in rabbits, sodium-phosphate cotransport is greatest in sucklings and decreased 87% in 3-month-old adults (271). The functional expression pattern of Pi absorption correlates well with the NaPi-IIb expression pattern in the small intestine. In mouse and rat, the expression levels of NaPi-IIb protein and mRNA are at the highest levels at the suckling stage, with NaPi-IIb protein and mRNA expression being threefold to fivefold higher than in adults (275,276). Furthermore, it is a well-known fact that a low-phosphate diet (LPD) stimulates intestinal sodium-coupled phosphate absorption. An LPD increases intestinal Na+-dependent Pi transport by ~3-fold in adult rats and 2.6-fold in adult mice (277,278). The molecular mechanism of LPD on intestinal Pi absorption involves regulation of NaPi-IIb expression at transcriptional or posttranscriptional levels, or both. An LPD stimulates intestinal Pi absorption in mice by enhancing NaPi-IIb mRNA and protein synthesis (279–282). An LPD stimulates intestinal Pi absorption in rats by stimulating apical NaPi-IIb protein expression, but not NaPi-IIb mRNA expression (280). The effect of LPD on Pi absorption in mice is independent of the vitamin D receptor, and may thus involve a nongenomic action of 1,25 (OH)2 vitamin D3 (279). In metabolic acidosis, the body accumulates excessive protons and acids, and thus the body requires more phosphate for buffering of the body fluids. In a rat model of metabolic acidosis, a decrease in renal NaPi-IIa expression and renal phosphate reabsorption were observed (283). A similar result was also observed in a mouse model of metabolic acidosis, where urinary fractional Pi excretion was increased twofold, whereas serum Pi levels remained normal. Interestingly, intestinal Na+-dependent Pi transport activity in these mice is increased 20-fold. Together with functional changes, the abundance of NaPi-IIb protein is induced approximately fivefold, but no change is detected in NaPi-IIb mRNA expression (284).

Pi absorption in mouse jejunum

Pi absorption in rat jejunum

2.0 1.5 1.0 0.5 0.0

A

120 Pi nMol/mg protein/10 sec.

Pi nMol/mg protein/10 sec.

2.5

2w

3w

8w

100 80 60 40 20 0

8-9 mo.

2w

3-4 mo.

B

FIG. 13-3. Sodium-dependent phosphate absorption in mouse and rat jejunum. (A) Sodiumdependent phosphate cotransport (NaPi) activity in mouse jejunum decreases with maturation: the greatest activity is seen in suckling rats, and then significantly decreases after weaning. (B) Sodiumdependent phosphate cotransport activity in rat jejunum decreases with maturation: the greatest activity is seen in suckling rats, and then significantly reduces in adult rats. (A: Modified from Arima and colleagues [275], by permission. B: Modified from Xu and colleagues [276], by permission.)

388 / CHAPTER 13 1,25(OH)2 vitamin D3 is a potent regulator of calcium and phosphate homeostasis, and it stimulates intestinal phosphate absorption via its effect on the sodium-coupled phosphate absorption process. Administration of 1,25(OH)2 vitamin D3 increases Na+-dependent Pi uptake by twofold in rabbit duodenum (285) and in suckling and adolescent rats (286). Similar results were observed in 1,25(OH)2 vitamin D3–treated adult rat jejuna (287). The mechanisms of 1,25(OH)2 vitamin D3 on intestinal Pi absorption involve transcriptional and posttranscriptional regulation of NaPiIIb expression, depending on the developmental stage of the animals. In adult rats and mice, 1,25(OH)2 vitamin D3 enhances intestinal sodium-dependent phosphate absorption by increasing the apical expression of NaPi-IIb transporter protein (276,280,282). In suckling rats, however, 1,25(OH)2 vitamin D3 stimulates intestinal sodium-dependent phosphate absorption by increasing NaPi-IIb mRNA and, concomitantly, protein expression (276). Endogenous glucocorticoid levels are low during early life and increase dramatically on weaning (288,289). Glucocorticoids are well known to reduce inhibit intestinal Pi absorption. In suckling rabbits, Na+-dependent Pi uptake is reduced 50% by methylprednisolone administration (290). Similar results were also seen in rat studies (291). The effect of glucocorticoids on intestinal Pi absorption is more significant during the neonatal period, with methylprednisolone administration decreasing intestinal Na-dependent Pi uptake by 70% to 83% in suckling and weanling mice, whereas no changes were observed in older animals. At the protein expression level, glucocorticoid treatment decreases apical NaPi-IIb abundance by fourfold in suckling mice, whereas NaPi-IIb mRNA abundance decreases more than threefold in suckling and weanling mice (275). Thus, the mechanism of glucocorticoid action on intestinal Pi uptake in suckling age involves inhibition of NaPi-IIb cotransporter expression in neonatal mice. Epidermal growth factor is a 53-amino-acid polypeptide that is secreted predominantly by salivary glands, with lower levels excreted by kidney and many other tissues. Epidermal growth factor receptors are present along the intestinal tract (292). As a growth factor, EFG has broad effects on cell division, DNA synthesis, tissue proliferation, cellular differentiation, and electrocyte and nutrient absorption (293,294). Epidermal growth factor reduces renal Pi uptake by inhibiting NaPi-IIa cotransporter mRNA and protein expression (295). Furthermore, in patients with intestinal ischemia injury, serum Pi levels (296) and EGF production and utilization are increased (297). These observations suggest that EGF may play an important role in Pi homeostasis by regulating renal and possibly intestinal Pi absorption. Studies on NaPi-IIb transporter expression show that NaPi-IIb mRNA abundance is decreased 50% by EGF administration in suckling rats and in human intestinal epithelial cells (Caco-2). This reduction may be due to inhibition of human NaPi-IIb promoter activity (298). The EGF response element has been identified as -AACTGG-, which is located between −739 and −734 bp of the human NaPi-IIb gene promoter. The trans-acting

factor that interacts with the EGF response element at the human NaPi-IIb gene promoter has been identified as the c-myb protein, a nuclear protein involved in regulation of cell growth, differentiation, and apoptosis (299). Estrogen is another important physiologic regulator of calcium and possibly phosphate homeostasis. Estrogen plays important roles not only in the regulation of intestinal Ca2+ absorption, but also in the maintenance of bone mineralization (300–302) and 1,25(OH)2 vitamin D3 activation (301, 303–306). Studies have shown that estrogen also plays a role in modulating intestinal Na+-dependent Pi uptake. In female adult rats, estrogen treatment increased Na+-dependent Pi absorption by ~45%, NaPi-IIb protein expression by ~75%, and NaPi-IIb mRNA abundance by ~50%. The activity of the human NaPi-IIb gene promoter also increased by ~36% after estrogen treatment (307). Absorption of Trace Mineral Elements Mammals have a greater absorptive capacity for many trace minerals during the neonatal period, which is likely because of the need for sufficient amounts of these key ions during peak growth and development. Trace minerals are easily absorbed from colostrums and milk (308). Kinetic studies have demonstrated that both a passive, linear and carrier-mediated processes are involved in the absorption of trace minerals in the neonatal intestine. Specific carriers have been identified over the past several years for iron (309), copper (310), zinc (311), and magnesium (312), but not for manganese (313). Interestingly, many of these metal ions can also be transported by a recently discovered iron transporter, divalent metal transporter 1 (Dmt1) (314), but whether this occurs in vivo is not known. Copper absorption is enhanced in the neonatal period, likely mediated by passive, nonsaturable transport localized predominantly in the ileum (310,315), which is replaced by a saturable pathway in more proximal gut segments later in life. Intestinal copper transport may be mediated by a recently identified copper transporter 1 (Ctr1) (316–318), but its developmental pattern of expression has not been described. The onset of saturable copper transport correlates with increased expression of metallothionein, which is an intracellular regulator of copper and zinc homeostasis (310,319). Similarly, iron absorption is also increased early in life, even in the setting of high body iron levels (309,320,321). Increased iron absorption is due, in part, to enhanced transport in the developing jejunum and ileum, whereas iron transport then occurs predominantly in the duodenum and proximal jejunum in adulthood. Furthermore, intestinal zinc transport has been unraveled at the molecular level with many intestinal zinc transporters recently identified (322), but their expression levels during ontogeny and their relative contribution to total intestinal zinc transport have not been determined. Intestinal iron absorptive transport systems are known to be induced by iron deprivation. This process plays a pivotal role in overall body iron homeostasis, and most known physiologic regulators of iron homeostasis alter gut

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 389 absorption (323). Knowledge of the molecular mechanisms of transepithelial iron transport has increased dramatically with the discovery of several of the key players (324–326). These include the brush-border–associated proteins duodenal cytochrome b (Dcytb) and divalent metal transporter 1 (Dmt1), which allow iron transport into intestinal epithelial cells. Other proteins are involved in iron extrusion into the interstitial space, namely hephaestin and iron-regulated gene 1 (Ireg1). Ferritins are iron-sequestering proteins found in the cytoplasm of enterocytes, and developmental changes in their expression have been reported (327). Although the developmental patterns of expression of most of these genes have not been studied extensively, their inducibility by iron deficiency across several postnatal developmental stages has been investigated. Collins and colleagues (328) used an oligonucleotide microarry approach to characterize gene expression patterns in the duodenal mucosa of iron-deprived rats over the course of rat ontogeny. These authors found strong induction of Dmt1, Dcytb, and transferrin receptor 1 (Tfr1) in suckling, weanling, adolescent, and adult rats. Other known iron transport protein-encoding genes were not as strongly or consistently regulated. Furthermore, induction of other novel genes potentially associated with intestinal metal iron homeostasis was reported, including the Menkes copper ATPase (Atp7a), metallothionein (Mt1), lipocalininteracting membrane receptor (Limr), and the sodiumdependent vitamin C transporter 2 (Svct2) (Table 13-3) (328). Interestingly, many of these known and novel genes were also similarly regulated in the jejunal gut segment. These data overall suggest that some aspects of intestinal

iron transport are similar across several developmental stages, including the neonatal period. Iron in milk is bound to lactoferrin, transferrin, or both, depending on which mammalian species is being considered (308), and it has been suggested that these interactions enhance iron transport in the neonatal period (329,330). Putative lactoferrin receptors have been identified in the fetal and neonatal human small intestine (331), whereas transferrin receptors are present in the apical membranes of enterocytes in the rat small intestinal epithelium (332). Putative brush-border membrane lactoferrin receptors are expressed at the highest levels in sucklings (333). Even though iron bound to lactoferrin is transported into intestinal epithelial cells (334), it is unclear whether this process plays an important role in developmental changes seen in iron uptake capacity (320). Ontogenic Regulation of Intestinal Transport of Water-Soluble Vitamins Water-soluble vitamins are essential micronutrients for the body, and they play critical roles in maintaining normal metabolism, energy, differentiation, and growth. These vitamins can not be synthesized by humans and mammals; therefore, they have to be obtained from exogenous sources via the intestinal absorptive processes. Exogenous sources of these vitamins are dietary sources and bacterial sources, whereby vitamins are produced by the normal microflora of the small and large intestines. Vitamins are transported in the intestine via a saturable, nonlinear process (when dietary

TABLE 13-3. Genes induced in rat duodenum at five different postnatal ages by dietary iron deprivation Gene name

Gene symbol

Aliases/Biological function

Divalent metal ion transporter 1, +/− IRE Duodenal cytochrome B (highly similar to mouse) Duodenal cytochrome B (same chromosomal region) Transferrin receptor Metallothionein 1 and 2 Tripartite motif protein 27 (similar to mouse) ATPase, Cu++-transporting, α polypeptide (Menkes) Divalent metal ion transporter 1, + IRE Metallothionein Glycerol-3-phosphate acyltransferase (similar to mouse) Factor-responsive smooth muscle protein Pyruvate carboxylase Phosphoglucomutase-related protein (same chromosomal region) Calponin 3, acidic Glutathione peroxidase 2 (gastrointestinal) Lipocalin-interacting membrane receptor (similar to mouse and human) Gap junction protein, β2 Retinoblastoma binding protein 7

DMT1 CYBRD1 CYBRD1 TFRC MT1/2 TRIM27 ATP7A DMT1 MT1 GPAM SM20 PC PGM-RP

Divalent metal ion transport Rat sproutin; ferric-reductase Ferric-reductase Iron ion homeostasis; transferrin-iron endocytosis Metal ion binding Oncogenesis; formation of cellular compartments Copper ion efflux from enterocytes Divalent metal ion transport Metal ion binding Glycerolipid synthesis Apoptosis; protein metabolism; oxidoreductase Gluconeogenesis; metabolism; lipid biosynthesis Acidulin; cell adhesion; carbohydrate metabolism Actin/Calmodulin binding Defense against oxidative stress Lipocalin receptor; possible cytoplasmic iron delivery Connexin 26; intercellular communication Interaction with histone deacetylases; corepressor complex Regulation of tissue calcification Cell adhesion; cell-surface–mediated signaling Ascorbic acid (vitamin C) transport

Progressive ankylosis (highly similar to mouse) Integrin, α6 Sodium-dependent vitamin C transporter 2

CNN3 GPX2 LIMR GJB2 RBBP7 ANK ITGA6 SVCT2

Ages studied include 8 days (suckling), 21 days (weanlings), 6 weeks (adolescent), and 12 and 36 weeks (adults). Modified from Collins and colleagues (328), by permission.

390 / CHAPTER 13 intake levels are low or normal) and by passive, linear diffusion (with high-intake levels). Saturable transport occurs via the action of specific transporters by facilitated diffusion or ATP-driven active transport (335). The ontogenic patterns of vitamin absorption have been investigated for only a few vitamins, but it is known that the developmental patterns are not identical for all vitamins. The intestinal transport of folate and riboflavin are carrier-mediated, Na+-dependent processes, the capacity of which decreases after the neonatal period (336,337). A large portion of folate in milk is bound to a high-affinity folate-binding protein that enhances folate uptake probably through a specific receptor on the membranes of enterocytes (337). Unbound folate is absorbed at much lower rates, by an endocytotic process that has greater capacity in the ileum than jejunum (338). Similarly, riboflavin absorption is also enhanced by milk factors (339). Biotin absorption has a different developmental pattern; adult rats absorb biotin preferentially in the jejunum, whereas suckling rats absorb biotin predominantly in the ileum. Furthermore, the nonsaturable pathway decreases in jejunum and ileum during postnatal development, and the capacity of saturable, carrier-mediated transport increases in the jejunum and decreases in the ileum (340,341). Milk-derived factors also strongly promote the transport of cobalamin (342). Cobalamin absorption occurs in all small intestinal gut segments and exhibits saturation kinetics. The intrinsic factor (IF), which promotes cobalamin absorption in the adult intestine, is distinct from milk factors that affect cobalamin absorption. The IF enhances transport only in the ileum and is more effective in weanling animals. In contrast, milk-derived factor(s) are more effective in promoting transport in suckling rats where IF/cobalamin receptor activity is low (342,343). Ontogenic development of IF and IF/cobalamin receptor activity is maximal in rats around weaning (343), and this timing correlates with enhanced production of IF in the stomach (344). In contrast, in human intestine, IF/cobalamin receptors are expressed in early gestation (345) and fetal parietal cells produce IF (346). Vitamin C is present in its reduced (ascorbic acid) and oxidized (dehydroascorbic acid [DHAA]) forms in the diet. Ascorbic acid is a cofactor for a number of enzymes involved in maintaining transition metal ions in their reduced forms, in the biosynthesis of extracellular matrix proteins and neurotransmitters, and in the regulation of iron intake. Deficiency of vitamin C leads to various clinic symptoms including scurvy, poor wound healing, and bone and connective tissue disorders. In the guinea pig and human ileum, the uptake for ascorbic acid is saturable, indicating a carrier mechanism, and the brush-border membrane uptake of ascorbic acid appears to involve Na+ movement (347–349). The Na+-dependent ascorbic acid uptake was confirmed later by using brush-border membrane vehicles isolated from guinea pig intestine (350–352). The carrier protein involving in the intestinal ascorbic acid absorption is SVCT1. Sodiumdependent vitamin C transporter 1 is highly expressed in the small intestine in human, mice, and rats. Sodium-dependent vitamin C transporter 1 mediates the transport of ascorbate,

the reduced form of vitamin C, in a Na+-dependent manner (353–355). Furthermore, DHAA uptake may occur through the GLUT proteins, which are facilitated diffusion glucose transporters (356,357). Vitamin C uptake decreases with age; there is a general decline of plasma ascorbic acid levels in elderly populations as a function of dietary intake compared with the total adult population. Over the entire range of ascorbic acid consumption, plasma vitamin C levels in the elderly are lower than those in young to middle-aged adults (358). These studies strongly suggest that dietary ascorbic acid uptake is impaired with age. A similar result is also reported in rats, where isolated hepatocytes from old rats showed a lower vitamin C uptake rate compared with cells isolated from young rats. The decline in uptake levels is correlated with the reduced SVCT1 mRNA levels (359). Biotin (vitamin H) is necessary for the metabolism of carbohydrates and fatty acids. Biotin cannot be synthesized by humans and other mammals, and therefore must be obtained from exogenous, dietary sources. Deficiency in biotin results in neurologic disorders, growth retardation, and dermal abnormalities (360). Studies have shown that the absorption mechanism of biotin in the small intestine involves a Na+-dependent carrier-mediated process (361). The carrier protein is localized at the apical membrane of the enterocytes of human, rat, and rabbit (362–364). The protein responsible for biotin transport in the intestine is identified as the sodium-dependent multivitamin transporter (SMVT). The intestinal SMVT cDNA has been cloned and characterized from rat, rabbit, and human (365–367). The SMVT protein transports not only biotin, but also pantothenate and lipoate (365,367,368). The activity of Na+-dependent biotin absorption is regulated during ontogeny and maturation of intestinal tract. Biotin uptake rates in the ileum are greater in suckling rats and not different between jejunum and ileum in weanling rats. In contrast, biotin transport is twofold greater in the jejunum than in ileum in adult rats (340,341). Considering the developmental regulation of the biotin absorptive process in the same region of the intestine, Na+-dependent biotin absorption shows an increase during postnatal development. In rat jejunum, the uptake rate in adult rats is about twofold greater than in suckling rats (369). These functional changes are correlated with the expression pattern of SMVT protein and mRNA. Sodium-dependent multivitamin transporter mRNA transcript in the jejunum of adults is 2.6-fold greater than in suckling rats, and nuclear run-on assays using nuclei isolated from suckling and adult rat jejunum indicated an approximately twofold higher nascent transcription rate for SMVT in adult compared with suckling rats (369). Folate is important for the formation of normal red blood cells and in the production of nucleic acids. Folate deficiency results in a serious anemia. Folate absorption occurs mainly in the proximal intestine; involves a pH-dependent, carrier-mediated process (360); and has been observed in isolated intestinal brush-border membrane vehicles from humans and rats (370–372). The molecular mechanism of

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 391 this absorption has been delineated; the protein now known to be responsible for folate uptake in the intestine is the reduced folate carrier (RFC). The cDNA-encoding RFC protein has been cloned from mouse and human intestine (373–375). Studies in animal models have shown that folate absorption in the intestine changes with age. Brush-border membrane vesicle (BBMV) uptake of folate decreases with age in rat jejunum, with the highest uptake being seen in sucklings (7-fold greater than adult), decreased uptake in weanlings (2.4-fold greater than adult), and the lowest uptake in adults (376). The decline in folate uptake with age is correlated with the RFC protein and mRNA expression, with protein abundance being 2.3-fold greater in suckling rats and 1.4-fold greater in weanling rats compared with adults. Reduced folate carrier mRNA abundance is also greater in suckling and lower in adult rats (376). Nuclear run-on assays confirmed a parallel decline in the RFC transcriptional initiation rate in jejunal epithelia with maturation; the transcriptional rate was 2-fold greater in sucklings and 1.6-fold greater in weanling rats compared with adults (376). Intestinal Transport of Lipid-Soluble Vitamins during Postnatal Development Lipid-soluble vitamin absorption is dependent on bile acids secreted from the liver, a process that is decreased in the neonatal period. Variations in bile acid secretion during the neonatal period thus likely explain some ontogenic differences reported for the absorption of lipid-soluble vitamins (377). Transport of retinol has been widely investigated and is discussed briefly here. Retinol is transported into intestinal epithelial cells by a carrier-mediated mechanism that decreases during ontogeny, and retinol absorption along the gut is higher in the jejunum and lower in the ileum in sucklings and in adults (378,379). Milk-derived factors have also been reported to increase retinol absorption (380). Retinol may also be complexed with retinol-binding protein, and the complex is transported via a specific, saturable, receptor-mediated process, a mechanism that is distinct from the transport of unbound retinol (381). Ontogenic patterns for the absorption of other lipid-soluble vitamins have not been reported to date. Intestinal Absorption of Bile Acids during Ontogeny Bile acids are an essential component of the process of solubilization and digestion of dietary lipids. The critical micellar concentration of bile acids in the duodenum determines, in part, the efficacy of lipid absorption through the gastrointestinal tract. Initial studies using brush-border membrane vesicles have shown that bile acid transport during the suckling period in the rat is markedly decreased, with a significant increase during the weanling period. Moreover, bile acid transport in the ileum has been shown to be enhanced by the administration of glucocorticoids. Advances in molecular cloning have allowed the identification of an apical

sodium-dependent bile acid cotransporter (ASBT clone). The ASBT is a 48-kDa protein, which is primarily expressed in the brush-border membranes of the ileum (382). Western and Northern blot analysis have confirmed the original observation that the ASBT expression is markedly increased during the weanling period (383,384). These findings explain the decrease in the bile acid pool size in the neonatal period and the relative decrease in fat absorption during early life. The second step in bile acid transport involves the cytocylic movement of bile acid. An ileal bile acid binding protein (IBABP), which is a 14-kDa protein, was identified as the major cytocylic bile acid binding protein, and again, this binding protein was shown to be markedly increased during the third postnatal week of rat development (385). Both ASBT and IBABP mRNA are precociously increased by early weaning and delayed by weaning prevention. Moreover, glucocorticoids administration induces both ASBT and IBABP mRNA. These findings suggest that a hardwired (preprogrammed) system together with dietary factors is involved in maintaining the enterohepatic circulation of bile acids.

ONTOGENY OF TRANSPORT FUNCTION ALONG THE VERTICAL AND HORIZONTAL GUT AXES Studies of gastrointestinal morphology and function have demonstrated that horizontal (proximal-to-distal) and vertical (crypt-to-villus) axes, where functional properties can vary greatly, exist in the mammalian intestine. These axes are first established during fetal development in precocial species, but in species with longer gestational periods such as pigs and humans, they are not fully established until several weeks after parturition (386–388). The physiologic factors that direct ontogenic development of these intestinal gradients have not been fully defined, but some studies suggest that predetermined genetic factors regulate development of intestinal gradients. However, other influences such as dietary factors may also be involved in this important physiologic process (149,389). The differences in gene expression patterns seen along these gut axes are regulated by a variety of trans-acting factors, which are expressed differentially in intestinal segments during postnatal development (390). The fetal and neonatal colon, where the most prominent developmental differences are noted, have distinct morphologic and functional features that are absent in adults. For example, the colons of neonates have villus-like structures (391,392) and active transport of some dietary components, which does not occur in the colon of adults. Furthermore, colonic Na+ absorption occurs by an electrogenic process during early postnatal development, whereas an electroneutral process predominates in adults, presumably mediated by NHE. In addition, other transport functions also vary along the length of the small intestine during postnatal development. Other notable differences are the distribution and

392 / CHAPTER 13 presence of vacuolated enterocytes and saturable absorption of bile acids and some vitamins, all of which are present only in early neonatal development. There are also distinct changes in transport function along the crypt-villus axis noted during development (81,149, 393). Some of these observations may be explained by the differential expression of genes encoding various transporters during early life, or by changes in rate of enterocyte migration and turnover. This latter possibility seems particularly relevant as cell replacement and mitotic activity is slower in the perinatal period, and it increases around the suckling/weaning transition (36). These observations may also be explained by the transcriptional repression of some transporter-encoding genes during neonatal life (394).

DEVELOPMENTAL REGULATION OF GASTROINTESTINAL FUNCTION The development of normal gastrointestinal function requires differentiation of four types of principal epithelial cells that include enterocytes, goblet cells, enteroendocrine cells, and M cells. All of these cells originate from common

M-cell

pluripotent stem cells in the intestine. The developmental regulation of gastrointestinal function involves complex interactions among diet, hormones, growth factors, and neurotransmitters (Fig. 13-4). The development of the multicellular gut depends on morphogenetic signals between cells that direct changes in gene expression. The signals that trigger age-dependent changes are originated from genetic and external factors, with both acting in concert to mediate intestinal development. An example of this type of regulation is that hormones or neurotransmitters deliver extracellular signals to nuclei by binding their cognate receptors on the cell surface, and transducer signals by binding to specific regulatory sequences in the DNA, thus inducing changes in expression of specific genes (395). Genetic hardwired signals predominantly determine organogenesis during early development (396). Studies of suckling and weanling rodents have shown that shifts in the activities of different intestinal enzymes and transporters are genetically programmed and relatively less influenced by dietary factors or hormones (397–400). One study showed that rats prevented from normal weaning by being maintained on powdered milk were generally similar to normal rats weaned onto chow. Notably, their fructose uptake still

Enteroendocrine cell

Goblet cell

Enterocytes

Basal lamina

Fibroblasts Sensory neurons

Immune cells Capillary Enteric neurons

Lamina propria

Myenteric ganglion

Luminal stimuli Paracrine effects Nervous stimuli Endocrine stimuli

FIG. 13-4. Signaling interactions that regulate nutrient absorption and secretion during ontogenic development of the intestine. Several physiologic factors that influence intestinal transport function are shown. Luminal effectors, paracrine effectors, nervous stimuli, and hormonal secretions all play a role in regulating transport function during postnatal development. Dietary (e.g., luminal) stimuli interact with different epithelial cells as shown. M cells have a modified basolateral surface that forms an invagination into which immune cells can migrate. Enteroendocrine cells and lymphocytes and other immune cells respond to stimuli by the release of signaling mediators that influence other cell types of the lamina propria through paracrine action. The enteric nervous system receives its input from paracrine effectors, from sensory neurons, and from the central nervous system. This information is processed in the enteric nervous plexus, which results in the activation of motor neurons that may control various mucosal functions. Lastly, hormonal or endocrine stimuli influence the cells of lamina propria, as well as cells of the epithelial layer. (Modified from Pacha [36], by permission.)

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 393 increased steeply on weaning, despite low dietary fructose levels, demonstrating hardwired programming of intestinal transporter development (138). Although internal hardwired programming mechanisms remain in control of development of gastrointestinal function during early developmental stages, external factors, including diet, hormones, growth factors, and neurotransmitters, absolutely play a role in the development of normal gastrointestinal function in the following stages. Many of these factors control the development of gastrointestinal function through transcriptional pathways. The transcriptional control of intestinal gene expression is regulated by multiple positive and negative cis-elements in gene promoters or enhancers (394). Cdx1 and Cdx2, caudal-related intestine-specific homeodomain transcription factors, are selectively localized in the mucosal epithelial cell nuclei in the small intestine and colon of the fetus and adults in both humans and mice (401,402). Whereas normal gastric mucosa does not express transcription factors Cdx1 or Cdx2, strong nuclear immunoreactivity for Cdx1 was detected in human gastric intestinal metaplastic mucosa (403). Furthermore, Cdx1 and Cdx2 are also expressed in human intestinal metaplastic mucosa (404,405). These findings suggest that Cdx1 and Cdx2 may play a pivotal role in the development of intestinal metaplasia in the human stomach. Hormonal factors also play an important role in the ontogeny of the gastrointestinal tract function (406). Much attention has been given to the role of glucocorticoid hormones on development of the intestinal function. Glucocorticoid administration of thyroxine to suckling animals has broad effects on many digestive functions, including sucrase, alkaline phosphatase, enteropeptidase, diamine oxidase, pepsin, and amylase expression (407–409). The cellular mechanism of the action has now become clear. These hormones bind to specific intracellular receptors (glucocorticoids receptors). The hormone/receptor complex undergoes conformational change, and then activates a transcriptional complex to bind to hormone-specific cis-elements on gene promoters or enhancers, thus inducing expression of genes that control intestinal development (410). Glucocorticoids receptor expression has been found in both suckling and adult animals, with decreased expression by age (411,412), which suggests a regulatory role of glucocorticoids on the developmental regulation of intestinal function at weaning (413). Both stimulatory and inhibitory effects of glucocorticoids on intestinal functions have been demonstrated for many genes. Glucocorticoids stimulate Na+-dependent glucose transport, bile acid transport, and Na+-K+-ATPase expression (185, 247,414–417), whereas inhibition is observed with Na+dependent phosphate transport and Ca2+ absorption (290,418). Although glucocorticoids have been demonstrated to have a primary role in the developmental regulation of gastrointestinal functions, many other regulatory peptides and hormones are an essential part of this regulatory machinery, including calcitriol, cholecystokinin, gastrin, secretin, insulin, and several growth factors (419–423). Two important growth factors are transforming growth factors (TGF) and EGFs.

Both TGF and EGF receptors have been found in fetal, neonatal, and adult intestine; however, age-dependent differential responses to intraluminal administration of EGF has been demonstrated, in which mucosal EGF-stimulated EGF receptor phosphorylation occurs in suckling jejunum, but not in weanling or adult jejunum (131,424,425). Although EGF receptor abundance was similar in 22-day-old fetal, 8-day-old suckling, and adult jejunum (425), adult animals exhibited a higher expression of EGF mRNA in comparison with suckling animals (426). It has been shown that milkborne growth factors provide important regulatory signals to the neonatal intestine under normal states (427,428). In contrast with EGF, strong expression of TGF mRNA was found in the small intestine of both adult and suckling rats, whereas rat milk is free of TGF. The small intestine is directly exposed to environmental factors as the result of daily dietary intake and is prepared to adapt its structure and function in response to variations in the diet. Food processed in the gastrointestinal tract induces a series of physiologic responses, including the release of trophic hormones, the stimulation of the enteric nervous system, and the activation of secretary, digestive, and absorptive functions. The normal diet also contains growth factors that directly stimulate the growth of intestinal mucosal cells independently of their nutritive role (429). Significant changes of diet occur during early developmental stages, such as during birth and the weaning period. The developmental patterns of the gastrointestinal tract at these stages are controlled by both genetic and dietary factors (430). Interactions between diet and intestinal maturation occur immediately after birth, by means of feeding in humans and in animals. In some species, the neonatal intestine undergoes rapid growth and reorganization, possibly as a result of programmed gene expression associated with the immediate response to milk (431). The stomach and intestines of newborn rats, rabbits, and dogs increase in mass within hours after the initiation of enteral nutrition (432). Milk-feeding in animals results in a greater DNA, RNA, and protein content in the stomach and intestine than in those with no milk consumption (433). This suggests that rapid growth of the small intestine in naturally fed animals is the result of the specific composition of natural milk, probably caused by stimulation of gene expression by milk-borne factors responsible for intestinal growth. The milk of mammals contains a range of trophic factors, hormones, and other biologically active substances that may cause growth-enhancing effects on the gastrointestinal mucosa (434,435). Differences in pancreatic and gastrointestinal hormone release between breast-fed and bottle-fed infants have also been described (436). Epidermal growth factor, insulin and insulin-like growth factors, polyamines, and glucocorticosteroids are important factors in milk necessary for proper intestinal development (94, 437,438). Luminal administration of EGF stimulates the proliferation and differentiation of enterocytes (439,440). The direct effect of nutrients on the postnatal shift in gene expression and processing of gene products has been implicated in the postnatal disappearance of glucose absorption

394 / CHAPTER 13 in the small intestine of lambs. As a consequence of the rumen maturation, which coincides with weaning, dietary carbohydrates are fermented into short-chain fatty acids by rumen microflora, the amount of hexoses entering the small intestine gradually decreases (441), and the capacity of Na+glucose cotransport declines (146). An example of diet-induced developmental regulation of gastrointestinal function is the dietary effect on lactase expression. Under normal circumstance, lactase activity decreases by age. However, the ontogenic decline in lactase activity can be promoted or delayed by changing the time when rats are switched from milk to the adult-type diet (442). Preweaned animals force-fed an artificial diet without lactose retained a high amount of lactase mRNA in the jejunum, whereas this transcript precociously decreased in the distal ileum. Conversely, prolonged nursing delayed the specific decrease of lactase mRNA levels in the latter segment. These results suggest that switching over from milk to the adulttype diet at weaning contributes to the modification of the longitudinal distribution of the lactase mRNA that normally occurs at this stage. Thus, although the ontogenic decline in intestinal lactase is intrinsically programmed, luminal factors modulate this process. The transition to the adult diet is required for the normal progression of events.

ACKNOWLEDGMENTS Our efforts in writing this chapter were supported by National Institutes of Health grants 1-R21-DK-06834 (J.F.C.), R01-DK-412174 (F.K.G.), R01-DK-33209 (F.K.G.), and R01-DK-063142 (L.B.).

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CHAPTER

14

Effect of Aging on the Gastrointestinal Tract Adhip P. N. Majumdar and Marc D. Basson The Aging Esophagus, 406 Structural and Functional Properties, 406 Barrett’s Esophagus, 407 Key Areas for Future Research, 407 The Aging Stomach, 407 Structural and Functional Properties, 407 Mucosal Injury and Repair, 408 Gastric Secretion and Digestion, 410 Key Areas for Future Research, 411 The Aging Intestine, 411 Structural and Functional Properties, 411 Immunologic Effects, 412 Gut Peptides and Other Key Molecules, 412 Contractility and Motility, 412 Mechanisms of Fecal Incontinence, 413 Key Areas for Future Research, 414 Aging and Gastrointestinal Cancers, 414 Epidemiologic Observations, 414 Epithelial Growth and Renewal as Potential Premalignant Events, 415

Inhibition of Apoptosis as a Potential Premalignant Event, 416 Changes in Susceptibility to Carcinogens, 417 Mutational Events, 418 Key Areas for Future Research, 419 Regulation of Mucosal Growth during Aging, 420 Intracellular Mechanisms, 420 Nutritional and Hormonal Factors, 420 Growth Factors and Protein Kinases, 421 Gene Expression Patterns, 423 Key Areas for Future Research, 423 Aging and Surgery of the Gastrointestinal Tract, 423 Adaptation to Fasting and Sepsis, 423 Adaptation to Bowel Resection, 423 Surgical Diseases of the Gut, 424 Angiodysplasia, 425 Key Areas for Future Research, 425 Acknowledgments, 425 References, 425

Census and other data suggest that persons older than 65 years constitute about 12.3% of the U.S. population, or 1 in 8 Americans. By 2030, the fraction of older Americans has been predicted to increase to 20%, with a total number of 71.5 million, more than twice as many as in 2000. The number of Americans older than 85 years has been predicted to increase from 4.2 million (based on the 2000 census) to

8.9 million in 2030. The predicted life expectancy of an American child born in 2000 is 76.9 years. Aging is defined as the progressive accumulation of changes over time that is associated with or is responsible for increasing susceptibility to disease and death, the final event of advancing age. A primary conceptual challenge in the study of mammalian aging is the occurrence of disease and the intricate relation of disease processes to aging. Aging is associated with marked changes in the structural and functional properties of the gastrointestinal (GI) organ system. Thus, consideration of the effects of aging on the GI tract is likely to become increasingly relevant as the population ages. This chapter therefore reviews the structural and functional changes in different parts of the GI tract with aging. In addition, how such changes disturb homeostatic mechanisms and predispose the gut to certain diseases is discussed. This latter discussion in particular focuses on malignant transformation.

A. P. N. Majumdar: John D. Dingell Veterans Affairs Medical Center, Department of Medicine, Karmanos Cancer Center, Wayne State University, Detroit, Michigan 48201. M. D. Basson: John D. Dingell Veterans Affairs Medical Center, Department of Surgical Service, Wayne State University, Detroit, Michigan 48201. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

405

406 / CHAPTER 14 THE AGING ESOPHAGUS Structural and Functional Properties Aging has been associated with a diversity of alterations in the oropharyngeal components of swallowing. In general, aging has been found to be associated with increased oropharyngeal segmental transit times (1,2), as well as increased pharyngeal peristaltic pressure wave activity and amplitude (1–4). Peak pharyngeal pressures were found to be greater in older subjects (5), whereas the anteroposterior diameter of the upper esophageal sphincter (UES) and the anterior hyoid bone were both shown to be significantly less in elderly subjects than in their younger counterparts, as is laryngeal excursion (2). Although the mean UES resting pressure tends to decrease with advancing age (5), possibly as the result of striated muscle degeneration, the UES residual pressure after deglutition is increased (6). Rapid anterior movements of the hyoid, normally observed in younger individuals, are found to be delayed in elderly subjects, as is the subsequent anticipated relaxation of the UES. Consequently, smooth passage of a luminal bolus from the oropharynx to the esophagus is likely to be compromised. During the esophageal phase of swallowing, the ingested bolus is propelled through the esophagus by primary peristaltic waves and is delivered into the stomach with a coordinated relaxation of the lower esophageal sphincter (LES). The normal esophageal motor activity consists of an orderly peristaltic contraction that propagates itself from the UES through the striated and smooth muscle sections and terminates in closure of the lower sphincter (7). There have been numerous studies examining the effects of aging on esophageal motility in patients with dysphagia, as well as in healthy asymptomatic control subjects. A variety of abnormalities have been described in both subgroups; however, the consistency of these aberrant findings has been the subject of debate. Earlier, Soergel and colleagues (8) evaluated the esophageal motor function of 15 nonagenarians by cineradiography, intraluminal manometry, and balloon kymography. In this group of subjects, disturbed esophageal motility consisted of disorganization of the response to deglutition, as a result of a defect in initiating relaxation of the LES, as well as primary peristalsis, whereas the incidence of secondary peristalsis was found to decrease less frequently. Numerous nonpropulsive, often repetitive, esophageal contractions were also seen. A similar observation was also made by Zboralske and coworkers (9), who studied 41 subjects, including 15 subjects aged between 90 and 98 years. The pathogenesis of this disorganization, however, is currently unknown. Ueda and colleagues (10) have studied the motor and electrical activities of feline esophagus by intraluminal pressure detectors and by chronically implanted platinum electrodes, before and after vagotomy. The prominent features after vagotomy were persistent weakness, incoordination, and repetitive responses in the lower esophagus preventing normal emptying of the organ. The significance of possible degenerative changes of vagal

innervation in the aged remains unknown. Grande and coworkers (11) studied 79 healthy subjects and noted an inverse correlation of age with LES pressure, as well as peristaltic wave amplitude and velocity. Alternatively, Richter and colleagues (12) did not detect any differences in LES pressure between young and elderly subjects using either the rapid or stationary pull-through methods. This finding has been confirmed by others (13) who further noted that the total length of the LES high-pressure zone was similar between young and elderly subjects. Several investigators have found an age-related decrease in the amplitude of peristaltic wave pressure (11,14). These studies also noted a decrease in peristaltic velocity and an increase in wave duration, whereas Hollis and Castell (15) found no age-related differences in these parameters. Secondary peristalsis, which is essentially a peristaltic wave that is not induced by a swallow, occurs less often in elderly subjects and may contribute to reduced clearance of residual, as well as refluxed, esophageal contents (14). Finally, several investigators have shown an increased frequency of tertiary or spontaneous nonperistaltic contractions in elderly subjects (10,11,16). The antireflux barrier at the gastroesophageal junction is generally attributed to the interaction of intrinsic LES tone, the crural diaphragm that exerts extrinsic pressure on the LES, and the length and thoracoabdominal distribution of the LES (17,18). Xie and coworkers (19) found that elderly subjects had increased gastroesophageal reflux events after pharyngeal water swallows significantly more often than their younger counterparts. This increase in reflux events was further associated with a decreased intraabdominal segment of the LES in the elderly. Other investigators (20–22) have noted an increased duration of reflux episodes in elderly patients. Interestingly, however, elderly patients seem to perceive reflux episodes less often than younger patients (15,22,23). The prevalence of Helicobacter pylori infection increases during aging (24). It has been estimated that 40% to 60% of elderly subjects may be infected with H. pylori (24). Because aging is also associated with an increased incidence of gastroesophageal reflux disease (GERD), it has been suggested that H. pylori infection may be a contributory factor for GERD. However, this relation has not been thoroughly investigated, particularly with respect to aging. Earlier studies by Liston and colleagues (25) failed to show a significant association between H. pylori and reflux esophagitis during aging. In a more recent study, Haruma and coworkers (26) observed a lower prevalence of H. pylori in subjects older than 60 years with reflux esophagitis. However, in a double-blind, placebocontrolled study, Schwizer and colleagues (27) observed in patients with GERD that H. pylori–positive patients relapsed earlier than those in whom H. pylori was eradicated. In contrast, Pilotto (28) reports that eradication of H. pylori infection in elderly patients did not influence the short- and long-term healing rates of esophagitis between young and elderly patients. Limited information is available with respect to agerelated changes of the esophageal mucosa. Furthermore, the significance of the development of Barrett’s esophagus with

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 407 advancing age remains unknown. Barrett’s esophagus, a premalignant lesion, requires the replacement of normal stratified squamous epithelium with intestinal metaplastic epithelium at the distal esophagus (29). The prevalence of Barrett’s esophagus increases with age and plateaus in the seventh decade of life (30). Although this increasing prevalence with aging certainly reflects the cumulative effect of protracted esophageal exposure to acid, aging may independently contribute to the evolution and progression of Barrett’s epithelium through age-related reduction in perception of acid reflux (22,23,31). Similarly, elderly patients would also be more likely to require medications that potentially diminish LES pressure, such as calcium channel blockers, β-adrenergic agonists, and nitrates, thereby enhancing the propensity for acid reflux disease (32).

Barrett’s Esophagus It is well recognized that Barrett’s esophagus is a premalignant lesion with the potential to transform into adenocarcinoma of the esophagus. The presence of Barrett’s esophagus is associated with a 30- to 40-fold increased risk for esophageal adenocarcinoma (33). The development of adenocarcinoma in Barrett’s esophagus is a multistep process during which the metaplastic epithelium ultimately transforms into low-grade and subsequently high-grade dysplasia. There is evidence that continuous acid exposure can induce differentiation and reduce proliferation, whereas short bursts of acid exposure increase proliferation (34). More importantly, normalization of intraesophageal pH can reduce esophageal mucosal cell proliferation. Interestingly, complete symptom eradication does not guarantee normalization of intraesophageal pH (35). Therefore, it is certainly conceivable that elderly individuals with intrinsically enhanced reflux but compromised sensitivity to reflux is particularly susceptible to induction of mucosal cell proliferation. Cyclooxygenase-2 (COX-2) or prostaglandin synthase 2, a membrane-bound protein involved in the generation of prostaglandins from arachidonic acid, mediates prostanoid release involved in inflammatory and tumorigenic responses (36). Cyclooxygenase-2 expression is restricted in certain tissues, but it can be induced in response to various stimuli (37,38). Increased expression of COX-2 has been observed in both animal models of inflammation and human inflammatory diseases (39–42). There is increasing evidence to implicate COX-2 in colorectal, gastric, and esophageal carcinomas (43–45). Moreover, it has been demonstrated that COX-2 protein expression is significantly higher in Barrett’s metaplastic and dysplastic epithelium, as well as adenocarcinoma, and is correlated with progressive morphologic changes (46). Certainly, nonselective COX inhibitors (nonsteroidal anti-inflammatory agents), as well as newer selective COX-2 inhibitors, may have a role in cancer chemoprevention for patients with Barrett’s esophagus. Finally, mutations in the tumor-suppressor gene p53, one of the most common genetic alterations in human cancer,

have been described in most Barrett’s esophagus cases with high-grade dysplasia or adenocarcinoma (47). Similarly, immunohistochemical staining of p53 has been demonstrated in low-grade dysplasia associated with Barrett’s esophagus and may be predictive of future evolution to high-grade dysplasia. Elderly patients with comorbid conditions precluding definitive surgical intervention for Barrett’s dysplasia are often considered for a variety of endoscopic ablative techniques. Despite the ability of many of these techniques to destroy Barrett’s mucosa, there is still evidence that the genetic aberrations associated with Barrett’s esophagus may persist (48).

Key Areas for Future Research Although the impact of aging on esophageal motility is clear, the mechanisms of these effects require further study, particularly with reference to the relative contributions of neural and hormonal influences. This is particularly important because the development of targeted modalities for the reconstitution of esophageal motility in the elderly could substantially reduce morbidity and mortality from aspiration pneumonia. The triggers for the development of Barrett’s esophagus and subsequent malignant degermation also require further study. Although repeated exposure to gastric refluxate is assumed to be pathogenic in this regard, the possibility that aging esophageal mucosa may be more susceptible to such refluxate at a cellular level is a fertile ground for investigation. Finally, the potential role of H. pylori in the pathogenesis of GERD, Barrett’s esophagus, and esophageal carcinoma requires further investigation.

THE AGING STOMACH Structural and Functional Properties Like other parts of the GI tract, the gastric mucosa is composed of numerous exocrine and endocrine cells (49), which renew at different rates. In the oxyntic gland area of the stomach, most of the newly produced cells migrate rapidly to the surface while differentiating into mucous cells. The migration time represents the time for replacement of the total cell population above the dividing zone. This migration time is about 3 days in adult rats (50) and 4 to 6 days in the adult dog or human (51). Most studies have indicated that parietal cells are unable to divide, and that some newly formed cells migrate slowly down the gland to differentiate into acid-producing cells (51). In the adult mouse, zymogen (chief) cells are replaced by mitosis (52), but after injury, they have been found to originate from undifferentiated cells. Aging is associated with marked changes in the structural and functional properties of the gastric mucosa. In rats, aging is found to be associated with gastric atrophy, as evidenced

408 / CHAPTER 14 by a significant reduction in mucosal glandular height, gland density (number of glands per square centimeter), and total mucosal DNA content (53,54), and it may lead to a reduction in the number of glandular epithelial cells of all types. In aged rats, gastric epithelial cells also show evidence of decreased secretory activity, with decreased number of intracytoplasmic secretory granules and decreased cell size (53). In contrast, the connective tissues of lamina propria of aged rats, especially between the glands and the muscularis, are found to be thickened due to massive collagen deposition (53). Electron microscopy studies in rats have further demonstrated ultrastructural degenerative changes in both parietal and chief cells (53,55). Although earlier studies have suggested that the gastric mucosa remains normal in older human subjects, several reports have documented a marked increase in the incidence of atrophic gastritis among the elderly (56,57). Earlier studies by Andrews and colleagues (56) have demonstrated some degree of chronic atrophic gastritis in 23 of 24 subjects older than 60 years. Bird and coworkers (57), who analyzed 201 gastric biopsy specimens from individuals between 65 and 90 years old, observed signs of atrophy in most of the specimens. Hradsky and colleagues (58) also found an increased incidence of gastritis with aging by comparing the gastric biopsy results in groups of patients 44 years or younger, between 45 and 59 years old, and between 60 and 84 years old. Although the underlying regulatory mechanism(s) for the development of gastric atrophy is unknown, one plausible explanation could be that the ability of the gastric mucosa to replenish the lost surface cells decreases with aging. Indeed, it has been demonstrated that in aged rats, reepithelialization of the gastric mucosa after hypertonic saline-induced mucosal erosion is markedly delayed compared with young animals (59).

Mucosal Injury and Repair Aging is also associated with increased susceptibility of the gastric mucosa to damaging agents (60). This, together with impediment for the repair process, may contribute to the increased incidence of gastric and duodenal ulcers observed among the elderly. Because intake of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is generally greater among the elderly, studies have been performed to determine the frequency of NSAID-induced injury in elderly subjects. Epidemiologic data suggest that aging is associated with increased NSAID-induced gastric mucosal injury (61). It is estimated that in the United States, approximately 16,000 deaths could be related to adverse events associated with NSAID use (62). Approximately 34% to 46% of patients receiving NSAID therapy are expected to have some manifestation of adverse GI events, and the overall risk for development of a serious GI complication is three times greater in NSAID users than nonusers (63). The risk tends to increase linearly with aging and becomes particularly high in the presence of disability (64), comorbidity

(congestive heart failure, hypertension, renal or hepatic insufficiency) (62), and comedication and concomitant intake of corticosteroids (65) and anticoagulants (66). Moreover, it has been demonstrated that both H. pylori infection and NSAID use independently and significantly increase the risk for peptic ulcer and bleeding (67). H. pylori infection increased the risk for peptic ulcer in NSAID users by about 3.5-fold in addition to the risk associated with NSAIDs (67–69). These observations are in agreement with pathophysiologic studies that demonstrated that aging, as well as H. pylori infection, may decrease gastric mucosal hydrophobicity (70) and gastric mucus gel formation (71), which is known to provide cytoprotection. Again, eradication of H. pylori infection before NSAID use has been shown to reduce the occurrence of NSAID-related peptic ulcer (72). Clearly, further studies are needed to evaluate the roles of various endogenous and exogenous factors in the age-related increase in the frequency of gastric mucosal injury. To determine the age-related susceptibility of the gastric mucosa to damaging agents and the subsequent reparative processes, we used a hypertonic saline-induced gastric mucosal injury model in Fischer-344 rats. Our investigation with hypertonic saline, which virtually eliminates the surface epithelium, showed a 67% higher lesion index (percentage of damaged area) in the gastric mucosa of 24-month-old Fischer-344 rats compared with their 4-month-old counterparts (73). Electron microscopic examination also showed that, although administration of hypertonic saline virtually eliminated the surface epithelium in both young and aged rats, the extent of injury in older animals extended beyond the surface epithelium (74). In aged rats, epithelial cells in deeper parts of the gastric glands demonstrated severe swelling with vacuolization and disintegration of the cell organelles, with dying and dead cells (74). Aspirin and ethanol have also been shown to produce a greater number of lesions in the gastric mucosa of aged than in young rats (75). Taken together, these observations suggest that aging is associated with reduction in mucosal protective mechanisms that may predispose aged animals to mucosal injury. Although the underlying biochemical mechanisms for this increased susceptibility are poorly understood, several investigators have demonstrated that gastric mucosal prostaglandin content, which provides cytoprotection to the surface epithelium, decreases with aging in humans (76,77). In addition, Gronbech and Lacy (78) have suggested that impaired mucosal defense and reduced restitution in aged rats is partly due to decreased density of calcitonin gene–related peptide in nerve fibers and the biosynthetic capacity of the mucosa in prostaglandins. A decline in mucosal blood flow in aged rats has also been suggested as another causative factor for the age-related decline of mucosal repair (79). In addition, the H. pylori infection, which increases among diabetic patients and the elderly, is thought to be an important causative factor for duodenal and gastric ulcers (80). Type II diabetes, the incidence of which increases with advancing age, has also been shown to affect the structural and functional integrity of many organ systems, including

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 409 the GI tract. Diabetes has been linked to “premature aging.” Although gastroparesis diabeticorum is the most common complication of diabetes in the digestive tract (81), clinical studies have demonstrated that diabetes ketoacidosis is often associated with abdominal pain, nausea, and vomiting (82–85). This could partly be the result of upper GI hemorrhage, the prevalence of which is increased in diabetic ketoacidosis (83,84). In addition, results from experimental models of diabetes have shown that diabetes is associated with spontaneous gastric ulceration and enhanced susceptibility of the gastric mucosa to exogenous damaging agents, as well as with stress-induced gastric ulcer (86–88). Food deprivation in streptozotocin-induced diabetic rats has been shown to produce severe gastric lesions (87,88). Although the underlying mechanisms remain to be elucidated, this could be partly due to the loss of surface mucosal cells resulting from spontaneous mucosal injury. Induction of diabetes mellitus by streptozotocin in rats has been shown to result in gastric mucosal atrophy with reduction in mucosal thickness (89). Whether these changes are worsened with aging, however, remains to be determined. However, the gastric mucosa of healthy adult animals possesses a remarkable capacity to repair promptly after an acute injury. Aging and diabetes are associated with decreased healing of the gastric mucosa (59,60,89). The regulatory mechanisms that direct this repair, as well as the potential modulating effects of aging or diabetes, are poorly understood. The process of mucosal reconstitution, which is accomplished by cell migration, begins within minutes after injury and is completed within a few hours. Earlier in vitro studies using frog mucosa in the Ussing chamber have demonstrated that the surface epithelium is restored within a few hours of exposure to 1 M NaCl (90,91). A similar phenomenon was also observed in rats (92). Although similar studies have not been performed during aging, we have demonstrated that, whereas the gastric mucosa of young rats shows signs of reepithelialization as early as 6 hours after hypertonic saline (2 M NaCl)-induced injury, which is essentially complete by 24 hours, in aged rats, only intermittent surface cells can be seen 24 hours after injury (59,60). The partial reepithelialization of the gastric mucosa of young rats that occurs within 6 hours after injury is thought to be the result of increased cell migration during this period. Aging appears to attenuate this process. A number of growth factors, including basic fibroblast growth factor (90,93), intestinal trefoil peptide (94–96), as well as epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) (97–101), all of which stimulate cell migration, are thought to play a role in this process in healthy young animals. However, their role in mucosal cell migration during aging remains to be evaluated. Similarly, polyamines, which are known to be essential for cell proliferation (102) and for repair of gastric and duodenal ulcers, have also been shown to stimulate cell migration in vitro (103). Therefore, it is tempting to speculate that they may also be involved in regulating healing of the gastric mucosa after injury during aging. To the best of our knowledge, no information is available about the role

of either growth factors or polyamines in regulating the reparative processes of the gastric mucosa with advancing age. However, in assessing the role of growth factors in mucosal restitutional process, Polk and colleagues (99) have demonstrated a marked increase in TGF-α, but not EGF, in the gastric mucosa of young rats within 30 minutes after an acute injury. They suggest that local production of TGF-α may have an important role in the early reparative phase of the gastric mucosa. In aged rats, we have also observed a marked increase in gastric mucosal TGF-α levels 30 minutes after hypertonic saline-induced injury (104). Yet, mucosal restitution, as determined by reepithelialization of the gastric mucosa, was attenuated (59). Because TGF-α exerts its physiologic action by activating the intrinsic tyrosine kinase of its receptor, the epidermal growth factor receptor (EGFR) (105), we postulated that decreased activation of EGFR tyrosine kinase might, in part, be responsible for the diminished mucosal repair in aging animals. The role of EGFR tyrosine kinase in gastric mucosal was initially evaluated by examining the effect of tyrphostin, an inhibitor of tyrosine kinases with a greater specificity for EGFR than other protein kinases (106,107), on gastric mucosal regeneration in 3- to 4-month-old rats at 24 hours after hypertonic salineinduced injury (108). In the absence of tyrphostin, the gastric mucosa showed signs of extensive regeneration, whereas in its presence, regeneration was greatly attenuated (108). These changes were accompanied by parallel alterations in the number of proliferating cell nuclear antigen (PCNA)– immunoreactive cells and EGFR tyrosine kinase activity in the gastric mucosa (108). The observation of increased PCNA immunoreactivity in the gastric mucosa of control rats (which were not treated with tyrphostin) at 24 hours after injury suggests that induction of cell proliferation is a necessary step for regeneration of the mucosa after injury. To further evaluate the role of EGFR tyrosine kinase in the early reparative phase of the gastric mucosa, we examined time course changes in total and EGFR tyrosine kinase activity in young adult rats over a period of 60 minutes after hypertonic saline-induced injury (109). Injury caused an immediate increase in both total and EGFR tyrosine kinase activity in the gastric mucosa, with the maximal stimulation occurring at 30 minutes after injury (109). When gastric mucosal EGFR tyrosine kinase activity was examined in young and aged rats at 30 minutes after injury, we observed that the enzyme activity and the expression of the receptor protein in the gastric mucosa of both age groups were significantly increased 30 minutes after injury; however, the magnitude of stimulation was lower in the gastric mucosa of aged than in young rats, when compared with the corresponding basal levels (109). A similar phenomenon was also noted for phospholipase C (PLC) and tyrosine phosphorylation of PLCγ (109). Taken together, the results suggest that TGF-α is less effective in enhancing activation of EGFR tyrosine kinase in aged rats. This could be attributed, in part, to increased sensitivity of the aged gastric mucosa to EGF family of peptides such that low doses are stimulatory, whereas high doses of these peptides may inhibit the physiologic

410 / CHAPTER 14 actions of these peptides in aged rats. The basis for this hypothesis comes from the observation that administration of a relatively high dose of EGF, which caused a significant stimulation in mucosal proliferative activity and tyrosine kinase activity in the gastric mucosa of young rats, produced a marked inhibition in aged rats (110,111). This was further substantiated by the observation that in isolated gastric mucosal membranes from aged rats, the concentration of TGF-α required to achieve the maximal stimulation of EGFR tyrosine kinase was several fold less than that required to attain the same stimulation in young animals (111). Because injury causes significant increase in TGF-α production in the gastric mucosa of both age groups (109), it is likely that increased levels of endogenous TGF-α or other EGFR ligand(s) may prevent the injury-induced stimulation of EGFR tyrosine kinase and the subsequent signaling events in aged rats through an autocrine/paracrine mechanism. Another possibility could be that the maximal stimulation in mucosal EGFR tyrosine kinase in aged rats after injury occurs at a different time than in young animals, and that it takes longer for complete regeneration of the mucosa in aged animals. Undoubtedly, further studies are needed to evaluate the participation of EGFR and other signaling pathways in early and late reparative phases of the gastric mucosa during aging.

Gastric Secretion and Digestion The primary function of the gastric mucosa is to continue the process of digestion initiated in the mouth through the secretion of acid and pepsin. Although the major constituents of the gastric juice are hydrogen ion, pepsin, mucus, and intrinsic factor, the only indispensable ingredient is intrinsic factor, which is required for absorption of vitamin B12 by the ileal mucosa. Pernicious anemia resulting from vitamin B12 deficiency has been observed in the aging population (112). A decreased mucosal cell mass and, in turn, a diminished production of intrinsic factor by the parietal cells could, in part, be responsible for the development of pernicious anemia observed in the aging population. However, an association of atrophic gastritis with pernicious anemia has been disputed. Considering that a severe reduction in intrinsic factor is required before vitamin B12 absorption is impaired by ileal mucosa, evidence has been presented to show that in atrophic gastritis appreciable quantities of intrinsic factor may still be produced when acid production is greatly diminished (112). However, pernicious anemia may occur in some patients with severe atrophic gastritis, and it is characterized by the presence of a megaloblastic anemia caused by vitamin B12 deficiency (113). At times, atrophic gastritis is also associated with intestinal metaplasia, and both conditions are age related and considered to be precancerous lesions (114). Thus, aged individuals with gastritis are statistically at risk for development of gastric cancer, but precise measurement of this risk has not been determined. Although chronic atrophic gastritis in the

aged often is found to be associated with H. pylori infection, the precise role of H. pylori in the initiation and progression of gastritis during aging is still controversial. Studies by Feldman and colleagues (115) on healthy volunteers suggest that aging was independently related to chronic gastritis, and several others have found that, although atrophic changes of the gastric mucosa were associated with H. pylori infection, these changes could not be related to aging. However, it has been demonstrated that in subjects infected with H. pylori, the intensity of mononuclear infiltration of chronic gastritis and the increase in intestinal metaplasia were higher in older subjects when compared with their younger counterparts, and that H. pylori eradication was beneficial in preventing the progression of atrophy and intestinal metaplasia regardless of age differences (116). In a study with 132 adult volunteers with gastric atrophy, a significant improvement of the ailment was observed after treatment for H. pylori infection (117). Moreover, in a study carried out in 22 elderly subjects from Finland who had H. pylori infection and moderate to severe atrophic gastritis, a significant improvement in histologic scores of inflammation, atrophy, and intestinal metaplasia in the corpus mucosa was observed 2.5 years after H. pylori eradication (118). Taken together, the results suggest that the structural and functional changes that occur in older subjects after H. pylori infection are no different from those observed in young patients, and that all age groups show similar beneficial effects after eradication of H. pylori. Because one of the main functions of the stomach is to secrete acid, considerable work has been done to examine the age-related changes in acid output by the gastric mucosa. Earlier studies have demonstrated a decline in both basaland histamine-stimulated acid output in aging humans (119), and this has been related to the degree of gastric mucosal atrophy and the parietal cell number. In Fischer-344 rats, we have also observed an age-related decline in basal and gastrin-induced acid secretion (54), which could be partly due to the reduced number of acid secretory cells (53,55). In contrast, some studies on healthy humans indicated no significant change in either basal or stimulated gastric acid output with aging (115,120). Similarly, the pentagastrininduced stimulation of acid output was found to be only slightly decreased among the elderly (121). Thus, the earlier studies, which reported a decline in both basal and maximal gastric acid output in aging human populations, could be the result of inclusion of subjects with atrophic gastritis, a condition that is recognized to be common among the elderly, affecting 25% of those older than 60 years (57,121). Another factor that may affect gastric acid secretion in humans is H. pylori infection (120,122), the incidence of which is also increased with aging (80,123). H. pylori has been recognized as a causative factor in the development of chronic antral gastritis (124). In rats, basal pepsin secretion and mucosal pepsinogen concentration were also found to be lower in aged than in young rats (54). The regulatory mechanism(s) for the agerelated decline in mucosal pepsinogen content in rats is

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 411 unknown. We have reported a decline in steady-state mRNA levels of several gastric proteases including pepsinogen C with aging (125). Whether this could result in decreased synthesis of the enzyme remains to be determined. In humans, aging has also been shown to decrease both basal and stimulated gastric pepsin output, and it was found to be independent of atrophic gastritis, H. pylori infection, or smoking (126,127). In contrast, serum pepsinogen I levels in normal female individuals, but not in male individuals, were shown to increase with age (128). A similar observation was also made in patients aged 50 or older with duodenal ulcer (129).

Key Areas for Future Research The phenomenon of gastric hyposecretion and hypochlorhydria in the aging stomach is well known, but its fundamental mechanisms await elucidation. The relative contributions of decreased expression of mucosal receptors for secretory hormones, abnormalities in intracellular signal transduction, and abnormalities in physiologic levels of the secretory hormones themselves should be studied further. The reduction in gastrin-induced acid secretion in the aging animal or human may be paradigmatic in this regard. This is particularly important because the achlorhydric stomach is associated with increased pernicious anemia and may also be at more risk for malignant degeneration, although the precise mechanism of this latter relation also awaits study. In addition, the impact of aging on mucosal healing also represents an opportunity for future research. It is becoming increasingly clear that growth factors and matrix proteins activate a complex cascade of intracellular signals during restitution that regulate the velocity and quality of mucosal healing. Signals at the focal adhesion complex such as focal adhesion kinase (FAK) and Src are likely to be of particular relevance in this regard. Surprisingly, there are little data available on the impact of aging on such signals or about changes in integrin expression or organization that may occur in the aging mucosa.

THE AGING INTESTINE Structural and Functional Properties Small intestinal mucosal permeability does not appear to change in aging humans, measured by lactulose/mannitol absorption (130), and the histology of the small mucosa is relatively well preserved in aging humans and animals. However, several investigators have described an increase in enterocyte proliferation and apoptosis with aging (131,132). Proliferating cell nuclear antigen immunoreactivity increases with advancing age in both the crypts and the villi, raising the possibility that abnormal absorptive function in the villi could coexist with normal proliferative biology in the crypts (133). The significance of this observation awaits study, but one

could question whether this hyperproliferative state might contribute to the increased likelihood of malignant degeneration or affect responses to various pathophysiologic stimuli, or both. Dietary restriction seems to potentiate small-bowel mucosal apoptosis in rats during aging (134). The density and distribution of mucosal neuroendocrine cells also seems to alter with aging in a complex fashion that seems to vary with the site in the GI tract and also the type of neuroendocrine cell being analyzed (135). Colonic mucosal proliferation is similarly enhanced in aging rats, with accelerated G1- to S-phase transition and progression through the S-phase, together with parallel alterations in several of the conventional mediators of the cell cycle, although colonocyte apoptosis may actually be decreased (136,137). An interesting question for further study is whether the disparity between increased apoptosis with aging in the small bowel and decreased apoptosis in the colon might contribute to the difference between the incidence of adenocarcinoma in the small and large bowels. In addition to differences in the histology of the mucosa itself, changes in the mucosal microenvironment may also occur with aging. The pH of the rat jejunal epithelial surface is significantly greater in senescent rats than in young adult rats, independent of the effects of sodium removal or amiloride. This may reflect the thinner mucous layer in these aged rats (138). Duodenal enterocytes from older rats exhibit decreased responsiveness to parathyroid hormone, possibly, at least in part, because of decreased expression of parathyroid hormone receptors (139). This correlates with age-related impairments in activation of PLC (140) and mitogen-activated protein kinase phosphorylation (141) in response to parathyroid hormone. Such signal loss may impair the regulation of intracellular calcium and proliferation in duodenal enterocytes (140,142). An age-dependent decrease in intestinal inorganic phosphate absorption has also been described and appears to correlate with a decrease in 1,25-dihydroxy vitamin D–dependent stimulation of expression of the type IIb sodiuminorganic phosphate cotransporter (143). 1,25-Dihydroxy vitamin D–dependent signals are similarly impaired in aging duodenal mucosal enterocytes, including protein kinase C activation and isozyme translocation, and the generation of inositol-1,4,5-trisphosphate and diacylglycerol (144–147). The role of steroids in supporting the small-bowel mucosa may also differ between younger and older rats. Adrenal resection results in mucosal atrophy, decreased crypt cell proliferation, and histologic disorganization in the proximal small intestinal epithelium of both young and old rats, together with increased numbers of Paneth cells. However, adrenal insufficiency in older rats also diminishes the activity of a broad spectrum of brush-border enzymes in the mucosa, including the disaccharidases, in contrast with the more specific patterns of loss observed in younger animals (148). In contrast, although ion transport in the kidney changes substantially with aging, it appears that mineralocorticoidsensitive, electrogenic sodium absorption in the rectosigmoid colon does not change substantially as humans age (149).

412 / CHAPTER 14 Intestinal mucosal L-DOPA and dopamine levels decrease in aging rats because of increased aromatic L-amino acid decarboxylase activity, but these changes do not correlate with inhibition of the sodium-potassium ATPase in the gut mucosa as clearly as they do in the aging kidney (150,151).

Immunologic Effects A substantial clinically significant effect of aging on small-bowel mucosal immunity has not been demonstrated, although two interesting articles have described an impairment of immunity to intraduodenal cholera toxin in older rats, and that orally administered kefir fermented milk enhances the intraduodenal immune response to cholera toxin in young but not senescent rats (152,153). Decreased specific antibody titers in intestinal lavage fluid from aging animals may correlate with compromised IgA immunoblast homing in the aging rat or monkey intestinal mucosa (152,154). Several more subtle deficiencies have also been described in animal models and humans. For instance, the repertoire of the mucosal gamma delta T cells, which constitute an important aspect of mucosal defense mechanisms, becomes increasingly restricted in aging humans and pigs, and it displays a striking variation along the length of the GI tract (155). Serum IgA decreases in humans with age (156). One investigator has suggested that lamina propria lymphocytes display an age-related loss of immune function (assessed by proliferation and interleukin-2 [IL-2] production) similar to peripheral blood lymphocytes, but intestinal epithelial lymphocytes appear to be spared from this decline with aging (156). Additional research is needed to elucidate the potential significance of such changes in immune function in aging humans. These alterations could also prove of greater significance in aging humans with clinical conditions directly or indirectly impairing other aspects of immune function.

of these cells also changes with aging in a complex and cell type-specific pattern (163). In general, proliferation of these cells does not seem to change, but apoptosis increases as the mice age (135). The density of murine colonic neuroendocrine cells immunoreactive for peptide YY (PYY), enteroglucagon, and serotonin displays a biphasic pattern, high in 1-month-old mice, decreased in 3-month-old mice, and then increasing again in 12- to 24-month-old mice (164). Although murine enteroendocrine cells resemble those of humans in many important ways (165), it should be noted that potentially relevant differences in the patterns of gut peptide appearance and change during development and aging are found when different species are compared. For instance, PYY cells increase with age in rodents, but this has not been demonstrated in humans (166). Human duodenal neuroendocrine cells have also been reported to vary with age in a complex fashion. For instance, gastrin/CCKimmunoreactive cells were found to be more numerous in 1- to 2-year-old and 60- to 69-year-old healthy humans than in 20- to 29-year-old humans, whereas somatostatinimmunoreactive cells were less common in 20- to 29-yearold humans than 40- to 49-year-old humans. The numbers of immunoreactive secretory granules within many of these cells was somewhat greater in 1- to 2-year-old than 20- to 29-year-old humans (167). Interestingly, aging also increased glutathione-S-transferase, but not glutathione peroxidase, superoxide dismutase, or lipid peroxidation within the small-bowel mucosa of aging Wistar rats. This pattern differs from that observed within the liver of these animals (168) and also seems to vary somewhat with the strain of rat studied (169), but its overall significance is not yet clear. Aging also alters the patterns in which brush-border enzyme expression alter in response to germ-free conditions (170), whereas small intestinal mucosal lipid esterification and absorption have been reported to appear altered in aging rats (171). In humans, most evidence suggests that lipid digestion and absorption are relatively preserved during aging (172). Thus, the significance of these interesting observations awaits further study.

Gut Peptides and Other Key Molecules Aging is correlated with complex changes in gut peptide levels (157). These have been best studied in the plasma, but they have also been characterized within bowel mucosa. Antral gastrin decreases in the aging rat mucosa (158,159), but neurotensin levels increase with age in the plasma, as well as in the ileal mucosa, of aging rats (160). Conversely, cholecystokinin (CCK) levels within the duodenal or jejunal mucosa increase with age in guinea pigs (161) and humans (162). Several investigators have characterized the density, morphology, proliferation, and apoptosis of neuroendocrine cells within the aging mouse duodenum, demonstrating a complex pattern in which some cells increase, whereas others decrease (135,163). For instance, cells immunoreactive for somatostatin decrease with age, whereas serotoninimmunoreactive cells may increase, and gastrin/CCKimmunoreactive cell density does not change. The morphology

Contractility and Motility Although numerous animal studies have addressed the effects of aging on GI tract motility, their relevance to human physiology and pathophysiology is not always clear. It seems a clinical truism that aging patients may complain more about bowel function. However, it is often difficult to distinguish the effects of aging on the gut per se from influences outside the gut, including altered states of health, diet, activity, and medication. In general, the effects of aging appear most pronounced at both ends, altering swallowing, esophageal contractility, and the mechanisms of fecal continence, whereas the effects of aging on midgut motility are more subtle and of less clear clinical significance. This section moves from the swallowing mechanism distally, considering each segment of the gut in turn.

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 413 Constipation seems common in elderly patients in clinical practice (173). However, the mouth-to-anus transit time does not appear significantly prolonged in older individuals, despite slower gastric emptying (174). Indeed, one study comparing young with middle-aged subjects actually suggested that both gastric and small intestinal transit are accelerated in middle-aged women compared with transit in their younger counterparts (175). The issue is complex, and differences in sex, dietary habits, ethnicity, and other factors may all contribute to subtle differences among studies. In general, the effects of healthy aging on intestinal motility appear relatively slight (176), compared with other factors that may influence this issue and may become more prevalent with age. For instance, pelvic radiation to treat prostate cancer may affect anorectal function (177), and this certainly occurs more commonly among the elderly. A recent metaanalysis (178) suggests a disparity between subjective reports of constipation and laxative use in the elderly and objective clinical assessments of constipation. It seems likely that a subgroup of deconditioned elderly patients with other medical issues may indeed have increasing constipation, together with slow rectosigmoid transit (178), but it is unclear whether this reflects aging itself or stems from alterations in diet, fluid intake, exercise, medication use, or surgical procedures such as cholecystectomy that may occur in many elderly individuals. Cigarette smoking, perhaps more common among older patients in some societies, is also associated with slowing of GI transit (179) and should also be considered in such evaluations. At least one study has demonstrated an increase in mouthto-cecum transit in the elderly independent of the effect of cholecystectomy on transit time (180). Studies of small intestinal motility and transit suggest an increased frequency of propagated clustered contractions and a slowing in the propagation velocity of phase III of the migrating motor complex, but the clinical significance of such findings is unclear (181,182). In general, small-bowel transit appears clinically nonproblematic in most elderly individuals without complicating factors. Conversely, age does not predict significant differences in the small-bowel motility of patients with unexplained bowel symptoms (183). However, it should be acknowledged that mouth-to-anus transit time has been noted to be prolonged in elderly patients with small intestinal bacterial overgrowth (184). In this subgroup of patients, such bacterial overgrowth may be associated with clinical consequences such as malabsorption and malnutrition. The overgrowth is effectively managed by antibiotic therapy rather than intervention directed at small intestinal motility. Interestingly, the development of disabilities that substantially decrease physical activity may be associated with an increased risk for small intestinal bacterial overgrowth (185), although the precise causal linkage here awaits elucidation. In the colon, studies in rats suggest a decrease in colonic transit time in senescent (22- to 28-month-old) rats, which is associated with decreased contractility in response to cholinergic stimulation, possibly related to decreased calcium influx

via membrane calcium channels in the rat colon myenteric plexus (186). Another study of rat colonic histology suggests that the density of myenteric neurons decreases in the sigmoid colon as rats age. Interestingly, this decrease seems to have been aborted by serosal application of the neurotoxic agent benzalkonium chloride (187). Such animal observations have not yet been confirmed in healthy elderly humans, although an age-related decline in inhibitory junction potentials has been described in the descending human colon independent of any age-related variation in immunoreactivity for vasoactive intestinal peptide, peptide histidine-methionine, met5enkaphalin, neuropeptide Y, or somatostatin (188). One recent study found no difference in small-bowel transit time between young and elderly women, but it found a decrease in colonic fermentation in the elderly (189). Many factors could alter colonic fermentation, including differences in dietary fiber intake, colonic bacteriology, luminal mixing or transit, and medication use. The key regulatory factors that influence colonic fermentation are a subject of continuing study. However, although the mechanism of age-related differences in colonic fermentation awaits clarification, such differences in colonic fermentation could well alter subjective perceptions of colonic function in aging patients even without a direct effect on colonic transit per se. Constipation in the elderly may also be associated with outlet obstruction, particularly in women. Elderly women more commonly display abnormal perineal descent, perhaps because of the cumulative effects of pregnancy, as well as general aging and failure of the anorectal angle to open with defecation (190). Either may cause constipation. Constipation in the elderly has also been attributed to decreased rectal sensation. The threshold for sensing rectal distension appears increased in aged but healthy humans, independently of rectal compliance or tone (191). One interesting study, although not specifically directed at aging patients, compared electrical stimulation therapy with biofeedback in patients with impaired rectal sensation and otherwise idiopathic chronic constipation. (This represented about one fifth of all patients seen with functional constipation by this group.) The investigators responsible for this study found that either electrical stimulation or biofeedback therapy improved overall symptoms. However, biofeedback appeared to specifically improve anal residual pressures, whereas electrical stimulation specifically improved rectal sensory thresholds and the maximum tolerated volume in the rectum (192).

Mechanisms of Fecal Incontinence Although many elderly patients report symptoms of constipation, other older patients describe fecal incontinence. Fecal incontinence is often underreported because patients are embarrassed to mention this symptom (193), and yet it represents an important factor that limits activity and reduces quality of life in the elderly (194). Although fecal incontinence is more prevalent among younger women than younger men, this sex gap may be reduced with aging,

414 / CHAPTER 14 as other factors associated with incontinence become more prevalent (195). A sonographic study suggests that the internal anal sphincter thickens with age, but becomes less elastic, because of increased fibrosis, whereas the external sphincter becomes thinner, and anal canal resting and squeeze pressures and rectal reservoir function decrease (196). However, most elderly individuals who display such findings remain continent, and the magnitude of these changes does not necessarily predict incontinence (197). Thus, as for constipation, incontinence of stool is a complex problem, often as much associated with comorbidities or dietary changes as with the direct effects of aging on sphincter function or anorectal sensation. For instance, rectal hyposensitivity (198), possibly related to occult pelvic or spinal nerve injury, has been implicated as possibly causative in some patients with fecal incontinence, chronic constipation, or functional bowel disorders (199). Continence is generally linked to the external sphincter, and fatty atrophy of this sphincter may play a role in incontinence after external sphincter repair procedures (200). However, abnormalities of the internal anal sphincter have also been linked to incontinence in some patients, with higher thresholds required to initiate the rectoanal inhibitory reflex and progressive increases in internal anal sphincter relaxation (201). Despite the details of anorectal physiology that may appear in the literature, it should be emphasized clinically that decreased mental status, chronic constipation with fecal overflow, diabetic neuropathy, and altered ability to reach the toilet in a timely fashion may all predispose to fecal incontinence in the elderly without the incontinence being the direct result of aging. Thus, although sacral nerve stimulation (201–203), biofeedback, radiofrequency energy application (193), and various surgical procedures including anterior levatorplasty and anal sphincteroplasty (204,205) have been advocated for the management of fecal incontinence, primary therapy should first be directed at thorough geriatric assessment of the patient’s diet, stool habits, and interaction with the environment, with manipulation of fecal consistency as indicated.

Key Areas for Future Research Although the aging intestinal mucosa exhibits complex variations in absorptive and secretory capacity, this seems not to have substantial clinical consequences in the healthy aging human because of the large surface area available and corresponding excess capacity. An important subject for future study, however, may be the impact of such changes in disease states in which this excess capacity has been reduced. At the most simplistic level, for instance, the question of how such changes impact small or large intestinal absorption and secretion after major bowel resection may merit additional investigation. In addition, the Scylla and Charybdis of constipation and fecal incontinence in the aging represents a major clinical problem. Improved understanding of the molecular

mechanisms of colonic absorption and motility may ultimately permit the development of targeted therapeutic interventions. In addition, more basic research is required at the physiologic level to understand the muscular mechanisms of continence and incontinence in younger and aging humans.

AGING AND GASTROINTESTINAL CANCERS Epidemiologic Observations Although the incidence of most human malignancies including cancer of the GI tract increases dramatically with advancing age, the precise role of aging in that increase remains a matter of continued controversy and raises a number of questions. For example, is the aging process itself inherently carcinogenic? Does aging enhance carcinogenic susceptibility? Or, is an increased cancer incidence with time merely reflective of a protracted exposure to carcinogenic agents (206)? Numerous endogenous processes inherent to both aging and carcinogenesis implicate potentially shared derangements in the pathway to tumor formation. These include, but are not limited to, alterations in DNA methylation, protooncogene expression, chromosomal fragile sites, DNA mismatch repair, immune system dysfunction, and free radical reaction damage (207). This section discusses these issues with respect to aging and its relation to carcinogenesis of the GI tract, with particular reference to gastric and colorectal cancers, the two most common GI malignancies, which have incidences that increase with aging. Aging has been shown to be associated with increased incidence of premalignant lesions in the stomach and colon. Yamaji and colleagues (208) have studied the effect of age on the malignant potential of colorectal polyps. They have observed that the occurrence of nonmalignant and malignant colorectal neoplasms increases with advancing age, and that the polyps of older persons show greater malignant potential than younger subjects regardless of the size of the lesion. With respect to gastric cancer, atrophic gastritis and its associated intestinal metaplasia are age-related lesions and are considered to be precancerous (209). Aged individuals with atrophic gastritis and intestinal metaplasia are statistically at risk for development of gastric cancer, but precise surveillance recommendations have not been determined. In addition, it has been reported that patients who had partial gastrectomy 15 to 25 years earlier for peptic ulcer disease are at increased risk for gastric cancer (210). Jaszewski and coworkers (211) have analyzed time-dependent changes in ornithine decarboxylase (ODC) activity, an indicator of mucosal proliferation, in the gastric mucosa of subjects who underwent Billroth I or II gastrectomy. They report that ODC activity was significantly higher in Billroth II patients who underwent gastrectomy more than 15 years earlier compared with those in whom gastrectomy had been performed less than 15 years earlier or in normal control subjects (211). They subsequently examined the frequency of H. pylori in postgastrectomy remnants and noted a 58%

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 415 frequency in Billroth II patients, which was associated with a more severe histologic gastritis (212). Atrophic gastritis and its associated intestinal metaplasia are age-related lesions and are considered to be precancerous (213). Baldini and colleagues (214) examined gastric biopsies from antrum and corpus of 117 healthy volunteers to determine a relation between age and H. pylori infection and risk for gastric cancer. They report that in the area with high gastric cancer rate, H. pylori infection was associated with atrophic gastritis and hyperproliferative state of the gastric mucosa, which were observed in both young and aged subjects, indicating a potential risk for development of gastric cancer (214). Ohkuma and colleagues (215) evaluated the effect of H. pylori infection and aging on atrophy and intestinal metaplasia of the gastric mucosa by examining 163 patients. Their results suggest that atrophic gastritis is not a normal aging process, but instead is likely to be the result of H. pylori infection, whereas intestinal metaplasia, a precancerous lesion, is associated with aging and H. pylori infection (215).

Epithelial Growth and Renewal as Potential Premalignant Events Many probable explanations for the age-related increase in cancer incidence have been offered including altered carcinogen metabolism and the cumulative effects of protracted exposure to cancer-causing agents. Neoplasia of the GI tract, including that of the stomach and colon, is a multistage process with hyperproliferation being central to the initiation of carcinogenesis. In view of this, this chapter discusses the growth-related processes of the mucosa of different parts of the GI tract during aging and the regulation of these processes. Cells of the GI tract mucosa are subject to a constant process of renewal, which in healthy adults reflects a balance

between proliferation of precursor cells and exfoliation of surface cells (216–218). The mucosa of the GI tract actually has one of the most rapid cell turnover rates of any tissue in the body. The continuous cell renewal is maintained by the sustained proliferative activity of a small number of mucosal stem cells (216,217,219). The proliferative zone is composed of actively dividing cells distinct from the stem cell, whereas in a larger nonproliferative zone, cells are undergoing progressive differentiation before being extruded into the lumen. Cell production not only compensates for cellular shedding from the surface epithelium, but also assures the renewal of certain specialized cells in the glandular tubes. Any dysfunction in this process may accelerate or diminish the growth rate, resulting in either hyperplasia/hypertrophy or atrophy of the organ, respectively. Knowledge of normal cell proliferation kinetics and regulation of GI mucosal growth during aging is, therefore, essential for the understanding of the pathogenesis of many GI mucosal diseases and disorders linked to aging including cancer. Although earlier observations in the mouse suggest that proliferative activity of the small intestine either decreases (220,221) or remains unchanged (222) with aging, some morphologic and biochemical studies from our and other laboratories have demonstrated that, in barrier-reared Fischer-344 rats, aging is associated with increased mucosal proliferative activity in the stomach and small and large intestines (53,132, 136,223–228). Representative figures (Figs. 14-1 and 14-2) show the age-related increase in proliferative activity in the mucosa of the stomach and different regions of the colon of Fischer-344 rats, as evidenced by increased immunoreactive PCNA. The age-related increase in mucosal proliferative activity, at least in the colon, is also found to be accompanied by an increase in the proliferative zone, a phenomenon that has been observed in patients with precancerous conditions, as well as in subjects with cancer of the colon, rectum, or both. For example, we have reported a marked increase in the proliferative zone of the disease-free colonic mucosa

Gastric mucosa 4 Mo

24 Mo

FIG. 14-1. Photomicrograph showing changes in gastric mucosal proliferative activity, as assessed by bromodeoxyuridine immunoreactivity, in 4- and 24-month-old Fischer-344 rats. (Reproduced from Majumdar and Jaszewski [235], by permission.)

416 / CHAPTER 14 COLON Proximal

Middle

Distal

4 mo

13 mo

22 mo

FIG. 14-2. Photomicrograph showing changes in mucosal proliferative activity in the proximal, middle, and distal colon of 4-, 13- and 22-month-old rats. (Reproduced from Xiao and colleagues [136], by permission.)

from subjects with adenomatous polyps, ulcerative colitis, and colorectal cancer, compared with control subjects (229). In experimental animals, administration of azoxymethane, a colonic carcinogen, has also been shown to cause widening of the proliferative zone of the colonic crypts (230,231). Morphologic studies of the colonic mucosa of human volunteers have further shown that, whereas cell proliferation in young subjects is confined to the lower two thirds of the crypt, with aging there is a major shift from the base to the middle and upper third of the gland (232–234). The observations of the age-related increase in mucosal proliferative activity, together with expansion of the proliferative zone in the colon, both of which are common occurrences in precancerous conditions and in cancer, have led us and others to postulate that aging may predispose the GI tract to neoplasia (132,224,228,235,236).

Inhibition of Apoptosis as a Potential Premalignant Event In addition to increasing proliferation of the mucosa of much of the GI tract, alterations in mucosal apoptosis (programmed cell death) may also contribute to age-related increases in predisposition of the tissues to carcinogenesis. Alterations in apoptosis are now recognized to be an important event in development, in normal cell turnover, in hormone-induced tissue atrophy, and in many pathologic

conditions including cancer (237–239). It has been demonstrated that cells derived from a variety of cancers, including colorectal cancer, show decreased apoptosis in response to physiologic stimuli (237,240,241). Additional evidence supporting the involvement of apoptosis in the development of colorectal neoplasia comes from the observations that transformation of colorectal epithelium to carcinoma is associated with progressive inhibition of apoptosis (240). Little information is available on age-related changes in apoptosis in tissues of the GI tract. To determine whether changes in apoptosis could contribute to the age-related increase in the incidence of gastric or colorectal carcinomas, or both, we examined the number of apoptotic cells in the gastric and colonic mucosa by TUNEL assay. We have observed that the number of apoptotic cells in the mucosa of the stomach and all regions of the colon of 22- to 24-month-old (aged) Fischer-344 rats that fasted overnight is lower than in the corresponding tissues from 4-month-old (young) counterparts (Table 14-1). Further support for the age-related decrease in apoptosis in GI mucosa comes from the observations that cleavage of 115-kDa full-length poly(ADP-ribose) polymerase, a substrate for caspase-3 and probably for other caspases, to the 85-kDa product was decreased by colonic mucosal lysates from 13- and 22-month-old Fischer-344 rats compared with their 4-month-old counterparts (136). These changes were accompanied by a concomitant reduction in the levels of 20-kDa active caspase-3 and -8, as well as 10-kDa active caspase-9 (136). To further determine the intracellular

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 417 TABLE 14-1. Changes in the number of apoptotic cells, as assessed by TUNEL assay, in the mucosa of the stomach and different regions of the colon of overnight-fasted 4- to 5-, 12- to 14-, and 22- to 24-month-old Fischer-344 rats Number of apoptotic cells per mm2 Tissue Stomach Colon Proximal Middle Distal

4–5 mo

12–14 mo

22–24 mo

15.2 ± 1.2

11.2 ± 1.5

10.6 ± 1.2a

8.0 ± 0.6 10.2 ± 0.8 8.2 ± 1.3

7.4 ± 1.1 7.3 ± 1.8 5.3 ± 0.6

6.7 ± 0.5a 5.3 ± 0.7a 4.9 ± 1.0a

Values represent mean ± standard error of the mean. ap < 0.01, compared with the corresponding values in 4- to 5-month-old rats.

events regulating the apoptotic processes in the colonic mucosa during aging, the levels of Bak, the induction of which promotes apoptosis, and BclxL, which promotes cell survival, were also examined in the colonic mucosa of 4- to 12- and 22-month-old Fischer-344 rats that fasted overnight. Whereas the levels of Bak were found to be about 50% lower, Bcl-xL levels were increased by about 50% in 22-month-old rats compared with 4- or 12-month-old animals (136). Lee and colleagues (242) have examined age-related changes in colonic mucosal apoptosis in Fischer-344 rats. In contrast with what we reported, immunohistochemical studies conducted by Lee and colleagues (242) show only a few apoptotic bodies in the colonic with no significant difference between young and aged rats. Although the discrepancy between the two studies is not fully understood, one possibility could be the nutritional status of the animals. Whereas Lee and colleagues (242) killed the animals in fed conditions, in our studies, the animals fasted for 24 hours to minimize the effect of food-induced stimulation of apoptosis. In fact, Lee and colleagues (242) report that the expression of Bax protein, which promotes apoptosis, is higher in the upper part of the colonic crypt of aged rats than in their younger counterparts, which they suggest increases the sensitivity of the colonic mucosa to apoptotic stimuli. However, in view of our observation of age-related decrease in apoptosis in the colonic mucosa, it is tempting to speculate that aberrant survival of a small group of cells or even a single cell in the colonic mucosa during aging could promote accumulation of secondary genetic changes, and thereby enhance susceptibility of the tissue to certain carcinogen(s) or tumor promoter(s). Although reduction of apoptosis was more modest in the gastric mucosa, that the marked increase in proliferative activity in the gastric mucosa in aged rats was not accompanied by a parallel increase in apoptosis suggests that aging may also promote accumulation of cells with secondary genetic changes in the gastric mucosa.

Changes in Susceptibility to Carcinogens Although many probable reasons have been suggested for the age-related increase in malignancies, including altered

carcinogen metabolism and cumulative effects of long-term exposure to cancer-causing agents (243–245), the possibility that aging may render target tissue(s) more susceptible to carcinogen(s) or tumor promoter(s), or both, has not been thoroughly investigated, and the results remain inconclusive in the studies that have been performed (246–248). Magnuson and colleagues (249) compared the susceptibility of the colon to azoxymethane, a colonic carcinogen, in 1- (young) and 12-month-old (old) Sprague–Dawley rats by examining the formation of aberrant crypt foci (ACF). Aberrant crypt foci, which were initially described by Bird (250) as lesions consisting of large, thick crypt in methylene blue–stained specimens of azoxymethane-treated mice, have since been detected in humans with colon cancer, as well as in benign adenomas, which are believed to be the precursors of colorectal cancer (251–254). Several investigators have demonstrated that patients with colon adenomas or cancer have significantly more ACF than control subjects (253). In addition, ACF showed 60% to 70% K-ras mutations (254–257). These and other observations have led to the postulation that ACF are neoplastic and likely are precursors of adenomas and carcinomas (258,259). In the azoxymethane model of colorectal cancer in mice, azoxymethane has been shown to produce a single to multiple aggregates of ACF, leading to the development of colonic adenocarcinomas (252,257). Magnuson and colleagues describe results that show a significantly greater number of ACF in older rats both at 6 and 14 weeks after azoxymethane treatment when compared with their corresponding younger counterparts (249). Moreover, they report that an increase in the number of ACF in older animals occurred in each size category and was not restricted to the small ACF only (249). A similar observation was also made by us in Fischer-344 rats, when 4- (young) and 24-month-old (aged) rats were treated with azoxymethane for 4 weeks to induce ACF formation in the colon (260). We observed that the number of ACF formed in aged Fischer-344 rats in response to azoxymethane treatment was markedly greater than in their 4-month-old counterparts (260). To further evaluate the age-related increase in susceptibility of the GI tract to cancer-causing agent(s), we examined the early events of colorectal neoplasia by measuring changes in proliferative activity in the colonic mucosa of young (4-month-old) and aged (24-month-old) Fischer-344 rats 5 days after a single injection of azoxymethane or vehicle (control animals). Morphometric analysis revealed the number of bromodeoxyuridine (BrdU)-immunoreactive cells (a measure of proliferative activity) per colonic crypt of control aged rats to be about 40% greater than in their younger counterparts (8.63 ± 2.3 in young control animals vs 12.3 ± 1.9 in aged rats; p < 0.025), indicating a higher basal proliferative activity in the colonic mucosa of aged rats (Fig. 14-3). Although azoxymethane markedly stimulated the number of BrdU-positive cells in both age groups, the magnitude of stimulation was found to be greater in aged than in young rats when compared with the corresponding control animals (see Fig. 14-3). In young rats, the number of BrdU-positive cells were increased by about 220% to an average of 28.2 ± 3.5 cells/crypt, whereas in aged rats, it was

418 / CHAPTER 14

A

B 4-mo Cont.

C 4-mo AOM

D 24-mo Cont.

24-mo AOM

FIG. 14-3. Photomicrograph showing bromodeoxyuridine-immunoreactive cells in the colonic mucosa of young (4-month-old) and aged (24-month-old) Fischer-344 rats 5 days after a single subcutaneous injection of the colonic carcinogen azoxymethane (AOM; 20 mg/kg).

increased by about 300% to an average of 49.8 ± 2.3 cells/ crypt when compared with the corresponding control animals. In addition, azoxymethane produced a change in distribution of BrdU-positive cells in the colonic mucosa. Whereas in young rats, most of the BrdU-positive cells were located at the bottom half of the crypt, in aged rats, they were distributed throughout the crypt with the exception of 10% to 20% of the upper region (see Fig. 14-3). The latter is similar to what we noted earlier in the colonic mucosa of patients with adenomatous polyps and ulcerative colitis (229), conditions that are considered precancerous (218). To further explore the age-related susceptibility of the colonic mucosa to carcinogens, we examined the in vitro changes in ODC and tyrosine kinase activities in the colonic mucosa in response to methylazoxymethanol, the active metabolite of the colonic carcinogen azoxymethane. We observed a greater degree of stimulation of ODC and tyrosine kinase activities in aged than in young colonic mucosa in response to methylazoxymethanol, the active component of azoxymethane (261), indicating an increased susceptibility of the colonic mucosa of aged rats to this carcinogen. A similar phenomenon was observed with TGF-α (262), a potent mitogen for mucosa of various parts of the GI tract including the colon (263,264). A growing body of evidence also suggests that TGF-α may play a critical role in the development of colorectal neoplasia through autocrine/ paracrine mechanisms. This is supported by the observation that cell lines derived from adenocarcinomas of various tissues, including the colon, show increased expression of TGF-α and its receptor, EGFR (263–269). Because TGF-α exerts its mitogenic action by activating the intrinsic tyrosine kinase activity of EGFR, we compared the changes in TGF-α-mediated activation of EGFR-associated tyrosine kinase between young and aged rats (262). Despite a higher basal EGFR tyrosine kinase activity level in the colonic mucosa of aged rats, TGF-α produced a significantly greater stimulation of EGFR tyrosine kinase activity and tyrosine phosphorylation of EGFR and several other mucosal proteins.

Because this occurred despite a relatively high basal EGFR tyrosine kinase in aged rats, we suggest that aging is associated with increased responsiveness of the colonic mucosa to TGF-α (262). A similar observation was also made with isolated colonocytes from azoxymethane-treated rats. Both EGF and TGF-α caused a comparatively greater degree of stimulation of EGFR tyrosine kinase in colonocytes isolated from rats 5 days after a single injection of azoxymethane than those from control animals (231). Taken together, these alterations in GI mucosal proliferation and differentiation strongly suggest that aging is associated with increased susceptibility of the GI tract not only to carcinogen, but also to certain growth factors, particularly the EGF family of peptides. These factors may play a role in malignant transformation of the GI tissues during aging.

Mutational Events Carcinogenesis, which is a multistep process, results from accumulation of mutations during progression from normal epithelium to carcinoma (270). Genetic changes that occur at different stages of epithelial cell carcinoma have been extensively studied by Fearon and Vogelstein in human colon cancer (270) and have been reviewed by others. However, at least for colon cancer, it has been suggested that the loss or inactivation of the tumor-suppressor gene in adenomatous polyposis coli (APC) initiates genomic instability that may produce the phenotypic appearance of an adenoma. Advanced tumors, however, possess mutation or deletion, or both, of a number of oncogenes and tumor-suppressor genes not seen early adenoma (270). Although such detailed analysis of genetic alterations has not been performed for gastric cancer, inactivation of several tumor-suppressor genes, including APC, p53, and deleted in colorectal cancer (DCC), has been observed in gastric cancer (271). However, little information is available as to whether aging, which is thought to predispose the GI tract to carcinogenesis, is associated with

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 419 increased inactivation of tumor-suppressor genes. If aging predisposes the GI tract to carcinogenesis, we postulate that aging may be associated with altered expression or mutations of genes involved in cell growth, or both. To test this hypothesis, we investigated the age-associated changes in mutations of APC, DCC, p53, and K-ras genes in the gastric mucosa of 19 healthy volunteers of varying ages (25–91 years) (272). Specifically, we studied the loss of heterozygosity (LOH) of these genes in the gastric mucosa. We observed that 3 of 19 subjects (16%) older than 60 years showed LOH of at least one of the tumor-suppressor genes. Among the subjects older than 60 years, the incidence of LOH was 38%, and two thirds of the subjects had mutations in more than one tumorsuppressor genes (272). In addition, we noted that in all three affected subjects, mutations in APC, DCC, or p53 were located in the body of the stomach, suggesting increasing susceptibility of this region to neoplastic changes (272). To further determine a link between genetic alterations and GI cancers during aging, Hastle and coworkers (273) examined telomere length in relation to age and colon adenomas. Telomeres are believed to be critical in the maintenance of chromosomal integrity and are composed of a sequence of six nucleotides (TTAGGG) that is repeated from several hundred to a thousand times (273,274). These sequences are synthesized by telomerase, a ribonucleoprotein enzyme composed of both protein and RNA. Telomerase makes a DNA copy of its own RNA sequence by reverse transcription. The extension of telomeres by telomerase is necessary to offset the normal shrinkage of chromosomes that occurs after each DNA replication. However, the telomeres shorten progressively with aging in somatic cells. In contrast, immortalized and some carcinoma cells show maintenance of telomere length despite repeated cell division due to the activity of telomerase (275). Hastle and colleagues (273) note a reduction in telomere length in normal colonic tissue of older compared with their younger individuals. Length reductions were also seen in colon adenomas and carcinomas. These authors hypothesize that telomere loss may render chromosomes less stable and impair viability. Alternatively, they may make chromosomes more prone to fusion bridge breakage cycles, leading to daughter cells that receive partly deleted and duplicated chromosomes. These alterations may have a role in generating loss of alleles of restriction fragment length polymorphisms that often occur in colorectal cancers (274). In a similar study, Furugori and colleagues (276) examined the telomere length in nonneoplastic gastric mucosal specimens from 38 human subjects aged between 0 and 99 years. They report an average annual loss rate of 46 base pairs. Telomere lengths in gastric carcinomas were also shorter than those for intestinal metaplasia (276). Issa and colleagues (277) studied the estrogen receptor (ER) in relation to aging and colorectal cancer. The ER may actually serve as a tumor-suppressor gene in that it is expressed in normal tissue, is altered in tumors, and suppresses growth in tumor cell lines and tissues. CpG islands located in the promoter region of genes are normally free of methylation, but methylation is common in islands of the ER in aging,

in colonic mucosa, and in colon tumors (277). Moreover, methylation of the ER in older healthy subjects or those with colon cancer is associated with inactivation of the gene. Folic acid is the primary methyl donor for DNA methylation and for producing the purines and pyrimidines required for DNA synthesis. Lack of folate or methyl group nutrients in the diet has been shown to cause DNA hypomethylation in both rats and humans, whereas an opposite phenomenon is noted with excess folic acid (278–280). Crott and coworkers (281) have studied the effects of dietary folate and aging on gene expression in the colonic mucosa of rats, and its implications in colon cancer. Gene expression was measured using oligonucleotide arrays. The authors observed that in folate-deficient rats, aging induced the down-regulation of immune-related genes, urokinase, p53, insulin-like growth factor binding protein-3 (IGF-BP3), and vav-1 oncogene, whereas folate supplementation caused down-regulation of vascular endothelial growth factor and caspase-2. They suggest that folate depletion with aging may increase the risk for development of colon cancer, and that folate supplementation may reduce the risk for age-associated cancers by suppressing deleterious changes in the expression of certain genes. Hypermethylation has been shown to cause functional inactivation of a number of tumor-suppressor genes (282). In some cases, CpG islands in the 5′ regions of tumorsuppressor genes are methylated, and their expressions are switched off. For example, in the breast cancer cell line MDA-MB-435, methylation of the promoter of connexin 26 has been shown to be associated with a concomitant loss of its expression (283). Hypermethylation has also been shown to cause functional inactivation of p16INK4A (284). In Fischer-344 rats, aging has been shown to be associated with increased expression and activation of EGFR in the GI mucosa (285,286), a phenomenon that has also been observed in preneoplastic and neoplastic lesions (229,230, 263,269). Results from several laboratories, including our own, have demonstrated that folic acid could be chemopreventive (287–290), and that this could be attributable, in part, to inhibition of EGFR activation (291). Indeed, we observed that supplemental folic acid and its metabolite 5-methyltetrahydrofolate greatly attenuated serum-induced activation of EGFR promoter activity in colon cancer cells in vitro (292). Supplemental 5-methyltetrahydrofolate also attenuated the basal EGFR promoter activity in colon cancer cells in vitro (292). However, whether the EGFR promoter function in the GI tissues during aging would also be similarly affected by supplemental folic acid or its metabolite(s) remains to be determined.

Key Areas for Future Research It is obvious that aging is associated with increased incidence of both premalignant and malignant gastric and colonic lesions. Interestingly, there is little evidence that the small intestine is similarly affected. Investigation of the disparate mechanisms of small intestinal malignant transformation

420 / CHAPTER 14 and colonic malignant transformation may therefore provide some clues as to why gastric and colonic cancers increase in the aging population. In addition, further research into the general biology of such tumors obviously is required. Increasing evidence suggests that the aging colonic mucosa is more susceptible to chemical carcinogens, but the mechanism of this effect is unknown. The changes in bacterial flora within the colonic lumen during aging are poorly characterized and may also contribute to the development of colonic cancer in the elderly. Finally, although somewhat outside the scope of basic physiology, it has become increasingly obvious that the real key to curing colon cancer is prevention. Thus, research into chemoprevention and improved screening modalities in high-risk elderly patients clearly is warranted.

REGULATION OF MUCOSAL GROWTH DURING AGING Intracellular Mechanisms Although aging, as described in the previous section, is associated with increased proliferation and decreased apoptosis in much of the GI tract, the intracellular events responsible for regulating these processes are poorly understood. With respect to the age-related increase in GI mucosal proliferative activity, however, it has been demonstrated that in Fischer-344 rats, aging is associated with a progressive increase in activity and protein levels of cyclin-dependent kinase (cdk2) in the gastric and colonic mucosa (137,293). Cyclin-dependent kinases are a highly conserved family of serine/threonine protein kinases that phosphorylates several molecules involved in cell cycle progression. Binding of cyclins to Cdks activates their intrinsic kinase activity, triggering events that induce progression of cells through different phases of the cell cycle (294,295). Moreover, activation of Cdks depends on the binding of a specific cyclin (295). For example, whereas Cdk4 and Cdk6 require D-type cyclins for their activation (296), Cdk2, which is involved in G1/S transition, is activated by cyclin E (297,298). In addition, Cdks are also regulated by a class of Cdk inhibitors. Of these, p21Waf1/Cip1 and p27kip1, which have been studied extensively, are considered universal inhibitors of different cyclin-Cdk complexes (299,300). The age-related increase in gastric and colonic mucosa was accompanied by a concomitant increase in cyclin D1 levels and reduction of p21Waf1/Cip1 (137,293). In addition, it was observed that the levels of phosphorylated retinoblastoma, a form that is involved in regulating progression of cells through the S-phase, are greater in both gastric and colonic mucosa of aged compared with young rats (137,293). Taken together, these results suggest that aging is associated with increased transition of G1- to S-phase, as well as progression through the S-phase of the cell cycle in both gastric and colonic mucosa.

Nutritional and Hormonal Factors Gastrointestinal mucosal cell proliferation is known to be under the regulation of a number of nutritional and hormonal factors. In general, deprivation of food results in decreased cell proliferation, and refeeding reverses the situation (301). Holt and Yeh (224,226) have compared the effect of fasting and refeeding on small and large intestinal proliferative activity in young (3- to 4-month-old) and aged (24- to 28month-old) Fischer-344 rats. In the mucosa of both small and large intestines, a 3-day fasting resulted in 40% to 60% reduction in proliferative activity in young rats, whereas it was decreased by only 10% to 20% in old rats when compared with their corresponding fed control animals (224,226). Although in both age groups refeeding increased small intestinal proliferative activity, in aged rats, this increase was associated with broadening of the proliferative zone (224,226). Moreover, in fasted aged rats, proliferative responsiveness of the large intestine to food was blunted (223–225). The latter observation suggests that nutritional modulation of mucosal cell proliferation is affected by aging. Age-associated changes in GI mucosal cell proliferation could be secondary to alterations in hormonal influences. Over the past few decades considerable evidence has appeared to show that a number of GI hormones/growth factors, specifically gastrin, bombesin (an amphibian peptide that is structurally and functionally analogous to gastrin-releasing peptide), and the EGF family of peptides regulate mucosal proliferation in much of the GI tract, including the stomach (235,302). However, responsiveness of the gastric mucosa to these peptides changes at different stages of life. We have reported earlier that in rats, the gastric mucosa becomes responsive to the growth-promoting action of gastrin around the third postnatal week of life (303). At this time, the parietal cells also become sensitive to gastrin and secrete acid in response to the hormone, when gastrin receptors also appear (304). In contrast, the functional properties are either decreased or remain unchanged during advancing age. For example, basal gastric acid and pepsin output decline during aging, as does gastrin secretion (54,158). In contrast, antral gastrin levels increase during this period, as does mucosal histidine decarboxylase activity (54,158). The age-related decline in gastrin secretion could be attributed partly to a higher ratio of somatostatin (D) to gastrin (G) cells in the antral mucosa (158). With aging there is also a progressive loss of gastric mucosal responsiveness to both acid secretory (54) and growth-promoting (158) actions of gastrin. Although the underlying mechanisms are unknown, one possibility could be the loss of functional receptors of gastrin. Singh and colleagues (305) report that the number of gastrin binding sites in the gastric mucosa of 24-month-old rats was lower than in their 3- and 6-month-old counterparts. Taken together, the results suggest a relation between gastrin receptor populations and gastric mucosal responsiveness to gastrin. Like gastrin, bombesin has also been shown to stimulate gastric mucosal cell proliferation in young animals (306,307).

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 421 To evaluate its role in GI mucosal cell proliferation during aging, we examined changes in gastric mucosal DNA synthesis and ODC activity in young (4-month-old) and aged (24-month-old) rats after continuous infusion of bombesin (300 ng/kg/h) for 2 weeks. We observed that whereas in 4-month-old rats bombesin infusion resulted in a significant twofold increase in gastric mucosal DNA synthesis and ODC activity, in aged rats, the peptide had no effect (308). Whether the lack of response of aged gastric mucosa to the growth-promoting effect of bombesin could be partly the result of loss of receptors of the peptide is unknown.

Growth Factors and Protein Kinases Epidermal growth factor family of peptides, specifically EGF and TGF-α, are known to regulate GI mucosal cell proliferation (235,263,264). Feldman and Aures (309) were the first to demonstrate that administration of EGF to suckling mice stimulates gastric and duodenal mucosal ODC activity. Subsequently, Malo and Menard (310) reported that EGF not only stimulates growth of the proximal, but also the distal small intestine. Similar observations were also made by us and other investigators (311–314). Using an organ culture system, we have demonstrated that EGF stimulates protein and DNA synthesis in both prenatal and neonatal rats (311) and ODC activity in the colonic mucosa of young mature rats (312). This observation not only indicates a direct growth-promoting effect of EGF on both small and large intestinal mucosa, but also shows that EGF may have a role in regulating mucosal growth from before birth to adulthood. Gastric mucosa also responds to the growthpromoting action of EGF. Dembinski and Johnson (314) report that in 10-day-old suckling rats, repeated injections of EGF for 5 days result in a significant increase in gastric mucosal DNA, RNA, and protein content. We have further demonstrated that in unweaned rats, daily injections of EGF at a dose of 20 µg/kg for 7 days significantly stimulate both gastric and small intestinal protein and nucleic acid content (110). Repeated injections of EGF for 2 days have also been shown to stimulate gastric mucosal DNA synthesis in young mature rats (111,315). More recently, we examined the responsiveness of gastric mucosa of 4- and 24-monthold Fischer-344 rats to TGF-α, the structural and functional analogue of EGF (315). We observed that pharmacologic doses of TGF-α, which stimulated gastric mucosal proliferative activity in 4-month-old rats, caused a marked inhibition of the same in their 24-month-old counterparts (315). We postulated that this inhibition is, in part, due to increased sensitivity of aged gastric mucosa to these peptides such that low doses of these peptides are stimulatory, whereas high doses inhibit proliferative processes (111). In support of this postulation, we have observed that the concentration of TGF-α needed to induce maximal stimulation in EGFR tyrosine kinase activity in gastric mucosal membrane preparations from aged rats is considerably less than that required

for the same induction in young rats (111). Likewise, Evers and colleagues (316) observed that whereas administration of neurotensin, a peptide present in the GI mucosa, increased the crypt cell density in the intestine of both young and aged rats, in aged rats, the peptide enhanced an increase in crypt depth and villous height. This observation is similar to observations after refeeding of rats that had been fasted for 72 hours (224,225,317) and after administration of the carcinogen dimethylhydrazine (318). In evaluating the age-related increase in GI mucosal proliferative activity, we have focused on the role of tyrosine kinases, which catalyze phosphorylation of tyrosine residues in proteins and play a crucial role in regulating proliferation, differentiation, and transformation of cells (319,320). The notion that tyrosine kinases may play a role in regulating GI mucosal cell proliferation comes from our earlier observation that the age-related increase in gastric mucosal proliferative activity is also accompanied by a parallel increase in overall mucosal tyrosine kinase activity and tyrosine phosphorylation of several membrane proteins (53,227). However, tyrosine kinases are associated with the products of many protooncogenes, as well as with receptors of several growth factors, including the EGFR, the common receptor for EGF and TGF-α (319,320), which is a 170-kDa transmembrane glycoprotein that spans the plasma membrane. It contains an amino-terminal extracellular ligand-binding domain, a single-transmembrane–anchoring region, and a carboxyl-terminal intracellular domain that has tyrosine kinase activity (105). High-affinity binding sites for the EGF family of peptides, specifically EGF and TGF-α, have been detected in much of the GI tract, with the greatest concentrations in the esophagus, stomach, and colon (321–324). Moreover, mRNA for TGF-α, as well as its receptor, has been detected in gastric and colonic mucosal cells (324–327). Overexpression of EGFR with increased tyrosine kinase activity has been associated with many human malignancies, including gastric and colonic cancers (264,328,329). Results from our laboratory have demonstrated that activation of EGFR tyrosine kinase is associated with increased GI mucosal proliferative activity. We have reported that increased mucosal proliferative activity in the colon in response to azoxymethane (a colonic carcinogen) and in the gastric mucosa after injury is accompanied by a concomitant increase in EGFR tyrosine kinase activity, and that these increases can be greatly attenuated by prior administration of tyrphostin, an inhibitor of EGFR tyrosine kinase (230,330). These and other relevant observations suggest a role for EGFR tyrosine kinase in GI mucosal cell proliferation. We have reported that an age-related increase in gastric and colonic mucosal cell proliferation is accompanied by a marked increase in expression and activation of several tyrosine kinases, including EGFR and its family member ErbB-2/HER-2 (262,285,331). Aging is also found to be associated with activation of EGFR in the gastric and colonic mucosa, as evidenced by increased tyrosine phosphorylation of EGFR (286). Ligand binding is one of the primary causes

422 / CHAPTER 14 for activation of intrinsic tyrosine kinase activity of EGFR, triggering a complex array of enzymatic and biological events leading to stimulation in cell proliferation. There is considerable evidence that shows that TGF-α, which is synthesized in mucosa of much of the GI tract including the stomach and colon, may play a key role in regulating EGFR tyrosine kinase activity in different pathophysiologic conditions. For example, cell lines derived from adenocarcinomas of the colon express both TGF-α and EGFR (264,328,329). Our observation that the azoxymethane-induced colonic mucosal EGFR tyrosine kinase activity is accompanied by a parallel increase in synthesis and secretion of TGF-α (230) suggests that TGF-α may be partly responsible for regulating this process through an autocrine/paracrine mechanism. However, results from cell-surface and biochemical studies have demonstrated that the presence of the transmembrane TGF-α is a normal consequence of TGF-α synthesis, and in most cases, the peptide is present on the cell surface in its precursor form, which can also activate EGFR (332–334). We have observed that the age-related increase in EGFR tyrosine kinase in the gastric mucosa is accompanied by a marked increase in 16- to 20-kDa precursor forms of TGF-α in the mucosal membrane fraction, but not in the cytosol (285). The colonic mucosa also shows a similar phenomenon (286). In addition, our recent in vitro studies show that the membrane-bound precursor form(s) of TGF-α from the gastric and colonic mucosa of aged rats induces a greater stimulation in EGFR tyrosine kinase activity than those from young rats (286). This could be attributed partly to a greater amount of TGF-α bound to its receptor, the EGFR (286). However, because TGF-α, but not EGF, is synthesized in much of the GI mucosa of normal adult rats (230,332,335), we postulate that TGF-α plays a key role in regulating gastric and colonic mucosa proliferation during aging, probably through an autocrine/ paracrine mechanism. Results from our laboratory also suggest that aging enhances sensitivity of the GI mucosa to both EGF and TGF-α such that low doses of the peptide stimulate the EGFR signaling cascades leading to stimulation in proliferation, whereas high doses inhibit these processes. This inhibition is found to be caused by enhanced internalization of the ligand-receptor complex (118). Likewise, responsiveness of the colonic mucosa to both EGF and TGF-α is also augmented after azoxymethane treatment (230,231). Activation of EGFR triggers a complex array of enzymatic events through activation of Ras, converging on mitogenactivated protein kinases, which after translocation to the nucleus activate transcription factors and induce activation of genes that promote growth (336–338). It has been demonstrated that with aging there is a marked activation of extracellular-signal regulated kinases and c-Jun NH2-terminal/ stress-activated kinases in the gastric mucosa of Fischer-344 rats, and that these changes are accompanied by a concomitant stimulation of the gastric mucosal DNA-binding activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) (339). In addition, it has been demonstrated that aging is associated with a marked induction of TGF-α-induced activation of AP-1 in the gastric mucosa (340). Because activation of

AP-1 and NF-κB is thought to result in induction of proliferation and inhibition of apoptosis (341,342), the age-related induction of transcriptional activity of AP-1 and NF-κB in the gastric mucosa suggests that they might be involved in regulating GI mucosal growth during aging. Although the precise regulatory mechanisms for the age-related increase in EGFR activation in the mucosa of the GI tract is unknown, our data suggest this could be partly the result of loss of ERRP (EGFR-related peptide), a novel “negative regulator,” which we have identified and characterized (343). Epidermal growth factor receptor–related peptide, a ~55-kDa secretory protein, possesses 85% to 90% homology to the external ligand binding domain of EGFR and 50% to 60% homology to the external domain of other members of the EGFR family (343). Expression of ERRP is high in benign human colon, stomach, liver, and pancreas but low in the respective invasive adenocarcinomas (343–348). We report that ERRP inhibits cellular growth by attenuating EGFR signaling processes (343,344). Overexpression of ERRP in colon cancer cell lines, HCT-116 and Caco2, or exposure of the cell lines to recombinant ERRP inhibits EGFR activation, as well as proliferation (343,344). In addition, intratumoral or subcutaneous injections of recombinant ERRP cause regression of palpable tumors in SCID mice xenograft of colon cancer cells (343). Because aging and carcinogenesis of GI tissues are associated with increased expression and activation of EGFR, we hypothesize that these increases could be partly the result of loss of ERRP, which we believe to be a suppressor of EGFR function. To test this hypothesis, we examined the relation between age-related changes in EGFR activation and expression of ERRP in the gastric mucosa of rats. Indeed, we observed that in the gastric mucosa of Fischer-344 rats, the age-related increase in EGFR activation was associated with marked reduction in ERRP levels (345). The same phenomenon was also noted in the colon, where the levels of ERRP in both the proximal and distal colon of the Fischer-344 rats were found to be lower in aged (21-month-old) than in young (6-month-old) animals (260). The age-related decrease in ERRP expression in the colonic mucosa was further decreased by administration of the colonic carcinogen azoxymethane (260). Because ERRP possesses a substantial homology to the ligand-binding extracellular domain of EGFR, we hypothesized that ERRP may compete with EGFR for the ligands. To test this hypothesis, we analyzed the amount of TGF-α bound to EGFR and ERRP in the gastric mucosa of 6- and 24-month-old Fischer-344 rats. We observed the amount of TGF-α bound to ERRP was about 50% less in the gastric mucosa of aged than in young rats (345). In contrast, the amount of TGF-α bound to EGFR in the gastric mucosa of aged rats was found to be about 70% greater than in young rats (345). These changes were accompanied by parallel alterations in EGFR and ERRP levels (345). We suggested that the higher amount of TGF-α bound to EGFR in the gastric mucosa of aged rats could be the consequence of a greater availability of TGF-α resulting from decreased amount of TGF-α bound to ERRP. The age-related decline in

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 423 ERRP levels, together with a concomitant reduction in the amount of precursor forms of TGF-α bound to ERRP, suggests that increased activation of EGFR, observed in the GI mucosa of aged rats, could be partly due to a greater availability of TGF-α and possibly other ligands of EGFR resulting from the loss of ERRP.

Gene Expression Patterns Advances in genomic techniques have allowed assessment of global changes in gene expression patterns. Using this technology, investigators have demonstrated that aging results in marked changes in the expression of IGF-I and IGFBPs in the colonic mucosa of rats (349). Because IGF-I plays a major role in the regulation of mucosal proliferation in the intestine (349), the age-related increase in IGF-I expression in the colon, it is tempting to speculate a role for IGF-I in modulating intestinal mucosal growth during aging.

Key Areas for Future Research Some research suggests that abnormalities in growth factors, growth factor receptors, and their regulators may play a major role in the regulation of these events. This is an important area for future basic science research because changes in mucosal growth affect the structural and functional properties of the mucosa and may also contribute to growth-related diseases such as cancer.

AGING AND SURGERY OF THE GASTROINTESTINAL TRACT As human life expectancy is prolonged and medical science advances, elderly patients increasingly have intraabdominal septic pathology or are increasingly subjected to the stresses of elective or urgent surgical procedures, or both (350,351). It therefore becomes important to begin to understand how aging affects the adaptation of the intestine to fasting and refeeding, sepsis, and bowel resection.

differences in mucosal composition and surface area, particularly if these studies include preadult humans or animals (354). Although the mechanisms of these various effects are therefore not all clear, they represent an important target for future study, because elucidation of the responsible factors may identify targets by which nutrition may be improved in elderly patients consuming marginal diets. Indeed, animal studies suggest that aging appears to alter intestinal responses to fasting and feeding in complex ways, often decreasing the response or inhibiting biological pathways. For instance, basal small intestinal ODC activity and polyamine synthesis appear higher in aging rats than young rats, and they are more dysregulated by fasting and refeeding in the older animals (355). Similarly, jejunal epithelial cells from aged mice demonstrate decreases in oxygen uptake in the absence and presence of exogenous substrate, glucose oxidation, entry of glucose and glutamine into the TCA cycle, and lower contribution of glucose and glutamine carbon to anaplerosis and subsequent synthetic compounds. These differences are greater in fed mice than fasted mice. These changes are all consistent with suppression of the role of the TCA cycle in biosynthesis and oxidative metabolism (356). Such results should be interpreted with caution, however, because another study found that age-related differences in oxidative metabolism in rats varied with the strain of rats studied (357). The induction of apolipoproteins A-1 and A-IV in the intestine and liver by fasting and refeeding has also been reported to be markedly blunted in 25- to 34-monthold mice compared with 6- to 9-month-old mice (358). Although the metabolic response to moderate surgical stress such as an elective colon resection may not differ between healthy younger humans and healthy older humans (359), it has long been a clinical observation that aging humans may be more susceptible to the severe stress of intraabdominal sepsis than younger humans (360). Murine studies seem to confirm this suspicion, even in the absence of human comorbidities. Although the mechanisms of the effect are not entirely clear, senescent mice die more rapidly and at lower bacterial thresholds in cecal ligation and puncture models, possibly related to an age-related defect in glucocorticoid inhibition of TNF-α (361,362).

Adaptation to Bowel Resection Adaptation to Fasting and Sepsis Intestinal absorption may be decreased in aging humans. For instance, ileal reabsorption of conjugated bile acids is decreased in older adults (352). Indeed, older patients may exhibit malabsorption of carbohydrates, lipids, amino acids, minerals, or vitamins (353). It remains unclear to what extent these defects represent aging per se and to what extent they reflect the development of comorbidities. For instance, although substantial abnormalities are found in lipid absorption in many elderly patients, these often correlate with intercurrent disease (172). The interpretation of studies of transport capacity may also be obscured by age-dependent

The adaptation to bowel resection poses particular challenges in postsurgical patients, because these patients must not only deal with the stress of surgery and a subsequent period of fasting due to ileus, but may also be required to adapt nutrient absorption to the demands of a short bowel syndrome thereafter. Although most clinical bowel resections are relatively well tolerated, some are more major. Such major bowel resections and the short gut syndrome that follows represent important animal models in which to study the stress of adaptation to bowel resection. Because many sick elderly patients undergoing surgery are already malnourished, the adaptation of the small bowel

424 / CHAPTER 14 to small-bowel resection is important for subsequent restoration of the normal nutritional state. At least one study suggests that the morphologic and proliferative adaptive response of the remnant small bowel to massive (70%) small-bowel resection is similar between young (2-month-old) and old (24-month-old) rats (363). Some studies suggest that the early adaptive response may be delayed in older animals, although adaptation is equivalent in the long term (364,365). Another investigator determined that the residual small bowel retained sufficient absorptive capacity even after 90% proximal small-bowel resection, although a delay in early response correlated with a decrease in ODC mRNA expression (364). In contrast, pancreatic hyperplasia after 90% proximal small-bowel resection appears inhibited in old rats, possibly because of an inadequate postresection increase in plasma enteroglucagon (366). Ileal-jejunal transposition, which creates a physiologic short gut syndrome without actually removing bowel, is associated with increases in mucosal mass, villus height, and crypt depth throughout the small bowel, as well as increased colonic and pancreatic weight and increased plasma neurotensin levels; but none of these parameters were observed to be affected by the age of the rats studied (367). It should also be pointed out that although many studies of intestinal adaptation focus on morphologic parameters, these may not necessarily correlate with gut mucosal function. For instance, duodenal adaptation to 70% distal small-bowel resection has been reported to induce equivalent morphologic adaptation in young and old rats, but sucrase-, maltase-, and lactase-specific activities in the duodenal mucosa were only found to increase in young rats (368). In addition, although the final morphologic results of adaptation may be similar between young and older animals, it is possible that the mechanisms by which these effects are achieved may differ. For instance, neurotensin has been hypothesized to induce colonic mucosal thickening in young rats by hyperplasia, but in older rats by hypertrophy (317). This may not be true for the small bowel (369). Delayed intestinal adaptation may nevertheless be more clinically important in the elderly because delayed adaptation may make the elderly less able to recover from malnutrition or combat other comorbidities. Neurotensin (363) and growth hormone, together with a high-protein diet (370), have each been reported to promote intestinal adaptation after massive bowel resection in aging rats, but neither has been demonstrated to be more effective in older animals than in younger animals. Furthermore, such agents may have systemic effects. One study of the effects of growth hormone on colonic anastomotic strength in old diabetic rats found no effect of the agent on anastomotic healing, but growth hormone administration doubled the overall mortality of the procedure (371). An interesting parallel case to the adaptation to smallbowel resection is the adaptation of a jejunal pouch to distal proctectomy and anastomosis between the new jejunal reservoir and the anus. Functional outcomes after distal proctectomy and coloanal reconstruction with a jejunal pouch seem reasonably equivalent between younger patients and selected

elderly patients, although reported constipation may be more common in the older patients (372). Interestingly, aging but otherwise healthy rats exhibit impaired healing of their abdominal wounds after colonic transection and anastomosis, but aging does not appear to impair colonic healing (373). However, although colonic anastomotic healing in rats is not generally affected by aging under optimal conditions, anastomotic bursting strength may be more substantially impaired by a low dose of the chemotherapeutic and antimitogenic agent 5-fluorouracil (374). In general, intestinal repair after injury by systemic alkylating agent toxicity is faster in younger animals than in older animals (375). Such observations raise the more general question of whether studies of responses to surgery and stress in otherwise healthy aging animals may adequately represent the pathophysiology of the responses to surgery and stress in aging humans whose status may be further compromised by other comorbidities.

Surgical Diseases of the Gut Many GI diseases of surgical significance increase in prevalence with age. The most obvious is malignancy. However, peptic ulcer disease is also more common among the elderly because of increasing NSAID use and increasing H. pylori colonization (376). Gastroesophageal reflux problems also become more prevalent, although it is difficult to distinguish the contribution of changes in GE junction physiology from the effects of longer exposure to refluxate, NSAID, aspirin or other barrier breakers, and obesity. Certain colonic diseases also become more prevalent with age. Aside from colon cancer, among the most obvious of these diseases are diverticular disease and arteriovenous malformations that cause lower GI bleeding. Either of these may cause patients to require surgery. In the Western world, aging and fiber-poor diets clearly predispose to the development of diverticulosis, defined as the presence of “false” diverticula of the colon as opposed to the rarer true colonic diverticulum that contains all layers of the colonic wall (377–379). Aging is associated with increased collagen deposition in the colonic wall (380), and a small electron microscopic study suggested that collagen fibrils in the left colon become smaller and more tightly packed than those in the right colon with age and especially in patients with diverticular disease (381). Indeed, tensile strength in the left colon also declines with age (382). Although a causal link has not been established, it seems plausible that these alterations in collagen support and mechanical properties could contribute to the development of diverticulosis as patients age. Interestingly, high-fiber diets appear to protect against age-related changes in collagen cross-linking, as well as the development of diverticulosis in a rat model (383), thus it is possible that either chemical or mechanical properties of the luminal contents may influence submucosal collagen metabolism. A high-fiber diet is expected to alter concentrations of luminal nutrients

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 425 and bioactive agents, including short-chain fatty acids (384,385) and bile acids (386), as well as pressure and strain within the colon (382,387). Either might exert direct effects on the colonic submucosal layers or indirect effects by influencing colonic epithelial cells to release paracrine factors (388,389). By comparison with Western white individuals, right-sided true diverticula may be more common among Asians (390). However, studies suggest that such diverticula are also increasing in incidence over time and with age, and may reflect environmental factors, as well as genetic background (391,392).

Angiodysplasia Colonic bleeding from angiodysplasia is also substantially more common among the elderly. Bleeding from such arteriovenous malformations may be more common in the right colon and has been attributed to degenerative changes in the colonic mucosa and vasculature (393).

Key Areas for Future Research The impact of aging on patients undergoing or adapting to GI surgery is an extremely important clinical problem that merits substantial additional basic and clinical investigation. It is clear from some of the work that has already been done that adaptation is slow, growth is less, and the regulation of these processes is abnormal. However, better elucidation of the mechanisms responsible for these events is required so that interventions may be designed to correct these abnormalities. As the population ages and perioperative management improves, more aging patients will undergo more complex GI surgery. Improved understanding of the impact of aging on the gut will facilitate their management and recovery.

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (5 R01 AG14343) and the Department of Veterans Affairs.

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355. Yoshinaga K, Ishizuka J, Evers BM, Townsend CM Jr, Thompson JC. Age-related changes in polyamine biosynthesis after fasting and refeeding. Exp Gerontol 1993;28:565–572. 356. Fleming SE, Kight CE. The TCA cycle as an oxidative and synthetic pathway is suppressed with aging in jejunal epithelial cells. Can J Physiol Pharmacol 1994;72:266–274. 357. Fleming SE, Fitch MD, Hudes M. Intestinal cell respiration is influenced by animal age, strain, and feeding status. J Gerontol 1994;49: B22–B30. 358. Araki S, Goto S. Age-associated changes in the serum level of apolipoproteins A-I and A-IV and the gene expression as revealed by fasting and refeeding in mice. Exp Gerontol 2003;38:499–506. 359. Watters JM, Redmond ML, Desai D, March RJ. Effects of age and body composition on the metabolic responses to elective colon resection. Ann Surg 1990;212:213–220. 360. Watters JM, Blakslee JM, March RJ, Redmond ML. The influence of age on the severity of peritonitis. Can J Surg 1996;39:142–146. 361. Hyde SR, McCallum RE. Lipopolysaccharide-tumor necrosis factorglucocorticoid interactions during cecal ligation and punctureinduced sepsis in mature versus senescent mice. Infect Immun 1992; 60:976–982. 362. Hyde SR, Stith RD, McCallum RE. Mortality and bacteriology of sepsis following cecal ligation and puncture in aged mice. Infect Immun 1990;58:619–624. 363. Thomas RP, Slogoff M, Smith FW, Evers BM. Effect of aging on the adaptive and proliferative capacity of the small bowel. J Gastrointest Surg 2003;7:88–95. 364. Sakamoto K, Fujiyama Y, Bamba T. Altered polyamine biosynthesis with aging after massive proximal small bowel resection in rat. J Gastroenterol 1996;31:338–346. 365. Poston GJ, Saydjari R, Lawrence J, Alexander RW, Townsend CM Jr, Thompson JC. The effect of age on small bowel adaptation and growth after proximal enterectomy. J Gerontol 1990;45:B220–B225. 366. Sasaki M, Sakamoto K, Fujiyama Y, Bamba T. Effect of ageing on pancreatic hyperplasia after 90% proximal small bowel resection. J Gastroenterol Hepatol 1997;12:376–381. 367. Tsuchiya T, Ishizuka J, Sato K, Shimoda I, Rajaraman S, Uchida T, Townsend CM Jr, Thompson JC. Effect of ileo-jejunal transposition on the growth of the GI tract and pancreas in young and aged rats. J Gerontol A Biol Sci Med Sci 1995;50:M155–M161. 368. Yoshinaga K, Ishizuka J, Townsend CM Jr, Thompson JC. Age-related changes in duodenal adaptation after distal small bowel resection in rat. Dig Dis Sci 1993;38:410–416. 369. Evers BM, Izukura M, Rajaraman S, Parekh D, Thakore K, Yoshinaga K, Uchida T, Townsend CM. Effect of aging on neurotensin-stimulated growth of rat small intestine. Am J Physiol 1994;267:G180–G186. 370. Fadrique B, Lopez JM, Bermudez R, Gomez de Segura IA, Vazquez I, De Miguel E. Growth hormone plus high protein diet promotes adaptation after massive bowel resection in aged rats. Exp Gerontol 2001; 36:1727–1737. 371. Seyer-Hansen M, Andreassen TT, Oxlund H. Strength of colonic anastomoses and skin incisional wounds in old rats—influence by diabetes and growth hormone. Growth Horm IGF Res 1999;9:254–261. 372. Dehni N, Schlegel D, Tiret E, Singland JD, Guiguet M, Parc R. Effects of aging on the functional outcome of coloanal anastomosis with colonic J-pouch. Am J Surg 1998;175:209–212. 373. Petersen TI, Kissmeyer-Nielsen P, Laurberg S, Christensen H. Impaired wound healing but unaltered colonic healing with increasing age: an experimental study in rats. Eur Surg Res 1995;27:250–257. 374. Stoop MJ, Dirksen R, Wobbes T, Hendriks T. Effects of early postoperative 5-fluorouracil and ageing on the healing capacity of experimental intestinal anastomoses. Br J Surg 1998;85:1535–1538. 375. Greenstein RJ. Age, as well as cell turnover kinetics, regulates brain/gut repair. Mech Ageing Dev 1993;69:219–231. 376. Jones JI, Hawkey CJ. Physiology and organ-related pathology of the elderly: stomach ulcers. Best Pract Res Clin Gastroenterol 2001; 15:943–961. 377. Kang JY, Melville D, Maxwell JD. Epidemiology and management of diverticular disease of the colon. Drugs Aging 2004;21:211–228. 378. Bassotti G, Chistolini F, Morelli A. Pathophysiological aspects of diverticular disease of colon and role of large bowel motility. World J Gastroenterol 2003;9:2140–2142. 379. Eastwood M. Colonic diverticula. Proc Nutr Soc 2003;62:31–36. 380. Yamajta A. Histopathological studies of the colon due to age. Jpn J Gastroenterol 1965;62:224–235.

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CHAPTER

15

Regulation of Gastrointestinal Normal Cell Growth Mark R. Hellmich and B. Mark Evers Overview, 435 Organization of the Gastrointestinal Mucosa, 436 Cell Cycle Regulation, 437 Apoptosis, 437 Signal Transduction Pathways Regulating Gastrointestinal Differentiation and Growth, 440 Wnt Signaling Pathways, 440 Phosphatidylinositol-3 Kinase/Protein Kinase B/Akt Pathway, 443 Mitogen-Activated Protein Kinase Pathways, 445 Role of Sonic Hedgehog Pathway in Gastric Gland Maintenance, 445 Transforming Growth Factor-β/SMAD Pathway, 446 Growth Factors and Receptor Tyrosine Kinases, 447 Growth Regulation by Luminal Nutrients and Secretions, 449

Amino Acids, 450 Short-Chain Fatty Acids, Fiber, 450 Nucleotides and Polyunsaturated Fatty Acids, 450 Pancreaticobiliary Secretions, 451 Growth Regulation by Small Peptide Hormones and Their Receptors, 451 Gastrin, 451 Glucagon-Like Peptides, 451 Glucagon-Like Peptide-2, 452 Peptide YY, 452 Neurotensin, 452 Bombesin/Gastrin-Releasing Peptide, 452 Somatostatin, 453 Future Perspectives/Clinical Applications, 453 References, 453

OVERVIEW

from populations of pluripotential stem cells residing in specific locations along the GI tract. In the stomach, the stem cells are located in the isthmus/neck region of the gastric glands. In the small intestine and colon, the stem cells are found in the base region of the crypts. Through a combination of symmetrical (or equivalent) and asymmetrical (or differential) mitoses, gut stem cells divide and differentiate through five main stages. In the first stage, pluripotential stem cells divide either symmetrically to produce new stem cells and/or asymmetrically to produce second-staged uncommitted or committed precursor cells. Precursor cells then either undergo equivalent mitosis to amplify the uncommitted cell population or produce transit cells by differential mitosis. The transit cells, which represent the third stage of epithelial cell development, gradually acquire specialized phenotypes by synthesizing new and increasingly cell type-specific gene products. Cells in the fourth stage have completed their maturation into the various fully differentiated cell types that perform region-specific functions within the gut. In the fifth stage, the terminally

The epithelium of the gastrointestinal (GI) tract proliferates, matures, and recycles constantly throughout the life of an individual. Although it is well known that multipotent stem cells fuel this process, the molecular mechanisms involved in stem cell proliferation, lineage determination, and epithelial cell differentiation remain largely undefined. Much of our current knowledge of normal GI growth and development has been obtained from studies using rodent models. In mice, the gut epithelium is first identifiable at embryonic day 7 (E7) as a single layer of proliferating endodermal cells (1). By E18, distinct functional regions of the gut are apparent. The specialized epithelial cells of the gut arise

M. R. Hellmich and B. M. Evers: Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

435

436 / CHAPTER 15 differentiated cells gradually deteriorate and eventually undergo cell death and elimination by various mechanisms. One of the fundamental challenges in GI cell biology is to define the molecular mechanisms that regulate stem/crypt cell proliferation, lineage determination, migration, and differentiation. This chapter reviews current knowledge of the molecular regulation of these complex processes. Our understanding of the molecular mechanisms mediating normal GI epithelial cell growth has accelerated in recent years due, in large part, to the development of genetically altered mouse models of gut development. These studies have identified several important pathways involved in the proliferation and/or differentiation of gut epithelial cells: (1) the Wnt/adenomatous polyposis coli (APC)/β-catenin/ T-cell factor (Tcf)/lymphoid-enhancing factor (Lef) pathway, (2) the phosphatidylinositol-3 kinase (PI3K)/Akt/glycogen synthase kinase-3 (GSK-3) pathway, and (3) the Hox Sonic hedgehog (Shh) gene pathway. In addition, a number of growth factors and peptide hormones expressed by intestinal mesenchymal cells and epithelial cells have been identified that regulate development, proliferation, and differentiation in normal and malignant GI tissues. They include members of the fibroblast growth factor (FGF) family, epidermal growth factor (EGF) family, transforming growth factor-β (TGF-β), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1) and -2, Shh and Indian hedgehog (Ihh), platelet-derived growth factor (PDGF), Wnt family ligands, and numerous

small peptide hormones, such as gastrin, glucagon-like peptide-2 (GLP-2), peptide YY (PYY), neurotensin (NT), bombesin (BBS)/gastrin-releasing peptide (GRP), and somatostatin. These pathways, growth factors, and peptide hormones are discussed in detail in other chapters of this textbook; therefore, this chapter focuses on their roles in normal GI epithelial cell proliferation, migration, differentiation, and apoptosis.

ORGANIZATION OF THE GASTROINTESTINAL MUCOSA The epithelial layer of the GI tract is a dynamic tissue composed of numerous cell types with important functions that include digestion, absorption, barrier and immune function, and peptide secretion (2,3). The mammalian intestinal mucosa undergoes a process of continual renewal characterized by active proliferation of stem cells localized near the base of the crypts, progression of these cells up the cryptvillus axis (in the small bowel) and crypt axis (in the colon), with cessation of proliferation and subsequent differentiation (4–7). Four primary differentiated cell types are found in the small intestine and include absorptive enterocytes, which make up the predominant population of differentiated cells; goblet cells; Paneth cells, which are located at the base of the crypts; and enteroendocrine cells (Fig. 15-1) (8–10). Several cell types are noted in the colonic epithelium, most

FIG. 15-1. Organization of the small intestinal epithelium. The epithelium is composed of crypts and villi (left). The lineage scheme (right) depicts the stem cell, the transit-amplifying cells, and the two differentiated branches. The right branch constitutes the enterocyte lineage; the left is the secretory lineage. Relative positions along the crypt-villus axis correspond to the schematic graph of the crypt in the center. (Reproduced from Radtke and Clevers [9], by permission.)

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 437 notably columnar absorptive cells and goblet cells. Enterocytes and colonocytes undergo a process of programmed cell death (i.e., apoptosis) and eventual extrusion into the intestinal lumen (8,10). In the small bowel, this entire process occurs over a 3- to 6-day period, depending on the species; in the colon, this process takes 3 to 8 days. The mechanisms regulating the tightly regimented processes of proliferation, differentiation, and apoptosis in the GI tract mucosa are complex (11) and likely involve the interplay of numerous peptide growth factors (both systemic and local), intestinal nutrients, and endogenous secretions.

CELL CYCLE REGULATION The mechanisms that determine whether cells continue to proliferate or cease dividing and differentiate appear to operate during the first Gap phase (G1) of the cell cycle (12,13). Regulation and progression through the cell cycle is regulated by a highly conserved family of serine-threonine protein kinases that are composed of a regulatory subunit (the cyclins) and a catalytic subunit (the cyclin-dependent kinases [Cdks]) (14). The association of cyclins with their corresponding Cdks leads to the activation and subsequent progression of the cell through the various phases of the cell cycle (Fig. 15-2). Cell cycle progression can be inhibited by a class of regulators, called the Cdk inhibitors (15–17). One family of Cdk inhibitors includes p21WAF1, p27KIP1, and p57KIP2, which are universal inhibitors of the cyclin-Cdk complexes (18); another family consists of p15INK4B, p16INK4A, p18INK4C, and p19INK4D, which selectively bind and inhibit Cdk4 and Cdk6. Progression through the G1-phase of the cell cycle requires association of D-type cyclins (D1, D2, and D3) with Cdk4 and Cdk6, whereas cyclin E binding to Cdk2 is required for the G1/S transition (19). p21WAF1 can be activated by the tumor-suppressor protein p53 directly or by p53-independent mechanisms. p27KIP1 can mediate cell cycle arrest in the G1-phase of the cell cycle by TGF-β. In addition, important targets of Cdk2 and Cdk4 include the retinoblastoma protein (pRb) and the pRb-related proteins, p107 and p130 (20–22). In their hypophosphorylated forms, the pRb family of proteins can sequester and inactivate the E2F family of transcription factors that regulate genes that participate in S-phase entry (23,24). Using the human colon cancer cell line Caco-2, which spontaneously differentiates to a small bowel-like phenotype after cell confluency, induction of p21WAK1 was noted as cell reached confluency and before the induction of differentiated genes such as sucrase-isomaltase (25,26). In addition, Caco-2 cells undergo a relative G1/S block and cessation of proliferation at 3 days after confluency, which was associated with suppression of Cdk2 and Cdk4 activities (27,28). In vivo findings demonstrate p21WAF1 expression in the fully differentiated columnar epithelium of the small bowel and stomach (29). Collectively, these data suggest a role for p21WAF1 in intestinal differentiation; however, subsequent studies using mice in which expression of the p21WAF1 gene was disrupted

FIG. 15-2. Cell cycle regulation. Cell cycle progression through four specific phases (G1, S, G2, and M) is controlled by protein complexes composed of cyclins and cyclindependent kinases (Cdks). These protein–protein interactions function to regulate progression through the cell cycle by phosphorylating downstream regulatory subunits such as retinoblastoma (pRb) protein. In the hypophosphorylated form, pRb sequesters transcription factors such as E2F. On phosphorylation, pRb releases E2F transcription factors, which are responsible for the transcription of cellular genes involved in cell cycle progression through the G1/S transition. Cell cycle arrest is controlled at various phases by the CIP/KIP and INK family of cyclin-dependent kinase inhibitors. PCNA, proliferating cell nuclear antigen. (Reproduced from Papaconstantinou HT, Ko TC. Cell cycle and apoptosis regulation in GI cancers. In: Evers BM, ed. Molecular mechanisms in gastrointestinal cancer. Austin: R.G. Landes Company, 1999;49–78, by permission.)

show no developmental defects in the intestines suggesting possible redundancy in the mechanism controlling inhibition of intestinal cell growth and initiation of differentiation (30).

APOPTOSIS The high turnover of epithelial cells in the GI tract necessitates a method to eliminate cells so that the delicate balance of proliferation can be maintained. Apoptosis, or programmed cell death, is recognized as an important process responsible for the maintenance of the cellular balance between proliferation and death (31–35). Alterations in proliferation or apoptosis resulting in an imbalance of either protein can lead to pathologic states in the intestine (e.g., tumor formation) (Fig. 15-3) (9,36–38). Apoptosis can be triggered by various factors, including withdrawal of hormones and growth factors, free radical formation, ionizing radiation, proinflammatory cytokines, and cell detachment from the extracellular matrix

438 / CHAPTER 15 Tissue homeostasis Proliferation

Apoptosis

Cell cycle

Apoptosis

Activation

Pro-apoptotic regulators

Inhibition

Anti-apoptotic regulators

A

ra

life

Pro

tion

sis

pto

Apo

sis

pto

Apo tion

fera

li Pro

Tumor formation

B

C

FIG. 15-3. The induction of tumor formation through alterations in cell cycle and apoptosis regulation. (A) Normal tissue homeostasis is achieved through a balance between cell proliferation and apoptosis. (B) Alterations of cell cycle regulation leading to increased activation or decreased inhibition results in uncontrolled proliferation. (C) A decrease in proapoptotic and an increase in antiapoptotic regulators results in increased cell survival and cell number. These events are implicated in the induction of tumor formation. (Reproduced from Papaconstantinou HT, Ko TC. Cell cycle and apoptosis regulation in GI cancers. In: Evers BM, ed. Molecular mechanisms in gastrointestinal cancer. Austin: R.G. Landes Company, 1999;49–78, by permission.)

(ECM) (Fig. 15-4) (39,40). There are two pathways that can lead to apoptosis: (1) the death receptor pathway activated by binding of death factors (tumor necrosis factor-α [TNF-α], Fas ligand, or TRAIL) to their receptors on the plasma membrane and activation of stress kinases, and (2) mitochondrial pathways activated by a number of stimuli. Both of these pathways then trigger intracellular events that lead to morphologic and biochemical changes that characterize apoptosis, including shrinking of the cytoplasm and detachment from neighboring cells, chromatin condensation, DNA fragmentation, and finally, apoptotic bodies that are phagocytized by neighboring cells and macrophages without inducing an inflammatory response. (This process is in contrast with cell necrosis that results in localized inflammation.) Activation of proteolytic enzymes called caspases (cysteine aspartate proteases) represents critical events in the conserved biochemical pathway that mediates apoptotic cell death (41–43). The caspases include two groups: (1) initiator caspase-8, -9, and -10, activated by proapoptotic stimuli; and (2) executioner caspase-3, -6, and -7, which execute the destruction and disassembly of cellular structures (44–46). The mitochondrial pathway is mediated by release of cytochrome c into the cytosol, which then triggers activation of caspase-9. This pathway is regulated by the expression of certain intracellular proteins belonging to the Bcl-2 family of genes, which includes proteins that are proapoptotic, as well as antiapoptotic (47). Once the cell-surface death receptors are activated, their cytosolic domains bind to adaptor proteins such as the Fas-associated death domain protein, which, in turn, links up with caspase-8 to form the death-induced signaling complex.

Caspase-8 can then either directly activate other members of the caspase family or cleave Bid to form active Bid (truncated Bid), which translocates to the mitochondria and induces release of cytochrome c, which, together with the apoptosisactivating factor 1 (APAF-1), activates caspase-9, which, in turn, activates other downstream caspases. The executioner caspases then target cytoplasmic proteins (e.g., gelsolin, vimentin, and focal adhesion kinase) (48,49). In addition, nuclear targets such as lamins and poly(ADP-ribose) polymerase (PARP) are targeted, resulting in DNA fragmentation and ultimately apoptosis. It is becoming increasingly apparent that the molecular mechanisms contributing to differentiation are also important for apoptosis, suggesting a continuation of this process. This link has been demonstrated using intestinal cell lines that induce p21WAF1 and p27KIP1 and down-regulation of Bcl-2 family members associated with enterocyte-like differentiation and subsequent apoptosis (50). Apoptosis can be detected in the crypts of healthy mice and humans, supporting the concept that spontaneous apoptosis involves damaged cells and may represent a mechanism to eliminate excess stem cells (32,36). The level of spontaneous apoptosis appears to be lower in the colon compared with the small intestine (4,51). A possible explanation may be that the antiapoptotic Bcl-2 protein appears to be expressed at higher levels in the base of the crypts of the colons of mice and humans (Fig. 15-5) (36,38). Conversely, expression of the proapoptotic proteins, Bax and Bak, is increased as cells ascend the vertical axis of the intestine (52). Controversy exists regarding the process of apoptosis at the tip of the small bowel villus and at the cuff of the crypts of the colon; however, it is clear that there is abundant DNA fragmentation

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 439

FIG. 15-4. Signaling pathways of apoptosis. (A) Activation of cell-surface death factor receptors (tumor necrosis factor receptor 1 [TNFR1], CD95/Fas receptor) by TNF-α or CD95 ligand or Fas ligand (CD95L/FasL) leads to receptor oligomerization, recruitment of adaptor proteins, and activation of initiator caspase-8, -9, or -10. c-Jun N-terminal kinase 1 or stress-activated protein kinase (JNK1/SAPK) and p38 kinase signal transduction pathways are involved in some steps of this process. Initiator caspases trigger activation of executioner caspases (-3, -6, and -7). In the mitochondrial pathway, the release of cytochrome c by proapoptotic stimuli leads to its binding to apoptosis protease activator protein 1 (APAF-1) and activation of procaspase-9, which, in turn, triggers the activation of executioner caspases. Activated executioner caspases cleave the nuclear and cytoskeletal proteins, leading to morphologic changes characteristic for apoptosis and ultimately to cell death. FADD/MORT1, Fas-associated death domain protein; PARP, poly(ADP-ribose) polymerase; RAIDD, RIP-associated protein with death domain; Rb, retinoblastoma protein; RIP, receptor-interacting protein; STAT1, signal transducer and activator of transcription 1; TRADD, TNFR-associated death domain. (B) Attachment of cells to the matrix occurs via focal adhesions. The integrin-triggered signals from the matrix are mediated via recruitment of focal adhesion proteins such as focal adhesion kinase (FAK), paxillin, Src, vinculin, β-catenin, α-actinin, and p130Cas proximal to the cytoplasmic tail of β1-integrins. Focal adhesions are important for prosurvival signaling through phosphatidylinositol-3 kinase (PI3K) and Akt (serine-threonine kinase also known as protein kinase B) signal transduction pathways, nuclear factor-κB (NF-κB) activation, and induction of inhibitors of apoptosis protein (IAPs). ECM, extracellular matrix. (Reproduced from Tarnawski and Szabo [40], by permission.)

440 / CHAPTER 15

FIG. 15-5. Schematic diagram summarizing views on distribution of stem cells, proliferative cells, and apoptosis gene products. Arrows indicate approximate cell positions over which an indicated protein or feature is observed. Wedges indicate gradients of protein expression when known. Asterisks denote observations made after irradiation. (Reproduced from Potten [36], by permission.)

in these cells, and the expression of the apoptotic protein Bax and death receptors and their natural ligands increase as cells approach the lumen (52–56). In addition, apoptosis of intestinal cells is associated with sequential activation of caspases (42). Cytotoxic drugs, chemicals, and ionizing radiation enhance apoptosis in the GI epithelium. Radiation-induced damage and subsequent apoptosis appears to be dependent on p53 in both the small intestine and colon. However, the apoptotic response in the colon appears to be less than in the small intestine, suggesting that damaged cells may persist for longer periods and may contribute to an increased incidence of carcinogenesis in the colon. Other studies of apoptosis in the intestinal tract have shown that intestinal trefoil factor (ITF), which is secreted by intestinal goblet cells, has a strong antiapoptotic action (57). This effect is mediated by Akt and requires functional PI3K and EGF receptor. Colonic apoptosis is increased in ITF-deficient mice, which is caused by lack of prosurvival signaling through the PI3K/Akt pathway. In another study, ITF activated nuclear factor-κB (NF-κB), which is an important survival pathway for intestinal epithelial cells (58). See Chapter 12 for a detailed discussion of apoptosis.

SIGNAL TRANSDUCTION PATHWAYS REGULATING GASTROINTESTINAL DIFFERENTIATION AND GROWTH Wnt Signaling Pathways Wnts Genetic evidence has established a central role for Wnt signaling pathways (Fig. 15-6A) in intestinal stem cell proliferation and differentiation (59). The Wnts are a family of secreted glycoproteins characterized by several conserved cysteine residues. Historically, they have been divided into two classes based on the intracellular pathway(s) they activate. Wnts of the canonical class (e.g., Wnt1, Wnt3A, and Wnt8) stimulate Tcf/Lef-mediated gene transcription by stabilized β-catenin. Wnts of the noncanonical class (e.g., Wnt4, Wnt5, and Wnt11) activate β-catenin– independent pathway regulation of phospholipase C, cytosolic Ca2+ levels, and protein kinase C (PKC) activity. A role for the canonical Wnt signaling pathway in regulating normal GI epithelial cell growth and differentiation is well established.

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 441

A

B Growth Factor

Wnt

Fz

GI Peptide Hormone

Nutrients

Lrp

APC Axin β-cat Ck1 gsk

APC Axin Ck1 gsk β-cat

PI3K

PKB/Akt

GSK-3 β-cat β-cat

β-cat β-cat β-cat

β-cat

mTor Protein Synthesis

+ ucho Gro TCF

β-cat

β-cat

TCF

TCF

D

C Growth Factor

GI Peptide Hormone

Growth

Crypt proliferation

Differentiation

+

E

Shh/Ihh

Patch

Smoothened

TGF-β or

Bmp Bmp P

MAPKKKs

P

Co-SMAD P

MAPKKs

R-SMAD

MAPKs

+

+ + BMP

AP-1

Growth or Differntation

Gastric differentiation

P

Normal Crypt Formation

FIG. 15-6. Pathways regulating intestinal epithelial cell proliferation and differentiation. (A) Wnt signaling pathways, (B) phosphatidylinositol-3 kinase/PKB/Akt pathway, (C) mitogen-activated protein kinases (MAPKs) pathways, (D) sonic hedgehog pathway, and (E) transforming growth factor-β (TGF-β)/SMAD pathway. APC, adenomatous polyposis coli; BMP, bone morphogenetic protein; Fz, Frizzled; GI, gastrointestinal; GSK, glycogen synthase kinase; Ihh, Indian hedgehog; LRP, low-density lipoprotein receptor-related protein; PI3K, phosphatidylinositol-3 kinase; PKB, protein kinase B; Shh, sonic hedgehog; Tcf, T-cell factor. (Modified from Radtke and Clevers [9], by permission.)

Wnt Receptors The receptor of Wnt proteins is a dimeric complex consisting of Frizzled (Fz) and low-density lipoprotein receptor– related protein (LRP). The Frizzled genes encode the family of receptor proteins that displays many of the characteristics of a G protein-coupled receptor (GPCR) (60). Indeed, hydropathy plots predict a protein with seven hydrophobic membrane-spanning domains, an extracellular N-terminal domain containing consensus sites for N-glycosylation, and an intracellular C-terminal domain possessing possible phosphorylation sites for protein kinase A (PKA), PKC, and casein kinase II. Biochemical data suggest coupling to intracellular G proteins. For example, when rat Fz2, but not Fz1, is expressed in zebra fish embryos, Wnt stimulation induces an increase in the concentration of free intracellular Ca2+ ([Ca2+]i) and activation of PKC. Treatment with pertussis toxin, which ADP-ribosylates and inactivates the α subunits of the heterotrimeric G proteins, Gαi, Gαo, and Gαt, inhibited Wntstimulated increase of [Ca2+]i and activation of PKC (61,62).

Productive Wnt signaling requires that Fz form a complex with a coreceptor. The Fz/LPR complex mediates Wnt activation of the canonical pathway described below, whereas Wnt signaling to the noncanonical pathway requires a receptor complex consisting of Fz and the proteoglycan coreceptor, Knypek (63). Canonical Wnt Signaling The transmission of Wnt signals via the canonical pathway occurs when Wnts act on target cells in a paracrine manner by binding to a receptor complex consisting of Fz and LRP. In the absence of Wnt family ligands, the cytoplasmic levels of β-catenin are maintained at a relatively low level by the β-catenin degradation complex consisting of the scaffolding proteins Axin and APC and the protein kinases, casein kinase 1 and GSK-3β. Casein kinase 1 and GSK-3β sequentially phosphorylate conserved serine and threonine residues near the N terminus of β-catenin. Once phosphorylated,

442 / CHAPTER 15 β-catenin is ubiquitinated and degraded via the proteasome complex. The mechanisms regulating the degradation of β-catenin by Wnt are unclear; however, it has recently been shown that the cellular levels of Axin are approximately 5000fold lower than other components of the destruction complex, suggesting that Axin may be a limiting component, and as such, may promote the rapid assembly and disassembly of the destruction complex to regulate cell levels of β-catenin (59). In the presence of Wnt, phosphorylation of β-catenin is inhibited, leading to its accumulation in the nucleus where it binds to and activates the transcription regulators Tcf/Lef. In addition, Axin is recruited by the activated Fz/LRP receptor complex to the plasma membrane, where it is ubiquitinated and degraded. The β-catenin/Tcf/Lef transcription complex enhances the expression of target genes, such as c-Myc, cyclin D1, c-Jun, Fra-1, and the uPA receptor. Negative Regulators of Wnt Wnt antagonists can be divided into two functional classes: the secreted Frizzled-related protein (sFRP) class and the Dickkopf (Dkk) class (64). Members of the sFRP class, which includes the sFRP family, Wnt inhibitory factor-1 (WIF-1) and Cerberus, bind to Wnts, preventing their interaction with Fz receptor. Dkk, in contrast, binds directly to the Fz coreceptor, LRP, causing its internalization, and thereby blocking formation of a functional Wnt/Fz/LRP complex. Mice and humans have multiple Dkk genes (64,65). In addition to LRP, Dkk interacts with another class of single-pass membrane receptors called Kremen-1 and -2 (66). Kremen, Dkk, and LRP form a ternary complex that disrupts Wnt/Fz/LRP signaling by promoting endocytosis and the removal of the LPR from the plasma membrane. Activation of the noncanonical Wnt pathway is inhibited by dominant-negative Fz, but not by either Dkk or dominant-negative LRP (67), suggesting that the antagonist effect of Dkk, mediated by LRP, is likely specific to the canonical Wnt/β-catenin pathway. The essential role that Wnt signaling plays in GI cell proliferation, differentiation, and apoptosis is evidenced by the observation that Wnt antagonists are often down-regulated in adenocarcinomas, whereas up-regulation leads to mucosal degeneration (68,69). Silencing of the sFRP promoter by hypermethylation has been found to occur with high frequency in primary colorectal cancers (68,69), whereas Hoffman and colleagues (69) show that forced overexpression of Dkk-1 in the intestine of mice was associated with a loss of proliferating crypts, eventual inflammation, and mucosal architectural degeneration (69), demonstrating a critical role for Wnt-regulated signal transduction in the homeostatic maintenance of the normal intestinal mucosa. T-Cell Factor-4: Regulation of Intestinal Cell Cycle Progression In the intestinal epithelium, Tcf-4 is the most prominently expressed TCF family member (70). Gene disruption has revealed that Tcf-4 is required to establish the proliferative

progenitors of the prospective crypts in the embryonic mouse small intestine (71). The basic architecture of the mouse intestine is established during mid to late gestation (72). From E13.5 to E18.5, the mouse gut endoderm transforms from a stratified to a simple columnar epithelium. By E16.5, the villi are formed and the functional division of the epithelium becomes apparent. Nondividing, differentiated epithelial cells are located on the villi, whereas the intervillous regions, which will form the crypts of the mature intestine, are populated by relatively undifferentiated, actively dividing cells. Disruption of the Tcf-4 gene (Tcf712−/−), by targeted gene knockout, showed that Tcf-4 is required for maintenance of the crypt stem cell population of the small intestine. Normally, the epithelium within the intervillous region of the developing small intestine has a pseudostratified appearance, with the individual epithelial cells exhibiting elongated nuclei and abundant free ribosomes and polyribosomes, but few mitochondria, Golgi complex, rough endoplasmic reticulum (ER), and lysomes. The undifferentiated morphology of the intervillous epithelium contrasts that of the villi, where the epithelial cells are tall and cylindrical with small, round nuclei located basally and possess abundant mitochondria, rough ER, Golgi, and a well-developed microvilli brush border on their apical surface. In the absence of Tcf-4, the intervillous epithelium resembles the villus epithelium in that it contains differentiated enterocytes and goblet cells, but not enteroendocrine cells (71). The lack of a proliferating cell population in TcF712−/− mice results in a decreased number of small intestine villi, a reduced number of cells in the intervillous region, extensive stretching and occasional tearing of the epithelial layer, and death of the animals within 24 hours after birth. van de Wetering (73) and colleagues have shown that disruption of β-catenin/Tcf-4 activity induces both a rapid arrest of the cell cycle at the G1 checkpoint and intestinal cell differentiation. The Tcf-4 target gene, c-Myc, plays a central role in switching the cellular program from proliferation to differentiation by directly repressing p21WAF1 promoter activity (73). Decreased β-catenin/Tcf-4 activity leads to a decrease in c-Myc expression, which, in turn, results in a release of c-Myc-mediated repression of the p21WAF1 promoter. Consequently, induction of p21WAF1 expression results in cell cycle arrest. T-Cell Factor-4: Regulation of Intestinal Cell Position along the Crypt-Villus Axis In addition to its role in cell cycle regulation, β-catenin/ Tcf regulates expression of the Eph/ephrin system that plays a central role in establishing the correct distribution of differentiated epithelial cell types along the crypt-villus axis. Eph receptors are members of the superfamily of receptor tyrosine kinases (RTKs) that regulate a variety of developmental processes, including cell migration, segmentation, axon guidance, topographic mapping, synaptogenesis, and angiogenesis. The Eph receptors are subdivided into two structurally distinct subclasses, designated EphA and EphB. EphA receptors

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 443 (EphA1-A8) bind glycosyl phosphatidyl-anchored ephrinA ligands (ephrinA1-A5), whereas EphB receptors (EphB1-B6) bind membrane-anchored ephrinB ligands (eprinB1-B3). Each position along the adult crypt axis is characterized by different levels of EphB and ephrinB ligand expression. The Eph2B receptor is expressed throughout the proliferative compartment with the highest levels associated with Ki67-positive cells (the presumptive stem cells) that intercalate between the Paneth cells at the bottom of the crypt, whereas the terminally differentiated Paneth cells are Eph2B negative. The levels of Eph2B decrease in a gradient toward the top of the crypt. Conversely, expression of the EphB ligands, ephrinB1 and B2, is highest at the upper cryptvillus junction and decreases toward the bottom of the crypt. Thus, proliferative cells of the crypt coexpress EphB2 receptors and their ligands in an inverse manner that is dependent on their position within the crypt and degree of differentiation. The EphB receptor and ephrinB ligands engage in bidirectional signaling between adjacent cells to regulate cell-cell repulsion. The cytoplasmic tail domain of ephrinB ligands are rapidly phosphorylated on tyrosine residues on its binding to the EphB receptors on a neighboring cell (74). The phosphorylated ephrinB ligand recruits the SH2/SH3 domain adaptor protein, Grb4, and induces the disassembly of actin-containing stress fibers, which culminates in cell type-specific changes in motility and adhesion. Batlle and coworkers (75) show that the β-catenin/Tcf transcription regulatory complex inversely controls the expression of EphB and ephrinB genes. With the exception of Paneth cells, the cells of the crypt that contained the highest levels of nuclear β-catenin expressed the highest levels of Eph and the lowest level of ephrinB ligand. As levels of nuclear β-catenin decrease, the cells ascend to the crypt-villus junction, and the levels of EphB also decrease, but the levels of ephrinB ligand increase, suggesting that the correct proportion of EphB and ephrinB is essential for the correct positioning of the epithelial cells along the crypt-villus axis. Support for this hypothesis is provided by the observation that, in adult EphB3 knockout mice, Paneth cells do not follow their normal downward migratory path, but rather scatter throughout the crypt. Also, in EphB2/EphB3 double-knockout mice, the proliferative and differentiated cell populations intermingle, indicating that signaling through these receptors normally restricts cell movement and regulates the allocation and topical distribution of cell populations along the crypt-villus axis. See Chapter 8 for a detailed discussion of Wnt signaling.

Phosphatidylinositol-3 Kinase/Protein Kinase B/Akt Pathway Phosphatidylinositol-3 Kinase The PI3K family of lipid kinases (see Fig. 15-6B) phosphorylate the 3′-OH on the inositol ring of phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the second

messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3). Phosphatidylinositol-3,4,5-trisphosphate can be converted back to PIP2 by the lipid phosphatase, PTEN, thus inhibiting PI3K-mediated signal transduction. The proliferative or survival responses, or both, induced by either growth factor or small peptide hormones are mediated, in part, by class I PI3Ks. Class I PI3K family members are heterodimers protein complexes consisting of a catalytic subunit and a regulatory subunit. The PI3Ks are further divided into the subclass IA kinases, which are activated by RTKs, and the subclass IB members activated by GPCRs. Three isoforms of the catalytic subunit (p110), named α, β, and γ, and seven regulatory subunits generated by alternative splicing of three genes (p85α, p85β, and p55γ) have been identified within PI3K class IA. Important downstream targets of PI3Ks are members of the protein kinase B (PKB)/Akt family of proteins. Protein Kinase B/Akt In humans, PKB/Akt was initially characterized after the isolation of two genes (akt1 and akt2), which were identified as the homologues of the viral oncogene v-akt. Subsequent studies revealed that the products of the akt genes encoded protein kinases bearing some resemblance to both PKC and PKA; hence, they were named PKB. To date, three members of the PKB family have been characterized: PKBα (Akt1), PKBβ (Akt2), and PKBγ (Akt3). Although they are produced by different genes, they share up to 80% of amino acid identity. Each PKB/Akt isoform contains an N-terminal plekstrin homology (PH) domain of approximately 100 amino acids, followed by a central kinase domain with structural similarities to PKA and PKC, and a C-terminal hydrophobic domain. Both the kinase and C-terminal domains contain serine and threonine residues that are phosphorylated in response to growth factors and other extracellular stimuli and are essential for maximal PKB/Akt activation. Activation of the Phosphatidylinositol-3 Kinase/Protein Kinase B/Akt Pathway Ligand engagement of either a PI3K-coupled RTK or GPCR results in PIP3 production by PI3K at the inner side of the plasma membrane (PM). Protein kinase B/Akt interacts with PIP3, causing its translocation to the PM, where 3′-phosphoinositide-dependent kinase-1 (PDK1) is located. 3′-Phosphoinositide–dependent kinase-1 phosphorylates PKB/Akt on a specific threonine residue (T308) within its kinase domain, leading to stabilization of the kinase domain activation loop in an active conformation. Although T308 phosphorylation by PKD1 is a requirement for kinase activation, phosphorylation of a serine residue (S473) located within the hydrophobic C-terminal region precedes T308 phosphorylation and is required for full PKB/Akt activation. The identification of the S473 kinase has been controversial; however, one study (76) has demonstrated that a complex of mammalian targets of rapamycin (mTOR) and rictor

444 / CHAPTER 15 phosphorylate S473 of PKB/Akt both in vivo and in vitro. On full activation, PKB/Akt phosphorylates cytoplasmic substrates and translocates to the nucleus through an unknown mechanism where additional substrates are localized. 3′-Phosphoinositide-dependent kinase-1 and PKB/Aktdirected phosphorylation modulates the function of numerous proteins involved in the regulation of epithelial cell survival, cell cycle progression, and cellular growth and differentiation. Activation of PI3K stimulated induction of intestinal mucosal proliferation after fasting and refeeding (77,78). Conversely, inhibition of PI3K/Akt by chemical inhibitors or PTEN augments intestinal differentiation (79). Targets of Phosphatidylinositol-3 Kinase/Protein Kinase B/Akt Signaling Glycogen synthase kinase-3β, phosphodiesterase-3B, mTOR, insulin receptor substrate-1 (IRS1), the Forkhead family member FKHR, and Cdk inhibitors p21WAF1 and p27KIP1 are all substrates for PKB/Akt and are involved in protein synthesis, glycogen metabolism, and cell cycle regulation. Protein kinase B/Akt modulation of specific substrate activity, such as β-catenin, cyclin D1, Mdm2, or Forkhead (FOXO) transcription factors, involves the phosphorylation-dependent regulation of their cytoplasmic or nuclear localization and protein stability. For example, PKB/Akt affects the cellular levels of cyclin D1 by phosphorylating GSK-3β, resulting in inhibition of its kinase activity. Inhibition of GSK-3β activity, in turn, leads to a reduction in the levels of phosphorylated β-catenin and cyclin D1, which increases their stability. The increased stability of β-catenin results in its nuclear accumulation and increased Tcf/Lefmediated transcription of downstream target genes, including the cyclin D1 gene. The combined effect of increased cyclin D messenger RNA (mRNA) expression and protein stabilization is to induce cell cycle progression via regulation of Rb hyperphosphorylation and inactivation. Protein kinase B/Akt also stimulates cell cycle progression by phosphorylating p21WAF1 and p27KIP1, which prevents their translocation to the nucleus where they function as key inhibitors of cell progression.

and p27KIP1, Rb, and signal transducer and activator of transcription 3 (STAT-3). Although the mechanism by which mTOR responds to nutrients is not completely known, studies suggest that intracellular amino acid levels are important, particularly the levels of branched chain amino acids such as leucine (80,81). This conclusion is based, in part, on the observation that amino acid alcohols, which cause accumulation of uncharged tRNA within the cell, inhibit mTOR signaling, suggesting that mTOR may measure amino acid concentrations indirectly by sensing levels of uncharged tRNA. Alternatively, nutrients may affect mTOR activity through effects on intracellular ATP levels. This hypothesis is supported by the finding that mTOR exhibits a relatively high Km (approximately 1 mM) for ATP (82), which may allow mTOR activity to respond to a cell’s energy/metabolic state. At the molecular level, proteins of the tumor-suppressor complex, TSC1TSC2, have been identified as upstream nutrient-sensitive negative regulators of mTOR. The proteins TSC1 (also known as hamartin) and TSC2 (also known as tuberin) form a heterodimeric complex (TSC1-TSC2) linked by interactions between their respective N-terminal domains (83,84). The TSC1-TSC2 complex is the functional unit in regulating mTOR signaling because, in cells lacking either TSC1 or TSC2, the phosphorylation state of the downstream target of mTOR, p70S6K, is resistant to amino acid deprivation. Conversely, increased levels of TSC1 and TSC2 prevent p70S6K phosphorylation even in the presence of abundant amino acids. Studies have identified the Ras-related GTPase, Rheb (Ras homologue enriched in brain), as a link between TSC1-TSC2 and mTOR. Rheb is inhibited by the GAP activity of TSC1-TSC2. Loss of function of the TSC1TSC2 complex yields constitutive unrestrained signaling from the cell growth machinery composed of Rheb, mTOR, and p70S6K. Constitutive activation of the Rheb/mTOR/ p70S6K signaling cassette, whether by genetic deletion of TSC1 or TSC2 or by ectopic expression of Rheb, is sufficient to induce insulin resistance (85). Finally, mTOR has been shown to form a nutrient-dependent complex with the protein raptor. Raptor is a 150-kDa scaffolding protein that tethers mTOR to its substrates 4EPB1/PHAS-1 and p70S6K, potentiating their phosphorylation (86–88).

Mammalian Target of Rapamycin A variety of cellular activities contribute to GI epithelial cell growth and differentiation, including nutrient uptake. The serine-threonine kinase, mTOR, is a key component of the cellular mechanisms regulating cell growth in response to nutrient stimulation. The mTOR inhibitor, rapamycin, has been a valuable tool for identifying downstream targets of mTOR. Some proteins identified in this manner include 40S ribosomal protein S6 Kinase (p70S6K), CLIP-170 (cytoplasmic linker protein-170), elongation factor 2 (EF2) kinase, glycogen synthase, hypoxia-inducible factor-1α (HIF-1α), eukaryotic initiation factor 4E (eIF4E)binding protein (4EBP1, also known as PHAS-I), PKCδ and PKCε, protein phosphatase 2A (PP2A), p21WAF1

Protein Kinase B/Akt and Mammalian Targets of Rapamycin/p70S6K Studies suggest that a link between PI3K and mTOR may occur through PKB/Akt-mediated phosphorylation of TSC2, which was found to disrupt and inactivate the TSC1-TSC2 complex. Manning and coworkers (89) used mass spectrometry to identify cellular proteins that immunoprecipitated with a phospho-specific antibody designed to recognize sites phosphorylated by PKB/Akt. One of the proteins identified in this manner was TSC2. Moreover, PKB/Akt phosphorylated TSC2 at several sites, including S939, S1130, and T1462 in its C-terminal region. Overexpressing TSC2 mutants with Ala substitutions at the PKB/Akt phosphorylation sites

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 445 blocked growth factor–induced activation of p70S6K, indicating that TSC1-TSC2 complex is downstream of growth factor–regulated PKB/Akt activity and upstream of mTOR/p70S6K. An additional level of cross talk between the PI3K and mTOR pathways occurs through a negative feedback loop involving mTOR-mediated inhibition of IRS1, an adapter protein required for PI3K activation by the insulin receptor. Activation of mTOR results in phosphorylation and subsequent proteasomal degradation of IRS1, leading to reduced PI3K signaling. Finally, the mTOR/rictor protein complex plays an essential role in fully activating PKB/Akt by phosphorylation of S473 as described earlier. Krüppel-like Transcription Factors: Positive and Negative Regulators of Epithelial Cell Growth GKLF (Krüppel-like transcription factor 4 [KLF-4]) is a member of the Krüppel family of transcriptional regulators characterized by the presence of three highly conserved carboxy-terminal Cys2/His2 zinc-finger DNAbinding domains. All Krüppel family members bind CACCC (GT-box) sequences on the DNA. Krüppel-like transcription factor 4 is expressed throughout the GI tract but is most abundantly expressed in the colonic epithelium, where it is found mainly in the nonproliferating/differentiating cells of the upper crypts and villus (90,91). Experiments in cell lines have demonstrated that KLF-4 regulates cell cycle arrest at the G1/S checkpoint, suggesting that KLF-4 may inhibit normal colonic epithelial cell growth and perhaps function as a tumor suppressor. Support for this hypothesis is provided by data showing that KLF-4 expression down-regulates β-catenin expression and inhibits cyclin-D1 expression in HT-29 colon cancer cells (92). In contrast with KLF-4, KLF-5 is expressed mainly in the proliferating cells of the crypt, suggesting it may regulate cell proliferation (93). Several lines of evidence indicate that KLF-5 exerts a stimulatory effect on normal epithelial cell growth. First, KLF-5 expression is increased in proliferating skin cells; second, mice heterozygous for the KLF-5 gene have shorter villi and less crypt cells than wild-type littermates (94); and third, expression of KLF-5 in the nontransformed rat intestinal epithelial cell line, IEC-18, increases the ability to form colonies in soft agar and enhances cyclin D1 promoter activity (95).

are carried out by phosphorylating downstream target proteins and by carrying information to the nucleus or cytoskeleton through translocation within the cell (96,97). Currently, six subfamilies of MAPK pathways have been identified, including the extracellular signal-regulated kinases (ERK1/2), the c-Jun N-terminal kinases (JNKs), the 38-kDa MAPKs (p38MAPKs), ERK6/p38-like kinase, Big MAPK, also called ERK5, and ERK3 (97). Mitogen-activated protein kinases are activated by a kinase cascade composed of three families of protein kinases acting in series. Each MAPK is activated by an upstream MAPK kinase (MAPKK), which, in turn, is activated by a MAPK kinase kinase (MAPKKK). All MAPKKs are dual-specificity kinases that activate MAPKs by phosphorylating both the tyrosine and threonine residues present in the consensus sequence (T-X-Y). Six MAPKK family members have been identified and are designated MEK1-5 and ERK3 kinase. Upstream of MAPKKs are the MAPKKKs, which include protein kinases such as Raf (-1, -A, -B, and -C), TGF-β–activated kinase (TAK), and thousand-and-one-amino acid kinase (TAO). Extracellular signal-regulated kinase-1 and -2 phosphorylate downstream components on a consensus Pro-X-Ser/Thr-Pro motif. Several kinases containing this motif have been identified. These include the serine-threonine kinases Rsk1-3, MAPKAP kinase-2 and -3, Mnk1, and Mnk2. Identified membrane protein substrates of ERK1, ERK2, or both include tyrosine kinase receptors, Gα-interacting protein (GAIP), regulator of G protein signaling (RGS) proteins that act as GTPaseactivating proteins for Gα subunits of heteromeric G proteins, and calnexin, an ER type 1 integral membrane protein. Cytosolic and cytoskeletal protein substrate of ERK1/2 include the cyclic adenosine monophosphate-specific phosphodiesterase 4D3 and the multifunctional docking protein paxillin. Nuclear substrates include components of the transcription ternary complex such as Elk1, c-Fos and c-Jun, the nuclear factor-activated T cells (NFATc), and members of the STAT family. Although the precise role played by MAPK family members in normal GI epithelial cell physiology is largely undefined, data from various in vitro models suggest that they regulate the proliferation and differentiation responses of cell to growth factors and peptide hormones. They also appear to function as signal integrators by mediating the “cross talk” between different extracellular stimuli (98).

Mitogen-Activated Protein Kinase Pathways Mitogen-activated protein kinases (MAPKs) are serinethreonine–directed kinases that are activated by a variety of stimuli, including growth factors, cytokines, neurotransmitters, hormones, ECM proteins, and cell stress (96) (see Fig. 15-6C). Mitogen-activated protein kinases regulate a number of cellular processes, including gene transcription, protein translation, metabolism, and the function of the cytoskeleton. As a result, MAPKs are involved in the control of cell growth, differentiation, and apoptosis. These actions

Role of Sonic Hedgehog Pathway in Gastric Gland Maintenance The first hedgehog gene was identified in Drosophila as a gene that regulated segment polarity. Since then, three hedgehog genes have been identified in vertebrates: Shh, Ihh, and desert hedgehog (Dhh). The products of all three hedgehog genes bind to the cell surface receptor, patched, which controls the activity of a second receptor, smoothened. The hedgehog family of genes plays an essential role in

446 / CHAPTER 15 regulating the embryonic gut development and maintenance of gastric glands in the adult stomach. During development, Shh is a critical endodermal signal in the epithelial-mesodermal signaling involved in specification of differentiation along the anteroposterior and radial axis of the vertebrate gut (see Fig. 15-6D). Sonic hedgehog knockout mice exhibit GI malformations, gastric mucosa hyperplasia, and signs of intestinal differentiation such as expression of the brush-border enzyme, alkaline phosphatase. Although the role of hedgehog in gut development is relatively well characterized, its role in regulating the growth and differentiation of the adult GI tract is only beginning to be uncovered. Experimental evidence suggests an important role for Shh in maintenance of the normal gastrin gland architecture. The glandular epithelium of the stomach is composed of tubular invaginations termed gastric glands or pits, which are subdivided into foveolas, isthmus, neck, and base regions. In the central stomach (corpus), each gastric pit contains approximately 200 cells representing the three predominant cell lineages: foveolar mucous cells (also called mucous pit cells), parietal cells, and zymogenic cells. The specialized zymogenic cells constitute the pepsinogen-secreting chief cells, histamine-producing enterochromaffin-like (ECL) cells, and the somatostatin-releasing D cells. Foveolar mucous cells are located on the mucosal surface and in the upper region of the foveolae. They contain tightly packed cytoplasmic mucous granules and elaborate mucus containing a small amount of bicarbonate-rich fluid that serves as a barrier to acid-peptic injury. The zygomatic cells are located at the base of the gastric glands. The different cell lineages arise to form common multipotent stem cells located in the central (isthmus) region of the gastric gland. One of the committed daughter cells, the granule-free prepit cell precursor cell, produces mucus-secreting pit cells that differentiate as they migrate up from the isthmus to the pit opening. Another daughter cell, the granule-free preneck cell precursor, gives rise to pepsinogen-producing neck cells that differentiate to zymogenic cells as they descend to the base to the gastric pit. The acid-producing parietal cells differentiate within the isthmus to form granule-free preparietal precursor cells, and then migrate either up or down the pit axis. Sonic hedgehog appears to be a critical negative regulator of gastric gland proliferation and an important determinant of gland asymmetry. This conclusion is based on the observations that treatment of mice with the Shh inhibitor, cyclopamine, resulted in the decreased expression of the Shh target genes, hepatocyte nuclear factor 3β, Islet-1 (Isl-1), bone morphogenetic protein 4 (BMP-4), and induced a significant increase in gastric epithelial cell proliferation. Compared with untreated control mice, cyclopamine-treated mice showed an increase of 169% in proliferating cell nuclear antigen–positive and 164% bromodeoxyuridine (BrdU)-positive gastric epithelial cells, respectively (99). The strong stimulation of gastric epithelial cell proliferation

on inhibition of Shh is surprising because activating mutations of the Shh pathway in skin and neural tissue exert an oncogenic effect (100), suggesting that Shh may exert apposing cell type-specific effects on proliferative pathways. See Chapter 9 for a detailed discussion of the hedgehog pathway.

Transforming Growth Factor-β/SMAD Pathway The TGF-β superfamily of growth factors regulates a plethora of biological processes, including embryonic development, wound healing, angiogenesis, cell proliferation, and differentiation (see Fig. 15-6E). In the GI tract, TGF-β plays an important role in inhibiting epithelial cell proliferation and as a mediator of the epithelial/mesenchymal cell interactions. The TGF-β family of ligands includes TGF-β family members, BMPs, and activins. These ligands bind to two surface receptors, the type I and II receptors. Each receptor is composed of an extracellular ligand-binding domain, a single-transmembrane-spanning domain, and an intracellular domain with intrinsic serine-threonine kinase activity. Transforming growth factor-β receptors signal to intracellular SMAD proteins. Three classes of SMAD proteins have been characterized: (1) the receptor-regulated SMADs (R-SMADs), which include SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8; (2) the common SMAD known as SMAD4; and (3) the inhibitory SMADs (I-SMADs) consisting of SMAD6 and SMAD7. SMAD4 is common to both the BMP and TGF-β signaling pathways. Transforming growth factor-β binding induces type II and type I receptor dimerization, autophosphorylation, and activation of R-SMADs. The phosphorylated R-SMADs associate with co-SMAD, SMAD4, and translocate to the nucleus where the SMAD complex interacts with either coactivators or corepressors of transcription in a cell type-specific manner to regulate expression of target genes. Transforming growth factor-β ligands signal preferentially through SMAD2 and SMAD3, whereas BMP signaling is mediated through SMAD1, SMAD5, and SMAD8. In adults, TGF-β is detected in all GI tract tissues and accessory organs. Within the small intestine, TGF-β is expressed in both the lamina propria cells and the epithelium, where it regulates cell proliferation and plays a role in inflammation and tissue repair. Interestingly, TGF-β null mice show no gross developmental abnormalities at birth; however, they die approximately 20 days after birth from multifocal inflammatory disease of the stomach and intestine. In humans, TGF-β expression increases after acute epithelial injury in vivo and in patients with active inflammatory bowel diseases, suggesting that TGF-β may play a role during periods of active inflammation of the intestinal mucosa. In addition, other TGF-β family ligands, including activin A, and their respective receptors, Act I and Act II, have been shown to be expressed within the intestine and to modulate intestinal epithelial cell function.

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 447 Runx3: Targets of Transforming Growth Factor-β Signaling Transforming growth factor-β inhibits gastric epithelial cell growth, in part, by activating Runt-related domain transcription factor, Runx3 proteins. Runx3 (previously called AML2/PEBP2αC/CBFA3) is a heterodimeric transcription factor composed of α and β subunits. The Runx family members contain an evolutionarily conserved DNA-binding domain called the Runt domain. Runx and R-SMADs work synergistically to regulate TGF-β target gene expression.

Growth Factors and Receptor Tyrosine Kinases Peptide growth factors are low-molecular-weight (<25 kDa) proteins that either act locally on adjacent cells in a paracrine manner or on the same cell in an autocrine fashion. They exert their biological effects by binding to specific high-affinity cell-surface receptors present on target cells. A number of peptide growth factors have been identified that regulate intestinal epithelial and nonepithelial cell proliferation, migration, and differentiation. These peptide growth factors have been cataloged into discrete families based on their overall structural homology. They include the EGF, TGF-β, IGF, FGF, and trefoil factor (TFF) families. In addition to these families, a small number of peptide growth factors, without structural similarities to other growth factors, have been identified in the GI tract, including HGF and PDGF. Epidermal Growth Factor Ligands and Receptor Epidermal growth factor and the related growth factors, such as TGF-α, heparin-binding EGF (HB-EGF), betacellulin (BTC), and amphiregulin (AR), bind to a family of receptors known as type I RTKs (101). The EGF receptor family is composed of four structurally related members: the EGF receptor (ErbB1/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) (102). Each receptor consists of an extracellular ligand-binding domain connected to the cytoplasmic protein tyrosine kinase domain by a singlemembrane-spanning segment. Activation of EGF receptors is controlled, in part, by the spatial and temporal expression of their ligands. ErbB ligands are synthesized as membraneanchored precursor proteins that are cleaved by cell-surface proteases to release the active growth factor. Ligand binding to ErbB induces homodimerization and heterodimerization of receptor monomers and subsequent autophosphorylation of specific tyrosine residues within their cytoplasmic tail domains. The phosphorylated tyrosine residues serve as docking sites for SH2-domain binding proteins, such as Grb2 and Gab1 (103), of which their recruitment leads to the activation of intracellular pathways, including PI3K- and MAPKregulated pathways (104). Epidermal growth factor and TGF-α are the prototype members of the EGF ligand family. The most common

receptor for EGF ligands in the GI tract is EGFR (ErbB1); however, mRNA encoding HER-2 and HER-3 has been detected in normal and fetal GI tissues, as well as in colon cancers. Within the GI tract, EGF is produced in submaxillary glands and Brunner’s glands in the duodenum (105). Also, a small amount is produced within the exocrine pancreas. Epidermal growth factor receptors have been reported on both the apical (brush-border) and basolateral surfaces of enterocytes. Evidence suggests that the apical EGFR regulate EGF effects on epithelial cell homeostasis, whereas the basolateral receptor is involved in EGF-mediated mucosal repair after injury. In addition, EGF ligands have been shown to be potent stimulators of intestinal cell proliferation, to modulate the expression of ornithine decarboxylase and thus polyamine production, to enhance nutrient transport in the enterocyte, to stimulate brush-border enzyme expression, and to promote intestinal healing (106). Insulin-like Growth Factor-1 Insulin-like growth factor-1 is produced by the liver and other tissues, including the intestine, where it plays an important role in regulating cell growth and nitrogen balance. Insulin-like growth factor peptides promote intestinal wound healing and exert trophic effects on normal cells of the intestine, as well as promote tumor growth through autocrine mechanisms. Insulin-like growth factor-1 treatment after 70% jejunoileal resection facilitates intestinal fat and amino acid absorption and increases total gut weight by up to 21% (107). Six high-affinity IGF-binding proteins (IGFBP1-6) regulate IGF-1 action. Insulin-like growth factor binding proteins can prolong IGF-1 action by extending its plasma half-life. More than 90% of circulating IGF-1 forms a 150-kDa complex with IGFBP-3 and an acid labile subunit. This high-molecular-weight complex cannot cross capillary membranes and may serve as a reservoir for circulating IGF-1 and prolong the plasma half-life of IGF-1. Locally, the presence of IGFBPs can limit IGF-1 action by sequestering IGF-1 from its receptor, and membrane or ECM-associated IGFBPs have been found to enhance IGF-1 action by slowing the presentation of growth factor to the IGF-1 receptor (IGF-1R), thus preventing the receptor desensitization that would occur with exposure to high concentrations of hormone. The IGF peptides exert their biological activities through interactions with at least three cell-surface receptors. Both type 1 and 2 IGF receptors are expressed in the GI epithelium. All IGF receptors are RTKs. Ligand binding to the IGF-1R activates multiple signal proteins and pathways, including phosphorylation of IRS-1, SH2-domain-containing protein, Shc, MAPKs, PI3K, and JAK/STAT pathways. Fibroblast Growth Factor The FGFs are a family of related 15- to 16-kDa proteins controlling normal growth and differentiation of mesenchymal,

448 / CHAPTER 15 epithelial, and neuroectodermal cell types. Fibroblast growth factor-1, or acidic FGF (aFGF), and FGF-2, or basic FGF (bFGF), are the best-characterized members of the FGF family. Although only limited data are available on the expression and biological activities of FGFs and their receptors in the GI tract, several reports have described the presence of FGF family peptides and FGF receptors in the intestine (108–110). Evidence supports the assumption that FGFs might serve as autocrine growth factors and stimulate intestinal epithelial cell proliferation (111,112). In addition, aFGF and bFGF promote intestinal epithelial restitution through a TGF-β-dependent pathway in vitro. Trefoil Factors The TFFs are a family of proteins expressed in a regionselective pattern throughout the GI tract, where they play an important role in the epithelial wound repair (113). Trefoil factor peptides are characterized by a highly conserved motif called the trefoil domain that consists of 38- to 39amino-acid residues that form 3 intrachain loops as a result of disulfide bonds between 3 pairs of cysteine residues (114). Three TFF family members have been identified: TFF1 (formerly pS2), TFF2 (formerly spasmolytic polypeptide), and TFF3 (previously ITF) (113). Trefoil factor 1 was first identified in human breast carcinoma cell lines and is expressed in the stomach, Brunner’s glands, and large-intestinal goblet cells. Trefoil factor 1 knockout mice exhibit severe hyperplasia and dysplasia of gastric cells (115), and somatic mutations in exon 1 and 2 of TFF1 have been found in gastric cancers (116,117), suggesting that TFF1 may be a specific tumor-suppressor gene for the stomach. Trefoil factor 2 is expressed in the mucous neck region of the antral and pyloric glands of the stomach and in the Brunner’s glands of the duodenum (118). In the rat stomach, TFF2 mRNA levels increase within 30 minutes after mucosal injury, and administration of exogenous TFF2 protects against ethanol- and indomethacin-induced gastric injury (119). Trefoil factor 3 is absent in the stomach, but is expressed in theca of mature goblet cells of the intestine and is secreted onto the mucosa surface throughout the small and large intestine. Both in vitro and in vivo studies indicate that TFF3 is important in protecting the intestinal mucosa from a variety of insults and is essential for injury repair. Mashimo and colleagues (120) showed that TFF3 knockout mice have impaired colonic mucosa restitution in response to dextran sodium sulphate-induced injury. Furthermore, this study showed that an effectual mucosal repair mechanism is essential to the health of the animal. Recent studies suggest that modulation of repair mechanisms by TFF peptides may be mediated by modulation of E-cadherin/β-catenin complex function (121,122). Preliminary studies indicate that TFF1 and TFF2 transcription is controlled, in part, by transcription factors regulated by the ERK1/2-MAPK pathway, and thus are coupled to EGF and perhaps GI peptide hormone receptors.

Additional evidence suggests a role for the gp130 receptor, which binds the inflammatory cytokines, IL-6 and IL-11, and signals through the JAK/STAT pathway. Hepatocyte Growth Factor Hepatocyte growth factor, also known as Scatter Factor, is a heparin-binding glycoprotein that stimulates cell motility and proliferation and tissue morphogenesis (123). Hepatocyte growth factor is produced primarily by mesenchymal cells and is secreted as pro-HGF, an inactive single-chain precursor molecule that is activated by proteolytic cleavage into α and β subunits linked by a disulfide bond (124). Hepatocyte growth factor activator is the serum proteinase that specifically converts an inactive single-chain form of HGF into an active two-chain form. The high-affinity receptor for HGF is the RTK, c-Met. The c-Met receptor is synthesized as a single polypeptide chain and cleaved into α and β chains on the cell surface. The c-Met receptor heterodimer consists of an extracellular α chain and a β chain that contains an extracellular domain, a single-transmembrane domain, and a cytoplasmic protein kinase domain (125). On ligand binding, c-Met is autophosphorylated on two critical tyrosine residues, Y1349 and Y1356. These two tyrosine residues function as docking sites for recruiting several signal transducers and adaptor molecules, including Grb2, Gab1, SHC, PI3K, and phospholipase Cγ (126). Hepatocyte growth factor–induced cell motility requires activation of PI3K (127) and the Ras-related GTPases, Rac and Rho (128), whereas HGF-stimulated proliferative responses require activation of the Ras-MAPK pathway through the Grb2-SOS complex (129). In the presence of HGF, a number of epithelial cell lines form complex and ordered three-dimensional structures, including branched tubules similar to those found in epithelial organs, such as the kidney or the mammary gland (130). There is evidence for a role for STAT3 in HGF-induced tubulogenesis. In response to HGF, STAT3 is phosphorylated and translocated to the nucleus. Blocking STAT3 activation abolishes the morphogenetic response to HGF. Immunoprecipitation experiments suggest that Gab1 may mediate HGF activation of STAT3 (131). Together, these studies demonstrate that HGF activates multiple signal transduction pathways involved in GI epithelial cell growth, migration, and differentiation. They also suggest that expression of distinctive subsets of signal transducers and adaptor proteins downstream of c-Met provides the molecular basis for the diversity of responses induced by HGF in different cell types. Keratinocyte Growth Factor Keratinocyte growth factor, a member of the FGF family, is secreted by stromal cells (132) and acts on GI epithelial cells to stimulate their proliferation and differentiation both in vivo (112,133) and in vitro (108,134). There is evidence suggesting that KGF may regulate differentiation in a cell

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 449 lineage-specific fashion. Iwakiri and Podolsky (135) have shown that KGF promotes differentiation of H2-UD intestinal epithelial cells to a goblet cell phenotype by inducing the expression of a subset of lineage-specific genes, including TFF3 and MUC2. Keratinocyte growth factor may also play a role in epithelium restitution as evidenced by its increased expression during inflammatory bowel disease (136–138). Keratinocyte growth factor binds to a RTK that is a splicing variant of the FGFR2 (139,140). After ligand binding, keratinocyte growth factor receptor (KGFR) is rapidly phosphorylated and undergoes clathrin-mediated endocytosis. Little is known about KGFR protein distribution within the intestinal mucosa; however, KGFR mRNA was found throughout the GI tract (112,136). In situ hybridization localized KGFR mRNA expression to the crypts of the mucosal epithelium (137). In a model of intestinal cell differentiation in Caco-2 cells, Visco and coworkers (141) show that KGFR was distributed on the basolateral membrane of polarized Caco-2 cells, and that the increased expression of KGFR was associated with confluence-induced cell differentiation. Furthermore, immunohistochemical analysis confirmed that KGFR is predominantly expressed on more differentiated colon epithelial cells in vivo. Interestingly, the differentiating colonocytes were still able to synthesize DNA and, therefore, to proliferate in response to the KGF, but not in response to the EGFR ligands TGF-α and EGF, suggesting that KGFR plays

a distinct role in the control of intestinal epithelial cell proliferation and differentiation (141).

GROWTH REGULATION BY LUMINAL NUTRIENTS AND SECRETIONS The GI mucosa is highly susceptible to changes in diet and intraluminal nutrients. For example, fasting produces a profound atrophy of both small bowel and colonic mucosa in rats and mice. This is noted by a decrease in BrdU incorporation after fasting. Refeeding results in a dramatic increase in BrdU incorporation, resulting in proliferation of the atrophic gut mucosa (Fig. 15-7) (78). Activation of the PI3K/Akt pathway appears to be of critical importance for the stimulated induction of intestinal proliferation after a fast. Similar effects are noted in humans deprived of enteral nutrients during periods of severe illness or gut disuse. Although total parenteral nutrition maintains the nutritional requirement of the individual, pronounced mucosal atrophy occurs in the absence of enteral nutrition. These findings demonstrate the importance of enteral delivery of nutrients to the intestinal mucosa for the maintenance of growth and proliferation. Enteral components, including certain amino acids (most notably glutamine), nucleotides, polyunsaturated fatty acids, and short-chain fatty acids (SCFAs; most notably

FIG. 15-7. Bromodeoxyuridine (BrdU) incorporation into the colon DNA in the fasting/refeeding model. (A) Bromodeoxyuridine incorporation into the colon genomic DNA from mice in control, fasting, and refeeding groups. All mice were injected with BrdU, and incorporated BrdU was visualized by the dot-blotting method. (B) Bromodeoxyuridine incorporation detected by immunohistochemistry (original magnification ×200). Representative fields are shown, and BrdU-positive nuclei are marked with arrows. (C, left). Results of densitometric analysis of BrdU incorporation measured by the dot-blotting method. (C, right) BrdU-labeling index calculated by immunohistochemistry. In both analyses, BrdU incorporation was calculated as a percentage of the value in the control group. Values are means ± standard error. (Reproduced from Ueda and colleagues [78], by permission.)

450 / CHAPTER 15 Diet

Threonine Goblet cells

Glutamate Glutamine Asparate Proline

Mucin Enterocytes CO2 Arginine Citrulline Polyamines

Nucleotides n-3 PUFA

gut or its function. In vitro studies using cultured enterocytes demonstrate cell proliferation, protein synthesis, expression of ornithine decarboxylase and immediate early genes (e.g., c-jun), and polyamine synthesis (159). In addition, glutamine suppresses enterocyte apoptosis, suggesting that it may also function as an antiapoptotic factor. Patients with short bowel syndrome after massive intestinal resection show improved nutrient absorption and indices of bowel function when provided with supplemental glutamine. Other amino acids that may play a role in the maintenance of gut mucosal proliferation include arginine, ornithine, and proline (160). Studies using formula-fed piglets suggest that threonine is important for intestinal growth as well; 60% of dietary threonine is used by the gut in the postprandial state (146).

Proliferation Anti-inflammation

Crypt cells

Glutamine Glucose SCFA

Blood

FIG. 15-8. Illustration of some of the key substrates and nutrients supplied to the gut by the diet and the blood that have been shown to play a role in oxidative metabolism, polyamine synthesis, cell proliferation, inflammation, and mucin production in the intestinal mucosa. PUFA, polyunsaturated fatty acid; SCFA, short-chain fatty acid. (Reproduced from Burrin and Stoll [142], by permission.)

butyrate), interact with peptide growth factors to stimulate proliferation or apoptosis (Fig. 15-8) (142–146).

Amino Acids Certain amino acids have been studied extensively and shown to be important, particularly for the maintenance of small intestinal mucosal proliferation (142,143,146,147). The amino acid glutamine is a unique amino acid that functions as a key fuel for rapidly proliferating cells such as enterocytes and lymphocytes, and also is a precursor of nucleic acids, nucleotides, and other compounds (148). The significance of glutamine on intestinal proliferation was first described by Windmueller and Spaeth (149) and, most recently, by in vivo studies in humans showing that 80% of dietary glutamine is used by the small intestine (148, 150–152). Although less characterized, glutamate likewise is essential for intestinal metabolism with approximately 60% utilization by the intestine (146,150,153,154). Glutamine supplementation improves and maintains structural integrity, growth, and function of the intestine during conditions of stress, small intestine disuse, or severe illness (155,156). The effects of both glutamine and glutamate appear most dramatic during periods of small bowel mucosal atrophy where these amino acids, given enterally, can maintain mucosal proliferation (148,157,158). In contrast, supplementation of glutamine had no effect on normal growth of the

Short-Chain Fatty Acids, Fiber Short-chain fatty acids, produced by fermentation of carbohydrates in the colon, represent the most important fuel source for the colon (161). The colonic epithelium derives 60% to 70% of its energy from SCFAs (acetate, propionate, and butyrate), with butyrate as the preferred oxidative fuel. The proliferative effects of SCFAs on the normal colon have been demonstrated extensively and appear to be both direct and indirect (142,162–165). Indirect proliferative effects of SCFAs include increased mucosal blood flow, altered GI motility, and release of certain proliferative gut hormones including GLP-2 and PYY. Short-chain fatty acids also decrease intestinal permeability and prevent gut barrier failure associated with parenteral nutrition in rodents. Systemic administration of SCFA to rats fed by total parenteral nutrition increases ileal proglucagon and ornithine decarboxylase gene expression and expression of the immediate early genes c-jun, c-fos, and c-myc (166–168). The absence of butyrate is associated with Bax protein induction and apoptosis in the proximal colon of the guinea pig (169). In contrast, butyrate inhibits proliferation of neoplastic colonic epithelial cells by inhibiting DNA synthesis and arresting colonocytes in the G1 cell cycle phase, inducing differentiation and eventual apoptosis. The trophic effects of dietary fiber (e.g., those components of plants that are resistant to digestion by endogenous enzymes) are, in part, mediated by SCFA released from luminal fermentation (170–174). Most fiber is fermented in the colon, primarily to SCFA, and of these, butyrate serves as the principal energy source for colonic epithelial cells. Fiber that causes fermentation appears to stimulate colonic mucosal growth more than those that are inert.

Nucleotides and Polyunsaturated Fatty Acids Nucleotides are ubiquitous low-molecular-weight compounds consisting of a purine or pyrimidine base that are important as precursors for nucleic acid synthesis in rapidly dividing cells (e.g., epithelial cells in the intestinal mucosa). Nucleotide supplementation has been shown to support intestinal mucosal immune function and intestinal cell growth

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 451 (142,175,176). In addition, nucleotide supplementation of rats given total parenteral nutrition can attenuate mucosal atrophy and stimulate proliferation of intestinal mucosa (177). There has been increased interest in the use long-chain fatty acids particularly the n-3 polyunsaturated fatty acids (e.g., ω3 fatty acids) for pediatric nutrition. Experimental and clinical studies have suggested that these fatty acids can reduce inflammatory effects of necrotizing enterocolitis, and thus may be useful for intestinal homeostasis (178,179). Kollman and colleagues (180) show that supplementation with ω3 fatty acids can enhance intestinal adaptation after small-bowel resection, possibly through YY levels that were noted to be increased.

Pancreaticobiliary Secretions Secretions from the pancreas and liver appear to contain trophic factors that maintain gut mucosal proliferation (181,182). Placement of these secretions into the distal ileum leads to mucosal hyperplasia (183). The increase in pancreaticobiliary secretions is thought to indirectly contribute to the trophic effect of certain intestinal hormones such as cholecystokinin (CCK) and may partially contribute to the proliferative effect of GRP/BBS.

GROWTH REGULATION BY SMALL PEPTIDE HORMONES AND THEIR RECEPTORS All known GI peptide hormones bind to receptors belonging to the GPCR superfamily. These receptors share common structural and functional characteristics, including seven-transmembrane-spanning α-helical domains and the ability to function as ligand-regulated guanine nucleotide exchange factors for intracellular heterotrimeric G proteins. Heterotrimeric G proteins consist of α, β, and γ subunits. The initial step in GI peptide hormone receptor-mediated signal transduction involves agonist-activated receptorcatalyzed exchange of GTP for GDP bound to the α subunit of the G protein and the dissociation of GTP-bound Gα subunit from its cognate Gβγ dimer. The activated GTP-Gα and Gβγ subunits, in turn, regulate the activity of various intracellular effector proteins, such as phospholipases, adenylyl cyclases, MAPKs, PI3K, membrane ion channels, and members of the Ras family of GTP-binding proteins. In addition to G protein-dependent signaling, GPCRs, in general, can also function as docking sites for adaptor proteins signaling through G protein–independent mechanisms analogous to RTKs (184).

Gastrin The actions of G17 in the GI tract are mediated, in part, by the cholecystokinin 2/gastrin receptor (CCK2R). In the normal stomach, G17 plays a role in the maintenance of the gastric mucosa by stimulating proliferation of ECL cells and

parietal cell differentiation (185–187). In the pancreas of rodents, G17 stimulates growth and differentiation of the endocrine pancreas, both during fetal development and in adults after injury. G17 stimulates the growth of some gastric, pancreatic, and colon cancer cells in vitro and in xenograft tumors in nude mice (188). Mice in which the gene for either gastrin or the CCK2 receptor have been deleted show reduced numbers of parietal and ECL cells (189–191), and transgenic mice that express gastrin under control of the insulin promoter in pancreatic β cells exhibit increased proliferation of the gastric and colonic mucosa. Using a nontransformed rat gastric epithelial cell line transfected with a human CCK2 receptor, Miyazaki and coworkers (192) showed that G17 stimulated an increase in [3H]thymidine incorporation into DNA. The increase in DNA synthesis was blocked by pretreating the cells with either a polyclonal antiserum to HB-EGF or the EGFR tyrosine kinase inhibitor, tyrphostin, suggesting that the CCK2 receptor-mediated mitogenic response requires EGFRmediated signaling. Support for this hypothesis was provided by Zhukova and coworkers (193) when they showed that CCK-8 induced a time- and dose-dependent increase in the level of tyrosine-phosphorylated EGFR in mouse Swiss 3T3 fibroblasts transfected with the CCK2 receptor. Cholecystokinin-8 also induced an increase in DNA synthesis ([3H]thymidine incorporation) that was comparable with low doses of either insulin or EGF alone. Combined treatment with CCK-8 and either insulin or EGF stimulated a synergistic increase in DNA synthesis. Western blot analysis revealed increased expression of cyclin D1, cyclin D3, cyclin E, and hyperphosphorylation of the tumor suppressor, pRb, which regulates transcription factors, such as E2F, that are critical for cell cycle progression from G1- to S-phase (194). Gastrin does not act directly on the proliferating cells (mucous neck cells) of the gastric glands because they do not express CCK2 receptors. Instead, gastrin stimulates ECL and parietal cells to secrete growth factors that include EGF, HB-EGF, TGF-α, AR, and Reg proteins. Gastrin and IL-1β stimulate the secretion of HB-EGF and AR, but not TGF-α from parietal cells. Gastrin and IL-1β induce a synergistic stimulation of growth factor secretion, and the PKC inhibitor staurosporine abolishes these stimulatory effects (195).

Glucagon-Like Peptides Glucagon-like peptide-1 is a 31-amino-acid residue polypeptide hormone secreted by the intestinal L cell in response to fat meals and carbohydrates (196). The effects of GLP-1 are mediated by the GLP-1 receptor (GLP-1R), which on agonist binding stimulates the expression of several immediate early genes and protooncogenes implicated in cell growth and the control of apoptosis, including c-fos, c-jun, junD, nur77, and PDX-1. Regulation of these transcription factors occurs through a PI3K/PKCξ-dependent pathway (197–199). Buteau and coworkers (200) show that GLP-1 induced activation of PI3K and promoted the proliferation of a derivative of the INS-1 cell line, called INS(832/13),

452 / CHAPTER 15 through transactivation of both EGFR and c-Src. Treatment with either the c-Src kinase inhibitor, PP1, or the EGFR inhibitor, tyrphostin, blocked GLP-1–induced [3H]thymidine and BrdU incorporation into DNA, whereas only tryphostin inhibited the proliferative action of the EGFR agonist, BTC. Furthermore, both PP1 and tryphostin suppressed GLP-1induced PI3K activation and the time-dependent increase in EGFR phosphorylation. Overexpression of a dominantnegative EGFR in INS(832/13) cells suppressed GLP-1induced cell proliferation, indicating, in a manner similar to G17, that the proliferative effects of GLP-1 require EGFR transactivation activation.

Glucagon-Like Peptide-2 Glucagon-like peptide-2 is produced with GLP-1 and also increases crypt cell proliferation and decreases enterocyte apoptosis in rats and mice (201,202). Glucagon-like peptide-2 decreases mucosal injury in indomethacin-induced colitis and chemical-induced inflammatory bowel disease. In premature pigs sustained by parenteral nutrition, GLP-2 stimulates intestinal growth. In patients with short bowel syndrome, GLP-2 increased villus height and crypt depth and improved nutrient absorption and nutritional status. Immunoneutralization of endogenous GLP-2 reduces the adaptive intestinal growth in diabetic rats. Glucagon-like peptide-2 administration in rodents increases small intestine weight, mucosal crypt, and villus height (203). The GLP-2–induced increase in small intestine weight and mucosal thickness is due, in part, to the stimulation of crypt cell proliferation, resulting expansion of the crypt compartment, and a lengthening of the villi (204,205). Together, these data suggest that GLP-2 is an important regulator of intestinal growth (206,207). The GLP-2 receptor, GPCR (208), is expressed predominantly in the GI tract localized to enteroendocrine cells (209). In the mouse, GLP-2 stimulates proliferation of columnar but not mucous progenitor cells. Increased proliferation is associated with activation of c-fos in crypt cells that do not express GLP-2 receptors (210), suggesting the involvement of an unidentified downstream mediator.

the Gαi/Gαo subfamily. When YR1 is expressed in the nontransformed rat intestinal epithelial cell line, IEC-6, PYY treatment stimulates an increase in cell proliferation and increased phosphorylation (activation) of the MAPKs, ERK1/2 (212). Epidermal growth factor, IGF-1, and TGF-α induce increased levels of serum PYY in a mouse and dog model (213), suggesting that the proliferative effects of these growth factors are mediated, in part, by PYY.

Neurotensin Neurotensin is a tridecapeptide synthesized in N cells of the small intestine, predominantly in the ileum (214,215). Neurotensin stimulates pancreatic and biliary secretions, inhibits small bowel and gastric motility, and stimulates the growth of various GI and pancreatic tissues. The trophic effects of NT are most prominent in the small intestine. Wood and colleagues (216) first reported that NT stimulates small bowel mucosa of rats fed a normal chow diet. Administration of NT prevents gut mucosal atrophy induced by an elemental diet and stimulates mucosal growth in defunctionalized, cell-emptying jejunoileal loops or isolated Thiry–Villa fistulas (TVFs) (217–219). Furthermore, NT restores mucosal integrity in rats after radiation-induced injury and prevents the translocation of indigenous bacteria (220). Several studies have shown that administration of NT can augment the normal adaptive hyperplasia of gut mucosa that is associated with massive small bowel resection in rats and rabbits (221–223). Enteroglucagon, which is released after NT administration, may contribute to the proliferative effects of NT (224). In the colon, NT has been shown to stimulate proliferation of rats fed a regular chow diet; however, the effects are less pronounced than in the small intestine (225). The effects of NT on various signal induction pathways have been evaluated in colon cancer cell lines, where it has been shown that NT treatment results in increased calcium mobilization, as well as activation of the ERK and JNK pathways, and induction of AP-1 transcription factor binding activity (226).

Bombesin/Gastrin-Releasing Peptide Peptide YY Peptide tyrosine-tyrosine (PYY) is a member of the pancreatic polypeptide family that also includes pancreatic polypeptide (PP) and neuropeptide Y (NYP). Peptide YY is synthesized and secreted by L cells of the distal ileum and colon (211). It has primarily been characterized as an inhibitor of gastric emptying, gut motility, and GI secretion. However, accumulating evidence suggests that PPY may also regulate intestinal epithelial cell growth. Data from cell lines expressing recombinant PYY receptor, Y1, provide the most direct demonstration of the growth-promoting effects of PYY. The Y1 receptor is a GPCR linked to the regulation of both adenylyl cyclases and MAPKs through G proteins of

Bombesin is a tetradecapeptide originally isolated from the skin of the frog, Bombina bombina, and is analogous to the mammalian GRP. In general, BBS/GRP may be thought of as a universal “on switch” with predominantly stimulatory effects (227). Bombesin/GRP stimulates pancreatic, gastric and intestinal secretion, and gut motility and release of all gut hormones. In addition, BBS/GRP is a potent trophic factor for the GI tract (228,229). In the small intestine, BBS stimulates intestinal mucosal growth in rats given an elemental diet (230,231). The trophic effects of BBS appear to be mediated by nonluminal and luminal (e.g., pancreaticobiliary secretions) factors as noted by construction of isolated TVFs (232). Bombesin also exhibits protective

REGULATION OF GASTROINTESTINAL NORMAL CELL GROWTH / 453 effects in the gut after injury with enhanced gut mucosal growth and significantly inhibited mortality in rats after administration of methotrexate, which leads to a severe enterocolitis in rats (233). Effects on colonic mucosal growth are limited, but similar to the small bowel, BBS appears to exert a trophic effect on the colon as well (230). These effects are less pronounced than in the small bowel or stomach.

Somatostatin Somatostatin is localized in the nerves and cell bodies of the central and peripheral nervous system and mucosa of the stomach and intestine (234). More than 90% of the somatostatin immunoreactivity in the human intestine is located within the mucosal endocrine D cells (235). In contrast with BBS/GRP, somatostatin is considered as the universal endocrine “off switch” that inhibits the release of growth hormone and somatomedin-C and all known GI hormones. Somatostatin inhibits GI secretion and motility and inhibits the growth of GI mucosa (236–238). Somatostatin inhibits the normal adaptive hyperplasia noted in rats after small bowel resection (239). These effects may be through either an indirect mechanism such as inhibition of other trophic hormones or a direct effect through interaction with the somatostatin receptor subtype 2. This somatostatin receptor subtype associates and stimulates tyrosine phosphatase Src homology 2-containing tyrosine phosphatase-1 activity, which, in turn, arrests cells in the G0/G1-phase of the cell cycle associated with up-regulation of p27KIP1 and an increase in the hypophosphorylated Rb protein (240). Although controversial, somatostatin may also function as an endogenous inhibitory factor for basal intestinal growth (241).

in rats (221–223). In a clinical trial assessing eight patients with short bowel syndrome, GLP-2 administered for 35 days resulted in improved intestinal energy absorption, decreased energy excretion, increased body weight and lean body mass, decreased fat mass, and enhanced urinary creatine excretion (243). These results are encouraging but limited and require a larger clinical series to reach definitive conclusions. Litvak and colleagues (244) demonstrated, in a murine model, that the combination of NT with GLP-2 was more effective in enhancing mucosal growth than either agent alone. Therefore, it seems likely that the combination of hormonal agents, with or without glutamine, may prove to be useful therapy in patients with diseases or conditions in which enhanced gut mucosal growth is advantageous. Another aspect that has been tested is the use of hormonal agents for ulcer healing. Using an experimental model, Schmassmann and Reubi (245) demonstrated improved wound healing in peptic ulcers of rats by inhibition of gastrin binding (during the early phase), and then administration of gastrin in the later phases to enhance healing rates. Although interesting, it is doubtful that these agents will prove more effective than what is currently available for patients with peptic ulcer disease. Finally, the use of hormonal agents as a method to stimulate the reparative phase during periods of inflammation has also been evaluated using experimental models. Both BBS and GLP-2 provide a protective effect for the GI mucosa during periods of gut injury and inflammation (201,233). In addition, nutrients that have a beneficial effect on the intestinal mucosa, such as glutamine and butyrate, have been shown to decrease inflammation in gut inflammatory models (142,143,145, 146,161,162). Therefore, short-term use of these agents in certain settings may provide an enhanced reparative effect and decrease inflammation in certain diseases of the intestinal mucosa.

FUTURE PERSPECTIVES/CLINICAL APPLICATIONS

REFERENCES

Many of the potential applications of hormonal agents in the stimulation of growth or mucosal repair remain in the experimental phases. However, it is anticipated that the advent of more specific and longer acting synthetic agonists could be beneficial for certain processes that require enhanced mucosal growth. For example, the potential use of agents to treat patients with short bowel syndrome has received considerable attention. Although controversial, some clinical studies have shown an advantage using a combination of glutamine with growth hormone as a method to enhance intestinal mucosal growth and successfully wean patients with short bowel syndrome off of total parenteral nutrition (242). In concept, the use of combination therapies is attractive, particularly hormones that specifically target the intestinal mucosa. Experimental studies using NT, BBS/GRP, and GLP-2 have shown augmentation of intestinal mucosal growth during periods of gut disuse or atrophy (203,216, 218,219,232). In addition, both NT and GLP-2 augment the adaptive hyperplasia associated with small bowel resection

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225. Evers BM, Izukura M, Chung DH, Parekh D, Yoshinaga K, Greeley GH Jr, Uchida T, Townsend CM Jr, Thompson JC. Neurotensin stimulates growth of colonic mucosa in young and aged rats. Gastroenterology 1992;103:86–91. 226. Ehlers RA 2nd, Bonnor RM, Wang X, Hellmich MR, Evers BM. Signal transduction mechanisms in neurotensin-mediated cellular regulation. Surgery 1998;124:239–247. 227. Greeley GH Jr, Newman J. Enteric bombesin-like peptides. In: Thompson JC, Greeley GH Jr, Rayford PL, Townsend CM Jr, eds. Gastrointestinal endocrinology. New York: McGraw-Hill, 1987; 322–329. 228. Lehy T, Puccio F, Chariot J, Labeille D. Stimulating effect of bombesin on the growth of gastrointestinal tract and pancreas in suckling rats. Gastroenterology 1986;90:1942–1949. 229. Thomas RP, Hellmich MR, Townsend CM Jr, Evers BM. Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues. Endocr Rev 2003;24:571–599. 230. Chu KU, Evers BM, Ishizuka J, Townsend CM Jr, Thompson JC. Role of bombesin on gut mucosal growth. Ann Surg 1995;222:94–100. 231. Puccio F, Lehy T. Bombesin ingestion stimulates epithelial digestive cell proliferation in suckling rats. Am J Physiol 1989;256:G328–G334. 232. Chu KU, Higashide S, Evers BM, Ishizuka J, Townsend CM Jr, Thompson JC. Bombesin stimulates mucosal growth in jejunal and ileal Thiry-Vella fistulas. Ann Surg 1995;221:602–611. 233. Chu KU, Higashide S, Evers BM, Rajaraman S, Ishizuka J, Townsend CM Jr, Thompson JC. Bombesin improves survival from methotrexate-induced enterocolitis. Ann Surg 1994;220:570–577. 234. Tulassay Z. Somatostatin and the gastrointestinal tract. Scand J Gastroenterol Suppl 1998;228:115–121. 235. Reisine T. Somatostatin. Cell Mol Neurobiol 1995;15:597–614. 236. Bass BL, Fischer BA, Richardson C, Harmon JW. Somatostatin analogue treatment inhibits post-resectional adaptation of the small bowel in rats. Am J Surg 1991;161:107–112. 237. Lehy T, Dubrasquet M, Bonfils S. Effect of somatostatin on normal and gastric-stimulated cell proliferation in the gastric and intestinal mucosae of the rat. Digestion 1979;19:99–109. 238. Rivard N, Guan D, Turkelson CM, Petitclerc D, Solomon TE, Morisset J. Negative control by Sandostatin on pancreatic and duodenal growth: a possible implication of insulin-like growth factor I. Regul Pept 1991;34:13–23. 239. Thompson JS, Nguyen BL, Harty RF. Somatostatin analogue inhibits intestinal regeneration. Arch Surg 1993;128:385–389. 240. Pages P, Benali N, Saint-Laurent N, Esteve JP, Schally AV, Tkaczuk J, Vaysse N, Susini C, Buscail L. sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of SHP-1. J Biol Chem 1999;274:15186–15193. 241. Parekh D, Izukura M, Yoshinaga K, Townsend CM Jr, Thompson JC. Endogenous somatostatin is an antitrophic factor for the gut and pancreas. Digestion 1990;46:84. 242. Scolapio JS. Treatment of short-bowel syndrome. Curr Opin Clin Nutr Metab Care 2001;4:557–560. 243. Jeppesen PB, Hartmann B, Thulesen J, Graff J, Lohmann J, Hansen BS, Tofteng F, Poulsen SS, Madsen JL, Holst JJ, Mortensen PB. Glucagonlike peptide 2 improves nutrient absorption and nutritional status in short-bowel patients with no colon. Gastroenterology 2001;120: 806–815. 244. Litvak DA, Evers BM, Hellmich MR, Townsend CM Jr. Enterotrophic effects of glucagon-like peptide 2 are enhanced by neurotensin. J Gastrointest Surg 1999;3:432–440. 245. Schmassmann A, Reubi JC. Cholecystokinin-B/gastrin receptors enhance wound healing in the rat gastric mucosa. J Clin Invest 2000; 106:1021–1029.

CHAPTER

16

Mucosal Repair and Restitution Mark R. Frey and D. Brent Polk Overview of Process, 460 Basic Mechanisms of Cellular Migration, 460 Comparison between Restitution and Chemotactic Migration, 461 Wound Healing by Distinct Phases of Cellular Migration and Proliferation, 462 Regulation of Epithelial Wound Healing by Extracellular Signals, 462 Role of Peptide Growth Factors and Cytokines, 462 Promotion of Wound Healing by Lipids and Other Nonpeptide Factors, 464 Regulation of Cellular Migration by Extracellular Matrix, 464

Intracellular Pathways Coordinating Migration, 465 Signal Transduction Pathways, 465 Gene Transcriptional Regulation, 466 Inside-Out Signaling, 467 Methodologies for Studying Gastrointestinal Cell Migration, 467 Cell Culture Models, 467 In Vivo Models of Restitution, 468 Relation of Altered Migration to Disease, 469 Future Challenges, 470 References, 470

The mammalian gastrointestinal tract plays a number of key roles in maintaining the health of the organism, including digestion, nutrient absorption, and barrier function. Efficient protection of the organism relies on both functional intestinal immune components and the integrity of the epithelial layer lining the gut. The epithelium is a rapidly renewing population forming a single-cell barrier along the entire length of the gastrointestinal tract. Thus, the mechanisms driving repair of this layer, which is constantly exposed to damage from luminal contents and pathogenic organisms, are critical for maintenance of a healthy intestine and organism. Intestinal epithelial repair after wounding generally involves several processes which, for the sake of this discussion, can be considered as at least partially independent (Fig. 16-1).

The immediate response to damage involves migration of surrounding epithelial cells to cover the denuded area (1–3); this part of the repair process is often termed restitution. A subsequent increase in proliferation of nearby cells aids in repopulating the damaged area and restoring normal architecture and cell density. For relatively small wounds, short restitution and proliferation phases may be sufficient to restore the monolayer. Depending on the depth and extent of the wound, substantial epithelial cell proliferation may also be required for complete coverage of the wound. Immunologic responses and deposition of protective granulation tissue may also be necessary to restore epithelial continuity when the wound is large. Significant progress has been made toward understanding the role of the early restitution phase of wound healing in maintaining a healthy intestinal barrier, as well as in delineating the signaling pathways involved in regulating epithelial cell migration into a wounded area and the interaction of restitution with other phases of intestinal repair. This chapter discusses the overall process of cell migration during restitution, the molecular control of the process as it is currently understood, and the basic experimental methods used to investigate epithelial cell migration. Perspectives on unanswered questions and possible future directions of investigation in the field also are discussed.

M. R. Frey: Department of Pediatric Gastroenterology, Hepatology, and Nutrition, Vanderbilt University, Nashville, Tennessee 37232. D. B. Polk: Departments of Cell and Developmental Biology and Pediatric Gastroenterology, Hepatology, and Nutrition, Vanderbilt University, Nashville, Tennessee 37232. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

459

460 / CHAPTER 16

A

B

C

D

E

F FIG. 16-1. The basic processes of intestinal epithelial wound healing. When an undamaged mucosal surface (A) is subjected to an insult that strips away epithelial cells (B), the first response is loss of polarization of cells near the wound margin and conversion to a migratory phenotype (C). Over a process of minutes to hours, depending on the extent of the wound, cells flatten and move to cover the denuded area, reestablishing the protective barrier (D). Leader and follower cells often move as a unified sheet, maintaining rudimentary attachments during the restitution process. Cell proliferation restores the epithelial population (E), allowing cells to reform normal junctional complexes and retrieve a polarized columnar phenotype (F).

OVERVIEW OF PROCESS Basic Mechanisms of Cellular Migration After the interruption of cell–cell junctions in an area denuded of epithelium, cells immediately surrounding the wound undergo a rapid loss of traditional epithelial cell columnar polarization. Cells dramatically reorganize their actin cytoskeleton, lose their microvilli and apicobasolateral orientation, and instead take on a flatter, broader appearance with polarization now defined from “leading” to “trailing” edge (2,4,5). This change in morphology has been described as dedifferentiation or “epithelial-mesenchymal transition,” but given that the cells first acquire a defined set of characteristics and morphology, and then regain their cell junctions and epithelial morphology as soon as the wound is closed, it may be more accurate to describe the phenomenon as a change in functional phenotype rather than a loss of epithelial differentiation. Immediately after loss of their neighbors, cells remaining at the wound margin flatten and extend lamellipodia at their leading edges into the denuded area (Fig. 16-2). This is accompanied by the formation of transient focal complexes, also at the leading edge, followed by a contraction of actinomyosin filaments, detachment of the rear of the cell, and retraction of a trailing edge (6,7).

The epithelial sheet then “crawls” in to close the wound through repeated cycles of protrusion, contraction, and detachment. All of these processes are thought to depend on extensive, dynamic remodeling of the actin cytoskeleton and the activity of a variety of regulatory signaling modules including small GTPases such as Rac and Rho (8–10), Ena/vasodilator stimulated phosphoprotein (VASP) proteins (11), and various tyrosine kinases (12–14). Interestingly, this is not the only wound closure or mass migration mechanism available to epithelial tissues. Developmental studies in several different organisms describe a model in which cells at the wound edge develop a unified actin cord along their leading edges, which then contracts, leading to a “purse-string” closing of the injured area (15–18). Although this model has primarily been described in embryonic wound healing and remodeling, studies in T84 (19) and CaCo-2BBE cells (20) demonstrate purse-string closure in nonembryonic cell lines. Furthermore, a few studies using corneal epithelial cell wound healing as a model system have also demonstrated purse-string closure. Danjo and colleagues (21) observed purse-string migration in vivo in wounded mouse corneas; this morphology was E-cadherin dependent, in that a neutralizing antibody to E-cadherin blocked formation of the actin cord and converted cells to a lamellipodial-style morphology. Taken together, these

MUCOSAL REPAIR AND RESTITUTION / 461

Chemical gradient, missing neighbors, etc.

A Cell alters orientation to allow for migration

B

Branched actin network supports protrusion

C Trailing edge detaches; filament contraction pulls rear of cell forward

Leading edge forms dynamic attachments

D

E FIG. 16-2. Lamellipodial crawling of epithelial cells. A cell sensing a chemical gradient or empty space (A) flattens (B) and extends lamellipodia, driven by organization of branched actin networks under the control of small GTPases (C). Highly dynamic focal attachments are formed as the cell extends. As attachments in the leading edge form, adhesions at the rear of the cell are removed (D) and the cytoskeleton contracts, pulling the trailing edge forward (E). Detailed models of cell crawling have been delineated in fibroblast and other epithelial cell types (for review see Ridley and colleagues [148]); available data suggest that these models will hold in intestinal epithelial cells.

results suggest that both mechanisms may play roles in adult intestinal epithelial wound healing. Some authors have speculated that the mechanism used to close a particular wound is likely determined by the size of the injured area, with large wounds tending to be closed by lamellipodial crawling and smaller wounds by purse-string closure (22). It is also possible that different mechanisms may be active at different phases of closure of a given wound.

Comparison between Restitution and Chemotactic Migration Chemotaxis, directed cell motility toward a chemical gradient, is often considered in contrast with chemokinesis, defined as stimulated migration in the absence of a chemical gradient. The portion of the restitution response regulated by chemotaxis versus chemokinesis in vivo is unclear, as is the extent to which mechanisms of wound-induced migration and chemotactic motility, as modeled in current cell culture models, are conserved. Because plentiful sources of potential chemotactic agents are present in the wound region in vivo, chemotaxis could play a role in wound healing. Underlying stroma or invading immune cells are rich sources of peptide growth factors and cytokines (23–25). Resident commensal bacteria can potentially release factors chemotactic for either the intestinal epithelium directly or for immune cells, and certain components of cellular debris (e.g., adenine nucleotides, phospholipids) (26,27) may also provide chemotactic stimuli. Furthermore, some of the same molecules that stimulate restitution in

cell culture models, such as epidermal growth factor receptor (EGFR) family ligands (28,29), hepatocyte growth factor (HGF)/scatter factor (30–32), lysophosphatidic acid (LPA) (27,33), and trefoil factor family peptides (TFFs) (34), also show activity in epithelial cell chemotaxis assays (35–39). However, several lines of evidence suggest that chemotaxis and restitutional migration are, at some level, different processes. First, the most widespread chemotaxis assay method, the Boyden chamber, measures movement of individual cells rather than sheets of cells as are observed in intestinal epithelial restitution (40). Furthermore, peptide factors such as EGF and HGF clearly can stimulate chemokinesis in assays where there is no chemical gradient (13,41), whereas also showing specifically directed activity in chemotactic assays (37,39). In addition, intermediate signaling pathways are not identical for chemokinetic versus chemotactic effects. For example, EGF-stimulated restitution of mouse small intestinal epithelial cells requires protein kinase C (PKC) activity (29), whereas chemotaxis to EGF in this cell model does not (37). Finally, basal restitution clearly occurs in in vitro model systems in the absence of any obvious chemotactic signal. In this regard, however, wounded intestinal epithelial cells shed EGFR family ligands via a matrix metalloproteinase (MMP)–dependent mechanism (42), and the physical segregation of growth factor receptors and ligands, normally provided by tight junction complexes in epithelial cells, is disrupted by wounding (43). This raises the possibility that cells may shed growth factors preferentially on their exposed face, thus creating a localized or limited autocrine chemotactic gradient.

462 / CHAPTER 16 TABLE 16-1. Molecules known to promote intestinal epithelial cell restitution Factor

Example gastrointestinal model systems

References

EGF/TGF-α Adenine nucleotides FGF family members HGF IL-2 IL-8 IGF-1 IFN-γ LPA Polyamines Short-chain fatty acids Trefoil factors TGF-β TNF-α Mucins

Organ culture; rat, mouse, and human intestinal epithelial cell lines Rat intestinal epithelial cell line Rat intestinal epithelial cell line; rat intestinal recovery from irradiation Cultured rat, mouse, and human intestinal epithelial cells Rat intestinal epithelial cells Human colon cancer cell lines Human colon cancer cell lines Rat intestinal epithelial cells TNBS colitis in vivo, IEC-6 cells Ischemic injury in rats, IEC-6 cells Human colon cancer cell lines Rat intestinal epithelial cells, human colon cancer cells, transgenics Rat intestinal epithelial cells Cultured human colon cancer, mouse intestinal epithelial cells Rat intestinal epithelial cell lines (cooperative with TFFs)

28, 29, 64 26 57, 58, 222 30, 56, 234 55, 108 111 161, 235 54, 108 121, 236 158, 178, 237, 238 118, 120 34, 64, 93, 94 54 46, 111 34

EGF, epidermal growth factor; FGF, fibroblast growth factors; HGF, hepatocyte growth factor; IEC-6, intestinal epithelial cell-6; IFN-γ, interferon-γ; IGF-1, insulin-like growth factor 1; IL, interleukin; LPA, lysophosphatidic acid; TFF, trefoil family factor; TGF-α, transforming growth factor-α; TNBS, 2,4,6-trinitrobenzene sulfonic acid.

Wound Healing by Distinct Phases of Cellular Migration and Proliferation

REGULATION OF EPITHELIAL WOUND HEALING BY EXTRACELLULAR SIGNALS

One interesting aspect of epithelial wound healing is the relation between cell migration and subsequent restorative proliferation. In epithelial cell culture wound closure models, restitution is followed by a wave of proliferation to repopulate the monolayer and allow flattened cells to regain their normal columnar epithelial phenotype (44). In vitro, these two phases of repair appear to be controlled by separate growth factor and extracellular matrix (ECM) signaling pathways (45). In restitution models, the proliferation phase is temporally and spatially distinct from migration, consistent with different morphologic needs and signal transduction pathways involved. Cells rounded up for mitosis are unlikely to be motile, and patterns of kinase activation and gene expression profiles for proliferation and migration may well be incompatible. In studies in our laboratory of young adult mouse colon (YAMC) cells, for example, wounding inhibits EGF-stimulated proliferation in a monolayer, and bromodeoxyuridine incorporation is essentially shut down in the migrating sheet about 20 cells deep surrounding a wound (46; also unpublished observations). On a signaling level, the activities of extracellular signal–regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs) promote proliferation and migration, respectively, and seem to oppose one another’s function (47). Although the situation in largely intact tissue may be more complicated, with multiple simultaneous foci of migration and proliferation across a wounded area, in vivo data showing rapid and immediate migration of wounded epithelia followed much later by a surge of proliferation certainly support temporal disassociation of migration and proliferation (3,4). Observations from wound healing models in developing flies and worms (see Martin and Parkhurst [22] for a review) further support a spatial and temporal segregation of these processes in vivo.

A wealth of different peptide growth factors, cytokines, and chemical agents are released by intestinal epithelial cells, underlying stromal cells, and immune cells in the microenvironment surrounding an epithelial wound. Intestinal restitution is regulated by these signals, as well as through interactions with the ECM, acting through cellular receptors to drive promigratory signal transduction. Many of these pathways have been tested with in vitro wound healing assays, and some have been used in clinical settings with some efficacy; for example, EGF given topically to ulcerative colitis patients promoted colonic epithelial ulcer healing and remission (48). Table 16-1 and Figure 16-3 list a selection of soluble extracellular molecules implicated in increased gastrointestinal epithelial restitution, the model systems in which their activity was defined, and their major sources in the gastrointestinal tract.

Role of Peptide Growth Factors and Cytokines Small peptides from a number of families with diverse structural and intracellular signaling mechanisms, loosely classified as peptide growth factors or cytokines, have been implicated in the regulation of intestinal restitution in both in vitro and in vivo studies. Among the best described mediators of epithelial cell migration in this class are the EGFR family ligands EGF and transforming growth factorα (TGF-α) (13,28,49,50), TFFs (34,51–53), and the antiproliferative peptide TGF-β, together with a number of cytokines such as interleukins (ILs), which may act through TGF-β release (54,55). Other growth factors that promote restitution in the intestinal epithelium include HGF, which enhances both motility and proliferation through the c-Met

MUCOSAL REPAIR AND RESTITUTION / 463

FIG. 16-3. Selected sources of epithelial restitution-promoting factors in the gastrointestinal tract. ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IFN-γ, interferon-γ; IL-8, interleukin-8; TFF, trefoil factor family; TGF-β, transforming growth factor-β; TNF, tumor necrosis factor.

receptor (30,32,56), and various fibroblast growth factor (FGF) family members (24,57–61). Available data suggest that each of these molecules has individual mechanisms for promoting migration at its disposal, as well as using a number of potentially shared signaling pathways that allow for cross talk in an interconnected regulatory web permitting robust and precise control over cell motility in the epithelial layer. Growth Factors EGFR family ligands, including EGF, TGF-α, betacellulin, amphiregulin, neuregulins, and heparin binding EGF, represent a major growth factor family that has a profound impact on intestinal epithelial cell restitution and proliferation. These ligands bind to, stimulate dimerization of, and thus activate members of the ErbB receptor tyrosine kinase family, of which EGFR is the canonical and best-described member (62,63). EGF and TGF-α have been shown to stimulate restitution of intestinal epithelial cells by several laboratories (13,28,64), and key elements of the downstream signaling required for this response have been described (see later). Interestingly, although TGF-α is expressed by intestinal epithelial cells themselves (65,66), the primary sources of EGF in the gastrointestinal tract are salivary glands, duodenal Brunner’s glands, and pancreatic secretions (67,68). Thus, available EGF in the gastrointestinal tract is largely luminal; however, the EGFR is primarily expressed in a basolateral pattern (69,70). These data suggest that TGF-α may be the primary EGFR ligand involved in normal gastrointestinal tissue function, whereas the role of EGF is more important when growth factor has abnormal access to receptor, such as in the case of injury to the epithelial monolayer. With regard to ligand availability in wounded cell models, studies on MMP function in cell migration have outlined a mechanism for release of EGFR ligands in wounded

epithelia (42,71–73) that may shed light on how epithelial monolayers can undergo restitution in the absence of exogenous growth factor. For example, Myhre and colleagues (42) show that wounded intestinal epithelial monolayers exhibit increased production of TGF-α in a metalloproteinase inhibitor–sensitive manner, that conditioned media from these wounded cells stimulate EGFR tyrosine phosphorylation, and that tyrosine kinase inhibitors decrease basal migration into the wound. The possibility that MMP activity may function as an early sensor of lost epithelial integrity, releasing ErbB ligands to stimulate migration, merits further study. In addition, MMP expression and activity appear to be sensitive to localization of cell–cell junctional proteins such as E-cadherin (74–77), implying a means of polarizing cellular MMP activity after wounding. TGF-β peptides in the gut are expressed primarily by epithelial cells (78,79), with additional contributions from the mesenchyme (23,25), recruited immune cells during disease or injury (80,81), and activated platelets (82–84). Epithelial TGF-β synthesis in cultured intestinal cell models is increased in the presence of basic FGF (bFGF), acidic FGF (aFGF), and IL-2 (54,55,58,85). Furthermore, cell stimulation by other factors (EGF, IL-1, γ-interferon) promotes release of the bioactive form of TGF-β, which is generated by posttranslational modification of the secreted propeptide (54). Bioactive TGF-β peptides bind to a complex of type II and type I serine-threonine kinase receptors; on binding, the type II receptor phosphorylates and activates the type I receptor. The type I receptor then initiates intracellular signaling through phosphorylation and activation of Smad proteins (86,87). In vitro wound assays in intestinal epithelial cell-6 (IEC-6) show that neutralizing antibodies to TGF-β1 impair both basal restitution and restitution in response to EGF, ILs, and γ-interferon (54). Thus, TGF-β1 appears to play a permissive role for migration in an environment with stimulation from any of a number of growth factors and cytokines, potentially allowing a cell to initiate or deny

464 / CHAPTER 16 promigratory signals by modulating one signaling cascade. In fact, TGF-β1 initiates ECM production and remodeling that may support wound closure indirectly (88,89). Trefoil Peptides In contrast with many other peptide factors that are thought to primarily require access to cells from the organism’s “inside” and act through receptors positioned at the basolateral membrane (though that distinction is an oversimplification in the case of migrating cells that have undergone a change in differentiation to acquire a migratory phenotype), TFFs are generally available from the gut lumen and interact with intestinal epithelial cells on their apical surface (see reviews by Taupin and Podolsky [90], Giraud [91], and Wong and colleagues [92]). TFFs 1-3 are expressed in distinct, spatially restricted patterns along the gastrointestinal tract, including Brunner’s glands and goblet cells. TFFs clearly play a major role in wound restitution in the intestinal epithelium. Knockout data on TFF3/intestinal trefoil factor (ITF) show little gastrointestinal phenotype in unchallenged animals, but a severe failure to heal wounds in the intestine (93). Similarly, TFF2-deficient mice show an increased susceptibility to nonsteroidal anti-inflammatory drug (NSAID)–induced gastric injury (94). Studies on TFFs to date indicate that they stimulate epithelial wound healing using mechanisms distinct from those activated by other peptides, involving interaction with epithelial cells in concert with mucin glycoproteins (95–97). TFFs stimulate migration in a TGF-β–independent manner (34), whereas, in contrast, many other peptide factors fail to promote restitution in the absence of bioactive TGF-β. Several studies implicate TFF3 in regulation of E-cadherin function in epithelial cells. TFF3 appears to stimulate redistribution of E-cadherin out of adherens junctions into the cytoplasm (98–100), thus inactivating the cell junction function of E-cadherin and disrupting cell–cell adhesion in a fashion permissive for cell motility. These data are particularly interesting in context of the reciprocal regulatory interactions between E-cadherin and EGFR (101–105). These pathways may provide a mechanism for coordination of multiple distinct factors promoting epithelial cell migration and wound healing. Cytokines In addition to peptide growth factors that directly stimulate migration-inducing signaling in intestinal epithelial cells, a host of cytokines released by the epithelium, immune cells, and submucosal stromal cells promote wound healing through both direct and indirect mechanisms. Macrophages and monocytes recruited to the wound area release IL-1 and -2 (106–108), which promote migration through increasing available bioactive TGF-β or increasing both its synthesis and availability, respectively. Injury and IL-1 also stimulate epithelial cell release of IL-8 (109,110), which, in turn, stimulates colonocyte motility in vitro (111). Furthermore, IL-8 is a chemoattractant for neutrophils, which themselves

potentially release TGF-α (112), TGF-β1 (80), and neutrophil elastase. In turn, elastase activates EGFR through cleavage of epithelial pro–TGF-α in keratinocyte and vascular smooth muscle cell models (113,114). Thus, multiple cascades of amplifying signals resulting in stimulated migration can result from initial production of a few cytokines in the wounded area. Macrophages and monocytes also produce tumor necrosis factor-α (TNF-α) (107,115,116), which exerts different effects on restitution, depending on the levels of TNF, and thus the TNF receptor activated. In vitro, modest TNF concentrations preferentially activate the high-affinity 75-kDa TNF receptor 2 (TNFR2) to stimulate restitution via a Src family tyrosine kinase–dependent pathway, whereas at high cytokine levels, the 55-kDa TNFR1 inhibits cell migration and proliferation (46). Of course, TNF is also a key inflammatory cytokine and exerts complex effects on cell proliferation and survival. As is the case for most cytokines and growth factors, TNF-induced effects on restitution represent only one facet of its activity both in epithelial cells in general and in the specific case of intestinal epithelial injury repair. Thus, translation of understanding of cellular migration responses to these factors into therapeutically relevant clinical approaches will require integration of this information with data from other areas of research.

Promotion of Wound Healing by Lipids and Other Nonpeptide Factors In addition to growth factors, cytokines, and trefoil factors, a variety of diverse organic molecules of varying sources have received attention as potential modulators of intracellular Ca2+ levels and cytoskeletal arrangement. Short-chain fatty acids (including butyrate) and polyamines are dietary components and metabolically utilized classes of molecules that can stimulate restitution (117–120), and they have evoked interest as potential therapeutic agents due to the relative ease with which they could be administered and their robustness in the environment of the gastrointestinal tract. Factors released from dead or dying cells, damaged in the injury process, that stimulate wound closure include adenine nucleotides (26) and phospholipid synthesis intermediates such as LPA (121,122). LPA is also released by activated platelets and growth factor–stimulated fibroblasts, and it stimulates Ca2+ mobilization and cytoskeletal rearrangement through activation of a G protein–coupled receptor (123,124).

Regulation of Cellular Migration by Extracellular Matrix The ECM functions as more than a surface or substrate for cell migration; it is a dynamic interface that modifies epithelial cell signaling and motility and is, in turn, modified by these cells. The immediate basement membrane is composed primarily of collagen IV, laminins (mostly laminin V),

MUCOSAL REPAIR AND RESTITUTION / 465 fibronectin, and proteoglycans (125–129). The underlying interstitial matrix contains collagens I and III, as well as some fibronectin, glycoproteins, and proteoglycans. Numerous studies have demonstrated the importance of ECM components in determining an intestinal cell migration response; in both cell and organ culture models, neutralizing antibodies against ECM components inhibit migration (89,130,131), and the same cell type moves at different rates on matrix of different composition (45,132). Furthermore, the composition of the matrix can determine not only a cell’s basal motility rate, but also its responsiveness to growth factors for accelerated migration. For example, CaCo-2 cell basal migration is more rapid on collagen I than laminin, but EGF and TGF-α stimulate migration only on laminin, not collagen I (45,132,133). Although other ECM-binding molecules such as dipeptidyl dipeptidase and the laminin receptor have been described (134,135), the best-characterized cellular receptors for ECM proteins are the integrins (see reviews of integrin biology by Berman and colleagues [136] and Schlaepfer and Hunter [137]). These transmembrane proteins are expressed as multiple α and β peptide chains, which form heterodimers of one molecule from each class, providing an enormous array of potential receptor combinations. Laminins primarily engage α6β4, other α6-containing integrins, and α3β1, as well as a few other β1-class dimers (138,139). Collagen is recognized by α1-containing dimers, largely α1β1, α2β1, and α3β1, whereas fibronectin can bind a variety of dimers containing β1 chains, αIIbβ3, αvβ6, and α4β7 (136,140), although most of the fibronectin-binding integrins with the exception of α5β1 (141) are expressed at low levels in the adult intestine. Interestingly, overexpression of α5β1 in CaCo-2 cells results in fibronectin-driven EGFR phosphorylation (142). Consistent with experiments blocking ECM constituents, neutralization of integrins can selectively block basal or growth factor–enhanced motility on specific matrix components (19,132,143). Studies of laminin-driven migration, for example, show that neutralizing antibodies to α3β1 and α6 integrin inhibit restitution of wounded T84 colorectal carcinoma cells on laminin (19), apparently by blocking formation of lamellipodia (144). Data from a number of systems have established that integrins are linked to the actin cytoskeleton through complexes involving α-actinin, talin, and vinculin (136,145). Integrindriven signaling through these complexes to the inside of the cell allows for transmission of extracellular data (e.g., force, composition of the matrix) to the regulatory mechanisms modulating the actin cytoskeleton remodeling to drive adhesion and motility (146–148). Intracellular focal adhesion– associated complexes containing focal adhesion kinase (FAK), Cas, and Src are involved in this signaling and also bridge integrins to MAPK- and small GTPase-mediated signaling to further modulate the intracellular mechanisms of motility. The importance of these linkages to intestinal restitution is demonstrated by work from Basson’s group (12,149) showing that restitution of CaCo-2 monolayers is associated with redistribution of cytoskeletal and cytoplasmic tyrosine

phosphorylation, and that tyrosine kinase inhibition in wounded cultures inhibits depolarization and onset of a migrating phenotype.

INTRACELLULAR PATHWAYS COORDINATING MIGRATION Maintenance of the migratory phenotype in epithelial cells and regulated cytoskeletal treadmilling to drive motility require continuous, Ca2+-dependent overall and localized cytoskeletal remodeling, with coordinated adhesion and release at the leading and trailing edges, respectively (148). To accomplish this task, the cell integrates outside-in signaling from ECM-integrin interactions and growth factor/cytokine/ receptor complexes with intracellular signaling, altered transcription, and inside-out feedback to the microenvironment.

Signal Transduction Pathways Cell migration initiated by integrins, cytokines, peptide factors, and other extracellular mediators involves mechanisms of cytoskeletal rearrangement and myosin motor engagement controlled by an extensive array of intracellular signaling pathways. The end point of many of these signaling cascades is regulation of Rho family small GTPase enzymes, which are involved in major aspects of the migratory phenotype in several ways (150,151). Data from other epithelial cell types and neuroblastoma cells, in which much of the basic migration machinery has been delineated, show that Ca2+-dependent activation of Rho leads to myosin lightchain phosphorylation and actinomyosin fiber contraction (152,153). Activation of the related Rac GTPase is essential for actin polymerization, which occurs in leading edge ruffles to form lamellipodia (154,155), whereas Cdc42 activity is required for filopodia formation (8,150). In the context of gastrointestinal wound healing, restitution of cultured colon carcinoma and rat small intestinal epithelial cells is abrogated by blockade of Rho family member activity (156). Furthermore, Rac1 activity is required for wound healing in IEC-6 cells and rescues the loss of migration seen in polyamine-depleted cultures (157,158), placing this GTPase as either the primary target or a rate-limiting downstream mediator of polyamine regulation of restitution. Upstream signaling molecules known to be associated with intestinal epithelial wound restitution include the EGFR tyrosine kinase (13,28,29,49,64) and its associated adapter proteins (e.g., Shc, Grb2), Src family nonreceptor tyrosine kinases (46,47,159), FAK (12,46,121,160), the phospholipase Cγ (PLCγ)/PKC cascade (13,29,161), phosphatidylinositol-3 kinase (PI3K) (29,161), and the p38 MAPK cascade (47,162,163). Our laboratory’s studies on YAMC cell migration have identified requirements for PLCγ, PI3K, and Src-induced p38 activity in EGFR-stimulated intestinal restitution in vitro (29,46,47). Egan and colleagues (164) have also placed nuclear factor-κB downstream of EGFR

466 / CHAPTER 16 signaling in restitution of rat intestinal epithelial cells. Two interesting facets of promigratory signaling are: (1) the apparent multiplicity of nonredundant pathways downstream of growth factor binding, each of which is required for complete accelerated restitution, and (2) conversely, the convergence of a number of extracellular signaling pathways on the EGFR and its mechanisms of migration promotion. After EGFR activation in YAMC cells, numerous signaling molecules are rapidly activated: Src family tyrosine kinases, p38 and ERK MAPKs, PI3K, AKT, PLCγ, and Rac. All of these effectors except ERK MAPK appear to be required for EGF-stimulated migration. However, these molecules do not lie in a simple linear pathway; p38 activation, for example, appears to be downstream of Src family kinases but not the targets of PI3K or PLCγ, PKC isoenzymes (47). Furthermore, inhibition of any one of these pathways with inhibitors or genetic disruption of Src, p38, PKC, or PI3K results in a loss of the ability of EGF to stimulate migration. In the case of p38, for example, inhibition of this MAPK actually increases EGF-stimulated ERK MAPK activation and shunts the cells to a proliferative program, possibly modeling the process by which a wave of cell division is repressed during restitution in vivo. In wounded CaCo-2 cells, p38 is activated, whereas ERK1/2 are inactivated (162), although this result is not consistent across cell lines. For example, wounded IEC-6 cells display increased activity of both p38 and ERK1/2 MAPK family members (163). Although the EGFR stimulates several equally critical signaling pathways that must be integrated downstream to enhance cellular migration, it is, in turn, a target of a variety of extracellular agents and other intracellular signaling pathways that use EGFR transactivation as a signal integrator. For example, prostaglandin E2–initiated PI3K activity and migration in LS174T colonocytes is dependent on Src-mediated transactivation of the EGFR (165). This effect is not apparently dependent on EGFR ligand release. In contrast, motility of bladder and kidney cancer cells is regulated by G protein–coupled receptor stimulation of metalloproteinases, leading to EGFR ligand release and subsequent activation of downstream signaling including Shc, ERK and p38 MAPKs, and c-Jun N-terminal kinase (JNK) (166). Other well-known modulators of restitution such as TFF peptides may also exert at least part of their effects through the EGFR; TFFs stimulate cytoplasmic translocation (inactivation) of E-cadherin (98–100), and E-cadherin, in turn, is a negative regulator of the EGFR (167). The complex ways in which apparently simple signaling pathways interact and integrate are likely to be key topics of investigation in the restitution field in upcoming years. As noted earlier, immune peptides and cytokines such as TNF are implicated in control of intestinal cell motility and wound healing. TNF initiates both distinct and overlapping cellular responses through TNFR1 and TNFR2 (reviewed by Chan and colleagues [168] and Locksley and colleagues [169]). Pathologic concentrations of TNF inhibit intestinal epithelial cell wound closure and proliferation through TNFR1 (46,70), whereas activation of TNFR2 by lower

concentrations of TNF leads to increased intestinal cell proliferation and migration (46,170,171). The role for cytokines, and TNF in particular, in these processes has only recently been appreciated despite the key role these molecules play in related aspects of epithelial biology such as acute and chronic inflammation and injury in disorders of the gastrointestinal tract (46,55), and the signaling pathways responsible for TNFR2-induced cell motility or TNFR1induced blockade of restitution are as yet unclear. In addition to the actions of peptide growth factors and cytokines on cell signaling for restitution, a growing literature is delineating the means by which ECM components signal through nonreceptor tyrosine kinases to alter cellular calcium levels and the activity of Rho family GTPases. Although little data on this issue currently are available in intestinal epithelial cells, potential mechanisms for ECMinduced signaling during restitution are suggested by data from other cell types. Studies in pancreatic cancer cells (172,173), Cos cells, and HeLa cells (174) show that integrininitiated FAK/Src/Cas complex signaling through Crk stimulates Rac activity in migrating cells. These and other results need to be confirmed in intestinal epithelial cells.

Gene Transcriptional Regulation Part of the ensemble of migratory responses of a cell involves up-regulating the expression of genes, the protein products of which sustain a migration phenotype, promote migration in neighboring cells, or both. EGFR activation in colonic epithelial cells stimulates expression of IL-8 gene product (175), which itself can stimulate colonocyte migration (111). Other migration-inducing factors such as IL-2, aFGF, and bFGF are thought to act through activating TGF-β transcription in intestinal epithelial cells (54,55,58,85). TGF-β, in turn, initiates Smad signaling in the epithelium and the underlying mesenchymal cells, stimulating expression of a number of genes including β5-class integrins (176) and fibronectin (89), further supporting restitution in the local environment. Gene regulation is also required during restitution in the absence of exogenous ligands; for example, wounded intestinal epithelial cells induce egr-1 and fos messenger RNA (mRNA) expression in a partially PKC- and ERK-dependent manner (177). Furthermore, forced expression of the colonocyte differentiation-associated transcription factor cdx2 in IEC-6 cells significantly increased restitution (178), underlining the importance of gene regulation for the migratory phenotype. Thus, intestinal mucosae regulate epithelial restitution through both conventional intracellular signaling and the modulation of gene expression profiles. A number of individual results such as the ones described earlier have been reported. However, progress to the next level of understanding exactly what comprises the “migratory phenotype” of intestinal epithelial cells will require large-scale efforts at complementary microarray and proteomic analysis of cells undergoing restitution compared with those in intact polarized monolayers.

MUCOSAL REPAIR AND RESTITUTION / 467 Inside-Out Signaling In addition to receiving integrin-mediated signals from the ECM and surrounding microenvironment, which are translated to changes in cell behavior, cells feed back information to the outside to modify their migrating environment. This inside-out signaling uses many of the same pathways that are themselves the targets of outside-in signaling (136,179). Integrin affinity and avidity for the ECM are regulated by physical stress from the cytoskeleton (180,181), modification by tyrosine kinase activity (182–184), paxillin binding (185), PLCγ (186) or PKC activation (187,188), as well as small GTPases (189). Many of these regulatory mechanisms, worked out in a variety of cell types, appear to be well conserved and are likely to be similar to the situation in intestinal epithelia in vivo, linked to the activity of known migration signaling pathways. EGFR ligands, for example, stimulate expression and partner reorganization of particular α-integrin subunits in a matrix-dependent manner (132,190). In addition, cells constantly modify their environment either by direct release of ECM components and ECM-digesting proteases, or by shedding peptides that stimulate ECM remodeling by nearby myofibroblasts. Cultured human intestinal crypt cells stimulated with a combination of TNF-α and interferon-γ produce increased levels of laminin-5 and -10 (191). TGF-β can stimulate production of basement membrane components and integrin subunits, as discussed earlier. Thus, intestinal epithelial cells involved in restitution can modify their own affinity for the basement membrane, as well as modify that matrix directly or modulate the function of mesenchymal cells to yield production of an appropriate ECM.

METHODOLOGIES FOR STUDYING GASTROINTESTINAL CELL MIGRATION A variety of methods are used to study wound restitution in the laboratory, both in cell culture and animal models. In addition, a number of methodologies are available that, although not directly modeling restitution per se, nonetheless uncover important details of cell motility that are likely applicable to epithelial wound healing. All of the available methodologies have distinct advantages and disadvantages, and often a combined approach may provide the best solution for a particular problem.

Cell Culture Models Cell culture models of intestinal restitution must address several general conditions, balancing effective modeling against the ease of investigation in an in vitro system. Key variables include the extent to which the physiologic ECM is modeled, the physical means of producing a wound, the culture conditions and panel of growth factors available to

cells, and the reproducibility and easy interpretation of results. Several useful models are described later. Scrape Wounding By far the most widely used cell culture model for intestinal epithelial restitution is the “scrape” wound model and its many variations. In the simplest form of this technique, a monolayer of cells is disturbed with a single-edge razor blade or some other implement. Observations are made at various times after wounding to determine the remaining width of the wound or the number of cells entering the denuded area, or both (29,192). This approach is both simple and powerful, and it allows rapid analysis of the influence genetic alterations to a cell line, growth factors, regulatory peptides, and chemical modulators of signal transduction have on wound closure. What is lacking in this model is a complete accounting for many potentially important features of the in vivo microenvironment: ECM, cell polarity, neural cell influence, stromal and immune cell contributions, physiologic levels of growth factors and cytokines, probiotic or pathogenic bacteria, and so on. In addition, the somewhat imprecise wound size and shape produced by this method may pose challenges for accurate quantification. Numerous modifications and improvements have been made to the method over the past several decades to address these issues. For example, many laboratories perform restitution assays on matrigel or fibronectin to provide a physiologically relevant substratum. Our laboratory plates cells on fibronectin or collagen and uses a modified wounding procedure involving a rotating silicone tip that removes cells from a reproducible circular area without destroying the underlying matrix (46,47). This modification has the added benefit of consistent circular wounds (Fig. 16-4), which are easily measured by time-lapse photography, thus providing an accurate measure of wound-closure rates. This method has been used to delineate roles for the EGFR, TNFR1 and TNFR2, and a variety of downstream signaling modules in intestinal epithelial restitution (46,47). Other modifications include scrape wounding one of the variety of polarized or semipolarized culture models available (193,194), or in the presence of cocultured support cells, generally either fibroblasts or immune cells, producing factors of interest (108,195). For biochemical or mRNA expression studies, scrape wounding generally is modified by making multiple wounds in a repeatable pattern on the culture monolayer, using the tips of a multichannel pipette, or rotating a minigel electrophoresis comb on the monolayer (42,47,196,197). Through these methods, large numbers of cells are at or near a wound/ culture interface, allowing for analysis of signaling cascades and gene expression activated under these circumstances. “Fence” Assay In the “fence” assay, cultures are plated on dishes with a removable ring or fence constraining cells to one portion of each dish. After cellular attachment, the fence is removed, and

468 / CHAPTER 16

Wound closure relative to control

A

C

B 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 Control

EGF

HGF

FIG. 16-4. “Drill-press” wound assay.Young adult mouse colon cells are subjected to multiple wounds with a rotating silicone tip and are photographed at various time points. (A) Initial wound. (B) Eight hours after wound closure in the presence of 10 ng/ml epidermal growth factor (EGF). (C) EGF and hepatocyte growth factor (HGF; 10 ng/ml) both stimulate wound closure ~50% in excess of control restitution (averages of 8 wounds; p < 0.001).

migration into the cell-free area is measured over time (132, 198). This model has the advantage that, for long experiments, the culture is more easily kept sterile than in a model where a foreign object is introduced after culturing. Similar to the rotating silicone disk model described earlier, the fence assay permits study of cells migrating on a defined matrix. Subconfluent Cultures In principle, subconfluent cultures may exhibit many of the same signaling and morphologic characteristics as cells migrating to close a wound. However, important differences make this model questionable. Most importantly, in contrast with a physiologic wound or a wounded culture monolayer where most cells have at least some attached neighbors (and may migrate in concert with them), many of the cells in a subconfluent monolayer have little or no cell–cell contact. Furthermore, in some cell lines, intrinsic cellular proliferation signals are specifically initiated in the subconfluent state, potentially confounding the study of migration responses to stimuli. Thus, this model represents a more complex situation than straightforward wound closure and is more useful for the study of individual cell migration. Polarized Cultures and Chemotaxis Assays One potential limitation of the assays described above is the lack of appropriate cell polarity in a typical epithelial monolayer cultured on plastic. Although the flattened phenotype

of many culture models is actually similar to the migratory phenotype in vivo, and thus offers a valid model for studying the postdepolarization parts of wound restitution in vitro, contributions (growth factor release, proliferation, and so forth) of still-polarized cells removed from the wound margin may be regulatory factors that are missed in this type of analysis, and the process of modifying the phenotype of wound edge cells is also not modeled in these cultures. Several polarized epithelial culture systems are available, including Madin– Darby canine kidney cocker spaniel cells, which can be induced easily to polarize in culture and are often used for both wound healing and apicobasolateral sorting studies (6,199), as are intestinal lines such as T84 and CaCo-2BBE, which will take on polarized morphologies under appropriate culture conditions (193,194). In addition, some general information about cell motility can be gleaned from chemotaxis assays, which can be performed on polarized or semipolarized monolayers. However, the caveats discussed earlier are important to consider when applying information from chemotaxis systems to restitution and wound healing problems.

In Vivo Models of Restitution Given the exceptionally complex nature of the restitution process and the array of cell types, soluble factors, and ECM interactions involved, the effort of translating and integrating lessons from cell culture studies into a larger understanding of in vivo processes is likely to be nontrivial. Several types of in vivo models have addressed this topic, however, and significant progress has been made. Chemical-Induced Direct Epithelial Injury Introducing a wound into the epithelium through topical application of a damaging salt solution, mild acid, or other chemical resulting in the direct loss of epithelia has been a powerful and informative approach to in vivo restitution studies. The wound introduced is an immediate, clearly defined, physical injury that, at least in early stages of healing, has little or no confounding involvement from inflammatory processes, though chronic or extended wounds of this sort do involve inflammatory responses. Parameters followed after wounding in these models include regional morphology and structure, total wounded area or length of colon involved, size of wounds, or migration of epithelial cells labeled in some manner into denuded areas. Early studies of restitution after bile salt-induced injury in pig, rat, and guinea pig colon used this approach to demonstrate a role in the healing process for rapid cell migration into the wound area (1,2,200). In both the porcine and rat colon, substantial cell migration into a salt-induced wound area was observed in the first 30 minutes after wounding, with partial reestablishment of polarized epithelium within the first few hours, clearly showing the ability of the tissue to repair barrier function on a much more rapid time scale than is explained by increased cell division. Variations on this technique using either salts or acid as the damaging agent have been used to

MUCOSAL REPAIR AND RESTITUTION / 469 characterize, among other things, the cytokines produced by mice after acute colorectal injury (201), age-dependent induction of EGFR activity after gastric ulceration in rats (202), and in vivo efficacy of EGF in acute duodenal ulcers (203). Other Chemical Injury Models in Rodents: Inflammation/Injury Models Injury to the intestinal epithelium has been accomplished through the use of other compounds such as the mouse dextran sulfate sodium (DSS) model and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis in rodents. These methods induce a much more heterogeneous response than in the models discussed earlier, because they apparently cause direct damage and induce indirect mechanisms of injury including inflammatory responses (204,205). The complexity of long-term response, with substantial inflammation, to these agents makes interpretation of data difficult for restitution studies, but the analysis of factors that ameliorate the initial damage—in particular, the analysis of susceptibility of animals with different genetic backgrounds to the early phases of injury, or the rapid induction of cytokines and growth factors—is quite useful. The DSS model has been used, for example, to show a role for various receptor systems in preserving intestinal mucosal homeostasis. Mice lacking the EP4 prostaglandin receptor show increased susceptibility to DSS colitis (206). Similarly, Rakoff-Nahoum and colleagues (207) demonstrated that mice deficient in toll-like receptor (TLR) signaling exhibit severe DSS colitis, and furthermore, that TLR signaling is required for commensal bacteria-mediated protection from epithelial damage in this model. Examples from the EGFR signaling field include the demonstration that TGF-α–overexpressing mice are resistant to DSS-induced colitis (208), and that TNBS-treated rat intestinal epithelia exhibit increased production of TGF-α within hours of the insult (209). Organ Culture A limited number of studies have yielded interesting results using organ bath culture of intestinal epithelia to model in vivo cell migration and restitution. For example, Moore and colleagues (130,210) used short-term in vitro maintenance of guinea pig intestinal epithelium to demonstrate rapid restitution of this tissue and to implicate matrix collagens as a requisite part of the process. Although the short survival time of partially intact gastrointestinal tissue outside the organism is a substantial limitation of this model, within the parameters of the rapid wound healing response this is a powerful technique that can yield much useful information and aid in bridging the gap between cell culture and in vivo studies. Visualization Systems: Two-Photon Microscopy A potential area of advance in the study of wound healing in vivo is through the development of improved microscopy systems that allow precise imaging of wound healing in

living animals. Studies have demonstrated the resolution to precisely track pH-marking dyes in the rodent duodenal epithelium in situ (211) and to measure renal microvascular permeability in real time (212). Application of these techniques to restitution and repair questions will finally allow real-time analysis of wound healing in vivo at levels of detail rivaling those available from in vitro models. Lessons from Other Model Systems: Wounding and Remodeling in Simple Animal Embryos and Data from Similar Tissues (e.g., skin, corneal epithelium) As is the case with most biological processes, a great deal of mechanistic conservation appears to hold across tissues and species for cell migration and wound healing. Thus, data from other tissues and organisms can be instructive in the study of gastrointestinal restitution. Wound healing and tissue remodeling in simple animal embryos have shown both striking similarities and distinct differences to the process of restitution-directed migration in the adult mammalian gastrointestinal tract (see reviews by Martin and Lewis [17] and Martin and Parkhurst [22]). Both lamellipodial and purse-string cell movement have been observed in remodeling and wound healing in Drosophila and Xenopus embryos, though the preferred mechanism appears to lean toward purse-string closure in the developing organism as opposed to the lamellipodial crawling described in the adult gastrointestinal cells and tissue. In addition, many of the same signaling pathways have been implicated in cell migration during development of worms, flies, and fish and in adult mammals, including Rho GTPases (18) and JNK (213). Parallels with gastrointestinal restitution can be seen during wound healing in other adult mammalian tissues, especially including the various epithelial layers such as skin and lung, which are at high risk for environmental damage. One model system that shows striking similarity and that also has been the subject of substantial effort is wound healing in corneal epithelial cells. Both the growth factors involved in promoting restitution and the signal transduction pathways involved in these two tissues are similar. For example, EGFR ligands, p38 MAPK, PKC, and HGF all appear to promote or support migration in both tissues (71,73,214–216). Taken together, these data suggest that mechanisms of epithelial wound healing are relatively well conserved, and they underline the value of studies in a variety of tissue types and organisms toward an understanding of gastrointestinal restitution.

RELATION OF ALTERED MIGRATION TO DISEASE Interest in intestinal restitution is driven, in part, by the wide variety of conditions that result in damaged intestinal epithelia, and that may thus benefit from cell migration/ restitution-directed therapy. Inflammatory diseases such as Crohn’s disease or ulcerative colitis induce substantial epithelial wounds in the gastrointestinal tract that must be repaired to maintain a healthy organism. Damage from luminal

470 / CHAPTER 16 contents and bacteria can also create a disease-like state requiring enhanced wound healing. Furthermore, a number of therapeutic regimens exhibit gastrointestinal toxicity as a major side effect. The ulcer-promoting properties of many NSAIDs are well documented (217), as are the gastrointestinal toxicities resulting from radiotherapy (218) and many chemotherapeutic drugs such as cisplatin (219,220), 5-fluorouracil alone (219,220) or in combination with leucovorin (221), and others. Such toxicities are not limited to the lower gastrointestinal tract; a limiting toxicity for some therapies is oral mucositis, which exerts a profound impact on patient quality of life, as well as on compliance. Clinical trials with agents that influence intestinal epithelial restitution have shown some efficacy, both directly in disease states and as protective agents in settings where the mucosa is damaged by other therapy. As discussed earlier, in clinical trials, EGF given topically is therapeutic for patients with ulcerative colitis (48). Similarly, trials with keratinocyte growth factor (FGF-7), which is up-regulated in intestine and skin after injury, have demonstrated protective activity against the development of oral mucositis in chemoradiotherapy patients (222). Clearly, the potential benefit of restitution- and repair-directed gastrointestinal therapy merits further study. In addition to a positive role in healing and protection from inflammatory damage, mechanisms of cellular migration may play a pathogenic role in the case of gastrointestinal cancers. Enhanced cell motility is a requirement for the invasion and metastasis of aggressive tumors, and a number of the signaling mediators responsible for restitution in the disease-free state are overexpressed or show altered activity in colorectal carcinomas. Increased levels of EGFR or EGF family ligands have been reported in a variety of neoplasias (223–226), as have intracellular mediators of mitogenic signaling such as Src (227,228), FAK (229,230), and Rho family small GTPases (231). Available data suggest that well-regulated cell motility and intestinal restitution mechanisms are critical for maintenance of a healthy gastrointestinal tract, especially in the face of pathogenic insults such as ulcerative colitis, Crohn’s disease, or Helicobacter pylori infection. In contrast, dysregulated or hyperactive cell motility may play a pathogenic role in some disease states, and many of the modulators of restitution can influence cell proliferation or apoptosis in disease. Thus, a thorough understanding of the regulatory signaling pathways stimulating cell migration, as well as the determination of which intracellular mechanisms may induce it selectively and restrict it to appropriate contexts, is key to the development of therapies directed toward intestinal wound healing. The importance of this issue is underscored by the apparent dichotomous role of the EGFR in ulcerative colitis patients. Whereas topical EGF application in these patients has been shown to have therapeutic effect, long-term activation of the EGFR is also associated with colorectal cancer, a disease for which ulcerative colitis patients are at increased risk (232,233). Untangling the good and bad parts of EGFR signaling for these patient populations will

be necessary for long-term use of therapies based on the restitution-promoting properties of this receptor system.

FUTURE CHALLENGES Although substantial literature now exists on the regulatory mechanisms directing intestinal epithelial cell migration and intestinal restitution, important questions remain. First, although a number of signaling pathways have been identified that can stimulate a wounded intestinal monolayer to close, the specific requirements for these in the absence of exogenous growth factors are relatively uncharacterized. Second, the wound-sensing mechanisms that tell a population of cells to begin to move are unknown, as are the termination signals for such a restitution response. Third, the relation of restitution to other forms of intestinal epithelial cell movement (e.g., remodeling during development, migration up the crypt onto the villus or surface epithelium) is unclear—Is it just more of the same or an entirely different process? Similarly, the related question of whether regulatory peptide-initiated wound healing represents an acceleration of basal migration or a different mechanism entirely is not yet well addressed. Finally, the effort to translate in vitro signaling data to the in vivo wound restitution process is in its early days. Integrating findings from the relatively simple epithelial cell culture models to a comprehensive understanding of in vivo restitution will be necessary to develop strategies for promotion of wound healing. Furthermore, such understanding should broaden approaches to inhibit inappropriate cellular migration such as epithelial invasion during tumorigenesis and metastasis. Many of the studies on signal transduction in wound restitution use restituting tissues or cell culture systems presented with saturating, likely nonphysiologic levels of regulatory peptides/growth factors, cytokines, or activators of signal transduction pathways such as phorbol esters. Similarly, overexpression studies of molecules hypothesized to be involved in cell migration have been widely used. Conversion of these data from a catalogue of what can promote or block migration to what does regulate restitution in vivo continues to be a major challenge in the field.

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CHAPTER

17

Mechanisms of Gastrointestinal Malignancies John Lynch and Anil K. Rustgi Principles of Oncogenesis, 477 Basic Mechanisms, 477 Gastrointestinal Carcinogenesis Is a Multistep Process, 480 Cardinal Features of Gastrointestinal Cancers, 481 Self-Sufficiency in Proliferation Signals, 481 Ignore Growth Inhibitory Signals, 482 Unlimited Replication, 484

Genetic Instability, 485 Chromosomal Instability, 486 Microsatellite Instability, 487 Base Excision Repair, 489 Epigenetic Mechanisms, 490 Acknowledgments, 492 References, 492

Gastrointestinal (GI) cancers represent a heterogeneous, complex array of disorders and diseases. They may be divided into rare inherited forms and more frequent sporadic forms. There is a critical interplay of genetic and environmental factors that foster the conversion of normal tissue to precursor, premalignant lesions and eventually to frank malignancy. Although it is apparent that certain genetic mechanisms are better appreciated in a cell-type– and tissue-type–specific context, there are nevertheless overarching shared features among GI cancers of different origins. To that end, this chapter approaches GI cancers by focusing on a dissection of the genetic basis of GI cancers and underlying molecular mechanisms. Thus, because of space constraints, it is not the intention of this chapter to elaborate on the causative mechanisms of each GI cancer, but rather to provide the reader with an understanding of the pivotal principals of oncogenesis and to use this as a platform for elucidation of the molecular steps involved in initiation, evolution, and progression of GI cancers. Finally, this chapter emphasizes the underpinnings of epithelial-based GI cancers given they represent the preponderant form of GI malignancy, but certainly GI malignancies can emanate from different cell types, and

these in aggregate constitute lymphomas, sarcomas or stromal tumors, and rare forms.

J. Lynch and A. K. Rustgi: Division of Gastroenterology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104.

Basic Mechanisms

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

PRINCIPLES OF ONCOGENESIS The term neoplasia refers to a pathologic process of abnormal and unregulated cell proliferation. Neoplastic cells have lost the ability to respond to the normal cues that dictate when a cell replicates, differentiates, migrates, and dies. Continuous and unconstrained cell replication ultimately leads to the formation of a mass or tumor called a neoplasm. Neoplasms can arise in any tissue of the GI tract. Whether they are benign or malignant, neoplasms can be life-threatening for those afflicted. GI malignancies are a leading cause of morbidity and mortality worldwide. Each year, there are three million new cases of GI cancers, resulting in two million deaths (1). This difficult burden of pain and suffering has prompted the expenditure of tremendous resources to find better ways to prevent and treat this complex disease. Thirty years of research into the biology of cancer has yielded new insights that are explored in this chapter.

Understanding the process of neoplastic transformation has been an important research focus. It has been determined that, at its most basic level, cancer is a genetic disease (Table 17-1).

477

478 / CHAPTER 17 TABLE 17-1. Classification of cancer-related genes Oncogenes Growth factor receptors Receptor and nonreceptor tyrosine kinases G proteins Serine-threonine kinases Nuclear transcription factors Tumor-suppressor genes Signal pathway suppressors Cell-cycle regulators Antiapoptotic mediators DNA mismatch repair genes Metastasis-related genes

Mutations in the genomic material of a cell disrupt critical gene functions that normally serve to regulate cell proliferation, programmed cell death (apoptosis), and cell differentiation (Table 17-2). The result is disordered, unregulated cell growth. Laboratory-based and clinical observations have identified several different processes that can cause neoplasia. They comprise the following: (1) hereditary predisposition to cancer; (2) exposure to carcinogens; (3) chronic inflammatory conditions; and (4) sporadic mutations that accumulate during a normal lifetime. Heredity A genetic predisposition for GI cancers exists in some families due to the germline or inherited transmission of a mutant gene from a parent to a child. Organs primarily affected by familial cancer syndromes include the esophagus, stomach, intestine and colon, and the pancreas. Despite the specific differences between the cancers and the genes involved, familial cancer syndromes do share certain features in common. First and foremost, they must have a significantly increased risk for a particular cancer in the absence of any other predisposing factors. Second, patients typically experience development of multiple tumors in various tissues and at a much younger age than in the general population. Penetrance can be variable, but it is typically greater than

TABLE 17-2. Mechanisms of activation or inactivation of cancer-related genes Oncogenes Proviral insertion Retroviral transduction Amplification Chromosomal rearrangement Point mutation Tumor-suppressor genes Loss of heterozygosity = allelic deletion Point mutation Microdeletion Promoter methylation DNA mismatch repair genes Point mutation Promoter methylation

50% in affected individuals. Lastly, the predisposition for cancer may affect a single organ or involve multiple tissues, but the pattern of involvement typically is conserved within a family kindred. The study of families with a preponderance of GI malignancies has provided novel insights into the mechanisms governing neoplastic transformation. Often, genes identified in familial cancer syndromes are found to be mutated or silenced in the more common sporadic forms of the cancers as well (Table 17-3). The inherited colon cancer familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) syndromes together account for approximately 5% of all colon cancers. However, identifying the predisposing inherited mutations illuminated gene pathways frequently mutated in the development of most sporadic colon cancers (2–4). The gene mutations responsible for the more common familial cancer syndromes have largely been identified, including the new human polyposis syndrome, MYH adenomatous polyposis (5–7). Interestingly,

TABLE 17-3. Hereditary basis for gastrointestinal cancers Esophageal Squamous cell carcinoma Tylosis palmeris et planteris: genetic linkage to chromosome 17 Gastric Adenocarcinoma Diffuse hereditary gastric cancer: germline E-cadherin mutations Pancreatic Adenocarcinoma Familial atypical mole and multiple melanoma: germline p16INK4a mutations Hereditary breast cancer syndromes: germline BRCA2 mutations Peutz–Jeghers syndrome: germline LKB1 mutations Colorectal Adenocarcinoma Familial adenomatous polyposis (FAP): germline adenomatous polyposis coli (APC) mutations MYH adenomatous polyposis: germline MYH mutations Hereditary nonpolyposis colorectal cancer (HNPCC): germline DNA mismatch Gene repair mutations Peutz–Jeghers syndrome: germline LKB1 mutations Juvenile polyposis syndrome: germline SMAD4, PTEN, bone morphogenetic protein (BMP) mutations Cowden syndrome: germline PTEN mutations Small intestinal Adenocarcinoma HNPCC FAP Peutz–Jeghers syndrome Stromal tumors Germline c-kit or platelet-derived growth factor receptor (PDGFR) mutations BRCA2, breast cancer gene 2; LKB1, serine threonine kinase 11; MYH, mut Y homologue; PTEN, phosphatase and tensin homologue; SMAD4, homosapiens mothers against decapentaplegic homologue 4.

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 479 several signaling or regulatory pathways are represented repeatedly in these familial syndromes—Wnt/adenomatous polyposis coli (APC)/β-catenin, transforming growth factorβ (TGF-β)/bone morphogenetic protein, PTEN, and the DNA mismatch repair (MMR) and base excision repair (BER) mechanisms. Furthermore, it is notable that uniformly these genes are members of the class known as tumor suppressors, because of their ability to prevent neoplastic transformation both in vitro and in vivo (8). The molecular events that precede cancer development, even in cells with an inherited predisposition, remain poorly characterized. The presence of the mutation by itself is not transforming; the second normal allele is thought to provide sufficient gene function to inhibit transformation in most cases. It is important for inactivation of the remaining normal allele to occur through allelic deletion, mutation, or DNA methylation. Thus, biallelic inactivation of tumor-suppressor genes is critical to the genesis of tumors (9); yet, other somatic alterations in other genes typically are needed. Several features of hereditary cancers remain difficult to explain. Although every cell in the body harbors the same mutation, the cancers typically develop in a limited set of tissues. This suggests these tissues may possess vulnerabilities not present in other tissues, but the mechanisms for this have yet to be determined. Moreover, cancer penetrance can vary between family kindreds, possibly because of the actions of unknown modifier genes. Therefore, much remains to be learned about the process of carcinogenesis, even in cells with a genetic predisposing mutation. Carcinogen Exposure DNA, the basic agent for transmission of genetic information, is a chemical. As such, its composition can be altered by physical and chemical reactions. Highly reactive chemical carcinogens, such as alkylating agents, can be directly genotoxic. However, most agents, called procarcinogens, must first be metabolized in a way that generates highly reactive electrophilic derivatives that can, in turn, react with DNA. The resulting chemical modifications to the DNA base can disrupt normal nucleotide pairing. If not corrected before cell division, daughter cells can permanently inherit sequence changes that alter gene function and expression. Given these risks, cells have evolved a number of mechanisms to protect DNA from environmental mutagens (10,11), as well as to identify and correct mutations when they occur (12,13). In addition, cells are programmed to undergo apoptosis if the DNA is severely mutated, thus protecting the organism from defective and potentially harmful cells. Despite these protective mechanisms, the GI tract is exposed continuously to chemical carcinogens, often due to ingestions. For example, Aspergillus molds contaminate a number of food staples, such as corn, peanuts, and rice, in developing countries. These molds can produce aflatoxin, a potent DNA mutagen, which when ingested and metabolized by hepatic cytochrome P450 enzymes becomes highly reactive and forms adducts with guanine nucleotides, thereby leading to

DNA mutagenesis. Significant dietary aflatoxin exposure in individuals with chronic-active hepatitis B infection yields a synergistic increase in hepatocellular cancer rates (14–16). Tobacco smoke contains more than 40 different chemical mutagens and carcinogens, including N-nitrosamines, polycyclic aromatic hydrocarbons, aromatic amines, ethylene oxide, and other agents that cause oxyradical damage to DNA nucleosides. Smoking has been strongly associated with increased rates of several GI cancers, including head and neck and esophageal squamous cell cancers (17). Nitrates, which are a common preservative found in processed meats and, until recently, beer, can be converted into the genotoxic N-nitrosamines by the actions of gastric bacteria and acid. High nitrate consumption has been associated in multiple studies with increased risk for gastric and esophageal cancers (18–21). Other cooking practices and methods of food preservation are also known to produce small amounts of carcinogenic compounds that may contribute to esophageal, gastric, and colorectal cancer (22). In summary, carcinogen exposure is an important cause of sporadic GI neoplasia. Inflammation Chronic inflammatory conditions have long been associated with increased rates of neoplasias (23). Typically, this risk increases the longer the inflammation is present, but it can be reduced if the inflammatory response is suppressed. The causes of chronic inflammation in GI tissues are varied and include infections such as Helicobacter pylori (chronic gastritis) or viral hepatitis B and C (chronic hepatitis), recurrent chemical (gastroesophageal reflux disease [GERD]) or enzymatic injury (recurrent pancreatitis), as well as autoimmune processes (Crohn’s disease, ulcerative colitis). Despite differences in the underlying processes, each of these chronic inflammatory conditions is known to increase the risk for cancer in patients with the particular disorder (24–26). The mechanism(s) by which chronic inflammation induces neoplastic transformation are unknown. The most attractive current hypotheses suggest that activated inflammatory cells elaborate high levels of reactive oxygen and nitrogen species, free radicals, and oxidants. These highly reactive oxygen and nitrogen species can, in turn, react with and damage DNA, RNA, lipids, and proteins, causing mutations and altered protein function, ultimately leading to neoplastic transformation (27,28). Alternatively, it has been proposed that the cytokines and chemokines secreted by activated inflammatory cells may promote carcinogenesis. Cytokines, such as tumor necrosis factor-α (TNF-α), and chemokines, such as MIF and macrophage inflammatory protein-1β, are secreted by inflammatory cells and in many studies appear to promote the growth and metastasis of tumors (29–33). These immune mediators can act in both a paracrine and an autocrine fashion to induce cell proliferation, inhibit apoptosis, promote cell migration and stromal degradation, and enhance new blood vessel growth (angiogenesis). Tumor-derived cytokines and chemokines can also modulate the immune response to inhibit normal surveillance mechanisms that target

480 / CHAPTER 17 neoplastic cells and promote leukocyte infiltration, causing degradation of stromal elements and enhanced neoplastic cell migration and metastasis (29,32–34). Inflammation and inflammatory cells also release eicosanoids, oxygenated lipids produced by arachidonic acid metabolism (35–37). Eicosanoids include the prostanoids (prostaglandins and thromboxane) and leukotrienes. Much like the cytokines and chemokines, eicosanoids have been associated with promoting transformation by enhancing cell proliferation and motility while reducing apoptosis (38–41). Eicosanoids also induce angiogenesis (42) and suppress normal immune surveillance mechanisms (43–46). Lastly, eicosanoids can also induce cytokine and chemokine synthesis and release, thereby further potentiating the inflammatory response (47–49). Because of these many beneficial effects on tumor growth and survival, increased eicosanoid biosynthesis, particularly of prostaglandins, can be seen often in many cancers. Given the critical role of chronic inflammation in GI carcinogenesis, treatment and resolution of the inflammatory condition has been a therapeutic focus for several years. It is important to treat chronic hepatitis C with interferon and ribavirin, to manage ulcerative colitis with mesalamine and immunomodulators, to eradicate H. pylori chronic gastritis with antibiotics, and to control acid reflux in patients with GERD with Barrett’s esophagus. Several of these therapies have been reported in clinical trials to reduce the risk for cancer (50–53). An appreciation of the critical role played by inflammation and immune mediators in GI carcinogenesis has and will continue to provide useful preventative and therapeutic targets. Sporadic Carcinogenesis Most GI cancers are sporadic in nature—that is, acquired during an individual’s lifetime. Discerning the mechanisms underlying sporadic GI cancers has therefore been difficult. Typically, sporadic cancers occur later in life. This has led to the hypothesis that such cancers develop from cells that have accumulated a lifetime’s worth of random DNA mutations culminating in neoplastic transformation (54,55). Although this remains an attractive hypothesis, there are now multiple observations reporting that certain exposures, habits, diets, and backgrounds increase or decrease the risk for certain GI cancers. Western high-fat diets are associated with increased risk for colorectal cancer, whereas high-salt diets increase the risk for gastric cancer (56–58). Obesity, in addition to the many other associated health concerns, also increases the risk for esophageal adenocarcinoma and colorectal cancer. Some practices can potentially be preventative. Several population-based studies have shown that frequent use of aspirin or nonsteroidal anti-inflammatory drugs can reduce rates of esophageal, gastric, colon, and possibly pancreatic cancer (59–63). Studies with animal models for GI cancers further buttress this observation (64–70). Folate supplementation likewise appears to reduce rates of colorectal cancer in a small number of patients (71,72). Lastly, genetic

polymorphisms may contribute to this process by modestly increasing susceptibility to dietary and environmental carcinogens, or altering the responsiveness of critical regulatory pathways. In summary, the molecular mechanisms responsible for sporadic neoplasias are likely to be highly complex and vary considerably between individuals.

Gastrointestinal Carcinogenesis Is a Multistep Process GI cancers typically are preceded by benign dysplastic intermediates: They do not arise directly from normal tissues. These dysplastic lesions can be distinguished morphologically and classified based on specific pathologic criteria. In the colon, the adenoma-carcinoma sequence describes this progression from normal mucosa through dysplastic intermediates to invasive carcinoma and has been well supported by many pathologic, epidemiologic, and animal studies (2,73–75). The earliest distinct lesions in this sequence are aberrant crypt foci (ACF). ACF are microscopic lesions that were first identified in methylene blue–stained colons of azoxymethane-treated mice as crypts that appeared larger and thicker, with an increased luminal diameter and an opening that was often slitlike or serrated (76–78). Most human ACF are hyperplastic (65–95%); however, a significant proportion of human ACF are dysplastic and similar in many ways to adenomatous polyps (79–81). Most intriguingly, genetic analysis of ACF has identified gene mutations commonly observed in adenomatous polyps (76,81–88). Similar multistep progression from normal tissue through dysplastic intermediates to cancer has been well described for human esophageal, gastric, and pancreatic cancers (89–92). In each case, cancers arise in dysplastic precursor lesions that are grossly or histologically apparent. Current models suggest intestinal-type gastric cancer is preceded first by atrophic gastritis, then intestinal-type metaplasia, before giving rise to the dysplasia and adenomas in which the carcinomas develop (89). In the esophagus, Barrett’s intestinaltype metaplasia is the earliest recognized tissue abnormality. The presence of dysplasia in Barrett’s epithelium is clinically worrisome; it suggests adenocarcinoma may develop in the future. Most recently, precursor lesions to pancreatic cancer have been formally agreed on and the characteristics necessary for their classification established (90,91). These criteria permit the classification of pancreatic lesions for both clinical and scientific uses. The ability to identify intermediate stages in the progression from normal tissues to cancer would seem to lead naturally to the hypothesis that carcinogenesis is a multistep process involving sequential genetic changes (54,55,89,93). This model has successfully supplanted an earlier model for oncogenesis, based largely on studies with chemical carcinogens, in which tumor initiation and progression were viewed as distinct processes (94). An important observation in support of the multistep hypothesis is that neoplasms are clonal, with each neoplastic cell derived from a single progenitor (54,95). This model proposes that the genetic

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 481 mutations required for neoplastic transformation are not obtained all at once, but rather gradually in stages. With each step in this process, the transforming cell acquires a new mutation that enhances cell proliferation or survival. By a process of natural selection or evolution, there is the emergence of a cell clone possessing all the features necessary for neoplastic transformation. Selection is a critical component of this process, because mutational events are random, and therefore only rare mutations lead to activation of growth-promoting and cell-survival pathways or inactivation of tumor suppressors and apoptotic pathways (96,97). It is these mutations that provide a selective growth and survival advantage to that cell and its progeny. This results in the expansion of that cell into a clonal population. Subsequent mutations occur in cells of that clonal population, and thereby endow a few rare cells with new advantages. These daughter cells undergo additional waves of clonal expansion. This process continues, building on wave after wave of clonal expansion, until a mass forms and neoplastic transformation has occurred (54,95,98). The specific number of somatically acquired gene mutations necessary for neoplastic transformation is unknown and varies depending on which genes and tissues are targeted. Nevertheless, it has been estimated that neoplastic transformation of human cells requires, on average, four to seven mutations (97). Given the length of time needed to acquire these random mutations, only stem cells remain long enough for this to occur. Therefore, it has been postulated that cancers may arise in stem cell populations (99,100). However, this assumes that a mutation does not alter population dynamics, permitting some cells to become long-residing members or altering patterns of cell migration and differentiation (101, 102). For these reasons, this remains an area of active research.

CARDINAL FEATURES OF GASTROINTESTINAL CANCERS Gene mutations that support carcinogenic transformation fall broadly into one of three classes (see Table 17-2): (1) gain-of-function events, generally activation of growthpromoting pathways including oncogenes; (2) loss-of-function events, primarily inactivation of tumor suppressors and apoptotic pathways; and (3) epigenetic alterations (DNA methylation patterns), which can lead to both aberrant gene expression and silencing (96). Cells can be identified as neoplastic if they have acquired certain well-defined characteristics or qualities from these mutational events. Although the processes whereby the mutations are acquired can be quite distinct, the features themselves are readily apparent. In a seminal review on the hallmark features of cancer, Hanahan and Weinberg (97) describe six cardinal features necessary for carcinogenesis, including self-sufficiency in proliferative signals, insensitivity to antiproliferative signals, evading normal apoptotic cues, limitless replication potential, self-sustaining angiogenesis, and, lastly, tissue invasion and metastasis (Table 17-4). Thus, neoplastic transformation

TABLE 17-4. Cardinal features necessary for carcinogenesis Self-sufficiency in proliferative signals Insensitivity to antiproliferative signals Evasion of normal apoptotic cues Limitless replication potential Self-sustaining angiogenesis Tissue invasion and metastasis

is a stepwise process in which DNA mutations and clonal selection result in the evolution of a cell that expresses the features common to all cancer cells (95).

Self-Sufficiency in Proliferation Signals The study of DNA viruses and retroviruses that cause tumors in humans and animals provided significant insight into gain-of-function processes that lead to carcinogenesis. In these studies, a single viral gene was often identified that could lead to neoplastic transformation of normal cells. These genes were called oncogenes. The first example of this was the v-src oncogene of the Rous sarcoma virus (103). Subsequently, it was learned that normal cells contain genes with sequences similar to the viral oncogenes. These normal genes were termed protooncogenes or cellular oncogenes and were determined to play roles in promoting cell proliferation and regulating differentiation. Unlike the viral proteins, the activity of these protooncogenes is tightly regulated in normal cells. More significantly, studies of human tumors showed that these protooncogenes were often mutated in cancers such that they were inappropriately active, much like their viral oncogene counterparts (104). The first confirmed human oncogene was Ha-ras, isolated from a human bladder cancer cell line (105), but since then more than 80 human oncogenes have been isolated, and new candidates are identified on an ongoing basis. Several different kinds of mutations can be predicted to induce protooncogene activation, and all have been observed in human neoplasias (96,104). These mutations lead to either (1) inappropriate protooncogene expression patterns, or (2) loss of normal mechanisms that regulate protooncogene function. Gene rearrangements and amplifications, point mutations, as well as insertion/deletion events can foster protooncogene activation by increasing its promoter-mediated expression or stabilizing protooncogene mRNA or protein levels, or both. Alternatively, these events can eliminate or inactivate protooncogene regulatory domains or alter protooncogene protein conformation in a way that leaves it in a perpetually active state. These processes ultimately can lead to inappropriate cell proliferation, delayed differentiation, and neoplastic transformation. Protooncogenes typically are proteins with important roles in signaling for cell proliferation. They fall into four broad categories: peptide growth factors (TGF-α/epidermal growth factor [EGF], insulin-like growth factor [IGF],

482 / CHAPTER 17 fibroblast growth factor, among others), receptor and nonreceptor protein tyrosine kinases (e.g., v-erb-B/EGFR, c-src, c-yes), signal-transducing proteins associated with the cell membrane (e.g., c-ras, BRAF), and nuclear transcription factors (e.g., c-myc, β-catenin, c-jun) (106). GI cancers often express several activated oncogenes simultaneously, but they remain exquisitely dependent on ongoing oncogene signaling. Interruption of oncogene stimulus not only inhibits growth, but often also precipitates cancer cell apoptosis and death (107). One therapeutic challenge that has arisen is that although every cancer will have several active oncogenes driving proliferation, the identity of the oncogenes involved can vary substantially, even within the same cancer type. Ki-RAS mutations resulting in an active oncogenic ras occur in nearly 90% of pancreatic cancers, but only 50% of colorectal cancers, and rarely in upper GI cancers. Critical growthsignaling pathways often will contain several protooncogenes in epistatic succession. Activating mutations in any of these oncogenes can produce a proliferative effect that is phenotypically similar if not identical to any of the others. For instance, the EGF growth factor, its receptor EGFR, Ki-RAS, c-jun, and c-myc are linked in the same signaling pathway. Thus, in the 90% of esophageal squamous cancers without an activating KRAS mutation, we might expect and do, in fact, observe increased EGFR expression and activation due to EGFR gene amplification. In summary, one critical feature of cancer cells, self-sufficiency in proliferative signals, typically is provided by gain-of-function mutations of protooncogenes, dissociating their growth-promoting function from normal regulatory mechanisms.

Ignore Growth Inhibitory Signals The existence of genes whose products regulate proliferation and prohibit the progression to neoplasia emerged from the statistical study of the pediatric cancer retinoblastoma. Alfred Knudson (108) postulated in 1971 that the genetics of the familial and sporadic versions of the disease were related (109). He hypothesized that retinoblastoma was caused by two mutations in a single gene. In the familial form of the disease, one mutation was inherited in the germline, and the second acquired somatically at a relatively high frequency. In the sporadic form of the cancer, both copies of the gene must be mutated in the same cell by somatic processes. The frequency of this occurring is quite low, hence the much lower incidence of the sporadic retinoblastoma cancer. Based on these observations, Comings first postulated that the gene mutated in familial cancer syndromes might encode a tumor-suppressing product. Their hypotheses were not fully confirmed until 1986, with the cloning of the retinoblastoma gene (Rb), the first of more than 20 suppressor genes to be identified (110). Unlike oncogenes, tumor-suppressor mutations are classic loss-of-function events. Both alleles must be inactivated or silenced for neoplastic progression to proceed. In familial

cancer syndromes, there is inheritance of a single, mutant, tumor-suppressor allele. Inactivation of the second normal allele is considered to be the initiating event for neoplastic transformation in these cases. The second allele typically is lost either by a loss of heterozygosity (LOH) or epigenetic gene silencing. LOH occurs when chromosomal missegregation or nonreciprocal translocations during cell division results in the loss of one of two gene alleles. In the case of familial cancer syndromes, one of the two alleles was nonfunctional because of a germline mutation. Loss then of the remaining normal allele by LOH fully inactivates the tumor suppressor, opening the door for neoplastic transformation. A number of different tools have been used to identify regions of chromosomes often deleted in familial and sporadic GI malignancies. A number of tumor-suppressor genes have been identified within these regions. An analysis of deletions in chromosome 18q common to colorectal and pancreatic cancers led to the recognition of DPC4/SMAD4 as an important tumor suppressor in these tissues (111,112). The tumor-suppressor p53 is located within a region of chromosome 17p with frequent LOH in many cancers (113–115). The APC tumor-suppressor gene is located in another hot spot for LOH in colon cancers on chromosome 5q (116), and in breast, prostate, and gastric cancers, the E-cadherin locus on chromosome 16q is a frequent target for LOH (117–120). More recently, promoter hypermethylation has been shown to be an important mechanism for silencing tumor-suppressor gene expression, one that in many cases can effectively substitute for LOH. A number of tumor suppressors have high rates of promoter hypermethylation in GI tract tumors, including the DNA repair gene MLH1 (colon, gastric, and esophageal cancers) (121,122), E-cadherin (gastric, colon, and hepatocellular) (123), INK4a/p16 (gastric, esophageal, and colon) (124), and the mitotic checkpoint gene Chfr (gastric, esophageal, and colon) (125). Candidate tumor suppressors have been identified by novel screens for genes epigenetically silenced in colorectal cancers (88). The secreted frizzledrelated proteins (SFRP) are a family of secreted Wnt receptor proteins that serve to dampen Wnt signaling (Fig. 17-1) and the proliferative response of intestinal epithelial cells. Expression of SFRP1, SFRP2, and SFRP5 is silenced often in colorectal cancers because of promoter hypermethylation. This silencing may be an early event in colorectal carcinogenesis because it can be detected in ACF. The tumor-suppressor genes themselves are a varied group, including cell-surface receptors, cell–cell adhesion proteins, lipid phosphatases, factors that modulate intracellular signaling pathways, and transcription factors. In addition to being the first tumor suppressor identified, Rb has proved to be one of the more important tumor suppressors discovered. Rb is a critical regulator of progression through the cell cycle, particularly the transition from the resting G1 phase to S-phase, when DNA replication occurs. In normal cells, Rb acts to inhibit a cell’s exit from G1 by binding several members of the E2F transcription factor family and

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 483 L R Frz P Dsh

Plasma membrane

Wnt SFRP hDLG

EB1 APC

β-cat

APC

Microtubules

Axin

Normal Wnt signaling or mutation of APC or β-catenin as occurs in colon cancer

GSK-3β Absence of Wnt signaling and Wild type APC and β-catenin

Stabilization and accumulation of β-catenin

TAK1, NLK Degradation of β-catenin

β-cat TCF-4

Nucleus 5’

c-Myc cyclin D1 PPARδ c-jun

FIG. 17-1. Wnt signaling, adenomatous polyposis coli (APC) protein, and β-catenin. Dsh, disheveled; Frz, frizzled; GSK-3β, glycogen synthase kinase-3β; hDLG, disk large homologue; LRP5/6, low-density lipoprotein receptor-related protein 5 or 6; NLK, nemo like kinase; PPARδ, peroxisome proliferative activated receptor delta; TAK1, TGF-β activated kinase 1; TCF, T-cell factor.

silencing the expression of E2F target genes (126,127). E2F transcriptional targets include a number of genes with products that are necessary for nucleotide synthesis and DNA replication, both of which are required S-phase activities (128–131). Phosphorylation of the Rb protein (pRb) by cyclin-dependent kinases (cdk) inactivates it by disrupting its interaction with E2F (128,131). Cdk function itself is regulated tightly by posttranslational mechanisms, as well as the presence of two families of cdk inhibitors (CDKI), the Cip/Kip and INK4a families of proteins. Loss of Rb function permits uninhibited progression through the G1-phase of the cell cycle and generally unrestricted cell division (132,133). Because of its central role regulating progression through the cell cycle, all mitogen and growth inhibitory signaling pathways ultimately converge on Rb to regulate its function. Thus, it is an attractive target for modulation by neoplastic processes. Although Rb itself is not often mutated in GI cancers, cancer cells have many alternative methods they use to inhibit Rb function (134). A number of viral oncoproteins directly bind and inactivate pRb, including adenovirus E1A, SV40 T antigen, and the E7 proteins of the human papillomaviruses (8,133). Moreover, the actions of a number of human oncogenes either induce cdk activity or limit the expression of the CDKIs, thereby leading to Rb inactivation (8,133). Alternatively, loss of the Cip/Kip and INK4a tumor suppressors p21, p27, and

p16 by mutations or gene silencing, as often occurs in GI malignancies, also contributes to Rb inactivation by permitting unopposed Cdk function. Another tumor suppressor with a pivotal role in preventing neoplastic transformation is p53. The importance of p53 rivals that of retinoblastoma for the frequency with which it is inactivated and the breadth of human cancers it is involved in. p53 was initially identified in a complex with SV40 T antigen (135,136). Shortly thereafter, it was observed that when p53 was coexpressed with a mutant, oncogenic Ras in primary rat embryonic fibroblasts, the cells acquired neoplastic features. This led to the early conclusion that p53 was an oncogene that promoted cell immortalization (137,138). It took an additional five years and several dozen other investigations before the true role of p53 as a tumor suppressor was fully revealed (139). Investigators then began finding p53 was often targeted by mutations or gene deletion in a variety of cancers, including GI cancers (140). Moreover, germline mutations in p53 give rise to the human condition Li-Fraumeni syndrome (141), which is largely composed of breast cancers and sarcomas. Some p53 mutations not only inactivated its function but, because p53 forms oligomeric complexes, inhibited the function of wild-type p53 in a dominant-negative fashion (142,143). The existence of dominant-negative mutants undoubtedly contributed to the early confusion over the role of p53 in carcinogenesis.

484 / CHAPTER 17 p53 is a sequence-specific transcription factor that is activated when the cell is stressed or damaged. On activation, p53 accumulates in the nucleus where it binds its cognate responsive element and induces the expression of targeted genes. The protein products of the targeted genes are the effectors of p53, inducing either cell growth arrest or apoptosis depending on the cellular context in which they are expressed (140,144). In the absence of stress or damage, p53 levels and activity are kept in check by a protooncogene called Mdm2 (140,144). Mdm2 binds p53 tightly and blocks its transcriptional activation domain, inhibiting p53 function. Furthermore, Mdm2 can function as an E3 ubiquitin ligase, attaching ubiquitin moieties to p53 that target it for proteosome degradation. Thus, Mdm2 is a potent inhibitor of p53 tumor-suppressor function. It is not surprising, then, that p53 function is abrogated in some cancers by Mdm2 gene amplification or overexpression (145). Moreover, Mdm2 is itself a p53 target gene, induced when p53 is activated to negatively feedback and suppress p53 levels. For this reason, Mdm2 must be inhibited to fully activate p53. There are three independent mechanisms by which p53 can be activated. The first is direct DNA damage, especially DNA double-strand breaks (DSBs). These often occur with telomere erosion or ionizing radiation, and they are potent stimulators of the protein kinases ATM (ataxia telangiectasia mutated) and Chk2. These kinases phosphorylate p53, disrupting p53 binding to Mdm2, thereby leading to stabilization of the p53 protein and enhancing transcriptional activity (146,147). In addition, they phosphorylate the Mdm2 protein, inhibiting Mdm2 function and further increasing p53 activity (148,149). A second mechanism for inducing p53 function is triggered by aberrant growth signals and oncogene activity. These signals stimulate the expression of the tumor-suppressor p14 ARF (150). Alternative reading frame (ARF) competes with p53 for Mdm2 binding and can sequester the Mdm2 away from the p53 protein (151). This stabilizes p53, enhancing its cellular levels and promoting cell growth suppression. Finally, a wide array of cell stressors including ultraviolet light, chemotherapeutic drugs, and hypoxia can activate p53 through a third, poorly characterized pathway that does not include ATM, Chk2, or ARF but may require other kinases, such as casein kinase II and ATR (ataxia telangiectasia related) (152,153). Therefore, all three mechanisms exploit different pathways, but in the end all promote similar end points: p53 protein is stabilized, and the interaction with the inhibitor Mdm2 is disrupted. Moreover, these pathways typically further stimulate p53 transcriptional activity by inducing posttranslational modification of p53 by the addition or removal of phosphate, acetyl, glycosyl, ribose, or sumo chemical groups. These chemical groups alter p53 protein conformation in a way that enhances its function as a transcription factor. Once activated, p53 binds DNA and activates the transcription of different genes. Several dozen genes have already been identified as transcription targets of p53, and many more remain undiscovered. Genes targeted by p53 typically contribute to tumor suppression by inhibiting cell-cycle

progression, promoting apoptosis, enhancing DNA repair, and blocking tumor-induced angiogenesis (55,140,144). One of the earliest effects of p53, and the best understood, is the ability of p53 to block progression of cells through the cell-division cycle. Most important to this effect is p53-mediated induction of the CDKI p21WAF1/CIP1 (154). p21WAF1/CIP1 is a potent inhibitor of cdk activity, which is required for orderly progression through the cell cycle. Inhibition of CDK activity by p21WAF1/CIP1 blocks progression of cells at two critical points, the G1-to-S and G2-to-M transitions. p53 induces other cell-cycle inhibitors, including Reprimo, 14-3-3σ, and GADD45, which inhibit progression at the G2-to-M transition only. In certain cells, p53 expression leads to the induction of programmed cell death. As with cell-cycle inhibition, it is mediated by several different target genes. p53 expression induces proteins such as BAX, NOXA, and P53AIP1 that, when in excess, disrupt mitochondrial function and precipitate cell death (55,140,144). In addition, p53 induces the expression of death-signal receptors such Fas, Killer/DR5, and PIDD, which can respond to extracellular signals and initiate apoptotic cell death. Lastly, p53 can directly stimulate production of highly toxic reactive oxygen species by the mitochondria, which, if severe enough, can hasten cell death (155,156). Beyond inhibiting cell proliferation or promoting the death of altered or mutant cells, p53 expression suppresses tumor growth in other important ways. Tumor growth beyond a few millimeters must be supported by the in growth of new blood vessels (angiogenesis). Tumor angiogenesis is suppressed by the actions of several p53 target genes including thrombospondin1 and maspin, among others (157,158). Less well characterized is the ability of p53 to foster genetic stability by inducing the expression of genes that repair damaged DNA and regulate chromosomal recombination and segregation during cell division. In summary, p53 is a critical regulator of many processes central to the suppression of neoplastic cell and tumor growth. For this reason, p53 or its vital downstream effectors are likely inactivated in every GI cancer.

Unlimited Replication Neoplastic cells are “immortalized.” Unlike normal cells, they can undergo an infinite number of cell divisions. In the laboratory, this means that cancer cells can be passaged many times without any ill effects. By contrast, normal human cells can only undergo a set number of cell divisions before permanently withdrawing from the cell cycle. This limit on the number of cell division is known as the Hayflick Limit, and it represents a powerful tumor-suppressing function inherent in normal human cells (159,160). The basis for the Hayflick Limit is the gradual erosion with each cell division cycle of the structures at chromosomal ends known as telomeres. Telomeres are nucleoprotein complexes at chromosome ends that function to maintain chromosomal integrity (161, 162). They are composed of a number of double-stranded

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 485 TTAGGG DNA repeats, a 3′ single-strand DNA overhang, and associated binding proteins. Telomeres evolved to solve two problems inherent in the maintenance and replication of the linear chromosomal DNA. First, DNA repair mechanisms recognize double-stranded DNA breaks and act to repair them when detected. Chromosomal ends would naturally trigger this response unless otherwise protected. In addition, DNA polymerase cannot effectively replicate the template strand at the 5′ end of the chromosome without a nucleotide primer. Telomeres provide this priming and permit faithful chromosomal replication by the DNA polymerase. The repetitive telomere sequence is synthesized at chromosomal ends by the actions of the enzyme telomerase reverse transcriptase (TERT) and the RNA component encoded by the telomerase RNA component (TERC). In human cells, telomerase activity correlates well with TERT expression levels. In adults, TERT expression is normally limited to activated lymphocytes and a subset of hematopoietic and epithelial stem cells (163–165). Limiting TERT expression yields an important benefit for human cells—telomere length functions as a potent tumor suppressor (161,166,167). In the absence of telomerase activity, telomere length is not maintained with cell division but is instead slowly eroded. After a number of divisions, the telomere erosion has reached a critical level, and the chromosome end becomes unprotected. These ends are treated by cells as DSBs of the DNA, leading to induction of DSB repair proteins and the tumor-suppressor proteins, such as p53 and p16. However, because eroded telomeres cannot be corrected by DSB repair mechanisms, normal cells respond by either irreversibly withdrawing from the cell cycle (senescence) or undergoing apoptosis. The molecular mechanisms governing senescence are not well established but are known to require p53 or p16 checkpoint function, or both, and can vary between tissue types (161,167). Nonetheless, in the absence of these two critical tumor suppressors, cells with shortened telomeres can enter a period of slow proliferation. After a few additional generations and complete telomere loss, the cells enter a period that is marked by increasing genomic instability and massive cell death known as “telomere crisis.” Rarely, a cell clone emerges from this crisis period with the ability to proliferate without limit. This cell is said to have become immortalized. Studies with primary human cells in culture and in vivo in yeast and mice fully support this model (168–174). Studies with human pathology tissues similarly advance this model with respect to neoplastic progression. Human cancer cells are uniformly immortalized and demonstrate the ability to maintain telomere integrity, although telomere length is generally not as great as that seen in normal tissues (175–179). Most cancers have reactivated TERT expression to levels sufficient for telomere maintenance, and typically this occurs as a late step in the transformation process. The remaining 10% of cancers that do not reactivate TERT have acquired an alternative means of telomere biosynthesis, termed alternative lengthening of telomeres (ALT) (180–182). This mechanism uses a recombination-based

interchromosomal exchange to reconstitute the telomere with successive cell division cycles. Moreover, studies with dysplastic tissues, such as colonic adenomas or PanIN lesions in the pancreas, have found features suggestive of telomere crisis in progress, with massive genomic instability, little TERT enzymatic activity, and loss of nearly all the telomere protecting chromosome ends (183). In summary, telomere length serves as an additional critical barrier to neoplastic transformation of normal cells. To become immortalized and proliferate indefinitely, cancer cells must acquire the means to protect their chromosome ends, and this is most commonly done by reactivation of TERT expression.

GENETIC INSTABILITY Common mutational events observed in human cancers include point mutations; altered DNA methylation; and gene rearrangements, amplifications, and deletions. One difficulty facing investigators has been in explaining the mechanisms causing this genetic variability. More critically, the role of genetic instability in the development and progression of GI cancers is not as straightforward as it would seem at first glance. We do know all cancers display increased genetic instability compared with normal cells (95,184,185). Genetic instability has been observed in the earliest stages of tumorigenesis in the colon, namely in ACF (82,88,186). Moreover, several familial cancer syndromes are due to germline mutations in “mutators,” that is, genes involved in DNA damage repair (HNPCC, MYH adenomatous polyposis, Li-Fraumeni syndrome) (9,55). Inactivation of a mutator allele by a combination of a germline mutation and a second somatic hit results in increased genetic instability and a rapid progression to cancer. Despite these specific observations, it is possible in the laboratory to induce a neoplastic phenotype in normal mammalian cell without the induction of genetic instability (187,188). In addition, it has been calculated that, in the case of sporadic tumors, the rate of somatic point mutations in normal cells is theoretically sufficient to generate a cancer without invoking the need for increased mutation rates (189,190). Despite this debate, it is clear that increased genetic instability is an important factor promoting cancer progression even if it is ultimately found innocent of causing tumorigenesis in the case of sporadic cancers. Several distinct forms of genetic instability have been identified (Table 17-5), including chromosomal instability (CIN), microsatellite instability (MIN), epigenetic instability, and

TABLE 17-5. Mechanisms of genetic instability Chromosomal instability Microsatellite instability Epigenetic instability Instability associated with inactivation of the nucleotide excision repair Instability associated with inactivation of the base excision repair

486 / CHAPTER 17

Chromosomal instability pathway: 85% of cancers APC / β-Catenin

Normal

COX2

K-Ras

Early adenoma

T=0

p53

Late adenoma

SMAD 4

Carcinoma

7–10 years

FIG. 17-2. Chromosomal instability pathway in colorectal carcinogenesis. APC, adenomatous polyposis coli; COX2, cyclooxygenase-2.

the instabilities associated with inactivation of the nucleotide excision repair or BER. Of these five mechanisms, four are commonly involved in the pathogenesis of GI cancers (184,191–193).

Chromosomal Instability Most human GI tumors are aneuploid. Aneuploidy is marked by gains or losses of whole chromosomes, a phenotype described as CIN (Fig. 17-2) (184). A common feature of CIN cancers is LOH often targeting tumor-suppressor genes. Common sites for LOH in GI malignancies include 5q (APC), 17p (p53), and 18p (DPC4/SMAD4). Chromosomal abnormalities of CIN tumors also typically include gene amplifications, commonly of growth-promoting cellular protooncogenes such as the EGFR or cyclin D1 (194–196). This action also promotes cell transformation and tumor growth. Critical features of the CIN phenotype have been defined and used to test models for CIN development. CIN is known to be present in all dysplastic lesions, but to worsen as the dysplasia advances. Cell fusion studies have indicated CIN is a dominant trait, implying it is not caused by the loss of a tumor suppressor. Lastly, the karyotypic profile of cancers is unique and has been rather difficult to replicate in the laboratory until relatively recently. The causes of CIN remain unknown, but significant advances have been made in this area. Undoubtedly, DNA damage, cell cycle, and spindle checkpoints play a role in this process (185,197,198). Human genes involved in the

DNA damage checkpoints that are known to play important roles in tumorigenesis include the breast-cancer related BRCA1 and BRCA2 genes, ATM, and p53 (9,185). Each of these gene products responds to DNA damage, particularly double-stranded DNA breaks, by inhibiting further progression through the cell cycle and induction of DNA repair, or if necessary, the induction of apoptosis if the damage is severe. Inactivation of these checkpoint controls yields cells more tolerant of severe DNA damage and permissive of neoplastic transformation and CIN events, as evidenced by the increased risk for cancer in families carrying germline mutations in these genes (193,199). Cell-cycle checkpoint genes are increasingly recognized for their role in fostering CIN in cancers. Recently, a gene product that regulates entry into the metaphase and chromosomal condensation, the checkpoint with forkhead associated and ring-finger gene (Chfr), was found to be absent in one-fourth to one-third of human colon, esophageal, and gastric cancers because of epigenetic gene silencing (125,200–202). The mitotic spindle, which equally segregates chromosomes between daughter cells during cell division, is monitored by spindle checkpoints (198). Our understanding of mammalian spindle checkpoints is limited, but abnormalities in them can be demonstrated in CIN colon cancers. In two colon cancer cell lines, inactivating mutations in the human homologue of the yeast spindle checkpoint Bub1 gene were identified (197). Another factor critically involved in chromosome segregation is the APC protein. APC binds microtubules (see Fig. 17-1) and links them to the chromosomal kinetochores during mitosis by binding kinetochore-anchored Bub1 and Bub3. APC

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 487 mutants, such as those commonly found in colon cancers, lack these binding sites and produce chromosomal segregation defects in vitro (203,204). Most recently, work investigating the role of telomere maintenance in both normal and neoplastic cells has provided novel insights into another potential mechanism for CIN instability, namely telomere crisis (161). As discussed earlier, telomere length is a critical barrier to neoplastic transformation and cell immortalization. It serves to indicate the natural ends of chromosomes and distinguish them from a DNA DSB. Because most cells do not express TERT, their telomeres are gradually eroded with each successive generation, ultimately leading to loss of telomere protection of chromosome ends. In normal cells with eroded telomeres, the DSB repair mechanism is activated, and critical cell-cycle regulators such as p53 or p16 INK4, or both, induce cell senescence or apoptosis. In the case where these tumor suppressors are inactivated, cell division proceeds at a slower pace. The DSB repair processes, including end-toend DNA fusion and exonucleolytic sequence degradation, are instead used by the cell with disastrous results. Dicentric chromosomes are formed by the fusion of two chromosome ends, which then initiates an ongoing CIN via breakagefusion-bridge cycles. Dicentric chromosomes pulled in opposite directions during mitosis can break at random points, resulting in nonreciprocal translocations. Moreover, the broken ends reactivate DSB repair and are fused again, possibly with another chromosome. The process is repeated with each cell division, resulting in massive genomic instability. This model is supported by many observations both in vitro and in vivo. Comparative genome hybridization studies and histologic studies reported increased rates of chromosomal rearrangements, nonreciprocal translocations, and anaphase bridging observed in adenomatous polyps (205). Clinicopathologic studies reveal that telomerase activity is low and telomere length is shortened in small- to moderatesized human colon polyps. Telomere length is stabilized and telomerase activity is increased in large polyps and colon cancers (175,206). In PanIN lesions, telomere fluorescence studies suggested telomere length is considerably shortened, even in the earliest PanIN-1A lesions (207). In addition, the degree of cytogenetic abnormalities correlated inversely with telomere length, with the greatest number and size of abnormalities arising in tumors with the shortest telomeres. Genetic studies in mice and yeast confirm this observation. In mice, disruption of the terc gene yielded cells with shortened telomeres and a high frequency of end-to-end chromosome fusions (176). The chromosomes that participated in the fusions were those with the shortest telomeres in the population (208). Disruption of telomere end-binding proteins or telomerase in yeast yields a similar phenotype, with marked chromosomal rearrangements and break points at chromosome ends. The mice with disruptions in their terc gene experienced premature aging in many organs and an increase in rates of neoplasia, primarily lymphomas, teratocarcinomas, and hepatomas. Breeding these mice with disruptions in their

p53 gene but not, surprisingly, in p16Ink4a, led to a dramatic increase in epithelial neoplasms including breast, squamous cell, and GI cancers (209). Analysis of these tumors found CIN with features nearly identical to human cancers, including chromosomal bridging, nonreciprocal translocations, chromosomal losses, regional amplifications, and deletions. Based on these many different observations, as well as the requirement of telomerase or ALT activation for tumor progression, telomerase dysfunction appears to be an important contributor to the CIN cancer phenotype and a source for much of the genetic instability associated with human cancer.

Microsatellite Instability Microsatellite instability (MSI or MIN) is perhaps the best understood of the genetic instabilities involved in GI cancer progression (Fig. 17-3) (9,191,210,211). The MMR system is a multiprotein complex of MutS (MSH2, MSH3, MSH6) and MutL (MLH1, PMS1, PMS2) homologues that recognizes and signals for repair of the base–base mismatches and short insertion/deletion mispairings that spontaneously occur with DNA replication. Short, repetitive DNA segments, known as microsatellites, are especially vulnerable to this type of error (9,184,191,210). In a certain proportion of colonic (approximately 15%), pancreatic (13%), and gastric cancers (10%), the MMR system is inactivated. In these cancers, there is an increased frequency of mutations in microsatellites. Although most microsatellite sequences are contained in noncoding regions of the DNA, some genes contain repetitive sequences in their coding regions and are therefore highly susceptible to inactivating mutations in MMR-deficient cells (212–215). MIN cancers, unlike CIN cancers, tend to be diploid or near diploid, resistant to DNA-damaging chemotherapeutic agents, and have a better prognosis overall (184,210). MIN instability can arise in either familial or sporadic GI cancers, although the mechanisms responsible for each are quite different. Nearly all mutations selected for MIN cancers tend to lead to inactivation of target genes. In the colon, approximately 70% of HNPCC cancers acquire frameshift mutations in APC or β-catenin (212,213). These mutations inactivate APC but activate β-catenin by mutation of a regulatory domain. The TGF-βRII and insulin-like growth factor II receptors (IGF-IIRs) are also often inactivated by mutations in MIN neoplasms (214,216,217). Approximately 90% of MIN colon cancers inactivate the TGF-βRII. These mutations most often occur with the transformation of a MIN adenoma to carcinoma (217). The p53 tumor suppressor is not often targeted in HNPCC colon cancers (218,219); however, p53-mediated apoptosis is significantly inhibited. BAX, an important p53 proapoptotic effector, is inactivated in 55% of MIN cancers (215,220). Other genes with products that are important for control of cell proliferation, DNA replication and repair, or apoptosis are often mutated in MIN cancers, including MED1 (221), ATM (222), the DNA

488 / CHAPTER 17

Microsatellite instability pathway: 15% of cancers

T=0

Normal

APC / β-Catenin

1–3 years

Late Adenoma

Early Adenoma

DNA mismach repair genes (hMSH2, hMLH1)

Carcinoma

TGF-βRII BAX, iGF-IIR E2F4, TCF-4

FIG. 17-3. Microsatellite instability pathway in colorectal carcinogenesis. APC, adenomatous polyposis coli; IGF-IIR, insulin-like growth factor II receptors; TCF, T-cell factor; TGF-βRII, transforming growth factor-β type II receptor.

helicase BLM (223) the transcription factors E2F-4 (224), the candidate tumor suppressor RIZ (pRb-interacting zinc finger) gene product (225), as well as caspase-5 (226). In summary, inhibition of DNA MMR promotes the accumulation of frameshift mutations that inactivate tumor suppressors, promote growth, and accelerate malignant transformation of colonic epithelial cells. This likely contributes to further diminished DNA repair, and therefore increased mutagenesis during the adenoma-carcinoma sequence. MIN was first recognized to be an important feature of HNPCC colon cancers in 1993. Early reports used a variety of markers and definitions for MIN cancers. Workshops and panels of experts have subsequently issued guidelines for the identification of MIN cancers and HNPCC kindreds (227, 228). Currently, a consensus panel of five mononucleotide and dinucleotide markers is used to define MIN neoplasms (229). Instability in two or more markers was designated as Microsatellite-High (MSI-H), with a high-degree of MIN. Instability in one marker was termed MSI-Low (MSI-L), and absence of instability was termed microsatellite-stable (MSS). HNPCC families, which together constitute about 3% to 5% of all colorectal cancers, often have germline mutations in hMLH1 and hMSH2. These inactivating germline mutations are observed in nearly 60% of the HNPCC families, particularly those with MSI-H cancers (230). These mutations do not cluster within specific domains and are distributed throughout the hMLH1 and hMSH2 genes (231). Germline mutations have been less often described for hPMS1, hPMS2, hMSH6, and hMLH3 (230,232–237).

Nonclassic HNPCC families, such as those with later onset of colonic malignancies or those with a predominance of extraintestinal malignancies such as endometrial cancer, are more often found to have germline mutations in their hMSH6, hPMS2, and hPMS1 genes (238). Lastly, about a third of HNPCC families have no identifiable germline mutation to explain their MIN cancer predisposition, suggesting the involvement of as yet unrecognized gene products in DNA MMR. As with all familial cancer syndromes, individuals with predisposing germline mutations in one of their MMR genes often acquire somatically a “second hit” mutation that inactivates the remaining normal allele and initiates neoplastic transformation. Generally, this occurs by LOH or somatic mutation of the normal allele. Epigenetic inactivation of the normal allele has also been described (239). Once the MMR gene is effectively inactivated in both alleles, MMR function is compromised, and sequence errors begin to accumulate in the genomes of daughter cells. Sporadic colon cancers with MIN constitute about 10% to 15% of all colon cancers (9,191). Like HNPCC colorectal neoplasms, sporadic MIN colon cancers tend to occur proximally; have a greater mucinous component; and be poorly differentiated, diploid, and associated with an improved survival for patients (240–242). The MIN instability also targets many of the same tumor-suppressor genes that are inactivated in HNPCC cancers, although the frequencies of inactivation may differ (214,221,243). Somatic mutations in hMLH1, hMSH2, and hPMS2 have been reported as a cause of

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 489 sporadic MIN cancers (244,245); however, the frequency of these mutations has been quite low. Sporadic MIN colorectal neoplasms with somatic mutations in hMLH1 and hMSH2 occur with frequencies of only 10% to 20% each. There has been a single reported case of a sporadic colon cancer with biallelic, inactivating mutations in hPMS2 (244), and hMSH6 and hPMS1 mutations have not been observed in sporadic colon neoplasms. Further studies with sporadic MIN colon cancers showed that, although coding sequences for hMLH1 were wild type, hMLH1 protein levels were markedly diminished. A careful analysis demonstrated that the loss of the hMLH1 protein was caused by inactivation of the hMLH1 promoter by an epigenetic mechanism (121,239,246). Thus, although the basic mechanism driving MIN instability in HNPCC and sporadic MIN colorectal neoplasias is the same loss of DNA MMR mechanisms, the underlying genetic events are quite different.

Base Excision Repair The DNA nucleotide bases are fairly stable chemically, although in the context of the cellular environment, random modifications can occur continually because of several processes. DNA can be chemically altered by reactive species such as reactive oxygen species or activated methyl and alkyl radicals, particularly in stressed cells. Less often, spontaneous nucleotide deaminations can occur to alter base chemistry. All of these modifications can alter the noncovalent interactions between nucleotides and lead to base mispairing and transition or transversion mutations (G→A or G→T, respectively) during DNA replication if not corrected (191). These single-base mutations can lead to missense and nonsense mutations if they occur in critical coding sequences. Missense mutations in coding regions can be inactivating (i.e., APC) or activating (i.e., Ki-RAS) (7,191,247). Nonsense mutations code for protein translational STOP codons. If they arise within the gene’s normal coding sequence they can cause the production of truncated proteins due to premature termination of translation. Typically the truncated proteins that are produced are nonfuunctional. The BER pathway is responsible for identifying these chemically modified bases and initiating their repair. Two components of the BER pathway have been implicated in GI cancer pathogenesis, MED1(MBD4) and hMYH (5–7,191,221,248,249). It has been observed that the CIN- and MIN-mediated genetic instabilities are mutually exclusive (184). BER defects can, however, be observed in CIN+ and MIN+ colon cancers, as well as a small subgroup of cancers that are CIN and MIN stable (191,250,251). MED1(MBD4) is a glycosylase involved in the repair of deaminations of methylcytosines. It is often mutated by MIN in MIN+ tumors, leading to increased C→T transitions (221,252). By contrast, germline mutations in another BER-related enzyme, hMYH, are reportedly the cause of the novel attenuated FAP-like familial colon cancer syndrome termed MYH adenomatous

polyposis (5–7). MYH adenomatous polyposis–associated colon cancers are recessive, indicating that unlike other familial cancer syndromes, two mutant hMYH alleles are inherited rather than one. MYH adenomatous polyposis colon cancers are diploid or near diploid, and they are stable genetically at the chromosomal and microsatellite level (CIN−/MIN−). Fundamentally, they are characterized by high rates of G→T transversion mutations. Oxidative damage to DNA can produce the stable guanine adduct 8-oxo-7,8-dihydroxy-2′-deoxyguanisine, which can mispair with adenine, leading to G→T transversions. hMYH, in conjunction with OGG1 and MTH1, identify and excise these oxidized guanines to permit their repair. In individuals who inherit two mutant hMYH alleles, a lifetime of accumulated oxidative damage to DNA leads to colon neoplasia that is in some ways quite distinctive genetically. Although the G→T transversions are themselves random events, certain mutations are selected for with great frequency by MYH adenomatous polyposis cancers. It has been reported that APC is inactivated by premature nonsense codons in up to half of the MYH adenomatous polyposis cancers, all due to G→T transversions, whereas LOH at the APC allele is quite infrequent. Moreover, nearly two thirds of the MYH adenomatous polyposis colon cancers possessed activating missense mutations in codon 12 of Ki-RAS caused by G→T mutation at a single guanine base, leading to inappropriate Ki-ras protein signaling (5–7,247). Conversely, the critical tumor suppressors p53, SMAD4, and TGF-βII receptor genes are not often mutated in MYH adenomatous polyposis colon cancers. Much remains to be learned about the mechanism of neoplastic transformation in MYH adenomatous polyposis cancers, particularly what other genes are often mutated by the loss of MYH-associated BER. In addition, it is unclear whether heterozygous carriers of hMYH mutations are at any increased risk for colon carcinogenesis, or if there is any role for inactivating MYH mutations in sporadic colon cancer pathogenesis. Ongoing studies may clarify these issues (249). Another DNA repair gene with a hypothesized role in GI neoplasia is methylguanine methyl transferase (MGMT) (250,251,253). MGMT is not a member of the BER, pathway but it performs a similar function in that it recognizes and repairs chemically altered DNA bases. Methylating agents preferentially target the N7 position of guanine, which is repaired by the BER system. About 10% of the time, these agents attack the O6 position of guanine or the O4 position of thymine. MGMT recognizes these modifications and removes the alkyl adduct through a “suicide reaction” in which the adduct is irreversibly transferred to the reactive site of the MGMT protein (253). It is the key enzyme protecting the cell from the cytotoxic effects of methylating agents such as temozolomide, streptozotocin, procarbazine, and dacarbazine, all of which are therapeutic agents used in cancer chemotherapy. It is also highly protective against carcinogenic agents that produce O6-guanine adducts such as methylnitrosourea (MNU), a breast carcinogen in rats; azoxymethane, a colon carcinogen in mice; and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),

490 / CHAPTER 17 a compound found in tobacco smoke and a potent lung carcinogen (253). If these alkyl groups are not removed before DNA replication, the guanine could mispair with thymidine, resulting in G→A transition mutations. The MGMT gene often is silenced by promoter hypermethylation in a number of different cancer types, including cancers of the colon, stomach, esophagus, prostate, and central nervous system (253–256). In tumors in which MGMT levels are reduced or absent, there is increased frequency of G→A transitions that, as with the transversions associated with hMYH deficiency, can either lead to oncogene activation or inhibition of tumor-suppressor function. Mutations leading to activation of K-ras function and inactivation of p53 are often observed in these tumors (257–259). Other gene targets remain unknown but are an area of investigation. In summary, repair of chemically modified DNA bases is a critical function that can be an important source of genetic instability in cells in which these pathways are disrupted.

Epigenetic Mechanisms Epigenetic mechanisms refer to processes, other than DNA sequencing, by which cellular information is inherited during cell division. Epigenetic mechanisms include the interrelated processes of DNA methylation, gene imprinting, and histone acetylation. These are normal cellular mechanisms used by cells for silencing gene expression and repression of viral and transposon transcriptional activity (260,261). They are based on regulated methylation of the cytosine bases of CpG dinucleotides. CpG dinucleotides are underrepresented in the genome; however, short stretches from 500 to several thousand nucleotides in length can be enriched for them. These regions are termed CpG islands and are commonly found in the proximal promoter regions of genes. Nearly 50% of genes identified contain CpG islands within their proximal promoters. Methylation of these CpG islands is a tightly regulated process in normal cells: Fully methylated CpG islands are rare and are found only in the promoters of genes on the inactivated X chromosome in female individuals and genes regulated by imprinting (262). Imprinting refers to the process whereby the alleles of certain genes are differentially methylated (263,264). This pattern is set in the zygote and gamete before fertilization and maintained during development to suppress expression of the maternal or paternal copy of an allele (264). It now appears disordered CpG island methylation and loss of imprinting are both involved in the pathogenesis of some GI cancers (123,124,246,265–268). The process of gene silencing by CpG methylation is a focus of ongoing investigations. Nonetheless, enough has been learned to allow us to formulate models. Currently, CpG methylation is believed to determine whether chromatin is in an open, transcriptionally competent conformation or is in a state in which it is compacted and closed off from transcriptional regulators. Cytosines in CpG dinucleotides are targeted for methylation by a class of enzymes

known as DNA methyltransferases (262,269). The regulation of these enzymes is poorly understood; however, once methylated, a family of proteins known as methyl-CpG binding domain proteins (MBDs) binds the methylated CpG dinucleotides. These MBDs in turn recruit other factors into a multiprotein complex. Included among these factors are histone deacetylases, which remove acetyl groups from histone lysine residues. Deacetylated histones form more compacted nucleosomes and alter DNA conformation into a closed, silent configuration (268). It is currently unknown how normal or neoplastic cells regulate their patterns of CpG island methylation. What is known is that the patterns of CpG methylation are highly conserved with cell division and are typically maintained in the daughter cells. For this reason it is a useful mechanism for permanent gene silencing that is used by both normal and cancer cells. Epigenetic contributions to neoplastic transformations can be seen in both aneuploid and diploid cancers. Although it is clear that epigenetic processes contribute to neoplastic progression, the exact nature of that contribution is uncertain. Loss of CpG dinucleotide methylation was, in fact, the first epigenetic abnormality associated with cancer (270). An examination in every tumor type revealed significantly reduced levels of CpG methylation overall when compared with normal cells. This is significant for two reasons. First, it can lead to the activation of protooncogenes and other genes required for the transformed phenotype. Second, DNA hypomethylation may be linked to chromosomal rearrangements and instability in tumors (271,272). Identification of the genes induced by promoter hypomethylation in neoplasia has been technically challenging, but several examples from human cancers are known. The signal transducing protein and protooncogene H-ras (colon cancer) (273), as well as the tumor-associated antigens MAGE (melanoma) (274) and CAGE (testicular cancer) (275), are all induced by promoter hypomethylation. Other candidates for induction with CpG island demethylation include cyclin D2 and maspin in gastric cancer, carbonic anhydrase 9 in renal cell carcinoma, S100A4 in colorectal cancer, and 14-3-3σ in pancreatic cancer (276–280). It has also been suggested by several observations that CIN may be fostered by DNA hypomethylation. A developmental disorder known as immunodeficiency, CIN, and facial anomalies syndrome (ICF) is caused by the mutation of the DNMT3B gene, a DNA methyltransferase (271,281). Cardinal features of this syndrome include loss of satellite DNA methylation and the formation of multiradial chromosomes containing the arms from chromosomes 1 and 16. Although patients with ICF are not at an increased risk for neoplasia, it has been noted that unbalanced translocations between chromosomes 1 and 16 were often present in Wilms’ tumors, as well as ovarian and breast cancers. The break points for these translocations typically were located in severely undermethylated pericentromeric regions. In addition, L1 retrotransposons have been shown to be hypomethylated in colon cancers (282). This may lead to their activation, which could, in theory, promote chromosomal rearrangements as well.

MECHANISMS OF GASTROINTESTINAL MALIGNANCIES / 491 An alternative mechanism by which CpG methylation contributes to tumor progression is by enhancing DNA mutagenesis. Methylated cytosines can undergo spontaneous deaminations. If not corrected before DNA replication, the deaminated methyl-cytosine will base pair with adenosine, thus causing C→T transition mutations. If this occurs in the coding sequence of a gene, it can cause missense and nonsense mutations that significantly alter gene function. Nearly 50% of the inactivating point mutations of the tumor suppressor p53 occur at methylated cytosines (283). In addition, methylated CpG dinucleotides are better targets for UV mutagenesis (284) and are the preferred target for G→T transversion mutations caused by the tobacco carcinogen benzo(a)pyrene diol epoxide (284,285). Although these processes undoubtedly contribute to neoplastic transformation, currently they are not thought to be the primary mechanism. Although cancer DNA is globally hypomethylated, there are now numerous examples in neoplastic cells where CpG islands are aberrantly hypermethylated, leading to faulty gene silencing. Typically, the genes silenced by CpG island hypermethylation in cancer are known or suspected tumor-suppressor and DNA repair genes. This aberrant silencing of tumor-suppressor and DNA repair genes currently is thought to be the predominant epigenetic mechanism contributing to carcinogenesis (286,287). The aberrant hypermethylation of CpG islands is acquired gradually. Thus, gene silencing can be detected early in the pathway to cancer, and it is often progressive. In fact, much of the CpG island hypermethylation that is observed is age related (288–290). It is not currently known whether this age-related CpG hypermethylation contributes significantly to neoplastic progression. However, it is clear that silencing of certain gene targets can endow a normal cell with the hallmark features of neoplastic cells including self-sufficiency in growth signals, loss of growth inhibitory signals, inhibition of apoptotic mechanisms, and promotion of metastasis and angiogenesis. The APC protein is an important suppressor of the Wnt signaling pathway by acting to reduce levels of the β-catenin protein. When APC protein expression is lost or its levels are reduced by promoter hypermethylation, the β-catenin protein is stabilized and cell proliferation is enhanced. More common is the silencing of antiproliferative factors such as SFRP, E-cadherin, and p16, as often occurs in colorectal, gastric, and pancreatic cancers, respectively (88,123,291). In addition, CpG island hypermethylation of the MDM2 inhibitor p14ARF can lead to reduced p53 response to oncogene activation (122,292,293). The VHL and thrombospondin-1 genes are also often silenced by promoter hypermethylation in GI cancers (294–297). In each case, angiogenesis is stimulated, fostering tumor growth. Lastly, tumor cell metastasis can be enhanced by disruption of the extracellular matrix or loss of cell–cell adhesion, as occurs with epigenetic silencing of TIMP3 and E-cadherin genes, respectively (254,291,298,299). Aberrant CpG promoter hypermethylation can also promote genetic instability by silencing critical checkpoint

factors and DNA repair pathways. As noted previously, the mitotic stress checkpoint gene Chfr is often silenced by promoter hypermethylation in colon, esophageal, and gastric cancers because of epigenetic gene silencing (125,200–202), which may foster CIN development. The genesis of sporadic MIN colorectal cancers, in contrast, is driven by hMLH1 promoter hypermethylation, which results in inactivation of the MMR system. The data supporting this hypothesis for the hMLH1 gene is particularly compelling. The hMLH1 promoter contains CpG islands that have been found to be heavily methylated in more than 80% of sporadic MIN colon cancers (300). Moreover, this promoter hypermethylation is biallelic, and reversal of the methylation by demethylating agents has led to hMLH1 protein reexpression in several MIN colon cancer cell lines (246). In one study comparing somatic mutations and LOH with hMLH1 protein levels and promoter hypermethylation, it was found that strikingly 83% of the sporadic colon cancers had significant hMLH1 promoter hypermethylation (300). In addition, whereas hMLH1 promoter hypermethylation was often present with either LOH or inactivating somatic mutations, suggesting they are cooperative processes, when somatic mutation and LOH occurred together in a single cancer, promoter hypermethylation was noticeably absent. Taken together, these observations clearly provide a role for hMLH1 promoter hypermethylation in inducting the MIN phenotype in a subset of sporadic colorectal cancers. O6-methylguanine methyltransferase is also often silenced by promoter hypermethylation in a number of different GI cancers (254–256), leading to increased rates of G→A transition mutations. In addition, a number of other genes are often silenced by promoter hypermethylation in human cancers, including ER, p16Ink4a, p14ARF, HPP1/TPEF, THBS1, APC, CDH1, RIZ-1, and RASSF1A (250,251,288,289). Studies of patterns of promoter hypermethylation initially in colorectal cancer, then later in other GI neoplasias (301), reveal that in a subgroup of cancers, multiple genes are simultaneously silenced by promoter hypermethylation. This has led to the proposal of a separate pathway for tumorigenesis called the CpG island methylator phenotype (CIMP) (250,251,288, 289,302–304). It has been hypothesized that aberrant silencing of multiple tumor suppressors and DNA stability genes simultaneously is the critical mechanism driving neoplastic transformation in this subgroup. Currently, there is no consensus as to how CIMP is defined. CIMP is loosely defined by the presence multiple genes simultaneously silenced by extensive promoter hypermethylation. Some groups are trying to develop criteria (301), but currently these preliminary standards are useful only with colon cancers. CIMP+ cancers tend to occur in older patients and are seen more commonly in women (301). They have been associated with low frequencies of p53 mutations but high rates of activating KRAS and BRAF mutations, such that nearly every CIMP+ neoplasm has activation of the oncogenic ras signaling pathway. CIMP+ cancers are a distinct group clinically as well. In the colon, CIMP+ cancers tend to occur proximally and are highly associated with MIN instability because of frequent

492 / CHAPTER 17 inactivation of hMLH1. The CIMP phenotype is highly associated with serrated adenomas and is commonly observed in a subset of hyperplastic polyps as well (250,251). This has led to the suggestion that hyperplastic polyps may not be entirely benign and without neoplastic potential. Many questions remain regarding the CIMP pathway, particularly what is driving this dramatic alteration in DNA methylation patterns. Until a consensus definition for the CIMP phenotype is developed, research into this interesting area will remain challenging.

ACKNOWLEDGMENTS This work was supported by RO1 DK47437 (JPL) and RO1 DK56645 (AKR).

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CHAPTER

18

Development of the Enteric Nervous System Michael D. Gershon and Elyanne M. Ratcliffe Origins of the Enteric Nervous System, 501 Crest-Derived Stem Cells Are Present in the Developing and Mature Bowel, 502 Defects in Early-Acting Factors Cause Extensive and Visible Abnormalities: Hirschsprung’s Disease, 503 Sox10, 504 Phox2b, 505 Glial Cell Line–Derived Neurotrophic Factor/GFR-α1/ Ret and Endothelin 3/Endothelin B Receptor, 505 Hand2, 507 Defects in Late-Acting Factors Cause Restricted Lesions of the Enteric Nervous System, 507 Neurotrophin-3/TrkC, 508

Mash1, 508 Neuropoietic Cytokines, 509 Bone Morphogenetic Proteins, 509 Serotonin, 510 Associated Pathology of Late-Acting Factors, 511 Other Factors in Enteric Nervous System Development, 511 Laminin and Proteoglycans, 511 Netrins, 512 β-Neurexin and Neuroligin, 513 Acknowledgments, 514 References, 514

Disorders of gastrointestinal motility are neither rare nor well understood. The word idiopathic is, unfortunately, but necessarily, associated with many of these disorders. These conditions are generally thought to be congenital when they occur in children, but that attribution has not yet proved to be helpful either in comprehending pathogenesis or in developing means of prevention or treatment. Similar gastrointestinal dysmotilities occurring in adults are normally considered acquired, but the possibility that adult dysmotilities might also be congenital in origin has not received a great deal of investigative attention. Most of the research that has been performed on the development of the enteric nervous system (ENS) has centered on the molecular explanation of the pathogenesis of Hirschsprung’s disease (1). This concentration is certainly understandable because Hirschsprung’s disease is by far the most obvious congenital

defect of the ENS (2–6). In Hirschsprung’s disease, ganglia are absent from varying lengths of intestine. The aganglionic region may be large or small so that long-segment or shortsegment forms of Hirschsprung’s disease have been defined (3–7); nevertheless, long or short, there is nothing subtle about the total absence of ganglion cells. The concentration of research effort on Hirschsprung’s disease has been beneficial in that it has led to an appreciable advance in understanding both the pathogenesis of that disorder and the development of the ENS (1,8–12). The recent progress made in understanding ENS development and the causes of aganglionosis, has not lessened the need to consider many congenital enteric dysmotilities idiopathic. Unfortunately, the mere presence of ganglia in the gut provides no assurance that these ganglia function normally. The ENS is not a binary system that is “there” or “not there.” Instead, the ENS is a complex nervous system. It is not a simple array of parasympathetic relay ganglia in the wall of the bowel, as has classically been taught (13); the ENS can, when called on to do so, control sophisticated “behaviors” of the gut even in the absence of input from the brain or spinal cord, or both (14–18). The consequent autonomy of the bowel often is overlooked because other organs lack a nervous system that is in any way comparable with the ENS and because the ENS and the central nervous system (CNS)

M. D. Gershon: Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032. E. M. Ratcliffe: Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Columbia University, New York, New York 10032. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

499

500 / CHAPTER 18 normally work in tandem with one another. The CNS has a powerful ability to perturb or quiet the bowel, and the gut has an equally remarkable ability to disturb the brain. The vagus nerves provide a conduit for the two-way communication between the ENS and the CNS. In fact, afferent nerve fibers far outnumber their efferent counterparts in the vagi (19). Some of the information sent by the bowel to the CNS by way of the vagus nerves is not fun to receive and is probably defensive and protective in function. Anyone who has watched a pet dog operate on city streets knows that vertebrates need a defense to prevent the ingestion of inappropriate substances or objects. Information from the gut thus leads to satiety, nausea, and vomiting (20,21), sensations that inhibit continuing ingestion and provide training that discourages repetition of the initiating stimulus. Pain is also one of the messages from the bowel that is perceived by the ENS, although it is probably delivered more by spinal than vagal nerve fibers (20,22,23). Still, not every message the CNS receives from the bowel is necessarily distressing. Some of what does not rise to consciousness may actually do the brain a great deal of good. This type of information is poorly understood, but vagus nerve stimulation has been used to treat epilepsy (24–26) and depression (25,27,28) and has been shown in both humans and animals to improve learning and memory (29). The relation of the CNS to the ENS is surprisingly analogous to that of a chief executive officer of a modern corporation to his or her staff. The detailed operation of the bowel is delegated to the local control of the ENS, but the brain retains the option of altering overall activity. The motor and secretory functions of the gut, moreover, are best described as “behaviors,” rather than reflexes, because they are complex and require a high level of integrative regulation to operate normally. This complexity of output is mirrored in the complexity and extraordinary size of the ENS. The ENS contains all of the classes of neurotransmitter found in the CNS, more types of neurons than any other set of peripheral ganglia, and a number of neurons that equals or exceeds the number found in the spinal cord (~108 in humans) (9,30–33). This number dwarfs that of motor axons in the vagus nerves (34). The ENS even projects centripetally to prevertebral ganglia, and thus can modulate the input it receives from the CNS (35–37). Because the ENS is so different from other autonomic ganglia, it follows that the mechanisms that govern the development of the ENS must also be different from those of other ganglia. These mechanisms must be able to encode the expression of enteric neuronal phenotypic diversity, the development of intrinsic microcircuits, and the formation of synapses between enteric interneurons. Circuits must be established that are able to integrate information received from the CNS with intrinsic afferent information transmitted to enteric neurons from local sensors responding to conditions in the lumen of the gut or the wall of the bowel. Despite the improved understanding of ENS development spawned by the concentration of effort devoted to explaining

the aganglionosis of Hirschsprung’s disease, little is known about the potentially subtle defects that may reside in the enteric ganglia of a dysmotile gut. Because there are many different subsets of enteric neurons in the mature ENS, one must expect that there are occasions when individual subsets fail to develop normally. Similarly, synapses between enteric neurons could be defective, CNS-ENS interactions may be abnormal, or both. Because so little is known about congenital defects of the ENS that are short of aganglionosis and equally little is known about how neuronal diversity, synapses, or neuronal projections arise, knowledge of the pathogenesis of any form of congenital neuromuscular disorder of the bowel, other than Hirschsprung’s disease, is deficient. Functional bowel diseases, however, do occur in children, even in very young children, and may well be congenital. If they are, these nonlethal conditions, which are not associated with pseudoobstruction, clearly do not arise from any of the defects responsible for Hirschsprung’s disease. Whatever abnormalities cause functional bowel disease, those abnormalities must be compatible with propulsive motility. The absence of pseudoobstruction in patients with a functional bowel disease means that propulsive motility occurs in these patients even if that motility is so abnormal that it is the cause of a disabling morbidity. A congenital ENS defect associated with abnormal motility, secretion, or both, but not pseudoobstruction, is likely to affect late-arising lineages of neural or glial precursors (38,39), rather than the early-appearing multipotent neural/ glial stem cells that, when perturbed, cause Hirschsprung’s disease (see earlier). Even late-arising defects, moreover, can potentially obstruct propulsive motility if they interfere with the function of one of the specific sets of neurons that are critical for peristaltic or secretory reflexes. Submucosal intrinsic primary afferent neurons (IPANs), for example, appear to be essential for the gut to manifest peristaltic and secretory reflexes in response to luminal stimuli (39–45). A defect in the signaling of a late-acting factor that is needed for development of submucosal IPANs, such as neurotrophin-3 (NT-3) or its preferred receptor TrkC (46,47), severely compromises motility but leaves a superficially normal-appearing ENS (48). It is tempting to speculate that a defect of this type, that is, the loss of a critical neuron, or even a set of synapses, underlies the pediatric dysmotilities that are still to be explained and more severe conditions, such as chronic intestinal pseudoobstruction, in which intestinal motility fails in the presence of enteric ganglia. This chapter seeks to provide a conceptual basis for understanding not only the pathogenesis of Hirschsprung’s disease, which is covered here, but also other disorders of motility that are not associated with aganglionosis. To do so, the development of the ENS is described not as a detailed catalogue of all known relevant phenomena, but to highlight genes that, when mutated, might cause defects in the ENS that would or would not be as obvious as those of Hirschsprung’s disease. Defects that are not anatomically

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 501 obvious might nevertheless be causes of abnormal intestinal motility, or even of its failure.

ORIGINS OF THE ENTERIC NERVOUS SYSTEM The ENS is derived from the neural crest (Fig. 18-1) (49). This conclusion was first drawn by Yntema and Hammond (50,51), who observed deficiencies in enteric ganglia when deletions were made in what they called the “anterior neural crest.” The work of Yntema and Hammond was more elegantly confirmed by Le Douarin and her colleagues, who, to identify the levels of neural crest that contribute to the ENS, took advantage of the distinctive nucleolar-associated heterochromatin of quail cells to track the migrations of crestderived cells after their transplantation into chick embryos (52,53). In these early studies, segments of chick neural crest were replaced with those of quail (or the reverse). In this experimental paradigm, to identify cells of the donor (chick or quail, depending on the particular experiment) is, in the resultant interspecies chimeras, tantamount to identifying cells of neural crest origin. Experiments with these interspecies chimeras indicated that the ENS is derived from both vagal (somites 1–7) and sacral (caudal to somite 28) axial levels (53). Unfortunately, the developmental origin of the ENS was not settled in the public mind by these experiments. A potential problem of work with interspecies chimeras is that the experiments are, of necessity, uncontrolled.

Vagal

Truncal

Colonizes the entire gut Contributes to the colonization of the esophagus

Sacral

Colonizes only the postumbilical bowel

FIG. 18-1. The gut is colonized by precursors that migrate from three regions of the neural crest. The bulk of the crest-derived émigrés migrates to the bowel from the sacral crest and colonizes the entire gut. A smaller set migrates from the sacral crest and colonizes only the postumbilical bowel. The esophagus receives a contribution of crest-derived precursors from the rostral truncal crest.

There really is no way to be sure that quail crest-derived cells are not more invasive when they migrate in a chick embryo than they would be if allowed to stay home and migrate in a quail embryo. Doing the experiment in reverse is not an adequate control because chick crest-derived cells could similarly become adventitiously invasive in a quail embryo. Allan and Newgreen (54) thus challenged the conclusions of Le Douarin and her colleagues that sacral as well as vagal cells participate in the formation of the ENS. These investigators tried to track the progression of cells that they recognized morphologically as neuroblasts or enteric neuronal precursors. Doing so, they discovered a proximodistal gradient in neuronal maturation, which they believed ruled out the possibility that sacral crest–derived cells could enter the bowel. The sacral crest–derived cells presumably would have to migrate in a distal to proximal direction, which the investigators did not observe. More recent studies, in which endogenous crest cells were traced by labeling them with a vital dye or a replication-deficient retrovirus, confirmed that both the avian and murine gut are, in fact, colonized by cells from the sacral as well as the vagal levels of the neural crest (55–57). The early studies of Le Douarin and her colleagues have now been redone, using a more objective method to identify quail cells in their chimeric hosts. Perhaps because of the data derived from studies using a vital dye or a replicationdeficient retrovirus, investigators who once objected to studies with interspecies chimeras as uncontrolled have now accepted them as valid (58); moreover, genetic means of tracking crest-derived cells have also been developed (59). In the newer studies of quail/chick interspecies chimeras, quail cells are identified by using an antibody that recognizes them, eliminating the need to identify the quail nuclear marker (60). Experiments with the anti-quail reagent have now definitively confirmed that the bowel is colonized by émigrés from the sacral crest and indicated that they migrate first to the extraenteric ganglion of Remak, and then subsequently enter the bowel (58,61,62). Because of this late arrival, the ascending sacral crest–derived cells mix with their more numerous descending vagal counterparts, and if unmarked, escape detection by morphologic observation (54). The mammalian bowel is similarly colonized by sacral crest–derived cells, and in an evolutionarily conserved manner, these cells also migrate first to extraenteric pelvic sites before they enter the gut to join the vagal cells that have already arrived in the distal bowel (59). These early sacral crest– derived émigrés might account for the development of neurons in explants of hindgut from mouse embryos at embryonic days 9.5 to 10 (E9.5–E10) (63). The experiments on the mammalian gut used the Wnt1 promoter to drive the expression of lacZ in crest-derived cells to facilitate tracing the migration of sacral ENS progenitors (59). The entire vagal crest does not contribute to the formation of the ENS; instead, the bulk of the enteric neuronal precursors evidently originate from only the portion of the vagal crest lying between somites 3 and 6 (64). In the mouse,

502 / CHAPTER 18 a third region of ENS precursors has been identified in the neural crest, the rostral truncal crest, which colonizes the nearby foregut (esophagus and adjacent stomach) (65). The importance of the contribution of the truncal crest to the mammalian ENS is that it appears to have needs and properties that differ from those that characterize the vagal and sacral crest–derived cells in the remainder of the bowel. The esophagus, for example, appears to be the only region of the gut in which the formation of enteric neurons is partially independent of Ret (65,66) and completely dependent on Mash1 (67,68), a proneural gene that is the mammalian equivalent of achaete-scute of Drosophila (69). Below the esophagus, only a subset of neurons (including all of those that are serotonergic) is Mash1 dependent (70) and all neurons are Ret dependent (71). A fascinating approach to the investigation of crest-derived cells colonizing the gut has been to use imaging of the migrating cells visualized in flagrante as they travel through isolated segments of fetal bowel in vitro (72). These cells were visualized by detecting the fluorescence of green fluorescent protein (GFP) expressed by crest-derived cells in transgenic mice under the control of the promoter for Ret. The crest-derived cells were found to migrate in long chains in contact with one another, in confirmation of an observation made earlier of migrating crest-derived cells identified in an avian gut with a replication-deficient retrovirus expressing LacZ (56). The GFP-labeled cells were found to undergo many changes of direction, and the early cells form a scaffold that is later traversed by the trailing cells of the population. Because isolated crest-derived cells migrate more slowly than those in contact with one another, it is possible that population pressure contributes to the ability of crest-derived cells to migrate proximodistally. Cells that migrate express both Ret, which enables them to be visualized, and Sox10, which is a neural crest marker. Migrating crest-derived cells, however, do not express any neuronal markers, indicating that migration and neuronal differentiation are incompatible. Once neurons differentiate from their crest-derived precursors, they do not migrate. This observation may be highly significant with respect to the pathogenesis of the aganglionic terminal bowel in mice that are deficient in endothelin 3 (Edn3) (73) and of Hirschsprung’s disease in patients carrying mutations in the gene encoding EDN3 (74), the endothelin-converting enzyme-1 (ECE-1) (75,76), or the Edn3 receptor, endothelin B (EDNRB) (77) (see later discussion). The crest-derived cells that colonize the gut are a heterogeneous population that changes progressively as a function of developmental age, both while precursor cells migrate to the bowel and after they arrive in the gut (10,11,38,78,79). Some premigratory crest cells are fate restricted, others are pluripotent, and some continue to be so even after they have migrated into the bowel. Premigratory crest cells, for example, can form melanocytes, but this potential is lost from the crest-derived cell population that traverses the caudal branchial arches to enter the gut (80). Crest-derived enteric neural/glia precursors become sorted into lineages, which

can be identified by the combination of transcription factors and growth factors on which they depend (Fig. 18-2). The sorting of cells into precursor lineages is mediated, in part, by interactions of these cells with the enteric microenvironment. Both intrinsic and extrinsic factors thus determine the fates of enteric neuronal/glial precursors. Cells require growth factors, such as glial cell line–derived neurotrophic factor (GDNF), to migrate in the bowel (66,81,82), proliferate, and survive (83–89), but no cell can respond to GDNF unless it is programmed to express Ret, the GDNF receptor (90). In the case of development, therefore, chance (encountering a required growth factor) favors the prepared cell (e.g., one that expresses the requisite receptors).

CREST-DERIVED STEM CELLS ARE PRESENT IN THE DEVELOPING AND MATURE BOWEL To study the effects of growth factors and other components of the enteric microenvironment on the development of enteric crest-derived neural/glia precursors, it is useful to be able to isolate these cells from the developing gut so that the environment in which crest-derived cells differentiate can be experimentally controlled. When multiple factors interact simultaneously, it is difficult to determine what any one of them does, particularly in nondefined media, when some of the interacting molecules are unknown. Crest-derived cells in the mammalian bowel all seem to express the common neurotrophin receptor, p75NTR, which also is not expressed by noncrest-derived cells (91–94). As a result, antibodies to p75NTR can be used to immunoselect crest-derived cells from the fetal gut, and because of this property, they can also be used for negative selection of a crest-depleted population of cells (95). When carefully done, positive and negative immunoselection with antibodies to p75NTR can provide a crest-enriched population of cells that does not express mesodermal markers and a crest-depleted population that fails to express crest markers (96,97). All enteric neuronal and glia precursors may express p75NTR, but p75NTR expression is not limited to stem cells; some committed neurons and glia are also p75NTR immunoreactive (91–94). Immunoselection from the fetal bowel with antibodies to p75NTR thus provides a mixed population of committed cells and stem cells of postmigratory neural crest origin (98,99). Immunoselection with a second surface antigen is thus needed to concentrate the subset of enteric p75NTR-expressing, crest-derived cells that are crest-derived stem cells. One such molecule has been found empirically to be an α4 integrin (100,101); therefore, flow cytometry can be used to immunoselect crest-derived stem cells from the bowel as cells that display high levels of p75NTR and α4 immunoreactivities. These stem cells are self-renewing and multipotent and can be found in the adult and the developing gut, albeit in smaller numbers in the adult bowel (101). Deficiencies in crest-derived stem cells have been postulated to account for the acquisition of Hirschsprung’s disease (102). In fact, mutations of genes

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 503 Neural/glial precursor Phox2b+/Sox10+ Sox10 Phox2b

Mash1

Glia Sox10+/Ret−

GDNF/GFRα1/Ret

Ret+ Mash1 (TC cells)

Submucosal IPANs

5-HT-, NOScontaining neurons

FIG. 18-2. Specific transcription and growth factors define stages in enteric nervous system (ENS) development. The crest-derived precursors that give rise to the ENS lose developmental potential as a function of ontogenetic age. Consequently, the deletion of factors required early in development results in the loss of all subsequent cells, thus causing major defects, aganglionosis, or both. Deletion of factors required later in ontogeny affect only restricted lineages, thus causing smaller defects. The early neural/glia precursors depend on their expression of Sox10 and Phox2b. These cells acquire Ret, which must be stimulated by glial cell line–derived neurotrophic factor (GDNF) in a complex with its coreceptor GFR-α1. Later cells express Mash1; some form neurons in the esophagus, whereas others transiently become catecholaminergic before giving rise to neurons, such as those that express serotonin (5-HT) or nitric oxide synthase (NOS). Intestinal precursors that are Mash1 independent give rise to late-born neurons, some of which express TrkC and require its stimulation by neurotrophin-3 (NT-3). The diagram indicates the subsequent lineages that are lost (ξ-) when a required factor is deficient. Arrows show the direction of development and/or the effects of the indicated factors, such as Sox10, which stimulates expression of Phox1b and Ret. IPAN, intrinsic primary afferent neuron; TC, transient catecholaminergic.

that are associated with Hirschsprung’s disease in animals do lead to a paucity of isolatable crest-derived stem cells in the terminal gut (102,103).

DEFECTS IN EARLY-ACTING FACTORS CAUSE EXTENSIVE AND VISIBLE ABNORMALITIES: HIRSCHSPRUNG’S DISEASE A multitude of early factors influence the fates of the crest-derived precursors of enteric neurons and glia. Among the most important of the early-acting factors that have been shown to be critically involved in the development of the ENS is the Sox10 transcription factor (104–107). Sox10 is member of a set of genes that resemble Sry, which was first identified for its role in the development of the human and mouse testes (108). The Sry set of genes is characterized by a conserved DNA-binding domain of about 80 amino acids with homology to the high mobility group (HMG) box DNA-binding domain of the transcription factor, UBF (109). The name Sox thus comes from Sry-like HMG box genes. Other important early-acting genes are Pax3, which works with Sox10 (110), the paired-like homeobox transcription factor, Phox2b (94,111–113), and ret (65,71,88,89,114,115).

Because Ret must be activated for enteric neurons to survive, expression of its ligand, GDNF (82,83,85,87,90,116–119), and coreceptor, GDNF family receptor (GFR)-α1 (120–122), are as important as Ret itself. Evidence suggests that the basic helix-loop-helix (bHLH) transcription factor, Hand-2, is also an essential proneural gene for enteric neurons (123–127). These factors have in common a critical early action in the development of enteric neurons and glia. Most have been found to be mutated in at least some patients with Hirschsprung’s disease (1). Genes that, when mutated, are associated with the development of Hirschsprung’s disease are illustrated in Figure 18-3, but these genes account for no more than ~30% of total patients (128). Most of these mutations are rare or components of complex syndromes, or both. RET (129,130), which is mutated in 3% to 35% of cases in which a gene has been identified, and EDNRB (77), which is mutated in 5% to 15% of cases, are the most common genes found to be associated with Hirschsprung’s disease. Hirschsprung’s disease is a relatively common birth defect, occurring in about 1 in 5000 live births (128). It is 100% heritable but is nonmendelian and a clear multigene condition. Mutations associated with Hirschsprung’s disease display a variable penetrance. This is partly because rare high-penetrance mutations in RET combine with mutations

504 / CHAPTER 18

FIG. 18-3. Mutations in a large number of genes have been associated with cases of Hirschsprung’s disease. Most of these mutations are rare. The gene most commonly affected is RET, followed by EDNRB. Less commonly seen are mutations in EDN3 or SOX10. The proportions of abnormalities that are frequent enough to quantify are indicated.

in other genes to determine the risk for occurrence. A common noncoding variant in RET in an enhancer sequence in the first intron makes a great contribution to risk. This mutation reduces the enhancer activity markedly, when assayed in vitro. The mutation displays a relatively low penetrance and exerts different effects in male and female individuals, perhaps accounting for the relatively greater frequency of Hirschsprung’s disease in male individuals (128). This newly discovered mutation thus shows that both rare high-penetrant mutations in the coding sequence of a critical gene and common but low-penetrant mutations in noncoding regions can contribute to the risk for a heritable disease. Both inherited and sporadic forms of Hirschsprung’s disease exist. Mutations in known genes account for less than half of cases of Hirschsprung’s disease (1). It seems likely that additional mutations will be found that modify the susceptibility of individuals to Hirschsprung’s disease. In contrast with RET, expression of EDNRB is not essential for the development of enteric neurons and glia, and thus is different from other genes associated with Hirschsprung’s disease. EDNRB is the human gene that encodes the endothelin B receptor (Ednrb), which is essential because it is the only endothelin receptor that is physiologically stimulated by endothelin 3 (Edn3) (131). Both enteric neurons and glia develop in mice that lack either Edn3 (132) or Ednrb (133). These animals do not have neurons in the distal colon, but enteric neurons are found in the remainder of the bowel. Ednrb is expressed in the fetal gut both by crest- and noncrestderived cells, and it is important for enabling crest-derived

cells to complete their colonization of the gut (73,98,103, 132–138). The contribution of Edn3/Ednrb to the colonization of the bowel is complex and is discussed further later in this chapter.

Sox10 Sox10 is expressed by all crest-derived precursors of enteric neurons and glia and is essential for their development (105). Heterozygous mice that carry a spontaneous mutation called dominant megacolon (Dom) in sox10 have an aganglionic terminal colon (307). Dom/Dom mice die during fetal life, and crest-derived cells from these animals undergo apoptosis before reaching the bowel. Sox10 has also been reported to be required for the initial expression of phox2b (139). Among its other interesting effects, Sox10 activates an enhancer of ednrb in enteric crest-derived precursors migrating in the bowel and approaching the colon (107). The effects of Sox10 on the expression of phox2b and ednrb illustrate the importance in ENS development of reinforcing interactions between the essential early-acting transcription and growth factors and their receptors. Ednrb also illustrates this theme because it promotes the expression and action of Ret (98,103,140,141). Sox10 expression is lost from enteric neurons during development but persists in glia (93) and is important in gliogenesis (142). The loss of Sox10 expression by crest-derived cells in the ENS appears to signal their transition from uncommitted neural/glial precursor to a progenitor in a neuronal lineage.

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 505 Phox2b Both vagal and sacral enteric crest–derived precursors express the paired homeodomain transcription factors Phox2a and Phox2b (111–113). Phox2b is expressed before Phox2a (112) and is the more critical of the two for the formation of the ENS. The development of all vagal and sacral enteric crest–derived precursors appears to be phox2bdependent because none of these cells survives to populate the bowel in mice that lack phox2b (111). Expression of phox2b has been postulated to be essential for the subsequent expression of Ret, a relation that is certainly consistent with the similarity of phenotypes of animals lacking phox2b to those of mice lacking ret. A polymorphism in PHOX2B has been associated with Hirschsprung’s disease (143). Mutations in PHOX2B are not specific for Hirschsprung’s disease, which is not surprising, because the gene plays an essential role in the development of virtually all autonomic derivatives (111). In addition to Hirschsprung’s disease, PHOX2B mutations are also associated with hypoventilation syndrome and the development of neuroblastomas (144).

Glial Cell Line–Derived Neurotrophic Factor/ GFR-α1/Ret and Endothelin 3/Endothelin B Receptor Ret is a receptor tyrosine kinase that is activated by the GDNF family of ligands, a group of relatives of transforming growth factor proteins, which activate Ret in a complex with one of its family of corresponding coreceptors, GFR-α1-4 (145,146). These ligands bind initially to the GFR-α1-4 coreceptors, but signal transduction is mediated by an activated Ret. The GDNF family of ligands and their corresponding coreceptors are GDNF itself, which preferentially binds to GFR-α1 (147,148), neurturin, which preferentially binds to GFR-α2 (149–151), artemin (GFR-α3) (148), and persephin (GFR-α4) (152,153). GDNF family ligands are relatively promiscuous in their interactions with coreceptors in vitro; however, in vivo, these ligands appear to be fairly faithful to their preferred GFR-α binding partner. To survive, develop, or both, vagal and sacral crest–derived precursors must express Ret and its ligand-preferring GFR-α coreceptor. Ret is stimulated on formation of a complex with the GDNF family receptor GFR-α1 and GDNF (38,145,154). In mice that lack Ret (71), GFR-α1 (121,147), or GDNF (116,155), there is a complete lack of an ENS below the esophagus/ proximal stomach (domain of the truncal crest–derived cells). The survival and development of the truncal precursors is not totally GDNF/GFR-α1/Ret dependent, nevertheless the number of esophageal neurons is severely reduced in mice that lack Ret (66). GDNF is expressed in the mesenchyme of the developing anterior foregut before the arrival of progenitors from the neural crest (156). It is then up-regulated in a proximodistal sequence peaking just ahead of the advancing front of vagal crest–derived cells migrating down the bowel.

GDNF promotes the migration of crest-derived enteric neural/glia precursors and acts as a chemoattractant for them (81,82,156). The chemoattractive effects of GDNF are mediated by Ret stimulation and are transduced by a signaling pathway that involves phosphatidylinositol-3 kinase and mitogen-activated protein (MAP) kinase (156). These observations have led to the proposal that attraction to the enteric mesenchymal source of GDNF is responsible for allowing crest-derived cells in the vagal pathway of migration to find the gut and also for inducing them to migrate proximodistally within the bowel. The crest-derived cells thus follow the distally receding peak of GDNF expression in the enteric mesenchyme (156). This hypothesis is consistent with the demonstrated Ret-dependent effects of GDNF and with the observed enteric expression pattern of GDNF; however, when the descending peak of GDNF expression reaches the cecum, a problem arises. The highest level of GDNF expression occurs in the cecum (156); thus, if nothing were to intervene, crest-derived cells could not migrate distal to the cecum because they would be pulled back to it by the chemoattractive effects of the cecal concentration of GDNF, which exceeds that of the colon. To get around this difficulty, an interaction of Ret and Edn3/Ednrb has been proposed (98). Edn3 inhibits the chemoattractive effects of GDNF on crest-derived cells (98,103); therefore, by inhibiting the cecal chemoattraction of crest-derived cells that have reached it, Edn3 might release these precursors from what would otherwise be a cecal trap and allow the crest-derived cells to enter the colon and complete their journey to the anal verge (98). The ability of Edn3 to oppose the chemoattractive effect of GDNF is due to an Ednrb-mediated reduction in the activity of protein kinase A (PKA) in migrating crest-derived cells. The idea that an Edn3/Endrb-mediated inhibition of GDNF chemoattraction is required for vagal crest–derived cells to colonize the colon posits a dynamic regulation of Edn3 expression in the developing gut. Indeed, the peak of Edn3 expression has been reported to be close to or ahead of the front of migrating enteric crest–derived cells (98). Edn3, however, is expressed in the rostral bowel, as well as in the cecum and colon; moreover, Edn3 also enhances the ability of GDNF to stimulate the proliferation of crest-derived cells in the gut (98) and impairs the self-renewal of enteric crest–derived stem cells, at least in vitro (103). Mice that lack ednrb have crest-derived stem cells in the rostral but not the distal gut. It is thus essential that the interaction of Ednrb with Ret be precisely orchestrated during development. If Ednrb were to oppose the chemoattractive action of GDNF in the rostral bowel, the gut could never be colonized and would run out of stem cells. This problem may be overcome by the spatiotemporal enhancement of Ednrb expression by Sox10 as crest-derived precursors approach the cecum (107). Even so, however, it is unclear how Ednrb stimulation can release crest-derived cells from the presumed “fatal” attraction to GDNF in the cecum (98) without at the same time exerting its other observed action to deplete the crest-derived pool of stem cells (103). If the crest-derived population were

506 / CHAPTER 18 to be enabled by Edn3 to migrate below the cecum at the expense of its content of stem cells, gangliogenesis would be expected to fail in the distal gut. In fact, as noted earlier, when Ednrb is deficient, the population of crest-derived cells that enters the colon is deficient in crest-derived stem cells (103). Although Ret and Endrb exert synergistic, stage-dependent effects on neural crest precursor proliferation and antagonistic effects on cell migration (157), it seems unlikely that these actions alone account for the colonization of the bowel by crest-derived precursors. Clearly, the interactive effects of Ednrb and Ret stimulation are complex; however, to understand how deficiencies of these factors contribute to the pathogenesis of aganglionosis of the terminal gut, all of their known effects and the contribution to the ENS of the sacral neural crest have to be taken into account. It is tempting to point out that the bulk of the ENS is derived from the vagal crest, and then to ignore the contribution made by the sacral crest. This temptation probably contributed to the past reluctance of some investigators to accept that a subset of enteric neurons is sacral crest derived (54,113,158). Compelling evidence has now overcome that reluctance (55–59,61,62), and it is thus necessary to explain why the terminal bowel in Hirschsprung’s disease (or in mice that lack Edn3 or Ednrb) lacks sacral crest– derived cells. Hirschsprung’s disease, furthermore, cannot be fully explained by defects in interactions of Edn3/Ednrb with GDNF/GFR-α1/Ret in the upper bowel or cecum (98,103). Abnormalities of these interactions may certainly contribute to pathogenesis, but the terminal bowel is aganglionic, not hypoganglionic, in Hirschsprung’s disease; therefore, the complete absence of neurons from the terminal bowel means that crest-derived cells of sacral and vagal origin have failed to colonize it. Sacral crest–derived cells ascend in the gut and do not traverse the foregut, midgut, or cecum; moreover, their ability to colonize the terminal bowel does not depend on the arrival of vagal crest–derived cells (59,61,62). Therefore, an explanation of the absence of sacral, as well as vagal, crest–derived cells from the defective bowel cannot be avoided to comprehend the pathogenesis of aganglionosis. It is apparent, both from studies of the effects of hypomorphic alleles in mice and from genome-wide association studies of human populations with Hirschsprung’s disease, that Ednrb and Ret do interact (98,103,140,141,157); nevertheless, the exact nature of this interaction remains speculative, and its effects, if any, on sacral crest–derived cells are unknown. Ednrb stimulation by Edn3 inhibits the differentiation of enteric crest–derived cells (98,138), but precursor proliferation and neuronal differentiation are enhanced by GDNF stimulation of Ret (83,85,115). These seemingly antagonistic actions of Ednrb and Ret may be functionally complementary. For example, one idea is that Edn3/Ednrb modulates the actions of GDNF/GFR-α1/Ret and prevents “irrational exuberance” among Ret-driven precursor cells (98). In vitro, addition of GDNF causes cells to leave explants of gut and migrate on the substrate, whereas addition of Edn3 prevents this phenomenon from occurring; thus, a potential function attributed to Edn3 is to act as a shepherd to keep

errant GDNF-stimulated, crest-derived cells from migrating out of the bowel in vivo. Indeed, ectopic ganglia have been found to develop outside of the gut in the pelvis of Edn3deficient mice (159,160). These ganglia, however, have also been interpreted possibly to represent sacral crest–derived cells that have ceased migrating too soon (i.e., before reaching the bowel) because of the deficiency of Edn3 (138). Direct observations of the migrating crest–derived cells marked in the bowel by the expression of GFP under the control of the Ret promoter (see earlier) indicate that migration of crest-derived cells ceases when these precursors differentiate as neurons (72). Crest-derived precursors thus migrate, but enteric neurons do not. By inhibiting premature differentiation of precursor cells into neurons, Edn3/Ednrb therefore would allow both vagal and sacral crest–derived precursors to continue to migrate until they complete the colonization of the entire bowel (138). Inhibition of differentiation of precursor cells does not imply that Edn3/Ednrb necessarily also enhances proliferation of crest-derived precursors; Edn3/Ednrb does not appear to do so. Because Edn3/Ednrb inhibits differentiation, however, the absence of Endrb stimulation in mice or human subjects that are deficient in Edn3 (EDN3), or that lack Ednrb (EDNRB), would allow GDNF/GFR-α1/Ret to promote neuronal differentiation without restraint. This unrestrained differentiation would be expected to deplete the pool of migration-competent precursors, resulting in the failure of colonization and aganglionosis of the terminal gut. Vagal crest–derived cells would form neurons only in the upper bowel, and sacral crest– derived cells would form neurons even before they reach the gut, explaining the presence of ectopic ganglia observed in mice that lack Edn3 (159,160). Ednrb is also expressed by smooth muscle precursors in the colon, and the absence of Endrb stimulation delays muscle differentiation (73); this delay leads to an increase in the secretion of laminin-1, which promotes neurogenesis. The accumulation of laminin-1 in the presumptive aganglionic region of the colon would be expected to add to the neurogenic drive provided by GDNF/GFR-α1/ Ret to the crest-derived precursors approaching this region and worsen the problem of premature differentiation in the Edn3/Ednrb-deficient colon. This mechanism would be expected to interfere with the colonization of the terminal bowel because that is the last region of the gut to be colonized either by vagal or sacral crest–derived precursors. One would not expect vagal crest–derived precursor cells to accumulate proximal to a specific site where migration suddenly becomes abnormal in animals deficient in Edn3/Ednrb. Instead, one would predict that migration would taper off gradually, leaving a hypoganglionic zone between the normal bowel and the aganglionic region. Also predicted would be a greater effect on the submucosal plexus than the myenteric because the submucosal plexus forms later than the myenteric and depends on the retention of migration-competent precursors in the myenteric layer of the gut to subsequently populate the submucosa (159). Each of these predictions has been borne out both in Edn3/Ednrb-deficient mice and in Hirschsprung’s disease.

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 507 The effects of GDNF on precursor proliferation decrease with age, but the ability of GDNF to support the development of enteric neurons nevertheless persists (83,115). The proliferative effects of GDNF on enteric neuronal precursors have led to the postulate that GDNF, by regulating precursor proliferation, controls the number of neurons in the ENS (85). This postulate, however, is a bit too simple because GDNF does not, by itself, control precursor proliferation. The proliferative action of GDNF is modified by other factors; for example, it is enhanced by and thus dependent on the presence of Edn3 (98,140,141). The ability of GDNF to stimulate proliferation, moreover, is sharply counteracted by effects of bone morphogenetic protein-2 (BMP-2) and BMP-4, which promote the differentiation of uncommitted precursors into postmitotic neurons (96). Many factors thus interact to sculpt the ultimate size, as well as the nature, of the ENS.

Hand2 The heart and neural crest derivatives expressed protein (Hand) set of bHLH factors comprises two factors, Hand1 (also known as eHand) and Hand2 (also known as dHand) (161). Hand genes are ubiquitous in animals and are represented in the genomes of a wide variety of organisms including flies and humans. Hand gene activity is essential for the specification or differentiation of both extraembryonic and intraembryonic structures. Hand-dependent extraembryonic structures include the yolk sac, placenta, and trophoblast. Hand-dependent intraembryonic structures are found in the heart, gut, and sympathetic ganglia, as well as in tissues that become populated by Hand-dependent, crest-derived cells, such as the developing vasculature, limbs, jaw, and teeth. Little is understood about the downstream target genes that Hand1 and Hand2 regulate or the mechanisms by which the Hand proteins modulate biological activity. Both Hand1 and Hand2 are expressed in the developing gut where they appear to be regulated by BMPs and GDNF (124,127). Hand2 is expressed in the bowel exclusively by crestderived cells, but these cells do not express Hand1 (124,127), which is expressed by mesenchymal cells that give rise to smooth muscle and interstitial cells of Cajal (124). Mice that lack Hand2 die of cardiovascular abnormalities at E10.5 (126); however, the murine foregut is colonized by crestderived cells at E9 to E9.5 (10,11,63,93,94). For this reason, the bowel can be removed at E9.5 from Hand2 null mice, and the effects of the gene knockout on the in vitro development of neurons can be studied (124). E9.5 explants of bowel from both wild-type and Hand2 null mice contain cells that express the neural crest markers, Phox2b, Sox10, Ret, and p75NTR. Crest-derived cells thus colonize the Hand2 null gut, as well as the bowel of their wild-type littermates; nevertheless, neurons develop only in cultured explants from wild-type mice and not in cultured explants from Hand2 null animals. Cells that express Phox2b, Ret, and p75NTR persist in cultures derived from mice that lack Hand2, but cells that express Sox10 almost disappear and no cells acquire a

neuronal marker. The absence of neurons in cultures of Hand2 null gut is compatible with the idea that Hand2 is necessary for the expression of a neuronal phenotype by enteric crest-derived cells. The maintenance of Phox2b- and Ret-expressing cells in Hand2 null cultures suggests that crest-derived cells do not undergo apoptosis because of the absence of Hand2. The disappearance of Sox10, which normally persists in glia, however, is consistent with the possibility that crest-derived cells developing as glia do not survive when Hand2 is not expressed. As noted earlier, Sox10 expression is important in gliogenesis (142) and persists in enteric glia but is lost in neurons (93); therefore, the behavior of Sox10-expressing cells suggests that the Hand2 null gut is colonized by uncommitted neural/glial precursors that express Sox10 (124). Sox10-expressing cells then are lost as these cells try unsuccessfully to differentiate as neurons or give rise to glia that cannot survive in the absence of Hand2, or both. The possibility that Hand2 functions in enteric neurogenesis is further supported by experiments that use small interfering RNA (siRNA) directed against Hand2. Crest-derived cells that have been isolated from the fetal mouse gut by immunoselection with antibodies directed against p75NTR and transfected with siRNA directed against Hand2 fail to develop as neurons (124). Hand2 expression may thus be essential both for the differentiation of enteric neurons and for the maintenance of enteric glia. Hand2 expression peaks in fetal life, after the maximal concentration of Phox2b is attained; however, although its expression is developmentally regulated, Hand2 continues to be expressed in the adult bowel. The role of Hand2 in the mature ENS remains to be determined, but it may be important in neuronal plasticity or maintenance, and thus, when abnormal, could play a role in the development of dysmotilities of the gut arising in human subjects who do have enteric ganglia.

DEFECTS IN LATE-ACTING FACTORS CAUSE RESTRICTED LESIONS OF THE ENTERIC NERVOUS SYSTEM As outlined earlier, defects in factors required early in ENS ontogeny tend to cause extensive abnormalities, such as aganglionosis, because these factors are required for the development of multipotent precursors that are common to all, or to a large subset of the ENS (38). In contrast, defects in factors that are required late in ENS development will produce smaller, much more restricted lesions, because these factors are required by cells that have a limited developmental potential (38,46,70). Late-acting factors that influence enteric neuronal development and their receptors include NT-3 and its high-affinity receptor TrkC (46,48,162), the bHLH factor, Mash1 (70), a neuropoietic cytokine that activates the ciliary neurotrophic factor (CNTF) receptor (169), the BMP factors (BMP-2 and -4; their receptors and antagonists) (96,163) and serotonin, which acts at 5-HT2B receptors (164).

508 / CHAPTER 18 Neurotrophin-3/TrkC Mice that are deficient in NT-3 or TrkC do not feed and die shortly after birth (48,162). The animals have severe sensory defects, which might explain these problems; however, NT-3 has also been found to be necessary for the development and maintenance of submucosal IPANs and for a subset of myenteric interneurons (46). These types of neuron are deficient when NT-3 or TrkC is deleted, and in adult mice, they demonstrate their TrkC/NT-3 responsivity by specifically taking up 125 I-NT-3 and subjecting it to retrograde axonal transport (46). The NT-3–linked defects in the ENS of mice lacking NT-3 or TrkC initially went unnoticed (48,162), but they are functionally significant. Submucosal IPANs, for example, are required for the initiation of peristaltic and secretory reflexes (45,165,166). The loss of this category of cell is probably sufficient to account for the inability of mice that lack NT-3 or TrkC to feed normally. In addition to the loss of function when NT-3 or TrkC is deleted, there is a gain of function when NT-3 is overexpressed in transgenic mice (46). The promoter for dopamine β-hydroxylase (DBH) targets transgene expression to the bowel (158,167,168), including that of NT-3 (46). In DBH/NT-3 transgenic mice, the gut becomes hyperganglionic, displaying more ganglia per unit area, more neurons and ganglion, and a significant increase in neuron size within enteric ganglia (46). NT-3 promotes the development of enteric neurons in vitro (47,169), but only if precursors are isolated from the gut after E14 (83). By this time, crest-derived cells have almost fully colonized the bowel (10,93,94), and GDNF is no longer able to stimulate their proliferation (83). The crest-derived precursors that cause early-developing neurons thus are unaffected by NT-3. Perhaps for this reason, the submucosal plexus, which develops later than the myenteric, is far more NT-3 dependent (46). NT-3/TrkC thus illustrates that a growth factor can exert an effect that is highly significant to the development of the ENS, and yet not cause an aganglionosis. Currently, no clinical condition has been associated with mutations in NT-3 or TrkC. However, the well-known tautology applies: The absence of evidence is not evidence of absence. It is possible that a defect in NT-3 or TrkC may be found to be associated with a human dysmotility; but if this occurs, studies with animals suggest that it will be a condition in which there is no aganglionosis.

Mash1 Mash1, which has been mentioned earlier in a different context, is, like the Hand genes, a bHLH transcription factor (69,170,171). Mash1 is critical for expression of the sympathetic neuronal phenotype, but it functions as a component of a network of cross-regulatory transcription factors, which include, in addition to Mash1, Phox2b, Phox2a, Hand2, and Gata3 (Gata2 in avians) (172,173). Mash1 and Phox2b expression are necessary and sufficient for manifestation of the noradrenergic sympathetic phenotype, but they also function in the development of other types of neuron, including those

of the ENS. The actions of Mash1 and Phox2b, therefore, must be supplemented by other factors to specifically generate a sympathetic or an enteric neuron. Phox2a, which is a paralogue of Phox2b, can activate promoters of tyrosine hydroxylase and DBH, which are components of the noradrenergic phenotype (174,175). With respect to the ENS, Mash1 was found to be expressed in enteric neuronal precursors (69,176), and its deletion was observed to lead to a deficient innervation of the esophagus, but not of the remainder of the bowel (67,68,177). Because the knockout of Ret was observed to cause aganglionosis of the bowel distal to the esophagus, whereas the knockout of Mash1 led to esophageal aganglionosis, Ret and Mash1 were conceived to play complementary roles in enteric neuronal development. Ret was thought to be required for the superior cervical ganglion and the portion of the ENS originating from vagal and sacral crest–derived cells, whereas Mash1 was believed to be necessary for the remainder of the sympathoadrenal system and the truncal crest–derived cells that colonize the esophagus (65). In fact, Mash1 turned out to be required for the development and survival of many neurons in the vagaland sacral crest–derived ENS (70). These Mash1-dependent cells have in common an early birthday (178) and the transient expression of a catecholaminergic phenotype (70). All of the serotonergic neurons and many nitric oxide synthase (NOS)–expressing neurons are examples of Mash1-dependent cells (68,70,93). In contrast, enteric dopaminergic neurons (179) and neurons that express calcitonin gene–related peptide (CGRP) (70) are late-born neurons that are derived from precursors that neither express Mash1 nor are transiently catecholaminergic. Two sublineages of enteric neurons can be distinguished by the properties of Mash1 dependence versus independence, origin from transiently catecholaminergic versus noncatecholaminergic precursors, and early versus late birthdays. The crest-derived cells that colonize the bowel probably arrive as a heterogeneous mixture of committed and uncommitted cells. The population as a whole is pluripotent, but individual cells within the population may not be. The observation that progenitors that ultimately become serotonergic and cholinergic neurons undergo their terminal mitosis (birthday) as early as E8.5 in mice suggests that the birth of some enteric neurons precedes their entry into the bowel (178). No crest-derived cells enter a primordial gut as early as E8.5 (see earlier). In contrast, many, even of the transiently catecholaminergic cells in the Mash1-dependent/early-born neuronal lineage, divide in the bowel (180). It is counterintuitive, and thus surprising, that the recently discovered dopaminergic neurons of the ENS, which are catecholaminergic cells, should develop from the set of precursors that are not themselves catecholaminergic; nevertheless, two waves of expression of tyrosine hydroxylase have been detected in the mouse and rat gut (179). The early one, which appears in the mouse foregut by E11 and disappears by E14, is absent in Mash1 null mice (70) and is a proliferating population of precursors (91,92,180). These transiently catecholaminergic cells lose tyrosine hydroxylase when they differentiate and express their final phenotype (serotonergic, NOS expressing, etc.). The second wave

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 509 appears perinatally and does not lose tyrosine hydroxylase but does retain it in the terminally differentiated dopaminergic neurons of the mature bowel (179). Most of these neurons are located in the submucosal plexus, and they develop normally in Mash1 null mice, which totally lack the serotonergic neurons that arise from transiently catecholaminergic precursors (70). Dopaminergic neurons, such as those that contain CGRP (the last type of enteric neuron to be born), are thus Mash1 independent.

Neuropoietic Cytokines Neuropoietic cytokines seem to play a role in both the neuronal and glial differentiation of enteric crest–derived cells (169). The identity of the neuropoietic cytokines that act in the mammalian ENS have not yet been established, but they appear to use the CNTF receptor complex. The complex is composed of three molecular components, only one of which, CNTFRα, actually binds CNTF (181). The two other receptor subunits, gp130 and leukemia inhibitory factor-β receptor (LIFRβ), are signal-transducing molecules that eventually activate the Jak tyrosine kinases (182). The latter two components also participate in mediating responses to other cytokines, such as interleukin-6 (IL-6) and LIF. The tripartite CNTFR is expressed in the fetal bowel (183), and the transducing subunits gp130 and LIFRβ are expressed both by noncrest and by crest-derived cells. Only crest-derived cells, however, express CNTFRα, the immunoreactivity of which is located in myenteric ganglia in the E16 to E18 fetal rat gut (169). Crest-derived cells thus express all of the receptor components to enable them to respond to CNTF, IL-6, and LIF. Both CNTF and LIF have been shown to promote enteric neuronal development in a dose-dependent manner, although neither CNTF nor LIF affects the proliferation of crestderived precursor cells (169). CNTF also has been demonstrated to increase the development of glia (169). CNTF and NT-3 seem to affect different subpopulations of neurons because simultaneous treatment with NT-3 and CNTF exerts additive effects; however, there may be some overlap in actions, because CNTF has been shown to promote the development of a NT-3–dependent subset of NOS-expressing neurons (169). CNTF, unlike NT-3, only seems to be required for the differentiation of neuronal precursors and not for their survival (169). It is unlikely that the critical enteric neuropoietic cytokine is either CNTF or LIF, because the knockout of either one does not affect ENS development. A role in the ENS of the recently discovered neuropoietic cytokine ligand for CNTFRα, cardiotrophin-like cytokine (184), is possible but has yet to be investigated.

Bone Morphogenetic Proteins Signaling by BMP-2 and -4 is critical in the early epithelial–mesenchymal interactions that occur during the morphogenesis of the bowel, and it is also important in the regionalization of the gut (185,186). The roles played by

the BMPs, however, do not end in early fetal life; they continue to be important in the mature bowel, where BMP expression occurs in the lamina propria and BMP signaling functions in the formation of intestinal crypts (187). BMP signaling also plays a role in the development of colonic epithelial cells (188). Abnormal BMP signaling in the colonic mucosa has been associated with the development of precancerous juvenile polyposis (187) and with the generation of adenomas of the colon (188). Later actions of BMP-2 and -4 help to regulate ENS development (96,163). BMP-2 and -4 (but not BMP-7), as well as the BMP receptors (BMPRs) IA, IB, and II, are expressed in the developing ENS (96). BMP-2 and -4 promote neuronal differentiation when applied to crest-derived cells isolated from the fetal gut with antibodies to p75NTR. BMPderived neurogenesis displays a bell-shaped concentration– effect relationship that suggests that it is necessary to control the concentrations of BMPs in the immediate microenvironments within narrow limits. Too little BMP would be ineffective, whereas too much BMP would be counterproductive and inhibitory. BMP-4 has been reported to increase the expression of Hand2 in the avian gut (127). Because of the evident importance of Hand2 in neuronal development (see earlier), it is possible that the ability of the BMPs to promote neuronal differentiation is mediated by a BMPinduced up-regulation of Hand2. The simultaneous expression in the fetal bowel of BMP antagonists (noggin, gremlin, follistatin, chordin), together with the BMPs and their receptors, supports the idea that the effective BMP concentration in the bowel is indeed controlled within strict limits (96). Moreover, the presence of the antagonists would seem to ensure that the actions of BMPs would have to be local. The antagonists would probably form inactivating complexes with secreted BMPs before they could move far from the cells that secrete them. The BMPs can act alone when applied in vitro, but they also interact with GDNF/GFR-α1/Ret, to enhance neuronal differentiation (96); however, by inducing differentiation of neurons, BMPs limit the extent of the GDNF-driven proliferation of crest-derived enteric neuronal precursors. As a result, BMPs will deplete the stem cell pool and, as noted earlier, limit the ultimate number of neurons generated within the developing ENS. GDNF-driven expansion has been postulated to be the major determinant of the number of neurons in the ENS (85); however, the effects of BDNF are trumped by BMPs and, indeed, the late down-regulation of BMP activity is associated with the development of hyperganglionosis, showing that BMPs are a physiologically important determinant of the number of neurons in the ENS (96). The BMPs do more than promote acquisition of a neuronal phenotype; they also specify the development of particular lineages of enteric neurons. One lineage that is promoted by BMPs is NT-3 dependent, and thus expresses TrkC (96). The physiologic significance of BMP signaling in ENS development was investigated in transgenic mice that overexpress the BMP antagonist, noggin, directed to the developing nervous system by the promoter for neuron-specific enolase

510 / CHAPTER 18 (NSE-noggin mice). It is impossible to study the effects of the knockout of BMPs on late events in ontogeny, because these molecules exert critical early actions on the morphogenesis of the bowel. Because NSE is not expressed by the early precursors of enteric neurons, but only by cells committed to the neuronal lineage (189), the overexpression of noggin in NSE-noggin transgenic mice is delayed until neurons begin to differentiate. No effects of the overexpression of noggin on gut morphogenesis thus occur in NSEnoggin transgenic mice. Enteric neurons are born over a long period that begins, as noted earlier, before the earliest émigrés enter the bowel and neurons continue to be born for at least the first 3 weeks of postnatal life (178). Because noggin expression is directed by the NSE promoter to the ENS, the NSE-noggin transgenic mice provide a means of evaluating the effects of endogenous BMP secretion on ENS development. Although experiments with NSE-noggin transgenic mice demonstrate that BMPs limit the ultimate size of the ENS (96), these experiments also indicate that BMPs physiologically enhance the development of the NT-3–dependent/TrkC-expressing subset of neurons (that gives rise to submucosal IPANs). BMPs also act to promote the development of enteric smooth muscle, which, like crest-derived precursors, expresses BMPRs IA, IB, and II.

Serotonin Serotonin is an important growth factor during development. It has been demonstrated, for example, to play a critical role in the determination of the right-left axis in both frog and chick embryos (190). Experiments with a panel of inhibitors suggest that this action is mediated by 5-HT3 and 5-HT4 receptors. Serotonin also appears to be an enteric growth factor. Serotonergic and cholinergic neurons are the first neurons to be born in the developing ENS (178). Cholinergic neurons, however, are born throughout a wide window of fetal time, whereas serotonergic neurons are generated within a much narrower period. The greater width of the window during which cholinergic neurons are born is undoubtedly because cholinergic neurons are a much more heterogeneous group of neurons than serotonergic cells; the multiple types of cholinergic neurons can be distinguished by the wide variety of molecules they coexpress with cholinergic markers (191–196). Serotonergic neurons, in contrast, represent a far more homogeneous set of neurons, and thus the population displays relatively uniform properties. Mucosal enterochromaffin (EC) cells contain the largest store of serotonin in the mammalian body (197,198). EC cells develop after serotonergic neurons have been generated (at E16 in the mouse), but neurons continue to be born after EC cells arise (199). The activity-dependent secretion of serotonin by early-developing serotonergic neurons or EC cells, or both, is thus in a position to affect the development and survival of subsets of neurons that develop after they do. Three serotonin receptor subtypes are expressed in the fetal ENS, 5-HT2A, 5-HT2B, and 5-HT4 (164,200–202).

Of these, the 5-HT2B receptor appears to be most important with regard to ENS development (164). Serotonin and (±)-2,5-dimethoxy-4-iodoamphetamine HCl (DOI), both of which stimulate all of the receptors of the 5-HT2 family, promote the in vitro development of enteric neurons in cultures of immunoselected crest-derived cells isolated from the E14 mouse gut. Methysergide (an antagonist at 5-HT1 and 5-HT2 receptors), the pan-5-HT2 antagonist ritanserin, and a 5-HT2B/2C–selective antagonist, SB206553, all block the effects of serotonin and DOI. In contrast, ketanserin, a 5-HT2A–selective antagonist, does not inhibit the developmental effects of 5-HT, suggesting that 5-HT2A receptors are not responsible for mediating the promotion of neuronal development by 5-HT or DOI. 5-HT provokes the nuclear translocation of MAP kinase, which, like the effect of 5-HT on neuronal development, is antagonized by ritanserin. Transduction of 5-HT2B–mediated effects on neuronal development therefore may involve MAP kinase. The effect of serotonin on neurogenesis must be a direct effect on precursor cells and not a secondary action mediated by nonneuronal cells in the wall of the bowel, because serotonergic agonists are able to promote neurogenesis and survival in cultures of isolated crest-derived precursors (164). Transcripts encoding the 5-HT2B receptor have been detected in the fetal gut; transcripts encoding the 5-HT2C receptor are rare. Because the 5-HT2C receptor is poorly expressed in the bowel, and the pharmacology is wrong for the 5-HT2A receptor, 5-HT–promoted enteric neurogenesis is almost certainly 5-HT2B mediated. In support of physiologic relevance of this effect, transcripts encoding the 5-HT2B receptor and receptor immunoreactivity are both abundant in developing (E15–E16) enteric ganglia; however, in the mature ENS, 5-HT2B receptors are present only in about a fourth of the ganglia. The immunoreactivity of the neuronal marker, protein gene product (PGP)9.5, is coexpressed in all 5-HT2B–immunoreactive cells of the fetal bowel; therefore, these cells are neurons or committed precursors of neurons. The coexpression in the fetal bowel of a developmentally regulated receptor, 5-HT2B, with serotonin makes it possible for serotonin to act as an ENS growth factor that promotes neuronal development through 5-HT2B stimulation. That serotonin is a neurotransmitter of an early-born set of neurons means that serotonergic neurons or EC cells can alter the fates of enteric neurons by the activity-dependent release of serotonin. Because neuronal activity and that of EC cells varies depending on the stimuli presented to the bowel, it is conceivable that serotonergic activity elicited by early experiences of the gut help to determine the final composition or nature of the ENS. Aganglionosis would not be a result of such a mechanism; in contrast, the limitation of serotoninmediated effects to the subset of precursors that expresses 5-HT2B receptors might well cause the subtle type of defect that one might expect to find in irritable bowel syndrome (IBS) or another “functional” intestinal dysmotility. Cardiac abnormalities occur in mice that lack 5-HT2B receptors (203). The ENS of these animals is deficient in serotonergic neurons (Fiorica-Howells and Gershon, personal

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 511 observations, 2005). In contrast with 5-HT2B null mice, the bowel of 5-HT2A null animals is normal at birth; however, enteric smooth muscle degenerates as HT2A null mice age (200). Enterocytes of 5-HT2A null animals are also smaller than normal, and Paneth cells in the small intestine are reduced in number. Because the 5-HT4 receptor is also expressed early in development and exerts developmental actions in frog and chick embryos (190), the possibility that 5-HT4–mediated effects occur during ENS ontogeny should be investigated. This is particularly true because of the effective therapeutic use being made of the 5-HT4 agonist, tegaserod, in the treatment of patients with constipation-predominant IBS and patients with chronic constipation (204–207). Although tegaserod is safe (208), it might be used by pregnant women, both because pregnancy is associated with constipation and because IBS is a female-predominant condition (209–211).

Associated Pathology of Late-Acting Factors Growth and transcription factors are required to act in a regular temporal sequence for enteric neuronal development to proceed normally. The extent of the defect that results from deficits in the functions of one or more of these factors depends not only on the magnitude of the resultant loss of function, but on the position of the abnormality in the temporal sequence in which the factors act. As discussed earlier, a mutation in a factor, such as GDNF or the receptors it stimulates, GFR-α1 and Ret, will obliterate all of lineages of cells that develop from the Ret-dependent precursors. Because these lineages encompass all of the neurons and glia of the bowel below the rostral foregut, Ret mutations can cause aganglionosis. One might wonder why, in this context, in the face of RET mutations in human subjects, only the terminal colon is aganglionic and not the entire bowel, as it is in mice that lack Ret (71). Unlike the Ret mutation in mice, which lack tyrosine kinase activity, RET-associated mutations in human subjects do not totally abolish the function of the receptor; they are partial loss-of-function mutations (140,142,212). Changes in the severity of mutations in Ret, alter the degree to which the bowel becomes aganglionic (98,213). Sex-dependent enhancers also play a role because they influence the degree to which RET is expressed (128). Other genes that modify the activity of RET are also important; thus, a defect in EDNRB can potentiate a hypomorphic RET (98,141). Ret, furthermore, profoundly affects proliferation of enteric crest–derived precursors early in development (83,85); therefore, the RET-deficient terminal bowel probably does not become colonized, because the migrating precursor pools of vagal and sacral crest–derived cells have not been sufficiently expanded in the proximal bowel and pelvic mesenchyme, respectively. The pools of vagal and sacral crest–derived cells thus each become depleted before either of them fills the colon. In contrast with aganglionosis, subtle abnormalities in the ENS, which result from deficiencies in NT-3/TrkC or other late-acting factors, occur only when the development of relatively small subsets of neurons

are affected. These mutations are unable to produce an aganglionosis. Most lineages of enteric neurons are TrkC independent; therefore, a mutation that results in the loss of function of NT-3 will not affect most enteric neurons, and thus the entire bowel will be ganglionated. Such a mutation might still be lethal, however, because, as in the case of NT-3/TrkC, the set of neurons that is affected is essential. The NT-3–dependent set of neurons includes submucosal IPANs that are required for propulsive and secretory reflexes (46). One might wonder whether a severe pediatric dysmotility, such as chronic intestinal pseudoobstruction, could be caused by a defect of this type. The bowel would be reported by a pathologist to contain an ENS, and a diagnosis of Hirschsprung’s disease would be rejected; nevertheless, a severely abnormal ENS could be masked by the presence of visible ganglia. Unfortunately, individual neuronal phenotypes traditionally have not been examined by pathologists, and thus constitute a major unknown. Similarly, a mutation that disturbs the function of a regulatory interneuron might cause a sublethal disturbance of motility, such as occurs in IBS or other functional disorders. The putative presence of defects in the ENS, in which anatomical and biochemical changes are neither obvious nor incompatible with life, has simply not yet been investigated.

OTHER FACTORS IN ENTERIC NERVOUS SYSTEM DEVELOPMENT Normal ENS development requires more than correct development of the enteric neurons themselves; it also necessitates that migration within the bowel be accomplished flawlessly, and that neurons connect with one anther to give rise to the complex microcircuits that characterize enteric ganglia and enable the bowel to operate independently of CNS input. In addition to GDNF, which has been discussed earlier, factors that help to guide the migration of crest-derived cells include laminin (214,215), slit proteins (216), and netrins (217). Adhesive interactions between β-neurexins and neuroligins, which play important roles in CNS synaptogenesis, may also be involved in synapse formation in the ENS (218).

Laminin and Proteoglycans Laminin is a favorable substrate for the adherence of crest-derived cells (219,220); moreover, the laminin-1 isoform also promotes the migration of cells away from the neural crest itself (221,222). In contrast, a monoclonal antibody called INO that blocks the effects of a laminin-heparan sulfate proteoglycan complex interferes with the migration of crest-derived cells in vitro (223,224), suggesting that crest cell migration is promoted by laminin in a complex with heparan sulfate proteoglycan. Antibodies to integrins that block attachment of crest-derived cells to laminin also inhibit cranial crest cell migration in vivo (225,226). Effects opposite

512 / CHAPTER 18 to those of laminin and heparan sulfate proteoglycans are exerted by chondroitin sulfate-rich proteoglycans such as aggrecan, versican, and a proteoglycan extracted from the truncal region of chick embryos (227–229). These molecules repel crest-derived cells and establish zones into which they will not migrate. Laminin-1 has three subunits, or peptide chains (α1β1γ1) (230). Laminin-1 exerts effects on the ENS that go beyond its abilities to act as an adhesion molecule and to promote the migration of crest-derived cells. Laminin-1 induces the differentiation of enteric crest–derived cells into neurons; this effect has been linked to the laminin-1 α1 chain (231). Promotion of neurogenesis by laminin-1 depends on an interaction of its α1 subunit with LBP110, a β-amyloid precursor protein (β-APP) that is expressed by migrating crest-derived cells in the gut (232). LBP110 interacts with a neurite extension-promoting region of α1 that has the sequence IKVAV (233–235). Different laminin isoforms are expressed in the gut at different times during development; however, early in development, laminin-1 is the primary isoform that is expressed (236–238). Laminin immunoreactivity in the fetal gut moreover is originally diffusely spread through the extracellular matrix, although it eventually becomes highly concentrated in basal laminae (239). Laminin-1–secreting cells have been located in the E12 fetal mouse intestine by demonstrating transcripts encoding the β1 subunit of laminin-1 by in situ hybridization (D’Autréaux, and Gershon, personal communication, 2005). Laminin β1–expressing cells are concentrated under the endoderm, in a distribution that is identical to that of cells containing mRNA encoding Ednrb (107). This coincident distribution of laminin-expressing mesenchymal cells with Ednrb is interesting in light of the evidence discussed earlier that the Ednrb ligand, Edn3, inhibits the expression of laminin-1, presumably by acting on the Ednrb-expressing smooth muscle cell precursors of the colon (73); therefore, laminin-1 accumulates in the presumptive aganglionic bowel of Edn3-deficient edn3ls mutant mice (239). These mice have a mutation in edn3 that prevents conversion of the inactive big Edn3 (132), which is secreted, to the active small peptide by ECE-1 and -2 (240,241). Only the small peptide, Edn3, is able to stimulate Ednrb (131); therefore, Ednrb signaling is disrupted in edn3ls mutant mice, which produce only big Edn3, and also by targeted mutations in ECE-1 (76). Moreover, the accumulation of laminin-1 is not caused by the absence of crest-derived cells from the presumptive aganglionic bowel because laminin-1 does not accumulate in the equally aganglionic gut of Ret null mice (73). When the colon of an edn3ls mutant mouse is explanted after E16, neurogenic crest–derived cells are abundant in the proximal region and migrate out of the bowel onto a surrounding collagen substrate (138). The crest-derived cells, however, do not migrate distally within the explants, which thus develop as an aganglionic “Hirschsprung’s-like” bowel in vitro. The addition of exogenous Edn3 changes the behavior of the crestderived cells in the proximal colon, which then enter what

had appeared to be a forbidden zone and migrate as far as, and even out of, the anal end of the explants. The ability of exogenous Edn3 to rescue the presumptive aganglionic gut in vitro strongly implies that the local stimulation of Ednrb in the colon is important in enabling crest-derived cells to colonize the bowel. The observation also implies that the absence of Edn3 in the small intestine and the failure of Ednrb stimulation to interact with Ret do not irreversibly impair the capacity of enteric crest–derived cells to complete their colonization of the colon (98,103,157). They do so when Edn3 is provided in vitro to bowel explanted after GDNF/GFRa1/Ret no longer drives their proliferation (83) and a deficiency in crest-derived stem cells migrating beyond the cecum cannot be rectified. It is also curious that Edn3, which inhibits the migration of enteric crest–derived cells (98), should, when applied to an Edn3-deficient gut in vitro, enable these cells to migrate into the terminal bowel (138). The rescue of the terminal colon by exogenous Edn3 in vitro is consistent with the hypothesis that Edn3 acts both on crest-derived cells and on the noncrest-derived cells of the colon into which they migrate to permit further migration to occur. The phenomenon could be explained by an Edn3-mediated postponement of neuronal differentiation and prevention of the excess secretion of laminin-1. Laminin is, as noted earlier, a favorable substrate for the migration of crest-derived cells, either alone or in a complex of laminin with heparan sulfate proteoglycan. The crestderived cells in the gut, however, are not identical to their premigratory ancestors in the neural crest. A soon as they start to migrate away from the crest and after their arrival in the gut, crest-derived cells find themselves in a series of changing microenvironments, which provide powerful developmental signals. In addition, as time goes by, crest-derived cells follow intrinsic programs that also change their properties. Vagal and sacral crest–derived cells, for example, do not normally give rise to neurons until they reach the gut, and because only precursors migrate (72), it is necessary that the formation of neurons be postponed to allow the crest-derived cell population to populate the gut. In the bowel, however, migration of crest-derived cells has to cease eventually for ganglia to develop at all levels and in the correct locations. Crest-derived cells may find the gut and migrate proximodistally in response to GDNF gradients (81,82,88,89,156,242), but they also have to turn perpendicularly from their trajectory in the outer gut mesenchyme to give rise to the submucosal plexus (159,243,244) and to migrate out of the gut to form the ganglia of the pancreas (245). These effects involve additional guidance and possibly also stop signals that involve laminin-1–mediated signaling.

Netrins Netrins are distant, but N-terminally homologous, relatives of laminin that attract or repel axons, respectively, toward or away midline structures (246–250). More recently, netrins

DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM / 513 have also been demonstrated to attract a variety of cells (251,252), promote neuronal survival (253,254), regulate vascularization (255,256), and act as adhesion molecules in basal laminae (250,257). Netrins bind to several molecules that act as receptors that mediate its actions, including deleted in colorectal cancer (DCC) (246,254,258,259), neogenin (260–263), the A2b adenosine receptor (264–266; but see also Stein and colleagues [259)]), and integrins (267,268). Netrins and DCC are expressed in the developing gut and pancreas of mouse and chick (217). DCC expression, but not that of netrins, is regulated developmentally and peaks at the time when crest-derived cells are colonizing the bowel. Netrins are expressed in the gut by the endodermal epithelium, and they become concentrated in the mucosal basal lamina; acinar cells express netrins in the pancreas. Enteric crest–derived cells migrate toward sources of netrin in vitro, and this directed migration is inhibited both by antibodies to DCC and by reagents that inhibit PKA. DCC signaling is affected by cyclic adenosine monophosphate and PKA (269,270). Crest-derived cells will also leave explants of gut in vitro to migrate toward cocultured explants of pancreas (217,245). Again, antibodies to DCC or PKA inhibitors block the gut-to-pancreas migration of enteric crest–derived cells (217). Mice that lack DCC also lack a submucosal plexus and pancreatic ganglia. Netrins, therefore, appear to be necessary for the perpendicular migrations of crest-derived cells to form the submucosal and pancreatic plexuses of ganglia, and the critical netrin receptor for both of these functions is DCC. The role of netrins/DCC in taking over the guidance of enteric crest–derived émigrés from GDNF/GFR-α1/Ret is clearly not experienced by all crest-derived cells, but only those that migrate into the submucosa and pancreas. Netrin/ DCC-mediated guidance is therefore a later and less global effect than that of GDNF/GFR-α1/Ret, which appears to influence the entire crest-derived cell population of the bowel. Because netrins are expressed in the mucosa, the question arises as to why crest-derived cells stop migrating in the submucosa and never enter the mucosa itself. It is interesting that the region in which these cells cease to migrate and in which they undergo neurogenesis is rich in laminin-1– expressing cells (see earlier) and secreted laminin, which in the developing gut is initially diffuse and only later restricted to basal laminae. Thus, there is a laminin gradient that radiates centrifugally from the mucosa that is in position to intercept the crest-derived cells as they approach the mucosa. By promoting neurogenesis in the submucosa, this subepithelial laminin-1 could stop the migrating crest–derived cells short of the mucosa itself. This putative role of laminin-1 is analogous to that proposed earlier for crest-derived cells approaching the presumptive aganglionic gut of mice deficient in Edn3 (and presumably that of human patients with Hirschsprung’s disease caused by a defect in EDNRB, EDN3, or ECE-1). Laminin has an additional action that would add to that of neurogenesis that would prevent crest-derived cells following a netrin gradient from reaching the mucosa.

Laminin converts the attraction mediated by the netrin-DCC interaction to repulsion (271). There is no evidence that netrins contribute to the pathogenesis of Hirschsprung’s disease; however, the submucosal plexus is much more affected by a defect in Edn3 signaling than the myenteric, and thus the length of bowel that lacks a submucosal plexus is longer than that in which the myenteric plexus is absent both in ednls mice (159) and in patients with Hirschsprung’s disease (272). There is evidence that netrins function in the innervation of the bowel by extrinsic vagal sensory axons (273). Growth cones of these axons express DCC in the bowel and grow selectively toward sources of netrins. As in the case of enteric crest–derived cells, this selective attraction is inhibited by antibodies to DCC. Similarly, axons grow from explants of nodose ganglia toward cocultured explants of gut; this directional growth is also inhibited by antibodies to DCC. Slit proteins and semaphorins are additional molecules that have been reported to influence the colonization of the gut by crest-derived cells. Slit proteins may oppose the colonization of the bowel by inappropriate crest-derived cells from the trunk (216). Slit proteins act on Robo receptors to exert effects that are usually repellant (274). Interestingly, Slit proteins also act on Robo receptors to silence the chemoattractive effects of netrins on DCC (275). Semaphorin D has been reported to act in chick embryos to prevent sacral crest–derived cells destined for the ganglion of Remak from entering the colorectum (276). β-Neurexin and Neuroligin Because the ENS contains interneurons and microcircuits, synaptic connections within the ENS are more abundant and complex than in other peripheral ganglia. For these synapses to be functional, a tight control must be exercised over their number, location, and function. Synaptic development involves an initial formation of appositions that are remodeled into defined synapses, which can then manifest plasticity, or they can be eliminated (277–279). Synapses are intrinsically asymmetric. Different scaffolds, with proteins that play roles in the assembly and maturations of synapses, line the cytosolic surfaces of presynaptic and postsynaptic membranes (280–282). Before forming synapses, axons grow to the vicinity of their targets in response to signals provided by short- or long-range guidance molecules, or both. On reaching the correct regions, cell-surface adhesion and signaling systems become active to coordinate the formation and assembly of presynaptic and postsynaptic membrane specializations. Interactions that are specific for particular synapses and general synaptogenic mechanisms, which are common to all or most synapses, appear to act synergistically during synapse formation (278). Matching cadherins or immunoglobulin-domain proteins, which interact in a homophilic manner, have been reported to promote specific axon-target synaptogenic events (283,284). These homotypic

514 / CHAPTER 18 cell adhesion molecules probably play critical roles in synaptic recognition and adhesion; nevertheless, the actions of heterophilic molecules are probably responsible for generating the asymmetry of synapses. In the CNS, neuroligins and neurexins have been demonstrated to function as components of a heterophilic mechanism of synapse formation that is common to many, if not all, synapses. The neuroligin and the neurexin families of proteins are both distributed widely in the brain. Neurexins are transmembrane molecules, which comprise an extremely large family. The α-neurexins are receptors for α-latrotoxin (black widow spider venom) (285). The β-neurexins include a subset that lacks an insertion in splice site 4, which binds to the neuroligins. Neuroligins are a family of transmembrane postsynaptic proteins (286,287) with sequence homology to cholinesterases (288). Despite their family resemblance to cholinesterases, neuroligins play roles in protein–protein interactions but lack enzyme activity. Neurexins are encoded by three genes in mice; these genes are each transcribed from two alternative promoters to give rise to six primary transcripts. Alternative splicing then generates more than 103 α- and β-neurexin isoforms from these primary transcripts (289). Neuroligins are encoded by three genes in rodents and by five genes in humans. Neuroligin-1 is located in the postsynaptic membranes of excitatory CNS synapses. The interaction between neuroligins and β-neurexins mediates adhesion at synapses (290,291) and is sufficient to induce presynaptic differentiation (218). Neuroligins and β-neurexins also interact with components of the transduction/effector machinery involved in synapse formation, which are cytosolic scaffolding proteins (292–295). Studies have just begun to be performed on the expression of β-neurexins and neuroligins in the bowel. Consequently, little or nothing is known about how synapses form in the gut. However, because there are many similarities between the CNS and the ENS, it is reasonable to propose that a general mechanism that functions in the formation of CNS synapses may also participate in the formation of ENS synapses. Making this hypothesis reasonable have been observations that the splice variants of β-neurexins that interact with neuroligins are expressed in the fetal rat gut, as also are each of the three rodent neuroligins (F. D’Autréaux, T. R. Gershon, and M. D. Gershon, personal observations, 2005). It is interesting that genes encoding neuroligins are mutated in patients with autism, and that the autism-associated mutations lead to a loss of function in the molecules (296–299). Children with an autism spectrum disorder often also have a coexistent functional or inflammatory gastrointestinal disease (300–306). It is thus conceivable that a neuroligin mutation that disturbs synapse formation in the CNS does so also in the ENS, and thereby contributes both to the pathogenesis of autism and the coexistent gastrointestinal disturbance. Because motility is a major defense of the bowel against infection, it is likely that a problem with enteric synaptogenesis could increase the likelihood of infection, even by commensal organisms, and lead to both a dysmotility and at least a low grade of inflammatory bowel disease. Such a problem could

underlie the condition that has been called a new variant of IBD or “autistic colitis” (305).

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant NS15547. E.R. is a St. Jude Children’s Research Hospital Fellow of the Pediatric Scientist Development Program (NICHD Grant Award K12-HD00850).

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CHAPTER

19

Cellular Physiology of Gastrointestinal Smooth Muscle Gabriel M. Makhlouf and Karnam S. Murthy Signal Transduction in Smooth Muscle of the Gut, 524 Signaling Pathways for Contraction, 524 Receptors and Endogenous Ligands, 524 G Proteins, Effector Enzymes, and Second Messengers Mediating the Initial Phase of Muscle Contraction, 524 G Proteins, Effector Enzymes, and Second Messengers Mediating the Sustained Phase of Muscle Contraction, 525 Desensitization of Receptor Function, 526 Signaling Pathways for Relaxation, 527 Receptors and Endogenous Ligands, 527 G Proteins, Effector Enzymes, and Second Messengers, 528

Regulation of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate: A Coordinated Interplay of Cyclases, Phosphodiesterases, and Protein Kinases, 528 Relaxation of the Initial Ca2+-Dependent Phase of Muscle Contraction, 529 Relaxation of the Sustained Ca2+-Independent Phase of Muscle Contraction, 529 Cross-Regulation: The Interplay of Signals in a Chemical Playground, 530 Acknowledgments, 531 References, 531

Gastrointestinal smooth muscle exhibits variable tone, on which are superimposed rhythmic contractions driven by electrical slow waves. The latter are cycles of membrane depolarization and repolarization that originate in a network of pacemaker cells, the interstitial cells of Cajal, located at the boundaries and within the substance of the circular muscle layer. Ca2+ influx during the depolarization phase of each slow wave triggers a transient contraction. Release of excitatory neurotransmitters, chiefly acetylcholine (ACh), causes further depolarization and Ca2+ influx and activates signaling cascades that result in Ca2+ release and greater contraction (1–3). Ion channel activities and the genesis and propagation of slow waves are discussed in greater detail in Chapter 20.

This chapter focuses on the signal transduction pathways that mediate tonic contraction and relaxation (i.e., inhibition of contraction) of gastric and intestinal smooth muscle by enteric neurotransmitters, hormones, and humoral substances. Signal transduction pathways in the esophageal body are different and have been reviewed extensively elsewhere (4). Phosphorylation of the 20-kDa regulatory myosin light chain (MLC20) is a prerequisite of smooth muscle contraction, and is thus the clearest quantitative biochemical index of contractile activity (1,5). The conformational change in the myosin head induced by MLC20 phosphorylation greatly enhances the ability of actin to activate myosin-Mg2+/ATPase and induce hydrolysis of adenosine triphosphate (ATP) bound to the myosin head. The interaction of actin and myosin results in generation of force and muscle contraction. A major advance during the last decade has been a clearer understanding of the pathways that lead to MLC20 phosphorylation. The extent of MLC20 phosphorylation reflects the balance between the activities of MLC kinase (MLCK) and MLC phosphatase (MLCP). MLC20 phosphorylation during the initial transient contraction induced by an agonist is mediated by Ca2+/calmodulin-dependent

G. M. Makhlouf: Department of Medicine, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298. K. S. Murthy: Departments of Medicine and Physiology, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

523

524 / CHAPTER 19 MLCK, whereas MLC20 phosphorylation during the subsequent sustained contraction is mediated by Ca2+-independent MLCK and inhibition of MLCP. Dephosphorylation of MLC20 via cyclic adenosine monophosphate (cAMP)– and cyclic guanosine monophosphate (cGMP)–dependent protein kinases (protein kinase A [PKA] and PKG) brings about relaxation of contracted muscle (1). The pathways that lead to these distinctive outcomes are discussed later in greater detail.

SIGNAL TRANSDUCTION IN SMOOTH MUSCLE OF THE GUT Signal transduction involves sequential activation of three membrane proteins consisting of a membrane-spanning receptor and a heterotrimeric G protein that couples the receptor to one or more effector enzymes; the latter act on membranebound or cytoplasmic precursors to generate regulatory signals that initiate cascades of protein phosphorylation and dephosphorylation. The signal is attenuated rapidly or terminated by mechanisms that target receptors (via phosphorylation by G protein–coupled receptor kinases [GRKs]), and G proteins (via enhancement of GTPase activity by regulators of G-protein signaling [RGS]) (6–8). Feedback phosphorylation by downstream kinases (e.g., PKC or PKA) also can participate in attenuating the signal by targeting specific receptors, G proteins, GRKs, and RGS proteins. The signaling pathways are determined by the repertoire of receptors, G proteins, GRKs, RGS proteins, and effector enzymes expressed in smooth muscle cells of the gut. Accordingly, the signaling pathways can resemble or markedly differ from those in other cell types.

SIGNALING PATHWAYS FOR CONTRACTION Receptors and Endogenous Ligands Table 19-1 provides a list of receptors, G proteins, effector enzymes, and intracellular messengers that mediate contraction of smooth muscle cells of the gut. Receptor tyrosine kinases (e.g., insulin-like growth factor type I and transforming growth factor-β receptors) that regulate smooth muscle growth and cytokine receptors (e.g., interleukin-1β and tumor necrosis factor-α receptors) that mediate inflammatory response are not included in this list, even though they can engage pathways that mediate contraction. Ligands for G protein–coupled receptors are variously derived from excitatory enteric nerves, smooth muscle cells, adventitious cells, or via the circulation (1–3). Ligands derived from smooth muscles are immediately available to cell-surface receptors. They include sphingosine 1-phosphate (S1P); lysophosphatidic acid (LPA); the endocannabinoids, anandamide and 2-arachidonyl glycerol; and various products of arachidonic acid and purine/pyrimidine metabolism (9,10). Other ligands, such as 5-HT, are present mainly in mucosal enterochromaffin cells and a few interneurons,

and they are not immediately available to activate 5-HT2 and 5-HT4 receptors expressed in smooth muscle cells. The receptors listed in Table 19-1 have been identified variously by reverse transcriptase-polymerase chain reaction, Northern blotting, Western blotting, or radioligand binding in freshly isolated or cultured gastric and intestinal smooth muscle cells. On reaching confluence, cultured gastric and intestinal smooth muscle cells resume their phenotype and express a full complement of receptors and other signaling molecules. Radioligand binding in enriched membranes derived from muscle tissue containing nerve fibers and other cell types cannot yield definitive evidence of receptor expression on smooth muscle cells. The receptors are expressed in cells from the circular and longitudinal muscle layers, except for sstr3, opioid µ, δ, and κ, and Y2 and Y4 receptors, which are absent from cells in the longitudinal muscle layer (11–13).

G Proteins, Effector Enzymes, and Second Messengers Mediating the Initial Phase of Muscle Contraction Receptors that mediate initial MLC20 phosphorylation and contraction of circular muscle are coupled to Gq, Gi, or both. Gq-coupled receptors activate phospholipase C-β1 (PLC-β1) via Gαq, whereas Gi-coupled receptors activate PLC-β3 via Gβγi (11–19). Both PLC-β1 and PLC-β3 preferentially hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to yield diacylglycerol, a membrane-bound activator of PKC, and inositol-1,4,5-trisphosphate (IP3), a diffusible messenger that interacts with sarcoplasmic IP3 receptors/Ca2+ channels to stimulate Ca2+ release and increase cytosolic Ca2+ ([Ca2+]i). Ca2+ binds to calmodulin and the complex activates MLCK, resulting in transient phosphorylation of MLC20 (Fig. 19-1). The Ca2+ transient is rapidly dissipated by reuptake of Ca2+ into the sarcoplasmic reticulum via a Ca2+-ATPase pump and by extrusion from the cells via plasmalemmal Na+-Ca2+ exchange or a Ca2+-ATPase pump. MLCK activity decreases coincidentally with the decrease in [Ca2+]i, and its decline is hastened by phosphorylation via Ca2+/calmodulin-dependent protein kinase II (CaMK II) or p21-activated kinase (PAK), or both (see Fig. 19-1) (17,20). Initial MLC20 phosphorylation and contraction mediated by Gq-coupled receptors in circular muscle are blocked by inhibitors of phosphoinositide (PI) hydrolysis (U73122), by Gαq or PLC-β1 antibodies, and by expression of Gαq minigene, whereas MLC20 phosphorylation and contraction mediated by Gi-coupled receptors are blocked by U73122, Gβ or PLC-β3 antibodies, and by expression of Gαi minigene (21). Downstream in the pathway, MLC20 phosphorylation and contraction are blocked by IP3 receptor inhibitors (heparin), calmodulin antagonists (calmidazolium), and MLCK inhibitors (ML-9) and by depletion of sarcoplasmic Ca2+ stores (21,22). Ca2+ release in intestinal longitudinal muscle exhibits an obligatory dependence on an initial step involving Ca2+ influx, and unlike Ca2+ release in circular muscle, it is abolished in Ca2+-free medium or by Ca2+ channel blockers. PIP2 hydrolysis

CELLULAR PHYSIOLOGY OF GASTROINTESTINAL SMOOTH MUSCLE / 525 TABLE 19-1. Ligands, receptors, G proteins, effector enzymes, and second messengers mediating the initial and sustained phases of muscle contraction Ligand

Receptor

G protein

Effector

Messenger

ACh

m3

CCK

CCK-A

Motilin

Motilin-R

Endothelin

ETA

5-HT NPY/PYY PP Histamine SP/NKA ATP/UTP ATP/UTP

ETB 5-HT2C Y2 Y4 H1 NK1/NK2 P2Y2 P2Y2

S1P

S1P2

S1P

S1P2/S1P1

Somatostatin

sstr3

Opioids

δ, µ, κ

Adenosine

A1

Gαq Gα13 Gαq Gα13 Gαq Gαq /Gα13 Gαq Gα13 Gαq Gαq Gαq Gαq Gαq Gαq Gαq Gβγi3 Gαi3 Gαq Gαq /Gα13 Gβγi Gαi Gβγi1 Gαi1 Gβγi2 Gαi2 Gβγi3 Gαi3

PLC-β1 F RhoA F PLC-β1 F RhoA F PLC-β1 F RhoA F PLC-β1 F RhoA F PLC-β1 F PLC-β1 F PLC-β1 F PLC-β1 F PLC-β1 F PLC-β1 F PLC-β1 F PLC-β3 F AC d PLC-β1 F RhoA F PLC-β3 F AC d PLC-β3 F AC d PLC-β3 F AC d PLC-β3 F AC d

IP3/DAG F ROK/DAG IP3/DAG F ROK/DAG IP3/DAG F ROK/DAG IP3/DAG F ROK/DAG IP3/DAG F IP3/DAG F IP3/DAG F IP3/DAG F IP3/DAG F IP3/DAG F IP3/DAG F IP3/DAG F cAMP d IP3/DAG F ROK/DAG IP3/DAG F cAMP ↓ IP3/DAG F cAMP d IP3/DAG F cAMP d IP3/DAG F cAMP d

F F F F

F

All receptors except for sstr3; opioids µ, δ, and κ; and Y2 and Y4 are expressed in circular and longitudinal muscle cells. Sustained contraction induced by somatostatin, opioid, and adenosine receptors is mediated by integrin-linked kinase (not shown). Also not shown are leukotriene receptors LTC4 and LTD4/LTE4, which are coexpressed in circular smooth muscle and are coupled to the Gq/inositol-1,4,5-trisphosphate (IP3)/diacylglycerol (DAG) pathway. AC, adenylyl cyclase; ACh, acetylcholine; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CCK, cholecystokinin; LTC, leukotriene receptor C4; NKA, neurokinin A; NK1, neurokinin receptor 1; NPY, neuropeptide Y; PLC, phospholipase C; PP, pancreatic polypeptide; PYY, peptide YY; ROK, Rho kinase; S1P, sphingosine 1-phosphate; SP, substance P; UTP, uredine triphosphate.

and IP3 formation are minimal in longitudinal muscle, and the sarcoplasmic Ca2+ channels are insensitive to IP3 or IP3 inhibitors, but they can be activated by nanomolar concentrations of Ca2+, ryanodine, or cyclic adenosine diphosphate ribose (23,24). The messenger that triggers Ca2+ influx in longitudinal muscle cells is arachidonic acid, a product of phosphatidylcholine hydrolysis by cytosolic phospholipase A2 (cPLA2). An initial transient in arachidonic acid formation, observed only in longitudinal muscle, triggers Ca2+ influx, leading to Ca2+-induced Ca2+ release via sarcoplasmic ryanodine receptors/Ca2+ channels. Agonist-induced cPLA2 activity is abolished by GDPβS and appears to be mediated by Gi. Downstream of Ca2+ release, the pathways in longitudinal muscle cells are identical to those in circular muscle cells.

G Proteins, Effector Enzymes, and Second Messengers Mediating the Sustained Phase of Muscle Contraction The same receptors that mediate initial MLC20 phosphorylation and contraction also mediate sustained MLC20 phosphorylation and contraction, but they engage a distinct G protein–dependent pathway (see Fig. 19-1). Receptors coupled

to Gq are also coupled to G13, and they initiate a pathway involving sequential activation of Gα13 (or both Gαq and Gα13) and RhoA. RhoA activates Rho kinase and phospholipase D (PLD). Hydrolysis of phosphatidylcholine by PLD yields phosphatidic acid, which is dephosphorylated to diacylglycerol, leading to sustained activation of various PKC isozymes (see Fig. 19-1). Rho kinase and PKC (usually PKC-ε) act to inhibit MLCP via distinct yet cooperative mechanisms. PKC phosphorylates CPI-17, a 17-kDa endogenous inhibitor of MLCP, greatly increasing its ability to inhibit MLCP (17). Rho kinase phosphorylates MYPT1, a 130-kDa myosin phosphatase targeting subunit that inhibits the catalytic activity of MLCP (25–29). Sustained MLC 20 phosphorylation and contraction are blocked by expression of Gα13 minigene, or both Gα13 and Gαq minigenes, by Clostridium Botulinum C3 exoenzyme, which inactivates RhoA, by expression of dominant-negative RhoA (RhoA[T19N]), and by inhibitors of Rho kinase (Y27632) and PKC (bisindolylmaleimide), but not by inhibitors of PI hydrolysis, calmodulin, or MLCK (17). The pathway outlined above has been corroborated for sustained MLC20 phosphorylation and contraction mediated

526 / CHAPTER 19 m2

m3

Gβγi3

IP3

m3

Gαq

Gβγi3

Gα13

+

+

+

+ PLC-β3

m2

Rac/Cdc42

PLC-β1

RhoA-GTP ROK

PLD

MYPT1-p

PA

PAK _

DAG

Ca2+/CaM

PI 3-K

MLC20-p

+

MLCK _

CaMK II

MLC20

_ MLC-Pase _ CPI-17-p

DAG PKC-ε

FIG. 19-1. Signaling pathways initiated by muscarinic m3 and m2 receptors in circular gastric and intestinal smooth muscle cells. Initial and sustained contractions are mediated by m3 receptors only. m2 receptors activate a parallel pathway that inactivates myosin light-chain kinase (MLCK) via p21-activated kinase 1 (PAK1). Sustained contraction is initiated by Gα13-dependent activation of RhoA, which triggers two pathways involving Rho kinase and protein kinase C (PKC; usually PKC-ε) that result in inhibition of MLC phosphatase activity. Some receptors, for example, sphingosine 1-phosphate (S1P2) and motilin receptors, activate RhoA via both Gαq and Gα13 (see Table 19-1). Ca2+ mobilization in longitudinal muscle differs from that in circular muscle (see G Proteins, Effector Enzymes, and Second Messengers Mediating the Initial Phase of Muscle Contraction). Otherwise, the signaling pathways mediating initial and sustained contractions in circular and longitudinal muscle are similar. CaMK II; calmodulin-dependent protein kinase II; DAG, diacylglycerol; GTP, guanosine triphosphate; IP3, inositol-1,4,5-trisphosphate; MLC20, 20-kDa regulatory myosin light chain; PI3K, phosphatidylinositol-3 kinase; PLC, phospholipase C; PLD, phospholipase D.

by m3, motilin, and S1P2 receptors. Endothelin type A (ETA) receptors engage G13 and RhoA and activate both RhoA and PLD/PKC, but appear to act only via MYPT1. CPI-17 is actively dephosphorylated by protein phosphatase 2A (PP2A) via a pathway involving sequential stimulation of p38 MAP kinase and PP2A by ETB receptors (Fig. 19-2) (19). Conversely, LPA3 receptors engage G12 and RhoA and activate Rho kinase and PKC, but appear to act only via CPI-17. The reason for the absence of Rho kinase–mediated MYPT1 phosphorylation has not been determined. Unlike Gq/G13-coupled receptors, Gi-coupled receptors initiate distinct signaling cascades involving sequential activation of Gβγi and phosphatidylinositol-3 kinase (PI3K). The latter can engage several pathways including a PIP3/ Akt/integrin-linked kinase (ILK) pathway, a c-Src kinase pathway, and a Cdc42/Rac1 pathway that leads to activation of PAK1 and p38 MAP kinase (see Fig. 19-1). Only the ILK pathway mediates sustained contraction and MLC20 phosphorylation by some Gi-coupled receptors (Gi1-coupled sstr3 receptors; Gi2-coupled opioid receptors; and Gi3-coupled A1 receptors). ILK causes both direct phosphorylation of MLC20 and indirect phosphorylation via CPI-17–mediated inhibition of MLCP (30–32). Three Gi-coupled receptors (muscarinic m2, cannabinoid CB1, and Y1 receptors) do not elicit contraction. Gi3-coupled m2 receptors initiate a pathway involving sequential activation of Gβγi3, PI3K, Cdc42 and Rac1, PAK1, and p38 MAP kinase; this pathway acts to inactivate MLCK (see Fig. 19-1)

and dephosphorylate CPI-17, thereby blocking initial and sustained MLC20 phosphorylation and contraction. CB1 and Y1 receptors are coupled to an atypical G protein comprising Gαi2 and Gβ5, with the latter bound to the Gγ-like domain of RGS6 (33). On activation of CB1 or Y1 receptors, Gαi2 dissociates from the Gβ5/RGS6 complex, which, unlike typical Gβγi, is incapable of signaling to PLC-β3 or PI3K.

Desensitization of Receptor Function Attenuation of receptor function (i.e., desensitization) is regulated by receptor phosphorylation via GRKs or second messenger–activated kinases, such as PKA and PKC (7,8,34). Homologous desensitization of agonist-occupied receptors is initiated by GRK-mediated phosphorylation of the receptors and binding to β-arrestin, which uncouples the receptors from the G protein, as a prelude to internalization (endocytosis), degradation, or recycling. β-Arrestin binds to clathrin-coated pits and acts as a scaffold for other proteins, including the terminal components of the MAP kinase system and the cytosolic tyrosine kinase c-Src; the latter stimulates tyrosine phosphorylation of the large GTPase, dynamin, and promotes its ability to cleave the clathrin-coated vesicles from the cell membrane (7,8). The vesicles fuse with endosomes where the receptors are dephosphorylated and either slowly or rapidly recycled to the surface.

CELLULAR PHYSIOLOGY OF GASTROINTESTINAL SMOOTH MUSCLE / 527 ETA

ETB

RhoA

p38 MAPK

Rho kinase

PLD

p-MYPT1 −

PKC

A second mechanism involves G-protein phosphorylation by PKC but is limited to Gαi1 and Gαi2, and requires activation of PKC via a separate agonist. Thus, concurrent stimulation of PKC by ACh inhibits the response to [Met]enkephalin, which is mediated by Gi2-coupled δ opioid receptors (35). The third mechanism is observed on successive stimulation with two different agonists that activate the same G protein (e.g., ACh and substance P). The G protein (i.e., Gαq) activated by the first agonist is sequestered by binding to caveolin-3 (36). The binding is transient, attains a peak within 10 minutes, and lasts about 30 minutes. During this period, stimulation with an agonist that activates the same G protein results in an attenuated response. Caveolinbinding motifs have been identified that bind G proteins to the cytoplasmic scaffolding domain of caveolin (37).

PP2A

MLCP

− p-CPI-17

+ CPI-17

p-MLC20 Sustained contraction

FIG. 19-2. Signaling by endothelin type A (ETA) and ETB receptors. Both ETA and ETB stimulate a Ca2+-dependent initial contraction in circular and longitudinal muscle similar to that depicted for m3 receptors in Figure 19-1. Sustained contraction in circular and longitudinal muscle cells is mediated via ETA receptors only and involves inhibition of myosin light-chain phosphatase (MLCP) via the MYPT1 pathway. The expected phosphorylation of CPI-17 via protein kinase C (PKC) is blocked via a pathway involving sequential activation of p38 mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) by ETB receptors. MLC20, 20-kDa regulatory myosin light chain; PLD, phospholipase D.

Heterologous desensitization can occur at receptor or G-protein level. Three mechanisms have been identified that target G proteins and limit their ability to transduce signals: inactivation of Gα subunits via RGS, selective phosphorylation of Gαi1/2, and transient sequestration of activated Gα subunits by binding to caveolin-3 (35,36). The first and most prevalent mechanism involves a family of RGS proteins that enhance the intrinsic GTPase activity of Gα.GTP and accelerate its deactivation by GTP hydrolysis. The process is more rapid (<2 minutes) than that involving receptor internalization (t1/2 = 10 minutes) and serves to attenuate response in the continued presence of an agonist, or terminate it rapidly on removal of the agonist (6).

SIGNALING PATHWAYS FOR RELAXATION Receptors and Endogenous Ligands Table 19-2 provides a list of receptors, G proteins, and effector enzymes that mediate relaxation of smooth muscle cells of the gut. The main ligands are the inhibitory neurotransmitters vasoactive intestinal peptide (VIP) and its homologues, peptide histidine isoleucine (PHI) and pituitary adenylate cyclase–activating peptide (PACAP), and nitric oxide (NO). VIP/PHI, which are synthesized by the same precursor, PACAP, and neuronal nitric oxide synthase (nNOS) are often colocalized in myenteric motor neurons that innervate mainly circular muscle. Their release is functionally linked, such that NO formed in nerve terminals regulates VIP and PACAP release; in turn, VIP and PACAP activate a distinct NOS isoform (eNOS) to generate NO in smooth muscle cells (38–41). In isolated myenteric ganglia devoid of smooth muscle, VIP release and NO production are blocked by NOS inhibitors, implying that VIP/PHI (and PACAP) release are mediated by NO. In dispersed muscle cells devoid of neural elements, VIP and PACAP stimulate NO and cGMP production. The effectiveness of NOS

TABLE 19-2. Ligands, receptors, G proteins, effector enzymes, and second messengers mediating relaxation Ligand

Receptor

G protein

Effector

Messenger

VIP/PACAP VIP/PACAP/ANP 5-HT Adenosine Histamine NO

VPAC2 NPR-C 5-HT4 A2b H2 sGC

Gαs Gαi/2 Gαs Gαs Gαs None

AC F eNOS/NO F AC F AC F AC F None

cAMP F cGMP F cAMP F cAMP F cAMP F cGMP F

Cyclic guanosine monophosphate (GMP) activates protein kinase G (PKG) only. Cyclic adenosine monophosphate (cAMP), which is produced in greater amounts (10- to 15-fold), activates PKA, and at higher concentrations can cross-activate PKG. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase–activating peptide (PACAP) stimulate both cAMP and cGMP: in the presence of cGMP, cAMP activates both PKA and PKG. PKA and PKG activate specific phosphodiesterases (PDEs) and inhibit adenylyl cyclase (AC) and soluble guanylyl cyclase (sGC), respectively, and are thus the main feedback regulators of cAMP and cGMP levels. Guinea pig tenia coli, which are devoid of endothelial nitric oxide synthase (eNOS), express PACAP-specific and VIP-specific receptors; only the latter activate AC. ANP, atrial natriuretic peptide; NO, nitric oxide; NPR-C, natriuretic peptide clearance receptor; VPAC2, VIP receptor 2.

528 / CHAPTER 19 inhibitors in blocking relaxation in innervated muscle tissue reflects their ability to inhibit NOS activity in myenteric motor neurons and smooth muscle cells, as well as their ability to inhibit NO-dependent VIP and PACAP release from the myenteric neurons. VIP/PACAP have been shown to stimulate NO production in muscle cells from various regions of the gut in all species examined, including human, dog, opossum, rabbit, guinea pig, rat, and mouse; the only exception is guinea pig tenia coli muscle cells, which are devoid of eNOS (1,38–43). Smooth muscle cells of the gut express mainly VIP receptor 2 (VPAC2) receptors, which exhibit equal affinity for both VIP and PACAP; the cells do not express VPAC1 or PACAPspecific PAC1 receptors (42,43). Guinea pig tenia coli are an exception, in that they express VIP-specific receptors that do not recognize PACAP, and PACAP-specific receptors that do not recognize VIP. The PACAP-specific receptors are probably a variant of PAC1 receptors that do not stimulate adenylyl cyclase, but that can cause relaxation by activating apamin-sensitive K+ channels (44). The VIP-specific receptor has now been cloned, and it differs from VPAC2 receptors by two adjacent phenylalanine residues in the ligand-binding NH2-terminal domain. Mutation of these two residues eliminates specificity for VIP and converts the receptor to a VPAC2 receptor. VIP and PACAP interact also with the natriuretic peptide clearance receptor (NPR-C); the latter is a singletransmembrane receptor abundantly expressed in smooth muscle cells of the gut (45). Interaction with this receptor is responsible for the ability of VIP and PACAP to stimulate NO production in smooth muscle cells (Fig. 19-3). G Proteins, Effector Enzymes, and Second Messengers VPAC2 receptors are coupled via Gs to activation of adenylyl cyclase, leading to cAMP formation and activation of PKA, whereas NPR-C receptors are coupled via Gi1/Gi2 to stimulation of Ca2+ influx and sequential activation of eNOS and soluble guanylyl cyclase, leading to cGMP formation and activation of PKG. Thus, in smooth muscle cells, VIP and PACAP stimulate concurrent formation of cAMP and cGMP and activation of PKA and PKG. A selective agonist of NPR-C, cyclic atrial natriuretic peptide (cANP4-23), stimulates NO and cGMP formation and activates PKG (45). The intracellular domain of NPR-C consists of 37-aminoacid residues and is devoid of guanylyl cyclase activity. A 17-amino-acid sequence, which selectively activates Gi1 and Gi2, has been identified in the middle region of the intracellular domain using synthetic peptides derived from various regions of the intracellular domain and by site-directed mutagenesis of the receptor in situ. The active sequence (469RRNHQEESNIGKHREL485R) is characterized by two NH2-terminal basic residues and a COOH-terminal motif BBXXB, where B and X represent basic and nonbasic residues, respectively (46) (see Fig. 19-3).

VIP/PACAP VAPC2

NPR-C

β α .GDP γ q

RRNHQEESNIGKHRELR β α .GDP i½ γ

Ca2+ Ca2+/CaM

Gαi½.GTP

Gαs.GTP

eNOS NO AC V/VI

sGC

cAMP

cGMP

PKA

PKG

Relaxation

FIG. 19-3. Signaling by vasoactive intestinal peptide (VIP) receptor 2 (VPAC2) and natriuretic peptide clearance receptors (NPR-C). In gastric and intestinal smooth muscle, VIP and pituitary adenylate cyclase–activating peptide (PACAP) activate cognate seven-transmembrane VPAC2 receptors coupled via Gs to adenylyl cyclase, as well as single-transmembrane NPR-C receptors coupled via Gi1 and Gi2 to Ca2+ influx and Ca2+/calmodulin activation of endothelial nitric oxide synthase (eNOS) expressed in smooth muscle cells. Activation of Gαi1/2 is mediated by a 17-amino-acid sequence in the middle region of the intracellular domain of NPR-C (inset). Cyclic guanosine monophosphate (cGMP) activates protein kinase G (PKG); cyclic adenosine monophosphate (cAMP) activates protein kinase A (PKA) and, in the presence of cGMP, cross-activates PKG. Both kinases are involved in relaxation of the initial and sustained phases of contraction (see G Proteins, Effector Enzymes, and Second Messengers). AC V/VI, adenylyl cyclase type V/VI; GRK, G protein–coupled receptor kinase; PDE, phosphodiesterase; sGC, soluble guanylyl cyclase.

Regulation of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate: A Coordinated Interplay of Cyclases, Phosphodiesterases, and Protein Kinases cGMP selectively activates PKG, whereas cAMP, which is formed in 10 to 15 times greater amounts than cGMP, selectively activates PKA, but at higher concentrations can cross-activate PKG (see Fig. 19-3). Autophosphorylation of PKG in the presence of cGMP greatly augments the affinity of cAMP for PKG. The ability of cAMP to cross-activate PKG is potentiated by concurrent generation of cGMP, and the more abundant cAMP becomes the main activator of both PKG and PKA (47–51). PKA and PKG are crucial feedback regulators of cAMP and cGMP levels. The kinases act upstream to induce

CELLULAR PHYSIOLOGY OF GASTROINTESTINAL SMOOTH MUSCLE / 529 −

GRK2

−

+

PDE3/4

+

VPAC2 R

NPR-C

AC V/VI

sGC

cAMP

cGMP

PKA

PKG

Relaxation of the Initial Ca2+-Dependent Phase of Muscle Contraction

−

−

+

PDE5

FIG. 19-4. Feedback regulation of receptors, cyclases, and phosphodiesterases by protein kinase A (PKA) and PKG. Both PKA and PKG act upstream to induce desensitization of vasoactive intestinal peptide (VIP) receptor 2 (VPAC2) and natriuretic peptide clearance receptors (NPR-C), inhibition of adenylyl cyclase type V/VI (AC) and soluble guanylyl cyclase (sGC), and activation of phosphodiesterases. PKA and PKG selectively phosphorylate and inhibit AC V/VI and sGC, respectively. PKG phosphorylates Thr466 in the intracellular domain of NPR-C, and PKA phosphorylates G protein–coupled receptor kinase 2 (GRK2) and enhances its ability to phosphorylate and internalize VPAC2 receptors (see Regulation of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate: A Coordinated Interplay of Cyclases, Phosphodiesterases, and Protein Kinases). cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; eNOS, endothelial nitric oxide synthase; GDP, guanosine diphosphate; GTP, guanosine triphosphate; NO, nitric oxide; PACAP, pituitary adenylate cyclase–activating peptide; PDE, phosphodiesterase.

(1) desensitization of VPAC2 and NPR-C receptors, (2) inhibition of adenylyl cyclase type V/VI and soluble guanylyl cyclase, and (3) activation of cAMP-specific phosphodiesterase 3A (PDE3A) and PDE4 and cGMP-specific PDE5 (52–54) (Fig. 19-4). PDE3A and PDE4 are activated (i.e., phosphorylated) by PKA only. PDE5 is activated by PKG, and in the presence of cGMP, by PKA also. In addition, cGMP inhibits PDE3A activity. Thus, in the presence of cAMP and cGMP, cGMP levels are decreased via PKG- and PKAdependent activation of PDE5, and cAMP levels are decreased via PKA-dependent activation of PDE3A and PDE4; the presence of cGMP attenuates the effects of PDE3A on cAMP levels (52–57). Further synthesis of cAMP is blocked by PKA-dependent phosphorylation of adenylyl cyclase, and further synthesis of cGMP is blocked by PKG-dependent phosphorylation of soluble guanylyl cyclase. Thus, synthesis of cAMP and cGMP by cyclases and their degradation by phosphodiesterases are coordinated by PKA and PKG. Cyclic AMP and cGMP are further attenuated on receptor internalization, a process that is also regulated by PKA and PKG. Phosphorylation of GRK2 by PKA (but not by PKG) augments the ability of GRK2 to phosphorylate VPAC2 receptors and induce internalization and desensitization (54). NPR-C phosphorylation and desensitization are not dependent on GRK2, but they can be induced via PKG-dependent phosphorylation of Thr466 in the intracellular domain of NPR-C, raising the possibility that feedback phosphorylation by PKG attenuates signaling by NPR-C.

In addition to regulation of upstream targets (i.e., receptors, GRKs, cyclases, and phosphodiesterases), PKA and PKG act on several downstream targets in the pathways that mediate initial and sustained muscle contraction, resulting in dephosphorylation of MLC20 and relaxation of contracted smooth muscle. These targets are depicted schematically in Figure 19-5. Relaxation of the initial Ca2+-dependent phase of contraction in circular smooth muscle by PKA and PKG correlates with the decrease in [Ca2+]i, which can be achieved by decreasing IP3 generation or IP3-dependent Ca2+ release, or both. Both PKA and PKG inhibit Gαq-dependent PLC-β1 activity, yet neither Gαq nor PLC-β1 is directly phosphorylated by PKA or PKG. Both kinases, however, phosphorylate RGS4 and GRK2, which contains an RGS-like domain, thereby enhancing their ability to accelerate deactivation of Gαq and to decrease activation of PLC-β1 and generation of IP3 (58). In addition, PKG, but not PKA, induces phosphorylation of type I IP3 receptors expressed in smooth muscle cells of the gut, resulting in inhibition of Ca2+ release (59,60). Two PKG isoforms, PKG-Iα and PKG-Iβ, are expressed in gastrointestinal smooth muscle, but only PKG-Iα is capable of phosphorylating type I IP3 receptors. Expression of dominant-negative PKG-Iα, but not PKG-Iβ, inhibits IP3-dependent Ca2+ release. That inhibition of IP3 generation is proximal to inhibition of IP3-dependent Ca2+ release suggests that it may be the dominant process in demobilization of Ca2+ in circular smooth muscle. Relaxation of the initial phase of contraction in longitudinal smooth muscle also correlates with the decrease in [Ca2+]i, but it is achieved by a different mechanism. Both PKG and PKA phosphorylate cPLA2 and inhibit its activity, suppressing the formation of arachidonic acid and inhibiting Ca2+ influx and Ca2+-induced Ca2+ release (61). It is probable that PKG also phosphorylates ryanodine receptors/Ca2+ channels and inhibits Ca2+ release. As in circular muscle, inhibition of the proximal and essential step in Ca2+ mobilization (i.e., arachidonic acid formation) may be the dominant process in demobilization of Ca2+ in longitudinal smooth muscle. Finally, PKG, but not PKA, can phosphorylate and inactivate the sarcoendoplasmic Ca2+/ATPase responsible for reuptake of Ca2+ into intracellular stores in both circular and longitudinal muscle. A sustained effect of PKG on this target would likely attenuate Ca2+-dependent signaling (62). Relaxation of the Sustained Ca2+-Independent Phase of Muscle Contraction PKA, PKG, or both can inhibit sustained MLC20 phosphorylation and contraction by targeting one or more steps in the RhoA pathway, including G13, RhoA, Rho kinase, PKC, MYPT1, and CPI-17. There is evidence that PKA can phosphorylate Thr203 of Gα13 in human platelets, but this has

530 / CHAPTER 19 Initial phase

Sustained phase Agonist

Agonist GTP.Gqαβγ

GTP.Gqα

Gα13

RGS4 +

PLC-β1 IP3 IP3RI

−

PKA/PKG

Rho kinase − +

DAG − SR

+

p-MLC

SERCA MLCK

RhoA

MLCP (cat.sub) MLC

−

[Ca2+]i

PLD

MYPT1

DAG

CPI-17

PKC

Relaxation

FIG. 19-5. Downstream targets of protein kinase A (PKA) and PKG. Both PKA and PKG phosphorylate regulators of G-protein signaling (RGS4), enhancing deactivation of Gαq and inhibiting inositol-1,4, 5-trisphosphate (IP3) formation. Only PKG can phosphorylate IP3 receptors, and thus inhibit Ca2+ release, and phosphorylate the sarcoendoplasmic Ca2+ATPase (SERCA) pump and augment Ca2+ reuptake into Ca2+ stores. These mechanisms act cooperatively to reduce [Ca2+]i and inhibit initial contraction and 20-kDa regulatory myosin light chain (MLC20) phosphorylation. In addition, both PKA and PKG phosphorylate RhoA, thereby blocking inhibition of myosin light-chain phosphatase (MLCP) via the PKC/CPI-17 and the Rho kinase/MYPT1 pathways. Both kinases also directly phosphorylate MYPT1 and block its ability to inhibit MLC phosphatase. These mechanisms act cooperatively to inhibit sustained MLC20 phosphorylation and contraction. DAG, diacylglycerol; GDP, guanosine diphosphate; GTP, guanosine triphosphate; IP3, inositol-1,4,5-trisphosphate; MLCK, myosin light-chain kinase; PLC-β1; phospholipase C-β1; PLD, phospholipase D.

not been examined in smooth muscle (63,64). There is, however, strong evidence that both PKA and PKG can phosphorylate activated, membrane-bound RhoA at Ser188. Phosphorylation of this residue in RhoA stimulates the translocation of RhoA back to the cytosol and inhibits its activity, causing a decrease in Rho kinase and PLD activities (see Figs. 19-1 and 19-5) (65,66). Inhibition of Rho kinase blocks phosphorylation of MYPT1 at Thr696, whereas inhibition of PLD decreases diacylglycerol formation and activation of PKC and blocks CPI-17 phosphorylation at Thr38. Blockade of the Rho kinase/MYPT1 and the PKC/ CPI-17 pathways enhances MLC phosphatase activity and leads to dephosphorylation of MLC20 and inhibition of sustained muscle contraction. Studies have shown that both PKA and PKG act by phosphorylating MYPT1 at Ser695, thereby blocking phosphorylation of the adjacent Rho kinase–phosphorylated site Thr696 (67). Thus, PKA and PKG can inhibit sustained contraction by acting on several targets in the RhoA pathway, including the most proximal target, RhoA. The effectiveness of blocking targets downstream of RhoA depends on their relative sensitivity to phosphorylation by PKA or PKG. In summary, dephosphorylation of MLC20 via PKA, PKG, or both is the essential mechanism for relaxation of contracted smooth muscle. During the initial phase of contraction, relaxation is induced by PKA- and PKG-mediated inhibition of IP3 formation in circular muscle (or arachidonic acid formation in longitudinal muscle) and by PKG-mediated inhibition of IP3-induced Ca2+ release in circular muscle

(or Ca2+-induced Ca2+ release in longitudinal muscle). The decrease in [Ca2+]i in both types of smooth muscle inhibits MLCK activity and induces MLC20 dephosphorylation. During the sustained phase of contraction, PKA and PKG inhibit RhoA activity directly and block Rho kinase– mediated phosphorylation of MYPT1, leading to increase in MLC phosphatase activity and decrease in MLC20 phosphorylation.

CROSS-REGULATION: THE INTERPLAY OF SIGNALS IN A CHEMICAL PLAYGROUND Physiologic activity reflects the predominant action of one agonist as influenced by other agonists released concurrently or successively. Thus, ACh acts simultaneously on two receptors, only one of which (m3) mediates contraction; m2 receptors, acting via PI3K, enhance the initial m3-mediated contraction by inhibiting RGS4 and attenuate the sustained contraction by activating RGS16. Successive release of an excitatory and inhibitory neurotransmitter (ACh and VIP) attenuates the relaxant effect of the latter (VIP): by stimulating PKC, ACh induces phosphorylation of Gαi1 and Gαi2, and blocks VIP-induced activation of smooth muscle eNOS and formation of NO and cGMP (see Fig. 19-3); in addition, PKC activates PDE4, thus attenuating cAMP formation. Successive release of two excitatory neurotransmitters, such as ACh and SP, that activate the same G protein (Gq) alters the response of each, because prior stimulation by one

CELLULAR PHYSIOLOGY OF GASTROINTESTINAL SMOOTH MUSCLE / 531 agonist leads to transient sequestration of Gαq by caveolin-3 and attenuation of the response to the other agonist. Finally, a single agonist, such as adenosine, can activate two receptors: A1, which is coupled to Gi3 and mediates contraction via Gβγi, and A2b, which is coupled to Gs and mediates relaxation via PKA. The initial Ca2+-dependent contraction mediated by A1 receptors is attenuated by the inhibitory effect of PKA on Ca2+ mobilization.

ACKNOWLEDGMENTS This work was supported by grants DK15564 and DK28300 from the National Institute of Diabetes, and Digestive and Kidney Diseases of the National Institutes of Health.

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532 / CHAPTER 19 42. Teng B-Q, Murthy KS, Kuemmerle JF, Grider JR, Makhlouf GM. Selective expression of VIP2/PACAP3 receptors in rabbit and guinea pig gastric and tenia coli smooth muscle cells. Reg Pep 1998;77: 124–134. 43. Teng B-Q, Grider JR, Murthy KS. Identification of VIP-specific receptor in guinea pig tenia coli. Am J Physiol 2001;281:G718–G725. 44. Jin JG, Katsoulis S, Schmidt WE, Grider JR. Inhibitory transmission in tenia coli mediated by distinct vasoactive intestinal peptide and apaminsensitive pituitary adenylyl cyclase activating peptide. J Pharmacol Exp Ther 1994;270:433–439. 45. Murthy KS, Jin JG, Teng BQ, Makhlouf GM. G protein-dependent activation of smooth muscle eNOS mediated by the natriuretic peptide-C receptor. Am J Physiol 1998;275:C1409–C1416. 46. Zhou H, Murthy KS. Identification of the G protein-activating sequence of the single-transmembrane natriuretic peptide receptor C (NPR-C). Am J Physiol 2003;284:C1255–C1261. 47. Francis SH, Corbin JD. Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 1999;36:275–328. 48. Murthy KS, Makhlouf GM. Interaction of cA-kinase and cG-kinase in mediating relaxation of dispersed smooth muscle cell. Am J Physiol 1995;268:C171–C180. 49. Jiang H, Colbran JL, Francis SH, Corbin JD. Direct evidence for crossactivation of cGMP-dependent protein kinase by cAMP in pig coronary arteries. J Biol Chem 1992;267:1015–1019. 50. Landgraf W, Hulin R, Gobel C, Hofmann F. Phosphorylation of cGMPdependent protein kinase increases the affinity for cAMP. Eur J Biochem 1986;154:113–117. 51. Lincoln TM, Dey N, Sellak H. cGMP-dependent protein kinase signaling mechanism in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 2001;91:1421–1430. 52. Zhou H, Murthy KS. PKA-dependent phosphorylation of GRK2 augments VPAC2 receptor phosphorylation, internalization, and homologous desensitization in smooth muscle cells. Gastroenterology 2004;126:A555. 53. Murthy KS. Activation of PDE5 and inhibition of guanylyl cyclase by cGMP-dependent protein kinase in smooth muscle. Biochem J 2001;360:199–208. 54. Murthy KS, Zhou H, Makhlouf GM. Regulation of phosphodiesterase 3 (PDE3) and adenylyl cyclase by cAMP-dependent protein kinase. Am J Physiol 2002;282:C508–C517. 55. Francis SH, Turko IV, Corbin JD. Cyclic nucleotide phosphodiesterases: relating structure and function. Prog Nucleic Acid Res Mol Biol 2001; 65:1–52. 56. Turko IV, Francis SH, Corbin JD. Binding of cGMP to both allosteric sites of cGMP-binding cGMP-specific phosphodiesterase (PDE5) is required for its phosphorylation. Biochem J 1998;329:505–510.

57. Rybalkin SD, Rybalkina IG, Feil R, Hofmann F, Beavo J. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem 2002;277:3310–3317. 58. Zhou H, Murthy KS. Relative contribution of RGS4 and RGS domain of GRK2 to inhibition of PLC-β activity by PKA: direct evidence from site-directed mutagenesis of RGS4 and GRK2. Gastroenterology 2003;124:A23. 59. Murthy KS, Zhou H. Selective phosphorylation of IP3R-I in vivo by cGMP-dependent protein kinase in smooth muscle. Am J Physiol 2003;284:G221–G230. 60. Komalavilas P, Lincoln TM. Phosphorylation of the inositol 1,4, 5-trisphosphate receptor. Cyclic GMP-dependent protein kinase mediates cAMP and cGMP dependent phosphorylation in the intact rat aorta. J Biol Chem 1996;271:21933–21938. 61. Murthy KS, Makhlouf GM. Differential regulation of phospholipase A2 (PLA2)-dependent Ca2+ signaling in smooth muscle by cAMP- and cGMP-dependent protein kinases. Inhibitory phosphorylation of PLA2 by cyclic nucleotide-dependent protein kinases. J Biol Chem 1998; 273:34519–34526. 62. Cornwell TL, Pryzwansky KB, Wyatt TA, Lincoln TM. Regulation of sarcoplasmic reticulum protein phosphorylation by localized cGMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 1991;40:923–931. 63. Manganello JM, Djellas Y, Borg C, Atonakis K, Le Breton GC. Cyclic AMP-dependent phosphorylation of thromboxane A2 receptor-associated Gα13. J Biol Chem 1999;274:28003–28010. 64. Manganello JM, Huang JS, Kozasa T, Voyno-Yasenetskaya, Le Breton GC. Protein kinase A-mediated phosphorylation of the Gα13 switch I region alters the Gαβγ13-G protein-coupled receptor complex and inhibits Rho activation. J Biol Chem 2003;278:124–130. 65. Murthy KS, Zhou H, Grider JR, Makhlouf GM. Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially meditated by phosphorylation of RhoA. Am J Physiol 2003;284: G1006–G1016. 66. Sauzeau V, Ve Jeune H, Cario-Toumaniantz C, Smolenski A, Lohmann SM, Bertoglio J, Chardin P, Pacaud P, Loriand G. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoAinduced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 2000;275:21722–21729. 67. Wooldridge AA, MacDonald JA, Erodi F, Ma C, Borman MA, Hartshorne DJ, Haystead TAJ. Smooth muscle phosphatase is regulated in vivo by exclusion of phosphorylation of threonine 696 of MYPT1 by phosphorylation of serine 695 in response to cyclic nucleotides. J Biol Chem 2004;279:34495–34504.

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20

Organization and Electrophysiology of Interstitial Cells of Cajal and Smooth Muscle Cells in the Gastrointestinal Tract Kenton M. Sanders, Sang Don Koh, and Sean M. Ward Electrical Activity in Gastrointestinal Muscles, 534 Role of Interstitial Cells of Cajal in Spontaneous Electrical Rhythmicity, 535 Interstitial Cells of Cajal Serve as Pacemakers in Gastrointestinal Muscles, 535 Mechanism of Electrical Rhythmicity in Interstitial Cells of Cajal, 538 Role of Interstitial Cells of Cajal in Active Propagation of Slow Waves, 542 Unitary Potentials, 544 Role of Interstitial Cells of Cajal in Neurotransmission, 546 Interstitial Cells of Cajal Are Functionally Innervated by Enteric Motor Neurons, 546 Intramuscular Interstitial Cells of Cajal Are Required for Nitrergic and Cholinergic Motor Neurotransmission, 547 Neural Control of Slow Wave Activity, 547

Mechanism of Action of Neurotransmitters on Intramuscular Interstitial Cells of Cajal, 547 Role of Interstitial Cells of Cajal as Stretch Receptors, 548 Motility Disorders Associated with Loss of Interstitial Cells of Cajal, 549 Animal Models to Study Loss of Interstitial Cells of Cajal, 549 Smooth Muscle Responses to Slow Waves and Neural Inputs, 550 Voltage-Dependent Inward Currents, 551 Voltage-Dependent Outward Currents, 556 Chloride Conductances, 565 Nonselective Cation Channels, 567 Integration of Electrical Activity in Gastrointestinal Muscles, 569 Acknowledgments, 569 References, 569

The functional role of the gastrointestinal (GI) tract is to digest and absorb nutrients. These processes are facilitated by the orchestrated movement of the luminal contents (i.e., food, enzymatic and fluid secretions, digestion products, and waste) from the mouth to the anus. This chapter discusses elements of the motor apparatus that generates GI motility. The main elements of the motor apparatus consist of three critical cell types (enteric neurons, interstitial cells of Cajal [ICC], and smooth muscle cells). There are elegant descriptions of enteric neurobiology elsewhere in this

textbook, so this chapter concentrates on the electrical features of ICC and smooth muscles that are at the heart of phasic and tonic motor patterns. Because ICC also have a critical role in neurotransmission, this chapter also discusses the connectivity between the terminals of enteric motor neurons and ICC. Smooth muscle cells are clearly the force-producing element through most of the GI tract, but like many motor systems in the body, these cells alone are incapable of producing the coordinated movements of GI motility. The first level of motor coordination comes from intrinsic electrical activity, known as slow waves, which can propagate over many centimeters and organize contractile events. One can think of this activity like the heartbeat that generates the periodic pumping of blood. In the gut, slow waves originate within specialized “pacemaker regions” that are populated with ICC. ICC are the active element within pacemaker regions

K. M. Sanders, S. D. Koh, and S. M. Ward: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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534 / CHAPTER 20 with unique intracellular timing mechanisms and ionic conductances that generate the pacemaker currents that underlie slow waves. ICC are coupled electrically to each other and to neighboring smooth muscle cells via gap junctions. Slow waves are generated in ICC, actively propagate in ICC networks, and conduct passively to smooth muscle cells. Smooth muscle cells do not have the apparatus to generate or propagate slow waves, but these cells express a number of voltage-dependent ion channels, most importantly voltage-dependent “L-type” Ca2+ channels that respond to slow wave depolarizations. Depolarization of smooth muscle cells increases the openings of Ca2+ channels, resulting in Ca2+ entry and contraction. Oscillations of the smooth muscle cell membrane potential in response to slow waves produce periods of low and increased Ca2+-channel open probability. Thus, the contractile behavior of smooth muscle cells in regions with slow wave activity is naturally periodic, leading to motility patterns such as peristalsis and segmentation. The amplitudes of slow waves and the force of contractions in response to each slow wave are regulated to a significant extent by the enteric nervous system. Both inhibitory and excitatory neural inputs occur and can change the smooth muscle contractile response to slow waves from weak to powerful. For example, excitatory neural input can increase the amplitudes of slow waves, increase Ca2+ entry, and enhance the force of contraction. Inhibitory neural inputs, via activation of K+ channels or suppression of inward current conductances in smooth muscle cells, reduce the amplitude of slow waves and reduce contractile force. Major excitatory and inhibitory neural control is mediated through a class of ICC that is intermingled within bundles of smooth muscle cells (intramuscular ICC [ICC-IM]). ICC-IM form synaptic structures with the varicose nerve terminals of enteric neurons and gap junctions with neighboring smooth muscle cells. Other substances that condition the response of smooth muscles to slow wave depolarizations include hormones, paracrine substances, and inflammatory mediators. The site(s) of action of some of these bioactive agents may also be at ICC, but the importance of ICC as targets for these substances currently is poorly understood. Several motility disorders have now been associated with loss of ICC; thus, the study of these cells has produced exciting new hypotheses about normal and abnormal GI functions.

ELECTRICAL ACTIVITY IN GASTROINTESTINAL MUSCLES Muscles in different regions of the GI tract display properties of phasic and tonic smooth muscles (see Szurszewski [1] and Golenhofen [2]). Phasic muscles display contractions that are heavily dependent on Ca2+ entry through voltagedependent Ca2+ channels for the initiation of contractions. Tonic muscles are also dependent on voltage-dependent mechanisms, but these muscles are also supplemented by robust pharmacomechanical mechanisms. The functions of phasic and tonic muscle tissues differ in GI motility, and the

mechanisms that elicit these contractile behaviors are also different. In actuality, many GI muscles are hybrid muscles with properties of both tonic and phasic muscles. In phasic GI muscles, there is often spontaneous electrical rhythmicity, known by various names,1 but referred to here as slow waves. The waveforms of slow waves vary from region to region and in different species, but the result in each instance is to vary membrane potential between the “resting membrane potential” or most polarized level, where the open probability for voltage-dependent Ca2+ channels is low (−80 to −55 mV) to a point of depolarization where open probability increases sufficiently to cause excitation– contraction coupling (e.g., −40 to −25 mV). During the period of increased Ca2+ channel opening (usually several seconds), Ca2+ enters smooth muscle cells and activates the contractile apparatus. In some cases, the slow wave depolarization initiates a regenerative response in smooth muscle cells resulting in one or more Ca2+ action potentials.

1 Regarding terminology used in this chapter, electrical slow waves of the GI tract have been referred to by a wide variety of names in the scientific literature. Among these are pacesetter potentials, electrical control activity, slow wave–like action potential, basic electrical rhythm, and slow wave. We have favored the term slow wave that was used by Ladd Prosser and Tadao Tomita and their colleagues for many years. With modern recording techniques and the ability to label cells from which recordings have been made, slow wave activity can now be monitored in at least four cell types within the tunica muscularis: pacemaker ICC (usually ICC between the circular and longitudinal muscle layers [ICC-MY]), longitudinal and circular smooth muscle cells, and ICC-IM. The amplitude and waveforms of slow waves in each of these cells is a function of the cable properties of the tissues and the active conductances present in each cell type. Some authors have chosen a different name for slow waves in each cell type where they record this activity. For example, initiating activity in ICC-MY has been called pacemaker potentials, the activity in circular muscle cells has been termed slow waves, the activity in longitudinal muscles has been referred to as follower potentials, and the activity of ICC-IM has been called regenerative potentials (15,16,48). There are also small, spontaneous, inward currents that result in noisy fluctuations in membrane potential that have been called “unitary potentials” (48). Unitary potentials can be recorded from either ICC-MY or ICC-IM and are likely to be the basic manifestation of pacemaker mechanism in ICC. Although there may be a reason to specifically name activity represented by unitary potentials, the manifestation of slow waves in circular and longitudinal muscle cells appears an electrotonic remnant of the slow waves generated in ICC (in addition to whatever supplementation occurs via local activation of voltage-dependent currents). We favor simplifying this terminology and refer in this review to all “slow wave activity” recorded in GI smooth muscle tissues as slow waves. However, the reader should be aware that there are clear differences in waveforms of the events recorded in different regions of the tunica muscularis. We believe the term unitary potentials needs to be retained and used to describe subthreshold or noncoordinated (nonpropagated) pacemaker events. These events are extremely important because they provide a window into the basic mechanisms of electrical rhythmicity in the gut.

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 535 Ca2+ action potentials greatly increase the entry of Ca2+ and the amplitude of contractions. There is usually sufficient time between slow waves for the excess Ca2+ that entered during the previous cycle to be taken up by intracellular stores or extruded from the cell, so contractions elicited by slow waves are phasic. Thus, slow waves naturally organize the contractile behavior of muscles that possess this mechanism into phasic contractions. There are several well-defined pacemaker regions in the GI tract. In the stomach, pacemaker activity is generated in the region between the circular and longitudinal muscle layer from the corpus through the pylorus. The corpus pacemaker along the greater curvature runs at the most rapid rate, so the pacemaker cells in this region dominate the other pacemakers through the distal stomach. In the small bowel, the dominant pacemaker also resides in cells between the circular and longitudinal muscle layers. However, the rate of small intestinal pacemakers and the slow propagation velocities of slow waves make it impossible for a dominant smallintestinal pacemaker to exist. Electrical activity in this organ is organized into segmental regions of excitation. The colon has two natural pacemaker regions: one between the circular and longitudinal muscle layers, and the other at the submucosal surface of the circular muscle. In some species, such as the dog, these pacemakers generate activity at different frequencies, and the two pacemaker events summate (3).

ROLE OF INTERSTITIAL CELLS OF CAJAL IN SPONTANEOUS ELECTRICAL RHYTHMICITY Each pacemaker region contains ICC, a cell type that has long been considered, from morphologic observations, to be the pacemaker (cf. Faussone Pellegrini and colleagues [4] and Thuneberg [5]) (Fig. 20-1). The region between the circular and longitudinal muscle layers in the GI tract contains the myenteric ganglia and fiber tracts. Thus, ICC within this region have been called ICC-MY or ICC-MP. ICC in pacemaker areas along the submucosal surface of the circular muscle layers are called ICC-SM. Both of these types of cells have morphologic features that could promote pacemaker function. ICC-MY and ICC-SM have fusiform cell bodies and multiple thin projections. Both cell types are rich in mitochondria and form electrical syncytia by making gap junction connections with other ICC. Thus, ICC form meshlike networks in the pacemaker regions of the GI tract (see Fig. 20-1A). The low-resistance connections between ICC-MY and ICC-SM make it easy for electrical events in one cell to conduct to the next and facilitate propagation of electrical events through the ICC networks. ICC-MY and ICC-SM also form low-resistance connections with nearby smooth muscle cells. These connections are also likely to be caused by gap junctions (ICC and smooth muscle cells express an abundance of gap junction proteins, and small gap junctions can be found; see Fig. 20-1D), but morphologic studies often have failed to identify the small

junctions between ICC and smooth muscle cells, in contrast with the large gap junctions observed between ICC. Lowresistance electrical connections between ICC and smooth muscle cells make it possible for events to conduct between the two types of cells. However, the relatively infrequent and small gap junctions formed with smooth muscle cells may be an important morphologic feature that facilitates pacemaker activity. If ICC and smooth muscle cells were too well coupled, the electrical sink caused by the relatively vast amount of smooth muscle membrane might prevent ICC from reaching threshold voltages necessary for propagation. ICC-MY and ICC-SM also have caveolae and a distinct basal lamina. Internal structures include an abundance of mitochondria that often is packed into the perinuclear region and is spatially close to the plasma membrane. Mitochondria often are closely associated with smooth endoplasmic reticulum, which often is referred to as sarcoplasmic reticulum, because ICC and smooth muscle cells develop from the same mesenchymal precursors (see Sanders and colleagues [6]). Although ICC express a variety of thin and intermediate filaments, they express few thick filaments. Thus, it is unlikely that ICC are designed for contraction and force development. For further details, refer to the many excellent reviews describing the ultrastructure of ICC and the cells in their environment (e.g., see Thuneberg [5], FaussonePellegrini [7,8], Komuro and colleagues [9], and Rumessen and Thuneberg [10]). Interstitial Cells of Cajal Serve as Pacemakers in Gastrointestinal Muscles Interstitial Cells of Cajal Are Spontaneously Rhythmic and Generate Slow Wave–like Activity For many years, slow wave activity was thought to be an intrinsic behavior of smooth muscle cells (see Tomita [11]), but when smooth muscle cells were isolated and studied with the patch clamp technique, slow wave activity was never recorded. Furthermore, dissection experiments revealed that specific regions within the thickness of the tunica muscularis generate slow wave activity (Fig. 20-2). These regions contained networks of ICC. As described earlier, ICC form gap junctions with each other and with neighboring smooth muscle cells. Thus, slow waves generated by ICC can conduct to the smooth muscle syncytium. When ICC were isolated, these cells were found to generate spontaneous depolarization with the same characteristics as slow waves (12). Primary cultures of ICC also produce spontaneous electrical activity that is strikingly similar to slow waves recorded from intact muscle (13,14) (see Fig. 20-4). Recordings from ICC in situ also revealed that the cells were spontaneously active, and that slow waves in ICC preceded the activity in nearby smooth muscle cells (15,16). Thus, it appears that ICC possess a mechanism capable of generating slow wave activity. This mechanism is unique to ICC and is not expressed by smooth muscle cells.

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FIG. 20-1. Features of interstitial cells of Cajal (ICC) in gastrointestinal (GI) muscles. (A) The ICC between the circular and longitudinal muscle layers (ICC-MY) network in the canine gastric antrum labeled with antibodies against the receptor tyrosine kinase, Kit (19). (B) Electron micrograph of ICCMY in the canine antrum. Cells contain few contractile filaments and an abundance of smooth endoplasmic reticulum, mitochondria, and caveolae. ICC-MY form gap junctions with each other and with neighboring circular smooth muscle cells. (C) Higher power electron micrograph of the area outlined in B. A small gap junction between ICC-MY and a circular smooth muscle cell is shown (arrow). (D) Close apposition between varicose nerve terminals and intramuscular ICC (ICC-IM) in the canine antrum. (E) Higher power image of the area outlined in D reveals synaptic-like contacts with presynaptic and postsynaptic membrane densifications in the nerve varicosity and in the ICC-IM. (F, G) Close apposition between vesicular acetylcholine transporter (vAChT) and nitric oxide synthase–like immunoreactive nerves and ICC-IM in the circular muscle layer of the murine antrum. Nerve fibers preferentially target ICC-IM (51,52). (See Color Plate 9.)

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 537

FIG. 20-2. Interstitial cells of Cajal (ICC) generate pacemaker activity in gastrointestinal (GI) muscles. In the experiment depicted in the slide, slow wave activity was recorded from two regions of a strip of circular muscle from the canine colon. In the top trace, each panel was recorded from a cell close to the submucosal surface of the circular muscle layer. The second trace was recorded from a cell within the thickness of the circular muscle layer. Slow waves from these recordings were always greatest in amplitude next to the submucosal border (cf. first and second traces). After the initial recordings were made, a thin strip of muscle along the submucosal border was removed. This region continued to generate slow wave activity (producing slow waves of similar magnitude and frequency), but the activity was gone in the bulk of the circular muscle layer. Dissection experiments like this one suggest that slow waves are generated in specific pacemaker regions in GI muscles.

Slow Waves Are Lost in Animals without Interstitial Cells of Cajal in “Pacemaker” Areas A major test of the hypothesis that ICC are responsible for generation of slow wave activity came when it was possible to selectively remove ICC from intact GI muscles. This was accomplished in two ways. Once it was recognized that ICC express Kit receptors, studies were undertaken to determine the importance of Kit signaling in the development of ICC. Maeda and coworkers (17) noticed aberrant contractile behavior in animals treated with a neutralizing Kit antibody and suggested that blocking Kit might negatively impact the pacemaker apparatus. Treatment of animals with neutralizing antibody caused disappearance of ICC and disrupted slow wave activity (18). Kit signaling is also compromised in animals with loss-of-function mutations in c-kit (e.g., White-spotting [W] mutants), the gene that encodes Kit receptors. Total ablation of Kit receptor function and signaling through this pathway is fatal, and animals usually die in utero. Partial function of Kit is preserved in compound heterozygotes, such as W/WV, and these animals lose ICCMY in the small intestine and ICC-IM in the stomach and sphincters. Other populations of ICC in the stomach (ICCMY), small bowel (ICC-DMP), and colon (all classes) are preserved in W/WV mutants. ICC-MY were hypothesized to

be the pacemaker cells in the small intestine, so loss of these cells in W/WV mice created the ideal model to test the role of ICC in generating slow waves. W/WV mutants fail to generate slow waves (19,20) (Fig. 20-3). Removal of ICC caused by treatment with neutralizing antibodies also showed the critical role of these cells in slow wave activity in gastric muscles (21). There are now a variety of developmental and pathophysiologic conditions in which ICC are lost, and when the lesion is extensive, there are defects in slow wave generation or propagation, or both (see Role of Interstitial Cells of Cajal as Stretch Receptors; Table 20-1). Together, these studies strongly support the hypothesis that ICC are responsible for pacemaker activity in GI muscles. Identification of ICC in situ (with labeled Kit antibodies) has made it possible to visualize and make electrical recordings directly from these cells and verify impalements. Dickens and coworkers (15) made recordings of this type and also recorded simultaneously from smooth muscle cells. Slow waves (referred to as “pacemaker potentials” in that study) occurred in ICC before occurring in nearby smooth muscle cells. This strongly supports the hypothesis that ICCMY are the source of slow waves. Optical techniques have been applied to measure Ca2+ transients in ICC and small bundles of attached muscle. These studies also show that the

538 / CHAPTER 20 A

Control (+/+)

−28 mV −62

B W/W v

−58mV 5 sec

FIG. 20-3. Loss of interstitial cells of Cajal (ICC) and slow waves in small intestinal muscles of W/WV mice. The definitive test of the hypothesis that ICC are pacemaker cells came when it was possible to obtain animals with defective ICC networks. In the small intestine of W/WV mice, nearly all ICC between the circular and longitudinal muscle layers (ICC-MY) are missing (19,20). Recordings from wild-type (+/+) mice showed typical slow wave activity (A), but muscles of W/WV mice lacked slow wave activity (B). Note that resting membrane potential is slightly depolarized in the W/WV muscle.

activity of ICC-MY precedes activation of neighboring smooth muscle cells (22).

Mechanism of Electrical Rhythmicity in Interstitial Cells of Cajal Cultured Interstitial Cells of Cajal Retain Mechanism of Rhythmicity It is difficult to obtain and identify freshly dispersed ICC in digestions of tunica muscularis. Thus, there are few studies of pacemaker activity in freshly isolated ICC. It appears that enzymes used to digest the tissues destroy extracellular epitopes for Kit antibodies. Cells labeled with antibodies in fresh digestions of tunica muscularis are often macrophages, a common cell type in the tunica muscularis that takes up labeled antibodies. The rhythmic phenotype of ICC is retained in short-term (i.e., 2–3 days) primary cultures of ICC (13,14), and this has been an extremely useful preparation for studies of the mechanism of electrical rhythmicity (Fig. 20-4). After disassociation of cells and 2 to 3 days in culture, ICC reform networks, express Kit receptors, and generate spontaneous electrical activity. Rhythmic electrical activity, similar in waveform to slow waves recorded from ICC in situ (16,23), can be recorded from cultured ICC under current clamp conditions (13). Under ideal conditions, the currents responsible for spontaneous electrical activity can be recorded under voltage-clamp conditions. The reversal potentials of the currents and weak voltage dependence of the frequency suggest that the pacemaker currents are due to a voltage-independent, nonselective cation conductance (13). Others, using pharmacologic approaches in experiments on intact muscles or while recording from ICC in situ, have concluded that the pacemaker conductance is caused by a Ca2+-activated Cl− current (24,25). This conclusion may be subject to further consideration, however, because of the poor specificity of most Cl− channel–blocking drugs.

Currently, there are two somewhat different mechanisms for the initiation of pacemaker activity in ICC described in the literature. These mechanisms and the critical elements proposed to be involved are discussed in the next section. Spontaneous Electrical Rhythmicity of Interstitial Cells of Cajal Ca2+ release from inositol-1,4,5-trisphosphate (IP3) receptor– operated stores is the event in ICC-MY that initiates spontaneous pacemaker currents. Ca2+ release from ryanodine receptors does not appear to be necessary for pacemaker activity. Obviously, if Ca2+ release is important, then Ca2+ sequestration into IP3 receptor–operated stores is fundamental for preloading and resetting of the pacemaker mechanism. No controversy appears to exist regarding the importance of IP3 receptors in pacemaker activity. Reasons to include IP3 receptor–operated stores as an essential element are based on pharmacologic experiments on cultured ICC using blockers of IP3 receptors (26), functional genomic experiments in which the type 1 isoform of IP3 receptors was knocked out and slow waves were lost in intact muscles (27), and pharmacologic experiments on intact GI muscles (26,28). How Ca2+ released from IP3 receptor–operated stores activates inward currents is more controversial. Some investigators believe that the Ca2+ released directly activates Ca2+-activated Cl− channels in the plasma membrane. Thus, in this concept, pacemaker activity is caused by spontaneous transient inward currents that occur in many smooth muscle cells and other cells that express spontaneous Ca2+ release mechanisms (cf. 29–31). A major problem with this hypothesis is that Ca2+-activated Cl− currents have not been found in either freshly dispersed (32,33) or cultured ICC (34). One report of Ca2+-activated Cl− currents in isolated ICC has not been confirmed by others (35). In most cells with Ca2+-activated Cl− conductances, depolarization to activate Ca2+ entry via voltage-dependent Ca2+ channels results in large inward tail currents on repolarization due to Ca2+-activated

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 539 TABLE 20-1. Human gastrointestinal motor disorders with loss of interstitial cells of Cajal

Disorders Pediatric disorders Chronic intestinal pseudoobstruction

Affected region

Technique of analysis

Small intestine

Chronic idiopathic intestinal pseudoobstruction

Small intestine

Kit IH and quantitative image analysis Kit IH

Neonatal pseudoobstruction

Small intestine

Kit-IH

Hirschsprung’s diseasea

Terminal colon

Kit IH

Hirschsprung’s disease

Rectosigmoid

Kit IH

Anorectal malformations

Colon

Kit IH

Internal anal sphincter achalasia

Internal anal sphincter

Kit IH

Adult disorders Achalasia Gastroparesis Diabetic

Extent of ICC loss (if quantified) ICC-MY were absent; ICC-SM were absent Abnormal distribution ICC-MY severely reduced Absent Absent or markedly reduced ICC-MY absent or sparse in AG bowel; ICC-IM reduced Abnormal in 7/12; absent in 1; severely depleted in 5 Absent or markedly reduced

Other pathology Abdominal distention, emesis, and constipation —

Delay in the development of ICC No peripherinpositive nerve fibers —

Constipation

Kit IH

Variable from absent to nearly normalb

Idiopathic

Antrum

Kit IH

Variable from absent to nearly normalb

Idiopathic

Antrum corpus (ICC-IM and ICCMY in both regions)

Kit IH and quantitative image analysis

14% of normal 17% of normal

Type 1 diabetes

Jejunum

ICC markedly decreased

Paraneoplastic motility disorders (gastroparesis, delayed smallintestinal transit) Megacolon

Small intestine biopsy

Kit IH and quantitative image analysis Kit IH

TEM

ICC-SM were absent

Colon

Sparse or disorganized ICC networks

References

1

307

1

308

2

309

4

310

8

311, 312

12

310

8

313

7

314

Tachygastria with more severe ICC loss Tachygastria with more severe ICC loss Hypoganglionosis, neuronal dysplasia; increase gastric compliance; antral hypomotility Diabetic gastroenteropathy; decreased NO neurons Small-cell lung carcinoma

9

315

4

315

1

316

1

98

1

317

Ganglion cells and nerve trunks reduced

1

318

Reduced peripherin-positive nerve fibers

TEM Antrum

No. of patients

Continued

540 / CHAPTER 20 TABLE 20-1. Human gastrointestinal motor disorders with loss of interstitial cells of Cajal—cont’d

Disorders

Affected region

Technique of analysis

Diabetes mellitus

Colon

Kit IH

Chronic idiopathic constipation

Colon

Kit IH

Slow transit constipation

Sigmoid colon

c-kit RT-PCR; Kit WB; Kit IH

Slow transit constipation Slow transit constipation

Colon

Kit IH

Pan-colonic

Kit-IH and quantitative image analysis

Colon

Kit IH

Infectious diseases Chagas disease

Extent of ICC loss (if quantified)

Other pathology

ICC reduced by 60% ICC reduced in each segment; ICC-MY not significant 41% reduction in Kit protein; reduction in ICC-IM, ICC-MY, ICC-SMB ICC reduced in all layers but LM ICC volume reduced in all regions

Colon cancer; 4/7 constipation Constipation for mean of 14 years; reduced myenteric ganglia —

ICC reduced in CM and LM; ICC-MY reduced

Megacolon

Moderate hypoganglionosis Constipation for at least 10 yr

No. of patients

References

7

319

14

320

12

321, 322

11

323

6

324, 325

6

326

Some of the names of diseases may overlap, but diagnoses are as given by authors. aSome authors have found contradictory results with Hirschsprung’s disease cases (327,328). bNotably, when interstitial cells of Cajal (ICC) were examined in NOD mice where large areas of muscle could be viewed in whole-mount preparations (97), there were patches where ICC were largely missing and patches where ICC were nearly as dense as in normal muscles. This was described as a “patchy lesion.” In the study by Forster and colleagues (315), fullthickness biopsies were obtained, and it is possible that this technique could find regions without normal numbers of ICC and regions where ICC appeared to be quite normally distributed. CM, circular muscle; ICC-IM, intramuscular ICC; ICC-MY, ICC between the circular and longitudinal muscle layers; ICC-SM, ICC along the submucosal surface of the circular muscle layers; Kit IH, immunohistochemistry for Kit receptors; Kit WB, Western blot for Kit receptors; LM, longitudinal muscle; RT-PCR, reverse transcriptase-polymerase chain reaction; TEM, transmission electron microscopy.

Cl− current (cf. 36). ICC express voltage-dependent Ca2+ conductances, but Ca2+ entry via this pathway does not activate inward tail currents in ICC (32,34). In our experiments, we have not been able to resolve Ca2+activated inward currents in ICC, but reduction in intracellular Ca2+ results in a large, inward, nonselective cation current that does not appear to have properties of a storedepletion conductance (e.g., thapsigargin does not activate such a current) (26). We proposed that the signal that activates the pacemaker conductance is actually the falling phase in Ca2+ in the space between the IP3 receptor–operated stores and the plasma membrane. We found that activation of pacemaker currents in ICC is preceded by a Ca2+ transient in mitochondria, and pacemaker activity in cells and slow waves in tissues are inhibited by a variety of drugs known to inhibit mitochondrial Ca2+ uptake (26). These data suggest that Ca2+ release from IP3 receptor–operated stores may initiate Ca2+ uptake by mitochondria that are closely associated with sarcoplasmic reticulum (the presumed Ca2+ store) in ICC. As Ca2+ falls in the space between the plasma

membrane and the Ca2+ store/mitochondrial complex, a nonselective cation conductance in the plasma membrane is activated. We have suggested that the resulting inward current is the primary pacemaker current in ICC (see Ward and colleagues [26] and Sanders and colleagues [37]). Buffering of intracellular Ca2+ to low levels in ICC activates a single-channel conductance of about 13 pS. Clusters of similar channels were rhythmically activated at the frequency of pacemaker activity during on-cell recordings (38). Excision of these membrane patches revealed channels of about 13 pS, and the open probabilities of these channels were dramatically inhibited by increasing Ca2+ from 10−7 to 10−6 M. Calmidizolium and W-7 (calmodulin inhibitors) increased the activity of the Ca2+-inhibited channels in on-cell and off-cell conditions, suggesting that gating is controlled by Ca2+/calmodulin binding. Channels with these properties have been described, and we compared the properties of the pacemaker conductance observed in ICC with homologously expressed murine TRPC4 channels (39), which are known to be negatively regulated by Ca2+/calmodulin binding (40).

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 541

A

B

C

D

20 µm

−33

−64

E

10 sec

5 sec −28

mV

−62

F

5 sec

FIG. 20-4. Spontaneous rhythmicity in cultured interstitial cells of Cajal (ICC) and in the intact small bowel of mouse. (A, C) Phase-contrast images of cultured ICC from the murine small intestine. (B, D) Panels confirm these cells are ICC because they label with Kit antibodies. ICC grow into coupled networks of cells in culture and retain the phenotype of electrical rhythmicity. (E, F) Recordings from cultured ICC and intact muscles, respectively.

The native pacemaker conductance in ICC is similar to TRPC4 channels in many ways: (1) similar single-channel conductance; (2) inhibition by increasing Ca2+; and (3) negatively regulation by Ca2+/calmodulin binding. The pacemaker conductance is not due to TRPC4 (or at least not homologous TRPC4 channels), however, because TRPC4−/− mice (a gift from V. Flockerzi) displayed normal intestinal slow waves and spontaneous pacemaker currents in ICC (unpublished observations). Another study used a novel preparation of freshly dispersed ICC from murine intestine (33). ICC were identified by Kit immunoreactivity, and Kit-positive cells displayed spontaneous, rhythmic potential oscillations. A large inward current (referred to as an “autonomous current”), with properties of a nonselective cation current, was activated on depolarization of the Kit-positive cells under voltage-clamp.

Once activated, the time course of this current was about 500 msec and exceeded the duration of the voltage-step. The current was blocked by removal of extracellular Ca2+. Although there is much to do to determine the nature of the autonomous current, its basic properties appear to be consistent with the pacemaker conductance identified in cultured cells. In summary, the identity and nature of the primary pacemaker conductance in ICC currently is controversial. From pharmacologic studies of tissues, some authors have hypothesized that the pacemaker is caused by release of Ca2+ from stores and direct activation of Ca2+-activated Cl− currents. Others have failed to find a Ca2+-activated Cl− conductance in ICC and suggest that a nonselective cation conductance, negatively regulated by intracellular Ca2+, is responsible for the pacemaker current. The latter requires Ca2+ handling by

542 / CHAPTER 20 mitochondria that are tightly associated with sarcoplasmic reticulum in ICC. Together with the ionic conductances in the adjacent region of plasma membrane, these structures form “pacemaker units” in ICC. Further experiments, most importantly on preparations of freshly dispersed ICC, are needed to resolve this controversy. It must be recognized that networks of ICC contain thousands of spontaneously active cells. This activity must be coordinated throughout a network of ICC to produce orderly slow wave propagation. Slow waves propagate at a relatively constant velocity (from 5–40 mm/sec) within a given region of tunica muscularis. Neither of the conductances proposed as pacemaker currents are voltage dependent. Thus, the only means of synchronizing the activation of the pacemaker conductance is to coordinate Ca2+ release events. Ca2+ waves, a phenomenon seen widely in biology, are possibly a means of synchronizing Ca2+ release, but such a mechanism, which is based solely on Ca2+ diffusion, is not consistent with the fact that slow waves propagate at rates more than 2 orders of magnitude faster than Ca2+ waves. A voltage-dependent event therefore is required to explain the coordination of pacemaker events throughout networks of ICC and the rates of slow wave propagation. The mechanism of coordination and propagation is discussed in the next section.

Role of Interstitial Cells of Cajal in Active Propagation of Slow Waves Slow waves can propagate for long distances in GI organs. An example of this is the propagation of slow waves from the dominant pacemaker of the stomach in the orad corpus around the circumference of the stomach and down the axis of the stomach to the pylorus. This long-distance propagation is responsible for gastric peristalsis and coordinated gastric motility. For many years slow wave activity and the propagation of slow waves were attributed to the smooth muscle syncytium. The realization that ICC are the pacemaker cells modified this concept slightly, but most investigators believed that once initiated, smooth muscle cells could sustain propagation of slow waves. This does not appear to be the case. Dissection, genetic, and pathophysiologic studies of intact muscle strips from the GI tract have shown that when ICC are removed from a region of muscle, slow waves fail to actively propagate into that region and decay in amplitude with distance as predicted by the cable properties of the smooth muscle syncytium (Fig. 20-5). Many studies of the electrical properties of smooth muscle cells in the GI tract have shown that although smooth muscle cells express a variety of voltage-dependent conductances, these cells do not possess the apparatus required to generate slow waves (see Horowitz and colleagues [41]). Taken together, these findings suggest that slow wave activity is generated and actively propagated within ICC networks, and slow waves passively spread to neighboring smooth muscle cells (41). Direct recording of slow waves from ICC in the murine small intestine and stomach show that ICC-MY generate

pacemaker activity and this is conducted to the smooth muscle cells (15,42,43) (Fig. 20-6). The importance of ICC in propagation of slow waves has implications in pathophysiologic situations in which part of an ICC network is damaged or lost. Slow waves may be generated in regions with intact ICC networks, but fail to propagate to all regions of the organ. Loss of slow waves in portions of an organ might adversely affect its motor performance (motility patterns). As described earlier, ionic conductances thought to be responsible for pacemaker currents are not voltage dependent. Thus, a mechanism to coordinate activation of these conductances is required. If the initiating event for pacemaker activity in ICC is release of Ca2+ from IP3 receptor–operated Ca2+ stores, then it seems logical to suggest that factors that regulate or lead to the regulation of the open probability of IP3 receptors may be central to the coordination of pacemaker activity. The most obvious way to enhance the open probability of IP3 receptors is to increase local concentrations of IP3. Certainly, agonists that increase IP3, such as muscarinic stimulation, can increase slow wave frequency (44). Some investigators have suggested that voltage-dependent regulation of the phospholipase C responsible for production of IP3 in ICC or voltage-dependent sensitization of IP3 receptor– operated Ca2+ release may be responsible for coordination of pacemaker activity (24). These authors suggest that ICC may possess a voltage sensor that regulates the synthesis of IP3 or the synthesis of a second messenger that regulates the sensitivity of cells to IP3-dependent mechanisms (42,43), as occurs in megakaryocytes and some smooth muscle cells (45). Currently, voltage-dependent regulation of IP3 production or another second messenger that increases the sensitivity of phospholipase C or other pathways linked to IP3 pathways has not been demonstrated in ICC. Another means to enhance the open probability of IP3 receptors is to increase Ca2+ concentration in the submembrane space between the plasma membrane and IP3 receptor– operated stores. IP3 receptors (particularly IP3R1, which is the receptor isoform involved in slow wave generation) (27) display a bell-shaped dependence on cytoplasmic Ca2+ (46). At low IP3 concentrations (as occurs under basal conditions in most cells), a small increase in cytoplasmic Ca2+ can increase the open probability of IP3 receptors (46), and IP3 receptors display Ca2+-induced Ca2+ release. We have suggested that voltage-dependent Ca2+ entry could entrain pacemaker activity in a network of ICC and produce active propagation of slow waves (34). We envision the following sequence of events: Random activation of pacemaker currents in cells within the ICC network generates unitary potentials (see later discussion of these events). Summation of unitary potentials causes depolarization and activation of voltage-dependent Ca2+ channels. When a threshold depolarization occurs, current through the voltage-dependent Ca2+ channels is regenerative (i.e., the ensuing depolarization activates more channels). This current is responsible for the sharp upstroke phase of slow waves. Ca2+ entry through the Ca2+-selective channels increases the Ca2+ activity near IP3

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 543 −42

Slow waves recorded from electrodes A and B

AB

mV −82 A

B

10 sec

Stimulator 11 mm

Recording electrodes

Stimulating electrodes Circular muscle Longitudinal muscle

−42 Slow waves recorded from electrodes A and B

A mV B A

−82

B 5 sec

Submucosa

6 mm

Recording electrodes

Circular muscle

Longitudinal muscle

FIG. 20-5. Intact interstitial cells of Cajal (ICC) networks in the pacemaker areas are essential for active propagation of slow waves. In the experiment depicted, two cells along the submucosal surface of the circular muscle layer (near the pacemaker region) were impaled simultaneously to record slow waves. Stimulating electrodes were used to pace slow waves from one end of the strip. Slow waves of equal amplitude and at a fixed latency were recorded with both electrodes (top: superimposed traces labeled A and B from the labeled electrodes). This demonstrates nondecremental (active) propagation. Then a small strip of muscle containing the pacemaker ICC was removed from the submucosal surface of the circular muscle layer. Only a small portion of the pacemaker region was left intact. Whether slow waves were actively paced or were generated spontaneously from these remaining pacemaker regions (as shown), slow waves decreased in amplitude as a function of distance. The decay in slow waves demonstrates passive conduction limited by the cable properties of the smooth muscle syncytium.

receptors, which are located in Ca2+ store membranes tens of nanometers from the plasma membrane. Small numbers of Ca2+ ions entering the space between the plasma membrane and the Ca2+ stores may be sufficient to increase the local Ca2+ concentration to a point where the open probability of IP3 receptors is enhanced and a Ca2+ release event is initiated. Thus, Ca2+ entry synchronizes IP3 receptor– operated Ca2+ release, and this initiates the pacemaker mechanism in affected cells. Slow waves are propagated cell to cell by this mechanism, leading to a wave of excitability through a network of ICC.

It is well known that generation and propagation of slow waves is not significantly affected by dihydropyridines in most GI muscles, thus the voltage-dependent Ca2+ conductance hypothesized to be involved in propagation must be due to a class of Ca2+ channels that is relatively resistant to these drugs (i.e., the mechanism responsible for propagation does not depend on classical “L-type” or CaV1.2 Ca2+ channels). Freshly dispersed ICC from the canine colon and cultured ICC from the murine small intestine both express a dihydropyridine-resistant, voltage-dependent Ca2+ conductance that might facilitate slow wave propagation (32,34).

544 / CHAPTER 20 2

−10

1 mV

0 Interstitial cell

−70 −10

mV Smooth muscle cell −70

FIG. 20-6. Slow waves in pacemaker interstitial cells of Cajal (ICC) and coupled smooth muscle cells. Simultaneous recordings from ICC and nearby smooth muscle cells in situ have shown that events occur first in the ICC and then in the smooth muscle cell (15,16,23). The activity of the ICC between the circular and longitudinal muscle layers (ICC-MY) (top trace) is characterized by a “diastolic” period in which activation of voltage-dependent Ca2+ channels (phase 0) leads to a threshold for a regenerative response, the upstroke potential (phase 1). The upstroke potential is due to regenerative activation of voltage-dependent Ca2+ channels. The Ca2+ channels responsible for the upstroke are resistant to block by dihydropyridines, but are blocked by Ni2+ and mibefradil (16,23). Ca2+ entry into ICC during the upstroke sets off the pacemaker conductance because of initiation of IP3 receptor–operated Ca2+ release (see Fig. 20-7 for details). Inward current through the pacemaker current channels causes a sustained “plateau” potential (phase 2). At the end of this current phase, membrane potential repolarizes to the maximum negative potential (resting membrane potential) and a new cycle begins. Responses of smooth muscle cells (bottom trace) are always smaller in amplitude because the pacemaker events conduct to the smooth muscle cells and are not regenerated by smooth muscle cells. The slow wave manifests that upstroke and plateau phases that are electrotonic remnants of the activity in ICC. If the slow wave is of great enough amplitude, L-type Ca2+ channels are activated in the smooth muscle cells. These can either result in Ca2+ action potentials superimposed on the slow wave or in sustained Ca2+ entry through the duration of the plateau phase. Either phenomenon can result in sufficient Ca2+ entry to accomplish excitation–contraction coupling.

The properties and pharmacology of this conductance are distinct from CaV1.2 channels. Dihydropyridine-resistant currents are blocked by Ni2+ and mibefradil, but they are activated at rather more positive potentials than typically observed for T-type currents (i.e., CaV3.X channels). The molecular entity responsible for the dihydropyridine-resistant channels in ICC has not yet been identified. These findings suggest that the slow wave upstroke and propagation is labile to (1) reduced extracellular Ca2+, (2) blocking voltage-dependent Ca2+ entry, and (3) drugs that inhibit the dihydropyridine-resistant current in ICC. Recordings from ICC-MY in intact small-intestinal muscles have shown that reducing external Ca2+, depolarization with increased K+, and treatment with Ni2+ or mibefradil reduce the upstroke velocity, reduce slow wave frequency, and uncouple pacemaker activity (16,23). In association with the blockade of propagation, enhanced resolution of unitary potentials occurred, indicating that the pacemaker mechanism itself was intact after blocking voltage-dependent Ca2+ entry. The appearance of unitary potentials is likely to be caused by the failure of these events to be coordinated when voltage-dependent Ca2+ entry is blocked. Another study found that slow wave propagation in canine antral muscle

strips was first slowed and then blocked when the driving force for Ca2+ entry or the availability of voltage-dependent, dihydropyridine-resistant Ca2+ channels were reduced (47). These data support the hypothesis that Ca2+ entry via dihydropyridine-resistant Ca2+ channels creates the initial upstroke depolarization of slow waves and provides a means for coordination of pacemaker activity and propagation of slow waves in ICC networks. Figure 20-7 summarizes the elements and mechanisms of slow wave generation and propagation.

Unitary Potentials Small-amplitude, noisy fluctuations in membrane potential referred to as unitary potentials can be recorded from small bundles of GI muscles or from direct impalements of ICC-MY (16,48). The inward currents underlying unitary potentials are likely to be the manifestation of basic pacemaker activity in ICC. We would attribute these events to the activation of the nonselective cation conductance described earlier; however, others (e.g., see Hirst and colleagues [24]) believe these events to be caused by

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 545 The parts:

(Interstitial cell of Cajal)n

(Interstitial cell of Cajal)1

Plasma membrane

Plasma membrane

Voltage-dependent Non-selective cation calcium channel channel ICC-to-ICC coupling via gap junctions

Voltage-dependent calcium channel

Non-selective cation channel

How they work:

1. Ca2+ is released from IP3 receptors In SR; 1st site where this occurs is the primary pacemaker. 2. Ca2+ released from IP3 receptors gates Ca2+ uptake into mitochondria. 3. Uptake of Ca2+ lowers Ca2+ activity in the space near the plasma membrane. Ca2+

Ca2+-inhibited

activates 4. Reduction in non-selective cation conductance.

5. Inward (pacemaker) current causes depolarization. 6. Depolarization spreads from primary pacemaker to other ICC via gap junctions. 7. Depolarization activates voltage-dependent Ca2+ channels in secondary pacemaker cells (ICCn). 8. Ca2+ entry phase-advances release of Ca2+ from IP3 receptors and regenerates mechanism.

FIG. 20-7. Model for slow wave generation and propagation (summarizing findings from References 19, 26, 27, 34, and 38). Two model “cells” are depicted connected together by a gap junction, low-resistance pathway (Rj). Other cable parameters are membrane capacitance (Cm), membrane resistance (Rm), and internal resistance (Ri). Pacemaker generation depends on close association between inositol-1,4,5-trisphosphate (IP3) receptor–operated stores (SR), mitochondria (M), and the plasma membrane. Areas of close association, as depicted, represent “pacemaker units” or the basic cellular structures necessary to generate pacemaker activity. The model cells are represented as 2 pacemaker units, but a given interstitial cell of Cajal (ICC) may contain many of these regions. A pacemaker unit on its own generates random “unitary currents” that result in small depolarization events in ICC networks (unitary potentials). Generation of pacemaker current in the leftmost cell (ICC1) results in the primary pacemaker event in this hypothetical case, and events in the model pacemaker unit and others in the same cell (not shown) entrain to produce a slow wave. The inward current in ICC1 depolarizes adjacent cells that are “driven” as subordinate pacemakers (ICCn). The proposed mechanism for these functions is described in the figure.

activation of Ca2+-activated Cl− channels. Unitary potentials result from the stochastic discharge of pacemaker currents in individual ICC (49). Unitary potentials activate voltagedependent inward current carried by the dihydropyridineresistant, voltage-dependent Ca2+ conductance in ICC-MY (see earlier). It has been shown that either depolarization or the break from a hyperpolarizing pulse enhance the frequency of unitary potentials and cause summation of these events in the gastric antrum. Thus, activation of dihydropyridineresistant, voltage-dependent Ca2+ channels and the depolarization that results drive summation of unitary potentials from many cells in a network of ICC, creating the plateau phase of slow waves. The threshold for activation of slow waves in ICC-MY is close to the resting potentials because unitary potentials are not easily detected in recordings from ICC-MY. Unitary potentials can be recorded from ICC-MY by reducing the availability of Ca2+ channels with blocking drugs or when the driving force for Ca2+ influx is reduced (23).

Thus, when the voltage-dependent mechanism responsible for entrainment of unitary potentials is inhibited, intrinsic pacemaker activity (i.e., unitary potential discharge) persists and becomes an uncoordinated discharge of “unitary” currents that are unable to reach threshold for generation of slow waves. In earlier reviews, we developed the idea that a division of labor exists among different classes of ICC (e.g., see Sanders [50]). ICC-MY (and ICC-SM) were thought to be pacemaker cells, and ICC-IM (and ICC-DMP) were thought to be involved in neurotransmission. This idea came from the observations that intestinal muscles were not electrically rhythmic without ICC-MY (19), and responses to nitrergic and cholinergic nerve stimulation in gastric muscles were greatly attenuated without ICC-IM (51,52). Although there is still no evidence for substantive innervation of ICC-MY, several studies have now identified pacemaker activity in ICC within the circular muscle layer. For example,

546 / CHAPTER 20 unitary potential discharge also occurs in ICC-IM (or in septal ICC [ICC-SEP] that lie between muscle bundles) because muscles without these cells do not display noisy fluctuations in membrane potential (51). The reason that ICC-MY are the dominant pacemakers under basal conditions is because ICC-IM are less able to summate (entrain) unitary potentials than are ICC-MY. As discussed later (see Role of Interstitial Cells of Cajal in Neurotransmission), stimulation of ICC-IM with neurotransmitters can increase the likelihood of unitary potential summation and cause these cells to become the dominant pacemakers. Less coordination may occur in the discharge of unitary potentials in ICC-IM because these cells may not form networks with each other (51,53). For example, in the fundus, solitary ICC-IM lie within bundles of smooth muscle cells and are connected to other ICC-IM only through gap junctions formed between ICC-IM and smooth muscle cells. Thus, unitary potentials would be attenuated as they conduct through the smooth muscle cell component of the muscle bundle syncytium. ICC-IM or ICC-SEP, via spontaneous discharge of unitary potentials, provide intrinsic pacemaker activity in small bundles of antral and pyloric muscles that lack ICC-MY (48,54). ICC-IM or ICC-SEP in the antrum possess the voltage-dependent mechanism responsible for coordination of unitary potential discharge in bundles of cells, as shown by Suzuki and Hirst (55). Notably, ICC-IM in the gastric fundus, a region that does not display slow wave activity, cannot be coordinated by changes in membrane potential (56). Thus, ICC in this region do not appear to express the voltage-dependent mechanism responsible for coordination of unitary potential discharge.

ROLE OF INTERSTITIAL CELLS OF CAJAL IN NEUROTRANSMISSION Interstitial Cells of Cajal Are Functionally Innervated by Enteric Motor Neurons Most autonomic neuroscientists have viewed the functional innervation of visceral smooth muscles to occur in an en passant fashion in which the neurotransmitter is released from varicose nerve terminals that lack specific contacts with postjunctional neuroeffector cells. In such a model, the neurotransmitter is free to diffuse through the interstitium and bind to receptors expressed by any cell within the vicinity. This concept requires relatively large amounts of transmitter to be released to reach effective concentrations at receptors tens or hundreds of nanometers from the site of release. This organization would make it difficult to direct neural inputs toward specific types of cells in an organ with a heterogeneous population of cells. In early morphologic descriptions of ICC, investigators report that these cells were closely associated with neurons and neuron processes (4,5,57–62). Now it has been recognized that the association between ICC and varicose nerve terminals is extremely close (often <20 nm) and preferential

in comparison with neural contacts with smooth muscle cells (63,64). Observations of this type led morphologists to suggest that ICC may play a role in enteric motor neurotransmission. In fact, the ultrastructural relation between enteric nerve terminals and intramuscular ICC-IM is highly specialized, and synaptic-like structures, with prejunctional and postjunctional densities, are apparent at points of contact between these cells. In essence, ICC-IM are physically interposed between nerve terminals and smooth muscle cells (65–67). ICC-IM are coupled via gap junctions to smooth muscle cells. Thus, the neuromuscular junction in GI muscles contains a third element, ICC-IM, which receive and transduce neuronal signals, and then convey stimuli to the smooth muscle syncytium. Because of the close synaptic contacts between nerve terminals and ICC-IM, tiny quantities of neurotransmitter may be sufficient to attain effective concentrations at postjunctional membranes. ICC-IM (and the equivalent cells in the small intestine, ICC-DMP) express a variety of receptors for excitatory and inhibitory neurotransmission (cf. 68) and are in association with varicosities containing both excitatory and inhibitory neurotransmitters. For example, varicosities containing substance P–like immunoreactivity (an indicator of excitatory motor nerve terminals) form synaptic contacts with ICC-DMP, and ICC-DMP express neurokinin-1 receptors (NK1Rs) (cf. 69–73) and somatostatin 2A receptors (74). The receptor for nitric oxide (NO), soluble guanylate cyclase, was also found to be expressed prominently in ICC-DMP, but only weakly in neighboring smooth muscle cells (75). The enzyme activated by cyclic guanosine monophosphate (cGMP) in response to nitrergic stimulation, cGMP-dependent kinase type I, was also found abundantly in ICC-DMP, but less intensively in the circular muscle layer. Because proteins translocate, receptors are internalized, and second messengers are produced in response to receptor binding, immunohistochemical techniques can be used to determine whether ICC are innervated functionally by enteric motor neurons. For example, when small-bowel muscles were stimulated by muscarinic agonists protein kinase C (PKC), translocation was observed. Electrical field stimulation or exogenous acetylcholine (ACh) caused translocation of PKCε-LI from the cytosol to a peripheral compartment in enteric neurons and ICC-DMP (76). Exposure of guinea pig small-intestinal muscles to exogenous substance P caused receptor internalization and aggregation into endosomes in ICC-DMP (72). Stimulation of intrinsic neurons caused tetrodotoxin (TTX)-sensitive receptor internalization in ICC-DMP, but no resolvable NK1R-LI in smooth muscle cells (77). In colonic muscles, stimulation with exogenous NO caused production of cGMP in ICC and smooth muscle cells, but stimulation of intrinsic neurons caused increase of cGMP only in ICC (78). Taken together, these observations suggest that ICC are selectively innervated and responsive to transmitter released from enteric neurons. These data also suggest that different populations of cells in GI muscles are stimulated by transmitters released from intrinsic neurons than

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 547 are stimulated by transmitter substances added to solutions bathing muscles in pharmacologic, “organ bath” experiments.

Intramuscular Interstitial Cells of Cajal Are Required for Nitrergic and Cholinergic Motor Neurotransmission It was difficult for many years to test the hypothesis put forward by morphologists regarding the role of ICC in neurotransmission. This is because ICC are small cells surrounded by smooth muscle cells, and it was difficult to selectively impale these cells to study their responses to nerve stimulation. Also, ICC are coupled electrically to smooth muscle cells, so it is difficult to know whether signals recorded in ICC are caused by stimulation of these cells or by responses conducted from smooth muscle cells. A significant advance came when it was possible to selectively remove ICC from GI muscles by interfering with the Kit signaling pathway (see Torihashi and colleagues [18]). W/WV mutants showed no loss of neural responses in the small intestine; however, intestinal muscles from these animals that lacked only ICC-MY and ICC-DMP were intact (19). In the stomach, however, ICC-IM are missing. The fundus has no ICC-MY in wild-type animals, so loss of ICC-IM in W/WV mice yielded muscles without ICC (51). No defects in terms of the density of motor neurons, release of neurotransmitter, and contractile responses to exogenous transmitters were observed in fundus muscles of these W/WV mice. Thus, the W/WV fundus was an ideal model to test the role of ICC-IM in neurotransmission. In wild-type mice, fundic ICC-IM are spindle-shaped cells that run parallel to the circular and longitudinal muscle bundles (Fig. 20-8). These cells are completely missing in stomachs of W/WV mice (51). Stimulation of intrinsic nerves in these muscles showed nearly a complete loss of postjunctional responses to nitrergic (51) and cholinergic (52) neurotransmission (see Fig. 20-8). All responses to neurotransmission are not lost in W/WV stomachs; there are remaining small responses attributed to the release of purinergic or peptidergic transmitters, or both, that are coreleased with NO and ACh. There are clearly receptors for ACh and NO expressed by smooth muscle cells in GI muscles. Why would release of transmitter not diffuse to smooth muscle receptors? The answer appears to lie in the close anatomic relation between varicose nerve terminals and ICC. The small synaptic volume may tend to restrict the spread of transmitter and metabolism, and dilution may further reduce concentrations to less than effective levels before contact can be made with smooth muscle receptors. When ACh metabolism was blocked by inhibitors of acetylcholinesterase, slow postjunctional muscarinic responses were resolved in W/WV fundus muscles (52). Close, synaptic-like contacts between enteric neurons and ICC also occur in the gastric fundus and antrum (79,80), in the deep muscular plexus of the small intestine (65,67), and in sphincter regions and colon (66) of animals and humans, suggesting that involvement of ICC in neurotransmission may be a common phenomenon throughout the GI tract.

Neural Control of Slow Wave Activity It is well known that nerve stimulation can phase-advance (stimulate premature) slow waves in GI muscles. The lack of apparent functional innervation of ICC-MY seems contradictory to this phenomenon. Hirst and coworkers (24) showed that unitary potentials could be stimulated to summate in small bundles of circular muscle containing ICC-IM (but no ICC-MY) by either electrical field stimulation of enteric nerves or by membrane depolarization. Nerve-evoked summation of unitary potentials had the same properties as summations evoked by depolarization (i.e., refractoriness, latency, threshold, and similar pharmacology). Nerve-evoked summation of unitary potentials was blocked by hyoscine, demonstrating that cholinergic nerve stimulation and postjunctional muscarinic receptors mediated the responses. The most important observation, however, came from dual-electrode recordings in which one electrode recorded from an ICC-MY, whereas another recorded from a smooth muscle bundle. As described previously (see also Dickens and colleagues [15]), slow waves in ICC-MY preceded the circular muscle slow waves under basal conditions. However, when nerves were stimulated, the event recorded within the smooth muscle bundle preceded the event in the ICC-MY. During a train of stimuli, the frequency of slow waves increased, and slow waves in the smooth muscle bundle consistently preceded the ICC-MY events. These authors suggest that neural activation of ICC-IM could reverse the normal ICC-MY to smooth muscle conduction of slow waves and cause ICC-IM to become the dominant pacemaker. This hypothesis was further tested in muscles from W/WV mice that lack ICC-IM (81). Antral muscles could be paced by activation of cholinergic motor neurons, and morphologic studies demonstrate patterns of innervation of ICC-IM by cholinergic neurons. Repetitive trains of pulses to stimulate intrinsic neurons could entrain slowwave activity to greater frequencies. These responses were completely blocked by muscarinic receptor antagonists, and neural entrainment was absent in muscles of W/WV mice, directly demonstrating the role of ICC-IM in neural regulation of slow wave frequency. Sustained cholinergic stimulation, as occurs during the gastric phase of digestion, enhances the intrinsic frequency of pacemaker activity in the antrum, and ICC-IM become the dominant pacemakers in antral muscles during prolonged cholinergic stimulation (82). This aspect of cholinergic neural input is mediated by ICC-IM, because the response is lost in muscles of W/WV mice.

Mechanism of Action of Neurotransmitters on Intramuscular Interstitial Cells of Cajal Unfortunately, few studies of the mechanisms of neurotransmitter action have been performed on isolated ICC-IM. One study investigated specific expression of neural receptors in ICC-IM of the fundus (68) and showed a substantial complement of excitatory and inhibitory receptors in

548 / CHAPTER 20 W/Wv

Wildtype

50 µm

A

B

Wildtype L-NA (100µM)

Control −35

−28

L-NA & Atropine (1µM)

mV −45 mV

−42 mV

−42

−57 (EFS; 0.5 ms pulse) W/Wv Control

L-NA (100µM) −50 mV

(EFS; 0.5 ms pulse)

L-NA & Atropine (1µM) −45 mV

−45 mV 5s

FIG. 20-8. Intramuscular interstitial cells of Cajal (ICC-IM) are present in the fundus region of wild-type (control) animals (A). ICC-IM are lost in the stomachs of W/WV mice (B). Neurotransmission is compromised in the absence of ICC-IM. The first set of traces shows typical responses of wild-type muscles to electrical field stimulation that is adjusted to selectively stimulate enteric motor neurons. Typical responses to a single pulse consisted of a fast depolarization (excitatory junction potential [EJP]) followed by a slower hyperpolarization (inhibitory junction potential [IJP]). The IJP was blocked by L-nitroarginine (L-NA), a blocker of nitric oxide synthase (NOS; second trace). The EJP was blocked by atropine (third trace), suggesting it was due to release of acetylcholine and activation of muscarinic receptors. In W/WV muscles (second line of traces), which lack ICC-IM, both excitatory and inhibitory responses were greatly reduced, and L-NA and atropine had no effect. These data suggest that ICC-IM are important postjunctional mediators of enteric excitatory and inhibitory neurotransmission mediated by acetylcholine and NO. EFS, electrical field stimulation.

these cells. Hirst and colleagues (83) investigated postjunctional responses to cholinergic and nitrergic stimulation in intact muscle bundles containing terminal processes of enteric motor neurons, ICC-IM, and smooth muscle cells. These authors conclude that cholinergic responses were caused by summation (coordination) of unitary potentials (as described in the previous section) (83), and nitrergic responses were caused by suppression of these events (84). It is also likely that NO can activate K+ conductances in postjunctional cells of the tunica muscularis (85,86). Cholinergic stimulation is likely to coordinate the occurrence of unitary potentials by increasing IP3 formation in stimulated ICC-IM. This should cause ICC-IM to discharge unitary potentials

in an approximately simultaneous manner and produce the summation of events (83). The mechanism by which NO regulates the occurrence of unitary potentials has not been resolved; however, cGMP-dependent pathways are used in these responses (84).

ROLE OF INTERSTITIAL CELLS OF CAJAL AS STRETCH RECEPTORS The morphology of ICC has also suggested that these cells could have a sensory role, possibly transducing responses to stretch (cf. 4). There are few physiologic tests

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 549 of this hypothesis available; however, one study suggests a prominent role for ICC-IM in responses to stretch in the stomach (87). A specialized apparatus was developed to precisely increase length of murine gastric antral muscles, and responses to stretch were recorded with intracellular electrophysiologic techniques and isometric force measurements. During application of length, ramps muscles were stretched to approximately 125% of resting length. This amount of stretch caused depolarization and increased slowwave frequency. These responses were nonneural and not affected by TTX or atropine. Increasing the ramp velocity enhanced the magnitude of the depolarization and the chronotropic response. Reduced extracellular Ca2+ (to 100 µM) and Ni2+ (250 µM) inhibited the chronotropic effect but did not block the depolarization. Indomethacin blocked both responses to stretch, suggesting that synthesis of eicosanoids may mediate responses. Muscles of W/WV mice (which lack ICC-IM) were unresponsive to stretch, suggesting that ICC-IM may express the transduction mechanism for these responses. Simultaneous recordings of slow waves in the corpus and antrum showed that stretch-induced changes in slow wave frequency uncouple the proximal-to-distal propagation of slow waves. These finding suggest that ICC-IM contain a stretch transducer that may couple to the synthesis of eicosanoids and produce membrane depolarization and positive chronotropic effects. Increasing antral frequency can result in uncoupling of corpus and antral pacemaker activity. Thus that antral stretch could be a factor in gastric dysrhythmias, possibly leading to incomplete gastric peristalsis and delayed gastric emptying. Other studies have suggested that ICC express mechanosensory ion channels. ICC of humans were reported to express TTX-resistant Na+ currents (88). The conductance responsible was suggested to be SCN5A, which was expressed by these cells. In experiments on isolated cells, the current amplitude of the TTX-resistant Na+ current was increased when the perfusion rate of the bath solution was increased. The authors of this study suggest that the Na+ conductance may be mechanosensitive; however, the physiologic significance of this conductance in the actions of ICC has not yet been determined.

MOTILITY DISORDERS ASSOCIATED WITH LOSS OF INTERSTITIAL CELLS OF CAJAL The mechanisms for many human disorders of GI motility have not been explained. Observations have suggested that ICC may be lost in a variety of human disorders, and this has created exciting new hypotheses for the causes of human GI motility diseases (see Table 20-1) (see reviews by Sanders and colleagues [6], Vanderwinden and Rumessen [89], Camilleri [90], and Hasler [91]). As described earlier, ICC have important roles in generating and propagating slow waves and mediating motor neurotransmission in GI muscles. Thus, loss of these cells is expected to result in major motor problems. Currently, the cause-and-effect relation between

loss of ICC and development of abnormal motility has yet to be established in most of the disorders in which ICC loss has been documented in biopsy or surgical specimens.

ANIMAL MODELS TO STUDY LOSS OF INTERSTITIAL CELLS OF CAJAL Animal models have been and will continue to be an important means of determining the consequences of ICC loss on motility (92). Animals in which ICC fail to develop or are lost in adulthood may provide models for studying human disorders. In human diseases, it is usually not possible to evaluate the status of ICC at multiple time points during the development of symptoms, thus cause-and-effect relations are difficult to establish. However, careful examination during development of diseases is possible with animal models and might provide a means of determining whether ICC defects are fundamental to the cause of motility disorders. Several animal models using genetic mutations, chemical treatments, surgery, or infections have been identified in which ICC populations are lost or damaged and motility disorders, including aberrant electrical activity, reduction in neurotransmission, or abnormal motility, are associated with these lesions. Defects in electrical activity and motility develop in parallel with the loss of ICC, and the motor problems in these animals may be analogous to some of the motility defects observed in human patients. The initial animals exploited as models of ICC loss were animals in which signaling via Kit receptors was compromised. This was accomplished either by blocking Kit receptors with neutralizing antibodies (17,18), by using animals carrying loss-of-function mutations in Kit or its ligand, stem cell factor (19,20,93), or by blocking downstream signaling molecules (94). In c-kit mutants (white-spotting mutants or W mutants) and stem cell factor mutants (steel or Sl mutants), ICC fail to develop because of defects in the Kit signaling pathway. Compound heterozygote animals are often bred for purposes of investigation (such as W/WV and Sl/Sld mutants) because homozygotes for these mutations often die in utero due to severe anemia. W/WV and Sl/Sld mutants do not have complete loss of function in the Kit signaling pathway, and these animals show only partial loss of ICC (cf. 19, 51, and 95). These animals have been excellent models to test the impact of losing specific types of ICC (e.g., loss of ICC-MY in the small bowel, or loss of ICC-IM in the stomach) or partial loss of other populations of ICC (e.g., ICC-MY in the stomach and general reduction of ICC in the colon). Some of these conditions might simulate patterns of ICC loss in human patients with partial loss of ICC. For example, electrical slow waves are absent in the small intestine of W/WV mice, and radiograms using barium demonstrate abnormal, slow, and uncoordinated motility in the small bowel (96). Nonobese diabetic mice (NOD) (type 1 diabetes model) lose patches of ICC in the antrum and have microscopic lesions in the tight synaptic relation between ICC-IM and nerve terminals (21). Studies on NOD mice

550 / CHAPTER 20 suggest the novel hypothesis that ICC loss might be a factor in GI motility problems arising from long-standing diabetes (97), and this hypothesis has now been supported by human studies (see Table 20-1) (98,99). Diabetes initiated by streptozotocin treatments also results in loss of ICC (100). The lethal spotting mice (ls/ls) have defects in endothelin B receptors and have a form of Hirschsprung’s disease (101). Although these animals do not apparently have lesions in ICC within the aganglionic section (102), they may be useful in determining the effects of distension above the aganglionic segment on ICC in the portion of the colon left after resection. Mice lacking functional dystrophin (mdx) also have microscopic lesions in ICC, and gastric dysrhythmias may be a consequence of this mutation (103). Surgical manipulations such as partial bowel obstruction (104) or bowel resection (105) have significant, but temporary, effects on ICC cells, and the condition reverses itself by healing or by relieving the obstruction. Several motility disorders follow bowel infections, and it may be possible to simulate some of these conditions with infections in animals. Infection with Nippostrongylus brasiliensis has been shown to affect ICC

populations in rats (106), but many other models could be developed to study postinfectious motility disorders and whether these conditions are associated with loss of ICC or long-term defects in the functions of ICC.

SMOOTH MUSCLE RESPONSES TO SLOW WAVES AND NEURAL INPUTS The smooth muscle cell response to electrical inputs from ICC underlies the motility patterns in GI organs (Fig. 20-9). Slow waves produce a cycle of depolarization and repolarization that conducts to smooth muscle cells and changes the open probabilities of the many voltage-dependent ion channels expressed by smooth muscle cells. Voltage-dependent ion channels form the backbone of the smooth muscle response to electrical rhythmicity, but metabotrophic and Ca2+-dependent ion channels also contribute to the integrated responses of these cells. Responses are further tuned to create observed motility patterns by paracrine, hormonal, and neural inputs to the smooth muscle/ICC syncytium.

Smooth muscle cells respond to slow wave depolarizations with increased Ca2+ channel open probability ICC generate and propagate slow waves

Smooth muscle cells Electrotonic conduction of slow waves

Varicosity

Pacemaker ICC Interstitial cell network in pacemaker region

Intramuscular ICC Enteric motorneuron

Neurotransmitters and hormones condition the smooth muscle syncytium to generate the desired response.

FIG. 20-9. Schematic summarizing some of the functions of interstitial cells of Cajal (ICC) and smooth muscle cells in gastrointestinal (GI) muscles. ICC in pacemaker regions (ICC between the circular and longitudinal muscle layers [ICC-MY] and ICC along the submucosal surface of the circular muscle layers [ICC-SM]) generate spontaneous slow wave activity. Intact networks of ICC are required to actively propagate this activity down and around GI organs. ICC are coupled electrically to circular and longitudinal muscle layers and conduct the slow waves to neighboring smooth muscle cells. Slow waves are not actively regenerated, so the amplitude of these events decays with distance from the pacemaker areas. In thicker GI muscles, septal ICC (ICC-SEP) may serve as active propagation pathways to deliver slow wave depolarizations deeper into the muscle bundles. Slow wave depolarizations cause activation of voltage-dependent channels in smooth muscle cells, such as voltage-dependent (L-type) Ca2+ channels. Ca2+ entry via this pathway results in increases in global Ca2+ and activation of the contractile apparatus. Increases in open probability of Ca2+ channels can result in the development of Ca2+ action potentials superimposed on the slow waves (as shown in the trace). The magnitude of the response of smooth muscle cells is regulated by neural, humoral, and paracrine factors. For proper motility, neural inputs are essential. As shown, varicose terminals of enteric motor neurons form close synaptic relations with ICC-IM. In the case of cholinergic and nitrergic neurotransmission, ICC-IM are vital for signal transduction. ICC-IM couple to smooth muscle cells via low-resistance gap junctions, and excitatory or inhibitory inputs from the motor neurons thus condition the smooth muscle syncytium for the appropriate response.

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 551 Many of the stereotypical motility responses of these cells also depend on modulations of ion channels. Our understanding of the variety and complexity of ion channel expression in GI smooth muscles has grown dramatically during the past 15 years. There is not space here for an exhaustive description of the channels and the properties and regulation of the conductances present in GI smooth muscles because there is considerable variation in channel types and mechanisms of regulation from region to region and from species to species. Two reviews (41,107) should be useful supplemental information together with the material described later. Common important participants in GI smooth muscle responses are emphasized in this section. Ion channel expression, as currently understood, is summarized in Table 20-2. This chapter emphasizes electrophysiologic mechanisms in ICC and smooth muscle cells; however, it must be noted that in addition to electromechanical coupling, smooth muscle cells display other modalities of excitation–contraction coupling. Stimuli that produce contractile responses without altering ionic fluxes across the plasma membrane initiate mechanisms that have been referred to as pharmacomechanical coupling (108). Mechanisms of pharmacomechanical coupling include: (1) binding of agonists to cell receptors, activation of phospholipase C (PLC), generation of IP3 and diacylglycerol (DAG), release of Ca2+ from IP3 receptor– operated stores, and activation of protein kinases; and (2) Rho kinase regulation of the Ca2+ sensitivity of the contractile apparatus. These are extremely important mechanisms for determining the contractile performance of smooth muscle cells and are addressed in other chapters of this text.

Voltage-Dependent Inward Currents CaV1.2 (dihydropyridine-sensitive “L-type”) Ca2+ Channels A significant portion of excitation–contraction coupling in GI muscles in response to slow wave depolarizations is caused by the activation of voltage-dependent Ca2+ channels. It seems that all GI smooth muscle cells express voltagedependent Ca2+ channels, and these channels show dynamic changes in open probability within the physiologic range of membrane potentials (Fig. 20-10). In some regions of the GI tract, slow wave depolarization causes activation of voltagedependent Ca2+ channels that is not immediately countered by activation of outward currents and Ca2+ action potentials occur. In other regions, threshold for Ca2+ action potentials is not attained during slow waves, but the sustained depolarization during the plateau phase of slow waves results in sustained, low-level activity of Ca2+ channels for several seconds. Both mechanisms provide an influx of Ca2+ that is sufficient for excitation–contraction coupling. In tonic muscles, hormonal or neuronal excitatory agonists can cause depolarization and activation of Ca2+ channels. Ca2+ influx during depolarization can result in tonic contractions.

Mitra and Morad (109) described typical voltage-dependent Ca2+ currents observed in GI smooth muscle cells. Rabbit and guinea pig gastric muscle cells from the gastric corpus were used for their studies. Ca2+ currents could be resolved in response to depolarizing steps positive to −30 mV. The currents reached amplitudes of 100 to 200 pA at +10 mV and reversed at +40 mV. The current was inhibited by removing extracellular Ca2+ and blocked by Co2+, Cd2+, or dihydropyridines. Another early article by Droogmans and Callewaert (110) describes similar inward currents in longitudinal muscle cells of guinea pig ileum. Voltage-dependent Ca2+ currents have been observed in many subsequent studies of GI muscle and are caused by L-type, dihydropyridine-sensitive conductances (32,110–125). This class of Ca2+ channels has also been described in the literature as “high threshold” because they activate to a resolvable level at potentials positive to −50 mV. Single channels with properties of L-type Ca2+ channels and conductances of about 10 and 30 pS have been measured in studies of a variety of GI smooth muscle cells (120,126–129). L-type Ca2+ channels are well suited for the mechanical activities of GI smooth muscles. With high thresholds for activation, they experience low open probabilities at the negative resting potentials of (greater than −60 mV) of smooth muscle cells in most phasic regions of the GI tract. Slow waves depolarize cells positive to −40 mV. Thus with each cycle, cells enter or just reach the voltage range where there is a steep increase in the open probability of L-type Ca2+ channels. L-type Ca2+ channels initiate excitation–contraction coupling in various motility patterns in the GI tract, and the high-threshold nature of these channels is compatible with the pattern of contraction and relaxation that occurs in segmentation and peristalsis. Another feature of L-type Ca2+ channels is voltagedependent inactivation (110,114,117). Depolarization initiates activation of Ca2+, but sustained depolarization results in inactivation. A portion of the inactivation is due to the entry of Ca2+ (i.e., Ca2+-dependent inactivation) (114,122), and a portion is due to voltage-dependent mechanisms. Inactivation, however, is incomplete at the potentials reached during slow waves, and there is a “window-current” (130,131) phenomenon in which channels activate but do not fully inactivate (110–112,117,123). The flux of Ca2+ ions entering cells in the window-current range (−50 to −20 mV) is small, but the number of Ca2+ ions relative to cell volume is sufficient to produce a significant change in concentration. The inward current during depolarizations equivalent to slow waves increases cell (Ca2+) to at least 10 µm (117). With cell Ca2+ buffering of approximately 100-fold, Ca2+ influx via L-type Ca2+ channels is sufficient to achieve excitation–contraction coupling. Direct measurements of changes in free [Ca2+]i with sensitive Ca2+ dyes in voltage-clamped, single smooth muscle cells demonstrate that Ca2+ influx is sufficient to increase basal cytoplasmic Ca2+ concentration to levels high enough to accomplish excitation–contraction coupling (see Vogalis and colleagues [121], Becker and colleagues [132], and

Unitary conductance

Jejunum/rabbit

If-like

Kir

KA

Esophagus, colon/human stomach, jejunum, colon/mouse, ileum/rat Esophagus/cat, ICC (colon, jejunum)/canine, ICC (jejunum)/mouse Small intestine, colon/canine, colon/mouse

Potassium-selective conductances KDR Colon/human, colon/canine, colon/mouse

Jejunum/human

Colon/human colon/mouse esophagus/cat Colon/human fundus/rat intestine/rat

TTX-r Na+

TTX-s Na+

T-type

Low-voltage activated (−55mV), fast inactivation

Strong inward rectification by Mg2+ or polyamine G-protein–activated inward rectifier current

10–32 pSa

25 pSa (60/ 150 mM)

High voltage activated (–40 mV), slow inactivation

Low-voltage activated, fast inactivation Low-voltage activated, fast inactivation (<1 msec) High-voltage activated, fast inactivation (>1 msec) Activated negative to −70 mV, time dependent

High-voltage activated, slow inactivation

Conductance properties

19 pS

10–20 pS

?

<10 pSa

7–15 pS

<10 pSa

Calcium- and sodium-selective conductances L-type Jejunum/human 10–30 pS colon/mouse stomach/guinea pig

Ion channel

Examples of distribution (cell/species)

PIP2,a Gβγa

?

?

VIP, isoproterenol

?

?

Veratridinea

ATP,a isoproterenola

Bay K8644, ACh, Erythromycin,a FPL64716a

Activators

Tertiapina

Ba2+

TEA, 4-AP (>3mM), dendrotoxin, margatoxin,a nuxiustoxina 4-AP (<3 mM), flecainide

Cesium

TTX (2–15 nM), lidocaine, saxitoxina TTX (2 µM) QX-314

DHP antagonists (nifedipine, diltiazem, nicardipine, verapamil) Ni2+, mibefradil, kurotoxina

Blockers

TABLE 20-2. Ion channels in gastrointestinal smooth muscle

Nav1.x (?)a

Cav3.x (M)

Cav1.2 (M)

Molecular candidate(s)

Kv4.3 (M/F), Kv4.2 (M), Kv4.1 (M), KChIPs (M) Kir2.1(M/F)

Kir3.1, Kir3.2 (M)

PKA ↓ (Kir2.1)a

PKC ↓a

Kv1.2 (M/F), Kv1.5 (M/F), Kv2.2 (M/F)

?

CaMKII (slowed inactivation)

PKA ↑, PKC ↓ (?)

?

Stretch Nav1.5 (M/F) (mechanosensitive)

?

?

PKA, PKC, PKG ↓ CaMKII ↑a

Regulation

187, 290, 291

13, 184, 185

157, 163, 172, 181, 289

158, 161, 162, 165, 166, 169, 179, 180

153

88,150

145, 148, 149, 287, 288

144–146, 286

12, 110, 113, 116, 118a, 140, 285

References

552 / CHAPTER 20

5–10 pS

SK

Nonselective cation conductances Muscarinic/ Ileum/guinea pig, peptide colon/canine activated 16–63 pSa

0.5–5 pS

Ca2+ activated

Esophagus/rabbit

1–400 pSa

Chloride-selective conductances Volume Colon/canine sensitive

Peak at −40 mV

Outward rectification with rapid deactivation at negative potentials

Outward rectification, I− > Br− > Cl− > F− > gluconate

Voltage independent

Weak-voltage dependence

35–40 pS

Ileum/mouse, colon/mouse

Voltage dependent and Ca2+ dependent

200–300 pS

14–38 pSa

Voltage-independent background current Voltage-independent background current

Fast recovery from inactivation and slow deactivation

5–12 pSa

95 pS (TREK)

Weak inward rectification

17–27 pS

Ca2+-activated K+ conductances BK Colon/canine, jejunum/rabbit, gall bladder/ guinea pig IK Ileum/mouse

Colon, small intestine (TASK)

Jejunum/human, stomach/rat, esophagus/ opossum, gall bladder/ guinea pig Colon/mouse (TREK-1)

ERG

K2P

Colon/canine, colon/mouse, gall bladder/ guinea pig

KATP

ACh, substance P, neurokinin A

Cd2+, Ni2+, Gd3+ La3+, SKF96365, 2-APB

Niflumic acid, DIDS, SITS, 9-AC

Tamoxifen, DIDS, NPPB,a DCPIBa

Charybdotoxin, TEA (>mM), maurotoxin,a TRAM-34a Apamin, bicuculline,a scyllatoxina

1-EBIO,a DC-EBIO,a NS309a 1-EBIO,a DC-EBIO,a NS309a

Charybdotoxin, iberiotoxin, TEA

Sulfur-containing AA, Zn2+ (>600 µM)a Lidocaine, bupivacaine, anandamide, Zn2+ (~12 µM)

E-4031, clofilium, MK-499, cisapride, dofetilide,a ergotoxina

Glibenclami de, tolbutamide

Tamoxifen, estrogen

Halothane, arachidonic acid,a coppera

Pinacidil, lemakalim, cromakalim, diazoxide, nicorandil RPR26023a

PKC ↓ Swelling, MAPK,a tyrosine kinase,a CaMKII ↑a Ca2+ ↑ CaMKII ↑a

Ca2+/ calmodulin ↑, CaMKII ↑

Ca2+/ calmodulin ↑

PKC ↓, PKG ↑, PLC/IP3 (STOC)

220, 221, 223, 224, 233, 242, 244, 297 252, 298, 299

217, 218, 296

215, 216, 295

200, 202, 205, 292, 293, 294

190, 191, 193, 195

Continued

265, 267–269, 272, 274

112, 262, 304

ClCA family,a bestrophin familya

TRPC family (?)a

260, 302, 303

ClC-3 (F/M)

KCa2.1, 247, 251, KCa2.2, 253, 300, KCa2.3 (M/F) 301

KCa3.1 (M/F)

KCa1.1 (M/F)

K2p3.1, 5.1 (M/F)

K2p2.1 (M/F)

Kv11.1 (M/F)

Hypoxia ↓ (?)a

Stretch, heat, NO or PKG ↑, PKA ↓ Acidic pH, hypoxia ↓

Kir6.1 or Kir6.2 and SUR2B (M/F)

PKA ↑, PKC ↓

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 553

Colon/canine

Stomach/ guinea pig, stomach/toad

Ion channel

P2X

Stretch activated

Ligand gated, inward rectificationa Outward rectification

~56 pSa 30–53 pS

Conductance properties

Unitary conductance PPADS, suramin, ANAPP3a

α,β-methlyene ATP Gd3+

Blockers

Activators

Stretch, Ca2+ ↑

Regulation

P2X2, P2X3, P2X4, P2X5 (?)a TRPC family (?)a

Molecular candidate(s)

280–284

278, 305, 306

References

not been tested in gastrointestinal (GI) tract. AA, amino acids; 9-AC, anthracene-9-carboxylic acid; ACh, acetylcholine; ANAPP3, arylazido aminopropionyl ATP; 2-APB, 2-aminoethoxydiphenyl borate; 4-AP, 4-aminopyridine; Bay K-8644, (−)-(S)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluromethylphenyl)-pyridine-5-carboxylate; CaMKII, Ca2+/calmoduline-dependent kinase II; DC-EBIO, 5,6-dichloro-1-ethyl-benzimidazolinone; DCPIB, 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl oxybutyric acid; DHP, dihydropyridine; DIDS, 4, 4′-diisothiocyanostilbene-2,2′-disulfonic acid; E-4031, 1-[2-(6-methyl-2-pyridyl)ethyl]-4-(methylsulfonyl-aminobenzoyl)piperidine; 1-EBIO, 1-ethyl-benzimidazolinone; FPL64716, 2,5-diemthyl-4-[2(phenylmethyl)benzoyl]-H-pyrrole-3-carboxylate; IP3, inositol-1,4,5-triphosphate; KChIPs, Kv channel–interacting proteins; MAPK, mitogenactivated protein kinase; M/F, molecular/functional study in the GI tract; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; NS309, 6,7-dichloro-1H-indole-2,3-dione 3-oxime; PIP2, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PPADS, pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid; QX-314, N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide; RPR26023, 3R,4R-4-[3-(6-methoxyquinolin4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid; SITS, 4-acetamide-4′-isothiocyanatostilbene-2,2′- disulfonic acid; STOC, spontaneous transient outward currents; TEA, tetraethylammonium; TRAM-34, 1-[2-chlorophenyl)diphenylmethyl]-1H-pyrazole; TTX, tetrodotoxin; VIP, vasoactive intestinal peptide; ?, data not known.

aHas

Examples of distribution (cell/species)

TABLE 20-2. Ion channels in gastrointestinal smooth muscle—cont’d

554 / CHAPTER 20

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 555 Human colonic myocytes

Murine colonic myocytes A

−100 mV

B Vh = −50 mV

−30 mV

3

Vh = −80 mV

+25

−15

1 −20 mV

2

3

−50

−80

1 80 pA

2 3

−10 mV

80 ms

1

1—Control 2—Bay K (µm) 3—Nifedipine (10 µm) + Bay K (1 µm)

2 3

0 mV

C

pA −30

2 3

+10 mV

−60

1 50 pA

100 ms

2 −60

−40

−20

0

20

40

60

D

−40 Amplitude of ICa (pA)

−120 −90 −70 −50 −30 −10 10 30 mV

80

−20 −60

−50 or −80

−80

−100 −120 −140 −160

Vh = −80 mV Vh = −50 mV Difference

−90

Membrane potential (mV) −100 −80 0

Internal CS-TEA 0

1

A

I-V relationship

Control Bay K (1 µm) Nifedipine (10 µm) + Bay K (1 µ)

−180

E

Comparison V1

V1 = −50 mV

V1 = −50 mV V1 = −35 mV

Difference

80 pA

V1 = −35 mV

80 ms

V1 = −15 mV

V1 = −15 mV

−200

B

−220

FIG. 20-10. Voltage-dependent inward currents expressed in human (left) and murine (right) colonic myocytes. Gastrointestinal (GI) muscles express L-type Ca2+ currents. Examples of this conductance are shown. (Left, A) Superimposed inward current traces recorded in absence (control), presence of 1 µM Bay K8644 (Bay K), and presence of 10 µM nifedipine plus 1 µM Bay K8644 (nifedipine + Bay K). (Left, B) I-V relation of ICa for data in A. (Right, A) Representative currents elicited from circular muscle cells of the mouse colon by test potentials from −50 to +25 mV. (Right, B) Representative currents elicited by test potentials from −90 to −15 mV (in 15-mV increments; Vh = −80 mV). (Right, C) I-V relation for currents obtained from holding potentials of −80 mV (open circles), −50 mV (solid circles), and difference currents (solid squares). (Right, D) Representative currents were recorded with conventional whole-cell voltage clamp in response to depolarizing steps to −50, −35, and −15 mV (V1) from two different holding potentials of −50 and −80 mV. The inset shows the voltage protocol used. (Right, E) Difference currents at each test potential were obtained by subtracting currents obtained from two holding potentials (−50 and −80 mV). A conductance besides the L-type Ca2+ current is evident from these recordings, and it was concluded that murine colonic myocytes also express a voltage-dependent, nonselective current. TEA, tetraethylammonium. (Left: Reproduced from Xiong and colleagues [118a], by permission; Right: Reproduced from Koh and colleagues [146], by permission).

Kamishima and McCarron [133]). Because of the steep voltage dependence of activation in the range of potentials achieved during slow waves and the window-current phenomenon, small changes in amplitude or duration of slow waves have significant impact on the magnitude of Ca2+ influx (121). This is the basis for the steep relation between voltage and contractile force in GI muscles (see Barajas-Lopez and Huizinga [134], Bauer and Sanders [135], Szurszewski [136], Morgan and Szurszewski [137], and Morgan and colleagues [138]). L-type Ca2+ currents in GI smooth muscles recover from inactivation within about 1 second after repolarization (122).

This relatively slow rate of recovery may explain the maximal rate of action potential firing in GI smooth muscles, but other processes must be responsible for the many second refractory periods experienced by the slowwave generating mechanism in the gut (i.e., greater than 3 seconds) (see Publicover and Sanders [139]). It has been noted that the time constant for recovery from inactivation of L-type Ca2+ channels is approximately the same as the time constant for restoration of resting levels of intracellular Ca2+ ([Ca2+]i) (122). Thus, the dominant feature regulating recovery from inactivation appears to be relieving the channels from Ca2+-dependent inactivation.

556 / CHAPTER 20 Because L-type Ca2+ channels provide an important role in initiating excitation–contraction coupling, regulation of these channels may provide an effective means of controlling motility. In fact, although there have been demonstrations of agonist regulation of L-type currents (e.g., particularly inhibition by cGMP-dependent mechanisms) (140,141), control of Ca2+ influx in GI muscles is mainly accomplished by membrane potential regulation of Ca2+ channel open probability. Membrane potential regulation is largely under the control of the plethora of K+ channels expressed by GI smooth muscle cells. There have been a few reports about the regulation of L-type Ca2+ channels by PKC-dependent mechanisms. Activators of PKC were reported to increase Ca2+ currents (142,143). Of interest is the paradoxical regulation of L-type Ca2+ channels by 3′,5′-cyclic adenosine monophosphate (cAMP)–dependent mechanisms. In the heart, this conductance is activated by protein kinase A (PKA), but in the GI tract, agonists linked to activation of adenylate cyclase, production of cAMP, and activation of PKA are inhibitory (e.g., β receptor agonists, vasoactive intestinal peptide [VIP], etc.). In fact, stimulation of cAMP-dependent pathways activates L-type Ca2+ current (140), thus other mechanisms such as parallel activation of K+ channels and pharmacomechanical mechanisms must be responsible for the overall inhibitory actions of the cAMP-dependent signaling pathway. CaV3 (“T-Type”) Ca2+ Channels There have been reports of “low-threshold” Ca2+ currents in GI muscles. Cat esophageal muscles express an inward current that activates at negative potentials, producing a characteristic “hump” in the I-V curve between −50 and −20 mV (144). Experiments on human colonic muscle cells revealed a fast-activating current that activated at negative potentials and inactivated when cells were held at potentials positive to −80 mV (145). This current was blocked by low concentrations of Ni2+ but was resistant to dihydropyridines. The authors conclude that the properties of the fast-activating current in human colonic cells had the properties of a T-type Ca2+ current. Experiments on murine colonic muscles revealed a similar low-threshold current in these cells (146). The lowthreshold current was resistant to dihydropyridines and was inhibited by Ni2+ and mibefradil. Although the properties of the murine conductance were similar to a T-type current, the current–voltage relation showed that the reversal potential occurred near 0 mV. The current was also reduced when either extracellular Ca2+ or Na+ were reduced. The low-threshold current in colonic cells had properties of a voltage-gated, nonselective cation current rather than T-type Ca2+ current. It is not fully clear what the role of a low-threshold current would be in GI muscles. These cells, in general, have resting potentials near −60 mV, and at these potentials the lowthreshold currents are substantially inactivated. During inhibitory stimulation, however, there are large inhibitory junction potentials (IJPs) that hyperpolarize the plasma membrane potential toward EK. Activation of low-threshold currents could be important as a means of active recovery of

membrane potential after IJPs. This hypothesis was tested in murine colonic muscles using blockers of the low-threshold nonselective cation conductance. IJPs had fast repolarization phases that were greatly decreased in velocity by blockers of the nonselective cation currents (147). Na+ Channels There have also been a few reports of Na+ currents in GI muscles. One study found a TTX-sensitive conductance in smooth muscle cells of the rat gastric fundus (148). This current was highly selective for Na+ and had a half inactivation value of −74 mV. Thus, at the resting potentials of fundus muscles (positive to −60 mV) (1), little of this current is available. The authors confirm this by showing that action potentials in fundus cells were not sensitive to TTX. TTXsensitive Na+ currents were also observed in rat intestine (149) and human colon (145). There has been a wide variation in IC50 values for TTX in studies reporting Na+-selective currents, suggesting several isoforms of Na+ channels may be expressed in GI muscles. Others have reported TTXinsensitive Na+ currents attributed to SCN5A (NaV1.5) channels in human intestinal smooth muscle cells (150). This conductance appears to have interactions with the cytoskeleton, perhaps conveying mechanosensitivity to the channels (151). The same authors have reported that SCN5A channels are expressed in human ICC, and suggest a role for this conductance in the regulation of slow wave activity. Lidocaine and QX-314, both blockers of SCN5A channels, decreased slow wave frequency, and stretch increased slow wave frequency (88). There is, however, some doubt about the expression of SCN5A channels in GI muscles (Sanders, unpublished). Inward Rectifier (If-like Currents) In the heart, spontaneous rhythmicity has been partially attributed to a hyperpolarization-activated conductance (If) (152). Few observations of this type of conductance have been made in GI smooth muscles. Benham and coworkers (153) report an inwardly rectifying cation current in rabbit jejunal longitudinal muscle cells. Hyperpolarization negative to −70 mV evoked a time-independent current and a current that developed over a period of several seconds. The time-dependent current reversed at about −30 mV and was blocked by cesium (1 mM). Others found hyperpolarizationactivated cation channels (64 pS) in toad stomach muscle cells, and this conductance was also activated by stretch (154).

Voltage-Dependent Outward Currents During the past two decades, substantial efforts have been made to identify and clone mammalian K+ channels. Now more than 60 mammalian pore-forming subunits have been cloned. Functional channels form as multimers of subunits and often are associated with auxiliary subunits that participate in regulation. K+ channels fall into three major structural

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 557 divisions: (1) pore-forming subunits with one ionic pore and six transmembrane segments (e.g., voltage-dependent K+ channels), (2) subunits with one ionic pore and two transmembrane segments (e.g., inward rectifier K+ channels and KATP channels), and (3) subunits with two ionic pore regions and four-transmembrane–spanning segments (e.g., two-pore K+ channels). Smooth muscle cells and ICC of the GI tract express representatives from each of these families. Thus, a broad spectrum K+ conductances tunes the smooth muscle response to slow wave depolarizations and agonist stimulation. Differences in K+ channel expression and availability result in the large variation in electrical activity in different regions of the GI tract and in different species. This section reviews the properties of the major K+ channels that have been identified in GI smooth muscle cells. There are other reviews that should be consulted for more detailed descriptions of the properties and expression of K+ channels in GI muscles (e.g., 41,107,155–157).

KV Family Channels Delayed Rectifier Channels Smooth muscle cells of the GI tract express a variety of voltage-activated K+ channels that are within the KV family of channels (Fig. 20-11). These channels have been termed delayed rectifiers because of their relatively slow time course of voltage-dependent activation (158–160). Voltageactivated K+ channels have low open probability at the negative resting potentials of many GI muscle cells, and open probability increases on depolarization. Studies with blockers suggest that KV channels participate to a small extent in setting resting membrane potential and to a greater extent in shaping the voltage responses of smooth muscle cells during excitable events. The complement of KV channels expressed in a given tissue region, which is often heterogeneous (161–163), helps to define the response of smooth muscles to slow wave depolarization and whether slow waves initiate action potentials or whether excitation–contraction coupling is determined by the amplitude and duration of the slow wave. There is considerable diversity in the expression of delayed rectifiers in different species and in different regions of the GI tract. For example, there are significant differences in delayed rectifier conductances even in two adjacent muscle layers, such as the circular and longitudinal muscles of the canine colon. The KV conductance in longitudinal cells activated at more negative potentials (half inactivation at −63 mV) compared with the equivalent conductance in the circular muscle (half inactivation at −36 mV) (164,165). The conductance in longitudinal cells was also less sensitive to 4-aminopyridine (4-AP) than in circular cells. Much has been done to identify the molecular species responsible for delayed rectifier channels in GI muscles. A complementary DNA (cDNA) encoding a delayed rectifier of the KV1.2 class was cloned from canine colonic circular smooth muscle and expressed in Xenopus oocytes (166).

The expressed channels generated a K+ current with voltagedependent activation and a single-channel conductance of 14 pS. KV1.2 channels were inhibited in a concentrationdependent manner by 4-AP (IC50 = 74 µM). Although these channels were widely expressed by GI muscles, tests failed to show expression in vascular muscle and uterine smooth muscles. KV1.5 transcripts were also identified in colonic smooth muscles with high homology to other species of KV1.5 but with only 74% to 82% homology in the NH2 and COOH terminal sequences (167). Functional expression of these 10 pS channels revealed voltage-dependent K+ channels that were resolved by depolarization positive to −50 mV. Expression of KV1.5 channels is widespread, and these authors concluded that KV1.5 encodes delayed rectifier K+ channels in both visceral and vascular smooth muscles. Functional channels of the KV family are composed of four pore-forming α subunits that coassemble into heterotetramers. The primary α subunits are also associated with β subunits that modulate the properties of native channels. The properties of these conductances in GI smooth muscles do not match perfectly the properties of homologously expressed channel species. Using the pharmacology of charybdotoxin (ChTX), it was shown KV1.2 and KV1.5 channels form heterotetramers when coexpressed (168). The presence of even one ChTX-insensitive subunit in final channel assemblies rendered heterotetrameric channels insensitive to ChTX. Expression of functional channels as heterotetramers is likely to explain the lack of sensitivity of native channels to ChTX and the differences between native and cloned KV channels. Besides the faster activating KV1 family channels in GI muscles, a more slowly activating component has been identified. This conductance is relatively insensitive to 10 mM 4-AP, but it is blocked by external tetraethylammonium (TEA). KV2.2 was cloned from human and canine colonic circular smooth muscle (169). Northern analysis showed that KV2.2 is expressed in all regions of the canine GI tract tested. Expression of KV2.2 resulted in 15 pS channels that were inhibited by TEA (IC50 = 2.6 mM) and quinine (IC50 = 13.7 µM). By testing quinine on intact colonic muscles, the authors suggest that KV2.2 channels contribute to the complement of delayed rectifier channels in GI smooth muscle cells. Accessory subunits associated with KV channels and the resulting 4α/4β complexes alter channel function (170). Several isoforms of β subunits (β1.1, β1.2, and β2.1) have been cloned that associate with KV1 family pore-forming α subunits and affect channel kinetics. β4 subunits associate with KV2.2. β1.1 and β4 subunits have been found in all regions of the canine GI tract, but other β subunits currently have not been detected (169). A-Type K+ Currents A-type currents are prominent in neurons where these conductances participate in the regulation of firing frequency (171). A-type currents also have been detected in GI smooth muscles (163,172–175; see also review by Amberg and

558 / CHAPTER 20 4-AP (5 mM)

Control +20 mV −80 mV

500 pA

A

B

20 ms Difference currents Peak currents (pA) 500 pA

C

I–V relationship

2500 2000

Control 4-AP

1500 1000 500 0

−80 −60 −40 −20 Voltage (mV)

D

0

20

20 ms

−80

b −40

+40 −40

1 nA 50 ms

+40 −40

E

Normalized value

a

0.5 Window current

0 −80 −60 −40 −20 mV

0

20

F

FIG. 20-11. Voltage-dependent K+ currents in murine gastrointestinal (GI) myocytes. Murine colonic myocytes express both delayed rectifier (KDR) and A-type (KA) currents. Outward currents were elicited by test potentials −70 and +20 mV from a holding potential of −80 mV. (A) Control. (B) 4-aminopyridine (4-AP; 5mM). (C) Subtracted currents: control minus currents with 4-AP present. (D) Summarized I-V relations in control (open circle) and 4-AP (closed circle). Asterisks denotes significant difference between current amplitudes in control and in the presence of 4-AP. (E) A-type currents from holding potentials of −80 mV (a) and −40 mV (b) recorded from murine antral myocytes. (F) Steady-state inactivation shown as a plot of normalized peak current (I/Imax) as a function of conditioning potential (closed circle) and fit with a Boltzmann function. Peak K+ currents at test potentials were converted into permeabilities using the Goldman–Hodgkin–Katz current equation for voltage dependence of activation. Permeabilities were normalized (P/Pmax), plotted as a function of test potential (open circle), and fit with a Boltzmann function. Dashed lines mark the voltages of half activation and inactivation. (D: Reproduced from Koh and colleagues [162], by permission; F: Reproduced from Amberg and colleagues [174], by permission.)

colleagues [157]). These currents rapidly activate and inactivate in response to depolarization. A-type currents are Ca2+ independent and are blocked by 4-AP. In murine colonic myocytes, A-type currents are also sensitive to low concentrations of flecainide (IC50 = 11 µM). The voltage dependence of activation and inactivation of A-type currents overlap in GI muscles, producing substantial window current near the resting potentials of GI muscle cells.

Despite the fast rates of activation and inactivation, the main contribution of A-type currents in GI muscles is to resting membrane potential. For example, in murine antral myocytes, 4-AP or flecainide depolarized membrane potential by 7 to 10 mV, and flecainide depolarized membrane potentials in intact muscles by 5 mV. Molecular and protein analyses have shown that the major species contributing to A-type currents in GI muscles is KV4

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 559 family channels (175,176; see also review by Amberg and colleagues [157]). Of this family, KV4.3, an isoform also found in the heart, is most common (175). K+ channel-interacting proteins (KChIPs), which are positive modulators of KV4mediated currents, are also expressed in GI muscles. Comparative studies of expression and function suggest that KV4.3, in association with KChIP1, are the major molecular correlates of A-type currents in murine colonic muscles and encode 19 pS K+ channels in these cells. Regulation of Voltage-Dependent K+ Channels KV family channels are regulated by a variety of receptordriven, intracellular signaling pathways in GI muscles. Delayed rectifiers in smooth muscles are enhanced by cAMP-dependent mechanisms (177,178), so agonists that are coupled to activation of adenylyl cyclase are generally stimulatory to this conductance and inhibitory toward excitability. For example, VIP increased the open probability, mean open time, and mean burst duration of 4-AP–sensitive, delayed rectifier K+ channels in canine proximal colon (179), and β-adrenergic stimulation increased delayed rectifier currents in vascular smooth muscles (177). Other studies have suggested that delayed rectifier currents are suppressed by PKC-dependent mechanisms (180). The open probabilities of both KV1.2 and KV1.5 were suppressed by cholinergic stimulation (via activation of M3 receptors) and PKC. Thus, part of the excitatory influence of agonists that activate phospholipase C may be through suppression of delayed rectifier conductances. A-type currents in murine colonic myocytes are regulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) (181). The rate of inactivation of A-type currents is decreased by CaMKII, and the inactivation rate is increased by inhibitors of calmodulin (W-7). Inhibitors of calcineurin (cyclosporin A and FK-506) and inhibitors of calcineurin/inhibitor-1/ protein phosphatase 1 cascade (okadaic acid) reduced the rate of inactivation (182). As described earlier, regulation of the A-type conductance may influence resting membrane potentials and basal excitability of GI muscles. Delayed Rectifier Conductance in Interstitial Cells of Cajal Most studies of ICC have concentrated on inward currents that might be involved in pacemaker activity (see Electrical Activity in Gastrointestinal Muscles). One study, however, concentrates on the outward currents in ICC-IM and reports that these cells express KV1.1 channels (183). The identity of ICC-IM was verified by molecular techniques showing that cells with KV1.1 currents were Kit positive. Immunohistochemical techniques showed that KV1.1 protein colocalized with Kit immunoreactivity in intact muscles. The mamba snake toxin dendrotoxin-K (DTX-K) is a specific blocker of KV1.1 channels cloned from a canine colonic cDNA library and expressed in Xenopus oocytes, and this toxin also blocked a component of outward currents in ICC.

DTX-K–sensitive currents might be a useful signature for ICC-IM in future electrophysiologic studies. Inward Rectifier K+ Channels Inward rectifier K+ channels are also expressed by GI muscle cells. These conductances, although important in regulation of resting potential and responses to a variety of agonists, have not received nearly as much investigation as KV and Ca2+-dependent conductances. Inward rectifiers have the property of conducting ions better at negative membrane potentials. Activation of these channels increases K+ permeability but often produces little outward current, because at negative membrane potentials there is little driving force on K+ ions. Thus, conductances of this type are particularly well suited for contributing to membrane potential and stabilizing membrane potential. Activation of inward rectifier conductances would tend to reduce the amplitude of conducting slow waves and reduce the tendency of slow waves to initiate Ca2+ action potentials in smooth muscle cells. Genes encoding members of Kir2, Kir3, and Kir6 subfamilies are expressed in GI muscles. Cells in the GI tracts of some species have very negative resting membrane potentials. For example, cells near the submucosal surface of the circular muscle layer in the canine colon have resting membrane potentials that approach EK (3). Cells along the submucosal surface express Kir2.1 as shown by reverse transcriptase-polymerase chain reaction (RT-PCR) (184). Low concentrations of Ba2+ (i.e., 10–100 µM) caused 16-mV depolarizations of cells in intact colonic muscles. Interestingly, the smooth muscle cells isolated from the colonic circular muscle layer showed little evidence for an inwardly rectifying K+ conductance, but ICC isolated from the submucosal surface expressed this conductance (184). ICC in the jejunum also expressed Ba2+-sensitive, inwardly rectifying K+ conductance, 10 to 100 µM Ba2+ depolarized ICC, and intact jejunal muscles (13). These data suggest that the negative influence of ICC on membrane potentials in some GI muscles (see Ward and colleagues [19]) may be caused by cell-specific expression of Kir channels. Inward rectifying K+ conductances have also been reported in feline esophageal smooth muscle cells (185). The current resulting from hyperpolarization of these cells was increased in amplitude as external K+ was increased and the reversal potential shifted in response to the shift in EK. The conductance was blocked Ba2+ and Cs+ with IC50s of 2.9 µM and 1.6 mM, respectively. Ba2+ (10 µM) depolarized membrane potentials by about 6 mV, suggesting the inward rectifier conductance contributed to resting membrane potential. Kir3.1/3.2 encode K+ channels regulated by muscarinic stimulation and G proteins in heart and brain (e.g., see Logothetis and colleagues [186]). This class of K+ channels has been found in smooth muscles of the canine small and large intestine (187), and immunohistochemistry demonstrated expression of Kir3.1 and Kir3.2 proteins in the circular and longitudinal muscle layers. Full-length cDNA for

560 / CHAPTER 20 Kir3.1 and Kir3.2 were cloned from murine colonic smooth muscle extracts and coexpressed in Xenopus oocytes with human M2 receptors. ACh (10 µM) activated Ba2+ sensitive, inwardly rectifying currents in these cells. Many GI muscles express KATP channels. These channels are heteromultimers consisting of two types of subunits: sulfonylurea receptors (SURs) and members of the inwardly rectifying potassium channel superfamily (Kir6). There are two Kir6 genes that encode subunits of KATP channels when coexpressed with SUR subunits, and the properties and conductances of different combinations of Kir6 and SUR subunits vary considerably. Unlike KV, Kir6 channels contain only two-membrane–spanning regions with a pore region between (188). These channels are inhibited by ATP at concentrations greater than 1 mM, thus metabolic problems where ATP levels decrease can initiation openings. Whether KATP channels are active under basal conditions is often controversial. However, KATP channels can be activated in most cells by hypoxia or ischemic conditions, and opening of these channels can dramatically affect membrane excitability. In GI muscles, the activity of KATP under basal conditions appears to vary. For example, glibenclamide (a KATP blocker) had no effect on resting potentials in taenia caeci (189) or canine colonic smooth muscles (190); however, KATP agonists (chromakalim and pinacidil) caused hyperpolarization that was blocked by glibenclamide. Lemakalim caused hyperpolarization of murine colonic muscles (191); however, in contrast with other muscles, glibenclamide alone caused depolarization (9 mV). Lemakalim induced outward current in murine colonic myocytes that was blocked by glibenclamide. Single channels with conductances of 27 pS were activated by KATP agonists (lemakalim and pinacidil) in murine colonic myocytes. RT-PCR showed expression of Kir6.2 and SUR2B transcripts in colonic smooth muscle cells, and transcripts for Kir6.1, SUR1, and SUR2A were not detected. This study has been controversial because the normal expression of Kir6.2 and SUR2B results in channels with a single-channel conductance of 80 pS (192). Jin and colleagues (193) revisited the question of Kir6 expression in murine colonic muscles and concluded that Kir channels in mouse colon are a product of expression of Kir6.1 and SUR2B. Interestingly, this report also finds an increase in expression of Kir6.1 and activity of KATP channels in a mouse model of experimental colitis. In addition to metabolic regulation, KATP currents are regulated by agonists in GI muscles. A study on gallbladder smooth muscles showed expression of KATP currents that were significantly activated by the adenylyl cyclase activator, forskolin, and treatment of intact muscles with forskolin caused hyperpolarization. The effects of forskolin were mimicked by calcitonin gene–related peptide (CGRP), the membrane-permeable adenosine cAMP analogue adenosine 3′,5′-cyclic monophosphothioate (Sp-cAMP[S]), and by the catalytic subunit of PKA. Okadaic acid, a phosphatase inhibitor, reduced the rate of recovery of basal currents after CGRP was removed. These findings show that KATP is regulated by cAMP-dependent PKA in GI muscles, and

therefore could be activated by a wide variety of endogenous agonists that are coupled to responses via activation of adenylyl cyclase. In contrast, drugs that activate PKC inhibit KATP in GI muscles. ACh and phorbol esters suppress KATP in smooth muscle cells of the rabbit esophageal muscularis mucosae (194). Others also have found suppression of KATP by PKC activation in guinea pig gallbladder (195) and murine colonic myocytes (196). In the latter study, phorbol 12,13-dibutyrate (PDBu) inhibited KATP current, and this effect was reduced by chelerythrine, an inhibitor of PKC. ACh also inhibited KATP current, and this effect was decreased by preincubation with a PKC inhibitor, calphostin C. Dialysis of cells with PKCε antibody blocked the effects of PDBu and ACh on KATP current. Ether-A-Go-Go–Related Currents Human ether-a-go-go–related gene (HERG) was cloned by homology to Drosophila K+ channels known as ether-ago-go (ERG) (197). HERG has six-membrane–spanning domains, a conserved pore domain, and a putative cyclic nucleotide binding domain. Expression of HERG channels produces an inwardly rectifying K+ current blocked by methanesulfonanilides, such as E-4031. The inwardly rectifying properties of ERG channels are due to fast voltagedependent inactivation. ERG channels result in IKr in cardiac muscles, of which defects in or blocking of can cause long QT syndrome and potentially fatal cardiac arrhythmias (198,199). An inwardly rectifying K+ selective with properties of HERG currents was found in smooth muscle cells of opossum esophageal circular muscle (200). The current was blocked by E-4031, cisapride, and La3+, all blockers of HERG. The voltage-dependent properties of these channels were such that in the range of −50 to −70 mV (near resting potential), the conductance did not fully inactivate. Thus, HERG channels may contribute to resting membrane potentials of GI muscles. A unique isoform of HERG channels was described and thought to contribute to expression of channels and the apparently unique properties of HERG in GI smooth muscles (201). The reported sequence, however, was later found to be an artifact due to contamination from murine (MERG) messenger RNA (mRNA) in samples (202,203). Three studies examined whether membrane potential and electrical activities of intact muscles were affected by blockers of ERG channels. Message for two variants of HERG was found in human jejunal muscles (202). These transcripts are consistent with the known structure of the cardiac form of HERG. HERG protein was also detected by immunohistochemical techniques in human circular and longitudinal smooth muscle cells, in enteric neurons, but not in ICC. In the presence of drugs to block neural effects, E-4031 and MK-499 (HERG channel blockers) (204) increased the amplitude of phasic contractions and enhanced the number of action potentials per slow wave in human jejunal muscles (202). Membrane potential was depolarized by 10 to 20 mV by E-4031. E-4031 and cisapride caused excitation, depolarized membrane potential, and either initiated contractile

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 561 activity or greatly potentiated contractions of guinea pig gallbladder muscles (205). Immunohistochemistry revealed expression of ERG channels in human, mouse, and guinea pig gallbladder smooth muscles. Others found ERG expression in membranes of jejunal muscles of the horse jejunum, ileum, cecum, large colon, and small colon (206). E-4031, clofilium, MK-499, and cisapride increased contractions of jejunal muscles, and the effects of E-4031 and cisapride were not additive. Taken together, data from these studies suggest that ERG channels contribute to resting membrane potential and regulation of membrane excitability in mammalian GI smooth muscles. Block of these channels may be an effective means to enhance GI motility in humans (202). Notably, cisapride was a widely used “prokinetic” drug given for a variety of human motility disorders (207–209). It was taken off the market because of life-threatening side effects attributed to its ability to block HERG channels and cause cardiac arrhythmias, such as torsades de pointes (210,211). Cisapride was thought to act either as a blocker of serotonin receptors (i.e., 5-HT3 receptors) or as an agonist of 5-HT4 receptors (212), and its stimulatory actions on GI motility (213) were suggested to be caused by these mechanisms. In clinical observations, cisapride treatment of patients with idiopathic gastroparesis invariably was associated with acceleration of gastric emptying (214). It is possible that the prokinetic actions of cisapride were, however, caused by its blocking function on ERG channels in GI smooth muscle cells (202). Although perhaps too dangerous to use in humans, prokinetic benzamides, such as cisapride, might be useful in treating motility disorders in animals, such as postoperative ileus in horses (206). Two-Pore K+ Channels Some work has identified a family of K+ channels in which individual subunits contain two-pore–forming domains (two-pore K+ channels). These channels generate currents that have been described as “background K+-selective currents.” Channels of this type may contribute to resting membrane potential and regulate cellular excitability in a variety of cells (215). Gating of two-pore K+ channels is affected by a variety endogenous agonists and agents used in clinical practice (such as volatile anesthetics), and the channels are also modulated by chemical and physical factors such as pH, O2, osmolarity, stretch, and temperature. Two-pore K+ channels activate in a quasi-instantaneous fashion and do not experience voltage-dependent inactivation. They are also largely insensitive to classical K+ channel–blocking drugs. It is now recognized that two-pore K+ channels are important functional channels in smooth muscle cells, but work to characterize the full spectrum of actions in visceral smooth muscles has really just begun. Several years ago, a K+ channel was identified in murine colonic smooth muscle cells that had a single-channel conductance of 80 to 90 pS and was activated by NO (85). Later, it was recognized that the same channels were activated by cell elongation (i.e., stretch

dependent) (216). During on-cell, single-channel recording, negative pressure applied to the outer surface of the cell membrane activated voltage-independent K+ channels with 95 pS slope conductance (Fig. 20-12). The effects of negative pressure were graded, suggesting that gating of the channels was stretch dependent. Stretching (elongation) of cells activated channels with the same properties. These channels were not regulated by Ca2+, nor were they blocked by TEA and 4-AP. NO donors and membrane-permeant cGMP analogues activated the stretch-dependent K+ channels. The authors speculate that stretch-dependent K+ channels may serve to stabilize membrane potential in portions of the GI tract with reservoir functions such as the proximal stomach and colon. Molecular analysis of colonic and other smooth muscle myocytes revealed expression of several members of the two-pore K+ channel family, including TREK-1 and -2 (KCNK2 and KCNK10) (86). TREK channels are known to be stretch dependent, and TREK-1 cloned from murine colon and expressed in COS cells formed 90 pS channels that were activated by stretch. TREK-1 channels were also activated by sodium nitroprusside and 8-bromo-cGMP. A consensus sequence for cGMP-dependent protein kinase (PKG) was found at serine 351. Mutation of this sequence (S351A) resulted in loss of responsiveness to sodium nitroprusside and 8-bromo-cGMP. This study suggests that TREK-1 encodes stretch-dependent K+ channels in GI muscles that are sensitive to nitrergic regulation. These channels are also expressed in ICC (S. D. Koh, unpublished observations), and thus may mediate part of the hyperpolarization response nitrergic nerve stimulation in GI muscles. An obstacle in determining the physiologic role of two-pore K+ channels is the lack of specific blockers for these ion channels. It was noted that some amino acids are capable of blocking hyperpolarization responses to nitrergic stimulation in some GI muscles, and a more thorough investigation of this phenomenon showed that sulfur-containing amino acids can produce relatively specific block of TREK-1 channels. L-cysteine, L-methionine or DL-homocysteine reduced the open probability of TREK-1 channels and stretch-dependent K+ channels in colonic myocytes (217). L-methionine was the most selective, having little or no effect on the other K+ conductances expressed by colonic myocytes. All of the sulfur-containing amino acids depolarized intact muscles and reduced the nitrergic portion of responses to enteric inhibitory nerve stimulation. These are the first physiologic data confirming the role of stretch-dependent K+ channels (TREK-1) in nitrergic inhibitory responses. The role of two-pore K+ channels also has been demonstrated in the maintenance of resting potentials in GI muscles. This is an important parameter because resting potential predetermines the excitability responses of GI muscles to slow wave depolarization. Although block of many K+ conductances (as described in this chapter) produces depolarization, the magnitude of the negative resting potential in GI muscles has not been fully accounted for by more standard K+. Molecular analysis of murine smooth muscles

562 / CHAPTER 20 −40 cm H2O 2 pA 5s

2 pA

A

1s

−40 cm H2O

4

NP0

3 2 1 0

B

0

20

40

60 Time (s)

80

100

120

−40 cm H2O 2 pA

Canine circular

2s

C A. mTERK1

B. non–transfected

C. I–V relationship

pA

3000 Transfected non-Transfected 2000 400pA 50ms

1000

−80 −60 −40−20

20 40 60 80 mV

D. control

E. stretch

F. I–V relationship 4000 Control Stretch pA

500pA 50ms

3000 2000 1000

−80 −60 −40−20

20 40 60 80 mV

FIG. 20-12. Stretch-dependent K+ (SDK) channels in murine colonic myocytes and mTREK-1 current expressed in COS cells. (Top, A) Application of negative pressure (−40 cm H2O) to patch pipettes increased channel activity in cell-attached patches of murine colonic myocytes. To remove contaminating currents, cells were held at 0 mV in asymmetrical K+ (5/140 mM). (Top, B) NPo plotted as a function of time in response to negative pipette pressure. Data points were tabulated every 2 seconds. (Top, C) Negative pipette pressure increased the open probability of channels with the same properties in canine colonic circular myocytes studied under identical conditions. (Bottom) Currents were recorded in whole-cell voltage clamp. The membrane potential was stepped from −80 to +70 mV in 10-mV increments for 400 msec. (Bottom, A) Representative currents from mTREK-1 transfected COS cells. (Bottom, B) Currents from nontransfected COS cells. (Bottom, C) Summarized I-V relations in transfected and nontransfected cells. (Bottom, D) Representative currents from mTREK-1 transfected COS cell in nonstretched condition. (Bottom, E) mTREK-1 currents from the same COS cell shown in D after cell elongation. (Bottom, F) Summarized I-V relations in control and after stretch. (Reproduced from Koh and Sanders [216], by permission.)

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 563 demonstrated expression of TASK-1 and -2 channels (KCNK3 and KCNK5) (218), a species of two-pore K+ channel that is inhibited by external acidification (219). TASK-2 channels cloned from murine intestinal myocytes and expressed in Xenopus oocytes resulted in a conductance that was timedependent, noninactivating, and blocked by acidic pH. A similar conductance was noted in native myocytes, and this current was blocked by local anesthetics, such as lidocaine, and acidic pH (218). Acidic pH and lidocaine caused significant depolarization of intact colonic and intestinal muscles, and these effects were additive when combined with blockers of other K+ conductances. The depolarization response to acidic pH was blocked by pretreatment with lidocaine. These data suggest a substantial contribution from TASK channels to the negative resting potentials of GI muscles. Ca2+-Activated K+ Channels Large-Conductance Ca2+-Activated K+ Channels Ca2+-dependent K+ conductances are ubiquitous in GI smooth muscles, and there is extensive literature on the properties and regulation of these conductances (for review, see Carl and colleagues [220]). By far the most widely studied channel type in GI muscles is the large-conductance, Ca2+-activated K+ channel, known as BK channels. Singer and Walsh (221,222) characterized BK channels in gastric smooth muscle cells of Bufo marinus. In symmetrical K+ gradients, these channels had a single-channel conductance of 250 pS and were activated by both voltage and increased Ca2+ concentration at the cytoplasmic surface of the membrane. Benham and colleagues (223) report similar channels in longitudinal muscle cells of the rabbit jejunum, although a somewhat smaller single-channel conductance for BK channels was observed in these cells (200 pS). An important property of BK channels is that they are activated by either Ca2+ or voltage. Sensitivity to Ca2+ can force repolarization or stabilization of membrane potential if intracellular Ca2+ is high. There is a steep relation between Ca2+ and open probability of BK channels (Fig. 20-13). For example, in studies of canine colonic and gastric smooth muscle cells, increasing [Ca2+]i from 100 nM to 1 µM was equivalent, in terms of increasing open probability, to a depolarization of 130 mV (224,225). Many GI muscles have quite negative resting membrane potentials (greater than −60 mV), and with resting cytoplasmic Ca2+ levels (<100 nM), the open probability of BK channels under these circumstances is exceedingly low. This means that BK channels are not typically a participant in resting potentials in GI muscles, and depolarization and influx of Ca2+ during excitable events (or a series of excitable events such as multiple action potentials) recruits BK channels. A comparative study of circular and longitudinal muscles of the canine colon illustrates how different muscles of the GI tract use BK channels (226). Cells of both circular and longitudinal muscle layers express BK channels and there was no difference observed in channel density, Ca2+ sensitivity, or inhibition by ChTX in circular and longitudinal myocytes. Unstimulated longitudinal muscle

strips were depolarized by ChTX, and the frequency of action potentials and the amplitude of contractions were increased. Unstimulated circular muscles were unaffected by ChTX, but slow waves were enhanced in circular muscle by this toxin after stimulation with ACh or Bay K 8644. Notably, however, some GI muscle cells express a large number of BK channel copies (e.g., 10,000). Thus, even with low open probability, occasional openings of these largeconductance channels might contribute to resting conductances. In tonic smooth muscles, which generally have less negative resting potentials and ongoing Ca2+ influx to maintain tone, BK channels may play an important role in balancing inward currents, as occurs in vascular smooth muscles (see Nelson and Quayle [227]). The sensitivity of BK channels to Ca2+ is greatly enhanced by β subunits that coassemble with the α pore-forming subunits (228,229). Regulation of BK channels may be an important means of controlling the excitability of GI muscles. Muscarinic stimulation of canine colonic smooth muscle cells resulted in suppression of BK channels, and these effects were mediated by pertussis toxin–sensitive G proteins (230,231). This regulation of BK channels may participate in the excitatory effects of muscarinic stimulation of colonic muscles. Part of the suppression of BK channels by muscarinic stimulation could be caused by activation of PKC, which decreases the Ca2+ sensitivity of BK channels (e.g., 232). NO and NO donors enhance openings of BK channels (233,234). Increased BK channel open probability in response to NO is caused by a negative shift in the voltage dependence of channel openings. Effects of NO are mediated by cGMP-dependent mechanisms and activation of protein kinase G (PKG). Exposure of the cytoplasmic surface of membrane patches to PKG shifted the voltage dependence of BK channels toward more negative potentials (235,236). Studies of the mechanism of PKG regulation of BK channel open probability were performed on cloned channels expressed in HEK293 cells (237). NO donors increased BK currents in whole-cell recordings and the open probability of BK channels in cellattached patches. The effects of NO donors were blocked by KT5823, a PKG inhibitor. Consistent with studies on native smooth muscle cells, exposure of the cytoplasmic surface of membranes with BK channels resulted in enhanced open probability. Point mutations of the α subunit (S1072A), within the consensus sequence for PKG phosphorylation, abolished the effects of PKG and NO donors. An important means of BK-channel regulation in many smooth muscle cells is activation via localized Ca2+ release from intracellular stores. Benham and Bolton (238) observed spontaneous transient outward currents (STOCs) in longitudinal muscle cells of the rabbit jejunum and vascular smooth muscle cells. These events were caused by activation of clusters of BK channels (calculated to be up to 100) and are thought to be because of spontaneous release of Ca2+ from intracellular stores (239,240). In vascular smooth muscles, Ca2+ release has been attributed to spontaneous activation of ryanodine receptors, and the release events have been termed Ca2+ sparks (241). Ca2+ sparks were suggested as a

564 / CHAPTER 20 30

A

N.p (open) 20

HP (mV) 1.0

100 mV

100

0.5

2+

0

Ca

0.1 −100

) m (µ

90 mV

B

70 mV

60 mV

20 pA 500 msec

1e-7

1e-6 Ca2+ (M)

1e-5

C Open probability

50 mV

120 100 80 60 40 20 0 −20 −40 −60 −80

VΩ

80 mV

1

0 0.1

1 Ca2+ (µm)

10

FIG. 20-13. Voltage and Ca2+ dependence of large-conductance, Ca2+-activated K+ channels (BK) in canine colonic myocytes. (Left) Open probability at negative to +50mV was low, but at more positive potentials, open probability increased and multiple channels were apparent in symmetrical K+ gradient (140/140 mM K+, 1 µM Ca2+) in a cell-attached patch. (A) Free Ca2+ in the bath was increased from 0.1 to 1 µM in steps of 0.025 µM in inside-out patches. Increasing [Ca2+] caused a parallel shift of activation curves to the left. Open probability [Np(open)] was measured using voltage ramps. Data were fitted with Boltzmann functions. (B) Voltage for half-maximal activation (V1/2) plotted as a function of log Ca2+ concentration. Data were obtained from inside-out patches (closed circle), artificial lipid bilayer (open circle), and a cloned BK channel from canine cSlo locus (open diamonds). (C) Ca2+ dependence of open probabilities. Data points were calculated from Boltzmann curves generated from measurements shown in B. All data are fit by Hill functions with a coefficient of 3.2. HP, holding potential. 䊊 = −100mV, 䊐 = −60 mV, 䉭 = −20 mV, 䉮 = +20 mV, 䉫 = +60 mV, = +100 mV. (Left: Reproduced from Carl and Sanders [224], by permission; Right: Reproduced from Carl and colleagues [220], by permission.)

regulatory mechanism to regulate vascular tone by transient activation of BK channels. Spontaneous Ca2+ release events also occur in GI smooth muscles; but in the GI tract, either ryanodine receptors (Ca2+ sparks) or IP3 receptors (Ca2+ puffs) can mediate localized Ca2+ transients. Several investigators have reported Ca2+ sparks in GI muscles and cells of both phasic and tonic muscles, such as guinea pig ileum smooth muscle cells (242), toad gastric muscle (243), guinea pig gallbladder smooth muscle cells (244), and feline esophageal smooth muscle cells. Kirber and colleagues (245) have shown that Ca2+ sparks are coupled to activation of BK channels. In tonic muscles, Ca2+ sparks may maintain a low level of BK-channel activation by initiating periodic sparks in different cells. This effect, like in vascular muscles, may

produce tonic net outward current and help to set resting potentials and regulate membrane excitability. Stimulation of STOCs may also provide an important feedback mechanism to reduce excitability in response to Ca2+ entry via enhanced Ca2+-induced Ca2+ release. Activation of BK channels (and small-conductance, Ca2+-activated K+ channels, SK; see later for additional discussion on SK channels) also occurs in response to IP3 receptor–dependent Ca2+ release. In murine colonic muscles, ryanodine receptor–mediated Ca2+ sparks were not found to be responsible for spontaneous Ca2+ transients. The events in murine colon were blocked by phospholipase C inhibitors and xestospongin C and were referred to as “Ca2+ puffs” because they were caused by release of Ca2+ from IP3

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 565 receptor–operated stores (246). STOCs, due to both ChTXsensitive and -insensitive K+ conductances, were induced by Ca2+ puffs (247). Purinoreceptor (P2Y) agonists, ATP and 2-methylthio-ATP, enhanced the occurrence of Ca2+ puffs and increased STOCs. Ca2+ release from ryanodine receptors was also recruited in response to P2Y receptor stimulation. Activation of K+ channels (STOCs) in response to P2Y receptor stimulation may mediate hyperpolarization and IJPs in response to purinergic stimulation in GI muscles. Stimulation by muscarinic agonists also leads to activation of phospholipase C and IP3 production. How excitatory and inhibitory responses can be achieved via the same pathway was explored by studying the effects of ACh on Ca2+ transients and STOCs in colonic myocytes (248). ACh was found to reduce localized Ca2+ transients and STOCs, and these effects were associated with an increase in basal cytosolic Ca2+. Blocking nonselective cation channels prevented the increase in basal cytosolic Ca2+ and increased the frequency and amplitude of Ca2+ transients in response to muscarinic stimulation. Similar results were obtained with substance P, another excitatory transmitter; but in the case of substance P, basal Ca2+ was increased via activation of L-type Ca2+ current (249). The degree to which Ca2+ transients couple to activation of BK channels in the plasma membrane is regulated in colonic myocytes by a Ca2+-dependent PKC (232). Inhibition of PKC greatly increased the amplitude and frequency of STOCs with no apparent change in Ca2+ transients. Single-channel studies also demonstrated that BK channels are inhibited by a Ca2+-dependent PKC expressed by colonic myocytes. β1 subunits of BK channels are required for regulation by PKC (250). PKC regulation of coupling strength may serve as a parallel excitatory mechanism in GI smooth muscle cells. Agonists that increase phospholipase C activation may lead to stimulation of PKC and reduce the sensitivity of BK channels to Ca2+. Small- and Intermediate-Conductance Ca2+-Activated K+ Channels Two additional types of Ca2+-activated K+ channels have been described in GI muscles (251–253). Small-conductance, Ca2+-activated K+ channels are encoded by the KCa2.1, KCa2.2, and KCa2.3 genes (KCNN gene family), and a related intermediate-conductance (IK) channel is encoded by KCa3.1 (see Stocker [254]). The topology of SK and IK channels is similar to KV channels with six-membrane–spanning regions and a pore region between the fifth and sixth segments. The S4 region, which conveys voltage-dependence in KV channels, has few positive charges in SK channels. This difference may explain the voltage independence of these channels. SK and IK channels are sensitive to cytosolic Ca2+ and, as described in the previous section, may participate in IJPs due to localized release of Ca2+ from IP3 receptor–mediated stores. Apamin, a blocker of SK channels, is a well-known inhibitor of the fast (purinergically mediated) IJP in GI muscles (for review, see Hoyle and

Burnstock [255]). Molecular studies of murine GI muscles suggest that SK2 channels are the dominant channels expressed (256). Ca2+ sensitivity of SK and IK channels is because of constitutive association of calmodulin with the channel α subunit in a Ca2+-dependent manner (257). Ca2+ binding to the calmodulin associated with SK and IK channels initiates conformation changes and gating of the α subunit. Regulation of open probability by Ca2+/CaMKII provides a secondary level of Ca2+ regulation of SK channels in GI muscles (258). Preactivated CaMKII increased the open probability of SK channels. Calmodulin inhibitors, KN-93 and W-7, decreased the open probability of SK channels in on-cell patches from murine colonic myocytes, but these effects were not observed in excised patches. In murine ileum smooth muscle cells, small-conductance (SK) channels with about 10 pS conductance were observed that were insensitive to TEA and blocked by 500 nM apamin (251). Channels (5 pS) with similar properties were also found in murine colonic smooth muscle cells (253). Apamin-sensitive outward currents were activated under whole-cell, on-cell, single-channel recording conditions by extracellular ATP, uridine 5′-triphosphate, and 2-methylthioadenosine 5′-triphosphate (2-MeS-ATP). Pyridoxal phosphate 6-azophenyl-2′,4′-disulfonic acid tetrasodium (PPADS), a nonselective P2 receptor antagonist, blocked the activation of SK channels by ATP and 2-MeS-ATP. ATP-induced hyperpolarizations and purine-mediated IJPs in GI muscles may be the result of P2Y receptor–induced release of Ca2+ from stores and activation of SK channels (246,253). Two channels were found to be sensitive to external TEA (BK channels and 40 pS IK channels) in mouse ileal circular smooth muscle cells (252). Increasing Ca2+ from 80 to 150 nM at the cytoplasmic face of membrane patches increased the open probability of IK channels substantially. IK channels were blocked by millimolar concentrations of external TEA, 100 nM ChTX, and 500 nM apamin. The sensitivity of IK channels to Ca2+ suggests these channels might contribute significantly to Ca2+-dependent currents in GI muscles. The role of IK channels in IJPs, however, is questionable because TEA and ChTX do not affect IJP amplitudes or P2Y-mediated hyperpolarization (259). Chloride Conductances Volume-Sensitive Cl− Conductances Volume-sensitive or swelling-activated Cl− currents have been reported in a variety of cell types, and there has been speculation that this conductance is encoded by the ClC-3 gene. ClC-3 is expressed in canine colonic myocytes, and these cells also displayed an outwardly rectifying Cl− current that is activated by swelling cells with reduced extracellular osmolarity (i.e., 300 to 250 mosM; Fig. 20-14) (260). The swelling-activated current in these cells was outwardly rectifying and dependent on the Cl− gradient. The current was blocked by tamoxifen or DIDS

566 / CHAPTER 20 A

B

Difference

3000

Hypotonic

1000 pA 100 mS

Current (pA)

Isotonic

+120 mV

2000

1000

ECl

0

1000

−100 −60 −20 20 60 100

0

−40 mV

−100 −60 −20 20 60 Test potential (mV)

−100 mV

C

2000

100

D 400

Difference

100

Hypotonic

100 pA 100 mS

+10 mV

200

−100 −200 −80 −60 −40 −20

−80 mV

0

0 −200 −80

−40 mV

ECl

0

Current (pA)

Isotonic

−60

−40

−20

0

Test potential (mV)

FIG. 20-14. Swelling-activated chloride currents in canine colonic myocytes. (A) Representative currents elicited by test potentials from 100 to +120 mV in 20-mV increments in isotonic and hypotonic solutions. Current activated by hypotonic solution is shown as difference current. (B) Summarized current-voltage (I-V) relations. Open and solid squares represent currents obtained in isotonic and hypotonic solutions, respectively. I-V relation for swelling-induced difference current is shown in inset of B. Reversal potential for difference current (dotted line) corresponds to Nernst equilibrium potential for Cl (ECl). (C) Representative tracings showing current elicited by from 80 to +10 mV in 6-mV increment in isotonic and hypotonic solutions. Swelling-activated current is also shown as difference currents. (D) Summarized I-V relations in isotonic and hypotonic solutions. Asterisks denote statistical differences between currents in isotonic (open squares) and hypotonic (solid squares) solutions. Inset shows summary I-V for difference current. (Reproduced from Dick and colleagues [260], by permission.)

(4,4-diisothiocyanato-stilbene-2,2′disulphonate) but unaffected by niflumic acid (25 µM), nicardipine, and removal of extracellular Ca2+. The swelling-activated Cl− current was regulated by PKC, and PDBu decreased the current. Extracellular ATP (at millimolar concentrations) inhibited the swelling-activated current. Currently, it is difficult to evaluate the importance of the swelling-activated Cl− conductance in GI muscles because its pharmacology overlaps with voltage-dependent cation channels. For example, tamoxifen and DIDS both inhibited L-type Ca2+ currents and delayed rectifier currents in colonic myocytes (261). Ca2+-Activated Cl − Conductances There has been little evidence for Ca2+-activated Cl− currents in GI smooth muscles. Such a conductance was found in rabbit esophageal muscularis mucosae muscles (112). Depolarization of cells dialyzed with solutions containing CsCl and no EGTA resulted in initial inward Ca2+ currents followed by substantial outward currents. On repolarization, the outward currents reversed to long-lasting inward

tail currents, a signature for the presence of Ca2+-activated Cl− currents. Outward currents were activated at more negative potentials when the equilibrium potential for Cl− ions was shifted to more negative potentials, suggesting that the outward current was carried by Cl−. Niflumic acid, a Cl− channel–blocking compound, reduced the outward current initiated by depolarization and the inward tail currents that occurred on repolarization. The inward tail currents were abolished when Ba2+ was used as the charge carrier or when cells were dialyzed with high concentrations of EGTA. These data suggest that an increase in [Ca2+]i is necessary for activation of the outward currents at depolarized potentials and the tail currents on return to negative potentials. The inward tail currents were not due to Na-Ca exchange, because replacement of external Na+ with TEA did not significantly alter their amplitude. A novel form of Ca2+-activated Cl− channels (ClCA4) was identified in murine GI muscles (262). This isoform was highly expressed in stomach and gastric muscles, but a current with the properties of a Ca2+-activated Cl− conductance has not been observed in muscle cells from these tissues.

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 567 potentials (e.g., −80 mV) but increases as a function of depolarization. IACh reaches a maximum near −40 mV and reverses at approximately 0 mV (272). IACh also carries divalent ions (267), but whether Ca2+ entry in physiologic gradients via these channels is an important source of Ca2+ for contraction is controversial (see Pacaud and Bolton [271]). Activation of IACh via muscarinic receptors is mediated by G proteins (269). Pertussis toxin rendered cells of the guinea pig ileum unresponsive to ACh. Dialysis of cells with ADPβS, which competes with GTP for G-protein binding sites, also inhibited activation of IACh in response to muscarinic agonists. Dialysis with GTPγS, however, activated a current with properties similar to IACh, and the effects of GTPγS dialysis and ACh application were not additive. Neurokinins (275) also activate nonselective cation currents in GI muscles (see Fig. 20-15C), but it should be noted that the properties of the nonselective cation conductance activated by substance P and neurokinin A (NKA) differed from those of IACh in canine colonic smooth muscle cells. Currents activated by NKA (or substance P) and ACH summed in response to cumulative application of the agonists (275). Thus, it is possible that each agonist regulates different populations of nonselective cation channels. Other agonists, coupled via Gq/G11, might also be expected to activate nonselective cation conductances in smooth muscle cells. IACh is facilitated by intracellular Ca2+ (220,268,271,272, 274). This is consistent with the observation that under

Nonselective Cation Channels Like all smooth muscles, GI muscles express nonselective cation conductances. These channels may be involved in a variety of functions, including responses to agonists, activation in response to changes in membrane potential, capacitative Ca2+ entry, responses to stretch, regulation of membrane potential, and responses to heat. However, these types of ion channels have received the least amount of attention during the past decade. Part of the difficulty in studying nonselective cation conductances is that multiple conductances are potentially expressed in GI muscle cells, but blockers of the channels show poor selectivity. Thus, it is hard to isolate a specific population of channels for study. Reviews by Albert and Large (263) and Inoue and Mori (264) are useful for detailed summaries of capacitative Ca2+ entry mechanisms in smooth muscles. Currents Activated by Muscarinic and Peptide Agonists One conductance that has been characterized in great detail is the nonselective cation conductance activated by muscarinic stimulation in GI muscles (265–274). The inward current activated by muscarinic stimulation is caused by a nonselective cation conductance, referred to as IACh (Fig. 20-15A and B). IACh is voltage dependent in many GI smooth muscle cells. The conductance is low at negative

A

SP

C 100 pA 60 ms

10−7

M

B

M

NKA

SP 10−6 M

150

Current (pA)

NKA 10−8

3×10−8 M

100 50

NKA

SP 10−5 M

10−7 M

0 −50 −80

−60

−40

−20

0

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20 pA

20 30 S

FIG. 20-15. Nonselective cation currents in canine colonic myocytes. (A) Difference currents calculated from currents generated before and in presence of acetylcholine (ACh; 10 µM). Cell was stepped to potentials ranging from −80 to +40 mV from a holding potential of −80 mV. (B) Summarized I-V relation for IACh. Data points represent current amplitude at end of depolarization pulses. IACh increased over range of −80 to −40 mV. (C) Substance P (SP) elicited a noisy nonselective cation current in a concentration-dependent manner at a holding potential of −70 mV (Cl− equilibrium potential). Responses were sustained for duration of SP exposure. Similar currents were activated under identical conditions by neurokinin A (NKA). (Reproduced from Lee and colleagues [274,275], by permission.)

568 / CHAPTER 20 current-clamp conditions, depolarization of guinea pig ileum muscle cells by ACh was greatly attenuated when the cells were exposed to Ca2+-free buffer (269). Early studies suggest that Ca2+ alone was not capable of directly activating the conductance (269); however, others have found that Ca2+ release in the absence of muscarinic stimulation can activate IACh (272). These findings may indicate differences in the properties and regulation of this conductance in different tissues or species. Depolarization-induced Ca2+ influx provides a source of Ca2+ that facilitates IACh, but release of Ca2+ from stores due to IP3 receptor–operated channels also acts to facilitate the current (271). Release of Ca2+ from stores may facilitate the initial activation of IACh that results in depolarization and activation of voltage-dependent Ca2+ channels. In contrast with IACh, nonselective cation currents activated by neurokinins in canine colonic smooth muscles lacked Ca2+ sensitivity (275). There have been difficulties in determining the contribution of IACh to physiologic responses in vivo or in intact muscles because of the lack of selective blockers for this conductance. Inoue (270) reports that low concentrations of divalent ions (i.e., Cd2+, Ni2+ with Kd of about 100 µM) reduced the amplitude of IACh in guinea pig ileal muscle cells; however, these blockers also inhibit voltage-dependent Ca2+ channels. The importance of IACh in physiologic responses has been questioned by findings that smooth muscle cells may not be directly innervated by excitatory cholinergic motor neurons and may not see significant concentrations of transmitter (see Role of Interstitial Cells of Cajal in Neurotransmission earlier in this chapter) (52). In the absence of ICC-IM there is little or no response to cholinergic nerve stimulation. It is not yet known whether ICC-IM and ICC-DMP (ICC that mediate neural inputs) express IACh, but Hirst and coworkers (83) have found that when unitary potential discharge is blocked in ICC-IM of guinea pig antral muscle bundles, the remaining excitatory junction potential (EJP; presumably due to activation of an inward current) is only a few millivolts in amplitude and is not enhanced by hyperpolarization. The latter suggests that a conductance other than a nonselective cation conductance might be responsible for the EJP. Additional studies are required to determine the ionic conductance(s) in ICC-IM and ICC-DMP that are regulated by muscarinic stimulation. Currents Activated by Adenosine Triphosphate Purines activate nonselective cation channels formed by P2X receptors in a variety of cells including many smooth muscles cells. In general, P2X receptors are less prominent in GI muscles because the response to purine transmitters in most instances is inhibitory, not excitatory. There are reports, however, of purinergic excitatory effects in GI muscles, and these responses are likely to be caused by activation of P2X receptors. P2X receptors are classic ligand-gated ion channels, and the channels in smooth muscles are typically rapidly desensitizing and activated by the putative P2Xselective agonist, α,β methylene ATP sensitive (276,277).

Nucleotides elicited contractions in the murine colon (but not in stomach and small intestine), and these responses were blocked by suramin and PPADS and showed no desensitization. Immunohistochemistry showed expression of P2X2 receptors in colonic circular and longitudinal muscles but not elsewhere in the GI tract (278). In the cat colon, stimulation of the lumbar colonic nerve (LCN) resulted in contractile responses due to α-adrenoceptor and P2X receptor stimulation (279). Desensitization with the putative P2X receptor agonist, α,β-methylene ATP, reduced the amplitude of LCN contractile responses. P2 antagonists, arylazido amino propyl adenosine triphosphate (ANAPP3) and suramin, also reduced LCN contractions. These data suggest that sympathetic neurons may innervate sites that express P2X receptors to elicit contractile responses in the colon. Little is known about the expression or characteristics of the P2X receptors activated in GI muscles. Studies have demonstrated that nonselective cation currents (IATP) can be activated in circular and longitudinal smooth muscle cells of the canine colon by ATP and purine agonists (H. K. Lee and K. M. Sanders, unpublished data). The currents in the two muscle layers had different properties. IATP in circular muscles was elicited by ATP but was only weakly sensitive to α,β-methylene ATP. Both agonists elicited currents in longitudinal muscle cells. I-V relations showed that IATP reversed at −13 mV and was carried mostly by Na+ ions in physiologic gradients. Circular muscle cells expressed mRNA encoding P2X2, P2X3, and P2X4, whereas longitudinal cells expressed P2X3 and P2X5. Functional and molecular data suggest differential expression of P2X subtypes in the different muscle layers. These channels may participate in the excitatory responses of colonic muscles to sympathetic nerve stimulation (279). Currents Activated by Stretch Some nonselective cation channels in GI muscles are activated by stretch, and these are potentially important in the regulation of smooth muscle responses. There have been few studies of these channels in mammalian GI muscles, but work on toad gastric muscle cells has revealed a variety of cation channels that are activated by negative pressure applied to cell-attached or excised, inside-out patches. Channels activated by negative pressure were permeable to cations and showed outward rectification and a slope conductance of 30 pS at negative potentials when divalent cations were absent from the pipette solution (280,281). Imaging techniques were used to show the impact of activating stretchactivated cation channels on global Ca2+ (282). With Ca2+ only in the pipette solution, a focal increase in Ca2+ was observed on membrane stretch, but when Ca2+ was present in the bath solution, the focal increase was amplified by depolarization and Ca2+ entry through voltage-gated Ca2+ channels. Entry of Ca2+ through stretch-activated channels was also amplified by Ca2+ release from stores. From measurements of total fluorescence, it was found that up to 18% of the current carried by stretch-activated channels in

ORGANIZATION AND ELECTROPHYSIOLOGY OF INTERSTITIAL CELLS / 569 physiologic ion gradients was carried by Ca2+ at negative resting potentials (283). This amount of Ca2+ was estimated to be high enough to activate local BK channels or to initiate release of Ca2+ from stores. Others reported two types of stretch-activated nonselective cation channels in guinea pig gastric muscles (284). Negative pressure applied to on-cell pipettes resulted in the activation of channels with short- and long-lasting opening kinetics and conductances of 33 and 53 pS, respectively. Both channels were blocked by inclusion of 100 µM Gd3+ in the pipette solution. Stretch-activated channels may be important in regulation of membrane potential or activation of muscles in response to stretch, or both. The molecular entities responsible for stretch-activated nonselective cation channels in GI muscles have not been identified.

INTEGRATION OF ELECTRICAL ACTIVITY IN GASTROINTESTINAL MUSCLES This chapter has reviewed many of the basic electrophysiologic functions and properties of the ICC and smooth muscle tissues that generate force in GI motility. Even in the absence of neural, hormonal, or paracrine substance influence, the ICC and smooth muscle syncytium accomplishes spontaneous electrical activity and excitation–contraction coupling. Figure 20-9 shows a schematic summarizing some of the functions of ICC and smooth muscle cells in GI muscles. ICC in pacemaker regions generate spontaneous pacemaker activity in the form of unitary potentials. Coordination (entrainment) of unitary potentials results in slow wave activity. A continuous network of ICC-MY (and probably ICC-SEP in animals with thick GI muscles) is required for active propagation of slow waves around and along GI organs. Slow waves conduct via low-resistance junctions to smooth muscle cells. Smooth muscle cells do not have the apparatus to generate or regenerate slow waves, thus slow waves decay as a function of distance from the ICC network. The rate of decay is dependent on the cable properties of the smooth muscle syncytium. The cable properties can be modulated via the plethora of ionic conductances expressed by smooth muscle cells. Slow wave depolarization elicits voltage-dependent responses in smooth muscle cells. Voltage-dependent, L-type Ca2+ channels are clearly the most important class of ion channels in terms of excitation– contraction coupling, but a variety of K+ channels are also activated during slow waves, and these conductances tune the smooth muscle response. Oscillation of membrane potential through the slow wave cycle results in periods of high and low open probability for Ca2+ channels, and this naturally organizes the contractile pattern into a series of phasic contractions contributing to motility patterns such as peristalsis and segmentation. The force and pattern of phasic contractions depends on coordination from the enteric nervous system. Both excitatory and inhibitory neural inputs are available to

regulate contractions. Neural inputs set the excitability of the smooth muscle cells by regulating resting membrane potential or by altering the responses of voltage-dependent channels to slow wave depolarizations. Inhibitory inputs from enteric motor neurons that release NO and ATP result in activation of K+ conductances, stabilization of membrane potential, and suppression of unitary potentials generated by ICC-IM. All of these responses tend to decrease depolarization responses to slow waves, reduce the tendency for action potential generation, and reduce excitation–contraction coupling. Excitatory neurons enhance inward currents or suppress outward currents, leading to enhanced depolarization responses in smooth muscle cells. Input from these neurons tends to increase the open probability of Ca2+ channels, enhance generation of Ca2+ action potentials, and increase excitation–contraction coupling. Superimposed on neural regulation are multiple layers of additional regulation caused by circulating hormones and paracrine substances that can also affect smooth muscle responsiveness. Thus, the final output of smooth muscle contraction and GI motility is a highly complex, integrated response involving multiple cell types and multiple regulatory levels. Other chapters in this textbook describe the contractile patterns of the gut and the various regulatory mechanisms that tune smooth muscle responses.

ACKNOWLEDGMENTS Preparation and the initial discovery of a considerable amount of the information in this chapter was supported by a Program Project Grant from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (P01 DK41315). Dr. Sanders also received support from a MERIT Award (DK40569), and Dr. Ward was also supported by a grant (DK 57236). The authors acknowledge significant contributions from the late Professor Burton Horowitz to the topics covered in this chapter and thank him for his friendship, energy, and insight.

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CHAPTER

21

Functional Histoanatomy of the Enteric Nervous System Simon J. H. Brookes and Marcello Costa Overview, 577 Insights from Structural Studies, 577 Enteric Ganglia and Plexuses, 578 Evidence for a Structural Basis for Intestinal Motility, 579 Characteristics for Distinguishing Classes of Enteric Neurons, 579 Intrinsic Innervation of the Gut Wall, 582 Intrinsic Sensory Neurons of the Gut Wall, 582 Enteric Motor Neurons, 583 Interneurons in the Gut Wall, 586 Viscerofugal Neurons, 588

Secretomotor and Vasomotor Neurons, 589 Interplexus Interneurons, 590 Extrinsic Innervation of the Gut Wall, 591 Parasympathetic Innervation of the Gut, 591 Sympathetic Innervation of the Gut, 592 Extrinsic Sensory Nerve Endings, 592 Vagal Afferents, 592 Spinal Afferents, 593 Heirarchies of Neurochemicals, 594 Summary and Conclusions, 595 Acknowledgments, 595 References, 595

OVERVIEW

The muscularis externa, consisting of smooth muscle cells and associated interstitial cells of Cajal (ICCs) also has significant integrative potential. It too can be considered as a quasi-independent control system in its own right. This chapter focuses on one of these control systems: the innervation of the gut wall. The enteric nervous system is classified as a separate division of the autonomic nervous system (1). Enteric neurons are targeted by axons of sympathetic and parasympathetic neurons originating outside the gut wall. Axons of extrinsic sensory neurons, originating from both spinal (dorsal root) ganglia and the ganglia of the vagus nerve (nodose and jugular ganglia), comprise another major source of innervation of the gut wall. Extrinsic nerve endings within the gut wall are included in this review because they contribute to the functioning of the enteric nervous system.

The process of ingesting food, subjecting it to chemical degradation, and absorbing the products is fraught with hazards. The acid, chemicals, and enzymes needed for breaking down food pose a threat of autodigestion. Toxins and pathogens are ingested with food, threatening to turn the consumer into the consumed. A number of control systems have evolved to allow the gut to achieve its function and simultaneously protect it from these dangers. Nerve cells form one major control system, acting on the muscularis externa, epithelial cells, and blood vessels. The gut also has a substantial endocrine system, with numerous types of enteroendocrine cells in the mucosa that strongly influence neurons and other cell types both within and outside the gut. The immune system in the gut, with mast cells, resident macrophages, and white blood cells, can be activated at short notice and forms another powerful control system.

Insights from Structural Studies Understanding how neural circuits control digestive processes requires knowledge of the nature of the cells that form the system, their location, distribution, and relationships, both with one another and with other cells and tissues in the gut wall. This “structural organization” provides a

S. J. H. Brookes and M. Costa: Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

577

578 / CHAPTER 21 framework to interpret how the system changes over time, commonly described as “function.” As Ramón y Cajal demonstrates in his masterly work (2), considerable understanding of function can be inferred from studies of structure. Every activity of a cell, tissue, or organ has a structural basis; the salient point is whether appropriate techniques are available to reveal the underlying structure. In assemblies of cells, complex responses may “emerge” from interactions among multiple elements. Such “emergent properties” may require sophisticated modeling techniques to understand their cellular basis. Nevertheless, studying the innervation of the gut with morphologic and histologic techniques has contributed significantly to our current understanding of how nerve cells control gut activity. This chapter reviews current views of the structure of the nervous system within the wall of the gut. A description of the general arrangement of the nervous tissue is followed by an analysis of the major types of enteric nerve cells on the basis of their shape, neurochemistry, biophysical properties, and connectivity.

Intravillous muscle

Muscularis mucosae

Submucous plexus

Enteric Ganglia and Plexuses The existence of numerous nerve cells within the wall of the gut has been known since the early studies of Meissner (3) and Auerbach (4). Using histologic stains, they described extensive networks of ganglionated neural plexuses between layers of the gut wall (Fig. 21-1). When the enteric nervous system develops, ganglia are laid out in a regular pattern along the gut, which varies greatly in appearance in different regions and species (5) (Fig. 21-2). It has been estimated that the guinea pig small intestine contains 2,750,000 myenteric neurons and 950,000 submucous neurons (6). The total numbers of enteric nerve cells vary with size across species (7); the mouse has about 400,000 myenteric and 330,000 submucous neurons, whereas the sheep has been estimated to have 30,000,000 myenteric and more than 50,000,000 submucous neurons. These counts of enteric neurons may, in fact, be underestimates, because they are based on a histochemical method that may not stain every nerve cell body (8,9). For example, a more recent estimate calculates the number of neurons in the guinea pig small intestine as about 6,000,000 (9). Every ganglion of the submucous and myenteric plexuses contains an intermingled selection of many functional classes of nerve cells, which means that location gives few clues about the functional identity of any particular enteric neuron. There are also numerous enteric glial cells that outnumber neurons in a ratio of up to 4:1 (10). These form dye-coupled and electrically coupled networks throughout the ganglia (11) and play a role inflammatory responses and damage in the gut wall (10,12,13). Running through the ganglia are nerve fibers arising from both enteric neurons and extrinsic afferent and efferent neurons. These do not form a defined neuropil, but rather course around nerve and glial cell bodies throughout the ganglion. In many cases,

Myenteric plexus

Circular muscle

Longitudinal muscle

FIG. 21-1. Cross section of guinea pig ileum stained for smooth muscle actin. The muscularis externa, muscularis mucosae, and intravillous muscle (an extension of the muscularis mucosae) are all clearly visible, with the myenteric and submucous ganglia visible between layers.

varicose swellings on these axons form close appositions or specialized synaptic contacts (with postsynaptic densities) on enteric nerve cell bodies or dendrites (14,15). The myenteric plexus is continuous, essentially running from the mid-esophagus to the anal sphincter in humans. In contrast, the submucous plexus is absent or sparse in the esophagus and stomach, but it is present throughout the small and large intestines. In small laboratory animals, such as the guinea pig and rat, the submucous plexus forms a single network, with all ganglia lying in the same plane. In some regions of gut, a nonganglionated plexus of nerve fibers has been described at the submucosal border of the circular muscle (16). In larger animals, the submucous plexus is divided into at least two distinct ganglionated layers. The inner submucous plexus (Meissner’s plexus) and outer submucous plexus (Schabadasch’s or Henle’s plexus) have been distinguished in the pig small intestine (17), horse jejunum (18) and human colon (19) (Fig. 21-3). At least in humans there is also a third intermediate submucous plexus. Individual ganglia within a plexus are not specialized; each ganglion contains a random selection of all of the different

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FIG. 21-2. Whole mounts of guinea pig ileum labeled to reveal neural tissue using antisera to protein gene product 9.5. The myenteric and submucous plexuses, with nerve processes innervating surrounding tissue are taken from the same region of a single preparation.

classes of neurons. However, the proportions of each class vary among the different plexuses in the submucosa (17,20,21).

Evidence for a Structural Basis for Intestinal Motility Studies on extrinsically denervated canine intestine (22) revealed reflexes that could not be mediated by central neuronal pathways. These observations led Bayliss and Starling (22) to write: “Local stimulation of the gut produces excitation above and inhibition below the excited spot. These effects are dependent on the activity of the local nervous mechanism.” Studies of isolated preparations of intestine confirmed that the enteric nervous system could produce integrated, adaptive responses to physiologic stimuli (23,24). It was readily deduced that some enteric neurons must be able to detect stimuli, such as distension, and hence function as sensory neurons. These must activate nerve cells that cause muscle contraction or relaxation (excitatory and inhibitory motor neurons, respectively). Bayliss and Starling (22) went on to state: Auerbach’s plexus is in fact a local nervous system with two reflexes, inhibition and augmentation, and one function, propulsion of food. The different time-relations of the two reflexes would lead one to guess that the system is composed of long path which conduct inhibitory impulses downward, and short paths in an upward direction. A histological testing of this hypothesis presents however considerable difficulties.

Identifying the enteric neurons in these pathways took many more years of study and, indeed, is still continuing today.

Apart from primary afferent neurons and inhibitory and excitatory motor neurons, there are also secretomotor neurons that activate ion fluxes and water movement across the mucosal epithelium and control acid output in the stomach. Vasomotor neurons control blood vessel diameter to adjust blood flow to the metabolic needs of the gut, particularly during secretory activity. Interneurons project up and down the gut; a specialized type of interneuron projects out of the gut wall to prevertebral sympathetic ganglia and is involved in gastrointestinal reflexes that do not require connections with the central nervous system (25). Characteristics for Distinguishing Classes of Enteric Neurons Morphology The earliest classification schemes for enteric neurons were based on soma-dendritic morphology using histologic stains (26,27). Typically, enteric neurons have simpler soma-dendritic morphology than many peripheral neurons (e.g., sympathetic neurons in prevertebral ganglia) and many central neurons, probably reflecting relatively simple synaptic integration. Dogiel identified three classes of cells; later schemes have identified up to seven (28,29). This approach has been powerful for distinguishing different populations of neurons, but it is not readily applicable to identify each single nerve cell, for example, after electrophysiologic recording. Substantial differences exist in the shapes of functional classes of cells among species (28). With the addition of cytoskeletal staining techniques using immunohistochemical

580 / CHAPTER 21 Mesenteric nerve trunk

Lymphatic vessel

Longitudinal muscle Myenteric plexus Tertiary plexus

Mesenteric artery & vein

Mesenteric membranes

Circular muscle Deep muscular plexus Inner circular muscle layer Outer submucous plexus

Submucosal venule & arteriole

Submucosa

Inner submucous plexus

Aboral

Mucosal villi

Muscularis mucosae

Mucosal plexus

Oral

FIG. 21-3. Diagrammatic layout of the human small intestine, with ganglionated and nonganglionated plexuses and muscle layers revealed.

labeling of neurofilament proteins, morphologic classification is becoming both more robust and more compatible with chemical coding studies (30,31). Histochemistry Another approach, distinguishing classes of enteric neurons by the combinations of neurochemicals found in their cell bodies (“chemical coding”) (32), allows many dozens of cell types to be reliably distinguished and quantified (Fig. 21-4). Combinations of neurochemicals in nerve cell bodies reflect those in the nerve endings, so nerve cell bodies can be related to the targets that they innervate. Combining three antisera allows 23 or eight classes of cells to be theoretically distinguished. With several dozen immunohistochemical labels available, enormous numbers of classes could be distinguished; the number of neurochemical classes consistently falls short of the mathematical maximum. When combined with retrograde labeling, or intracellular recording and dye filling, it becomes possible to deduce the probable functional roles of classes. Chemical coding has its limitations. In practice, only two to four neurochemicals usually can be localized simultaneously. Signal-to-noise ratio and variability in the levels of expression can also complicate analysis. It also has become clear that the unique chemical codes in one preparation often show considerable variability among regions of gut and among different species (33,34).

Anyone who is not actively involved in studying neurochemistry could well be forgiven for feeling confused by the mass of information about transmitters, modulators, peptides, enzymes, and other proteins used to distinguish classes of enteric nerve cells. Many dozens of substances have been localized in enteric neurons, either singly or in combinations (33,35). Exhaustive accounts of the classes of enteric neurons have been worked out for only a few model preparations in laboratory animals (34,36–40), among which the guinea pig small intestine is still the most intensively studied. Availability and technical problems have so far restricted the number of studies performed on human tissue, which lags far behind our understanding of animal models. Extrapolation from guinea pig (or rat, mouse, or pig) tissue to human tissue can be quite misleading (33,34). Given this, the value of chemical coding studies has reasonably been questioned (41). There is no doubt that much of our current understanding of the organizational principles of the enteric nervous system has come from small laboratory animals. The task for the future is to build a comparable account of the human enteric nervous system. The technical approaches that have been developed so successfully in laboratory animals will help this endeavor. Biophysical Characteristics Features of neurons recorded electrophysiologically have been widely used to classify enteric neurons since the first

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 581

FIG. 21-4. Basis for quantitative immunohistochemical analysis of enteric neurons in guinea pig ileum. A single myenteric ganglion has been double labeled to reveal all nerve cell bodies (NCB) antiserum (36), against which calretinin-immunoreactive (solid arrows) and nitric oxide synthase (NOS)– immunoreactive cell bodies (open arrows) can be quantified.

intracellular and extracellular recordings (42–44). Type I/S cells have prominent large, fast excitatory postsynaptic potentials and sometimes fire repeatedly during depolarization, whereas type II/afterhyperpolarization (AH) cells generally lack large fast excitatory postsynaptic potentials (45–49) but often have a prominent long AH after soma action potentials. AH cells also tend to fire fewer action potentials during depolarization and have a pronounced inflection on their action potentials that has been proposed to be a more robust distinguishing feature (50). The limitation of electrophysiologic classifications is that the firing properties of neurons are powerfully affected by damage caused by microelectrodes. Longitudinal muscle

In addition, synaptic activation can alter electrical characteristics for long periods (51). Overall, the number of independently varying characteristics recorded electrophysiologically appears to be relatively small. Thus, in most schemes, only two or three major electrophysiologic classes can be distinguished reliably (52,53). The best approach to classify enteric neurons is to combine all of these different characterizations into a single scheme. In addition to morphology, neurochemistry, and electrophysiologic characteristics, retrograde labeling in organ culture has provided another powerful tool to complete the structural description of enteric neurons (Fig. 21-5).

Tertiary plexus Myenteric plexus

Dil-filled motor neurons

Oral

Dil application site on tertiary plexus

Anal

FIG. 21-5. Methods used for retrograde labeling in vitro. A whole-mount specimen of gut tissue is dissected to reveal the target layer to which a glass bead, coated in the water-insoluble carbocyanine dye, DiI, is applied. Over 2 to 4 days in organotypic culture, axons that contact the DiI take it up, selectively labeling a small group of motor neuron cell bodies.

582 / CHAPTER 21 INTRINSIC INNERVATION OF THE GUT WALL Intrinsic Sensory Neurons of the Gut Wall Over the past decade, several studies have provided compelling evidence about the identity of enteric sensory neurons, which we have been urged to call “intrinsic primary afferent neurons” (IPANs) because they are intrinsic to the gut wall and transduce physical stimuli into electrical activity (54,55). This is reasonable, although the word intrinsic may be misleading, because the heart also contains “intrinsic” primary afferent neurons (56). The term IPAN now appears to have gained some acceptance and is used in this review for enteric neurons that detect mechanical or chemical stimuli (see Chapter 23, AH- and S-Type Neurons). Submucosal Intrinsic Primary Afferent Neurons The first direct evidence for the identity of IPANs was based on the expression of the immediate early gene fos to identify neurons that were activated by small bubbles of nitrogen or cholera toxin applied to the mucosa (57). Activated cells were immunoreactive for substance P (SP) and for the calcium-binding protein calbindin. The same class previously had been shown to be immunoreactive for choline acetyltransferase (ChAT), the enzyme involved in synthesis of the transmitter acetylcholine, and hence a marker for functionally cholinergic neurons (58). Importantly, these submucous neurons project both to myenteric ganglia (59,60) and within submucous ganglia (61). They have large smooth cell bodies with multiple long processes (“multipolar neurons”) (61), in common with primary afferent neurons in dorsal root ganglia, trigeminal ganglia, and nodose ganglia. Myenteric Intrinsic Primary Afferent Neurons May Detect Other Stimuli The neurons Kirchgessner and colleagues (57) suggested to be IPANs were located exclusively in the submucous ganglia; mucosal stimuli failed to activate directly any myenteric neurons in their study. However, there is a large population of similar cells in the myenteric plexus that have large smooth cell bodies, multiple long processes, and immunoreactivity for ChAT, tachykinins, and the calciumbinding protein calbindin. Their distinctive morphology was first described by Dogiel (26), and they are widely called “Dogiel type II” cells. It was shown by intracellular recording from their cell bodies that many of these neurons were activated by chemical stimuli applied to the mucosa (62). Short-chain fatty acids, acid, and alkaline solution all activated Dogiel type II cells, even when synaptic transmission was blocked by low [Ca2+] (63). This does not rule out the possibility that IPANs may also be activated indirectly, for example, by 5-hydroxytryptamine (5-HT, or serotonin) or purines released from enteroendocrine cells in the mucosa (57,64–68). A subset of myenteric Dogiel type II neurons are

also activated by stretch of the gut wall (69), although tension, rather than length per se, may be the critical variable in their activation (70). Dogiel type II cells have several long processes arising from broad, smooth, tapering initial segments that narrow down to form fine varicose branching axons that ramify through enteric ganglia (46,48). Some axons run circumferentially, in secondary branches of the myenteric plexus, and then penetrate through the circular muscle into the submucous plexus. Here they provide both varicose endings in submucous ganglia and other branches to the mucosa proper (71,72); all myenteric Dogiel type II neurons project to the mucosa (73). Dogiel type II neurons have been retrogradely labeled from the mucosa of the guinea pig ileum (74), colon (47,75), porcine ileum (76), and human small intestine (77), but possibly not in adult human colon (78). Dogiel type II neurons have been shown to make slow synaptic contacts with other classes of enteric neurons in myenteric ganglia (79); their profuse varicose axons in enteric ganglia are the source of these synaptic connections. Ultrastructural studies using calbindin immunoreactivity as a marker support this suggestion (80–83). If these fine varicose axons represent outputs from Dogiel type II neurons, this raises the question about where their transduction sites may be. Endings in the mucosa are the sites at which chemical stimuli are detected, although they may also function as transmitter release sites onto epithelial cells. However, mechanotransduction sites are much less certain. Using von Frey hairs, Kunze and colleagues suggested that there were punctate sites within myenteric ganglia that were particularly sensitive to vertical compression (84), but there were no distinctive morphologic specializations. Such stimuli have been used to identify specialized transduction sites of extrinsic tension-sensitive mechanoreceptors in the gut wall (85,86). In small and large intestines of guinea pigs and mice, IPANs neurons have AH cell electrophysiologic characteristics (46,48,49,75,87–91). Studies have shown that in the rat and guinea pig the current responsible for the long AH is mediated by intermediate-conductance, calcium-dependent potassium (IK) channels, which can be detected immunohistochemically on Dogiel type II neurons in rat (92), mouse, and human enteric nervous system (93). However, Dogiel type II neurons are not unique in having long afterhyperpolarizing potentials (94) and, in the pig, many Dogiel type II neurons lack an AH (95,96) but may have fast synaptic inputs (97). The function of the AH is not entirely clear. There is no reason to believe that it is essential to function as a primary afferent neuron. The AH is restricted to the cell bodies of Dogiel type II neurons and probably plays no role in sensory transduction directly, but most probably affects the integrative properties of IPANs. It has been suggested that the AH in the soma may act as a “gate” mechanism, preventing the conduction of action potentials across the soma of the cell, thus restricting the number of synaptic outputs activated (98). Direct electrophysiologic evidence to support this hypothesis has been provided (99). If this is the case, the “gate” would be opened after slow synaptic input

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 583 to Dogiel type II neurons. As Dogiel type II neurons synapse onto other Dogiel type II neurons, this could lead to positive feedback within enteric circuits, leading to bursts of excitation in assemblies of AH neurons and generating highly nonlinear responses (98,100). Calbindin immunoreactivity is detectable in only a subset of Dogiel type II neurons in the guinea pig ileum (49) and is present in many unipolar type I cells in the guinea pig colon (38,88). Other immunohistochemical markers are present in Dogiel type II neurons including ChAT (the synthetic enzyme for acetylcholine) (101,102), tachykinins (74), neuromedin U (NMU) (71), calcitonin gene–related peptide (CGRP) in the pig and human (103,104), and calretinin in the rat (92,105,106). Like calbindin, none of these immunohistochemical markers is unique to Dogiel type II neurons, making their unequivocal identification problematic. Probably the best diagnostic criterion for identifying IPANs is their Dogiel type II morphology. Exactly how many classes of multipolar IPANs exist is uncertain. Three classes can be distinguished by responses to mucosal mechanical stimuli (submucous IPANs), mucosal chemical stimuli (most myenteric IPANs), and intramural tension (a subset of myenteric IPANs). A morphologic class with distinctive soma-dendritic structure, an aborally directed projection, and sparser-than-average local projections has been demonstrated (46). These cells may include stretch-activated IPANs, but like other IPANs, they all appear to have mucosal projections as well (73). It is likely that some IPANs respond to both mechanical and chemical stimuli. Could Other Cells Also Function as Intrinsic Sensory Neurons? Evidence suggests that other classes of neurons, including some unipolar interneurons, can be activated directly by mechanical distortion of the gut wall (107). In guinea pig colon, Spencer and colleagues (108,109) have demonstrated stretch-activated bursts of junction potentials in both muscle layers at either end of specimens. Recordings from enteric neurons have shown that Dogiel type II AH cells located at many points in the preparation show little firing. However, unipolar Dogiel type I interneurons, with S-cell electrophysiologic characteristics, fire bursts of action potentials that persist in calcium-free solution (which blocks synaptic activity). This is powerful evidence that some interneurons are intrinsically stretch sensitive and may contribute significantly to stretch-activated reflex activity in the gut wall (107). It also has been reported that viscerofugal neurons, with Dogiel type I morphology (110,111), which project to prevertebral ganglia, can also function as mechanically sensitive primary afferent neurons (112,113). It also has been suggested that extrinsic primary afferent neurons may activate enteric reflex pathways via collaterals within enteric ganglia (53). It is well established that peptidergic extrinsic afferent axons, detectable by CGRP immunoreactivity, provide varicose branching axons within enteric ganglia (114). Stimulation of these nerves, with either

the selective neurotoxin, capsaicin, or with electrical stimulation, activates enteric neuronal circuitry (115,116). It has been reported that in the rat colon, extrinsic denervation abolishes stretch-activated reflexes, suggesting that mechanosensitive extrinsic afferents play an essential role (117). However, extrinsic denervation does not block stretch-activated reflexes in the guinea pig small intestine (118). Whether the apparent discrepancy between these reports reflects real differences among regions of gut, among species, or among experimental designs remains to be determined. Nevertheless, it is likely that extrinsic afferent collaterals play a role in activating enteric neuronal circuitry under some circumstances. Functional Implications IPANs of the gut wall play a key role in activating enteric neuronal circuitry in response to chemical and mechanical stimulation. Several classes of IPANs share common features of multipolar Dogiel type II morphology, AH cell electrophysiology, and cholinergic neurochemistry, at least in small laboratory animals. These cells have extensive outputs onto most other classes of enteric neurons, including other neurons of their own class. These outputs are likely to activate enteric neuronal circuitry. Interconnections between IPANs may lead to positive feedback and coordinated firing as assemblies of cells, providing an anatomic basis for nonlinear responses to graded stimulation. Other classes of enteric neurons, and extrinsic sensory neuron collaterals, may also contribute to stretch-activated reflexes in the gut.

Enteric Motor Neurons Since the earliest studies on isolated or denervated preparations of intestine (22–24,119) it has been clear that there are intrinsic motor neurons within the enteric nervous system. Mechanical activity of gut smooth muscle can be stimulated electrically (120) to make pharmacologic preparations that are affected by an enormous range of drugs and transmitters (121). Axons of motor neurons project ramifying varicose processes for many millimeters parallel to the muscle fibers of the longitudinal or circular layers of muscularis externa and muscularis mucosae. The motor axons often are concentrated within particular layers; in the small intestine, a deep muscular plexus is formed between a thin inner layer of specialized smooth muscle cells and the bulk of the circular muscle (122). In large intestine, there is a dense submuscular plexus on the inner face of the circular muscle. Varicosities, which are transmitter release sites, often form close appositions with ICCs. It is now clear that much of the effect of transmitters released by motor neurons is mediated via ICCs. In mutant animals that are deficient in ICCs, both excitatory and inhibitory transmission to the muscle is impaired (123–127). Most cell bodies of motor neurons lie in myenteric ganglia; in guinea pig ileum, all motor fibers in the circular muscle

584 / CHAPTER 21 disappear after removal of the myenteric plexus (128). However, in humans and larger animals, substantial numbers of circular muscle motor neurons are located in the outer submucous (Schabadasch’s) plexus (129,130). Motor neurons usually have ovoid cell bodies, with short lamellar or sometimes filamentous dendrites and a single axon that travels within the plexus before entering the muscle where it narrows, becomes varicose, and starts to branch extensively. Enteric motor neurons have not been demonstrated to make synaptic contacts with other classes of neurons in the enteric ganglia; their axons only innervate smooth muscle. The motor neurons to the muscularis mucosae are substantially located in the submucous plexuses. Circular Muscle Motor Neurons Inhibitory and excitatory circular muscle motor neurons can be readily identified by the presence of neurochemical markers for inhibitory neurotransmitters such as nitric oxide

synthase (NOS) and vasoactive intestinal polypeptide (VIP) and markers for excitatory neurotransmitters such as ChAT and tachykinins. Inhibitory motor neurons always project aborally to the muscle that they innervate, whereas excitatory motor neurons project orally, or sometimes locally, to the circular muscle (Fig. 21-6). This has been shown for the lower esophageal sphincter (131) gastric corpus (132), gastric fundus (133), small intestine (134), and proximal colon (135). In all but one of these regions, excitatory motor neurons comprise significantly more than half of the total population of motor neurons; the exception is the lower esophageal sphincter where inhibitory motor neurons predominate (131). The polarity of projections of inhibitory and excitatory motor neurons reflects the polarity of reflexes that Bayliss and Starling (1899) described when they wrote: “Excitation at any point of the gut excites contraction above, inhibition below. This is the law of the intestine” (22). Despite the numeric predominance of excitatory motor neurons, during simultaneous activation of both

FIG. 21-6. Retrograde labeling of circular muscle motor neurons in guinea pig ileum. Nerve cell bodies of motor neurons were labeled in organotypic culture by the carbocyanine dye, DiI, applied to the circular muscle. (Top) An inhibitory motor neuron (arrows) located 14 mm oral to the DiI (i.e., with an aborally projecting axon) was immunoreactive for nitric oxide synthase (NOS) but not for choline acetyltransferase (ChAT). (Bottom) An excitatory motor neuron, located 2 mm aboral to the DiI, was immunoreactive for ChAT but not for NOS.

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 585 types of motor neuron (e.g., by electrical field stimulation), inhibition typically dominates; although at the end of the stimulation period, a substantial rebound excitation often occurs. Inhibitory motor neurons tend to have longer projections than those of excitatory motor neurons; the longest inhibitory motor neuron projections are about 25 mm in length (134,136) compared with 8 mm for excitatory motor neurons in the guinea pig ileum. This difference in overall length of projection does not, however, apply to human gut, where inhibitory and excitatory motor neurons have similar maximum lengths of projection (78,137). On average, inhibitory motor neuron soma-dendritic areas tend to be larger than their excitatory counterparts (see Fig. 21-6); this may reflect a larger field of innervation. Motor neurons often contain a variety of neurochemicals: long, descending inhibitory motor neurons in the guinea pig ileum are immunoreactive for VIP, NOS, and gastrin-releasing peptide (GRP); and short inhibitory motor neurons are immunoreactive for VIP, NOS, enkephalin (ENK) and neuropeptide Y (NPY) (134), γ-aminobutyric acid (GABA) (138), and galanin (139). There are also differences between short and long excitatory motor neurons with more long motor neurons containing immunoreactivity for ENK than short motor neurons. However, all inhibitory motor neurons appear to use nitric oxide as a transmitter, and all excitatory motor neurons are cholinergic, based on the presence of NOS and ChAT, respectively. Thus, it is mostly modulatory substances, rather than the primary transmitters, that distinguish different subtypes of motor neurons. In this regard, it is interesting that tachykinins are present in nearly all cholinergic excitatory circular muscle motor neurons in guinea pig small intestine but only in a small proportion of human cholinergic colonic excitatory motor neurons (140). This suggests that noncholinergic excitation is mediated by some or all cholinergic motor neurons (134). Functional Implications The stereotyped polarization of inhibitory and excitatory motor neurons provides a starting point for the polarized reflex pathways described as “the law of the intestine.” Inhibitory and excitatory motor neurons can also be distinguished by the presence of enzymes involved in cholinergic and nitrergic neurotransmission, although they also contain a number of other modulatory neurochemicals. Longitudinal Muscle Motor Neurons In larger mammals, including humans, axons of longitudinal muscle motor neurons project within the longitudinal muscle itself, running in parallel with the bundles of muscle. In smaller species, such as the guinea pig and other rodents, the axons of motor neurons are confined to a tertiary plexus on the innermost face of the muscle layer (122,141). As with the circular muscle layer, varicosities along the axons are the transmitter release sites responsible for the excitatory junction potential (142).

Cell bodies of motor neurons to the longitudinal muscle layer are generally located within the myenteric plexus, although there may be some in submucous ganglia in the porcine ileum (143). They form a separate population from the circular muscle motor neurons. This makes possible the independent activation of the two muscle layers during physiologic control of motility. Longitudinal muscle motor neurons branch extensively within the tertiary plexus, close to their cell bodies (89,144,145), and they are highly excitable S cells electrophysiologically (146). Using retrograde tracing techniques, motor neurons to the longitudinal muscle show rather less polarized projections than circular muscle motor neurons. In the guinea pig small intestine, nearly all longitudinal muscle motor neurons (greater than 97%) were cholinergic, and hence excitatory (147). This is consistent with relatively small inhibitory junction potentials in this region (148), suggesting that the longitudinal muscle layer receives relatively little direct inhibitory innervation. Varicose NOS- (149) and VIP-immunoreactive (150,151) nerve fibers in the tertiary plexus are relatively sparse. However, electrical stimulation does evoke relaxations of the longitudinal muscle (152,153), indicating either that the 3% of motor neurons can still be functionally important, or that overflow from the circular muscle can occur after repetitive, synchronous activation of large numbers of axons. A functional inhibitory innervation has been demonstrated in the longitudinal muscle of many regions of guinea pig gut (154) and in the small intestine of rat, rabbit (155,156), and human (157). In the guinea pig gastric corpus and proximal colon, inhibitory motor neurons to the longitudinal muscle layer have descending projections (135,158), but in the sheep rumen, projections are not so polarized (159). Thus, longitudinal muscle layer tends to be innervated by local neurons, and polarization of inhibitory and excitatory pathways is more variable than the stereotyped pattern of circular muscle. About half of the cholinergic longitudinal muscle motor neurons in the guinea pig ileum are immunoreactive for tachykinins (147). The longitudinal muscle of the guinea pig ileum receives a potent noncholinergic excitatory innervation (160), mediated by SP (161,162). Excitation by tachykinins, in the presence of muscarinic antagonists, follows repetitive electrical stimulation; single-shot stimuli evoke twitches that are almost entirely cholinergic (120,163). The anatomic data from retrograde tracing studies indicate that tachykinins are released from a subset of cholinergic motor neurons. The apparent frequency dependence probably reflects release of transmitters stored in different vesicles in the same nerve endings (164). Longitudinal muscle motor neurons in the guinea pig ileum are typically small, simple cells, which make up about a fourth of all neurons in the myenteric ganglia of the guinea pig ileum. This seems rather surprising given the thinness of the muscle layer. Comparable figures have not been calculated for other species or regions, but in several studies, longitudinal muscle motor neurons were also small, simple, and abundant (78,135,158,159,165). It is possible that large numbers of small neurons, each innervating a relatively small

586 / CHAPTER 21 region of muscle, allow for finely graded control of motility. Despite longitudinal and circular muscle motor neurons forming distinct populations, a number of reports indicate that they may be synchronously activated during stretch (109,166–168) and that reciprocal innervation of the two layers postulated by Kottegoda (169,170) may be the exception rather than the rule. Functional Implications The longitudinal muscle layer is innervated separately from the circular muscle, allowing independent activation under some conditions. Longitudinal muscle motor neurons are numerous and project locally to their target tissue. They are well suited to providing graded motor control of muscle in the vicinity of a stimulus, but they may also be activated by long ascending or descending pathways during motility patterns coordinated over longer distances. Specialized Enteric Motor Neurons During development, smooth muscle in the rodent esophagus undergoes a transdifferentiation to form striated muscle (171). The striated muscle of adult animals is innervated by cholinergic motor neurons located in the nucleus ambiguus, which make conventional motor end plates onto muscle fibers (172). However, motor end plates receive a secondary innervation by nerve endings that contain NADPH diaphorase and are immunoreactive for NOS (173) and arise from enteric nerve cell bodies (174). These unusual endings have been shown to inhibit neuromuscular transmission by motor end plates (175), and thus can be considered a specialized type of enteric motor neuron.

Interneurons in the Gut Wall The gut is clearly capable of expressing a diverse range of motor patterns, including migrating myoelectric complexes (MMCs), irregular mixing patterns of activity during digestion, and powerfully propulsive contractions during peristalsis. This suggests the existence of additional classes of neurons to the IPANs and motor neurons to allow this behavioral repertoire. The neurons responsible for this flexibility are likely to be the enteric interneurons. In the best characterized preparations of small laboratory animals, all interneuron classes described to date have a single axon and a cell body with short lamellar or filamentous dendrites. These neurons receive synaptic inputs from IPANs, from extrinsic neurons (sympathetic and parasympathetic neurons), and from other interneurons in the gut wall and make synaptic outputs onto other classes of enteric neurons. One striking feature of the arrangement of interneurons within the gut wall is that they all make preferential connections with other cells of the same class further along the gut; that is, they are organized into functional chains. This arrangement has two consequences for the functioning of the circuitry.

First, it probably allows simple reflex circuits to extend further up or down the gut than would otherwise occur. This is well illustrated by the single class of ascending interneurons in the guinea pig small intestine. These cholinergic neurons make prominent fast nicotinic excitatory synaptic contacts onto one another (176), which are functionally involved in spreading ascending excitatory reflexes up to 40 mm up the gut wall. It can be readily calculated that the longest oral projection of an IPAN is about 1 mm, the longest projection of an excitatory motor neurons is about 8.5 mm, and the longest projection of an ascending interneuron is about 14 mm. Thus, at least two interneurons must be involved in the pathways that underlie excitation of circular muscle 40 mm oral to a distension stimulus (177,178). The second consequence is more speculative. Activation of a chain of cells stretching out along the gut, each with outputs to a defined set of enteric neurons, could modulate enteric circuitry to favor particular patterns of activity. Thus, chains of interneurons could be the basis for switching between motility patterns. It is well established that inputs from extrinsic pathways to the gut can powerfully modulate gut motility; for example, vagal efferents play a role in pattern switching from fasting to fed behavior after a meal (179). One could speculate that interneuronal chains could be key targets for such modulation. Indeed, it has been reported that serotonergic interneurons are a preferential target for input from sympathetic neurons projecting to the gut wall (110,180). Currently, it is unclear whether vagal efferent (parasympathetic) neurons make similar selective contact with any classes of enteric interneurons. Ascending Interneurons In the guinea pig ileum, there is one class of ascending interneuron and at least three classes of descending interneurons, most of which are cholinergic (on the basis of ChAT immunoreactivity), but which can be readily distinguished on the basis of the cotransmitters and peptide modulators that they contain. Ascending interneurons are short, orally directed cells that are immunoreactive for ChAT, tachykinins, enkephalin, dynorphin calretinin, and neurofilament protein triplet (Fig. 21-7) (176,181) and comprise about 5% of all myenteric neurons (181). They make synaptic contacts with other ascending interneurons (82,83), with excitatory motor neurons to both circular and longitudinal muscle, and possibly with other classes of enteric neurons (see Fig. 21-9). Their organization into functional chains, linked by cholinergic nicotinic synapses (176), is compatible with ascending interneuronal pathways being substantially inhibited by nicotinic blockers (177,182–184). However, some ascending excitation persists in the presence of nicotinic blockers (183), possibly mediated by the tachykinins in ascending interneurons. This reflex pathway shares elements with distensioninduced peristaltic contractions (185), which are also substantially inhibited by hexamethonium (167,186). The opiate receptor antagonist naloxone can restore peristalsis in the continuing presence of hexamethonium (187,188),

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 587

FIG. 21-7. Retrograde labeling combined with immunohistochemical double labeling reveals multiple cell types including ascending interneurons. DiI was applied to the myenteric plexus 7 mm oral to these micrographs, and the preparation was stained for calretinin and neurofilament protein triplet (NFP) immunoreactivity. Five types of neurons can be distinguished. Two ascending interneurons immunoreactive for both calretinin and NFP are present; one is labeled by DiI (arrow), the other is not (arrowhead). An excitatory motor neuron to the longitudinal muscle layer is distinguished by immunoreactivity for calretinin but not NFP (open arrow). An excitatory motor neuron to the circular muscle contains DiI but lacks both calretinin and NFP (open arrowhead). Lastly, an unidentifiable interneuron or motor neuron without DiI is immunoreactive for NFP but not calretinin (double arrow).

possibly by removing the inhibition of endogenous opioids, including enkephalin, which are present in both excitatory motor neurons and in ascending interneurons (176). Ascending interneurons in the guinea pig distal colon have a more filamentous morphology than in the small intestine (89), and their chemical coding also appears to be rather variable (38). In both rat and mouse, tachykinin-containing ascending pathways have been described (40,189,190). In the human gut, ascending interneurons have been described that contain tachykinin immunoreactivity and that are mostly cholinergic (191). Functional Implications Ascending interneurons are organized into chains that project up the gut, which thus can spread excitation from a localized stimulus to other classes of cells, especially excitatory motor neurons, to allow coordinated motor patterns to spread over long distances within the wall of the intestine. Descending Interneurons One class of descending interneurons in the guinea pig ileum contains somatostatin immunoreactivity. These cells have long filamentous dendrites and a single long aborally projecting axon, which makes branching varicose endings in nearby ganglia (94,192). These cholinergic neurons (101) also project to the submucous plexus (94,193), where they mediate nonadrenergic inhibitory synaptic potentials (194). Somatostatin-containing interneurons receive little of their

synaptic input from IPANs, but they receive substantial input from other neurons of the same class (192,195,196). They make outputs onto another class of interneurons (NOS containing) and onto inhibitory motor neurons. On the basis of their connectivity, it has been speculated that these cells may be involved in MMCs (197), but direct functional evidence currently is lacking. Somatostatin is not restricted to descending pathways; in the guinea pig, distal colon somatostatin is present in ascending interneuronal pathways (38). Another class of descending interneurons, also cholinergic (101) and immunoreactive for 5-HT (serotonin), is found in the small intestine (198,199), stomach (200), and distal colon (201). These cells may either accumulate 5-HT using a 5-HT transporter or may synthesize 5-HT de novo (41). They have typical Dogiel type I morphology, with short lamellar dendrites and a single axon. Like the somatostatin-containing interneurons, these cells project aborally to both myenteric and submucous ganglia (193), causing numerous 5-HT– immunoreactive varicosities surrounding many neurons, including other cells of their class. The role that 5-HT– containing interneurons play in gut function is not readily deduced from their connections, or from physiologic studies. Exogenous 5-HT acts on many classes of receptors located on enteric neurons, smooth muscle, epithelium, and enteroendocrine cells; but in many cases, it is unclear whether endogenous 5-HT actually reaches the potential targets in adequate concentrations to elicit a response (121). Most of the 5-HT in the gut wall is contained within the enterochromaffin cells, and it is 5-HT from this source that may mediate many of the effects revealed by 5-HT receptor antagonists.

588 / CHAPTER 21 However, there is evidence that 5-HT may contribute to fast excitatory synaptic transmission, via 5-HT3 receptors (202,203), probably from 5-HT–containing interneurons. However, the major target of these interneurons (apart from other 5-HT–containing interneurons) is unclear but is probably not the inhibitory motor neurons (204). One report suggests that when excitatory synaptic potentials are pharmacologically blocked, inhibitory synaptic potentials, mediated by endogenous 5-HT acting on 5-HT1A receptors, are revealed the myenteric plexus (205). The remaining descending interneurons belong to a heterogeneous group of cells that are immunoreactive for combinations of NOS, GRP, NPY, galanin, alkaline phosphatase, pituitary adenylyl cyclase–activating polypeptide (PACAP), and VIP, which have axons that provide varicosities in enteric ganglia (39,139,150,151,206–209). Many, but probably not all, of these cells are cholinergic (36). The degree of overlap of these markers is variable (210). All of these interneurons have Dogiel type I morphology and make close appositions with other interneurons of the same class, inhibitory motor neurons, somatostatin-containing interneurons, and possibly ascending interneurons (196,211). Functional studies indicate that some of these descending projections are mediated via ATP released onto P2x receptors (212,213), probably from NOS/GRP/VIP interneurons. Currently, there are no reliable methods for visualizing nerves or nerve terminals that release ATP as a transmitter; thus, the source of ATP is uncertain. Interestingly, a role for purinergic transmission is clear in descending inhibitory reflexes in the ileum; but in the colon, the equivalent pathways appears to be cholinergic (214).

Pathways underlying polarized reflexes are summarized in Figures 21-8, 21-9, and 21-10. Functional Implications The presence of at least three classes of descending interneuron provides a substrate for multiple patterns of coordinated activity in the gut wall. At least one of these pathways may be involved in spreading descending inhibitory reflexes beyond the projections of individual inhibitory motor neurons. The other interneurons may be involved in diverse functions such as switching motor activity between fed and fasting behavior, coordinating MMCs, mediating descending excitation, and coordinating mucosal and muscular function.

Viscerofugal Neurons Reflex arcs from the gut to sympathetic ganglia that do not involve the central nervous system are well established (25). Although collaterals from spinal afferent neurons may mediate some of these effects (215,216), there are also interneurons within the enteric nervous system that project out of the gut (217) (see Chapter 22, Morphology of Viscerofugal Neurons). The majority of such viscerofugal neurons are located in the myenteric ganglia, but some are in the submucous ganglia (218,219). These cells have Dogiel type I morphology, with short lamellar or filamentous dendrites and a single axon that projects out of the gut wall without apparently providing collaterals within the gut wall (110,111).

FIG. 21-8. Summary diagram of functional classes of nerve cell bodies identified in the guinea pig ileum, with letters corresponding to those used in Table 21-1, updated from Costa and colleagues (36). Major classes of cells in both myenteric and submucous ganglia are shown; some minor populations of “displaced submucous neurons” have been omitted from the myenteric ganglion for clarity (D and F).

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 589 +

+

In the esophagus and stomach, neurons projecting into the vagus nerve have been described (85,86,172,227), and some of these reach the brainstem (227). In the distal colon of the rat, a population of rectospinal neurons has been described in the myenteric plexus, which project to the spinal cord (228,229).

Excitatory motor neurons

Ascending interneuron

Functional Implications Viscerofugal neurons provide a pathway to activate sympathetic inputs to the gastrointestinal tract, depending on mechanical and chemical stimuli in the gut. These are likely to be involved in reflexes that coordinate activity among distant regions of gut, such as enterogastric and intestinointestinal reflexes.

Intrinsic primary afferent neuron

Descending interneuron

Secretomotor and Vasomotor Neurons

Inhibitory motor neurons −

Chemical and mechanical stimuli activate secretomotor reflexes in the gut wall (230–232) and vasomotor reflexes (233) (see Chapter 27). The neurons responsible for controlling these mucosal functions are located primarily in the submucous ganglia, although a smaller number are located in myenteric ganglia. Broadly, they can be divided into vasomotor neurons, which influence blood vessel diameter in the submucosa and mucosa, and secretomotor neurons, which influence secretory activity of epithelial cells. However, many submucous neurons project to both epithelia and blood vessels and are likely to coordinate control of both targets. In the guinea pig small intestine, five classes of submucous neurons currently have been distinguished. One of these is the submucosal IPAN described earlier, which uses acetylcholine and tachykinins as neurotransmitters and also contains calbindin immunoreactivity. Of the remaining classes, two are cholinergic and can be distinguished by the combinations of neuropeptides that they contain. The remaining 45% of submucous neurons in this preparation are noncholinergic, VIP-containing neurons (58,234).

−

FIG. 21-9. Simple circuit underlying polarized motor reflexes are summarized. An intrinsic primary afferent neuron, responding to a localized stimulus, excites cell bodies of excitatory and inhibitory motor neurons and ascending and descending interneurons within its ganglion of origin, leading to excitation orally and inhibition aborally. Interneurons also excite motor neurons in ascending and descending pathways, extending the distance over which reflexes operate.

Although they comprise less than 1% of ileal neurons (36), they are found in increasing numbers distally (220–222). Viscerofugal neurons are cholinergic (223) but also contain a variable mix of NOS (224), VIP and GRP (223), CGRP, or SP (225). Viscerofugal neurons receive fast synaptic inputs (111) from cholinergic descending interneurons (226). Some of these neurons may also be directly mechanosensitive (112,113). Viscerofugal neurons that project to targets other than the prevertebral ganglia also have been described. +

+

− Oral

+

−

+

−

+

−

+

−

+

−

−

− Aboral

FIG. 21-10. Multiple connected circuits along the intestine allow polarized activity from a localized stimulus to occur at any point in the gut, despite every enteric ganglion containing a mixture of the different cell types.

590 / CHAPTER 21 Secretomotor Neurons Cholinergic neurons, immunoreactive for the calciumbinding protein calretinin (181) have been estimated to comprise 12% of all submucous neurons (181). They project to submucous arterioles and are likely to contribute to the cholinergic vasodilation evoked by electrical stimulation of ganglia or single neurons (235). Calretinin-immunoreactive varicosities are also found in the muscularis mucosa and around the base of the mucosal crypts, but they do not make endings in submucous ganglia. Therefore, this class is also likely to contribute to cholinergic secretion and possibly to motor activity of the muscularis mucosa. The other major classes of cholinergic submucous neurons contain immunoreactivity for NPY/cholecystokinin (CCK)/ CGRP/NMU/somatostatin/galanin (58) and dynorphin1-8 (236). They provide varicose endings in the mucosa, in the subepithelial plexus, and around the base of the glands, but not on submucous blood vessels. They comprise up to a third of all submucous neurons in the guinea pig small intestine (234) and are the major class of cholinergic secretomotor neurons. There is a small class (less than 1%) of cells in the myenteric plexus with identical chemical coding, which also project directly to the mucosa (74,237). The major class of noncholinergic neurons in the submucosa of the guinea pig small intestine contains VIP immunoreactivity, together with galanin (139), NMU (71), and the opioid peptides dynorphin1-8, dynorphin1-17, dynorphin B, and α neo endorphin (236). They make extensive projections to the subepithelial plexus of the mucosa, to the muscularis mucosae, and to submucous arterioles (238). These nerve cells project locally to the mucosa and do not show any polarity along the ileum (234). A small population of neurons in myenteric ganglia with similar chemical coding also projects to the mucosa (74). The secretomotor effects of VIP have been studied extensively (239), and it is clear that these populations of myenteric and submucous neurons are responsible for much of the neurally mediated noncholinergic increase in secretion recorded in Ussing chamber studies (240). They receive synaptic inputs from a diverse range of sources including inhibition from extrinsic sympathetic efferent neurons (241) and from somatostatin-containing interneurons (194) and excitation from myenteric neurons (242,243), from nitric oxide–immunoreactive interneurons (244), from 5-HT–containing interneurons (193), and from submucous and myenteric IPANs (61). The chemical coding and polarities of submucous secretomotor pathways show considerable variability both between different regions and in different species. In the guinea pig small intestine, cholinergic and noncholinergic secretomotor are not polarized, whereas in both proximal and distal colon of the guinea pig, noncholinergic pathways have a significant aboral projection, and cholinergic pathways project orally (245,246). A VIP antagonist blocks descending pathways to the mucosa more effectively than ascending pathways, which are instead blocked by atropine (246). Likewise, in the guinea pig gastric corpus, cholinergic secretomotor pathways

have a significant orally directed polarity. The gastric corpus mucosa also receives a NOS-immunoreactive input from myenteric neurons with descending (aborally directed) axons (247,248). The combinations of peptides and transmitters also vary considerably among species. In the rat small intestine, nearly all submucous neurons are cholinergic (102), presumably including VIP-containing neurons, many of which also contain NPY (102,189,249,250). The secretomotor neurons in the stomach are of particular interest, given their key role in the control of gastric acid release. GRP is effective in releasing gastrin from G cells in the stomach and has been localized immunohistochemically in the antral mucosa of the guinea pig stomach (251). Retrograde labeling studies have shown that GRP-containing neurons to the mucosa are mostly cholinergic and many contain NPY immunoreactivity (252). These cells, like most other gastric enteric neurons (253), have a high probability of receiving excitatory synaptic drive from vagal efferent fibers. Functional Implications Cholinergic and VIP-containing submucous neurons are the major pathways controlling secretion from the gastrointestinal epithelium. Some of these neurons share secretomotor and vasodilator functions, probably helping to ensure that blood flow matches secretory demand. Submucous neurons integrate synaptic inputs from multiple sources both within the gut wall and extrinsic to it. Patterns of expression of peptides are variable among species, as are polarities of projections, although the physiologic significance of this remains to be determined.

Interplexus Interneurons A small proportion of VIP-immunoreactive submucous neurons (<1% of the total) project to the myenteric plexus (60). Retrograde tracing studies have shown that dye applied to the myenteric plexus of the guinea pig ileum labels submucous cell bodies, including tachykinin- and calbindinimmunoreactive IPANs (59,60). However, they also label a substantially larger number of VIP-immunoreactive neurons that appear to project directly to the myenteric plexus and do not have collaterals to the mucosa or submucous blood vessels (59,60). The projections and neurochemistry of the major classes of enteric neurons are summarized in Figure 21-8 and Table 21-1. Functional Implications These cells probably function as interplexus interneurons, perhaps being involved in the coordination of motor and mucosal functions between the myenteric and submucous plexuses. As such, they may complement the descending interneuronal pathways and IPANs that innervate both myenteric and submucous ganglia (242,243).

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 591 TABLE 21-1. Classes of myenteric and submucous neurons in the guinea pig ileum Function

Chemical coding

Shape

Percentage

Class

Submucous IPANs Myenteric IPANs Inhibitory motor neurons to CM Inhibitory motor neurons to CM Excitatory motor neurons to CM Inhibitory motor neurons to LM Excitatory motor neurons to LM Ascending interneurons Descending interneurons Descending interneurons Descending interneurons Viscerofugal interneurons Secretomotor/vasomotor neurons Secretomotor/vasomotor neurons Secretomotor neurons Interplexus interneurons

ChAT/SP /±NMU/±Calb ChAT/±SP /±NMU/±Calb NOS/VIP/Dyn/±GRP/±NFP/+Enk NOS/VIP/±Dyn/Enk/±NFP ChAT/SP/±Enk/NFP NOS/±VIP ChAT/±SP/±Calret ChAT/SP/Enk/Calret/NFP ChAT/Som ChAT/5-HT/NFP NOS/±ChAT/±VIP/Dyn/±GRP/±NPY ChAT/NOS/±VIP/±GRP/±CCK ChAT/Calret VIP/Dyn ChAT/NPY/Som/CGRP/CCK VIP/Dyn

Type II Type II Type I Type I Type I Type I Type I Type I Filamentous Type I Type I Type I Type III Type III Type III Type III

11–20% SMP 30% MP ≤17% MP 12% MP 12% MP <1% MP 25% MP 5% MP 4% MP 2% MP <8% MP ≤1% MP 12% SMP <1% MP, 45–52% SMP <1% MP, 19–29% SMP <1% SMP

P M A B K Q IJL H G E AC N O D F R

Neurochemical classes with function and morphologic classification in guinea pig small intestine, updated from Costa and colleagues (36), with classes named as in Figure 21-8. 5-HT, 5-hydroxytryptamine; Calb, calbindin; Calret, calretinin; CCK, cholecystokinin; ChAT, choline acetyltransferase; CM, circular muscle; Dyn, dynorphin; Enk, enkephalin; GRP, gastrin-releasing peptide; IPAN, intrinsic primary afferent neuron; LM, longitudinal muscle; MP, myenteric plexus; NFP, neurofilament protein triplet; NMU, neuromedin U; NOS, nitric oxide synthase; NPY, neuropeptide Y; SMP, submucous plexus; Som, somatostatin; SP, substance P or related tachykinins; VIP, vasoactive intestinal polypeptide.

EXTRINSIC INNERVATION OF THE GUT WALL The enteric nervous system receives functionally important inputs from sympathetic, parasympathetic, and extrinsic sensory neurons (both spinal and vagal). All of these classes provide varicose fibers within the enteric ganglia. Spinal afferents and sympathetic efferent fibers also directly innervate the muscle layers of the gut wall, mucosal glands, and blood vessels. The overall number of sympathetic, parasympathetic, and sensory neurons involved is relatively small compared with the many millions of enteric neurons, but they exert a disproportionate influence on gut function.

Parasympathetic Innervation of the Gut Parasympathetic nerves run to the gut via the vagus nerve and the pelvic nerves/pelvic plexus. Parasympathetic efferents, which make cholinergic motor end plates on the striated muscle of the esophagus, arise from cell bodies in the nucleus ambiguus (172). Efferents to esophageal ganglia and to the rest of the gut arise from the dorsal motor nucleus of the vagus and are mostly, but not solely, cholinergic (172,254). Tracing studies have shown that cells from the lateral and medial subnuclei of the dorsal motor nucleus target different regions of gut (255,256). Anterograde labeling studies have shown that vagal efferents primarily innervate myenteric ganglia, giving few branches to the submucosa or mucosa (257). Within the ganglia, vagal efferent nerve endings become varicose and run through the ganglia, providing many side branches, which contact enteric neurons. An early

study suggested that vagal efferents may only contact a small population of “command neurons” in the stomach (258). However, later studies that achieved more complete fills revealed branching varicose endings apparently contacting most enteric neurons in the stomach (227). Detection of fos or phosphorylated cyclic adenosine monophosphate response element binding protein has shown that most gastric myenteric neurons are activated by vagal efferent neurons (259,260). Likewise, intracellular recordings from gastric neurons indicated that a high proportion of gastric enteric neurons receive cholinergic excitatory synaptic input from the vagus nerve (253,261). Stimulation of vagal efferents causes both inhibitory and excitatory effects in the stomach. Gastric acid secretion is increased, as is motor activity in the antrum. However, the upper stomach typically relaxes during vagal stimulation (262,263), which is an important component of gastric accommodation (264). Because all effects of vagal efferents on enteric neurons are excitatory (253), vagal efferents to the upper stomach must primarily contact inhibitory motor neurons, whereas those to the distal stomach must predominantly contact excitatory motor neurons (265). There is evidence that vagal efferents to inhibitory pathways are located more caudally in the dorsal motor nucleus than those to excitatory pathways (266). The rectum and distal colon receive a powerful parasympathetic input via the pelvic nerves, pelvic plexus, and rectal nerves (267,268). The cholinergic nerve cell bodies responsible for this input are located within the sacral parasympathetic nucleus of the spinal cord (269,270) and within the pelvic ganglia (271). The major target of the nerves entering the rectum is the myenteric plexus, with some fibers branching

592 / CHAPTER 21 within the smooth muscle layers, but relatively few fibers running to the submucous ganglia or mucosal layers (272). Within the myenteric ganglia, sacral parasympathetic nerve endings are fine varicose axons that branch extensively within the ganglia, apparently making close contact with a high proportion of nerve cells within the ganglia (273). Functional Implications Parasympathetic inputs to the gut are widely distributed but are concentrated in the proximal and distal gastrointestinal tract, which are innervated by separate populations of vagal and sacral pathways, respectively. With their varicose endings coursing extensively throughout the ganglia, they are well suited to making functional contacts with many, perhaps most, enteric neurons. Parasympathetic outputs are excitatory, mostly nicotinic, and probably contribute to an ongoing background of synaptic activity throughout enteric circuits, thus potentiating local reflex responses. In addition, when strongly activated, they trigger stereotyped motor patterns involved in emesis and defecation.

Sympathetic Innervation of the Gut Between the stomach and the rectum, the predominant extrinsic input to the gut comes from the sympathetic nervous system (274). Sympathetic nerve cell bodies innervating the gut wall arise mostly from prevertebral ganglia (coeliac, superior mesenteric, inferior mesenteric, and intermesenteric ganglia) and provide a dense network of varicose branching axons that surround all nerve cell bodies within myenteric and submucous ganglia (Fig. 21-11). Unlike parasympathetic efferents, they also supply axons directly to the smooth muscle, particularly in the sphincteric regions (275–277), to blood vessels within the gut wall, and to the mucosa proper. These different targets are innervated by different populations of sympathetic neurons; axons supplying blood vessels contain tyrosine hydroxylase (TH) and NPY, those that innervate the submucous plexus contain TH and somatostatin, whereas those innervating myenteric ganglia contain TH alone (278). This suggests that motility, secretion, and blood flow can be independently controlled by sympathetic pathways. Some sympathetic neurons innervating blood vessels are located in the paravertebral, sympathetic chain ganglia (279,280). Sympathetic varicosities in enteric ganglia primarily release noradrenaline, which causes presynaptic inhibition of transmitter release (281,282) via α2-adrenoceptors (121). It also causes direct, postsynaptic hyperpolarizing inhibitory junction potentials in VIP-containing secretomotor neurons in the submucous plexus (241). Noradrenaline, acting via α1 receptors also can have excitatory effects on enteric neurons (121). Although every neuron in each enteric ganglion is surrounded by sympathetic axons with varicose transmitter release sites, some neurons are surrounded by particularly dense baskets of endings. In the guinea pig ileum, these

appear to be the 5-HT–accumulating, cholinergic, descending interneurons (110,283). Functional Implications Sympathetic neurons make abundant varicose transmitter release sites throughout enteric ganglia, surrounding all enteric neurons. They release noradrenaline, which causes potent presynaptic inhibition, and thus are well suited to “damping down” spontaneous activity within enteric neuronal circuits and extrinsic inputs from parasympathetic pathways (284) and suppressing reflex pathways. In addition, noradrenaline released from these endings is likely to target at least one class of descending interneurons in the myenteric plexus, although it is unclear whether this would lead to excitation or inhibition.

EXTRINSIC SENSORY NERVE ENDINGS Vagal Afferents Primary afferent neurons with cell bodies in the nodose ganglia travel to the gut wall via the vagus nerves. There is also an extensive extrinsic sensory innervation by neurons with cell bodies in spinal (dorsal root) ganglia. Functionally, these two pathways are quite distinct; vagal pathways are considered to signal physiologic states of the gut, whereas nociception is generally believed to be mediated by spinal afferents (285,286). Vagal afferents make three types of endings within the wall of the gut. Axons in the mucosal layers of the stomach and small intestine run near the basal lamina, crypts, and lamina propria, often relatively close to enteroendocrine cells of the gut wall (287–289). They are activated by enterochromaffin cells releasing 5-HT either spontaneously (290) or in response to lumenal chemicals (291). The second class of vagal afferent nerve ending consists of arrays of branching flattened or varicose fibers extending within large areas of longitudinal or circular smooth muscle (289). These endings, called intramuscular arrays, are dense in the upper stomach and in the sphincters of the upper gut. Their function has not been determined, but they may be a type of mechanoreceptor, perhaps sensitive to length (292). The third type of ending, described originally using histologic stains (293–295), form intraganglionic laminar endings (IGLEs) in the myenteric ganglia of the upper gut. They are present in large numbers in the esophagus and stomach, decrease in density along the small and large intestines, and are not found in the rectum (296,297). IGLEs form flattened, leaflike plates that lie close to the connective tissue sheath surrounding myenteric ganglia. Ultrastructurally, these endings are in close apposition with enteric neurons, and in some cases appear to have presynaptic clusters of vesicles (298). Functional studies have shown that IGLEs are the mechanosensitive transduction sites of vagal tension receptors (85,86). They transduce mechanical stimuli on a time scale of a few milliseconds, in the absence of extracellular

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 593

FIG. 21-11. Extrinsic innervation of guinea pig colon revealed by anterogradely transported biotinamide (Biot) in vitro. The preparation is also labeled for tyrosine hydroxylase (TH) immunoreactivity to reveal sympathetic nerve terminals in the ganglion. Some anterogradely labeled varicose fibers are immunoreactive for TH (arrows), but many are not (open arrows).

calcium ions, and thus detect mechanical stimuli directly, probably via stretch-activated ion channels and not via chemical transmission from other cell types (299).

Spinal Afferents

flattened branching structures called rectal IGLEs (Fig. 21-12). These endings function as mechanotransduction sites for a specialized class of low-threshold, slowly adapting mechanoreceptors (311). The existence of such endings runs contrary to the widespread belief that spinal afferents are morphologically unspecialized in the gut wall (285,286) and raises the

Endings of spinal afferent neurons within the wall of the gut have not been described anatomically as thoroughly as vagal afferents. Anterograde filling of spinal afferents by injecting dye into dorsal root ganglia has been achieved in only a handful of studies (300–302), and comprehensive morphologic descriptions of their endings are not available. Chemical coding studies, using CGRP and tachykinin immunoreactivity (114), have shown their axons coursing through myenteric ganglia, making varicose endings around enteric neurons, projecting through to the submucosa and mucosa, densely innervating blood vessels and the subepithelial plexus. VR1 immunoreactivity also labels spinal afferent axons in the gut (303) and reveals branching varicose endings in close contact with myenteric and submucous cell bodies, as well as endings within the smooth muscle, lamina propria, and blood vessels. Most, but not all, VR1-containing axons are immunoreactive for CGRP, and some CGRP-containing axons lack VR1 (304). It is widely believed that spinal afferents make only unspecialized endings within the gut wall. However, CGRP, tachykinins, and VR1 do not label all dorsal root ganglion cells that project to the gut or accessory organs (305–310). It is likely that nonpeptidergic spinal afferent innervation of the gut remains to be described. Studies have identified a population of sacral spinal afferents to the guinea pig rectum that lack CGRP immunoreactivity (273). These neurons make morphologically specialized endings within myenteric ganglia (273), which have sculpted

FIG. 21-12. Confocal image of anterogradely labeled specialized ending of a spinal mechanoreceptor in guinea pig rectum.This specialized structure, called a rectal intraganglionic laminar ending, consists of flattened platelike endings near the surface of a myenteric ganglion and corresponds to the mechanotransduction site of a low-threshold mechanoreceptor in the wall of the rectum (311).

594 / CHAPTER 21 possibility that other classes of spinal afferents may also have specialized endings. Functional Implications Vagal and sacral afferent neurons have specialized endings within enteric ganglia that are mechanotransduction sites, which may also be modulated by enteric neuronal activity. They signal information about the state of the gut wall to the central nervous system and are responsible for activating neural pathways that cause all sensation from the gut and also trigger centrally mediated reflexes. Spinal afferent axon collaterals within the enteric nervous system and in sympathetic ganglia provide an anatomic substrate for these neurons to activate enteric neurons, independent of central nervous system pathways.

HEIRARCHIES OF NEUROCHEMICALS This review has given an account of the major types of nerve cells that have cell bodies or processes, or both, within the wall of the gut and their morphologic and neurochemical specializations. Throughout this review, combinations of neurochemicals expressed in neurons have been used as a tool to classify enteric neurons. However, in many cases, these neurochemicals are themselves functionally important. In combination with data on cell projections, morphology, and electrophysiologic features, it is possible to make deductions about the roles of various cell classes. A picture emerges of a modest number of functional classes of neurons within enteric ganglia (see Fig. 21-8) that receive contacts from one another and from extrinsic nerves in a highly organized fashion. Within any preparation, each class of nerve cell contains a fixed combination of neurotransmitters, transmitter-related enzymes, peptide modulators, structural proteins, and other proteins that define its “chemical coding.” However, homologous cells in different regions of gut or in different species often have quite different chemical codes. The extent of this variability and its significance deserve some consideration. Chemical coding is not random; patterns can be discerned within the detail. In functional terms, acetylcholine and nitric oxide are arguably the predominant neurotransmitters in the enteric nervous system. One (or both) of the corresponding neurochemical markers, ChAT and NOS, is present as a neurochemical marker in nearly every enteric neuron (36,37). These markers appear to be among the best conserved among homologous cells. Thus, ChAT is found in excitatory motor neurons with local or orally directed processes within the enteric nervous system, whereas NOS is found in inhibitory motor neurons with descending projections (131, 132,135,137,165,312). There is obvious functional significance in excitatory motor neurons not containing NOS and inhibitory motor neurons not containing enzymes involved in excitatory neuromuscular transmission. However, these are not absolute “rules.” NOS and ChAT do coexist in some

interneurons in the wall of the gut (36,191), and there have been reports that in some preparations inhibitory motor neurons may express ChAT (313–315). After acetylcholine and nitric oxide, a number of other neurotransmitters are also functionally significant, but in many cases may be slightly less crucial. VIP and SP stand out as examples. In many regions of gut, these peptides (and their close relatives) have been shown to play a role in transmission, but generally only when the occlusive effects of the primary transmitters, acetylcholine and nitric oxide, have been blocked. There are exceptions to this: tachykinins may have significant roles in peristalsis (316), and VIP may play a role in relaxation of canine muscularis mucosa (317) and rat gastric fundus (318). A third group of neurochemicals can be loosely considered to act as neuromodulators, influencing neurotransmitter release presynaptically and sometimes contributing a minor component to postsynaptic excitability. These include 5-HT, or serotonin, and many peptides, including NPY, somatostatin, CCK, enkephalin, and dynorphin. In many cases, it has been experimentally challenging to identify a role for the endogenous compounds, despite that they are present, are released during stimulation, and have functional receptors in the tissue. The fourth and last group of neurochemicals used in “chemical coding” studies includes calcium-binding proteins, structural proteins, and an assortment of other proteins; these may not play any direct role in functioning of neuronal circuits, but they may affect individual cell properties. It should be stressed that this classification of neurochemicals is intended only as a generalization; in a particular synapse, junction- or behaviorrelative contributions may differ significantly from the overall scheme. When comparing homologous neurons in different regions of gut or among species, the variability in chemical coding is not evenly distributed among the groups of neurochemicals outlined earlier. ChAT and NOS tend to be highly conserved, perhaps because biologically, there are no alternatives to these molecules. Secondary transmitters often are expressed in a more variable fashion. For example, tachykinins are present in all excitatory circular muscle motor neurons in guinea pig ileum, but only in a subset of those in human colon (134,140). However, they have not been reported in inhibitory motor neurons. There are several functional alternatives to SP as a secondary excitatory junctional transmitter, including neurokinins A and B. Adenosine triphosphate, peptide histidine isoleucine, PACAP, and carbon monoxide can all be released with VIP and nitric oxide from inhibitory motor neurons. All contribute to smooth muscle relaxation, via multiple intracellular pathways, and each can vary greatly in relative importance in different preparations. Neuromodulators, which act to cause presynaptic inhibition or slow postsynaptic changes in excitability, tend to be even more variable in chemical coding studies. A number of modulators can cause presynaptic inhibition in enteric ganglia, including GABA (319), NPY (320,321), 5-HT (322), opioids (323), adenosine (324), and nitric oxide (325). Postsynaptic inhibition can be mediated

FUNCTIONAL HISTOANATOMY OF THE ENTERIC NERVOUS SYSTEM / 595 by enkephalin (326), 5-HT (327), somatostatin (328), galanin (329), and adenosine (330). The slow, depolarizing, postsynaptic modulatory mechanism is substantially mediated by endogenous tachykinins (205) but may have components mediated by 5-HT (331), GRP and VIP (332), CCK (333), CGRP (334), and PACAP (335). Thus, for each type of modulatory mechanism, there are a number of alternative neurochemicals. This could explain why neuromodulators show such variability in expression in chemical coding studies.

SUMMARY AND CONCLUSIONS This chapter has reviewed our current understanding of the structure of nerves within the wall of the gut and some of the functional implications that can be inferred from this. The account given here is only a beginning, largely based on a few model systems that have been studied intensely. Our understanding of the functional anatomy of the human enteric nervous system lags far behind. A number of neurochemicals that may function as transmitters or modulators need to be integrated into the account. Thus, ATP, dopamine, glutamate, and glycine may all play a role in gut physiology, but their cellular sources and targets largely remain to be determined. To understand the functional neuronal circuitry of the gut, we need to accomplish the following: 1. identify all functional classes of nerve cells, 2. characterize the neurochemical and electrophysiologic properties of each class, 3. establish the connectivity between functional classes of neurons, 4. record their activity during the behavior of interest, 5. model the activity of the circuitry and determine the extent to which the data in items 1 to 4 above explain the observed behavior, and 6. perturb the functions of specific neurons or synapses using drugs or molecular or genomic approaches to assess their contribution to circuitry. There is still much to be learned about the dynamics of how enteric neurons control gut function. To date, most functional studies have been performed on simple and relatively artificial reflex pathways. The challenge for the future will be to record and understand neuronal activity during normal gut function. This probably will require recording from multiple neurons simultaneously, ideally when the preparation is minimally restrained. Approaches using optical recording methods, based on calcium- or voltagesensitive dyes, hold promise (336,337). To bridge the gap between individual cells, tissues, and organ function, methods that allow high-resolution monitoring of the whole preparation, such as spatiotemporal mapping (166,338), may be valuable, when combined with single-cell recording. To date, the structural studies reviewed here have contributed to the first three requirements; the final three will require considerable ingenuity and dedication. Understanding the

neuronal control of human gut function will have significant implications for diagnosis of many gastrointestinal disorders and is likely to identify novel targets for future pharmacologic or molecular interventions.

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (National Institute of Diabetes and Digestive and Kidney Diseases, grant 56986). Simon J. H. Brookes is a senior research fellow of the National Health and Medical Research Council, Australia.

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CHAPTER

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Physiology of Prevertebral Sympathetic Ganglia Joseph H. Szurszewski and Steven M. Miller Overview, 603 Morphology, 605 Innervation of Prevertebral Ganglia, 607 Chemical Coding of Prevertebral Ganglion Neurons, 610 Chemical Coding of Nerve Fibers in Prevertebral Ganglia, 611 Substance P, 611 Nitric Oxide, 611 Organization of Sympathetic Motor Innervation to the Gut, 611 Overview, 611 Guinea Pig, 612 Rat, 612 Pig, 613 Dog, 613 Cat, 613

Targets in the Gut of Sympathetic Neurons in Prevertebral and Pelvic Ganglia, 614 Visceral Afferent Neurons, 614 Morphology of Viscerofugal Neurons, 616 Chemical Coding of Viscerofugal Neurons, 619 Guinea Pig, 619 Rat, 619 Pig, 620 Dog, 620 Populations of Mechanosensory Viscerofugal Neurons, 620 Electrophysiology of Prevertebral Ganglion Neurons, 620 Pacemaker Neurons, 622 Concluding Remarks, 623 Acknowledgments, 623 References, 624

OVERVIEW

images in Figures 22-1 through 22-4 and 22-8 through 22-11 are volume-rendered, three-dimensional (3D) reconstructions of neurons using ANALYZE (Biomedical Imaging Resource, Department of Physiology and Biomedical Engineering, Mayo Foundation, Rochester, Minnesota) software running on a PC. The full rotational sequences around the three major axes of these neurons and the spatial distribution of receptors and apposing structures on these neurons, as well as of other PVG neurons not illustrated in this chapter, can be found by accessing the Gastrointestinal Physiology Lab section of the Mayo Clinic College of Medicine Web site at: http://mayoresearch.mayo.edu/mayo/research/gi_phys_lab and clicking on the tab bar “Physiology of Prevertebral Ganglia.” Two other reviews of PVGs have recently been published (3,4). PVG neurons are motor neurons that lie entirely outside the central nervous system and are located in the celiac, superior mesenteric, and inferior mesenteric ganglia. They provide sympathetic input to the gastrointestinal tract, spleen, liver, pancreas, and urogenital organs (e.g., kidney, bladder, ureters, ovaries, uterus, vas deferens). PVG neurons are essential for control of transit through the gastrointestinal tract, secretion, absorption, and gastrointestinal blood flow.

In two previous reviews on this topic (1,2) we detailed the anatomy, histology, ultrastructure, electrophysiology, neurochemistry, and innervation of the abdominal prevertebral ganglia (PVGs). We also reviewed the historical background that led to the hypothesis that the PVGs are integrating centers where two independent nervous systems (the enteric nervous system and the central nervous system) meet and act on or communicate with each other. In this chapter, we focus most of our attention to advances made since 1990. We reference the older literature when it is needed to place the newer data in better perspective. The single-slice confocal J. H. Szurszewski: Department of Physiology and Biomedical Engineering, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. S. M. Miller: Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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604 / CHAPTER 22 Sympathetic innervation of different regions of the gastrointestinal tract is centered in specific PVGs and, in some instances, in specific regions of a specific ganglion. In general, the more cranial PVGs innervate proximal gut regions, whereas caudal ganglia innervate more distal regions. In ganglia of large animals such as the pig, functionally similar neurons within a specific ganglion tend to gather to form a neuronal center, whereas in small animals such as the guinea pig, they are not specifically arranged or localized to any particular region of the ganglion. Like the paravertebral sympathetic chain ganglia, PVGs receive preganglionic input from the intermediolateral column of the spinal cord. Unlike neurons in paravertebral ganglia, most neurons in PVGs also receive convergent synaptic input from neurons located in the enteric nervous system, from collaterals of unmyelinated afferent nerves with cell bodies in the dorsal root ganglia, and in some instances, from other PVG neurons. It may no longer be appropriate to refer to PVGs as sympathetic, because some neurons in these ganglia receive varicose-like structures from the dorsal motor nucleus of the vagus. PVG neurons that innervate and regulate the vasculature in the wall of the gastrointestinal tract, referred to as vasomotor neurons, are generally innervated by one to five preganglionic axons that form a strong synapse and by a variable small number of axons that form weaker synapses. A strong synaptic input is one that always initiates a postsynaptic spike, the outgoing signal, when it is activated. At strong synapses, incoming cholinergic signals are merely translated into outgoing adrenergic signals. In contrast with vasomotor neurons, PVG neurons that regulate secretion and motility, referred to as visceromotor neurons, are generally innervated by a relatively large number of weak excitatory synaptic inputs from the gastrointestinal tract and central nervous system. A weak synaptic input is one that initiates an excitatory postsynaptic response that is subthreshold for generating an action potential. Subthreshold excitatory postsynaptic potentials can reach threshold for an action potential by fluctuation in amplitude, summation, facilitation, or modulation by another neurotransmitter/neuromodulator agent such as an excitatory neuropeptide. At weak synapses, summation of incoming cholinergic signals is translated into outgoing adrenergic signals. PVGs do not have stereotypical patterns of motor activity stored in their circuitry as do ganglia in the enteric nervous system. Instead, axons of PVG motor neurons make synaptic contact with neuronal structures in the enteric nervous system to modify programmed enteric circuits. Here, they suppress the release of neurotransmitter agents from excitatory motor neurons in the myenteric plexus and inhibitory neurotransmitters from secretomotor motor neurons in the submucous plexus. PVG can be thought of as extended neural networks that coordinate enteric neuronal activity in different parts of the gastrointestinal tract. A unique feature of PVG neurons is that they receive convergent fast excitatory synaptic input from a unique subset of enteric ganglion neurons. These afferent neurons are referred to as viscerofugal neurons. Viscerofugal neurons in the guinea pig, mouse, rat, rabbit, cat, and dog use acetylcholine for

fast synaptic transmission. In porcine PVGs, it may also be 5-hydroxytryptamine (5-HT), because 5-HT–immunoreactive (IR) nerve terminals of peripheral origin surround tyrosine hydroxylase (TH)–IR neurons. Most of the viscerofugal neurons that project to PVGs are located in the distal portion of the intestinal tract. Thus, PVGs interconnect lower regions of the intestinal tract with more proximal regions. The gut wall is equipped with a broad array of mechanosensory nerves. AH/Dogiel type II neurons (also referred to in the literature as intrinsic primary afferent neurons) in the myenteric plexus are low-threshold mechanosensors. Their discharge is dependent on smooth muscle tone and tension rather than stretch. Slow excitatory peptidergic neurotransmission appears to be the principal mode of communication among AH neurons and between AH neurons and internuncial S/Dogiel type I neurons. New evidence indicates that S neurons have mechanosensitive somas and mechanosensitive dendrites. There are two types of vagal afferent nerves that are low-threshold mechanosensors. The intramuscular arrays (IMAs) are arranged parallel to the longitudinal muscle layer, but they appear to function as “in-series” receptors and respond to contraction and changes in muscle tension. Intraganglionic laminar endings (IGLEs) are found in enteric ganglia in the upper and lower ends of the gastrointestinal tract and are structurally specialized. IGLEs appear to function as tension receptors. Spinal afferent mechanosensory nerves with cell bodies in the dorsal root ganglia have terminal fields in the mesenteric, serosal, mucosal, and muscle layers. Spinal afferents with terminal fields in the muscle layers send axon collaterals to PVGs before entering the spinal cord. Mechanosensory spinal afferent nerves appear to be arranged in series to the contracting muscle layer and are high-threshold tension detectors that respond dynamically across a wide range of stimulus intensity extending to the noxious range. The final set of mechanosensory nerves are the viscerofugal neurons. Viscerofugal neurons are inparallel stretch receptors and encode information about the degree of stretch of the circular muscle layer. They function as slowly adapting mechanoreceptors that detect changes in volume. The ongoing synaptic input that these neurons supply to PVG neurons and the ease in activating viscerofugal neurons in vitro suggest, or at least raise the possibility, that viscerofugal neurons are likely to be active most of the time in the gut wall in vivo. The input these neurons provide to PVG neurons and the return output of the latter neurons to the gut wall is an example of an ongoing reflex in the peripheral nervous system that controls normal body function. It is unlikely that viscerofugal neurons discharge together in unison as vasomotor neurons do with cardiac and respiratory rhythms or under times of stress. The different arrangement of mechanosensory nerves allows the gut to differentiate between in-series tension receptors and in-parallel stretch receptors. Viscerofugal neurons allow the gut to fill, IGLEs and IMAs allow the gut to respond to tension, AH/Dogiel type II neurons initiate tone-dependent peristaltic contractions, S/Dogiel type I neurons initiate tone-independent stretch-activated reflex contractions, and primary afferent neurons signal pain.

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 605 PVG neurons that modulate contractions, absorption, and secretion have all the attributes of an integrating center. PVG neurons receive weak convergent synaptic input from peripheral and central preganglionic nerves. With the exception of mouse PVGs, a large number of neuropeptides are found in varicose nerve fibers that make contact with the soma and dendrites. Receptors for neurotransmitters, for neuropeptides that function as neuromodulators, and for gas transmitters are distributed presynaptically and postsynaptically. The multiplicity of neurotransmitters and neuromodulators allows for excitation, inhibition, and various levels in between. Although peptide neuromodulators may not by themselves evoke action potentials, they do serve to modulate neuronal excitability to incoming synaptic input. Slow excitatory synaptic potentials without action potentials sustain nervous regulation over long periods. The coexistence of the rapidly acting neurotransmitter acetylcholine and long-acting neuropeptides such as vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase–activating peptide (PACAP), and substance P (SP) suggest multimessage cotransmission. Acetylcholine is used to organize fast reflex activity, whereas the neuropeptides are used to modulate neuronal excitability long term. Whether excitatory or inhibitory, slow synaptic potentials provide a mechanism for temporal and/or spatial modulation of fast excitatory postsynaptic potentials, thereby increasing or decreasing, respectively, the probability of action potential generation in ganglion cells. A weak synapse may be made a functionally strong synapse by an excitatory neuropeptide. Neuropeptide modulators may also act presynaptically to influence the release of the classical neurotransmitter acetylcholine, as well as other neuropeptides. One of the unique features of PVGs is that synaptic modulation of primary afferent nerves can take place within the PVGs, as well as in the gut wall. Conventionally, synaptic modulation of primary afferent neurons takes place in the brain and spinal cord. Neurotransmission in PVGs can be homosynaptic or heterosynaptic. Homosynaptic transmission is a characteristic feature of wiring transmission in which there is one-to-one transmission and a close spatial correspondence between the release site for a ligand and its receptor. Heterosynaptic transmission is a characteristic feature of volume transmission in which there is a spatial mismatch between the release site of a ligand and the postsynaptic location of its receptor. Fast cholinergic nicotinic transmission is an example of the former, whereas slow transmission mediated by SP is an example of the latter. All neurons are not created equal. Nerve cells in PVGs may be distinct in their electrical properties, expression of neuropeptides, morphology and complexity of the dendritic tree, and suite of ion channels, and they may exhibit different patterns of endogenous electrical activity. Some neurons are tonic discharging, some are phasic discharging, and some have a multisecond-long after-spike hyperpolarization. Some neurons express an M-current to suppress discharge, whereas an A-current facilitates firing in other neurons. Visceromotor neurons appear to have similar ionic conductances and similar electrophysiologic characteristics. They may be coactivated or tuned during synaptic activation from peripheral and

central sources. The patterns of firing, however, are not necessarily fixed, but they are subject to modulation resulting from synaptic stimulation by fast-acting neurotransmitters and slow-acting neuromodulators. Modulation of PVG neurons by neuropeptide modulators allows ganglion neurons to adapt their output in the face of a continually changing environment. A number of ionic currents, intracellular second messengers, and neuromodulators, as well as their receptors, are at the basis of neuromodulation. There is no Rosetta stone for neuromodulation. A substantial number of neurons in the inferior mesenteric ganglion (IMG) of the cat and celiac ganglion (CG) of the rabbit are either pacemaker neurons or regularly discharging neurons that have a firing pattern that is pacemaker-like. Because preganglionic input to visceromotor neurons can also be tonically active, a large proportion of visceromotor neurons in vivo would be expected to exert sustained inhibitory control of their target tissue. In summary, PVGs are coordinating, distributing, and integrating centers. The visceromotor neurons in these ganglia have all the essentials for an integrating center: convergence, modulation, weak synaptic inputs, plurichemical transmission, mosaic of receptors, and a suite of ionic currents. The remainder of this chapter provides details about the main points summarized thus far.

MORPHOLOGY Cell bodies of IMG neurons are arranged orderly in layers, and the dendritic arbor of any one neuron is confined to a plane with a thickness that does not exceed the thickness of its parent cell body (5) (Fig. 22-1). These structural features suggest that IMG neurons do not make extensive connections with neurons in other layers. A similar structural arrangement of dendritic arbors has been observed in mouse superior mesenteric ganglia (SMG) (6), guinea pig enteric ganglia (7), and lumbar sympathetic ganglia (8), raising the possibility that neuronal connectivity in peripheral autonomic ganglia is highly structured and organized. What has yet to be determined is whether IMG and SMG neurons in the same plane have the same or different phenotype. It is also unclear whether preganglionic nerves and viscerofugal nerves, which are known to diverge on entering the IMG and SMG, make synaptic contact with neurons in the same plane or with neurons located in different planes. Based on 3D reconstruction of single neurons in the pelvic ganglia (PGs), there appears to be two populations of neurons. One population is mononucleated or binucleated and has twoto seven-long primary dendrites and up to seven secondary dendrites. The other population is mononucleated and has one to three relatively short unbranched dendrites (5). Neurons with several long processes may be sympathetic because their morphology is similar to sympathetic neurons in the IMG. Neurons with short processes may be parasympathetic (5,9). A general principle for peripheral autonomic motor neurons is that the size of the dendritic field determines the number of available sites for synaptogenesis (10). In comparison

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FIG. 22-1. Spatial arrangement of neurons in guinea pig inferior mesenteric ganglion (IMG). Sixteen neurons in the same IMG were microinjected with biocytin, visualized with streptavidin-Cy5, and volume rendered. The visualized neurons (false-colored) are viewed from 0-degree rotation in A. A planar arrangement of the neurons was observed when the entire data set was rotated 90 degrees along the long axis (x-axis) of the ganglion (B). (C, D) Images were made at higher magnification of three neurons located in the top left of A. (B, D) Note the mingling of dendritic arbors of neurons located side by side in the same plane, whereas dendritic arbors of neurons located in different layers did not intermingle. The volume-reconstructed images were made combining 32 sets of images (375-µm/side). Each set contained 78 sequential confocal images recorded at 1.2-µm-depth increments. The total number of sequential consecutive confocal sections used to render and volume reconstruct the neurons was 2496. Each neuron was reconstructed separately, tiled, and placed in the same space it occupied in the intact ganglion relative to the other neurons. Scale bars = 1 mm (A, B); 200 µm (C, D). (See Color Plate 10.) (Reproduced from Ermilov and colleagues [5], by permission.)

with vasomotor neurons, visceromotor neurons in the guinea pig CG have a three times larger dendritic arbor, four times as many functional inputs, and four to five times as many synaptic profiles per 100 µm (11). Although visceromotor neurons receive input from two independent populations of neurons (central nervous system and enteric nervous system), there is no evidence to suggest that there are separate domains on the dendrites for each convergent input. As befits their role as integrators of incoming synaptic signals, visceromotor neurons in the CG, SMG, and IMG have a dendritic tree considerably more extensive and complex than neurons in the PG that act as relay neurons (Figs. 22-2 and 22-3). The number of primary dendrites range from 3 to 15 (5,6,8,12–16), and the dendritic length ranges from a few hundred microns to, in some instances, several millimeters (5,17,18). The ratio of total path length to vector length, a measure of the tortuosity of the path of the dendrite from its base at the somal surface to the visible terminal end, is 2.2 (5).

Dendrites modulate synaptic strength by interposing a variable electrotonic distance between the site of the input and the region of the cell (axon hillock) that initiates an action potential (19,20). The longer the dendrites are, the more likely that they play a significant role in the electrical integration of converging synaptic input. The length constant for dendrites of sympathetic neurons is estimated to range from 200 to 1000 µm (21,22). Therefore, a postsynaptic potential generated at the far end of a dendrite longer than the length constant of the dendrite is likely to be attenuated by the time it reaches the soma and axon hillock. A postsynaptic potential generated within the length constant would be expected to reach the soma and axon hillock with little attenuation. In either case, subthreshold postsynaptic potentials also likely alter neuronal excitability indirectly by altering the cytoplasmic concentration of intracellular messengers such as calcium and other second messengers stimulated by synaptic activity. Dendrites therefore may also function as segregated cytoplasmic domains that partition changes

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FIG. 22-2. Three-dimensional volume-reconstructed images of a neuron in guinea pig and spatial distribution of monoclonal antibody 35 (MAb35), a marker for nicotinic acetylcholine receptors. The inferior mesenteric ganglion (IMG) neuron was filled by microinjection with Lucifer Yellow, imaged by confocal microscopy, volume reconstructed using ANALYZE software, and viewed at 0- (left) and 180-degree rotation (right). The majority (57%) of cell-surface–associated sites of MAb35 immunoreactivity (putative nicotinic acetylcholine receptors colored white) are distributed on the dendritic arbor. This neuron had 11 primary dendrites and an extensive dendritic arbor. Scale bar = 50 µm.

in concentration of calcium and other second messengers stimulated by synaptic activity (8). The large and complex dendritic tree characteristic of neurons that receive input from viscerofugal neurons adds to the extended surface area of the neuron. The ratio of the measured surface area of dendrites to the measured surface area of the parent cell body ranges from 8:1 to 11:1 for guinea pig IMG neurons (5) and 5:1 to 6:1 for mouse SMG neurons (6). This is to be compared with Dogiel type I and II neurons that have ratios of 2:1 (7). PG neurons that function as relays and have few but strong synaptic inputs have a ratio of 0.6:1 (5). The estimated ratio for giant cells of the reticulum is 5:1 (23), and 25:1 for pyramidal neurons of the somatosensory cortex (24). Apposing boutons, which may signify active synaptic regions, occupy 1% to 2% of the total membrane surface area in PVG and PG neurons including the somatic and dendritic areas (8,18,25). Although the density of apposing boutons is similar in different sympathetic ganglia, the total number of putative synapses on the soma and dendritic tree appears to be proportional to the size of the neurons (8). Approximately 75% of nicotinic acetylcholine receptors are located on the dendritic membrane, a structural arrangement

that supports the notion based on electrophysiologic data that the majority of synaptic inputs to IMG neurons occur on dendrites (2,18,26). The spatial distribution of presynaptic VIP and PACAP appositions are also distributed along secondary and tertiary dendritic branches (18) (Fig. 22-4). This similarity in spatial distribution between nicotinic and peptidergic synaptic regions provides a structural basis whereby the efficacy of fast, weak, nicotinic cholinergic transmission can be modulated by the increase in membrane input resistance by PACAP (18) and VIP (27). The similarity in spatial distribution of cholinergic and peptidergic synaptic regions is consistent with immunohistochemical studies showing colocalization of acetylcholine and neuropeptides in enteric neurons which project to PVGs (28,29).

INNERVATION OF PREVERTEBRAL GANGLIA Virtually all PVG neurons receive central preganglionic input from neurons located in the intermediate zone of the thoracolumbar spinal cord. Visceromotor neurons in the CG,

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FIG. 22-3. Three-dimensional volume-reconstructed image of a neuron in the mouse superior mesenteric ganglion (SMG) and spatial distribution of appositions with vesicular acetylcholine transporter (VAChT)–like immunoreactivity (IR; white) on the surface of the neuron. VAChT is required for the transport of acetylcholine from the cytoplasm into synaptic vesicles. Thus, VAChT–like-IR is a marker of presynaptic nerve endings containing acetylcholine. The neuron was microinfected with Lucifer Yellow, imaged by confocal microscopy, and volume reconstructed using ANALYZE software. This neuron had an extensive dendritic arbor. Scale bar = 50 µm.

SMG, and IMG that target enteric ganglion neurons also receive input from viscerofugal neurons. Axons of spinal afferent neurons with cell bodies in dorsal root ganglia cause collaterals as they pass through the PVGs. They represent a small proportion of the endings, approximately 10% (30). A subpopulation of neurons in cat IMG receives input from neighboring neurons in the IMG (31). Lastly, a small proportion of neurons in rat CG has pericellular boutons that originate from the dorsal motor nucleus of the vagus (32,33). After each preganglionic fiber enters its target ganglion it ramifies or diverges to provide input to many neurons. Divergence allows for synaptic and spatial amplification of incoming signals. The degree of divergence varies among the different ganglia and among species for the same ganglion (34–37). The ratio of central preganglionic nerves to postganglionic neurons ranges from 1 to 200 in sympathetic ganglia (38). In the cat IMG, the ratio of incoming fibers in the peripheral lumbar colonic nerve to ganglion neurons ranges from 1:19 to 1:38 (37). The ratio of central

preganglionic nerves to ganglion neurons in parasympathetic ganglia is 1:3 (38). The preganglionic neuron that supplies input and the neurons it innervates can be thought of as a neural unit (39). Neurons in PVGs also receive input from more than one incoming fiber. The number of converging inputs correlates with the geometric complexity of the target cell. Neurons in guinea pig and mouse PVGs that have extensive dendritic arbors receive 40 to 60 inputs (1,2,5,18,40). Neurons that have few dendrites, as do many neurons in PGs, receive one to five inputs from the pelvic nerve and one to five inputs from the hypogastric nerve (1,2,5,40). Most PVG neurons receive strong and weak synaptic input (2). A strong excitatory synapse is one that leads to a large-amplitude, fast excitatory synaptic potential that invariably exceeds threshold for action potential generation and the action potential has a sharp, rising phase. Strong inputs mediate invariant synaptic gain. There is a one-to-one transfer of the signal from presynaptic input to postsynaptic neuronal firing. The safety factor for transmission of a

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FIG. 22-4. Spatial (three-dimensional) distribution of presynaptic vasoactive intestinal peptide (VIP)–like-immunoreactive (IR), vesicular acetylcholine transporter (VAChT)–like-IR appositions (yellow, A and B, respectively), and monoclonal antibody 35 (MAb35)–IR (putative nicotinic acetylcholine receptors [nAChRs], red) on the surface (C) and inside the same neuron (D) in guinea pig inferior mesenteric ganglion (IMG). Note that the majority of VIP–like-IR presynaptic endings (55.9 ± 5.7% of the total neuron-associated, VIP–like-IR–containing structures) are on secondary and tertiary dendritic branches of the neuron, and that the majority of VAChT–like-IR (marker of the presynaptic acetylcholine-containing terminals) and MAb35 sites (marker of nAChRs) also are on secondary and tertiary dendrites. The presence of MAb35–like-IR within the IMG neuron (D) most likely represents nAChR subunits associated with intracellular organelles during biosynthesis and trafficking of nAChRs. IMG neurons were intracellularly injected with Lucifer Yellow (LY), immunostained with appropriate antibodies, visualized with either Cy3 or Cy5, and imaged using a confocal microscope. Images of the LY-filled neurons (gray) and associated immunoreactive sites (red) were volume reconstructed and superimposed using ANALYZE software. Scale bars = 50 µm. (See Color Plate 11.) (From Ermilov and colleagues [18], by permission.)

strong synapse is high. Strong synapses have a large quantal content and presumably more connecting varicosities with the target neuron. The number of quanta released is vastly greater than that required to depolarize the neuron to elicit an action potential. Vasomotor neurons located in the lateral regions of CGs receive one to five strong converging preganglionic inputs (41–46). A weak excitatory input, in contrast, evokes a subthreshold excitatory postsynaptic potential. Weak synapses mediate activity-dependent synaptic gain (47). Weak synapses are ineffective unless they summate, facilitate each

other, or are modulated by neuropeptides. In visceromotor neurons, converging fibers from central and peripheral sources evoke a postsynaptic spike only through temporal and spatial summation of fast and slow excitatory postsynaptic potentials. In weak synapses, the number of quanta released is less than for strong synapses (48), and the probability of release is lower. At weak synapses, the probability of release may be as low as 0.2, whereas it is approximately 1.0 at strong synapses (8,49). The advantage of weak synaptic input to the target cell is that it provides the cell with a

610 / CHAPTER 22 mechanism for rapidly sampling and integrating many such inputs over a restricted period and over selected pathways. Convergence of weak synaptic inputs is the sine qua non of integration. Some progress has been made regarding the underlying molecular differences between weak and strong synapses. In CGs, mean levels of immunoreactivity to the t-SNARE protein synaptosome-associated protein of 25 kDa (SNAP25), choline acetyltransferase (ChAT), and vesicular acetylcholine transporter are significantly greater in preganglionic terminals (strong input) than in terminals of viscerofugal neurons (weak input) (50). In fact, some boutons of viscerofugal neurons have no detectable SNAP-25, yet these boutons make conventional synapses with conventional ultrastructural appearance (50). If effective transmission takes place only at boutons with SNAP-25, then synapses lacking the t-SNARE protein would have low-release permeability. It has been estimated that less than a third of terminals of viscerofugal neurons in the medial region of the CG are involved in fast cholinergic transmission. Thus, weak synaptic strength at terminals of viscerofugal neurons may be partly due to a low proportion of boutons that express SNAP-25 and other synaptic proteins (50). There appears to be a correlation among the extent of labeling of SNAP-25, syntaxin, and Ca channels at a release site; the store of docked vesicles; and the probability of spontaneous and evoked quantal release (51). There may also be differences in the type and density of Ca channels in weak synapses. In paravertebral ganglia, where preganglionic inputs are strong, N-type Ca channels are prominent in terminals of high-strength inputs (50,52), whereas P/Q-type Ca channels are prominent in weak inputs (50,53). Approximately 50% of the boutons observed at the ultrastructural level lack any detectable presynaptic specialization (8), and all are encapsulated by a thinly spread Schwann cell (glial cell). The presence of the Schwann cell covering would be expected by conventional thinking to limit the diffusion of transmitter from the release site in the covered bouton should any transmitter be released. However, it has been suggested that Schwann cell layers may not present a significant barrier, and that neuropeptides released from boutons fully enclosed by a Schwann cell process may diffuse throughout the neuropile and reach potential target cells (25). Furthermore, the binding site of a neurotransmitter/ neuromodulator and its release site may not be spatially correlated. For example, the binding sites for BoltonHunter–labeled 125I-SP and the potential sites of SP release have relatively poor spatial correlation (54), raising the possibility that SP may modulate ganglionic transmission through heterosynaptic and homosynaptic actions on neurokinin-1 (NK1) receptors (55). The possibility that endogenously released SP diffuses over relatively large distances throughout the ganglion with actions on those neurons expressing NK1 receptors, the observation that many of the boutons do not have synaptic specialization at the ultrastructural level, and the spatial mismatch between receptor and ligand raise the possibility that volume transmission is a mode for

synaptic transmission in PVGs. Volume transmission is much slower, uses lower concentration of effector, and spreads over larger populations of cells than does wiring transmission (release of neurotransmitters/neuromodulators at conventional synaptic contacts) (56,57). Volume transmission submits populations of cells to a common global control. Ensembles of neurons that possess a common receptor for a diffusing neuropeptide (signal) would be affected at the same time. In the case of CG neurons, visceromotor and vasomotor neurons are located in distinctly separate regions of the ganglia. Thus, a neuromodulatory substance such as SP released from boutons in one of the regions would be expected to affect receptors only on dendrites extending from a group of neurons with similar neurochemistry and similar functional roles (25,58,59).

CHEMICAL CODING OF PREVERTEBRAL GANGLION NEURONS In PVGs of mammals, most neurons are noradrenergic (NA), that is, they are TH-IR or dopamine β-hydroxylase-IR as shown in IMG of guinea pig (60) and pig (61,62). Immunoreactivities to neuropeptide Y (NPY) or somatostatin (SOM) also have been commonly found in sympathetic noradrenergic postganglionic PVG neurons of mammals, including human CG-SMG (63), rat CG-SMG (64), guinea pig CG-SMG (41,54,64) and IMG (41,54,60,65,66), pig IMG (61,62), dog IMG (67), and cat CGs (68). In human SMG and CGs, there are, however, only a small number of SOMIR neurons compared with the number found in CGs and SMG of various animal species. In guinea pig, about 20% to 65% of CG-SMG neurons are estimated to be NPY IR (69,70), whereas in rats, most CG-SMG neurons are NPY IR (64). In contrast, about 12% of rat CG-SMG neurons are IR for SOM (64) compared with estimates of 25% of CG-SMG neurons and 45% to 75% of IMG neurons being SOM-IR in guinea pig (41,60,69,70). Nonadrenergic (i.e., TH-negative) neurons also are found in guinea pig CG (69), guinea pig IMG (60,65), rat CGSMG (71), and pig IMG (62). They comprise about 2% to 5% of neurons in guinea pig IMG (60,65) but a substantially larger proportion, about 10% to 15%, of IMG neurons in rat and pig (61,62). Some TH-negative prevertebral ganglion neurons in guinea pig and pig are IR for ChAT-IR and are presumed to be cholinergic neurons (62,65). In guinea pig IMG, most cholinergic neurons are also NPY-IR, are located in the caudal lobe of the ganglion, and are presumed to project to the rectum via the hypogastric nerves (65). Small subpopulations of PVG cells are immunoreactive for VIP in human CG-SMG (63), guinea pig IMG (60), and rat (64,72); for galanin (GAL) in pig IMG (62); for nitric oxide synthase (NOS) in guinea pig IMG (60) and pig IMG (62,73); for calcitonin gene–related peptide (CGRP) in pig IMG (61); or for enkephalin (ENK) in cat CG (74) and cat IMG (75).

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 611 CHEMICAL CODING OF NERVE FIBERS IN PREVERTEBRAL GANGLIA Substance P In PVGs of guinea pig, SP-IR fibers contain immunoreactivity to CGRP and represent collaterals of capsaicin-sensitive, unmyelinated sensory neurons with cell bodies that are located in dorsal root ganglia. Exogenously applied SP has excitatory effects on PVG neurons, and evidence suggests that SP can mediate slow synaptic potentials in PVG neurons (76,77). In CGs of guinea pig, these effects are mediated by NK1 and NK3 tachykinin receptors, which are present on both phasic and tonic neurons. NK1, NK2, and NK3 receptors are expressed in rat PVGs as demonstrated by in situ hybridization experiments (78). In guinea pig IMG and CG, there is a dense and uniform distribution of varicose SP-IR fibers, and most of these also contain CGRP immunoreactivity (54). SP binding sites with pharmacologic characteristics of NK1 tachykinin receptors are present in guinea pig IMG and CG and occur with greatest density in regions of the ganglia that contain predominantly SOM-IR neurons, that is, visceromotor neurons (54). Consistent with the presence of SP binding sites with NK1 characteristics in guinea pig CGs, electrophysiologic studies show that effects of tachykinins on guinea pig CG neurons are mostly mediated by NK1 receptors, but there is also some contribution by NK3 receptors to the electrophysiologic effects of SP in the ganglion (76,77).

Nitric Oxide NOS IR terminals are present in the PVGs of guinea pig, rat, and mouse. NOS-containing cell bodies are few in guinea pig and rat (79) and appear to be rare or absent in mouse (80,81). There are three sources of NOS terminals: sympathetic preganglionic neurons in the intermediolateral column and dorsal commisural nucleus from spinal cord segments T12 to L2; primary afferent sensory nerves with cell bodies in the dorsal root ganglia; and viscerofugal neurons (28,81–88). NOS terminals from preganglionic nerves are found throughout the ganglia, suggesting axodendritic contacts, whereas NOS terminals from viscerofugal neurons form pericellular baskets, suggesting axosomatic contacts (82). In rat, approximately 22% to 33% of colonic viscerofugal neurons that project to the IMG are NOS immunopositive (89); in guinea pig, approximately 62% of colonic viscerofugal neurons that project to the CG are NOS immunopositive (82,83). Nitric oxide (NO) should be considered a candidate neurotransmitter or neuromodulator. The amplitude of the late slow excitatory synaptic potential is significantly enhanced in the presence of an NOS inhibitor, and exogenous NO generated from sodium nitroprusside hyperpolarizes sympathetic neurons in mouse SMG (81). NO may be the mediator

of slow, inhibitory, postsynaptic potentials observed during distension of the colon (90) and inferior mesenteric vein (91) and in the CG during distension of the stomach and duodenum (92). In rabbit CG, NO appears to be generated in and released from preganglionic nerve terminals and from terminals of gastric viscerofugal neurons (3,93–96). Endogenously released NO appears to act presynaptically to either facilitate or inhibit fast cholinergic nicotinic synaptic activation. Repetitive supramaximal electrical stimulation of the splanchnic nerves facilitates nicotinic fast synaptic activation, whereas NO released from mechanosensory gastric viscerofugal neurons inhibits cholinergic nicotinic transmission (93,94,96). In both instances, activation of muscarinic receptors is a prerequisite to the activation of the NO pathway (97). The mechanism mediating the facilitatory and inhibitory effects has not been determined, nor has the location of the muscarinic receptors that lead to NO release. The inhibitory gating effect of NO released from viscerofugal nerves during gastric distension is unusual because release of NO is action potential–independent and there are no changes in postsynaptic electrical activity. It has been suggested that activation of gastric mechanoreceptors induces an intracellular pathway that activates, at a distance, a NOS producing NO, which then is released into the CG. In summary, NO appears to be a subtle neuromodulator of the membrane potential of PVG neurons, and it appears to gate cholinergic nicotinic transmission. When inputs from viscerofugal nerves decrease input from central preganglionic nerves, modulation of visceral targets supplied by CG neurons is mainly exerted by the CG. When central preganglionic input modulates viscerofugal input, it is the central nervous system that exerts control.

ORGANIZATION OF SYMPATHETIC MOTOR INNERVATION TO THE GUT Overview Injection of retrograde tracer into the wall of the gut and other organs has been used to positively identify the neurons that project to these regions. Sympathetic innervation of the gastrointestinal tract is predominantly from neurons located in PVG with a smaller population from the paravertebral ganglia (64,98–101). Innervation from the paravertebral chain ganglia is from the lower part of the thoracic chain and is not arranged organotropically. Projections from the different PVG are arranged organotropically. In general, more cranial (cephalad) PVG innervate proximal gut regions, whereas more caudal ganglia innervate more distal regions. The main targets of the CG are the stomach and small intestine (102). The SMG supplies the small intestine, and the IMG supplies the colon (102). The PG supplies the distal colon and rectum (100,103–105). Although the main target organ of the CG is the stomach and small intestine (including the ileum),

612 / CHAPTER 22 it, together with the SMG, also supplies innervation to the ileum and proximal colon in rat and guinea pig (85,106). Sympathetic neurons within a PVG can be arranged topographically with respect to their target tissue. In general, neurons in PVG of larger animals tend to gather to form a neuronal center, whereas in small animals they are not specifically arranged or localized to any particular region of the ganglion (62). Thus, in pig PVG, ileal-projecting neurons are predominantly located in the left and right SMG near the entrance of the splanchnic and intermesenteric nerves (107). Colon-projecting neurons in the pig are found exclusively in the cranial part of the IMG along the dorsolateral border, whereas neurons supplying the rectum form a long stripe along the entire length of the lateral border of the ganglion (62). In cat IMG, neurons supplying the proximal colon are located in the proximal part of the ganglion, whereas neurons supplying the rectum are located in the distal lobe of the ganglion (37). In dog IMG, colon-targeting neurons are more numerous at the emergence of the lumbar colonic nerve, whereas rectal-targeting neurons are more numerous at the emergence of the hypogastric nerve (67). In rat, neurons targeting the ileum and proximal colon are spread diffusely throughout the CG and SMG (98,101,103). In guinea pig IMG, neurons supplying the colon are evenly distributed throughout the ganglion (108). In rat PGs, neurons targeting the colon and rectum are located in the dorsal part of the ganglion near the pelvic nerve (104).

Guinea Pig In guinea pig, CG neurons project to the stomach, small intestine, cecum, and proximal colon, but not to the distal colon (106). Stomach-projecting CG neurons and those projecting to the cecum are mostly located in the lateral poles of the ganglion, and proximal colon-projecting neurons are mostly located in the medial part of the ganglion, whereas small intestine–projecting CG neurons are scattered throughout the ganglion (106). Most proximal colon-projecting CG neurons are SOM-IR, and a majority projecting to the small intestine are NPY-IR, whereas the majority projecting to the stomach are neither SOM-IR nor NPY-IR. SMG neurons project to the cecum, proximal colon, and distal colon. A small number of SMG neurons project to the small intestine, but only a few project to the stomach (106). Distal colonprojecting neurons in the SMG are mostly located in the caudal pole of the ganglion, whereas neurons projecting to the proximal colon and cecum are scattered throughout the ganglion. Most of the SMG neurons projecting to the cecum or proximal colon are SOM-IR or NPY-IR, whereas those projecting to the distal colon comprise about equal proportions of NPY-IR neurons, SOM-IR neurons, or neurons that lack immunoreactivity for either peptide. IMG neurons project to the cecum, proximal colon, and distal colon, with a few projecting to the small intestine, but none projecting to the stomach (106). These cells are scattered throughout the ganglion. NPY-IR or SOM-IR cells comprise most of the

IMG neurons projecting to the cecum. Nearly half of the distal colon-projecting neurons in the IMG are NPY-IR, one-fifth are SOM-IR, and one-third are not IR for either peptide (106). Neurons in the anterior major PG of guinea pig project to the rectum. They contain immunoreactivity for NPY and are sometimes surrounded by methionine-enkephalin (METENK)-IR varicose nerve fibers (109). These PG neurons appear to be nonadrenergic because they lack immunoreactivity for TH. Consistent with these findings, anterograde labeling in vitro with tracer placed on the cut ends of the rectal nerves showed that most extrinsic fibers in the rectum lack TH-IR (110). Neurons in the guinea pig anterior major PG do not project into the hypogastric nerves (111).

Rat PVGs in rats are organized differently than in other species. The ganglia comprise, in addition to the CG, SMG and IMG, a pair of splanchnic ganglia on the greater splanchnic nerves and a single interrenal ganglion located on the intermesenteric nerve between the SMG and IMG (85). CG and SMG neurons project to the stomach, small intestine, and proximal colon, with relatively few projecting to the middle colon and none to the distal colon (85,98,101). IMG neurons project mainly to the middle and distal colon and rectum, with a few projecting to the proximal colon and distal ileum (112,113). Splanchnic ganglion neurons innervate the stomach, small intestine, and proximal colon. Neurons in the interrenal ganglion project mostly to the middle and distal colon, with a small number projecting to the proximal colon and ileum. Rat PG neurons project to the bowel via the rectal nerves, pelvic nerves, and penile nerves (104,105). PG neurons project to the distal colon and rectum (86,112), with only a small number to the middle colon and none projecting to the proximal colon or ileum (100,101,103–105). PGs are mixed ganglia containing both sympathetic and parasympathetic neurons (113,114). Approximately equal numbers of both types of neurons are distal colon projecting (100). Most of the distal colon-projecting PG neurons are located in the dorsal part of the ganglion near the entrance of the pelvic nerve (103,104). Anterograde labeling with tracer injected into rat PG showed that the majority of projections of PG neurons to the bowel were to the myenteric plexus, with some projections to serosal ganglia and circular muscle (105). Importantly, there are only rare PG neuron projections to submucous ganglia and none to submucosal blood vessels, mucosa, or longitudinal muscle. Specific information from other species on the cellular targets in the bowel of PG neurons is not yet available. It is clear from the discussion above that PG neurons do not innervate the same range of structures as do PVG neurons, which supply both myenteric and submucous ganglia, both longitudinal and circular muscle layers, submucosal blood vessels, muscularis mucosae, and mucosa. Therefore, PVG and PG are functionally similar in that both control motility, but the

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 613 ganglia are different in that PVGs also control secretion and blood flow. In summary, in rat, like in other species, there is a rostrocaudal sympathetic innervation of the gut from PVG and PG: The CG and SMG supply the majority of innervation to the ileum and proximal colon, and the IMG and PG supply the majority of innervation to the middle and distal colon. Projections from PVGs to the different parts of the gut are organized somatotropically; that is, the rostral ganglia (CG, SMG, and splanchnic ganglia) innervate all regions of the gastrointestinal tract, whereas the caudal ganglia (interrenal ganglia and IMG) innervate the distal ileum and colon (85). The stomach, duodenum, and proximal ileum are innervated mainly by CG neurons. The distal ileum and proximal colon mainly receive innervation from neurons in CG and SMG (85).

Pig Similar to guinea pig and rat, a large number of CG and SMG neurons in pig project to the ileum (73). The majority of ileum-projecting neurons in the pig CG-SMG complex are located in the left and right superior mesenteric poles of the ganglion, near the entrance of the splanchnic and intermesenteric nerves. A majority of them (77%) are TH-IR (i.e., they are NA) and contain also immunoreactivity for NPY, SOM, or GAL. The distribution and chemical coding of IMG neurons projecting to the ascending colon and rectum in pig was investigated by Pidsudko and colleagues (62). Many IMG neurons were found to project to the ascending colon or rectum. The IMG of the pig has two symmetric lobes: right and left. Colon-projecting neurons are located in the cranial half of the ganglion in two distinct clusters: one along the dorsolateral border of each lobe, and the other located in the middle half of the ganglion close to the emergence of the caudal colonic nerves. Rectal-projecting neurons are mostly located in a band extending longitudinally along the entire dorsolateral border of each lobe. An additional group of rectal-projecting neurons is located near the caudal colonic nerves in the right lobe only. Nearly all (about 90%) of the colon- and rectal-projecting neurons in pig IMG are TH-IR. All TH-negative, colonprojecting neurons and most TH-negative, rectal-projecting neurons in pig IMG are not ChAT-IR, suggesting they are nonadrenergic and noncholinergic cells. However, a minor subpopulation of the TH-negative, rectal-projecting neurons is ChAT-IR, suggesting they are cholinergic cells. Many of the TH-IR, colon-projecting IMG neurons are also SOM-IR, a small number are NPY-IR, and a few are IR for GAL. In contrast, many TH-IR, rectal-projecting neurons are NPY-IR, small numbers are SOM-IR, and a few are GAL-IR. TH-negative colon-projecting neurons often are SOM-IR or NPY-IR, and TH rectal-projecting neurons also often are NPY-IR. Some TH-negative/ChAT-negative rectalprojecting neurons are SOM-IR, GAL-IR, NOS-IR, or

VIP-IR. Colon- or rectal-projecting neurons are not SP-IR, CGRP, or Leu-enkephalin (LEU-ENK). In terms of their innervation, both colon- and rectal-projecting IMG neurons receive moderate LEU-ENK-IR or ChAT IR and a poor VIP-IR or SOM-IR nerve supply (62). Transection of the caudal colonic nerves was found to increase the number of GAL-IR and PACAP-IR IMG neurons projecting to the distal colon (115).

Dog Studies on the projections of PVG neurons to the stomach and small intestine in dog are lacking. CG and SMG neurons in this animal project to all regions of the colon, with a majority projecting to the proximal (ascending) colon (116). IMG neurons also project to all regions of the colon and also to the rectum (67,116). Colon-projecting IMG neurons tend to be located near the emergence of the lumbar colonic nerves, whereas rectal-projecting IMG neurons tend to be located near the emergence of the hypogastric nerves (67). Colon- and rectal-projecting IMG neurons in dog comprise populations of calbindin/TH-IR and NPY/TH-IR cells. A greater proportion of rectal-projecting neurons are calbindin-IR compared with distal colon-projecting neurons (67). A minor proportion of rectal- or distal colon-projecting neurons are VIP- and TH-IR. Calbindin/TH-IR distal colonand rectal-projecting IMG neurons innervate myenteric and submucous ganglia and the muscle layers, predominantly the longitudinal layer. In contrast, NPY/TH-IR IMG neurons mainly innervate intestinal blood vessels (67). Unlike small animals such as rat and guinea pig, where the PG consist of a distinct major PG and a few small, accessory ganglia, the situation in dog and other large animals is more complex. In dog, the PG is more diffuse, consisting of a complex of 70 to 100 ganglia of varying sizes (117). In dog, unlike in the rat, PG neurons project to all regions of the large intestine and to the distal ileum (116,117). Rectalprojecting neurons are located in ganglia at the dorsocaudal part of the PG complex (117). About 60% of rectal-projecting PG neurons are calbindin-IR and 30% are NPY-IR, whereas none are NPY-IR, NOS-IR, VIP or TH-IR. Rectal-projecting PG neurons are surrounded by baskets of ENK-IR varicose nerve fibers (117).

Cat IMG neurons in cat project to the colon and rectum (35,37,75). Neurons that supply the distal colon are scattered throughout the ganglion and some are IR for LEU-ENK-IR (75). IMG neurons with axons that project into the colonic nerves are located in all parts of the ganglion except the distal lobe close to the origin of the hypogastric nerve (37), and those that project their axons into the hypogastric nerve are located only in the distal lobe of the ganglion (35).

614 / CHAPTER 22 TARGETS IN THE GUT OF SYMPATHETIC NEURONS IN PREVERTEBRAL AND PELVIC GANGLIA

the IMG are found in the myenteric plexus (60). NA/- fibers are considered inhibitory to neurons essential for enteric reflex motor activity. Sympathetic terminals form basket endings around cholinergic motor neurons, as well as around 5-HT handling neurons (121–123). It has been known for some time that noradrenaline acts presynaptically to inhibit release of neurotransmitters in the enteric plexuses (102,122, 124,125). Noradrenaline does not hyperpolarize myenteric neurons directly (126,127).

It was mentioned earlier in this chapter that sympathetic neurons innervating the gastrointestinal tract are found predominantly in PVGs and PGs, with a small proportion located in paravertebral ganglia. Colocalization of neurotransmitters and neuropeptides provide markers of the likely destination of a neuron. NA/NPY neurons are considered vasomotor because NPY-positive terminals surround arterial vessels located in the gut wall (118,119). NA/SOM and NA/neurons are considered visceromotor neurons because the terminals of these neurons surround neurons in the enteric plexuses. NA/SOM fibers are thought to inhibit secretomotor reflexes because NA/SOM-positive fibers surround submucosal ganglion neurons known to control absorption and secretion (41,102,118), whereas fibers that are NA/- are thought to modulate or inhibit motility because they are found around myenteric ganglion neurons. However, the projections of NA/SOM neurons appear to differ between the stomach and the small and large intestines. In the guinea pig stomach and small intestine (41,120), NA/SOM fibers innervate primarily the submucous plexus. In the colon, NA/SOM fibers from

Medulla oblongata

VISCERAL AFFERENT NEURONS The gut is equipped with four groups of afferent neurons: those with cell bodies in cranial and dorsal root ganglia, those with cell bodies in the wall of the gut, those with cell bodies in the wall of the gut that project their axons to PVGs and PGs (128), and those with cell bodies located in serosal ganglia and myenteric plexus of the most distal regions of the rectum (Fig. 22-5). Sensation from the gut is mediated via primary afferent neurons with transduction sites in the gut and nerve cell bodies in the cranial or spinal ganglia (129,130) (see Chapter 25). Endings of primary afferent mechanosensory nerves located in the serosa, muscle layers,

Spinal cord

PSN

NG

Rectospinal pathway DRG + PVG

+

Serosal and mucosal mechanoreceptors

+

“In series receptor” LM +

MP AH

−

(−) VFN −

CM “In parallel receptor”

“In series receptor”

Mucosa

FIG. 22-5. Schematic diagram of the possible arrangement of mechanosensory afferent nerves in the gut wall. Viscerofugal neurons (VFN) function as an “in-parallel” arrangement to the circular muscle (CM) layer, whereas intrinsic primary afferent neurons (AH) function as “in-series” arrangement. Receptors labeled “in-series” receptors in longitudinal muscle (LM) layer and in myenteric plexus (MP) are intramuscular arrays (IMAs) and intraganglionic laminar ending (IGLEs). They function as “in-series” tension receptors. IGLEs in the rectum are not shown. The physiologic significance of rectospinal neurons has not been identified; they may participate in the defecation reflex. DRG, dorsal root ganglion; NG, nodose ganglion; PSN, preganglionic sympathetic neuron; PVG, prevertebral ganglion. (Modified from Szurszewski and colleagues [128], by permission.)

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 615 mucosa, and mesentery are predominantly high threshold (131,132). They are silent at rest and respond dynamically across a wide range of stimulus intensity extending to the noxious range. These high-threshold fibers are generally considered to be nociceptors. Vagal and pelvic mechanosensory afferent nerves, IMAs and IGLEs, are low-threshold tension detectors activated at physiologic levels of distension (133,134). IMAs are fine varicosed nerve fibers running parallel to longitudinal muscle fibers. They appear, however, to function as “in-series” receptors and are sensitive to contraction and tension (135). IGLEs of the vagus and pelvic nerves are flattened lamellar endings that function as mechanotransducers. Their endings are in myenteric ganglia and are low-threshold, slowly adapting mechanoreceptors, and they appear to be sensitive to both distension and muscle contraction (110,133–140). Enteric AH/Dogiel type II neurons are an important group of mechanosensory neurons that regulate normal gastrointestinal motility. They are entirely contained within the gut wall. Their discharge is dependent on muscle tone and tension rather than stretch. They interconnect with each other to form a self-reinforcing network (141). S/Dogiel type I neurons also are mechanosensory (142,143). Discharge of action potentials in these neurons is stretch activated by muscle tone–independent contractions. It has been suggested that AH/Dogiel type II neurons underlie peristaltic contractions, and S/Dogiel type I neurons are responsible for ongoing contractions (144). Rectospinal neurons are a distinct class of enteric neurons that project directly without interruption to the spinal cord through L6/S1 pathways (105,145), providing a direct link between the enteric nervous system and the central nervous system. They are multipolar/multidendritic. They are a heterogenous population, with some containing only VIP, some only CGRP, some only calbindin, and some VIP and calbindin (145,146). Their cell bodies appear to receive synaptic contacts from other enteric neurons. Long ago it had been

Celiac ganglion

Superior mesenteric ganglion 10% A

17% A 50% B

17% C

speculated that “internuncial” neurons in the enteric nervous system communicate directly with central networks (147). The stimulus that these neurons encode currently is unknown. Viscerofugal neurons are a unique set of enteric ganglion neurons that relay mechanosensory information to PVG neurons. They are found throughout the gut in guinea pig, mouse, rat, rabbit, cat, dog, and pig. Although we previously referred to these neurons as intestinofugal afferent neurons (128), the term viscerofugal neurons is used in this chapter to recognize that there is a population of enteric neurons in the cardiac (138) and antral (148) regions of guinea pig and rat stomach (149,150) that send axons to the CG. In guinea pig, the density is 3 cells/cm2 in the proximal duodenum, increasing to 138 cells/cm2 in the rectum (151). In rat, there are 6.6 cells/cm2 in the glandular stomach increasing to 62.1 cells/cm2 in the colon (152). A proximodistal gradient also exists in pig (153,154), with the highest density at the end of the ascending colon. Approximately 90% of all viscerofugal neurons projecting to the CG, SMG, and IMG have their cell bodies in the colon (151) (Fig. 22-6). The approximately even distribution within the gut wall of efferent sympathetic nerve endings from the PVGs suggests that more distal regions of the gut control regions more proximal (151) (Fig. 22-7). In guinea pig, mouse, rat, rabbit, and dog, viscerofugal neurons use acetylcholine as the primary fast excitatory neurotransmitter (2). Activation of viscerofugal neurons by electrical stimulation or distension of a gut segment attached to a PVG evoke fast excitatory postsynaptic potentials that are blocked by nicotinic receptor antagonists (2). Electrical stimulation and colon distension release acetylcholine in the PVGs as measured by radio-thin layer chromatography (155). Viscerofugal neurons in rat gut are ChAT-IR (152). Viscerofugal neurons in guinea pig distal colon are immunopositive for monoclonal antibody 35 (MAb35), an antibody that recognizes non–α-bungarotoxin–sensitive neuronal nicotinic acetylcholine receptors (156) (Fig. 22-8).

16% D

A. Stomach, small intestine B. Proximal colon C. Distal colon D. Rectum

35% B

25% C 30% D

A. Small intestine B. Proximal colon C. Distal colon D. Rectum

Inferior mesenteric ganglion

30% C

22% B

48% D

B. Proximal colon C. Distal colon D. Rectum

FIG. 22-6. Distribution of gastric and intestinofugal neurons to prevertebral ganglion neurons in guinea pig. Data are taken from Messenger and Furness (151).

616 / CHAPTER 22

FIG. 22-7. The majority of viscerofugal neurons supplying neurons in the celiac and superior mesenteric ganglia (CG/SMG) (cf. Fig. 22-6) arise from the colon. Because visceromotor neurons located in the CG/SMG supply an approximately even innervation within the upper gastrointestinal tract, mechanoactivation of viscerofugal neurons in the colon will reflexly modulate activity in the stomach and small intestine. The majority of viscerofugal input to visceromotor neurons in the inferior and superior mesenteric ganglia (SMG/IMG) arises in the distal colon and rectum (cf. Fig. 22-6). Because visceromotor neurons in the SMG/IMG innervate the colon, mechanoactivation of viscerofugal neurons in the colon and rectum reflexly modulate activity in the colon. The functional significance of these reflex arcs is that motor activity in distal gut regions regulates motor and secretory activity in proximal gut regions, ensuring that bulk and fluid content of material in more proximal regions are appropriate and that intraluminal content arrives at the colon in time. The reflex arc provides a protective mechanism against large increases in tone and intraluminal pressure.

Immunoreactivity for nicotinic acetylcholine receptors is located on secondary and tertiary dendrites of neurons in guinea pig IMG (18) (see Figs. 22-2 and 22-4), and nicotinic receptor antagonists abolish electrically and colonic distension–evoked excitatory postsynaptic currents (157). Although electrical recordings have not been made from porcine PVGs, it appears, based on immunohistochemistry, that, unlike guinea pig, mouse, rat, rabbit, cat, and dog, 5-HT may play a major role in mediating fast synaptic transmission (153,154).

MORPHOLOGY OF VISCEROFUGAL NEURONS The morphology of viscerofugal neurons has been assessed from the appearance of the neurons retrogradely labeled with Fast Blue, Fluorogold, biotinamide, or DiI injected into PVGs or PGs of guinea pig, rat, and pig. In the colon of guinea pig, most retrogradely labeled viscerofugal neurons had smooth cell bodies with multiple fine processes and

were classified as Dogiel type II enteric neurons (148,151,158). However, a sizable proportion appear to have irregularly shaped cell bodies with broad (lamellar) dendrites typical of enteric neurons of the Dogiel type I class (148,151,159). Studies using intracellular injection of biocytin or Lucifer Yellow into retrogradely labeled viscerofugal neurons have more precisely revealed their morphologies (156,160). In guinea pig colon, viscerofugal neurons projecting to the IMG that were injected with biocytin were found to have irregular-shaped cell bodies (mean dimensions of about 40 × 20 µm), a single axon, short lamellar dendrites, and often additional larger, irregularshaped dendrites (160). These morphologies correspond to the Dogiel type I class of enteric neurons. In a later study (156), Lucifer Yellow was injected intracellularly into viscerofugal neurons in guinea pig distal colon that had been retrogradely labeled from the IMG with the lipophilic dye, DiI. The results showed a sizable proportion (30%) of injected cells had Dogiel type II–like morphology (Fig. 22-9), as well as viscerofugal neurons having Dogiel type I morphology (Fig. 22-10).

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 617

A

B

C

D

E

F

FIG. 22-8. Spatial distribution of monoclonal antibody 35 (MAb35)-LI immunoreactivity (IR) (a marker for nicotinic acetylcholine receptors, red) in two viscerofugal neurons in guinea pig distal colon retrogradely labeled with DiI and filled by microinjection with Lucifer Yellow. (A–C) The viscerofugal neuron has Dogiel type I morphology; (D–F) the viscerofugal neuron has Dogiel type II morphology. In both neurons, putative nicotinic acetylcholine receptors are located on the surface membrane (B, E) and clusters of putative receptors covered the cell bodies and processes (A, D). Cytoplasmic staining also can be observed in both types of neurons (C, F). The three-dimensional volume-reconstructed images were made of neurons in whole-mount preparations of guinea pig distal colon. Viscerofugal neurons were intracellularly injected with Lucifer Yellow after retrograde labeling with DiI, immunostained labeled with MAb35, and imaged using a confocal microscope. Images were superimposed using ANALYZE software. Scale bars = 25 µm. (See Color Plate 12.) (Reproduced from Ermilov and colleagues [156], by permission.)

In the rat stomach, small intestine, and large intestine, retrogradely labeled viscerofugal neurons with the appearance of Dogiel type I morphology were the predominate type (89,152,161). Similarly, in pig, retrogradely labeled viscerofugal neurons in the small or large intestine are mostly Dogiel type I–like in appearance (153,154). However, it is possible that not all viscerofugal neurons in these studies were sufficiently well labeled with tracer to be confident about their morphology. Retrograde labeling using biotinamide or DiI applied to the cut ends of mesenteric nerves in vitro also has been used to examine the morphology of viscerofugal neurons in guinea pig small intestine (122), distal colon (156), rectum (110), and mouse distal colon (162). These procedures effectively label viscerofugal neurons and reveal morphologic detail comparable with that obtained by direct intracellular injection of tracer. In guinea pig small intestine, viscerofugal neurons were found to have round or ovalshaped cell bodies (26 × 10 µm in mean dimension), a single axon, and a variable number of short lamellar or filamentous dendrites (122). Their morphology is consistent with a

Dogiel type I classification. In guinea pig distal colon, two morphologic types were found (156). The major one had an irregular cell body shape, a single axon, several short lamellar dendrites, and occasionally additional filamentous dendrites (Dogiel type I–like morphology). The other type had a smooth cell body and two or more long, smooth processes (Dogiel type II–like morphology). Confocal microscopy and 3D reconstructions of serial optical sections made through viscerofugal neurons in the guinea pig distal colon provide quantitative information on their surface area and volume (156). The mean surface area and volume of the cell bodies of Dogiel type II–like viscerofugal neurons (2200 µm2 and 4500 µm3, respectively) is significantly larger than the surface area and volume of Dogiel type I–like viscerofugal neurons (1600 µm2 and 2800 µm3, respectively). Notably, for both types of viscerofugal neurons, the surface area of the dendritic processes was much larger than the surface area of their respective cell bodies. Viscerofugal neurons in guinea pig rectum are mostly of Dogiel type I morphology with lamellar dendrites and a single axon (110). In mouse distal colon, viscerofugal neurons retrogradely labeled with DiI in vitro

618 / CHAPTER 22

FIG. 22-9. Three-dimensional reconstruction of a viscerofugal neuron with Dogiel type II morphology retrogradely labeled with DiI, imaged by confocal microscopy, volume reconstructed using ANALYZE software, and viewed at 0- and 180-degree rotation. Three-dimensional volume-reconstructed images were made of the neuron in a whole-mount preparation of guinea pig distal colon. Note that the long axis of the neuron is oriented parallel to the circular muscle layer axis. Asterisks in lower left and right panels indicate axon; asterisks in upper left and right panels indicate cat ends of dendrites. (Courtesy of Leonid Ermilov and Philip Schmalz.)

FIG. 22-10. Three-dimensional reconstruction of a viscerofugal neuron with Dogiel type I morphology retrogradely labeled with DiI, imaged by confocal microscopy, volume reconstructed using ANALYZE software, and viewed at 0- and 180-degree rotation. Three-dimensional volume-reconstructed images were made of the neuron in a whole-mount preparation of guinea pig distal colon. (Courtesy of Leonid Ermilov and Philip Schmalz.)

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 619 were mostly of the Dogiel type I morphologic class with a single axon and short lamellar dendrites (162). A small number of viscerofugal neurons have a mixture of short lamellar and long filamentous dendrites, whereas a sizable proportion have a smooth cell body and long filamentous processes (Dogiel type II–like morphology). The mean dimensions of the cell bodies (about 29 × 16 µm) are similar for the different morphologic types observed.

CHEMICAL CODING OF VISCEROFUGAL NEURONS Guinea Pig Viscerofugal neurons contain both cholinergic and noncholinergic components. In the small and large intestines, all viscerofugal neurons studied that project to the CG and IMG are ChAT-IR (28,160). In both regions, the majority of viscerofugal neurons projecting to the CG, SMG, and IMG are also IR for VIP, bombesin (BOM-IR), or both (148,151,158,159). Most viscerofugal neurons in the large intestine that project to the CG or IMG are calbindin-IR or neuronal NOS-IR, whereas viscerofugal neurons in the small intestine are not immunoreactive for either calbindin or NOS (28,82,148,151,158,159). PACAP-like immunoreactivity is present in viscerofugal neurons in the colon and in varicose nerve fibers surrounding IMG neurons (18). PACAP peptides are released in the

A

ganglion during physiologic levels of colonic distension, act presynaptically to enhance release of acetylcholine, and act directly on PVG neurons to increase membrane input resistance, thereby increasing the efficacy of fast cholinergic nicotinic neurotransmission. The resulting increase in output firing frequency inhibits colonic motility. Using confocal microscopy and 3D reconstruction of Lucifer Yellow–filled neurons, PACAP-preferring receptors were found on secondary and tertiary dendrites, a spatial distribution similar to the distribution of nicotinc receptors (Fig. 22-11). This spatial arrangement provides a structural mechanism whereby the efficacy of fast cholinergic nicotinc transmission can be modulated by the release of PACAP peptides. Immunohistochemical characterization of viscerofugal neurons projecting to the PGs in guinea pig has not been reported.

Rat Viscerofugal neurons projecting from the stomach, small intestine, or cecum to the CGs and SMG are all ChAT-IR, implying that they are cholinergic (152). In proximal colon, about 20% of the viscerofugal neurons projecting to the CGs and SMG are NOS-IR; however, viscerofugal neurons in the small intestine that project to the ganglia are not NOS-IR (163). Most viscerofugal neurons projecting to the IMG or PG are calbindin-IR, and many also contain BOM, NOS, or both (89). Almost half of the viscerofugal neurons projecting from the large intestine to PG are VIP-IR, compared

B

FIG. 22-11. Spatial (three-dimensional) distribution of presynaptic pituitary adenylate cyclase– activating peptide (PACAP)-LI immunoreactivity (IR) (A) and neuronal type I PACAP receptor (PAC1)-LI-IR (B) on guinea pig inferior mesenteric ganglion (IMG) neurons. The IMG neurons were intracellularly injected with Lucifer Yellow, and the preparations were subsequently immunostained with appropriate antibodies visualized with either Cy3 or Cy5 fluorescent dyes. Preparations were imaged using a confocal microscope. The images were volume reconstructed and superimposed using ANALYZE software. Voxel counting revealed that the majority (54.0 ± 9.4%, n = 6 neurons) of PACAP–like-IR appositions (red, A) and the majority (66.7 ± 4.9%, n = 7 neurons) of PAC1-receptor–like IR (red, B) were distributed along secondary and tertiary dendrites of IMG neurons (gray). Scale bars = 50 µm. (See Color Plate 13.) (Reproduced from Ermilov and colleagues [18], by permission.)

620 / CHAPTER 22 with only a minor proportion of VIP-IR viscerofugal neurons projecting to the IMG. A significantly greater proportion of viscerofugal neurons projecting to the PGs are NOS-IR compared with viscerofugal neurons that project to the IMG (89). Viscerofugal neurons that are VIP- or BOM-IR project from the stomach to CGs or SMG (149,150).

Pig In the porcine small intestine, viscerofugal neurons projecting from the myenteric and submucous plexuses to the superior caudal mesenteric ganglia (CMG) are 5-HT-IR (153). In the colon, a considerable proportion of viscerofugal neurons projecting from the myenteric or submucous plexus to the superior CMG are CGRP-IR, whereas viscerofugal neurons in the submucous, but not myenteric, plexus are 5-HT-IR (154). In contrast with the guinea pig, rat, and dog (see later), viscerofugal neurons in the pig small intestine and colon are not VIP-IR (154). Only a small proportion of the viscerofugal neurons projecting to the caudal IMG from myenteric and outer submucosal plexus ganglia of the pig colon and rectum are NOS-IR (164).

Dog In myenteric ganglia of dog distal colon and rectum, viscerofugal neurons that project to the IMG are immunoreactive for VIP, dynorphin, CGRP, ENK, SP, or BOM (67). However, unlike in the guinea pig and rat, viscerofugal neurons in dog distal colon and rectum are not IR for calbindin. Whereas SP-IR is present in viscerofugal neurons of dog large intestine, this has not been demonstrated in guinea pig, rat, or pig.

POPULATIONS OF MECHANOSENSORY VISCEROFUGAL NEURONS There are two general populations of viscerofugal neurons. Primary viscerofugal neurons project without synaptic interruption to PVGs, and second or higher order viscerofugal neurons receive fast synaptic input from other enteric neurons before exiting the bowel wall. This view rests on the observations that blockade of nicotinic synaptic transmission in the gut wall abolishes the sustained responses to colonic distension, leaving an initial, transient (phasic) discharge in PVG neurons (2,15,165–167), and on the finding that nicotinic receptor blockade in the gut wall reduces, but does not completely abolish, the release of acetylcholine in PVGs (155). The second or higher order neuron is ChAT-IR in guinea pig colon (152,160,168) and ileum (28,122). Fiber tract stimulation evokes large-amplitude fast-excitatory synaptic potentials in retrogradely labeled viscerofugal neurons in guinea pig colon (160). In addition, colonic viscerofugal

neurons with Dogiel type I– and Dogiel type II–like morphology retrogradely labeled with DiI and injected with Lucifer Yellow, immunostained for nicotinic acetylcholine receptors, and volume reconstructed to generate 3D images revealed the presence of immunoreactivity for cholinergic nicotinic receptors in both the cell membrane and cytoplasm (156) (see Fig. 22-8). Notably, the processes of colonic viscerofugal neurons with Dogiel type II morphology are arranged parallel to the circular muscle axis (see Fig. 22-9), and these neurons are immunopositive for cholinergic nicotinic receptors, suggesting that activity in this population of visceromotor neurons also can be modulated by cholinergic input from other enteric neurons (156). In summary, primary and second or higher order viscerofugal neurons are endowed with nicotinic acetylcholine receptors. Confocal and 3D imaging studies that show that some viscerofugal neurons have Dogiel type II–like morphology verify an earlier insightful study that centripetal axons in mesenteric nerves in rabbits and cats were axonal projections of Dogiel type II myenteric neurons (169). Viscerofugal neurons are distension sensitive (2,16,128) (Fig. 22-12). They are slowly adapting or nonadapting neurons that respond directly to stretch of the circular muscle layer with a bust of action potentials (165). They function as volume receptors (2,165,170). They are arranged “in parallel” to the circular muscle layer because an increase in volume and circumference of the gut wall increases the frequency of fast excitatory synaptic input to PVG neurons, whereas synaptic input is virtually absent when the circular muscle layer is contracted and the circumference of the wall is decreased (128,165) (Fig. 22-13). When the circular muscle of the colon wall contracts to empty the segment, the mechanoreceptors are unloaded and synaptic input decreases.

ELECTROPHYSIOLOGY OF PREVERTEBRAL GANGLION NEURONS Three patterns of firing have been described in CG neurons in response to an inward depolarizing current injection delivered through a recording microelectrode or patch clamp electrode (46). One pattern is characterized by an initial repetitive discharge of action potentials that is not maintained but is succeeded by a period of quiescence. This pattern is referred to as phasic firing. The second pattern is characterized by a continuous, repetitive discharge of action potentials and is referred to as tonic firing. The third pattern is characterized by a single action potential followed by an unusually long after-spike hyperpolarization and is maintained quiescence for as long as the depolarizing current pulse is maintained. Neurons responding in this manner are classified as LAH for long after-spike hyperpolarization. The proportion of neurons in the CG that fire phasically ranges in different species from 14% (41) to as high as 52%

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 621

50 µm

23.5 cm H2O Colonic pressure 3.5

−27 mV −47 Electrical activity 5 sec

Distend colon (+ 0.05 mL)

FIG. 22-12. Morphology of a mouse superior mesenteric ganglion (SMG) neuron and the response of the neuron to colonic distension. Recording was made in vitro. Colonic distension evoked an immediate increase in the frequency of fast excitatory synaptic potentials and action potentials (bottom). The neuron was injected with Lucifer Yellow (top left) and three-dimensional reconstructed from serial confocal sections (top right) after assessing its response to colonic distension. The neuron has multiple dendrites and its axon (asterisk) is projected caudally into the intermesenteric nerve and toward the distal colon.

(171,172). Tonic firing neurons comprise from 35% (41) to 60% (171) in the different species studied. These proportions for the CG are to be compared with the IMG where the percentages are 18% and more than 80%, respectively (31,44,171,173). Phasic firing neurons with a LAH in the CG comprise approximately 49% of the population of neurons tested (41) and only approximately 2% in the IMG (41,44,174). In sympathetic paravertebral chain ganglia, almost all neurons are phasic firing (44). There is a correlation between the firing properties of a neuron and its neuropeptide content. NA/NPY neurons tend to be phasic firing neurons. NA/SOM and NA/- neurons are tonic firing neurons (41,46). There also is a correlation between the firing properties of a neuron and the strength of synaptic input to the neuron. Typically, phasic firing and LAH neurons receive strong synaptic input, whereas tonic firing neurons receive few, if any, strong inputs but many weak inputs. Another differentiating characteristic is that phasic firing and LAH neurons rarely receive synaptic input from viscerofugal neurons, whereas most tonic firing neurons do. The firing properties of a neuron are determined by the palette of voltage-sensitive potassium ion currents that are

expressed and Ca2+-dependent potassium ion currents that are exhibited by the neuron (44). The potassium channel currents help to set the firing rate of neurons. Phasic firing neurons have a prominent M-current (IM) that is absent or small in tonic firing neurons. The M-current is a slowly developing, noninactivating, time- and voltage-dependent potassium current that was initially described in bullfrog sympathetic ganglia (175). It carries the nomenclature Mcurrent because muscarinic agonists block this potassium current (176). The M-current exerts a major influence on the firing properties of the neuron by reducing the rate of action potential generation. Most of the M channels are closed at the resting membrane potential of the neuron and opened at potentials more depolarized than −60 mV. Thus, the outward current limits the ability of the neuron to fire repetitively. Whereas the initially high discharge in phasic firing neurons can be explained by the absence of the A-current (see later), the termination of firing seems to be due to IM. Tonic firing is attributed to the combined effects of a transient potassium current (called the A-current, IA), which is initiated by depolarization and the lack of a significant M-current. The delay in action potential discharge at the onset of a suprathreshold depolarizing current pulse is due to the transient outward

622 / CHAPTER 22 Circular muscle tension

Intraluminal pressure

Afferent synaptic input

A

Circular m.

A B

B Mechanoreceptor:

Adapted from: Gabella, G., 1987

FIG. 22-13. Relation among spontaneous circular muscle contraction, intraluminal pressure, and mechanosensory afferent synaptic input to a neuron in the mouse superior mesenteric ganglion (SMG) attached to a segment of distal colon in vitro. Note that during the decrease in intraluminal pressure there was an increase in the frequency of fast excitatory synaptic input, whereas the frequency of the input markedly decreased at peak intraluminal pressure and contraction. These data suggest that the mechanosensory nerves function as “in-parallel” stretch receptors in the circular muscle layer as shown schematically in the panels below the recordings. Circular m., circular muscle. (Diagrams modified from Szurszewski and colleagues [128], by permission. Photomicrograph reproduced from Gabella G. Dynamic aspects of the morphology of the intestinal muscle coat. In: Szurszewski JH, ed. Cellular physiology and clinical studies of gastrointestinal smooth muscle. Amsterdam: Elsevier Science, 1987;5–31, by permission.)

potassium current (IA). The A-current is inactivated at resting membrane potentials typically found in phasic firing neurons. The Ca2+-activated potassium conductance following the action potential not only slows the firing frequency, but it also helps to hold the neuron hyperpolarized, increasing the A-current reactivation and potentiating its effects. LAH neurons have two Ca2+-activated potassium conductances with differing kinetics (44,46,177,178) that underlie the after hyperpolarization.

PACEMAKER NEURONS A number of studies have raised the possibility that there is a population of neurons in PVGs that are pacemaker neurons that generate a rhythmic discharge of action potentials. It has long been suspected that PVGs harbor pacemaker neurons and that they may be the basis for tonic inhibitory influence on intestinal motility (179–182). Using intracellular recording methods, approximately 10% of the neurons in the rostral lobe of the cat IMG exhibited an ongoing rhythmic discharge of action potentials that had a frequency ranging from 0.5 to 22 sec−1 in the different neurons that were tested (31). Each action potential was

preceded by a slow, diastolic-like depolarization. The rhythmic discharge was unaffected by nicotinic, muscarinic, α, and β-adrenoceptor antagonists. Furthermore, a low-Ca2+, high-Mg2+ solution to block release of noncholinergic and nonadrenergic neurotransmitters and neuromodulators had no effect on the rhythmic discharge. Injection of a hyperpolarizing current pulse blocked the discharge, whereas a depolarizing current pulse increased the frequency of discharge. Interpolation of an action potential due to injection of a brief but suprathreshold depolarizing current pulse reset the endogenous rhythm. A similar discharge pattern was observed in approximately 5% of the neurons tested in dog IMG (183). Based on observations made in cat IMG, it has been suggested that a population of neurons have the intrinsic ability to generate sustained rhythmic activity, and that they may be legitimately referred to as pacemaker or autogenic neurons. Using patch-clamp recording methods to record from nondissociated sympathetic neurons of rabbit CG, approximately 37% of neurons examined exhibited rhythmic discharge of action potentials (184). The basis of the rhythmic discharge was attributed to a quasi–steady-state current– voltage relation that displays a negative slope between −80 and −60 mV (185,186). Because of this N-shaped relation,

PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 623 the membrane potential cannot remain stable (187). The persistent voltage-dependent inward current at peak level is approximately 22 pA (184,185). This current, which occurs across a membrane input resistance of approximately 580 mΩ (184), brings the null voltage to −41 mV, which results in continuous firing. Thus, based on the N-shaped characteristics of the current–voltage relation, it was suggested that these neurons in rabbit CGs are pacemaker neurons. If the voltage- and time-dependent, low-threshold inward current is self-regenerating, then, indeed, these neurons can be classified as pacemaker neurons. However, both “spontaneous pacemaker activity” (187) and nerve-evoked, pacemakerlike rhythmic firing of action potentials in silent neurons have been shown to be due to selective tonic activation of a voltage dependent inward current carried mainly by sodium ions (INa, M) (186). This current is limited and blocked at voltages of −20 mV or more positive. It is negatively affected by intracellular Ca2+ and by muscarinic antagonists. Repetitive and single splanchnic nerve stimulation induces a slow muscarinic excitatory postsynaptic potential that was large enough to elicit a sustained action potential discharge similar to that seen in spontaneously occurring pacemaker neurons in the same ganglion (187). It has been suggested, therefore, that ongoing pacemaker-like activity and nerveinduced pacemaker-like activity in rabbit CGs are due to selective activation of a muscarinic voltage-dependent inward current (186,187). The mechanism that leads to the continuous release of acetylcholine has yet to be clarified. Similar nerve-induced, sustained, pacemaker-like activity also has been recorded from sympathetic neurons of guinea pig IMG (188). In guinea pig IMG, benzohexonium did not affect the discharge, but atropine and transection of the lumbar colonic nerve that connected a segment of colon to the ganglion resulted in complete disappearance of pacemakerlike activity (188). The physiologic importance of pacemaker neurons and of neurons that exhibit pacemaker-like activity is not insignificant. Both sets of neurons would provide tonic sympathetic inhibitory input to their visceral targets. Their discharge frequency, and hence degree of inhibition, would vary with the degree of muscarinic receptor activation because PVG neurons receive ongoing cholinergic input from enteric neurons. Because visceromotor neurons express muscarinic receptors (1,2), the enteric PVG reflex pathway would function as a feed-forward system. Increased cholinergic input would lead to increased sympathetic activity in sympathetic visceromotor neurons and an increase in inhibitory modulation of enteric neurocircuitry.

CONCLUDING REMARKS Our knowledge of PVGs is becoming quite complete. We know a lot about PVGs as an entire system—about the morphology of the neurons in the ganglia, about the way the neurons innervate the bowel wall, and about the distribution of viscerofugal neurons that provide peripheral input

to PVGs. Impressive advances have been made in marking nerves with antibodies to peptides or neurotransmitters and their synthesizing enzymes. Relatively recent work has shed light on the sensitivities of PVG neurons to certain neurotransmitters and neuromodulators, the time course and ionic mechanisms of their action, but we do not have this information for viscerofugal neurons. There are other gaps between our increasingly exquisite knowledge of PVG and viscerofugal neurons. For example, do viscerofugal neurons encode only mechanosensory information? There is reason to believe that there are sensory modalities other than distension that activate viscerofugal neurons (167), but the nature of the stimulus has not been determined. There is preliminary evidence for an enteroenteric reflex controlling secretion of water and electrolyte in the small intestine that is dependent on a nerve pathway through PVGs (189,190). There are no data on the extracellular and intracellular anchors of the mechanosensory apparatus. Although preliminary data suggest that the epithelial sodium channel and stomatin, integral membrane proteins found in other well-studied mechanosensory neurons, are present in viscerofugal neurons (191), more data and more work are required to assign these proteins to the mechanotransduction process. Nothing is known about the ultrastructure of the transduction apparatus—where it is located and how mechanical stimuli lead to channel opening—and the receptive field of a single mechanosensory viscerofugal neuron is unknown. Although we know that viscerofugal neurons make synaptic contact only with visceromotor neurons, the guiding forces and growth agents that cause the axons of viscerofugal neurons to seek out specific targets are unknown. Although small in number, there are viscerofugal neurons in the upper gut that send their axons into the vagus nerve (192) and viscerofugal (rectospinal) neurons that send their axons into the pelvic nerve. There also are a small number of neurons in mouse SMG that send their axons into the splanchnic nerves (6). The central targets of these three populations of neurons are unknown, and there is no information on the stimulus modality they encode. Finally, we echo a comment made in our last review of this topic. There is no information about the clinical importance of viscerofugal neurons in humans. This area of enteric neurosciences will mature substantially when the clinical relevance of viscerofugal neurons becomes more apparent.

ACKNOWLEDGMENTS The authors thank the many colleagues who worked in their laboratory, most especially Leonid Ermilov and Philip Schmalz who provided 3D imaging data. The authors are grateful to Jan Applequist for her skilled technical assistance in the preparation of the chapter. The authors are also grateful to the National Institutes of Health and the National Institute of Diabetes and Digestive and Kidney Diseases for support through Public Health Services Grant DK 17632.

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PHYSIOLOGY OF PREVERTEBRAL SYMPATHETIC GANGLIA / 627 145. Neuhuber WL, Appelt M, Polak JM, Baier-Kustermann W, Abelli L, Ferri GL. Rectospinal neurons: cell bodies, pathways, immunocytochemistry and ultrastructure. Neuroscience 1993;56:367–378. 146. Doerffler-Melly J, Neuhuber WL. Rectospinal neurons: evidence for a direct projection from the enteric to the central nervous system in the rat. Neurosci Lett 1988;92:121–125. 147. Wood JD. Neurophysiological theory of intestinal motility. Nippon Heikatsukin Gakkai Zasshi 1987;23:143–186. 148. Messenger JP, Furness JB. Distribution of enteric nerve cells that project to the coeliac ganglion of the guinea-pig. Cell Tissue Res 1992;269:119–132. 149. Lee Y, Shiosaka S, Hayashi N, Tohyama M. The presence of vasoactive intestinal polypeptide-like immunoreactive structures projecting from the myenteric ganglion of the stomach to the celiac ganglion revealed by a double-labelling technique. Brain Res 1986;382: 392–394. 150. Hamaji M, Kawai Y, Kawashima Y, Tohyama M. Projections of bombesin-like immunoreactive fibers from the rat stomach to the celiac ganglion revealed by a double-labeling technique. Brain Res 1987;416:192–194. 151. Messenger JP, Furness JB. Distribution of enteric nerve cells projecting to the superior and inferior mesenteric ganglia of the guinea-pig. Cell Tissue Res 1993;271:333–339. 152. Furness JB, Koopmans HS, Robbins HL, Lin HC. Identification of intestinofugal neurons projecting to the coeliac and superior mesenteric ganglia in the rat. Auton Neurosci 2000;83(1-2):81–85. 153. Timmermans JP, Barbiers M, Scheuermann DW, Stach W, Adriaensen D, Groodt-Lasseel MH. Occurrence, distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig. Cell Tissue Res 1993;272:49–58. 154. Barbiers M, Timmermans JP, Adriaensen D, Groodt-Lasseel MH, Scheuermann DW. Topographical distribution and immunocytochemical features of colonic neurons that project to the cranial mesenteric ganglion in the pig. J Auton Nerv Syst 1993;44(2-3):119–127. 155. Parkman HP, Ma RC, Stapelfeldt WH, Szurszewski JH. Direct and indirect mechanosensory pathways from the colon to the inferior mesenteric ganglion. Am J Physiol 1993;265(3 pt 1):G499–G505. 156. Ermilov LG, Miller SM, Schmalz PF, Hanani M, Lennon VA, Szurszewski JH. Morphological characteristics and immunohistochemical detection of nicotinic acetylcholine receptors on intestinofugal afferent neurones in guinea-pig colon. Neurogastroenterol Motil 2003;15:289–298. 157. Skok VI, Farrugia G, Ermilov LG, Miller SM, Szurszewski JH. Patchclamp recordings of membrane currents evoked during natural synaptic activity in sympathetic neurons. Neuroscience 1998;87:509–517. 158. Kuramoto H, Furness JB. Distribution of enteric nerve cells that project from the small intestine to the coeliac ganglion in the guinea-pig. J Auton Nerv Syst 1989;27:241–248. 159. Furness JB, Kuramoto H, Messenger JP. Morphological and chemical identification of neurons that project from the colon to the inferior mesenteric ganglia in the guinea-pig. J Auton Nerv Syst 1990;31: 203–210. 160. Sharkey KA, Lomax AE, Bertrand PP, Furness JB. Electrophysiology, shape, and chemistry of neurons that project from guinea pig colon to inferior mesenteric ganglia. Gastroenterology 1998;115:909–918. 161. Luckensmeyer GB, Keast JR. Distribution and morphological characterization of viscerofugal projections from the large intestine to the inferior mesenteric and pelvic ganglia of the male rat. Neuroscience 1995;66:663–671. 162. Miller SM, Szurszewski JH. Relationship between colonic motility and cholinergic mechanosensory afferent synaptic input to mouse superior mesenteric ganglion. Neurogastroenterol Motil 2002;14: 339–348. 163. Domoto T, Teramoto M, Tanigawa K, Tamura K, Yasui Y. Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion. Cell Tissue Res 1995;281:215–221. 164. Barbiers M, Timmermans JP, Scheuermann DW, Adriaensen D, Mayer B, Groodt-Lasseel MH. Nitric oxide synthase-containing neurons in the pig large intestine: topography, morphology, and viscerofugal projections. Microsc Res Tech 1994;29:72–78. 165. Miller SM, Szurszewski JH. Circumferential, not longitudinal, colonic stretch increases synaptic input to mouse prevertebral ganglion neurons. Am J Physiol Gastrointest Liver Physiol 2003;285:G1129–G1138. 166. Bywater RA. Activity following colonic distension in enteric sensory fibres projecting to the inferior mesenteric ganglion in the guinea pig. J Auton Nerv Syst 1994;46(1-2):19–26.

167. Stebbing MJ, Bornstein JC. Electrophysiological analysis of the convergence of peripheral inputs onto neurons of the coeliac ganglion in the guinea pig. J Auton Nerv Syst 1994;46(1-2):93–105. 168. Lomax AE, Zhang JY, Furness JB. Origins of cholinergic inputs to the cell bodies of intestinofugal neurons in the guinea pig distal colon. J Comp Neurol 2000;416:451–460. 169. Pilipenko VI. Materials on functional morphology of the peripheral nervous system. II. Functional nature of type II Dogiel’s cells. Biull Eksp Biol Med 1956;41:70–74. 170. Anthony TL, Kreulen DL. Volume-sensitive synaptic input to neurons in guinea pig inferior mesenteric ganglion. Am J Physiol 1990;259 (3 pt 1):G490–G497. 171. Kreulen DL, Szurszewski JH. Nerve pathways in celiac plexus of the guinea pig. Am J Physiol 1979;237:E90–E97. 172. Decktor DL, Weems WA. An intracellular characterization of neurones and neural connexions within the left coeliac ganglion of cats. J Physiol 1983;341:197–211. 173. Weems WA, Szurszewski JH. An intracellular analysis of some intrinsic factors controlling neural output from inferior mesenteric ganglion of guinea pigs. J Neurophysiol 1978;41:305–321. 174. Jobling P, Gibbins IL, Lewis RJ, Morris JL. Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs. Neuroscience 2004;127:455–466. 175. Brown DA, Adams PR. Muscarinic suppression of a novel voltagesensitive K+ current in a vertebrate neurone. Nature 1980;283: 673–676. 176. Adams PR, Brown DA, Constanti A. M-currents and other potassium currents in bullfrog sympathetic neurones. J Physiol 1982;330: 537–572. 177. Cassell JF, McLachlan EM. Two calcium-activated potassium conductances in a subpopulation of coeliac neurones of guinea-pig and rabbit. J Physiol 1987;394:331–349. 178. Jobling P, McLachlan EM, Sah P. Calcium induced calcium release is involved in the afterhyperpolarization in one class of guinea pig sympathetic neurone. J Auton Nerv Syst 1993;42:251–257. 179. de Groat WC, Krier J. The central control of the lumbar sympathetic pathway to the large intestine of the cat. J Physiol 1979;289:449–468. 180. Iggo A, Vogt M. Preganglionic sympathetic activity in normal and in reserpine-treated cats. J Physiol 1960;150:114–133. 181. Beacham WS, Perl ER. Background and reflex discharge of sympathetic preganglionic neurones in the spinal cat. J Physiol 1964;172: 400–416. 182. Anthony TL, Kreulen DL. A physiologically-evoked M1-muscarinic depolarization in guinea-pig inferior mesenteric ganglion neurons. J Auton Nerv Syst 1994;46:207–215. 183. King BF, Szurszewski JH. An electrophysiological study of inferior mesenteric ganglion of the dog. J Neurophysiol 1984;51:607–615. 184. Gola M, Niel JP. Electrical and integrative properties of rabbit sympathetic neurones re-evaluated by patch clamping non-dissociated cells. J Physiol 1993;460:327–349. 185. Gola M, Delmas P, Chagneux H. Encoding properties induced by a persistent voltage-gated muscarinic sodium current in rabbit sympathetic neurones. J Physiol 1998;510(pt 2):387–399. 186. Delmas P, Niel JP, Gola M. Muscarinic activation of a novel voltagesensitive inward current in rabbit prevertebral sympathetic neurons. Eur J Neurosci 1996;8:598–610. 187. Niel JP, Delmas P, Gola M. Synaptically activated low-threshold muscarinic inward current sustains tonic firing in rabbit prevertebral sympathetic neurons. Eur J Neurosci 1996;8:611–620. 188. Pasichnichenko OM, Skok VI. Background and reflex activity of guinea pig caudal mesenteric ganglion neurons. Neurophysiology 1999; 31:323–329. 189. Furness JB. Intestinofugal neurons and sympathetic reflexes that bypass the central nervous system. J Comp Neurol 2003;455: 281–284. 190. Quinson N, Furness JB. Intestino-intestinal secretomotor reflexes in the rat. Proc Aust Neurosci Soc 2002;13:102 (Abstract). 191. Ermilov L, Miller SM, Farrugia G, Szurszewski JH. Intestinofugal afferent neurons in the guinea pig distal colon are mechanosensory and utilize ENAC-like proteins. Gastroenterology 2004;126:A429 (Abstract). 192. Berthoud HR, Jedrzejewska A, Powley TL. Simultaneous labeling of vagal innervation of the gut and afferent projections from the visceral forebrain with dil injected into the dorsal vagal complex in the rat. J Comp Neurol 1990;301:65–79.

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CHAPTER

23

Cellular Neurophysiology of Enteric Neurons Jackie D. Wood The Enteric Nervous System, 629 Conceptual Model, 630 Histoanatomy, 631 Structure–Function Relations with Other Autonomic Ganglia, 632 Morphology of Enteric Neurons, 632 Enteric Sensory Neurons, 633 Sensory Chemoreception, 636 Enteric Interneurons, 636 Enteric Motor Neurons, 637 AH- and S-Type Enteric Neurons, 642

AH-Type Neurons, 642 S-Type Enteric Neurons, 645 Synaptic Transmission, 646 Fast Excitatory Postsynaptic Potentials, 646 Slow Synaptic, Endocrine, and Paracrine Excitation, 648 Slow Excitatory Postsynaptic Potentials, 648 Inhibitory Postsynaptic Potentials, 654 Presynaptic Facilitation, 655 Presynaptic Inhibition, 656 References, 657

THE ENTERIC NERVOUS SYSTEM

The ENS integrates glandular secretion, contractile state of the musculature, and intramural blood flow into specific patterns of digestive behavior, each of which accomplishes a specialized function, such as postprandial mixing or reversed propulsion during emesis. ENS control circuits may evoke contraction or inhibit contractile activity in selective muscles (e.g., longitudinal and circular intestinal muscle coats) as required for the organization of specific patterns of organ motility. The ENS is responsible for minute-to-minute organization of absorption from and glandular secretion into the intestinal lumen and integrates these functions with motility. In concert with the control of motility and secretion, integrative microcircuitry in the ENS controls local blood flow in the gut wall and the distribution of flow to the musculature, mucosa, and lamina propria. Malfunctions of integrative ENS control of the effector systems of the gut are becoming increasingly recognized as underlying factors in gastrointestinal disorders, especially in the functional gastrointestinal disorders (1–4). The effector systems of the digestive tract are the musculature, mucosal secretory epithelium, and blood and lymphatic vasculature. Minute-to-minute behavior of any of the specialized compartments of the gut (e.g., stomach, small intestine, and large intestine) reflects neurally integrated activity of the effector systems. The ENS not only controls the activity of each effector, it automatically coordinates the activity of each one to achieve organized behavior of the whole organ.

There are as many neurons in the enteric nervous system (ENS) as in the spinal cord. About 100 million neurons are required for programmatic control of the gastrointestinal functions that account for several different digestive states (e.g., postprandial state, interdigestive state, and vomiting). The volume occupied by the central nervous system (CNS) in the cranium and spinal cord would be greatly expanded if the required numbers of specialized types of neurons, together with accompanying glia, were present in the CNS. As an alternative to packing the enteric neural control circuits exclusively within the skull or vertebral column and transmitting control signals over long, unreliable transmission lines to the gut, vertebrate animals have evolved with the neural control networks distributed along the length of the digestive tract in close apposition to the muscles, glands, and blood vessels that must be controlled and their activity integrated for effective function of the whole organ (e.g., stomach) in relation to neighboring organs (e.g., the upper small intestine).

J. D. Wood: Departments of Physiology and Biology and Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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630 / CHAPTER 23 Timing of excitatory or inhibitory neural inputs, or both, to each of the individual effector systems achieves coordination of activity. Coordination of mucosal glandular secretion and contractile behavior of the musculature in a timed sequence during intestinal propulsion of luminal contents is one of many examples of ENS integrative function. Secretion of H2O, electrolytes, and mucus occurs first, followed by propulsive contractions in the musculature. A simultaneous increase in neurally controlled blood flow to the secretory glands supports the enhanced secretory response. The ENS is a “minibrain” that, similar to a computer hard drive, stores a library of programs for a variety of patterns of small or large intestinal functional behaviors. Output of one of the programs determines motor behavior in the postprandial state and another establishes the pattern of intestinal motility that characterizes the fasting state. The specialized propulsive motility seen in the upper third of the small intestine during emesis reflects output of another of the programs in the library. During emesis, peristaltic propulsion in the upper third of the small intestine is reversed for rapid movement of the luminal contents toward the stomach. The emetic program can be “called up” from the library either by commands from the CNS or by local sensory detection of threatening substances in the lumen (5). An overlay of signals released from enteric inflammatory/immune cells brings up a different program, which when running in the distal intestinal tract, rapidly removes threats (e.g., parasites, food antigens, and toxins) that have appeared in the lumen (6).

Conceptual Model The heuristic model for the ENS (Fig. 23-1) is the same as for all integrative nervous systems, whether in vertebrate or invertebrate animals. Like other integrative nervous systems

(e.g., spinal cord and brainstem), the ENS functions with sensory neurons, interneurons, and motor neurons. Sensory neurons have, at their terminals in the gut, receptors specialized for detecting changes in stimulus energy (see Chapter 25). Sensory receptors in the digestive tract are classified according to the kinds of energy they detect. These include thermoreceptors, chemoreceptors, and mechanoreceptors. The receptor regions transform changes in stimulus energy into signals coded by action potentials that subsequently are transmitted for processing along sensory nerve fibers to other points in the nervous system. Interneurons are interconnected one with another to form networks that process incoming information from sensory neurons and control the behavior of motor neurons to effector systems (see Fig. 23-1). Multiple synaptic connections among interneurons organize the cell bodies, dendrites, and axons of the neurons into “logic” circuits that decipher action potential codes from sensory neurons and neural signals from elsewhere in the nervous system. Some of the interneuronal circuits are integrative; others are reflex circuits. Integrative circuits are circuits that process incoming information and reconfigure it into functional output signals that are not direct transforms of the input signal. Plasticity resides in integrative circuits. Reflex circuits, in contrast, are “hardwired,” much like spinal motor reflexes (e.g., knee jerk reflex), to generate stereotyped outputs in response to input from specific kinds of sensory receptors. Motor neurons are the final common pathways for transmission of control signals from integrative and reflex circuits to the effector systems (see Fig. 23-1). Motor neurons may be stimulated to fire by excitatory synaptic input derived from interneuronal circuits, or their activity may be suppressed by inhibitory synaptic input from the interneuronal circuitry. In the digestive tract, motor neurons may release excitatory or inhibitory neurotransmitters at their junctions with the

Brain

Effector systems Muscle Sensory neurons

Interneurons

Enteric nervous system

Motor neurons

Secretory glands Blood vessels

Organ system behavior FIG. 23-1. Sensory neurons, interneurons, and motor neurons are interconnected synaptically to form the integrated microcircuits of the enteric nervous system (ENS). Like the central nervous system (CNS), sensory neurons, interneurons, and motor neurons are connected synaptically for flow of information from sensory neurons to interneuronal integrative networks to motor neurons to effector systems. The ENS organizes and coordinates the activity of each effector system into meaningful behavior of the whole organ. Bidirectional communication occurs between the CNS and the ENS.

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 631 muscles, thereby actively exciting muscular contraction or actively inhibiting contraction.

Histoanatomy Cell bodies of ENS neurons are in ganglia obscured inside the walls of most of the digestive tract. The ganglia are interconnected by interganglionic fiber tracts to form ganglionated plexuses (Fig. 23-2). Interganglionic fiber tracts contain projections from ganglion cell bodies in one ganglion that connect synaptically with neurons in neighboring ganglia. They also contain primary sensory afferent fibers that are projections from cell bodies in dorsal root spinal ganglia or nodose ganglia (i.e., vagal afferents), as well as sympathetic nerve fibers projecting from prevertebral ganglia (see Chapters 22 and 25) and efferent fibers of vagal or sacral parasympathetic nerves, or both. The ganglionated plexuses form a distributed continuum around the circumference and along the length of each of the specialized organs that make up the digestive tract. Myenteric and submucosal plexuses are the prominent ganglionated plexuses of the ENS (see Fig. 23-2). The myenteric plexus, known also as Auerbach’s plexus, is the most expansive plexus of the ENS. It is located between the longitudinal and circular muscle coats of most regions of the gastrointestinal tract. It is a two-dimensional array of flat disklike neurons, ganglia, and interganglionic

fiber tracts sandwiched between the longitudinal and circular muscle coats of most regions and under and between the taenia coli of the colon of humans and some other species (see Fig. 23-2). Unlike other autonomic ganglia, the cell bodies of the ganglion cells are not in “grapelike” clusters. They lay edge to edge like a single layer of coins placed in a two-dimensional plane (7). The single-layered, twodimensional organization is postulated to be an adaptation for sustaining the neural networks in a functional state as the ENS is subjected to the mechanical forces and deformations that occur during contraction of the musculature or expansion and stretching of the wall as the lumen fills. Most of the motor neurons that innervate the circular and longitudinal muscle coats reside in the myenteric plexus (8,9). The submucosal plexus is a ganglionated plexus situated throughout the submucosal space between the mucosa and circular muscle coat (see Fig. 23-2). It is most prominent as a ganglionated network in the small and large intestines. No ganglionated submucosal plexus exists in the esophagus, and ganglia are sparse in the submucosal space of the stomach. In larger mammals (e.g., pig and humans), the submucosal plexus consists of an inner submucosal network (Meissner’s plexus) located at the serosal side of the muscularis mucosae and an outer plexus (Schabadasch’s plexus) adjacent to the luminal side of the circular muscle coat. In human small and large bowels, a third intermediate plexus lies between Meissner’s and Schabadasch’s plexuses (10).

Longitudinal muscle

lar

rcu

Ci

Myenteric plexus

cle

s mu

Submucosal plexus

Mucosa

A

B

Enteric nervous system

Longitudinal axis

Longitudinal axis

Submucosal plexus

Myenteric plexus C

FIG. 23-2. Histoanatomic organization of the enteric nervous system (ENS). (A) Ganglia and interganglionic fiber tracts of the myenteric and submucosal plexuses are prominent features of the ENS. The myenteric plexus is distributed between the circular and longitudinal muscle coats; the submucous plexus is between the mucosa and circular muscle coat. (B) Tyrosine hydroxylase stain of the submucosal plexus as it appears in a preparation obtained from guinea pig small intestine. (C) Tyrosine hydroxylase stain of the myenteric plexus as it appears on the underside of the longitudinal muscle coat after microdissection from guinea pig small intestine. The long dimension of myenteric ganglia in the small intestine is in the circumferential direction; no such orientation occurs for the submucosal plexus. Asterisks identify ganglia; diamonds identify interganglionic fiber tracts. (See Color Plate 14.)

632 / CHAPTER 23 Ganglion cells in the submucosal plexus supply motor innervation to the intestinal crypts and villi and possibly to the muscularis mucosae. Neurons in submucosal ganglia project fibers to the myenteric plexus and also receive synaptic input from axons projecting from the myenteric plexus (11–13). The interplexus connections link the two networks into a functionally integrated nervous system. Structure, function, and neurochemistry of enteric ganglia differ significantly from other autonomic ganglia. Unlike other autonomic ganglia where function is mainly as relay distribution centers for signals transmitted from the CNS, enteric ganglia are interconnected to form a nervous system with mechanisms for integration and processing of information such as those found in the brain and spinal cord.

Structure–Function Relations with Other Autonomic Ganglia Most structural and functional properties of the ENS are similar to those of the CNS. Some examples are as follows: 1. Complex integrative functions: Similar to the brain, the ENS executes complex integrative functions that control single effector systems and coordinate the activity of multiple systems into patterns of overall behavior that achieve homeostasis. Program libraries are present in both the ENS and CNS, but not in other autonomic ganglia. 2. Sensory neurons: Sensory neurons transmit information to the neural circuits of the ENS for processing in the same manner that the CNS receives and processes sensory signals. 3. Interneurons: Interneurons are interconnected synaptically into information-processing circuits in both the ENS and CNS, but generally not in other autonomic ganglia. 4. Motor neurons: Enteric motor neurons are the final common pathways for flow of information from interneuronal-processing circuits to the effector systems. The function of enteric motor neurons is analogous to that of α and γ motor neurons from the spinal cord to skeletal muscles. 5. Glial elements: The supporting cells of the ENS are similar to glial cells in the brain, but unlike the Schwann cells found associated with other autonomic ganglia. Enteric glial elements structurally and chemically resemble astroglia of the brain (14,15). 6. Synaptic neuropil: Synaptic neuropils are present in both enteric ganglia and the CNS. Neuropils are tangled meshworks of fine, nonmyelinated nerve fibers that are segregated in regions of the ganglia away from the cell bodies that give rise to the fibers. The presence of a neuropil is significant because in all integrative nervous systems, most of the information processing occurs in microcircuits within a synaptic neuropil. 7. Multiple synaptic mechanisms: As in the brain and spinal cord, the neural circuits of the ENS express

8.

9.

10.

11.

multiple forms of neurotransmission that are involved in the handling of information. Multiple neurotransmitters: Most of the neurotransmitters found in the brain and spinal cord are also expressed in the ENS. Absence of connective tissue: Collagen, which forms part of the connective tissue for the structural support of autonomic ganglia, is absent from the interior of enteric ganglia, as it is in the CNS. Reduced extracellular space: Unlike other autonomic ganglia, extracellular space is reduced by close packing of glial support elements. Isolation from blood vessels: No blood vessels enter enteric ganglia of the intestine, and a blood–ganglion barrier analogous to the blood–brain barrier is inserted between the vasculature and the synaptic circuits of the ganglia.

Morphology of Enteric Neurons Dogiel types I and II neurons are prominent morphologic classes of neurons in the ENS. The German neuroanatomist A. S. Dogiel described two morphologic types of enteric ganglion cells that are named for him. These are Dogiel types I and II, both of which are found in myenteric and submucosal plexuses. Both types of neurons are distributed in a two-dimensional plane (i.e., the cell bodies are not stacked one on the other). Dogiel type I neurons have cell bodies with multiple short processes and a single long process (Fig. 23-3). These are flat neurons with the processes extending for short distances from the cell body in the circumferential and longitudinal planes of the wall. The short processes are dendrites, which receive synaptic input; the single long process is an axon, which transmits information in the form of action potentials away from the cell body. Strictly defined, Dogiel type I neurons have short, club-shaped dendrites. Other uniaxonal enteric neurons have multiple short processes with a variety of morphologic appearances, an example being the filamentous uniaxonal neurons that Furness and colleagues (16) have described. Axons of Dogiel Type I neurons project for relatively long distances through interganglionic fiber tracts and many rows of ganglia. Still, the projections are not long in absolute length; the longest known projections of any enteric ganglion cell are only about 2 to 3 cm. Dogiel type I and other uniaxonal neurons projecting in a specific direction are identified by expression of specific neurotransmitters (see Chapter 21). Aborally projecting uniaxonal neurons release nitric oxide and vasoactive intestinal polypeptide as neurotransmitters; those projecting in the oral direction release substance P and acetylcholine. Some of the uniaxonal neurons are motor neurons to the musculature and secretory epithelium; others are interneurons. The cell bodies of ENS neurons with Dogiel type II morphology have smooth surfaces with long and short processes arising in a variety of configurations (see Fig. 23-3).

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 633 Uniaxonal

Dogiel type II

FIG. 23-3. Neurons with uniaxonal morphology and neurons with Dogiel type II multipolar morphology are the two primary morphologic categories of ENS neurons. Examples of the two kinds of neurons were obtained by injecting the marker biocytin from the microelectrode during intracellular electrophysiologic studies. The neurons with uniaxonal morphology exhibit S-type electrophysiologic behavior and slow synaptic excitation mediated by G protein receptor coupling to phospholipase and are either interneurons or motor neurons. The neurons with Dogiel type II multipolar morphology exhibit AH-type electrophysiologic behavior and slow synaptic excitation mediated by G protein receptor coupling to adenylate cyclase and are interneurons. Calibration bars = 20 µm.

The long processes may extend through interganglionic fiber tracts across several rows of ganglia in circumferential, oral, and aboral directions. Shorter processes may only project within the home ganglion. Almost all Dogiel type II neurons in the myenteric plexus of the small intestine of mouse and guinea pig project a process to the submucosa/ mucosa (17,18). Terminals of Dogiel type II projections into the mucosal regions are fired by mechanical and chemical stimulation of the mucosa (Fig. 23-4A). Dogiel type II neurons are involved at an early stage in the handling of sensory information, as well as interneuronal functions in the microcircuits of the ENS. They are identified by expression of immunoreactivity (IR) for the calcium-binding protein, calbindin, in guinea pig, mouse, and sheep (17,19,20)

and the neurotransmitters substance P, acetylcholine, and calcitonin gene–related peptide.

Enteric Sensory Neurons Sensory neurons are one of the three functional categories of enteric neurons illustrated in Figure 23-1. Sensory afferent fibers with neuronal cell bodies in nodose and dorsal root spinal ganglia transmit information from the gastrointestinal tract and gallbladder to the CNS for processing. These afferents also transmit information to the local processing circuits in the ENS and to prevertebral sympathetic ganglia.

634 / CHAPTER 23 Slow excitatory input

Microelectrode Myenteric neurons

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FIG. 23-4. Gating function of the cell bodies of AH-type interneurons direct excitatory traffic in the enteric nervous system (ENS) microcircuits. (A) Most AH neurons in the myenteric plexus project one of their neurites to the mucosa. 5-Hydroxytryptamine (5-HT) released from mucosal enterochromaffin cells acts at 5-HT3 receptors on the terminals in the mucosa to fire action potentials in the terminal. Bidirectional conduction (arrows) occurs in the mucosal projection with propagation away from the cell body in the direction of mucosa and propagation from the terminal toward the submucosal plexus and the cell body in the myenteric plexus. The level of prevailing excitability in the cell body determines how inbound information from the mucosal terminals is handled. Firing of the cell body by slow excitatory input occludes inbound spikes from the mucosal terminals. (B) Cell body of an AH myenteric neuron in an intermediate state of excitability fires a single action potential during injection of a depolarizing current pulse. (C) An overlay of histamine moves excitability of the same neuron to a hyperactive state that is reflected by repetitive spike discharge to the same depolarizing current pulse. (D) Cell body of an AH myenteric neuron in an inexcitable state. Intraneuronal injection of depolarizing current evoked no action potential discharge. (E) Model for an AH neuron shows how the somal gate for transfer of spike information across the cell body is closed when the neuron is in the inexcitable state. Slow excitatory inputs of either synaptic or paracrine origin increase the excitability of the somal membrane and “open” the gate for transfer of inbound spike information to other neurites distributed around the perimeter of the cell body. (F) Stimulation of a slow excitatory postsynaptic potential in the same neuron as D moves excitability to a hyperactive state that is reflected by repetitive spike discharge to the same depolarizing current pulse. (G) Inbound spikes from a neurite invading the cell body of an AH-type interneuron. The spikes arrive in bursts (arrows). Only the first spike of a burst fires the soma due to fractionation of the inbound spikes by postspike hyperpolarization of the membrane potential. (H) One of the inbound spike bursts from G recorded with an expanded time base. (I) The first spike of the burst in H recorded with an expanded time base shows how the invading spike from the neurite (arrow) triggers the somal spike by depolarizing the somal membrane potential to spike threshold. (Reproduced from Wood [6], by permission.)

Mechanoreceptors, chemoreceptors, and thermoreceptors are present in the ENS. Mechanoreceptors sense changes in mechanical forces in the mucosa, musculature, serosal surface, and mesentery. They supply both the ENS and the CNS with information on stretch-related tension and muscle length in the wall and on the movement of luminal contents as they brush past the mucosal surface. Mesenteric receptors generate coded information on gross movements of the organ. Chemoreceptors generate information on the concentration of nutrients, osmolarity, and pH in the luminal contents.

Recording of nerve impulses in afferent fibers as they exit the gut shows most sensory receptors to be multimodal in that they respond to both mechanical and chemical stimuli (see Chapter 25). Thermoreceptors supply the brain with deep-body temperature data used in body temperature regulation and probably account also for the sensations of temperature change in the lumen. Presence in the gastrointestinal tract of pain receptors (nociceptors) equivalent to those connected with C-fibers and A-delta fibers elsewhere in the body is likely, but is not unequivocally confirmed except for the gallbladder (21).

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 635 The biophysical principles of sensory function in the gastrointestinal tract are similar to those found elsewhere in the body. Each sensory neuron has at least one neurite with a generator region that converts a change in stimulus energy into an action potential code. The frequency of action potential discharge is a code for transmission of information on intensity of the stimulus to processing circuits in the ENS and CNS. Sensory Mechanoreception Information on contractile state of the musculature and distension of the gut wall is coded by mechanoreceptors. Whether the cell bodies of intramuscular and mucosal mechanoreceptors reside in dorsal root ganglia, enteric ganglia, or in both places is an unresolved and mildly controversial question. Furness and colleagues (22,23) and Pan and Gershon (24) suggest that enteric neurons with Dogiel type II morphology and AH-type electrophysiologic and synaptic behavior are primary sensory afferent neurons and use the acronym IPAN as a shorthand name for these neurons (22–24). Nevertheless, the complex behavior of the so-called IPANs (i.e., AH/Dogiel type II enteric neurons) is not at all like the sensory neurons that have their cell bodies in dorsal root and nodose ganglia and sensory terminals in the gut wall (see accounts of enteric sensory neurons in Chapter 25 and later discussion in this chapter of the cellular neurophysiology of AH neurons). Because the neurophysiology of AH-type neurons does not fit long-standing neurophysiologic definitions for sensory neurons, scholarly confusion could be avoided by abandoning the term IPAN. Low- and High-Threshold Mechanoreceptors Low- and high-threshold mechanoreceptors code mechanosensory information from the gastrointestinal and biliary tracts (see Chapter 25). Low-threshold splanchnic afferents respond to innocuous levels of distension; highthreshold afferents respond to distending volumes (e.g., balloon distension) only at strengths above a set threshold. The latter high-threshold afferents are predominantly unmyelinated C-fibers. Low-threshold afferents are myelinated fibers of the A-delta class. Low-threshold mechanoreceptors are believed to be the afferent component of autonomic regulatory reflexes. Whether some degree of activity in low-threshold pathways reaches the level of conscious perception is uncertain; nevertheless, it is likely that some nonpainful sensations such as abdominal fullness and the presence of gas are derived from the low-threshold–related activity. High-threshold afferents are thought to be the sensory correlates of sharp, localized pain in organs such as the gallbladder where pain is the only consciously perceived sensation (26). Combined involvement of both low- and high-threshold fibers in nociperception cannot be excluded. Cervero and Janig (21) suggest for organs such as the urinary bladder and colon, where distension can evoke sensations ranging from mild fullness to intense pain, that activation of differing proportions of low- and

high-threshold mechanoreceptors account for the gradation of sensations. Visceral Pain Acute visceral pain may emerge from activation of highthreshold nociceptive fibers, whereas chronic forms of visceral pain may be attributed to sensitization of both types of mechanoreceptors by inflammatory conditions or ischemia. Application of acetic acid or other irritants to the large intestinal mucosa in animals reduces the threshold and sensitizes both the high- and low-threshold distension-sensitive afferents. Involvement of another class of splanchnic afferents termed silent nociceptors is also suspect in chronic abdominal pain. Silent nociceptors are splanchnic afferents that do not discharge impulses in response to the strongest of distending stimuli. Inflammatory mediators sensitize these normally silent receptors. Spontaneous firing and responses to normally innocuous mechanical distension can be recorded in previously unresponsive splanchnic afferents after the bowel is inflamed by experimentally applied irritants or inflammatory substances. Visceral Hypersensitivity in Irritable Bowel Syndrome Reports that a significant fraction of patients with the irritable bowel syndrome (IBS) progress to the development of IBS-like symptoms after an acute bout of infectious enteritis are reminiscent of the basic scientific findings that sensory afferents become sensitized in inflammatory states (27). Gwee and colleagues (28) report that 23% of patients with acute gastroenteritis progressed to IBS-like symptoms within 3 months. Nevertheless, the question of whether the association between acute infectious enteritis and IBS reflects low-level inflammation (e.g., microscopic enteritis) and chronic sensitization of intestinal afferents by inflammatory mediators is not fully resolved. Primary sensory afferent terminals in the intestine express receptors for several different messenger substances, including inflammatory mediators. Receptors for 5-hydroxytryptamine (5-HT), bradykinin, adenosine triphosphate (ATP), adenosine, prostaglandins, leukotrienes, and proteases are among those expressed by the afferent terminals (29). Accumulation of any of these substances has potential for increasing the sensitivity of intestinal afferents, especially in disordered conditions such as inflammation and ischemia. Mediators released during mast cell degranulation can sensitize “silent” nociceptors in the large intestine. In animal models, mast cell degranulation results in a reduced threshold for pain responses to balloon distension, and treatment with mast cell stabilizing drugs prevents this decrease of the pain threshold (30). Primary sensory spinal afferents, which are stretch sensitive, have pathophysiologic importance because a significant proportion of patients diagnosed with IBS experience sensitivity to intestinal distension that is perceived as abdominal pain (31,32). The primary causes of abdominal pain of digestive tract origin are distension and exaggerated

636 / CHAPTER 23 muscular contraction. Stimuli such as pinching and burning of the mucosa do not evoke pain. Hypersensitivity of the mechanosensors for stretch (distension) and contractile tension are implicated as pain factors in patients diagnosed with IBS. Heightened sensitivity to distension and conscious awareness of gastrointestinal sensations experienced by patients with IBS are generalized phenomena that extend throughout their digestive tract, including the esophagus (33). The locus of neurologic disturbance responsible for the hypersensitivity in patients with IBS is not fully understood. Disordered neurophysiology in either the peripheral nervous system or CNS, or both, is suggested by the available evidence. A peripheral neural hypothesis states that mechanosensitive afferents in the gut wall become hypersensitive to mechanical stimuli and, as a result, transmit erroneous information to the CNS (see Chapter 25). The alternative hypothesis is for the mechanosensors to be transmitting accurate information, with misinterpretation in the central processing circuits being responsible for the perception of abnormal sensations from the gut (see Chapter 26). Results obtained with modern methods of brain imaging suggest abnormal processing of sensory information in the amygdala, anterior cingulate cortex, and prefrontal cortex in patients with IBS (34–36). A logical suggestion is that sensory physiology may be disordered at peripheral or central levels, or both, of neural organization in patients with IBS who experience gastrointestinal hypersensitivity to mechanical stimuli.

Sensory Chemoreception Sensory transduction of chemical information related to luminal nutrient concentrations in the small bowel involves paracrine signaling from enteroendocrine cells to spinal and vagal afferent terminals in the intestinal wall. Mucosal enteroendocrine cells “taste” the luminal contents and release cholecystokinin (CCK) in direct proportion to the concentration of products of protein digestion and lipids. Cholecystokinin Receptors on Vagal Afferents The amounts of CCK released from enteroendocrine cells are a transform of the nutrient concentration, which, in turn, acts to evoke concentration-dependent firing in vagal afferent neurons. Vagal afferents in the stomach and upper small bowel bifurcate for simultaneous transmission of the same information to the CNS and ENS (37). The firing frequency transmitted by the afferents becomes a transform of the stimulus strength for simultaneous decoding in the brain and enteric processing circuitry. The neuronal receptors for CCK belong to the CCK-A subtype (38,39). Vagal sensory mechanisms involving CCK are implicated in the functions involved in the sensations of fullness, satiety, and control of food intake (39). Disordered vagal sensory function is suspect in the pathophysiology of functional dyspepsia (40,41).

Serotonergic 5-Hydroxytryptamine-3 Receptors Firing of impulses in vagal afferent terminals, spinal afferent terminals, and the terminals of the projections of myenteric Dogiel type II neurons in the intestinal mucosa can be initiated by the action of 5-HT released from mucosal enterochromaffin cells (24,42). Distortion of the mucosal surface by mechanical shearing forces or the presence of noxious chemical agents stimulate the enterochromaffin cells to release 5-HT, which then becomes a paracrine signal to serotonergic receptors on spinal afferent and myenteric Dogiel type II terminals in the mucosa and elsewhere in the gut wall (see Fig. 23-4A). The serotonergic receptors on vagal afferent terminals, on spinal afferent terminals, and on the mucosal terminals of myenteric Dogiel type II neurons belong to the 5-HT3 serotonergic receptor subtype (43,44). Stimulation of the 5-HT3 receptors on vagal afferent terminals is a trigger for nausea and emesis and accounts for the efficacy of 5-HT3 antagonists (e.g., odansetron) as antiemetics. Serotonergic 5-Hydroxytryptamine-3 Receptors and the Irritable Bowel Syndrome Histologic examination of mucosal biopsies removed from the colon of patients with IBS showed increased numbers of enterochromaffin cells and mucosal mast cells, both of which are paracrine sources of serotonin (45–47). Measurements of increased postprandial concentrations of serotonin in the hepatic portal blood of patients with IBS suggest that increased release and excessive stimulation of 5-HT3 receptors on intestinal afferent nerve terminals might account for the abdominal pain and discomfort experienced by these patients. Efficacy of 5-HT3 receptor antagonists (e.g., alosetron) in the treatment of abdominal pain in women with the diarrheapredominant form of IBS adds to the findings that suggest a role for disordered release of serotonin and its action to stimulate firing in sensory afferents as underlying pathologic factors in IBS (48).

Enteric Interneurons Interneurons are the second of the three functional categories of enteric neurons illustrated in Figure 23-1. Enteric interneurons are synaptically interconnected into integrated circuits that process sensory information and control the activity of motor neurons to the musculature, secretory epithelium, and blood vasculature. They have individualized properties including specific morphologic characteristics of the cell somas, directionality of projections, length of axonal projections, electrical and synaptic behavior, and neurotransmitter codes (see Chapter 21). Multiple synaptic connections contact the cell bodies of interneurons and their axons and dendrites in the ganglionic neuropil. Synaptic connections are made between axons and cell bodies, between axons and dendrites, and from axon to axon. Whereas the bulk of information processing in

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 637 integrative nervous systems occurs in the synaptic neuropil, only the cell bodies of enteric neurons currently are accessible for electrophysiologic study of electrical and synaptic behavior. Like in the CNS, microcircuits formed from synaptically interconnected interneurons account for the higher functions that are required for integrated control and coordination of the behavior of effector systems within the intact organ.

(i.e., mucus-secreting goblet cells, Brunner’s glands, and crypts of Lieberkühn) (Fig. 23-5). They are uniaxonal neurons with multiple short processes existing in a variety of shapes. Firing of secretomotor neurons releases acetylcholine or vasoactive intestinal polypeptide, or both, as neurotransmitters at their junctions with the secretory epithelium. Secretomotor axons bifurcate to simultaneously innervate the intestinal crypt and submucosal arterioles (see Fig. 23-5). Collateral innervation of the blood vessels links blood flow to secretion by releasing acetylcholine simultaneously at neuroepithelial and neurovascular junctions. Acetylcholine acts at the blood vessels to release nitric oxide from the endothelium, which, in turn, dilates the vessels and increases blood flow in support of stimulated secretion (49). Secretomotor neurons have receptors that receive excitatory and inhibitory synaptic input from other neurons in the integrative circuitry of the ENS and from sympathetic postganglionic neurons. They are influenced also by paracrine chemical messages from nonneural cell types in the mucosa and submucosa (e.g., enterochromaffin cells and immune/ inflammatory cells). Activation of the excitatory receptors on secretomotor neurons stimulates the neurons to fire and release their transmitters at the junctions with the epithelium of the crypts and surrounding blood vessels.

Enteric Motor Neurons Motor neurons are the final common pathways from the ENS to the effector systems of the gut (see Fig. 23-1). The pool of motor neurons consists of both excitatory and inhibitory neurons. Excitatory motor neurons release neurotransmitters that stimulate muscle contraction and glandular secretion. Inhibitory motor neurons release neurotransmitters that suppress muscular contraction. Secretomotor Neurons Secretomotor neurons are excitatory motor neurons in the ENS that innervate the gastrointestinal secretory glands

Inflammatory mediators

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FIG. 23-5. Intestinal crypts of Lieberkühn are innervated by secretomotor neurons in the submucosal plexus. Neurotransmitters (e.g., acetylcholine [ACh] and vasoactive intestinal peptide [VIP]) that evoke secretion are released at the junctions with the crypts when secretomotor neurons fire. Simultaneous firing of axon collaterals to submucosal arterioles dilates the blood vessels to increase blood flow in support of stimulated secretion. Inhibitory input from sympathetic postganglionic fibers suppresses firing of secretomotor neurons, thereby inhibiting secretion. Paracrine mediators released from inflammatory/immune cells act at receptors on the cell body to increase excitability of secretomotor neurons and at presynaptic inhibitory receptors to suppress release of norepinephrine from inhibitory sympathetic nerves and somatostatin from inhibitory intrinsic neurons. Stimulation of secretomotor neurons by paracrine signals is expected to evoke secretion from mucosal crypts and can account for neurogenic secretory diarrhea. Presynaptic inhibition of norepinephrine and somatostatin release facilitates secretion by removing the braking action of sympathetic and intrinsic nerves, and this may also contribute to diarrheal symptoms. Inset shows an inhibitory postsynaptic potential (IPSP) evoked in a secretomotor neuron by electrical stimulation of sympathetic nerves and release of norepinephrine.

638 / CHAPTER 23 Excitatory receptors on guinea pig intestinal secretomotor neurons include nicotinic, muscarinic M1, and purinergic P2Y1 subtypes. Secretomotor neurons in the submucosal plexus of guinea pig receive purinergic excitatory synaptic input from neurons in the myenteric plexus and neighboring neurons in the submucosal plexus and sympathetic postganglionic fibers (13). The overall result of secretomotor neuronal firing is stimulation of secretion of H2O, NaCl, H2CO3, and mucus from the crypts into the intestinal lumen.

release of norepinephrine from the postganglionic sympathetic axons that provide inhibitory input to secretomotor neurons (50). Two pathologic factors are therefore involved in the production of neurogenic secretory diarrhea. One is hyperstimulation of secretomotor neuronal firing and the other is presynaptic suppression of norepinephrine release from sympathetic postganglionic neurons that results in removal of the sympathetic braking action on the neurons (see Fig. 23-5).

Inhibitory Input to Secretomotor Neurons

Excitatory Receptors Expressed by Secretomotor Neurons

Inhibitory inputs, seen electrophysiologically as inhibitory postsynaptic potentials (IPSPs), hyperpolarize the membrane potential of secretomotor neurons, thereby decreasing the probability of firing (see Fig. 23-5). The physiologic effect of inhibiting secretomotor firing is suppression of mucosal secretion. Postganglionic neurons of the sympathetic nervous system are an important source of inhibitory input to the secretomotor neurons. Norepinephrine released from sympathetic axons acts at α2a noradrenergic receptors to inhibit firing of the secretomotor neurons. Inhibition of secretomotor firing reduces the release of excitatory neurotransmitters at the junctions with the secretory epithelium. The end result is reduced secretion of water and electrolytes. Suppression of secretion in this manner is part of the mechanism involved in sympathetic nervous “shutdown” of gut function in homeostatic states where blood is shunted from the splanchnic to systemic circulation.

Secretomotor neurons have excitatory receptors for several neurotransmitters including acetylcholine acting at nicotinic receptors and the muscarinic M1 subtype (51); ATP acting at the P2Y1 receptor subtype (13); substance P acting at neurokinin-1 (NK1) or NK3 receptor subtypes, or both (52–54); serotonin acting at 5-HT3; and perhaps 5-HT2 receptor subtypes (55,56). Efficacy of blockade of 5-HT3 receptors by a 5-HT3 antagonist in the treatment of diarrhea in the diarrhea-predominant population of women with IBS (48,57) suggests that overstimulation of secretomotor neurons by serotonin is a significant factor in diarrhea-predominant IBS. Observations that IBS symptoms of cramping abdominal pain, diarrhea, and fecal urgency are commonly exacerbated in the postprandial state (58,59), and evidence for increased postprandial release of serotonin (60) raises suspicion that hyperactive release of serotonin from the enterochromaffin cell population in the mucosa underlies the diarrheal symptoms of IBS. This is reinforced by evidence for increased numbers of serotonin-containing enterochromaffin cells and also mast cells in the mucosa of patients with IBS (46,61). Once released from enterochromaffin cells or serotonergic neurons, the action of 5-HT at its multiple receptor subtypes is terminated by enzymatic degradation and by movement into the blood and binding to blood platelets and by a serotonin reuptake transporter (SERT) expressed by the mucosal epithelium and enteric neurons (62). SERT has pathophysiologic significance in that it is suppressed in inflammatory states of the bowel. Suppression of SERT allows accumulation of 5-HT and might account for some of the disordered mucosal secretion and intestinal motility associated with inflammatory conditions in the intestine (63).

Secretomotor Neurons Determine Liquidity of Intestinal Contents Insight into the neurobiology of submucosal secretomotor neurons is essential for understanding the pathophysiology of secretory diarrhea, as well as constipation. In general, increased activity is associated with increased secretion that is manifest as neurogenic secretory diarrhea when secretomotor activity is sufficiently intense. Suppression of secretomotor firing is associated with decreased secretion, reduced liquidity of the luminal contents, and a constipated state, if severe. Suppression of secretomotor firing by antidiarrheal agents (e.g., opiates, clonidine, and somatostatin analogs) is manifest as harder, drier stools. Stimulation by chemical mediators, such as vasoactive intestinal peptide (VIP), serotonin, and histamine, is manifest as more liquid stools (see Fig. 23-5). Several different pathophysiologic events can cause watery diarrhea. For example, secretomotor neurons may be stimulated by excessive serotonin release from mucosal enterochromaffin cells or histamine release from inflammatory/ immune cells in the mucosa/submucosa or circulating VIP released from a VIPoma outside the bowel (i.e., Zollinger– Ellison syndrome). Release of histamine or other inflammatory mediators associated with diarrhea not only stimulates the firing of secretomotor neurons, but also simultaneously acts at presynaptic inhibitory receptors to suppress the

Excitatory Musculomotor Neurons Acetylcholine and substance P are the principal neurotransmitters released from excitatory motor neurons to evoke contraction of gastrointestinal smooth muscles. The receptors for acetylcholine on the musculature belong to the muscarinic M1 receptor subtype (64). Receptors for substance P at neuromuscular junctions are the NK1 subtype (65–67). Axons of excitatory musculomotor neurons to the intestinal circular muscle coat project in the orad direction, and excitatory musculomotor axons to the longitudinal muscle coat project in the aborad direction (see Chapter 21) (68,69).

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 639 Axons of excitatory gastric motor neurons project in the orad direction to both the musculature and mucosa (70,71). Inhibitory Musculomotor Neurons Inhibitory motor neurons release neurotransmitters that suppress contractile activity of the smooth musculature. Early evidence implicated a purine nucleotide, possibly ATP, as the inhibitory transmitter released by enteric inhibitory musculomotor neurons (72). Consequently, for the gut, the terms purinergic neuron and nonadrenergic, noncholinergic (NANC) neuron temporarily became synonymous terms for inhibitory motor neuron. The discovery of purinergic inhibitory motor neurons was highly significant because before 1970 all neurally mediated inhibition was erroneously assumed to be mediated by catecholamines released from the extrinsic sympathetic innervation. In fact, there is little noradrenergic innervation of intestinal smooth muscle outside of the sphincters; most of the sympathetic input is to the ENS. The P2Y1 purinergic receptor subtype mediates the inhibitory action of ATP in the intestinal circular muscle coat of the guinea pig (73). ATP was joined subsequently by VIP, pituitary adenylate cyclase–activating peptide, and nitric oxide as candidate inhibitory transmitters released by enteric inhibitory musculomotor neurons (67–71,74–78). Enteric inhibitory musculomotor neurons that express VIP or nitric oxide synthase, or both, innervate the circular muscle coats of the stomach, intestines, gall bladder, and various smooth muscle sphincters. Inhibitory musculomotor neurons appear as an evolutionary adaptation for neural control of the specialized self-excitatory properties of the musculature (see Chapter 24). Electrically conducting junctions (i.e., gap junctions) connect the smooth muscle fibers one to another to form a functional electrical syncytium that is reminiscent of cardiac muscle. Like cardiac muscle, action potentials and pacemaker potentials spread from muscle fiber to muscle fiber in three dimensions and trigger a contraction as they enter each successive muscle fiber. An interconnected network of nonneuronal pacemaker cells, known as interstitial cells of Cajal (ICCs) extends around the circumference and throughout the longitudinal axis of the small and large intestines (79). The ICC networks generate electrical pacemaker potentials, also called electrical slow waves, which trigger action potentials and their associated contractions in the intestinal circular muscle coat. The pacemaker potentials spreading from the ICC networks become an extrinsic excitatory stimulus to which the circular muscle responds. The physiologic characteristics of the musculature as a self-excitable electrical syncytium suggests that the pacemaker network should continuously evoke contractions that spread in three dimensions throughout the extent of the syncytium, which, in effect, is the entire length of intestine. Nevertheless, in the normal bowel, long stretches of intestine can exhibit no contractile activity and appear as if the musculature were in a paralytic state. Ongoing firing of impulses by enteric inhibitory motor neurons accounts for

the occurrence of contractile silence (i.e., physiologic ileus) in the self-excitable electrical syncytium that forms the intestinal circular muscle coat. The circular muscle is able to respond to a pacemaker potential only when the inhibitory musculomotor neurons in a segment of intestine are switched to an inactive state by input from other neurons in the interneuronal control circuits. When the inhibitory motor neurons are firing and releasing their inhibitory neurotransmitters at the neuromuscular junctions, the muscle cannot be excited to contractile threshold by the omnipresent ICC pacemaker activity. Likewise, action potentials and associated contractions can only propagate into regions of the musculature where the inhibitory innervation is “turned off.” Consideration of the physiologic properties of the musculature suggests that inhibitory musculomotor neurons and control of their activity by the integrative microcircuits of the ENS have evolved as the mechanism that determines when the ongoing slow waves initiate a contraction, as well as the distance and direction of propagation once the contraction has been initiated. The inhibitory motor neurons to the circular muscle discharge continuously. Consequently, action potentials and contractions in the muscle can occur only when the inhibitory neurons are “switched off ” by inhibitory input from interneurons in the control circuits. In the various smooth muscle sphincters found along the digestive tract, the inhibitory motor neurons are normally quiescent and are switched to an active state with timing appropriate for coordination of the opening of the sphincter with physiologic events in adjacent regions. When this occurs, the inhibitory neurotransmitter relaxes ongoing muscle contraction in the sphincteric muscle and prevents excitation-contraction in the adjacent muscle from spreading into and closing the sphincter. In nonsphincteric circular muscle, the state of activity of inhibitory motor neurons determines the length of a contracting segment by controlling the distance of spread of action potentials within the three-dimensional electrical geometry of the syncytium. Contraction can occur in segments in which ongoing inhibition has been switched off, whereas adjacent segments with continuing inhibitory neuronal input cannot contract. The boundaries of the contracted segment reflect the transition zone from inactive to active inhibitory musculomotor neurons. The directional sequence in which the inhibitory motor neurons are switched off establishes the direction of propagation of the contraction within the smooth muscle syncytium. Normally, they are switched off in the aboral direction, resulting in contractile activity that propagates in the aboral direction. In the abnormal conditions associated with emesis, the interneuronal microcircuitry must switch off the inhibitory musculomotor neurons in a reverse sequence to account for small intestinal propulsion that travels in the retrograde direction toward the stomach (5). The first indication that enteric inhibitory motor neurons were continuously firing was the discovery that neuronal blockade of neural activity with local anesthetics or tetrodotoxin resulted in dramatic increases in action potential discharge and associated contractile activity of the small intestinal circular muscle of the cat (80). Before neural blockade,

640 / CHAPTER 23 muscle action potentials are difficult to evoke, and when evoked do not propagate over more than a few millimeters of bowel. After neural blockade, evoked action potentials and associated contraction of the circular muscle propagate over distances of several centimeters (80). Observations of continuous patterned discharge of action potentials by some of the myenteric neurons of dog, cat, guinea pig, and rabbit small intestine were early evidence for the continuous neuronal inhibition of the circular muscle (81,82). Continuous release of the inhibitory neurotransmitters, VIP and nitric oxide, occurs coincident with the continuous inhibitory neural activity in the canine small intestine (83,84). Spontaneously occurring inhibitory junction potentials that reflect the ongoing release of the inhibitory neurotransmitter can sometimes be recorded from the muscle. Nevertheless, in most preparations studied electrophysiologically in vitro, the ongoing inhibitory activity is manifest in the muscle as a steady hyperpolarization of the membrane potential and decreased input resistance. This reduces the probability that electrical pacemaker current from the ICCs will depolarize the muscle to the threshold for action potential discharge and the accompanying contraction. The steady inhibitory hyperpolarization probably reflects smoothing of many inhibitory junction potentials due to: (1) release of transmitter from multiple sites during asynchronous discharge of different nerve fibers, (2) long diffusion distances from transmitter release sites to the muscle, (3) electrical syncytial properties of the muscle, and (4) inhibitory neural input to ICCs (85). In general, any treatment or condition that ablates enteric inhibitory musculomotor neurons results in tonic contracture and “achalasia” of the intestinal circular muscle. Several circumstances that involve functional ablation of the intrinsic inhibitory neurons are associated with conversion from a hypoirritable condition of the circular muscle to a hyperirritable state. These circumstances include: (1) experimental application of local anesthetics or nerve-blocking drugs (80); (2) hypoxic vascular perfusion of an intestinal segment (86); (3) surgical ablation (87); (4) congenital absence, as in Hirschsprung’s disease (88); (5) paraneoplastic syndrome (89); and (6) inflammatory enteric neuropathy (90). Most of the evidence suggests that subpopulations of inhibitory musculomotor neurons are tonically active, and that blockade or ablation releases the circular muscle from the inhibitory influence. The behavior of the muscle in these cases is tonic contracture and disorganized phasic contractile activity reminiscent of fibrillation in cardiac muscle. Disinhibitory Motor Disorders The neuromuscular physiology of the intestine predicts that spasticity and achalasia will accompany any condition in which ablation of inhibitory musculomotor neurons occurs. Without inhibitory control, the self-excitable syncytium of nonsphincteric regions will contract continuously and behave as an obstruction. This happens because the muscle is freed to respond to the ICC pacemakers with contractions that propagate without amplitude, distance, or directional control.

Contractions spreading in the uncontrolled syncytium collide randomly, resulting in fibrillation-like behavior in the affected intestinal segment. Loss or malfunction of inhibitory musculomotor neurons is the pathophysiologic basis of disinhibitory motor disease. It underlies several forms of chronic intestinal pseudoobstruction and sphincteric achalasia. Neuropathic degeneration in the ENS is a progressive disease that, in its earlier stages, can be manifest as symptoms that may be confused with IBS. Chronic Intestinal Pseudo-obstruction Intestinal pseudo-obstruction is failure of intestinal propulsive motility in the absence of any mechanical form of obstruction. Both myopathic and neuropathic forms of chronic intestinal pseudo-obstruction are recognized. Degenerative changes in the musculature underlie the myopathic form, whereas neuropathic forms arise from degenerative changes in the ENS. Failure of propulsive motility in the affected length of bowel reflects loss of the enteric neural microcircuits that program and control the repertoire of motility patterns required for the necessary functions of that region of bowel. Pseudo-obstruction occurs partly because contractile behavior of the circular muscle is hyperactive but disorganized in the denervated regions (91). Hyperactive, disorganized contractile activity, recorded with manometric catheters, is a diagnostic sign of the neuropathic form of chronic small-bowel pseudo-obstruction in humans. The hyperactive and disorganized contractile behavior reflects the absence of inhibitory nervous control of the muscles that are self-excitable (i.e., autogenic) when released from the braking action imposed by inhibitory motor neurons. Chronic pseudo-obstruction is therefore symptomatic of the advanced stage of a progressive enteric neuropathy. Retrospective review of patients’ records and full-thickness biopsies removed from patients diagnosed with IBS suggest that some IBSlike symptoms can be an expression of early stages of the neuropathy (92–95). Degenerative noninflammatory and inflammatory ENS neuropathies are two kinds of disinhibitory motor disorders that culminate in pseudo-obstruction. Noninflammatory neuropathies can be either familial or sporadic (96). The mode of inheritance can be autosomal recessive or dominant. In the former, the neuropathologic findings include a marked reduction in the number of neurons in both myenteric and submucosal plexuses, and the presence of round, eosinophilic intranuclear inclusions in about 30% of the residual neurons. Histochemical and ultrastructural evaluations reveal the inclusions not to be viral particles, but rather proteinaceous material forming filaments (97,98). Members of two families have been described with intestinal pseudo-obstruction associated with the autosomal dominant form of ENS neuropathy (99,100). The numbers of enteric neurons were decreased in these patients, with no alterations found in the CNS or parts of the autonomic nervous system outside the gut.

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 641 Degenerative inflammatory ENS neuropathies are characterized by a dense inflammatory infiltrate confined to enteric ganglia. Paraneoplastic syndrome, Chagas’ disease, and idiopathic degenerative disease are recognizable forms of pseudo-obstruction related to inflammatory neuropathies. Paraneoplastic Syndrome, Chagas’ Disease, and Idiopathic Enteric Neuropathy Paraneoplastic syndrome is a form of pseudo-obstruction where commonality of antigens between small-cell carcinoma of the lungs and enteric neurons leads to autoimmune attack, which results in loss of neurons (89). Most patients with symptoms of pseudo-obstruction in combination with small-cell lung carcinoma have IgG autoantibodies that react with their enteric neurons (101). These antibodies react with a variety of molecules expressed on the nuclear membrane of neurons, including Hu and Ri proteins, and with cytoplasmic antigens (i.e., Yo protein) of Purkinje cells in the cerebellum (102–105). Immunostaining with sera from paraneoplastic patients shows a characteristic pattern of staining in enteric neurons (102). The detection of anti-enteric neuronal antibodies is a means to a specific diagnosis; nevertheless, the mechanisms by which the antibodies damage the neurons are unresolved. The association of enteric neuronal loss and symptoms of pseudo-obstruction in Chagas’ disease also reflects autoimmune attack on the neurons with accompanying symptoms that mimic the situation in lower esophageal sphincter achalasia and paraneoplastic syndrome. Trypanosoma cruzi, the blood-borne parasite that causes Chagas’ disease, has antigenic epitopes similar to enteric neuronal antigens (107). This antigenic commonality activates the immune system to assault the ENS coincident with its attack on the parasite. Idiopathic, inflammatory, degenerative ENS neuropathy occurs unrelated to neoplasms, infectious conditions, or other known diseases (92,94). De Giorgio and colleagues (92) and Smith and colleagues (94) describe two small groups of patients with early reports of symptoms similar to IBS that progressively worsened and were later diagnosed as idiopathic, inflammatory, degenerative neuropathy as determined by full-thickness biopsies taken during exploratory laparotomy that revealed chronic neuropathic intestinal pseudoobstruction. Each patient had inflammatory infiltrates localized to the myenteric plexus. Sera from the two cases reported by Smith and colleagues (94) contained antibodies against enteric neurons similar to those found in secondary inflammatory neuropathies (i.e., anti-Hu), but with different immunolabeling patterns characterized by prominent cytoplasmic rather than nuclear staining. Recognition of the CNS-like functions of the ENS leads to a conclusion that early neuropathic changes are expected to be manifest as functional symptoms that worsen with progressive neuronal loss. In diagnostic motility studies (e.g., manometry), degenerative loss of enteric neurons is reflected by hypermotility and spasticity when it occurs in the small or large intestine, because inhibitory

motor neurons are included in the missing neuronal population (91). Sphincteric Achalasia Sphincteric achalasia is one of the disinhibitory gastrointestinal motility disorders related to enteric neuropathophysiology. Smooth muscle sphincters separate the various specialized compartments of the digestive tract. The lower esophageal sphincter isolates the esophagus from the acidic environment of the stomach. The pyloric sphincter is a barrier to reflux from the duodenum into the stomach. The sphincter of Oddi guards against reflux from the duodenum into the biliary and pancreatic ducts. The internal anal sphincter contributes to the maintenance of fecal continence. Tonic contracture, which is an inherent myogenic property of the sphincteric musculature, maintains closure of the orifices that separate the compartments. Enteric inhibitory musculomotor neurons innervate the smooth musculature that forms the sphincters. The function of the inhibitory innervation is to release inhibitory transmitters (e.g., ATP, VIP, and nitric oxide) that act on the muscle to relax contractile tone, thereby opening the sphincter. Inhibitory musculomotor neurons are the motor component of lower esophageal sphincter relaxation during swallowing, relaxation of the sphincter of Oddi to admit bile and pancreatic secretions into the duodenum, and internal anal sphincter relaxation for defecation and passing of flatus. Inhibitory enteric neural control of the sphincteric musculature differs from the tonically active inhibitory outflow to the intestinal musculature. Inhibitory musculomotor neurons to the sphincters are normally silent and are activated transiently with appropriate timing for opening of the sphincter and passage of luminal contents from one compartment to another. The sphincter is opened when the inhibitory musculomotor neurons are stimulated to firing threshold by excitatory synaptic input from interneurons in the ENS microcircuits. Closure of the sphincter returns when termination of the excitatory input and halt in firing of the inhibitory neurons occurs. The requirement for inhibitory musculomotor neurons to open sphincters predicts that ablation of the ENS will lead to sphincteric achalasia. Failure of development of the fetal ENS, including its population of inhibitory musculomotor neurons, accounts for the constricted terminal large intestinal segment and failure of relaxation of the internal anal sphincter in congenital megacolon (i.e., Hirschsprung’s disease). Achalasia in the lower esophageal sphincter leads to dilatation of the esophageal body and dysphagia. Achalasia in this group of patients is associated with loss of the inhibitory innervation of the sphincter and the presence of antimyenteric antibodies in the patient’s serum (108). The situation is similar for achalasia in the sphincter of Oddi, which leads to distension in the biliary tree and the characteristic pain localized in the right upper body quadrant. The hormone CCK can be used as an effective test of the functional state of the inhibitory innervation of the sphincter

642 / CHAPTER 23 of Oddi in patients with biliary pain of undiagnosed origin (109,110). Endoscopic placement of manometric catheters in the sphincter records changes in resting pressure in response to intravenous injection of CCK. Administration of CCK evokes relaxation in a normally innervated sphincter because excitatory receptors for CCK are expressed by the inhibitory musculomotor neurons that innervate the sphincteric smooth muscle. In contrast, CCK evokes a paradoxical contraction and increase in intraluminal pressure in a denervated sphincter. This occurs because the smooth muscle also expresses excitatory receptors for CCK, and contraction of the sphincter results from direct stimulation of the muscle in the absence of strong input from inhibitory musculomotor neurons to the sphincter.

AH- AND S-TYPE ENTERIC NEURONS Two types of enteric neurons are distinguished by their electrophysiologic behavior in studies where electrical activity is recorded with intracellular microelectrodes. The two types are designated as AH- or S-type. AH-type neurons have Dogiel type II multipolar morphology, and S-type neurons are uniaxonal with multiple short dendrites (see Fig. 23-3).

AH-Type Neurons AH-type neurons are interneurons that are synaptically interconnected for feed-forward excitation of neighboring AH neurons in driver circuits (Fig. 23-6). This is a form of synaptic connectivity in which the neurons of the circuit make recurrent excitatory synaptic connections one with another (111). These kinds of neural circuits are called positive feed-forward circuits. A feed-forward circuit is one in which the synaptic connectivity causes excitation to build rapidly to firing threshold in each of the members of the driver circuit. Rapid buildup of firing in individual neurons in the circuit ensures simultaneous activation of the entire network around the circumference and along the length of a segment of bowel. Output of the circuit is excitatory synaptic input to pools of motor neurons around the circumference and along the length of the intestinal segment (112,113). The driver circuit output simultaneous activates pools of musculomotor neurons to accomplish effective spatial distribution of the contractile forces in the musculature that are necessary for organized propulsion of the luminal contents. Driver circuit output to a pool of secretomotor neurons synchronizes mucosal secretion within a segment of intestine. Neural connections between myenteric and submucosal plexuses coordinate mucosal secretion with contractile behavior of the musculature (13). Mucosal Projections of Myenteric AH-Type Neurons Cell bodies of AH-type neurons in the myenteric plexus are flat, coinlike disks with multiple neurites exiting around

Driver circuit AH neurons Excitatory input

Excitatory output Presynaptic inhibitory receptor

Motor neurons

FIG. 23-6. Driver circuits are positive feed-forward synaptic circuits formed by neurons with AH-type electrophysiologic behavior and Dogiel type II multipolar morphology. Neurons in the circuit make recurrent excitatory synaptic connections with one another, which results in positive feedback flow of synaptic excitation that leads to rapid buildup of firing within the population of driver neurons. Slow synaptic excitation accounts for escalation of excitation in the individual neurons and prolonged firing in the circuit, the output of which activates simultaneous pools of motor neurons. Presynaptic inhibitory receptors at the synapses in the driver circuit maintain a braking action that prevents “runaway” excitation in the circuit. Inhibitory synaptic input to the neurons, which form the circuit (not shown), is postulated to stop neuronal firing and halt excitatory output from the circuit to the motor neuronal pool.

the perimeter (see Fig. 23-3). Figure 23-4 shows how each AH neuron in the myenteric plexus projects one of its neurites through the intestinal circular muscle and lamina propria into the mucosal epithelium (114). The endings in the mucosa express serotonergic 5-HT3 receptors. Serotonin released from enterochromaffin cells during application of mechanical or chemical stimuli to the mucosa binds to the 5-HT3 receptors and stimulates the ending in the mucosa to fire (115–117). AH Neuronal Gating Function Excitation of the cell bodies of AH neurons by paracrine mediators or slow excitatory synaptic input from neighboring neurons in the neural network, or both, is a central component of their gating function. The gating properties of the cell body control spread of excitation from neuron to neuron in the recurrent synaptic networks formed by AH neurons (see Figs. 23-4 and 23-6). The state of the somal gate (i.e., open, closed, or partially open) determines the output behavior of the circuit in which they reside. All AH neurons in the circuit fire synchronously when their somal gates are fully opened by excitatory synaptic input or by an overlay of a paracrine message. At the opposite extreme, the gates are closed when the cell somas are in their inexcitable state. Gating controls the propagation of action potentials among the multiple neurites that arise around the perimeter of the AH multipolar cell bodies. Slow excitatory events (see later in this chapter) are an integral part of the

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 643 gating mechanism. Figure 23-4 illustrates how the gating mechanism works in AH-type neurons. The membrane of the cell body of AH neurons may exist in an inexcitable, hyperexcitable, or intermediate state of excitability (see Figs. 23-4D to F). The somal membrane cannot be fired by injection of depolarizing electrical current from action potentials spreading into the cell body from its neurites when in the inexcitable state (see Figs. 23-4G to I). The state of somal inexcitability reflects the absence of stimulation by slow excitatory mediators (e.g., excitatory neurotransmitters or inflammatory mediators) or it might reflect activation of inhibitory input to the neuron. Hyperexcitability reflects exposure to increased concentrations of one or more excitatory mediator and intermediate states reflect exposure to intermediate concentrations of one or more of several known

Action potential AH

excitatory mediators. If the cell soma is in its inexcitable state, action potentials traveling toward it along one of its neurites cannot fire the soma when they arrive (see Figs. 23-4G to I). The gate to other neurites around the soma’s periphery is closed when the soma is inexcitable. In intermediate states of excitability, firing of the somal membrane by an inbound spike in one of its neurites is followed by the postspike afterhyperpolarization that is a characteristic of AH-type neurons (Fig. 23-7A). The prolonged refractory period during the AH prevents greater frequency of firing of the somal membrane by additional incoming spikes (see Fig. 23-7D). In this state, the “partially open” somal gate fractionates the transfer of information across the cell body so that only a fraction of the inbound spikes cross the cell soma fire to fire other neurites around the somal perimeter (see Figs. 23-4G to I).

"Shoulder"

4s 2 ms

A

B

2 ms

C Recording electrode

(1)

(4)

Stimulating electrode

(2) (3) Fiber tract

5 ms

D FIG. 23-7. Properties of AH-type neurons. (A) Action potential in an AH neuron in its low-excitability state is followed by characteristic long-lasting afterhyperpolarization. Downward deflections are electrotonic potentials evoked by repetitive intraneuronal injection of hyperpolarizing current pulses. Decreased amplitude of the electrotonic potentials during the AH reflect decreased input resistance due to opening of Ca2+-gated K+ channels. (B) Action potential in an AH neuron recorded with a fast time base shows characteristic “shoulder” on the falling phase of the spike. “Shoulders” appear on the spikes when AH neurons are in states of reduced excitability; they reflect the opening of voltageactivated Ca2+ channels. (C) Superimposed action potentials in an AH neuron in its state of hyperexcitability do not have a “shoulder” on the falling phase. (D) Postspike afterhyperpolarizing potentials prolong the refractory period for firing of the cell soma in AH neurons. Twin extracellular stimulus pulses were applied to an interganglionic connective (inset). Each of the two stimulus pulses fired one of the neuron’s projections in the fiber tract and the action potential in the projecting fiber propagated to the cell soma, where it depolarized the somal membrane to firing threshold. The time interval between the two stimulus pulses was progressively shortened until first the somal action potential and then the electrotonic invasions from the projection in the fiber tract were not evoked by the second stimulus pulse. The superimposed traces show that the refractory period for the somal action potential was longer than for the spike conducted in the neurite in the fiber tract. (1) Superimposed traces of the somal action potential evoked by the first of the twin stimulus pulses. (2) Stimulus artifact for second stimulus pulses was sufficiently close to the first in time that both the cell soma and its projection in the fiber tract were refractory. (3) The time interval between the two stimulus pulses was sufficiently long for the second pulse to evoke a spike that invaded the cell soma without firing the soma, because the timing of the second stimulus pulse fell within the refractory period produced by the AH. (4) Action potentials evoked in the cell body by electrotonic invasion from its neurite when the timing between the two pulses was longer than the duration of the AH.

644 / CHAPTER 23 Each inbound neurite spike fires the cell soma when it is in its hyperexcitable state. This occurs because excitability of the somal membrane is enhanced, membrane resistance and space constant are increased, and after-spike hyperpolarizing potentials are suppressed during slow synaptic or paracrine excitation (see later discussion). The gate is “wide open” in this state, and each arriving action potential in any given neurite is conducted across the cell body to activate spike discharge in all neurites around the perimeter of the multipolar neuron, including its projection to the mucosa (see Fig. 23-4A). When this happens, output from the cell body is distributed to multiple synaptic sites in the circuitry by the extensive ramifications of the Dogiel type II neurites within and among ganglia (see Fig. 23-3). The level of somal excitability determines how action potentials that are inbound to the cell body from the mucosa are “gated” (see Fig. 23-4E). Inbound spike information from the mucosa does not cross the cell soma when it is in the inexcitable state, and synaptic output from the cell body to neighboring neurons does not occur. Figure 23-4A illustrates how inbound information from the mucosa may participate in axonal reflexes that are independent of the cell body when it is in its inexcitable state. Intermediate levels of excitability permit only a fraction of the inbound spikes to fire the cell soma, and thereby transfer differently encoded information to neighboring neurons in the circuit. Figures 23-4G to I show how fractionation by the cell soma of inbound spike information in one of its neurites occurs. Hyperexcitability and repetitive firing of the cell soma preclude transfer of spike information from the mucosal endings, because outbound firing in the mucosal projection would collide with and occlude transfer of the local mucosal information either into the myenteric or submucosal plexus (see Fig. 23-4A).

AH-type neurons have distinguishing electrophysiologic characteristics that include: (1) greater resting membrane potentials associated with lower input resistance than found in S-type neurons; (2) low excitability in the resting state reflected by inability to evoke action potential discharge by injection of depolarizing electrical current (see Fig. 23-4D); (3) absence of anodal-break excitation at the offset of injection of hyperpolarizing current pulses; (4) prolonged postspike afterhyperpolarization (see Fig. 23-7A); (5) a “shoulder” on the falling phase of the action potential that reflects opening of voltage-dependent Ca2+ channels (see Fig. 23-7B); (6) failure of blockade of voltage-dependent sodium channels to abolish the action potentials; (7) exposure to multivalent cationic Ca2+ entry blockers (e.g., Mn2+, Mg2+, or Cd2+) depolarizes the membrane potential coincident with increased input resistance and enhanced excitability; and (8) activation of adenylate cyclase and increase of intracellular cyclic adenosine 3′,5′-phosphate (cAMP) depolarizes the membrane potential, increases input resistance and augments excitability. Aside from the electrophysiologic properties, most AH-type neurons (i.e., ≈80%), but not S-type neurons, express the calcium-binding protein, calbindin in guinea pigs. Neurons with AH-type electrophysiologic properties comprise the largest proportion of neurons in the intestinal myenteric plexus of the guinea pig (i.e., ≈70%) and the smallest proportion of submucosal plexus neurons (i.e., <10%).

Terminology for AH-Type Neurons

Resting Membrane Potentials in AH-Type Neurons

IPAN is an acronym for “intrinsic primary afferent neuron,” which was proposed as another name for AH-type neurons (116–121). The term is inaccurate, and therefore a source of confusion, because AH neurons are not sensory afferent neurons. They do not meet the traditional rigorous criteria for sensory afferent function as applied for spinal, cranial, and vagal sensory neurons in all textbooks of sensory neurophysiology (see Chapter 25). The main justification given for renaming AH neurons as sensory afferent neurons was discovery of the paracrine action of serotonin on the endings in the mucosa (see Fig. 23-4A). Serotonergic paracrine signaling to AH neurons is not the single most important factor that makes these neurons unique and is not a criterion for function as a sensory receptor. Paracrine release of multiple mediators from nonserotonergic cell types (e.g., histamine and proteases from mast cells) excites AH neurons in addition to multiple kinds of slow and fast synaptic inputs (122,123). AH neurons are neurons with truly unique behavioral properties in the vertebrate nervous system in general; nevertheless, the properties do not include coding of sensory information as defined in traditional neurophysiology.

Sodium-potassium ATPases maintain ionic gradients of high K+/low Na+ inside enteric neurons. Potassium conductance is the main ionic determinant of the resting membrane potential. The resting potential is usually 20 to 30 mV less negative than the potassium equilibrium potential, which is approximately −90 mV. In AH neurons, a component of the resting potassium conductance and consequently the resting potential are dependent on the concentration of free intracellular Ca2+ (126–129). Three kinds of Ca2+-activated K+ channels are known to occur in neurons in general. One type is a large-conductance channel (i.e., 200–400 pS) that requires free intraneuronal Ca2+ and membrane depolarization for activation. These channels are called maxiK or BK channels. The second type of Ca2+-activated K+ channel has a lower single-channel conductance in the range of 2 to 20 pS and is called SK for small-conductance channel. The third type of Ca2+-activated K+ channel has an intermediate singlechannel conductance in the range of 20 to 100 pS and is called an intermediate conductance or IK channel. IK channels are voltage insensitive, but gated by increases in cytosolic Ca2+. Measurements of Ca2+-activated K+ current in voltage-clamp

Confusion can be avoided by abandoning the term IPAN and falling back to the term enteric AH-type neuron, as was applied by the investigators who were the first to name the neurons (124,125). Cellular Physiology of AH-Type Enteric Neurons

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 645 studies and immunohistochemical localization with an antibody selective for IK channels suggest the IK channel might be the primary functional Ca2+-activated K+ channel in AH neurons (130–133). A steady transmembrane influx of Ca2+ is responsible for increased intraneuronal levels that hold the IK channels in an open state. This is reflected by high K+ conductance, decreased input resistance, and hyperpolarized membrane potential in AH neurons in the absence of any exposure to excitatory chemical signals. Neurotransmitters and paracrine neuromodulators act to increase or decrease the Ca2+-activated K+ conductance, and this is the key mechanism for up- and down-modulation of excitability and input–output relations in AH neurons (127). The functional significance of resting potentials less than the K+ equilibrium potential is provision of a mechanism whereby the membrane potential can be modulated in the hyperpolarizing or depolarizing direction as determined by whether the messenger substance acts to increase or decrease the K+ conductance. In enteric neurons, inhibitory signal substances such as opioid peptides, galanin, and adenosine decrease neuronal excitability by increasing K+ conductance and hyperpolarizing the membrane (134–136). Excitatory messengers, such as substance P, serotonin, and histamine, decrease resting potassium conductance, depolarize the membrane, and enhance excitability (137,138). Action Potentials in AH-Type Neurons Action potential generation in the cell bodies of AH neurons differs from spike-generating mechanisms in the cell bodies of S neurons and the AH neurons’ own neurites. The ionic mechanism of action potential generation in the cell bodies of AH neurons includes conductance changes for Ca2+, Cl−, Na+, and K+. Both Na+ and Ca2+ carry the inward current of the rising phase of the spike. A characteristic “shoulder,” reminiscent of the plateau on cardiac action potentials, is present at the onset of the falling phase of the spike in AH neurons (see Fig. 23-7B). The “shoulder” reflects activation of voltage-gated Ca2+ conductance in N-type, high-voltage– activated Ca2+ channels (139) as the membrane is depolarized by an inward Na+ current. The Na+ channels are typical of tetrodotoxin-sensitive channels found in neurons elsewhere (140). Application of tetrodotoxin reduces the rate of rise, the amplitude, and the threshold, but it does not abolish the action potentials in AH neurons. The rate of rise of the spike in tetrodotoxin is increased by elevation of external Ca2+, and the pure Ca2+ spike in this case is abolished by multivalent ions that block Ca2+ entry (141–143). Dihydropyridines and other organic Ca2+ entry blockers that suppress Ca2+ conductance in smooth and cardiac muscles do not affect the Ca2+ component of the spike in AH neurons (143). AH-type neurons possess the full complement of voltagegated K+ currents generally found in neurons elsewhere. These include A-type, delayed rectifier, hyperpolarization-activated cation current (Ih) and inwardly rectifying currents (144,145). The falling phase of the spike is associated with time- and voltage-dependent activation of delayed rectifier K+ channels.

Long-lasting hyperpolarizing afterpotentials are the hallmark of the action potentials in AH neurons and the basis for the term AH-type neuron (see Fig. 23-7A). The afterhyperpolarization activates slowly beginning from 45 to 80 msec after termination of the spike and lasts for up to 30 seconds. The amplitude of the AH summates when two or more spikes are fired in close sequence. An increase in membrane conductance reflected by a decrease in the input resistance occurs during the hyperpolarizing afterpotentials. The amplitude of the afterpotential is increased by elevation of extracellular Ca2+ and is suppressed by multivalent ions that block Ca2+ entry. The amplitude is reduced and the duration of the hyperpolarizing afterpotential is shortened in bathing media with reduced Ca2+ (142). The polarity of the AH is reversed with current clamp of the membrane potentials to values greater than the K+ equilibrium potential of approximately −90 mV. These early findings were evidence that an outward current carried by K+ ions generated the hyperpolarizing afterpotential. The evidence suggests that BK and intermediateconductance IK Ca2+-activated K+ channels carry the outward K+ current responsible for the hyperpolarizing afterpotential (130–133). Earlier evidence for Ca2+-activated K+ channels was obtained from voltage-clamp results, which suggest that the slow outward current is coupled temporally to inward Ca2+ currents that are activated by depolarizing voltage steps (146). The amplitude of the current responsible for the hyperpolarizing afterpotential is a direct function of the amount of Ca2+ that enters the cell during depolarizing clamp steps or as the result of discharge of action potentials. Further evidence for the Ca2+ dependence of K+ current activation is the suppression of the current and the associated hyperpolarization produced by application of multivalent cations that block Ca2+ entry. This is consistent with the results, obtained with optical imaging of intraneuronal Ca2+, that show increases of intraneuronal free Ca2+ during the hyperpolarizing afterpotential (147–149). The hyperpolarizing afterpotentials function to lengthen the refractory period after the spike, and this automatically limits the frequency of spike discharge by the cell body (see Figs. 23-4G to I and 23-7D). During slow synaptic excitation (see later) the neurotransmitter or paracrine modulator acts to reduce the hyperpolarizing afterpotential, and this allows the somal membrane to fire repetitively at a greater frequency. Modulation of the afterpotential by chemical messengers is part of the overall mechanisms by which the excitability and input–output relations of AH neurons are governed (150).

S-Type Enteric Neurons Whereas AH-type neurons were named for their characteristic postspike after hyperpolarizing potentials, S-type neurons (i.e., synaptic-type neurons) were so named because nicotinic fast excitatory postsynaptic potentials (EPSPs) were found in virtually all S-type enteric neurons, and only restricted subpopulations of AH-type neurons express nicotinic receptors and fast depolarizing responses to application of acetylcholine.

646 / CHAPTER 23 S-type enteric neurons are distinguished by: (1) lower resting membrane potentials and greater input resistance than found on average in AH neurons; (2) increased excitability that is reflected by spontaneous discharge of action potentials and by repetitive spike discharge when the membrane potential is depolarized by injecting electrical current through the microelectrode; (3) increase in the frequency of repetitive firing in direct relation to the degree of membrane depolarization, unlike AH neurons; (4) anodal-break excitation (i.e., action potential discharge) at the offset of intraneuronally injected hyperpolarizing current pulses, which is another reflection of increased excitability that does not occur in AH neurons at rest; (5) abolition of somal spikes by agents (e.g., tetrodotoxin) that block voltage-activated sodium channels; (6) insensitivity to stimulation of adenylate cyclase by forskolin and increase of cAMP; and (7) insensitivity to multivalent cationic Ca2+ entry blockers. Fast (<50 msec in duration) nicotinic EPSPs can be evoked experimentally in the majority of S-type neurons. S-type neurons are more likely to show spontaneously occurring spike discharge than AH neurons. Nevertheless, AH neurons discharge spontaneously when activated by excitatory neurotransmitters or paracrine signals (see the next section). Action Potentials in S-Type Neurons Somal action potentials in S-type neurons and spike generation by neurites of both AH- and S-type neurons are generated by classic mechanisms of activation and inactivation of time- and voltage-dependent Na+ and K+ conductance channels. The spikes are always abolished by sufficient concentrations of tetrodotoxin and the rate of rise and amplitude are reduced in depleted Na+. Unlike AH neurons, mechanisms for generation of repetitive spike discharge are always activated, and the cells often will discharge spontaneously, with the spikes preceded by ramplike prepotentials. In contrast with AH neurons, S neurons fire continuously during longlasting depolarizing current pulses, and the frequency of discharge is related directly to the size of the depolarizing pulse. Prominent positive afterpotentials are associated with the spikes and are not followed by Ca2+-dependent, after-spike hyperpolarizing potentials such as those in AH neurons. Unlike AH neurons, depletion of extracellular Ca2+ or activation of adenylate cyclase by forskolin does not increase excitability. S-type electrophysiologic behavior is a characteristic of musculomotor and secretomotor neurons and interneurons that have uniaxonal morphology (see Fig. 23-3).

Ca2+ channels. Once released, enteric neurotransmitters bind to their specific postsynaptic receptors to evoke ionotropic or metabotropic synaptic events. When the receptors are directly coupled to the ionic channel, they are classified as “ionotropic.” They are “metabotropic receptors” when their effects to open or close ionic channels are indirectly mediated by guanosine triphosphate (GTP)–binding proteins and the induction of cytoplasmic second messengers (e.g., cAMP, inositol trisphosphate, and diacylglycerol). Synaptic events in the ENS are basically the same as in the brain and spinal cord. EPSPs, IPSPs, and presynaptic inhibition and facilitation are the principal synaptic events in the ENS. An enteric neuron may express mechanisms for both slow and fast synaptic neurotransmission. Fast synaptic potentials have durations in the millisecond range; slow synaptic potentials last for several seconds, minutes, or longer. Fast synaptic potential are usually EPSPs. The slow synaptic events may be either EPSPs or IPSPs.

Fast Excitatory Postsynaptic Potentials Fast EPSPs were reported for the earliest intracellular studies of myenteric neurons but were found only in S neurons in the early work of Hirst and colleagues (125,151) and Nishi and North (152). Fast EPSPs are depolarizing responses with durations less than 50 msec (Fig. 23-8A). Fast EPSPs were later reported to occur in AH and S neurons in both myenteric and submucosal plexuses. They appear to be the sole mechanism of transmission between vagal efferents and enteric neurons. Most of the fast EPSPs are mediated by acetylcholine acting at nicotinic postsynaptic receptors. The actions of 5-HT at the 5-HT3 serotonergic receptor subtype and purine nucleotides at P2X purinergic receptors behave much like fast EPSPs, and it is possible that some fast EPSPs are purely serotonergic or purinergic or reflect a summation of purinergic and serotonergic input (153–158). Nicotinic Receptors Different combinations of α and β subunits assemble in different combinations to form nicotinic receptors. Functional receptors are formed by five subunits. Eight different α subunits (i.e., α2-9) and nine β subunits (i.e., β2-4) have been identified. The properties of a specific nicotinic receptor are determined by the kinds of subunits that form the pentameric receptor. The main mediator of nicotinic fast EPSP-like responses in enteric neurons is a receptor composed of α3 and β4 subunits (157).

SYNAPTIC TRANSMISSION Fundamental mechanisms for chemically mediated synaptic transmission in the ENS are the same as elsewhere in the nervous system. Synaptic transmitters are released by Ca2+-triggered exocytosis from stores localized in vesicles at axonal terminals or transaxonal varicosities. Release is triggered by the depolarizing action of action potentials when they arrive at the release site and open voltage-activated

Purinergic Receptors P2X receptors are trimeric proteins formed by subunits with two transmembrane domains. There are at least seven subtypes of P2X receptors ranging from P2X1 to P2X7. Immunohistochemical studies using specific P2X receptor antibodies found at least three P2X receptor subunits in the ENS that were identified as P2X2, P2X3, and P2X7

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 647 Action potential

Stimulus artifact

Stimulus artifact

IPSP

EPSPs 10 mV 10 mV

A

10 msec

0.5 Sec

B

C

On Off Stimulus

5-HT3 response

20 sec

5-HT1P Slow EPSP-like response

2 sec

D

“Puff 5-HT”

FIG. 23-8. Fast and slow excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) are the principal kinds of synaptic events in enteric neurons of guinea pig small intestine. (A) Two fast EPSPs in an S-type myenteric neuron were evoked by successive stimuli and are shown as superimposed traces on the oscilloscope. Only one of the EPSPs reached threshold for discharge of an action potential. (B) Slow IPSP evoked by stimulation of a sympathetic noradrenergic input to a secretomotor neuron in the submucosal plexus. (C) Slow EPSP evoked by repetitive electrical stimulation of the synaptic input to an AH-type myenteric neuron. (D) Mimicry of fast and slow neurotransmission by 5-hydroxytryptamine (5-HT) in an AH-type myenteric neuron as an example of a paracrine mediator released from enterochromaffin cells. 5-HT was applied as a 20-msec pulse by micropressure ejection from a fine-tipped pipette. The initial “fast” response reflects stimulation of the 5-HT3 serotonergic receptor subtype and opening of ligand-gated ion channels. The delayed, long-lasting, slow EPSP-like response reflects activation of the 5-HT1P serotonergic receptor subtype.

(159–164). The P2X2 receptor is expressed in neurons that express immunoreactivity (IR) for the chemical codes of inhibitory musculomotor neurons, noncholinergic secretomotor neurons, and calbindin-immunoreactive neurons (160). Loss of the purinergic component of fast EPSPs in P2X2 knockout mice supports the suggestion that the P2X2 receptor is a major player in purinergic fast transmission in enteric neurons (156). P2X3 subunits form heteromers with P2X2 subunits in aborally projecting inhibitory musculomotor neurons. Calbindin-immunoreactive enteric neurons do not express P2X3 receptor IR (162,163). Serotonergic 5-Hydroxytryptamine-3 Receptors Responses mediated by 5-HT3 receptors are found on neurons in both plexuses throughout the gastrointestinal tract, including the stomach (165). Neither purinergic nor serotonergic fast EPSPs occur in each and every class of neurons (i.e., based on chemical codes) in the guinea pig colon and elsewhere (158). Results obtained with patch-clamp recording methods show that the nicotinic and 5-HT3 serotonergic receptors connect directly to nonselective cationic channels. Opening of these channels is responsible for the depolarizing event (166).

Rapid desensitization is a characteristic of both the nicotinicand 5-HT3–operated channels, both of which are ionotropic. Purinergic “fast” depolarizing responses, like serotonergic 5-HT3 receptor–mediated responses, reflect opening of ligand-gated, nonselective, cationic channels (157,167). Fast Excitatory Postsynaptic Potential Rundown Amplitudes of nicotinic fast EPSPs in the intestine become progressively smaller when they are evoked repetitively by focal electrical stimulation applied to the surface of the ganglion or interganglionic fiber tract. Decrease in EPSP amplitude occurs at stimulus frequencies as low as 0.1 Hz, and the rate of decline is a direct function of stimulus frequency. Rundown of this nature does not occur at the synapses in the stomach (168,169) or gallbladder (170). The rundown phenomenon reflects presynaptic inhibition of acetylcholine release by additional transmitter substances broadly released by the electrical stimulus or by negative feedback involving autoinhibition of acetylcholine release mediated by presynaptic inhibitory muscarinic receptors (52). Rundown cannot be attributed to postsynaptic changes, because no decrease in the amplitude of the EPSPs occurs during repetitive applications of acetylcholine from microejection pipettes.

648 / CHAPTER 23 Significance of Fast Excitatory Postsynaptic Potentials The importance of fast EPSPs emerges from their function in the rapid transfer and transformation of neurally coded information between axons and neuronal cell bodies and axons and dendrites that form the enteric microcircuitry. They are the bytes of information in the information-processing operations of the logic circuits. Fast EPSPs may or may not depolarize the membrane to its threshold for discharge of an action potential. Summation of multiple inputs increases the probability of reaching firing threshold. Fast EPSPs do not reach threshold when the neuronal membranes are hyperpolarized during slow IPSPs. They are most likely to reach spike threshold when the membranes are depolarized during slow EPSPs or when depolarizing action of modulators are released in paracrine fashion from nonneuronal cells. This effect of slow EPSPs and of slow EPSP-like paracrine mediators is an example of neuromodulation whereby the input–output relations of a neuron to one input (i.e., fast EPSPs) is modified by a second synaptic or other kind of modulatory input.

Slow Synaptic, Endocrine, and Paracrine Excitation Neurocrine, endocrine, and paracrine are the primary forms of slow excitatory chemical signaling to the cell bodies of enteric neurons. Neurocrine signaling refers to chemical transmission from neuron to neuron (i.e., synaptic transmission). Slow neurocrine signaling may be in the form of inhibitory synaptic potentials (IPSPs) or slow synaptic excitation (slow EPSPs). Endocrine signals are hormones transported into the vicinity of the neurons by the blood. Paracrine signals are chemical substances that reach the neurons by diffusion in the extracellular milieu after release by nonneuronal neighboring cells in proximity to the neurons. Enterochromaffin cells, lymphocytes, macrophages, polymorphonuclear leukocytes, and mast cells are sources of paracrine signals to the ENS. Slow synaptic transmission and most paracrine signal transmission in the ENS are mediated by metabotropic receptors. Paracrine signal substances mimic slow synaptic excitation when applied to ENS neurons in vitro or when released in vivo during degranulation of enteric mast cells in antigen-sensitized animal models (123,171,172). Focal electrical stimulation applied with microelectrodes to interganglionic fiber tracts or to the surfaces of ganglia in the myenteric or submucosal plexus evokes slow EPSPs and IPSPs in ENS neurons with AH-type electrophysiologic behavior and multipolar Dogiel type II morphology and neurons with S-type electrophysiologic behavior and uniaxonal morphology (see Figs. 23-8B and C). Slow EPSPs are found in neurons in the small and large intestines and gastric antrum, but not the gastric corpus, gallbladder, or pancreas (143,150,168–170,173–175). Slow EPSPs are restricted to the enteric microcircuits of specialized digestive compartments where peristaltic propulsive motility is a significant function. Prominent slow IPSPs are evoked in secretomotor neurons in the submucosal plexus (see Fig. 23-5), and less prominent IPSPs occur in neurons in the myenteric plexus.

Slow Excitatory Postsynaptic Potentials Slowly activating membrane depolarization, which continues for several seconds to minutes and sometimes hours after termination of stimulation and release of the neurotransmitter from the presynaptic terminal, is a characteristic of slow EPSPs (see Fig. 23-8C). Enhanced excitability reflected by prolonged trains of action potentials is associated with the depolarizing response. Enhanced excitability during experimentally evoked slow EPSPs is apparent as repetitive spike discharge during intraneuronal injections of depolarizing current pulses in both AH- and S-type neurons and as anodalbreak excitation at the offset of hyperpolarizing current pulses in both kinds of neurons. AH neurons, which will not discharge at all or will fire only a single spike at the beginning of a depolarizing current pulse in the inactivated state and which show no anodal-break excitation, will fire repetitively in response to depolarizing pulses and discharge at the offset of hyperpolarizing pulses when the slow EPSP is in effect (see Figs. 23-4D and F). When activated by either slow synaptic or paracrine inputs, electrophysiologic behavior of AH neurons is much like S neurons and may be confused as such, if the AH neurons happen to be in an activated state because of ongoing release of the transmitter or a paracrine mediator (e.g., inflammatory/immune mediators). The “shoulder” on the falling phase of the action potential and characteristic postspike hyperpolarization in AH neurons is suppressed or abolished during slow EPSPs. Suppression of the afterhyperpolarization is part of the mechanism that permits repetitive spike discharge at increased frequencies during the enhanced state of excitability. The changes in electrophysiologic behavior during the slow EPSP underlie opening of the “somal gate” and relay of inbound spike information in a neurite across the cell body to output projections elsewhere around the perimeter of the multipolar Dogiel II neurons (see Fig. 23-4E). Slow EPSPs differ for neurons with AH-type electrophysiologic behavior and multipolar Dogiel type II morphology and neurons with S-type electrophysiologic behavior and uniaxonal morphology. Stimulus-evoked slow EPSPs and responses to excitatory paracrine mediators in AH neurons generally are associated with an increase in neuronal input resistance that reflects closure of ionic channels and decreased membrane ionic conductance. In S neurons, a large proportion of which are musculomotor or secretomotor neurons, stimulus-evoked slow EPSPs and responses to excitatory paracrine mediators are associated with a decrease in neuronal input resistance that reflects opening of ionic channels and increased membrane ionic conductance. Ionic Mechanisms in AH-Type Neurons Conductance changes in several kinds of ionic channels underlie the slow EPSPs in AH neurons. The depolarization phase occurs when Ca2+ channels that are normally open at rest are closed (127,128,135,176). Closure of the Ca2+ channels reduces intraneuronal Ca2+, which, in turn, leads to closure

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 649 of Ca2+-activated K+ channels (i.e., IK channels). Closure of the K+ channels accounts for the increased input resistance seen in electrophysiologic records from the neuron during the depolarization phase of the slow EPSP. Changes in input resistance are often biphasic, with an increase followed by a decrease as the depolarization progresses. Increased Cl− conductance accounts for the second phase of decreased input resistance. Bertrand and Galligan (177) estimate that 90% of the conductance change during slow EPSPs and the EPSP-like responses to an NK3 agonist were accounted for by closure of Ca2+-activated K+ channels, and the remaining 10% by increased Cl− conductance. Chloride channels also contribute to the conductance changes associated with histamine-evoked slow EPSP-like depolarizing responses (178). Inward current in N-type Ca2+ channels during the rising phase of the action potential, which in the resting state would inject Ca2+ and lead to postspike increase in Ca2+-activated K+ conductance, is suppressed during slow excitatory responses in AH neurons (130,139). This accounts for suppression of postspike hyperpolarization (i.e., the AH) during the response. Enhanced excitability, seen as repetitive spike discharge and anodal-break excitation during stimulus-evoked EPSPs and during exposure to paracrine mediators, is related to suppression of A-type and delayed rectifier K+ currents (144). Ionic Mechanisms in S-Type Neurons The ionic mechanism for the depolarization phase of slow EPSPs and slow EPSP-like responses to paracrine mediators in S-type neurons is opening of cationic conductance channels and is opposite to the mechanism in AH-type neurons. Increased Na+ and Ca2+ conductance accounts for most of the depolarization phase and decreased input resistance seen during the EPSP and paracrine-evoked excitatory responses in S neurons (13,122). Depletion of extraneuronal Ca2+ in in vitro studies has minimal effect on slow EPSPs mediated by purinergic P2Y1 receptors or on slow EPSP-like depolarizing responses to paracrine mediators, whereas signification suppression occurs with depletion of Na+ in the bathing medium (24,179). IR for the transient receptor potential channel 6 (TRP-6 channel) is expressed in the submucosal plexus in uniaxonal neurons with S-type electrophysiologic behavior, slow IPSPs, and chemical codes for secretomotor neurons including VIP, choline acetyltransferase, and neuropeptide Y (180). This suggests that the cationic conductance channels responsible for the depolarizing responses during slow EPSPs and responses to slow EPSP mimetics in S-type neurons might be TRP-6 channels. Mediators of Slow Excitatory Postsynaptic Potentials Several messenger substances found in neurons, endocrine cells, or immune/inflammatory cells mimic slow EPSPs when applied experimentally to enteric neurons. Receptors for more than one of the messenger substances may be present

on the same neuron. Table 23-1 is a partial listing of the substances together with receptor subtypes. Substance P, serotonin, ATP, and acetylcholine fulfill criteria for function as neurotransmitters for metabotropic slow EPSPs in enteric neurons. Other substances are implicated mainly by their presence in enteric neurons, by mimicry of the slow EPSP when applied experimentally, or both. Receptors for these substances, as well as combinations of receptors for some of the substances listed in Table 23-1, can be coexpressed by the same enteric neuronal cell body. Serotonin, histamine, interleukin-1β (IL-1β), bradykinin, and mast cell proteases are examples of slow EPSP mimetics of paracrine origin. Histamine, IL-1β, and mast cell proteases are released from intestinal mast cells to become neuromodulatory signals that are decoded by the enteric microcircuits. The evidence for 5-HT as an enteric signal substance, which includes synthesis, storage, and release from enteric neurons and the activity of reuptake transporters for termination of action, is relatively complete (181). Nevertheless, the strongest evidence is that agents such as N-acetyl-5-hydroxytryptophyl5-hydroxytryptophan amide, renzapride, and anti-idiotypic antibodies to 5-HT block both the slow EPSP-like actions of 5-HT and the slow EPSP in the same AH neurons (181,182). Aside from its role as a putative enteric neurotransmitter, 5-HT also has a paracrine signaling function. A large fraction of the body’s 5-HT, which is estimated to be as high as 95%, is stored in enterochromaffin cells interspersed in the intestinal epithelial lining. Mechanical stimulation (e.g., shearing forces) at the mucosal surface (183) or noxious stimulation (e.g., stimulant laxatives such as sennosides) releases TABLE 23-1. Slow excitatory postsynaptic potential mimetics 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Acetylcholine (muscarinic/M1) Cholecystokinin (A) Bombesin Calcitonin gene–related peptide Thyrotropin-releasing hormone 5-Hydroxytryptamine [5-HT1P] Norepinephrine Pituitary adenylate cyclase–activating peptide Interleukin-1β (IL-1β) Histamine (H2) Adenosine (A2) Vasoactive intestinal peptide Cerulein Gastrin-releasing peptide Tachykinins (neurokinin-3 (NK3)/NK1) Corticotropin-releasing hormone γ-Aminobutyric acid Motilin Glutamate (group I metabotropic) IL-6 Platelet-activating factor Bradykinin (B2) Corticotropin-releasing factor (CRF1) Proteases (protease-activated receptors 1 [PAR-1],-2, and -4) 25. ATP (P2Y1) 26. Prostaglandin E

650 / CHAPTER 23 the 5-HT, which may then reach receptors on AH neuronal projections in the mucosa, other enteric neural elements, and spinal and vagal sensory afferents (25,120,181,184). Bornstein and colleagues (185) reviewed five criteria for transmitter function that were fulfilled by substance P: (1) pharmacologic evidence obtained with NK antagonists suggests the intramural release of substance P within intestinal segments; (2) the K+ conductance decrease produced by substance P and the slow EPSP is the same; (3) chymotrypsin, which digests substance P, reduces both the response to the peptide and the slow EPSP; (4) the widespread occurrence of slow EPSPs implies that terminals of the responsible axons synapse with most cell bodies in the ganglion, as is the case for the multipolar AH-Dogiel type II neurons that express substance P; and (5) slow EPSPs can be evoked in myenteric neurons within ganglia that contained substance P but no immunocytochemically demonstrable 5-HT–containing fibers. The evidence suggests that slow EPSPs can be initiated by substance P released from AH neurons or from collateral projections of spinal afferents inside the intestinal wall. Extensive ramifications of substance P–immunoreactive spinal afferents are present in the myenteric and submucosal plexuses, and electrical stimulation of the afferent nerves in the intestinal mesentery, as well as application of capsaicin, evokes slow EPSPs in myenteric neurons (186). In fact, some of the spinal afferent terminals in the intestinal wall might be the mechanoreceptors that monitor the mechanical state of the musculature and function to initiate axon reflexes that underlie the distension-evoked peristaltic reflex in isolated intestinal preparations (187). NK3 and NK1 receptors are implicated as mediators of the action of substance P on enteric neurons (185,188–190). Both NK1 and NK3 receptor subtypes become internalized by endocytosis, and then are recycled back to neuronal surface membrane after their binding with the NK ligand (191,192). Endocytosis of the NK1 receptor is independent of endocytosis of the NK3 and vice versa (192). Quantitative description of increased endocytosis of the NK1 receptor initiated by mechanical stimulation of the small intestinal mucosa in guinea pigs can be extrapolated as a quantitative indicator of the release of substance P evoked by movement of the villi (193). The slow EPSP-like action of 5-HT is related to a serotonergic receptor designated 5-HP1P, so named by M. D. Gershon and coworkers (194,195) based on binding characteristics of 3H-labeled 5-HT. The P is for periphery to distinguish 3 H-5-HT1 receptor binding in the gut from 3H-5-HT1 receptor binding in the brain. The so-called 5-HT1P receptor has not been cloned, and a true molecular characterization of this important receptor subtype remains elusive. Slow EPSP mimetic action of acetylcholine is mediated by the M1 muscarinic receptor subtype (52,196). The action of histamine occurs at the H2 receptor subtype, and proteaseactivated receptors (PARs), PAR-1, -2, and -4, mediate the slow EPSP-like responses evoked by mast cell proteases (122,197,198).

Motilin is a slow EPSP mimetic of particular interest because it is implicated as an endocrine messenger in the initiation of the small intestinal migrating motor complex in the interdigestive period (199). Additional interest emerges from findings that macrolide antibiotics (e.g., erythromycin) might act at motilin receptors to facilitate gastric emptying in disorders such as diabetic gastroparesis (200). Slow EPSP-like actions of motilin are prominent in AH neurons of the gastric antrum (201). Slow Excitatory Postsynaptic Potential Metabotropic Signal Transduction Slowly developing activation and the prolonged time course of slow EPSPs and slow EPSP-like responses to applied mimetics offer clues to the mechanism of signal transduction. A 30-msec experimental “puff ” of a slow EPSP mimetic from a micropressure ejection pipette or electrical stimulation of synaptic inputs for the slow EPSP evokes changes in excitability that last for several minutes (143,202). This happens in experiments where the exposure is limited to the 30-msec duration of the “puff ” by a high flow rate of bathing solution in the tissue chamber. The slow EPSP mimetics act transiently at their localized receptor sites on the outside of the somal membrane, whereas receptor-mediated ionic conductance changes including closure of the Ca2+activated K+ channels in AH neurons and opening of cation channels in S neurons occur globally around the circumference of the cell body. Simultaneous activation for the whole cell suggests involvement of intraneuronal second messenger systems that connect localized surface receptors (i.e., metabotropic receptors) to biochemical processes in the neuronal interior, and ultimately to globally distributed ion channels. Receptor occupancy by messenger substances in these cases stimulate intraneuronal synthesis or release of a second messenger, which, in turn, initiates the neurochemical reactions and molecular conformational changes responsible for transformation of the somal membrane from hypoexcitability to hyperexcitability. Metabotropic Transduction in AH-Type Neurons Most evidence points to receptor-mediated activation of adenylate cyclase, increase of intraneuronal cAMP, and activation of protein kinase A (PKA) as steps in the signal transduction process for the slow EPSP in AH neurons. Forskolin, a substance that directly activates adenylate cyclase, is a useful tool for the study of signal transduction in AH neurons. Application of forskolin increases cAMP in enteric ganglia (203) and mimics the slow EPSP in AH neurons, but it has no effect on excitability in S-type neurons (204). Likewise, other treatments that increase cAMP such as intraneuronal injection of cAMP, application of membrane-permeant analogs of cAMP, and treatment with phosphodiesterase inhibitors each evoke slow EPSP-like behavior in AH neurons (205).

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 651 Exposure of guinea pig myenteric ganglia to 5-HT (206,207) or histamine (208), both of which are slow EPSP mimetics, stimulates cAMP formation. Stimulation of cAMP by 5-HT is mediated by 5-HT1P receptors, and histamine action is mediated by the H2 receptor subtype. This supports the hypothesis that cAMP is an intracellular second messenger in the signal transduction process in AH neurons. Occupancy of receptors for the slow EPSP leads to activation of adenylate cyclase, which, in turn, leads to synthesis of cAMP, phosphorylation of protein kinases and membrane channel proteins (Fig. 23-9), and eventually to the dramatic changes in neuronal excitability and suppression of postspike afterhyperpolarization that occur during the slow EPSP in AH neurons. Suppression of the postspike afterhyperpolarization during slow EPSPs reflects phosphorylation by PKA of the cytoplasmic tail of the Ca2+-activated K+ channels (IK channels) that underlie the postspike hyperpolarization (209). Agents that inhibit the intraneuronal phosphatase, calcineurin, prevent dephosphorylation of the IK channels and prolong suppression of the postspike after hyperpolarizing responses in AH neurons during the slow EPSP (210). G proteins couple receptors for multiple signal substances to adenylate cyclase or phospholipase C, or both, to initiate postreceptor cascades of reactions that culminate in the slow EPSP in AH neurons. Bertrand and Galligan (211) suggest that G protein coupling for the slow EPSP-like action of the

Slow EPSP mimetics Examples: Serotonin (5-HT1P) Substance P (NK-3) Histamine (H2) Proteases (PARS1, 2, 4) Forskolin

AH-Neurons

NK3 receptor agonist, senktide, did not involve a pertussis toxin–sensitive G protein in AH neurons. In contrast, Pan and colleagues (212) report evidence that pertussis toxin–sensitive Gαo coupled the 5-HT1P receptor to the signaling cascade for the slow EPSP-like responses to 5-HT. Strong evidence supports the hypothesis that activation of adenylate cyclase, stimulation of formation of cAMP, and activation of PKA are steps in the signal transduction cascade for the increasedresistance slow EPSP in AH neurons (see Fig. 23-9). Nevertheless, the evidence of Bertrand and Galligan (211) and Pan and colleagues (212) suggests that G protein coupling to phosphatidylcholine phospholipase C (PC-PLC), formation of diacylglycerol, and activation of protein kinase C (PKC) might be components of the transduction mechanism. If it is indeed operational, the PKC transduction pathway undoubtedly depends on PC-PLC rather than phosphatidylinositolspecific PLC (PI-PLC), because liberation of inositol-1,4, -5-trisphosphate (IP3) by PI-PLC would be expected to release Ca2+ from intraneuronal stores, which, in turn, would open Ca2+-gated K+ channels, decrease input resistance, and oppose depolarization of the membrane potential. Pan and colleagues (212) postulate for the cascade mediated by Gαo→PC-PLC that activation of PKC phosphorylates adenylate cyclase, thereby stimulating formation of cAMP and activation of PKA. In this scenario, the receptors do not couple directly to adenylate cyclase through Gαs protein, as in other cell types.

Slow EPSP mimetics Examples: Proteases (PARS1, 2, 4) Eicosanoids ATP (P2Y1)

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FIG. 23-9. Postulated metabotropic signal transduction cascades for slow excitatory postsynaptic potentials (EPSPs) and paracrine mediators, which evoke slowly activating depolarizing responses associated with increased input resistance (i.e., channel closures and decreased conductance) in AH-type neurons and slowly activating depolarizing responses associated with decreased input resistance (i.e., channel openings and increased conductance). 5-HT, 5-hydroxytryptamine; ATP, adenosine triphosphate; cyclic AMP, cyclic adenosine 3′,5′-phosphate; IP3, inositol-1,4,-5-trisphosphate; NK-3, neurokinin-3; PAR, protease-activated receptor; PIP2, phosphatidylinositol 4,5-bisphosphate. (Reproduced from Wood JD, Kirchgessner A. [282].)

652 / CHAPTER 23 Pan and colleagues (212) identified PKCα and PKCδ as the predominant PKC isoforms in myenteric neurons, whereas Poole and colleagues (213) found PKCα localized to enteric glial cells and PKCδ in intestinal smooth muscle. Poole and colleagues (213) report that the PKC isoforms γ, η, θ, λ, and ε are those expressed in guinea pig enteric neurons. Stimulation of PKC with a phorbol ester mimics most, but not all, perimeters of slow synaptic excitation in AH neurons (214). Kawai and colleagues (214) report that, unlike stimulusevoked slow EPSPs and stimulation of adenylate cyclase by forskolin, the Ca2+ component of the action potential was unaffected by stimulation of PKC. A consensus sequence with serine residues where phosphorylation by PKC or PKA might occur is present in the cytoplasmic tail of the IK channel in AH neurons. Three PKC isoforms (γ, η, and θ) are expressed in AH-type multipolar Dogiel II neurons that express IR for calbindin, and not in S-type uniaxonal neurons (213). These particular isoforms are dependent on diacylglycerol for activation, but they are not dependent on increased Ca2+ in concert with diacylglycerol for activation (215), as would be expected if they are involved in signal transduction in AH neurons, where free Ca2+ concentrations decrease during slow synaptic excitation. Adenosine A1 Receptors Tonic release and accumulation of endogenous adenosine is a property of the guinea pig intestinal preparations that are commonly used in microelectrode studies of electrophysiologic behavior and synaptic transmission in enteric neurons (216). Whether released spontaneously or applied experimentally, adenosine acts to suppress initiation of the increased resistance slow EPSP in AH neurons and to abort the responses once they are evoked (136,217,218). Suppression by adenosine of an EPSP in progress is evidence that adenosine acts at postsynaptic receptors. Adenosine or selective adenosine A1 receptor agonists suppress slow EPSPs and the slow EPSP-like actions of forskolin, histamine, bombesin, gastrin-releasing peptide, VIP, or CCK, but they do not suppress slow EPSP-like responses to 5-HT, calcitonin gene–related peptide, or substance P in AH neurons (219). Slow EPSP-like responses to blockade of Ca2+ entry, intraneuronal injection of cAMP, or application of membranepermeant analogs of cAMP is unaffected by adenosine. Lack of suppression of effects of increase of intraneuronal cAMP suggests that the action of adenosine is direct inhibition of adenylate cyclase. Blockade of the inhibitory action of adenosine by preincubation with pertussis toxin suggests that Gαi protein links the inhibitory A1 adenosine receptors to adenylate cyclase in AH neurons (220). The ionic mechanisms responsible for the increasedresistance slow EPSPs in AH neurons and for the slow EPSP-like actions of the large number of substances that mimic the EPSPs appear to be the same. This raises the question of how 5-HT, substance P, and calcitonin gene–related peptide evoke the same kind of response as forskolin, VIP, CCK, pituitary adenylate cyclase–activating peptide, and

histamine, and yet unlike the latter, are insensitive to the inhibitory action of adenosine A1 receptor agonists in the same neurons. Exposure to adenosine in the culture medium suppresses stimulation of formation of cAMP by histamine, but not by 5-HT or substance P in dissociated myenteric ganglia (221). A possible explanation is that the postreceptor signal transduction mechanism for 5-HT, calcitonin gene–related peptide, and substance P might not involve the PKA pathway. Findings of Pan and colleagues (212) that inhibition of PKA suppressed the slow EPSP-like responses to 5-HT and reports that 5-HT stimulates production of cAMP in myenteric ganglia (206,207) argue against a hypothesis that the PKA pathway is not involved. The general conclusion is that an unequivocal explanation for the differential action of adenosine A1 receptor activation on responses to slow EPSP mimetics currently is unavailable. Suppression of Resting Ca2+ Influx in AH-Type Neurons The slow EPSP-like excitation, which results from blocking resting influx of Ca2+, adds insight into mechanisms underlying slow EPSPs in AH neurons. Treatments that block Ca2+ channels evoke responses that mimic all aspects of slow EPSPs in AH- but not S-type neurons (127,222). Exposure to solutions with increased Mg2+ and depleted Ca2+ or to multivalent cations that impede transmembrane influx of Ca2+ and suppress Ca2+-dependent processes (e.g., Mn2+, Cd2+, or La2+) results in slowly activating membrane depolarization associated with increased input resistance, suppression of postspike hyperpolarizing potentials, and enhanced membrane excitability, which are reflected by repetitive spike discharge during injection of depolarizing current pulses and anodal-break excitation at the offset of hyperpolarizing current pulses. These effects of blockade of Ca2+ suggest that part of the downstream effects of activation of the variety of metabotropic receptors that lead to the EPSPs or EPSP-like responses to mimetic agents is suppression of resting Ca2+ influx, reduction of free intraneuronal Ca2+, and closure of Ca2+-gated K+ channels. Metabotropic Transduction in S-Type Neurons Characteristics of slow EPSPs in enteric neurons with S-type electrophysiologic behavior and uniaxonal morphology differ from the slow EPSPs in AH neurons. The EPSPs in both types of neurons are characterized by slowly activating depolarization of the membrane potential. Slow EPSPs in S-type neurons are distinguished from those in AH neurons by association of decreased input resistance (i.e., increased ionic conductance) with the depolarizing responses (Fig. 23-10A). When a putative signal substance evokes a response in an AH neuron, the depolarizing response is associated with an increase in input resistance and a reversal potential approaching the K+ equilibrium potential of −90 mV (see Fig. 23-10B). Exposure of S-type neurons to the same signal substance evokes depolarizing responses

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 653 +5 mV 0 mV

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FIG. 23-10. Paracrine-like exposure to the mast cell mediator, trypsin, illustrates how different ionic mechanisms underlie slow EPSP-like excitation when evoked by the same mediator in S- and AH-type neurons. (A) Micropressure application of 10-µM trypsin evoked slowly activating membrane depolarization associated with decreased input resistance (not shown) and augmented excitability in a uniaxonal S-type neuron (inset) in the myenteric plexus. The amplitude of each evoked depolarizing response decreased as the resting membrane potential was current-clamped in the depolarizing direction starting at −79 mV and continuing in steps that stopped at 5 mV. Data obtained from five S-type neurons in which the depolarizing responses to trypsin were associated with decreased input resistance extrapolate to a reversal potential near 0 mV. (B) Micropressure application of 10-µM trypsin evoked slowly activating membrane depolarization associated with increased input resistance (not shown) in a multipolar AH-type neuron (inset) in the myenteric plexus. The amplitude of each evoked depolarizing response decreased as the resting membrane potential was current-clamped in the hyperpolarizing direction starting at −40 mV and continuing in steps that stopped at −100 mV. Data obtained from five AH-type neurons in which the depolarizing responses to trypsin were associated with increased input resistance extrapolate to a reversal potential near −90 mV. (Reproduced from Wood JD, Kirchgessner A. [282].)

associated with decreased input resistance and reversal potentials in the range of −20 to 0 mV (see Fig. 23-10A). The available evidence suggests that metabotropic signal transduction for the decreased-resistance slow EPSPs in S neurons differs from the transduction cascade responsible for the increased-resistance slow EPSP in AH neurons (see Fig. 23-9). Whereas the transduction mechanism in AH neurons is dependent on activation of adenylate cyclase and second messenger function of cAMP, the transduction mechanism in S-type uniaxonal neurons involves activation of PLC and a Ca2+-calmodulin second messenger system (13,122,179,223–226). Receptors for the slow EPSP in S neurons are G protein coupled to activation of PI-PLC. PI-PLC catalyzes the formation of IP3 and diacylglycerol. Once released, IP3 acts as a second messenger to release Ca2+ from intraneuronal membrane stores. Binding of the released Ca2+ to calmodulin activates calmodulin kinases II (CaMKII) and IV. Diacylglycerol, together with Ca2+ and anionic phospholipids, activates conventional isoforms of

PKC (i.e., α, β I, β II, and γ isoforms). PKC phosphorylates cation conductance channels that open, when phosphorylated, to increase cationic conductance, thereby depolarizing the membrane potential. Limited evidence suggests that the cationic conductance channels might be TRP-6 channels (180). Opening of the cationic conductance channels accounts for the decreased input resistance and reversal potential near zero that are observed while recording with microelectrodes during the depolarization phase of the EPSP (see Fig. 23-10A). Termination of the EPSP results from activation of the intraneuronal phosphatase, calcineurin, which catalyzes dephosphorylation of the cationic channels (225,226). Pharmacologic evidence, obtained for decreased-resistance slow EPSPs in S-type neurons and for mimetics of the EPSPs, supports the hypothesis that the signal transduction cascade for the decreased-resistance slow EPSP is G protein coupling of multiple kinds of receptors to a PI-PLC transduction pathway. Purinergic P2Y1 receptors initiate

654 / CHAPTER 23 increased-resistance slow EPSP-like responses in AH neurons and decreased-resistance slow EPSP in S neurons. Exposure to ATP likewise mimics either increased- or decreased-resistance slow EPSP-like responses, depending on whether the neuron belongs to the AH- or S-type (13). Application of trypsin, which is a PAR agonist, also evokes depolarizing responses that mimic decreased-resistance slow EPSPs in S neurons and increased-resistance slow EPSPs in AH neurons (122,198). Inhibition of PI-PLC with U73122 or blockade of intraneuronal IP3 by 2-APB suppresses both the stimulus-evoked slow EPSPs and the slow EPSP-like actions of ATP and the PAR agonist trypsin in uniaxonal S-type neurons (13,122). The calmodulin inhibitor W-7 abolishes stimulus-evoked slow EPSPs in S-type uniaxonal neurons in both the myenteric and submucosal plexuses. Inhibition of CaMKIIa with KN-62 also suppresses the amplitude of the depolarizing responses of slow EPSPs that are associated with decreased input resistance in S-type neurons (225,226). IR for CaMKIIa is expressed exclusively in uniaxonal enteric neurons with S-type electrophysiologic behavior in the guinea pig. Most of the submucosal neurons that express CaMKIIa-IR in the guinea pig, rat, and human also express VIP-IR, which is a marker for secretomotor neurons (226). Significance of Slow Excitatory Postsynaptic Potentials in the Integrated System The functional significance of slow EPSPs in AH neurons is twofold. First is an increased probability of impulse discharge in the AH neuron that is then transformed into excitation or inhibition at either the next order neuron or an effector, such as the musculature or secretory epithelium. Intestinal peristaltic propulsion, for example, requires sustained discharge by neuronal elements in the microcircuits to account for the sustained neural drive of several seconds’ duration that occurs in the musculature. The behavior of AH neurons during slow EPSPs fits the requirements for a neuronal element of which the functional significance in the circuit would be production of either prolonged excitation or inhibition of the intestinal circular or longitudinal muscle layers or a prolonged glandular secretory response. Application of the putative neurotransmitters for the slow EPSP stimulates the release of the neuromuscular or neuroepithelial neurotransmitters, acetylcholine, tachykinins, VIP, and nitric oxide, from neurons in myenteric and submucosal plexuses of intestinal segments in vivo and in vitro (227–229). This is consistent with the hypothesis that AH neurons are interneurons in feed-forward synaptic circuits that supply coordinated synaptic drive to the excitatory and inhibitory motor innervation of the musculature and excitatory drive to the secretory epithelium (see Fig. 23-6). The extensive ramifications of the processes of the AH-Dogiel type II neurons to innervate large numbers of neurons in the same and neighboring ganglia (see Fig. 23-3) and the localization of substance P and calcitonin gene–related peptide in these neurons (17,189,230) are consistent with this function.

Inhibitory Postsynaptic Potentials Slow IPSPs are hyperpolarizing synaptic potentials found in both myenteric and submucosal ganglion cell somas of the small and large intestines and in myenteric neurons of the gastric antrum (143,169,231–233). IPSPs activate slowly and continue for several seconds after termination of the stimulation (see Fig. 23-8B). Slow IPSPs are hyperpolarizing responses associated with decreased input resistance and suppression of excitability. Reduction in the membrane resistance and excitability, together with membrane hyperpolarization, decreases the probability of action potential discharge that might occur spontaneously or in response to excitatory synaptic inputs or to inbound action potentials in neurites of multipolar AH-Dogiel type II neurons. IPSPs are more readily demonstrated in the submucosal than in the myenteric plexus of in vitro preparations. IPSPs are found readily in submucosal secretomotor neurons, where they have much sharper activation kinetics than in myenteric neurons. Earlier studies found slow IPSPs in less than 10% of small intestinal myenteric neurons (143,234), whereas more than 80% of submucosal neurons show IPSPs in response to electrical stimulation (231). Interaction with slow EPSPs counteracts the inhibitory inputs and is a factor accounting for the low incidence of IPSPs in myenteric plexus studies. Experimental stimulation of interganglionic connectives activates both excitatory and inhibitory inputs, and the excitatory inputs predominate to a variable degree. Selective blockade of slow synaptic inputs by adenosine A1 receptor agonists uncovers and enhances stimulus-evoked slow IPSPs in myenteric AH-type neurons (216). Likewise, blockade of stimulusevoked slow EPSPs with selective NK1 or NK3 tachykinergic antagonists uncovers slow IPSPs that are mediated by the 5-HT1A receptor subtype in AH neurons (52,236). Ionic Mechanisms for Slow Inhibitory Postsynaptic Potentials The decrease in input resistance observed during slow synaptic inhibition reflects increased conductance in K+ channels (231,237–239). Of the variety of potassium channels found in the membranes of enteric neurons, inwardly rectifying potassium channels are the ones opened by a variety of inhibitory neurotransmitters to account for the increased conductance and hyperpolarization of the membrane potential during the IPSP (237,238). The Ca2+-activated potassium channels that both contribute to the resting membrane potential and account for postspike hyperpolarization are not involved in the generation of the slow IPSPs (238–240). Pertussis toxin– sensitive GTP-binding proteins are involved in direct coupling of the receptors to the potassium channels that underlie slow IPSPs in submucosal uniaxonal S-type neurons (13,240,241). Mediators of Slow Inhibitory Postsynaptic Potentials Several putative neurotransmitters and paracrine signal substances evoke slow IPSP-like responses when applied

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 655 experimentally to enteric neurons. Some of these substances are peptides, others are purinergic compounds, and another important one is norepinephrine (Table 23-2). Receptors for two or more of these substances may be localized to the cell body of the same neuron. Opiate/Opioid Receptors Opiates and opioid compounds (e.g., enkephalins, dynorphin, and morphine) all mimic the IPSPs when applied to enteric neurons. Actions of these substances are mediated by specific subtypes of opiate receptors and are limited to subpopulations of neurons. Opiate receptors of the µ subtype predominate on myenteric neurons, whereas the receptors on submucosal neurons are the δ-opioid subtype (242). Morphine addiction and effects of withdrawal in enteric neurons is observed as high-frequency discharge of impulses on the addition of the opiate receptor antagonist, naloxone, during chronic exposure to morphine (243,244). IPSP-like actions of nociceptin involve an “orphan” opioid receptor-like (ORL1) receptor that is distinct from typical naloxone-sensitive opioid receptors (245). Norepinephrine, Galanin, Somatostatin, and Purinergic Mediators Norepinephrine binds to α2a-adrenoceptors to evoke IPSPs. Noradrenergic IPSPs occur primarily in secretomotor neurons of the intestinal submucosal plexus (13,232,239). The noradrenergic inputs come from postganglionic projections from sympathetic prevertebral ganglia that are involved in inhibition of secretomotor activity. Galanin is a 29-amino-acid polypeptide that mimics slow IPSPs by opening K+ channels in almost all of the neurons of the intestinal myenteric and submucosal plexuses (246–249). CCK does this also, but the effect is seen only in a subpopulation of 10% to 15% of myenteric neurons in the small intestine or gastric antrum (250–252). Application of adenosine, ATP, or other purinergic analogs simulates the slow IPSP in intestinal myenteric neurons (136). This is seen in nearly all AH neurons and results from the suppression of adenylate cyclase activity and reduction in intraneuronal levels of cAMP (221). Both the degradative enzyme, adenosine deaminase, and a selective adenosine A1 TABLE 23-2. Slow inhibitory postsynaptic potential mimetics 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Acetylcholine 5-Hydroxytryptamine (5-HT1A) Neurotensin Somatostatin Adenosine (A1) Nociceptin Opioid peptides Norepinephrine (α2) Cholecystokinin Adenosine triphosphate (ATP) Galanin (GAL1)

receptor antagonist potentiate slow EPSPs in vitro (216). Potentiation of the slow EPSPs reflects reduction of ongoing inhibitory action of endogenously released adenosine and its accumulation in the intestinal wall. Somatostatin is implicated as the endogenous inhibitory transmitter responsible for the intrinsic inhibitory inputs to submucosal ganglion cells (see Fig. 23-5). Hirst and Silinsky (253) describe IPSPs in the submucosal plexus that persisted after sympathectomy, and therefore could not be attributed to activation of norepinephrine release from sympathetic postganglionic fibers. This IPSP, unlike the noradrenergic IPSPs, requires repetitive stimulation and occurs with a more slowly developing time course than noradrenergic IPSPs (254). Somatostatin-containing nerve terminals and the nonadrenergic IPSP disappear in parallel after destruction of the myenteric plexus and its projections to the submucosal plexus (255). Significance of Slow Inhibitory Postsynaptic Potentials in the Integrated System A reduction in the membrane resistance and excitability of the somal membranes, together with membrane hyperpolarization during the slow IPSPs, decrease the probability of action potential discharge. The probability that the somal membrane will be fired by spikes invading the soma electrotonically from one or the other of its neurites or during fast excitatory synaptic input is reduced during slow IPSPs (see Figs. 23-4 and 23-6). This influence is the inverse of the slow EPSP and acts to close the gate for transfer of spike information across the multipolar soma of AH neurons (see Fig. 23-4E). Slow synaptic inhibition probably functions to terminate the excitatory state of slow synaptic excitation and reestablish the low-excitability state in the ganglion cell soma of AH neurons. In the intact animal, there may also be inhibitory substances of endocrine or paracrine origin that function in particular situations to lock the somal membranes in a low-excitability state.

Presynaptic Facilitation Presynaptic facilitation refers to enhancement of synaptic transmission, which results from actions of chemical signal substances or drugs at neurotransmitter release sites on enteric axons. This is known to occur at fast excitatory synapses in the myenteric plexus of the small intestine and gastric antrum and at noradrenergic inhibitory synapses in the submucosal plexus. CCK mediates presynaptic facilitation in gallbladder ganglia (256). Presynaptic facilitation is evident as an increase in amplitude of fast EPSPs at nicotinic synapses, where it reflects enhanced release of acetylcholine from axonal release sites. At noradrenergic inhibitory synapses in the submucosal plexus, presynaptic facilitation appears as enhancement of the hyperpolarizing responses to stimulation of sympathetic postganglionic fibers and is associated with increase of cAMP in the sympathetic nerve terminals (233).

656 / CHAPTER 23 Drugs that facilitate the release of acetylcholine at nicotinic synapses in the ENS are efficacious in treatment of certain gastrointestinal disorders. Cisapride (Prepulsid) and tegaserod (Zelnorm) are examples of therapeutic agents used to treat disorders of gastrointestinal transit and constipation-predominant IBS that act in this way (257–259). Cisapride acts to suppress opening of K+ channels, which slows repolarization of the presynaptic terminal during an action potential, thereby enhancing the release of acetylcholine (260). Side effects of the action of cisapride on K+ channels in cardiac muscle were cardiac dysrhythmias that restricted the use of the drug for gastrointestinal propulsive disorders (261). Tegaserod acts as a partial agonist at presynaptic 5-HT4 receptors to facilitate the release of acetylcholine at nicotinic synapses in the ENS (262).

Presynaptic Inhibition Presynaptic inhibition refers to mechanisms that suppress release of neurotransmitters from axon terminals or varicosities. It involves binding of chemical messengers to inhibitory receptors at transmitter release sites on the axon. Presynaptic inhibition is a significant synaptic event within the enteric microcircuits of the gastric corpus and antrum, as well as the small and large intestines and rectum of the guinea pig (150,161,168,169,263). It is a mechanism that prevents runaway excitation in feed-forward synaptic circuits (see Fig. 23-6; see also AH-Type Neurons earlier in this chapter). Presynaptic inhibitory receptors are found at fast and slow excitatory synapses, at inhibitory synapses, and at neuromuscular junctions. Presynaptic inhibition in many cases involves axo-axonal transmission where release of a neurotransmitter from one axon acts at receptors on another axon to suppress release of transmitter from the second axon. Presynaptic inhibition also takes the form of autoinhibition and can occur at both neural synapses and neuroeffector junctions. Autoinhibition occurs when the transmitter released from the same enteric neuron accumulates in the vicinity of the release site and activates presynaptic inhibitory receptors to suppress further release. It functions in this way as a negative feedback mechanism that automatically regulates the concentration of neurotransmitter within the synaptic or junctional space. Substances of nonneural origin, released in a paracrine or endocrine manner into the milieu surrounding the synapse, are known to act presynaptically to suppress neurotransmission. Mast and enteroendocrine cells are sources of nonneuronal messages to presynaptic inhibitory receptors in the ENS (123).

is released from sympathetic postganglionic nerve terminals in the myenteric and submucosal plexus. Sympathetic postganglionic fibers heavily innervate the ENS. Norepinephrine Norepinephrine acts at presynaptic α2a receptors to suppress transmission at both slow and fast excitatory synapses and at excitatory neuroeffector junctions (239,264). The presynaptic inhibitory actions of norepinephrine are significant because norepinephrine is the neurotransmitter released at the synaptic interface between postganglionic sympathetic axons and the ENS (265). Electrical stimulation of sympathetic postganglionic fibers in the periarterial mesenteric nerves suppresses fast EPSPs in myenteric ganglion cells without altering the responses to exogenously applied acetylcholine, which fulfills the criteria for presynaptic inhibition (152,266). Norepinephrine also fulfills the criteria for a presynaptic action by suppressing stimulus-evoked slow EPSPs without effect on the postsynaptic actions of substance P, serotonin, or acetylcholine (264). As expected for a presynaptic action, norepinephrine does not abort a slow EPSP in progress, but does block the ability to evoke subsequent EPSPs (264). Norepinephrine reduces release of 5-HT and substance P from isolated intestinal segments, which is consistent with presynaptic suppression of slow synaptic excitation (267,268). Axo-axonal synapses with vesicular profiles characteristic of noradrenergic synapses are found at the ultrastructural level in the guinea pig myenteric plexus (269). Observations of inhibitory effects of norepinephrine on acetylcholine release from intestinal segments in vitro add to the evidence for its presynaptic action (270–272). Significance of Sympathetic Noradrenergic Presynaptic Inhibition Sympathetic nervous transmission to the gastrointestinal tract increases during physical exertion, environmental stress, and psychogenic stress. Increased sympathetic activity acts to shunt blood from the splanchnic to the systemic circulation. This is significant because as much as 25% of the cardiac output can be perfusing the splanchnic circulation. Shunting of blood from the splanchnic circulation occurs coincident with suppression of digestive functions including motility and secretion. The sympathetic presynaptic inhibitory action of norepinephrine suppresses intestinal motility by preventing synaptic transmission in the microcircuits that initiate and control motility. Noradrenergic inhibitory synapses on secretomotor neurons “shuts down” secretion in the face of reduced intestinal blood flow.

Mediators of Presynaptic Inhibition Several messenger substances found in neurons, enteroendocrine cells, or immune cells of the bowel produce presynaptic inhibition when applied experimentally to enteric synapses. Important among these is norepinephrine, which

Immune/Inflammatory Mediators Histamine, IL-1β, IL-6, and platelet-activating factor receive attention because of their roles in neuroimmune communication between mast cells and the ENS program circuits.

CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 657 Histamine, like other listed immune/inflammatory messengers, acts at presynaptic receptors on cholinergic axons to suppress fast EPSPs at nicotinic synapses and at sympathetic nerve terminals (see Fig. 23-5) to suppress the release of norepinephrine in the ENS microcircuits (51,273,274). This action of histamine is blocked by selective histamine H3 receptor antagonists and mimicked by selective histamine H3 agonists. Evidence indicates that presynaptic action of histamine is at the H3 receptor subtype (51,275). Histamine released during antigenic degranulation of intestinal mast cells (e.g., food allergies or parasitic infections) acts in a similar manner (123,171,172). 5-Hydroxytryptamine Application of 5-HT to enteric synapses reduces the amplitude of the fast EPSPs without effects on nicotinic postsynaptic responses to acetylcholine, thereby fulfilling criteria for serotonergic presynaptic inhibition (263). The receptor for the presynaptic inhibitory action is not unequivocally identified, but it fits the profile of the 5-HT1 receptor in the gastric antrum and small intestine (165,276,277) or the putative 5-HT4 receptor subtype in the colon (57). Acetylcholine The presynaptic inhibitory action of acetylcholine on nicotinic fast EPSPs occurs at muscarinic receptors in the myenteric and submucosal plexuses of the small and large intestines (52,150) and at myenteric synapses in the gastric antrum (169). The affinity of the presynaptic receptors for selective agonists and antagonists is suggestive of the M2 class of muscarinic receptors (52). The presynaptic muscarinic receptors at fast nicotinic synapses are components of autoinhibitory mechanisms that function in negative feedback regulation of the amount of acetylcholine released by the arrival of the action potential at the release site. Inhibition of acetylcholine esterase by drugs such as eserine results in suppression of stimulusevoked fast EPSPs, and this effect is reversed by application of atropine. In this situation, inhibition of the choline esterase permits accumulation of acetylcholine, which feeds back on the presynaptic muscarinic receptors to suppress further release of acetylcholine. Atropine blocks this presynaptic influence of the esterase and restores the EPSP (150). Pancreatic Polypeptide, Peptide YY, and Neuropeptide Y Members of the pancreatic polypeptide family of messenger peptides (e.g., neuropeptide Y [NPY], peptide YY, pancreatic polypeptide) are reported to act presynaptically to suppress transmission at nicotinic synapses in the microcircuits of the stomach (278). The presynaptic inhibitory action of NPY is significant because it is colocalized with norepinephrine in sympathetic postganglionic axons, as well as being found in enteric neurons. This suggests that both the CNS and the

ENS may use the long-lasting inhibitory action of this mediator to selectively inactivate synapses within the microcircuits. Presynaptic NPY receptors appear to be present on vagal efferent fibers in the gastric corpus and participate in circuit functions by which the ENS microcircuitry of the corpus might ignore its vagal input (278). Adenosine Presynaptic inhibition by adenosine occurs at the A1-type of P1 purinoreceptor (216,279,280). This occurs at fast nicotinic synapses, slow excitatory synapses, and noradrenergic inhibitory synapses. Suppression of fast EPSPs occurs in the gastric and intestinal microcircuits, whereas presynaptic inhibition of IPSPs is seen in the intestinal submucosal plexus. Galanin Presynaptic inhibition by galanin in the submucosal plexus of guinea pig small intestine occurs at the galanin-1 (GAL1) receptor subtype, not at GAL2 or GAL3 (281). Exposure to galanin suppresses both noradrenergic slow IPSPs and fast nicotinic EPSPs through action at presynaptic inhibitory receptors. Suppression of slow EPSPs by galanin involves both presynaptic and postsynaptic inhibitory actions.

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CELLULAR NEUROPHYSIOLOGY OF ENTERIC NEURONS / 663 265. Wood JD. Neurotransmission at the interface of sympathetic and enteric divisions of the autonomic nervous system. Chin J Physiol 1999;42:201–210. 266. Hirst GD, McKirdy HC. Presynaptic inhibition at mammalian peripheral synapse? Nature (Lond) 1974;250:430–431. 267. Bartho L, Holzer P, Lembeck F. Sympathetic control of substance P releasing enteric neurones in the guinea pig ileum. Neurosci Lett 1983;38:291–296. 268. Jonakait GM, Tamir H, Gintzler AR, Gershon MD. Release of [3H] serotonin and its binding protein from enteric neurons. Brain Res 1979;174:55–69. 269. Manber L, Gershon MD. A reciprocal adrenergic-cholinergic axoaxonic synapse in the mammalian gut. Am J Physiol 1979;5:E738–E745. 270. Del Tacca M, Soldani G, Selli M, Crema A. Action of catecholamines on release of acetylcholine from human taenia coli. Eur J Pharmacol 1970;9:80–84. 271. Knoll J, Vizi ES. Presynaptic inhibition of acetylcholine release by endogenous and exogenous noradrenaline at high rate of stimulation. Br J Pharmacol 1970;40:400–412. 272. Paton WDM, Vizi ES. The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip. Br J Pharmacol 1969;35:10–28. 273. Wood JD. Histamine signals in enteric neuroimmune interactions. Ann N Y Acad Sci 1991;664:275–283. 274. Wood JD. Physiological and pathophysiological paracrine functions of intestinal mast cells in enteric neuroimmune signaling. In: Singer MF,

275. 276. 277. 278. 279. 280. 281. 282.

Ziegler R, Rohr G, eds. Gastrointestinal tract and endocrine system. London: Kluwer Academic Press, 1995;254–263. Tamura K, Palmer JM, Wood JD. Presynaptic inhibition produced by histamine at nicotinic synapses in enteric ganglia. Neuroscience 1987; 25:171–179. Galligan JJ, Surprenant A, Tonini M, North RA. Differential localization of 5-HT1 receptors on myenteric and submucosal neurons. Am J Physiol 1988;255:G603–G611. Surprenant A, Crist J. Electrophysiological characterization of functionally distinct 5-hydroxytryptamine receptors on guinea-pig submucous plexus. Neuroscience 1988;24:283–295. Schemann M, Tamura K. Presynaptic inhibitory effects of the peptides NPY, PYY, and PP on nicotinic EPSPs in guinea-pig gastric myenteric neurones. J Physiol (Lond) 1992;451:79–89. Barajas-Lopez C, Surprenant A, North RA. Adenosine A1 and A2 receptors mediate presynaptic inhibition and postsynaptic excitation in guinea pig submucosal neurons. J Pharmacol Exp Ther 1991;258:490–495. Christofi FL, Tack J, Wood JD. Suppression of nicotinic synaptic transmission by adenosine in myenteric ganglia of the guinea-pig gastric antrum. Eur J Pharmacol 1992;216:17–22. Liu S, Hu H-Z, Gao C, Gao N, Xia Y, Wood JD. Actions of galanin on neurotransmission in the submucous plexus of guinea pig small intestine. Eur J Pharmacol 2003;471:49–58. Wood JD, Kirchgessner A. Slow excitatory metabotropic signal transmission in the enteric nervous system. J Neurogasterol Mot 2004; 16(suppl 1):1–10.

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CHAPTER

24

Integrative Functions of the Enteric Nervous System Jackie D. Wood Integrated Control of the Stomach, 666 Brainstem/Enteric Nervous System: Cooperative Integration of Gastric Function, 666 The Antral Pump, 667 Neural Control of the Gastric Reservoir, 668 Gastric Emptying, 669 Integrated Control of the Small and Large Intestines, 669 Musculature, 670 Peristaltic Propulsion, 670 Peristaltic Retropulsion, 673 Physiologic Ileus, 673 Intestinal Power Propulsion, 674 Plasticity in the Enteric Nervous System, 674 Gastric Reflexes, 674

Secretomotor Control, 674 Hirschsprung’s Disease, 674 Microcircuit Level, 675 Integrated Control of the Anal Canal and Pelvic Floor, 678 Pelvic Floor Musculature, 678 Sensory Innervation, 679 Rectoanal Reflex, 680 Spinal Motor Control, 680 Supraspinal Motor Control, 681 Integrative Mechanisms for Defecation, 681 Epilogue, 682 References, 682

The digestive tract does not work without the integrative functions of the enteric nervous system (ENS). Normal functioning of the neural networks of the ENS is necessary for effective motility, secretion, and blood flow, and coordination of these functions into organized patterns of behavior at the level of the integrated organ system. The ENS, unlike other divisions of the autonomic nervous system, is known to be an independent integrative nervous system that shares most of the properties of the central nervous system (CNS). Its function is to integrate contraction of the musculature, glandular secretion and intramural blood flow into organized patterns of digestive behavior. The neural networks of the ENS may either evoke contractions of the gut musculature or inhibit contractile activity as required for the organization of specific patterns of intestinal motility.

The ENS also organizes absorption from and secretion into the intestinal lumen and integrates these functions with motility. Furthermore, intramural blood flow and the distribution of flow to the musculature, mucosa, and lamina propria are controlled by the integrative microcircuitry of the ENS. As might be expected, malfunctions of integrative ENS control of the effector systems of the gut are increasingly recognized as underlying factors in gastrointestinal disorders, especially in the functional gastrointestinal disorders, and are therefore a target for drug therapy (1–3). The musculature, mucosal secretory glands and blood and lymphatic vasculature are the effector systems of the gut. Moment-to-moment behavior in any of the specialized compartments of the gut (e.g., stomach, small intestine, and large intestine) reflects neurally integrated activity of the effector systems. The ENS not only controls the activity of each effector, it coordinates the activity of each to achieve organized behavior of the whole organ. Coordinated activity is achieved through the timing of excitatory or inhibitory neural inputs, or both, to each effector system individually. Coordination of mucosal secretion and muscle contractile activity in a timed sequence during intestinal propulsion of

J. D. Wood: Departments of Physiology and Biology and Internal Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

665

666 / CHAPTER 24 luminal contents is an example of ENS integrative function. Secretion of H2O, electrolytes, and mucus occurs first, followed by propulsive contractions in the musculature (4). A simultaneous increase in neurally controlled blood flow to the secretory glands supports the enhanced secretory response. The ENS is a “minibrain” with a library of programs for multiple patterns of small or large intestinal behavior (Fig. 24-1). A specific program determines mixing pattern of motor behavior in the postprandial state, another establishes the pattern of interdigestive intestinal motility that characterizes the fasting state (i.e., migrating motor complex), and still another acts in defense against infectious invaders, enterotoxins, or food allergins (see Chapter 38). In general, the gut does not work without the ENS. Sphincters do not relax, organized propulsion does not occur, and liquidity of the luminal contents is disordered.

INTEGRATED CONTROL OF THE STOMACH The named anatomic regions of the stomach (i.e., fundus, corpus, antrum, and pylorus), which are under integrative neural control, are not the same as the functionally defined regions. Functionally, the stomach is divided into a proximal reservoir and distal antral pump on the basis of distinct differences in neurally organized motility between the two regions. The reservoir incorporates the anatomically defined fundus and about one third of the corpus; the antral pump includes the caudal two thirds of the corpus, the antrum, and the pylorus. Differences in motility between the reservoir and antral pump reflect adaptations for different functions. The muscles

of the proximal stomach are specialized for maintaining continuous contractile tone (tonic contraction) and do not contract phasically. Phasic contractions are generated by the musculature of the antral pump. Aboral spread of strong phasic contractions in the region of the antral pump propels the gastric contents toward the gastroduodenal junction. Strong propulsive waves do not occur in the reservoir.

Brainstem/Enteric Nervous System: Cooperative Integration of Gastric Function Unlike in the small and large intestines, the ENS of the stomach lacks sufficient computing capacity for independent control of the minute-to-minute changes in gastric motility required for coordinated ingestive and emptying behavior. The lack of computational capacity in the ENS is compensated by “more intelligent” neural networks in the brainstem that are integrated with the ENS. Cooperative input from the brainstem to the gastric ENS is derived from the dorsal motor nucleus of the vagal nerves (DMVN). Vagal efferent nerve fibers transmit the information from the DMVN to the ENS. Nevertheless, only 10% of the fibers in the vagal nerves are efferent; the other 90% are sensory afferents that transmit from the stomach to the brain. Consequently, the data stream from the brain to the large number of neural elements that form the ENS microcircuits is transmitted by a much small number of efferent vagal fibers (5). Each vagal efferent fiber projects extensively within the gastric myenteric plexus, where synaptic contact is made with the excitatory and inhibitory motor neurons that innervate the gastric musculature (6). Most of the vagal efferents release acetylcholine

Enteric nervous system program library (Small intestine)

(Small intestine)

Postprandial program (mixing pattern)

(Migrating motor complex pattern)

(Small and large intestine)

(Small intestine)

Aboral power propulsion program

Interdigestive program

Programed defense

Emetic program (Oral power propulsion)

(Small and large intestine)

(Large intestine)

Physiological ileus (Contraction absent)

Haustral program (Haustra)

FIG. 24-1. The enteric nervous system (ENS) contains a library of programs for specific patterns of intestinal motor behavior that are adaptive for one or the other of the intestinal digestive states. In the postprandial state, the ENS “runs” the motor program for mixing movements, whereas other programs are inoperative. At the completion of digestion and absorption of the nutrients of a meal, the postprandial program is terminated and the system “calls up” the interdigestive program, which when running programs for motor behavior described as the migrating motor complex and also as the interdigestive housekeeper. The ENS runs defensive programs characterized by powerful propulsive motility when threatening agents (toxins, infective agents, and food allergins) find their way into the intestinal lumen.

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 667 to stimulate nicotinic excitatory receptors on the postsynaptic myenteric neurons. Electrical stimulation of vagal efferents in animal studies evokes a mixture of contraction and relaxation as a consequence of activating parallel pathways to excitatory and inhibitory motor neurons. Contractile responses to vagal stimulation are largely cholinergic and are blocked by drugs that act at muscarinic receptors on the musculature. Vagally evoked relaxation is mediated by release of nitric oxide, ATP, and vasoactive intestinal peptide from the inhibitory motor innervation of the musculature (7). Vagally mediated excitatory or inhibitory gastric reflexes therefore arise from selective activation or suppression of populations of dorsal vagal motor neurons, which project either to inhibitory or excitatory motor neurons in the ENS. Vagovagal Reflexes Sensory afferent information for vagovagal reflex involvement in control of gastric motor function is processed in the brainstem. Spontaneously discharged impulses recorded in efferent vagal fibers reflect ongoing generation of neural activity in the brainstem nuclei (8–10). The spontaneous activity may be generated by DVMN neurons themselves or result from activity in circuitry elsewhere in the brainstem or in higher brain centers that supply excitatory synaptic input to the DVMN neurons. These connections with higher brain centers underlie emotional and behavioral influences on vagal outflow to the gut and, in particular, the effects of stress on gastrointestinal function that involves central mediators, which include thyrotropin-releasing hormone, corticotropin-releasing hormone, and cholecystokinin (see Chapters 28, 29, and 30). Because efferent vagal outflow connects to both excitatory and inhibitory motor neurons in the gastric ENS, the outflow pathways are suggested to be reciprocally controlled such that contraction of the gastric musculature arises from activation of cholinergic pathways and simultaneous inhibition of inhibitory pathways. Accordingly, vagally evoked relaxation of the musculature would involve simultaneous activation of inhibitory pathways and suppression of excitatory pathways. The integrative circuits in the brainstem behave as if they are hardwired for reciprocal control with a complex interplay of inhibitory and excitatory synaptic mechanisms (9,11,12). Some afferent fibers in the sensory vagus connect monosynaptically with the cell bodies of vagal efferent neurons in the DMVN. Nevertheless, most vagal afferents project to second-order neurons in the nucleus tractus solitarius (13), and much of the sensory information from the stomach is processed in polysynaptic circuits in the brainstem. Multiple subnuclei form the nucleus tractus solitarius. The subnucleus gelatinosus and the medial and commissural nuclei are the principal recipients of sensory information from the stomach. A projection from the stomach to the area postrema transmits sensory information that triggers emesis (14). Neurons in the nucleus tractus solitarius project to other nuclei within the brainstem (e.g., DMVN) that are involved in the vagovagal reflex integration of gastric function. Other gastric sensory pathways ascend through the midbrain and

reticular nuclei to innervate higher brain centers, in particular, to the hypothalamic nuclei involved in mechanisms of satiety and regulation of food intake.

The Antral Pump Gastric action potentials determine the duration, strength, and aboral direction of travel of the phasic contractions in the antral pump. Gastric action potentials are initiated by a dominant pacemaker located aboral to the midregion of the corpus. After starting at the pacemaker site, an action potential propagates rapidly around the gastric circumference and triggers a ringlike contraction. The action potential and associated ringlike contraction then travel more slowly until they terminate at the gastroduodenal junction. Gap junctions and electrical syncytial properties of the myogenic gastric musculature account for the propagation of the action potentials from muscle fiber to muscle fiber away from the pacemaker site toward the gastroduodenal junction. The gastric action potential lasts about 5 seconds and has a rising depolarization phase, a plateau phase, and a repolarization falling phase (15). The gastric action potential is responsible for two components of the propulsive contractions in the antral pump. A leading contraction, with a relatively constant amplitude associated with the rising phase of the action potential, and a trailing contraction, of variable amplitude, is associated with the plateau phase (Fig. 24-2) (16). The pacemaker generates action potentials continuously; nevertheless, the action potentials trigger a trailing contraction only when the plateau phase is above threshold voltage. Strength of the trailing contraction increases in direct relation to increases in the amplitude of the plateau potential beyond threshold. The leading contractions produced by the rising phase of the gastric action potential have minimal amplitude that remains constant as they propagate to the pylorus. As the rising phase reaches the terminal antrum and spreads into the pylorus, contraction of the pyloric muscle closes the partition between the stomach and duodenum. The trailing contraction follows the leading contraction. As the trailing contraction approaches the closed pylorus, the gastric contents are forced into an antral compartment with ever-decreasing volume and progressively increasing intraluminal pressure. This results in jetlike retropulsion through the orifice formed by the trailing contraction. Trituration and reduction in particle size occur as the material is forcibly retropulsed through the advancing orifice and back into the gastric reservoir to wait for the next propulsive cycle. Repetition at 3 cycles/min in humans reduces particle size to the 1- to 7-mm range that is necessary before a particle can be emptied into the duodenum. Neural Control of the Antral Pump The action potentials in the gastric reservoir are myogenic (i.e., an inherent property of the muscle) and occur in the absence of release of any neurotransmitters from gastric

668 / CHAPTER 24 Gastric action potential Rising phase

Leading contraction

Plateau phase

A

Trailing contraction

AP

10 sec e

Contractions:

Tim

g lin ai Tr g in ad

Le

e

Tim

AP

AP = Gastric action potential

AP

B

AP

FIG. 24-2. A pacemaker in the midstomach generates gastric action potentials (AP) that evoke ringlike contractions as they propagate to the gastroduodenal junction. (A) Gastric action potentials are characterized by an initial rapidly rising upstroke, followed by a plateau phase, and then a falling phase back to the baseline membrane potential. (B) Integrated neural control of the action potential determines the contractile behavior of the antral pump. The rising phase of the gastric action potential accounts for a leading contraction that propagates toward the pylorus during one propulsive cycle. The plateau phase accounts for the trailing contraction of the cycle. The strength of the leading contraction is relatively constant; the strength of the trailing contraction is variable and increases in direct relation to neurally evoked increases in amplitude of the plateau phase of the action potential. (Modified from Szurszewski [16], by permission.)

enteric motor neurons. However, the myogenic characteristics of the action potential are modulated by motor neurons in the gastric ENS. Neurotransmitters released by the enteric motor neurons determine the amplitude of the plateau phase of the action potential, and thereby control the strength of the contractile event triggered by the plateau phase. Neurotransmitters (e.g., acetylcholine) released from excitatory motor neurons increase the amplitude of the plateau phase and that of the contraction that is initiated by the plateau phase. Inhibitory neurotransmitters (e.g., norepinephrine, vasoactive intestinal peptide, or nitric oxide) decrease the amplitude of the plateau and the strength of its associated contraction. The magnitude of the excitatory or inhibitory actions of neurotransmitters increases with the presence of increasing concentration of the transmitter substance at receptors on the gastric musculature. Progressively higher frequencies of impulse discharge by enteric motor neurons progressively release greater quantities of neurotransmitter. In this way, firing behavior of motor neurons determine, through the actions of their neurotransmitters on the plateau phase, whether the trailing contraction of the propulsive complex of the distal stomach occurs. With sufficient release of transmitter, the plateau exceeds the threshold for contraction. Beyond threshold, the strength of contraction is determined by the amount of neurotransmitter released and present at its receptors on the musculature. Dysrhythmia of electrical and contractile behavior, which is reminiscent of appearance of ectopic excitatory foci and fibrillation in the heart, can occur in pathophysiologic states

of the musculature of the antral pump or its innervation, or both. Antral pump dysrhythmias in humans are associated with epigastric pain, nausea and vomiting, delayed gastric emptying, and gastroparesis (17–19).

Neural Control of the Gastric Reservoir The gastric reservoir performs two primary functions. One function is accommodation for the arrival of a meal without a significant increase in intragastric pressure and intramural tension. Failure of this mechanism can lead to the uncomfortable sensations of bloating, epigastric pain, and nausea. The second reservoir function maintains constant compressive forces on the contents of the reservoir, which press the contents into the 3 cycles/min motor activity of the antral pump. Pharmacologically induced relaxation of the musculature of the gastric reservoir (e.g., by injections of insulin or glucagon) neutralizes this function and suppresses gastric emptying. The musculature of the gastric reservoir is innervated by enteric excitatory and inhibitory motor neurons. Integrative interactions of vagal efferent nerves with intramural enteric neural networks control the firing frequencies of the motor neurons. Changes in the firing frequencies of the motor neurons and coordination of the activity in excitatory and inhibitory motor neurons function to adjust the volume and pressure within the reservoir to the amount of solid or liquid, or both, present while simultaneously maintaining

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 669 compressive forces on the contents. Integrative neural control of this nature continuously adjusts and readjusts the volume and pressure within the reservoir as required during ingestion and emptying of a meal. Increasing the firing frequency of excitatory motor neurons, when coordinated with decreased firing of inhibitory motor neurons, results in increased intramural contractile tension in the reservoir, a decrease in its volume, and an increase in intraluminal pressure. Increasing the firing frequency of inhibitory motor neurons, when coordinated with decreased activity of excitatory motor neurons, results in decreased intramural contractile tone in the reservoir, expansion of its volume, and a decrease in intraluminal pressure. Inhibitory motor neurons in the gastric reservoir express the 5-hydroxytryptamine receptor 1 (5-HT1) subtype for serotonin, which, when activated, stimulates neuronal firing and release of inhibitory neurotransmitters that relax the musculature of the reservoir. This basic discovery led to the therapeutic application of the 5-HT1 receptor agonist, sumatriptan, which proved effective in treatment of dyspeptic symptoms of early postprandial fullness and satiety (20).

which, in turn, increases sensitivity of stretch receptors to distension of the reservoir (21,22). Increased sensitivity is reflected by an abnormal increase in firing frequency in vagal afferents in response to distension, which results in transmission of erroneous information to processing centers in the brain. Decreased compliance of the gastric wall after vagotomy is revealed as increased steepness in the slopes of intragastric pressure–volume curves in studies with balloon distension in the stomach. The loss of adaptive relaxation after vagotomy is associated with a reduced threshold for sensations of fullness and epigastric pain during filling of the gastric reservoir by a meal in humans. Increased sensitivity to gastric distension in these cases is explained by increased stiffness of the wall and consequent sensitization of the mechanoreceptors that sense distension. These effects of vagotomy underscore the importance of sensory detection in the gastric wall and processing of the sensory information in the dorsal vagal complex and help to explain disordered gastric sensations in diseases that have a component of vagus nerve pathology (e.g., autonomic neuropathy in diabetes mellitus or iatrogenic surgical damage).

Gastric Reservoir Relaxation Neurally mediated decreases in tonic contracture of the musculature are responsible for relaxation of the reservoir, which includes increasing wall compliance, increasing the volume and, reducing intraluminal pressure. Three kinds of relaxation are recognized. One kind is called receptive relaxation. Receptive relaxation is initiated by the act of swallowing. It is a reflex triggered by stimulation of mechanoreceptors in the pharynx followed by transmission of the mechanosensory information over sensory afferent fibers to the dorsal vagal complex in the brainstem and subsequent activation of efferent vagal fibers to stimulate inhibitory motor neurons in the gastric ENS. The second kind of relaxation is adaptive relaxation. It is triggered by distension of the gastric reservoir, which normally results from intake of a meal. It is a vagovagal reflex that involves stimulation of stretch receptors in the reservoir wall, transmission of the mechanosensory information over vagal afferents to the dorsal vagal complex, processing of the sensory information, and activation of efferent vagal outflow to inhibitory motor neurons in the gastric ENS. The third kind of neurally integrated reservoir relaxation is feedback relaxation. Feedback relaxation is triggered by the presence of nutrients in the small intestine. This form of reservoir relaxation can involve both local reflex connections between receptors in the small intestine and the gastric ENS, or hormones (e.g., cholecystokinin) that are released from endocrine cells in the small intestine and transported by the blood to stimulate receptors on vagal afferent terminals, which then activates a vagovagal reflex that relaxes the reservoir. Adaptive relaxation is impaired in patients who suffer injury to the vagus nerves during laparoscopic fundoplication surgery for correction of pathologic reflux of gastric contents into the esophagus (19). After a vagotomy, increased tone in the musculature of the reservoir decreases the wall compliance,

Gastric Emptying The orderly delivery of gastric chyme to the duodenum at a rate that does not overload the digestive and absorptive functions of the small intestine is another function that requires neural integration of gastric motility. Integrative neural control compensates for minute-to-minute variations in the volume, composition, and physical state of the duodenal contents by adjusting the rate of delivery into the duodenum. This is necessary because the intraluminal milieu of the small intestine is different from that of the stomach, and undiluted gastric contents have a composition that is poorly tolerated by the duodenum. Neural control of gastric emptying automatically adjusts the delivery of gastric chyme to an optimal rate for the small intestine and guards against overloading the small intestinal mechanisms for the neutralization of acid, dilution to isoosmolality, and enzymatic digestion of the foodstuff. Some of the moment-to-moment neural control of the rate of gastric emptying involves feedback regulation of the gastric reservoir. An example is the powerful actions of lipids in the duodenum to slow gastric emptying. In this case, cholecystokinin released from enteroendocrine cells in the intestinal mucosa acts as a hormone to activate cholecystokinin receptors on vagal gastric afferents, thereby initiating vagovagal reflex relaxation of the reservoir (see Chapter 4).

INTEGRATED CONTROL OF THE SMALL AND LARGE INTESTINES Intestinal motility refers to wall movements or lack thereof in the small and large bowels. Integrated function of the musculature and nervous system is necessary for generation

670 / CHAPTER 24 of the various patterns of motility that reflect operation of the various motor programs stored in the program library of the ENS (see Fig. 24-1). Intestinal motor movements involve the neurally controlled application of forces of muscle contraction to mix and propel material present in the lumen.

Musculature The physiologic properties of the intestinal musculature dictate the kind of neural control required for adequate generation of specialized patterns of motility. The musculature of the stomach and intestines is unitary-type smooth muscle. Unitary-type smooth muscles contract spontaneously in the absence of neural or endocrine influence and contract in response to stretch. There are no structured nerve–muscle junctions like in skeletal muscle and neurotransmitters diffuse over extended distances to influence relatively large numbers of muscle fibers. The muscle fibers in unitary-type smooth muscles are connected to their neighbors by gap junctions (23). Because gap junctions are selectively permeable to ions, they transmit electrical current from muscle fiber to muscle fiber. Electrical connectivity, without cytoplasmic continuity from fiber to fiber, accounts for the electrical syncytial properties of unitary-type smooth muscle. Gap junctions confer electrical behavior analogous to that of cardiac muscle and account for the spread of action potentials and accompanying myogenic contractions from points of initiation (e.g., pacemaker regions) in three dimensions within the muscle (24). Networks of specialized pacemaker cells, called interstitial cells of Cajal (ICCs), are associated with the intestinal smooth musculature (see Chapter 20). Pacemaker potentials (i.e., electrical slow waves) originate in the ICC networks and appear as rhythmic oscillations consisting of depolarization and repolarization phases. Gap junctions connect the ICC networks to the intestinal circular muscle coat. During the depolarizing phase of each slow wave in the ICC network, electrical current flows across the gap junctions to depolarize the membrane potential of the muscle fibers to their threshold for action potential discharge and an associated action potential–triggered contraction. Neural Control by the Enteric Nervous System Neural networks in the ENS organize intestinal muscular activity into functional patterns of wall behavior (2,25). Motility patterns in the small and large intestines are organized to fulfill the specialized functions of the individual organ. The motility patterns in each of the different organs are specific for each digestive state and reflect neurally coordinated contractions and relaxations of the smooth muscle. Examples of intestinal motility patterns are the fed state with a mixing motility pattern, interdigestive state with the migrating motor complex (see Chapter 38), and the powerful propulsive pattern that is part of a defensive response to infectious agents, food antigens, or enterotoxins (see Fig. 24-1).

Contractions in each motor state are organized to produce the propulsive forces that move the contents along the intestine, mix ingested foodstuffs with luminal digestive enzymes, and bring nutrients into contact with mucosal brush-border enzymes and transporters for efficient digestion and absorption. ENS-mediated relaxation of spontaneous smooth muscle tone allows sphincters to open and influx of material to be accommodated in reservoirs of the stomach and large intestine. The ENS, together with its interactions with the CNS, organizes motility into patterns of efficient behavior suited for the differing digestive states, as well as pathophysiologic patterns such as occur during vomiting.

Peristaltic Propulsion The peristaltic reflex is a polysynaptic reflex circuit in the ENS that underlies all of the propulsive motility patterns found in the small and large intestines and esophagus. It is the intestinal analog of spinal motor reflexes (e.g., monosynaptic patellar and Achilles tendon reflexes and polysynaptic withdrawal reflexes). Spinal reflexes are investigator-evoked artifacts arising from connections of stretch receptors in the muscle or nociceptors in the skin that activate α spinal motor neurons to evoke contractions/twitches in particular somatic muscles (e.g., the quadriceps muscle in a patellar tendon reflex). Monosynaptic spinal reflexes reflect the effects of abrupt activation of stretch receptors (i.e., muscle spindles) in the muscle and have little relevance for full understanding of the complexity of neural control of posture and movement. The peristaltic reflex is much the same in that it is a fixed response evoked by investigational stretching of the intestinal wall or stroking of the mucosa. It is like a polysynaptic spinal reflex in that it is a motor response to sensory stimulation that is repeated the same way each time the “hardwired” reflex circuit is activated. The peristaltic reflex circuit is “wired” such that it evokes relaxation of the circumferentially oriented muscle layer and contraction of the longitudinal muscle below the point of stimulation and contraction of the circumferentially oriented muscle layer above the point of stimulation. Like spinal reflexes, the peristaltic reflex is positioned at the lowest level of the hierarchical organization of neural control of intestinal motility and undoubtedly underlies each of the various patterns of propulsive motility that impart functionality to the intestine during daily life. As with a spinal motor reflex, the sequencing of the pattern of behavior of the intestinal longitudinal and circular muscles is hardwired into the circuitry, whereas the strength of each motor component of the pattern and the repetition rate of the pattern are adjusted by sensory feedback or other commands to automatically compensate for local loads and higher functional demands on the intestine as a whole. The distance over which propulsion occurs and the direction in which it occurs in the specific patterns of motility, which characterize the various digestive states, are additional factors requiring a higher order of neural control. Short-distance propulsion

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 671 in the postprandial digestive state, propulsion over intermediate distances during interdigestive motility (i.e., migrating motor complex), and long-distance power propulsion, all in the orthograde direction, and long-distance retropulsion during emesis are neural control requirements that are met by the integrative microcircuitry of the ENS. Further improvement in understanding of the neural integration of intestinal motility will require moving forward from the “overused” concept of the peristaltic reflex and on to investigation of microcircuits in positions at levels of organization beyond the reflex “hardwiring” that faithfully reproduces the muscle behavior each time the investigator stretches the intestinal wall or strokes the mucosa. The muscle layers of the intestine contract and relax in a stereotyped pattern during peristaltic propulsion (Fig. 24-3). This pattern is determined by the sequence in which the peristaltic polysynaptic reflex circuit activates excitatory and inhibitory motor neurons to the longitudinal and circular muscle layers. During propulsion, the longitudinal muscle layer in the segment ahead of the advancing intraluminal contents contracts in response to activation of its excitatory motor innervation, while at the same time, the circular muscle layer relaxes in response to activation of its

inhibitory motor innervation. The intestinal tube behaves geometrically like a cylinder with constant surface area (26). Shortening of the longitudinal axis of the cylinder during contraction of the longitudinal muscle is accompanied by a widening of the cross-sectional diameter. The simultaneous shortening of the longitudinal axis and relaxation of the circular muscle results in expansion of the lumen, which prepares a receiving segment (see Fig. 24-3) for the forwardmoving intraluminal contents during peristaltic propulsion. Organization of a receiving segment constitutes half of propulsive peristaltic reflex behavior. The other half is contraction of the circular muscle in the segment behind the advancing intraluminal contents. The longitudinal muscle layer in this segment relaxes at the same time that the circular muscle contracts, resulting in the conversion of this region to a propulsive segment that propels the luminal contents ahead, into the receiving segment (see Fig. 24-3). The propulsive segment is formed when neural connections in the reflex circuit “turn off ” both the excitatory innervation to the longitudinal muscle and the inhibitory innervation to the circular muscle. Omnipresent electrical slow waves evoke contraction of the circular muscle in the propulsive segment, whereas the inhibitory innervation to the segment

Inlet Stereotypic peristaltic behavior

Physiological ileus

Time = × s Outlet Anal

Oral Time = 2 × s

Propulsive segment

Receiving segment Propulsive segment Time = 3 × s Luminal contents

Muscle behavior Circ. Muscle: Long. Muscle:

Relaxed Relaxed

Contracted Relaxed

Receiving segment

Contracted

Time = 5 × s

Direction of propulsion

A

Time = 4 × s

Relaxed

B

FIG. 24-3. Integrated control of contractile behavior of the longitudinal and circular muscle coats forms propulsive and receiving segments in stereotypic fashion during peristaltic propulsion in the intestine. (A) The circumferential and longitudinal muscle layers of the intestine behave in a stereotypical pattern during peristaltic propulsion. A “hardwired” reflex circuit in the enteric nervous system determines the pattern of behavior of the two muscle layers. During peristaltic propulsion, the longitudinal muscle layer in the segment ahead of the advancing intraluminal contents contracts while the circumferential muscle layer relaxes simultaneously. Simultaneous shortening of the longitudinal intestinal axis and relaxation of the circumferential muscle in the same segment results in expansion of the lumen, which becomes a receiving segment for the forward moving contents. The second component of the reflex is contraction of the circular muscle in the segment behind the advancing intraluminal contents. The longitudinal muscle layer in the same segment relaxes simultaneously with contraction of the circular muscle, which results in conversion of this region to a propulsive segment that propels the luminal contents ahead into the receiving segment. (B) Motor behavior of the intestinal wall during the peristaltic reflex in a segment of guinea pig ileum in response to distension by infusion of saline at time = xs. Shortening of the longitudinal axis and expansion of the lumen are seen in the receiving segment, and contraction of the circular muscle and reduction in the diameter of the receiving segment are apparent at time = 3xs, as propulsion proceeds to empty the lumen through the outlet. (B: Redrawn from photographic images provided by Professor M. Takaki, Department of Physiology, Nara Medical University, Nara University, Japan.)

672 / CHAPTER 24 remaining on either side of the resection. Consequently, organized propulsion is not impaired after resection of various lengths of bowel. Synaptic gates connect the blocks of basic circuitry in the heuristic model and contribute to a mechanism for controlling the distance over which the complex of propulsive and receiving segments travels (see Fig. 24-4). When the gates are opened, neural signals pass between successive blocks of the basic circuit, resulting in propagation of the peristaltic event over extended distances. Long-distance propulsion is prevented when all gates are closed. Well-understood presynaptic facilitatory and inhibitory mechanisms in the microcircuitry of the ENS are presumed to be involved in gating the transfer of signals between sequentially positioned blocks of reflex circuitry in the heuristic model (see Presynaptic Facilitation and Presynaptic Inhibition sections in Chapter 23). Synapses formed by the interneurons that transmit excitatory signals to the next downstream block of circuitry are gating points for controlling the distance over which peristaltic propulsion travels (see Fig. 24-4). Messenger substances that act presynaptically to inhibit the release of transmitter at the excitatory synapses stop transmission and close the entrance gates to the next downstream block of circuitry, thereby determining the distance of propagation. Drugs that facilitate the release of neurotransmitters at the excitatory synapses (e.g., cisapride and tegaserod)

is silenced. Contractions recorded by sensing devices implanted on the bowel during digestive and interdigestive intestinal motor behavior reflect the formation of propulsive segments. They occur at the frequency of the electrical slow waves because removal of inhibition from the circular muscle allows it to respond to the electrical current flowing from the ICC network during each slow wave cycle. In contrast, formation of the propulsive segment during power propulsion (see Intestinal Power Propulsion later in this chapter) occurs unrelated to the frequency of the electrical slow waves and involves much stronger contraction of the circular muscle in the propulsive segment than occurs during peristaltic propulsion in the digestive and interdigestive motor patterns. The heuristic model for peristaltic propulsion has blocks of the basic polysynaptic reflex circuit connected “in series” along the length of the small and large intestines (Fig. 24-4). A block of the basic polysynaptic circuit is formed by synaptic connections between interneurons and motor neurons (Fig. 24-5). Propulsion occurs over extended lengths of intestine as blocks of the basic circuit are recruited to activity in consecutive segments. In this respect, the intestine is like the spinal cord, where connections for polysynaptic reflexes remain regardless of the destruction of adjacent regions of the spinal cord. Resection of an intestinal segment does not alter the reflex circuitry in the two segments

Peristaltic reflex circuit

tic ap syn itory e r P hib tor in ep rec

Interneuron Peristaltic reflex circuit

Synaptic gate

A Propagated propulsion

Gating synapses uninhibited: Synaptic gates open No propagated propulsion

B

Gating synapses inhibited: Synaptic gates closed

FIG. 24-4. Synaptic gates determine distance of propagation of peristaltic propulsive motility. Presynaptic mechanisms gate the transfer of signals between sequentially positioned blocks of polysynaptic peristaltic reflex circuitry. Synapses between the neurons, which carry activating signals to each successive block of circuitry, function as gating points for control of the distance over which peristaltic propulsion travels. Messenger substances that act presynaptically to inhibit the release of transmitter at the excitatory synapses close the gates for transfer of information, and thereby determine the distance of propagation. Drugs that facilitate the release of neurotransmitters at the excitatory synapses (e.g., cisapride and 5-HT4 partial agonists) have therapeutic propulsive application by increasing the probability of information transfer at the synaptic gates, thereby enhancing propulsive motility.

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 673 Propulsive segment

Receiving segment Distension

Long.muscle (Relaxed) +

Long.muscle (Contracted) +

Mechanoreceptors

Motor neurons

+

Excitatory

+ Interneurons

Inhibitory

− +

Inactive Active

+

− −

− + Circular muscle (Contracted)

(5-HT) Enterochromaffin cell

−

+ + − + Circular muscle (Relaxed)

Mucosal distortion FIG. 24-5. A “hardwired” polysynaptic peristaltic reflex circuit in the enteric nervous system underlies intestinal propulsive motility. Two kinds of input activate the circuit. One is distension of the intestinal wall and activation of mechanoreceptors; the second is stimulation of release of 5-hydroxytryptamine from enterochromaffin cells by mechanical stimulation of the mucosa. When the reflex circuit is active, excitatory motor neurons to the longitudinal muscle coat and inhibitory motor neurons to the circular muscle coat are activated to form the receiving segment below the point of stimulation (see Fig. 24-3). At the same time, activation of excitatory motor neurons to the circular muscle coat and inactivation of inhibitory motor neurons to the circular muscle coat occur in the propulsive segment above the point of stimulation.

(27,28) have therapeutic application by increasing the probability of information transfer at the synaptic gates, thereby enhancing propulsive motility.

Peristaltic Retropulsion Microcircuits of the ENS can be programmed to control for peristaltic propulsion in either direction along the intestine. For example, if forward passage of the intraluminal contents is impeded in the large intestine of the Hirschsprung’s mouse model, reverse peristalsis propels the bolus over a variable distance away from the obstructed segment. Retroperistalsis then stops, and forward peristalsis propels the bolus again in the direction of the obstruction (29). During the act of vomiting, retroperistalsis in the form of power propulsion starts in the middle of the small intestine and rapidly transports the luminal contents toward the open gastric pylorus (30). In this case, as well as in the obstructed intestine, the coordinated muscle behavior required for effective propulsion is the same except that it is organized by the ENS to travel in the oral direction.

Physiologic Ileus Ileus is defined clinically as mechanical, dynamic, or adynamic obstruction of the bowel. Adynamic ileus is pathologic obstruction of the bowel caused by failure of the smooth muscle to contract. Dynamic ileus is pathologic intestinal obstruction caused by spastic contraction (i.e., failure to relax)

of the circular musculature in a segment of bowel. Mechanical obstruction and adynamic and dynamic ileus are accompanied by severe colicky pain, abdominal distension, vomiting, and constipation (31). Physiologic ileus is a term used to portray the normal absence of motility and propulsion in the small and large intestines. It is a fundamental behavioral state of the bowel in which the ENS programs quiescence of motor function. The state of physiologic ileus disappears after destruction or anesthesia of the ENS. Disorganized and nonpropulsive contractile behavior occur continuously (i.e., dynamic ileus) because of the myogenic contractile properties of unitary-type smooth muscle when enteric neural functions are anesthetized or destroyed by pathologic processes. Quiescence of the intestinal circular muscle reflects the operation of a neural program in which almost all of the synaptic gates within and between basic peristaltic circuits are held shut. In this state, the inhibitory motor innervation of the circular muscle remains in a continuously active state, and responsiveness of the circular muscle to electrical slowwave generation by the ICC networks is suppressed. Physiologic ileus, which is a normal condition, is in effect for varying periods in different intestinal regions, depending on factors such as the time after a meal. The normal state of motor quiescence becomes pathologic when the gates for the particular motor patterns are rendered inoperative for abnormally long periods. In this state of adynamic ileus, sometimes called paralytic ileus, the basic circuits are locked in an inoperable state, whereas unremitting activity of the inhibitory motor neurons suppresses myogenic activity.

674 / CHAPTER 24 Intestinal Power Propulsion Power propulsion is peristaltic reflex-based propulsion characterized by strong, long-lasting contractions of the circular muscle that propagate for extended distances along the small and large intestines. The contractions, when recorded by sensing devices placed on or in the intestine, reflect formation of the propulsive segment of the peristaltic reflex and are sometimes referred to as giant migrating contractions because they are considerably stronger than the phasic contractions during the migrating motor complex or mixing pattern (32). Giant migrating contractions last 18 to 20 seconds and span several cycles of the electrical slow waves. They are a component of a highly efficient propulsive mechanism that rapidly strips the lumen clean as it travels at about 1 cm/sec over long lengths of intestine. Intestinal power propulsion differs from peristaltic propulsion during the migrating motor complex and shortsegment mixing movements in that circular contraction in the propulsive segment are much stronger, and more open synaptic gates permit propagation over longer stretches of intestine. The circular muscle contractions are not time locked to the electrical slow waves and reflect strong activation of the muscle by release of acetylcholine and substance P from excitatory motor neurons. Power propulsion occurs in the retrograde direction during emesis in the small intestine and in the orthograde direction in response to noxious stimulation in both the small and the large intestines. Abdominal cramping pain sensations and sometimes diarrhea are associated with this motor behavior (33). Application of irritants to the mucosa, the introduction of luminal parasites, enterotoxins from pathogenic bacteria, allergic reactions, and exposure to ionizing radiation each trigger the neural program for the powerful propulsive response. These characteristics suggest that power propulsion is a defensive adaptation for rapid clearance of undesirable contents from the intestinal lumen (34). It also accomplishes mass movement of intraluminal material in normal states, especially in the large intestine.

PLASTICITY IN THE ENTERIC NERVOUS SYSTEM Bullock and Horridge (35) define plasticity in nervous systems as “Ability to be modified or changed.” Usually, plasticity is an inferred property (mechanisms unspecified) of a nervous system that can make an adaptive change in activity after removal of some of its parts. Different types of plasticity are recognized. In one, there is immediate adaptive change in pattern of motor movement; in a second, there is slow “relearning” or recovery believed to involve changes in central synaptic connections. The occurrence of immediate adaptive change suggests that existing neural connections can meet new demands, and that this should be called apparent plasticity. Consideration of the ENS as a brain-inthe-gut, with most of the inherent properties of an independent

integrative nervous system, raises the question of whether plasticity as defined by Bullock and Horridge is a property of the ENS.

Gastric Reflexes Recovery over time of effective gastric motor function after vagotomy is an ENS example suggestive of plasticity. The gradual return of functional gastric reflexes after extrinsic denervation may be analogous to the progressive return of spinal motor reflexes as “spinal shock” subsides after disconnection of the spinal microcircuits from the brain. Nevertheless, whether functional reconfiguration occurs in the enteric microcircuits in ways that can be related to recovery of reflex behavior in regions of the spinal cord below an injury remains an unanswered question.

Secretomotor Control Remodeling of the circuitry in the intestinal submucosal plexus, which controls neurogenic secretion, appears to happen after transection of the sympathetic postganglionic nerves to the intestine. Firing rates of secretomotor neurons that innervate the intestinal crypts of Lieberkühn are suppressed by inhibitory inputs from both the sympathetic nervous system and somatostatinergic interneurons in the ENS (36,37). The overall effect for the colon, as an example, is drier stools with less mucus. Normally, sympathetic nervous input to secretomotor neurons predominates with less inhibitory input derived from interneurons in the control networks (37). After sympathectomy, a significant increase in nonadrenergic inhibitory input occurs (38) that is suggestive of sprouting and formation of new connections from neurons intrinsic to the ENS.

Hirschsprung’s Disease Observations in patients with Hirschsprung’s disease suggest that neural plasticity as defined by Bullock and Horridge (35) for other nervous systems may be present in the ENS of the large intestine. The characteristic rectoanal inhibitory reflex relaxation of the internal anal sphincter found in healthy individuals is absent in Hirschsprung’s disease and reflects congenital absence of the ENS from a variable length of terminal large intestine. Pediatric surgeons treat Hirschsprung’s disease by resecting the affected region of terminal bowel and reattaching more proximal colon at the anal margin. Holschneider and colleagues (39) repeated the standard balloon-distension protocol for demonstration of the rectoanal reflex in a group of patients with Hirschsprung’s disease after corrective surgery. These authors found that, after 1 to 6 years in 66% of 426 patients, internal anal sphincter relaxation responses evoked by rectal balloon distension appeared similar to

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 675 a normal rectoanal reflex. It appeared that over time the repositioned proximal colon was capable of developing motor behavior similar to that of the rectosigmoid. Results of Holschneider and colleagues (39) suggest that reconfiguring of the ENS control circuits was initiated in the segment of more proximal colon after transposition into direct communication with the anus. In effect, a more proximal colonic segment assumed behavioral characteristics of the rectosigmoid. This would fit Bullock and Horridge’s (35) definition of plasticity as an adaptive change in activity and “relearning” in a nervous system after removal of some of its parts.

Microcircuit Level All integrative nervous systems, whether in vertebrate or invertebrate animals, contain neural networks that are programmed to generate timing and phasing signals for repetitive, rhythmic movements. Walking, swimming, flying, and copulation are examples of repetitive movements controlled by neural networks. Repetitive activation of peristalsis within the activity front of the migrating motor complex, mixing movements occurring rhythmically at multiple sites along the small intestine during the digestive state, and rhythmic cycles of secretion from the mucosa in response to food antigens all reflect rhythmic output from enteric neural networks (34). Earlier concepts of neural networks taught that each kind of somatomotor behavior generated by an integrated muscle group required a different “hardwired” circuit for each specific behavior (35). Later findings suggest otherwise! Current evidence shows that a neural network can be polymorphic, with the same circuit being capable of generating a variety of different motor behavioral patterns. Katz and Harris-Warrick (40) describe this kind of circuit as a defined anatomic network consisting of a library of components that can be reconfigured in different ways to form a variety of different functional circuits. Neuromodulatory signals determine the configuration, and thereby determine the output of the network. This is accomplished through the selective release of a neuromodulatory substance that blankets the circuit and acts to alter the electrical and synaptic behavior of the neural elements in the circuit. A neuromodulatory substance is defined as one that alters the input–output relations for a single neuron or an integrated circuit. An example of neuromodulatory reconfiguration of a neural circuit occurs in the spiny lobster, where 14 neurons in the stomatogastric ganglion are connected synaptically into a network that controls for different patterns of rhythmic movements in the foregut as digestion proceeds (41). An overlay of a particular neuromodulator on the 14-neuron circuit reconfigures it for a different motor pattern by changing the strengths of synapses in the network and altering the intrinsic firing properties of the neurons in the network. In the lobster, an overlay of 5-HT evokes a specific pattern of motor behavior, and an overlay of dopamine evokes another. The signaling by 5-HT and dopamine is mediated by

a second intraneuronal messenger system using cyclic adenosine monophosphate (cAMP) (42). Comparable mechanisms operate in the selection of programs from the enteric neural networks in mammals, where neurons, endocrine cells, and immune/inflammatory cells store and release several different neuromodulatory messengers. Like the example for the invertebrate nervous system, the ENS also contains a library of programs for specific patterns of intestinal motor behavior (see Fig. 24-1). Each program adapts the bowel for a specific digestive state or, in some cases, an adverse condition in the intestinal lumen (e.g., inflammation, infectious agents, or food allergins). One or the other program may be “called up” by endocrine or paracrine release of a neuromodulator that floods over the circuit or by neuromodulatory inputs from the CNS. Histaminergic Neuromodulation Histamine release from enteric mast cells and the overlay of histamine on the neural network in the intestinal submucosal plexus presents a clear example of how a neuromodulator can reconfigure the network to generate a rhythmic pattern of secretion and muscle contraction. Experimental application of histamine to simulate degranulation of mast cells or actual degranulation of mast cells in the intestine of antigen-sensitized animal models evokes cyclical bursts of secretion of electrolytes, H2O, and mucus coordinated with contraction of the musculature (43–45). The secretory behavior is activated for about 1 minute, and then inactivated for a few minutes before the next cycle (Fig. 24-6A). As each secretory cycle peaks, contraction of the muscularis externa occurs (4). The neural requirement for this behavior is confirmed by the application of neural-blocking drugs, which prevents the patterned response to histamine. The effects of paracrine release of histamine from enteric mast cells is reminiscent of the neuromodulatory endocrinelike effects of dopamine and 5-HT for each to evoke a different specific output of patterned behavior from the neural network described earlier for the lobster. Like the lobster neurons, second-messenger function of cAMP is involved in the neurons of the enteric network. Histamine activates histamine H2 receptors to stimulate the formation of cAMP in the enteric neurons (46). Two neuromodulatory actions of histamine at the single neuron level account for the emergent pattern of enteric network output that is subsequently transformed into a rhythmic pattern of behavior at the level of the secretory epithelium and musculature. One neuromodulatory action is dramatic increases in the excitability of the cell bodies of neurons that make up the circuit (Fig. 24-7). This results in long-lasting firing of action potentials and mimics slow synaptic excitation (see Slow Synaptic, Endocrine, and Paracrine Excitation section in Chapter 23). The neuronal excitatory action of histamine is mediated by histamine H2 receptors in the guinea pig intestine (47,48). The second neuromodulatory action of histamine is at synaptic connections between the neurons that form the

676 / CHAPTER 24 Histamine

Secretion

Muscle contraction

A

5 min Histamine Presynaptic inhibition Histamine H3 antagonist Secretion

B

2 min

FIG. 24-6. Activation of an enteric nervous system (ENS) program for coordinated mucosal secretion and muscular contraction in the guinea pig colon. (A) An overlay of histamine on the microcircuits of the submucosal plexus evokes repetitive cycles of mucosal secretion and coordinated contractions of the muscularis externa. The full-thickness preparation of intestinal wall was placed in an Ussing chamber with a small strain gauge attached to the serosal surface. Upper trace records mucosal secretion reflected as increases in transmucosal short-circuit chloride current; the lower trace is muscle contraction recorded by the strain gauge. (B) Blockade of the presynaptic inhibitory action of histamine with a selective histamine H3 receptor antagonist enhances the amplitude of the mucosal secretory cycles. (Courtesy of Professor H. J. Cooke, Ohio State University College of Medicine and Public Health, Columbus, Ohio; A, bottom trace: modified from Cooke and colleagues [4], by permission.)

integrative networks. Histamine acts at receptors on the presynaptic terminals to suppress the release of neurotransmitter, thereby suppressing transmission at the synapses (see Fig. 24-7). The presynaptic inhibitory action of histamine at enteric cholinergic synapses is mediated by the histamine H3 receptor subtype in the guinea pig (49).

Serotonergic Neuromodulation 5-HT is another neuromodulator, which is released in paracrine fashion from enterochromaffin cells in the intestinal mucosa. It differs from histamine in that an overlay of 5-HT on the ENS microcircuits does not “call up” the

Presynaptic receptors Histamine3 inhibitory receptors

Fast EPSP Control

Histamine (0.1 µM) 20 ms

A Somal receptors

Histamine2 slow excitatory receptors

B

Slow EPSP

Histamine

“Puff” histamine

FIG. 24-7. Histamine acts at two sites on elements of the enteric nervous system (ENS). (A) Action at presynaptic histamine H3 inhibitory receptors suppresses the release of acetylcholine at nicotinic excitatory synapses, thereby suppressing fast excitatory postsynaptic potentials (fast EPSPs) in the microcircuits of the ENS. (B) Action at histamine H2 excitatory receptors on the cell bodies of enteric neurons evokes slow EPSP-like excitation characterized by prolonged discharge of action potentials.

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 677 program for the rhythmic pattern of coupled secretion and propulsive motility. Nevertheless, receptors for 5-HT are present on secretomotor neurons and other neural elements of the microcircuits (50). Mention is made relative to histamine because two of the actions of 5-HT at the single neuron level are the same as those for histamine. Action at 5-HT1P and perhaps 5-HT7 receptors on the cell bodies of enteric neurons mimics the long-lasting excitability increases seen during slow synaptic excitation (51–53). Inhibitory actions, similar to those of histamine, at presynaptic receptors on enteric cholinergic nerve terminals are prevalent (54) and mediated by receptors tentatively identified as the 5-HT1A subtype (55).

Positive feed-forward excitatory circuit Motor neurons Circuit output

Motor neurons Circuit output

Interneuron Presynaptic inhibitory receptor

Excitatory synapse

Neuromodulatory overlay

Presynaptic Inhibitory Modulation The neuromodulatory actions of histamine and 5-HT raise questions about the significance of suppressing synaptic transmission in an ENS neural circuit via one receptor subtype while simultaneously activating the neuronal cell bodies in the circuit to high levels of excitability via a different receptor subtype when the circuit is blanketed by the same signal substance. Clues for an answer emerge from the actions of selective histamine H3 receptor–blocking drugs on the rhythmic secretory activity evoked by flooding the circuits with histamine. Application of a histamine H3 receptor antagonist greatly enhances the secretory cycles evoked by the presence of histamine (see Fig. 24-6B). Figure 24-8 illustrates how presynaptic inhibitory receptors might act to maintain a braking action that prevents “runaway” excitation in the circuit when excitability is increased to high levels in cell bodies of the neurons that are interconnected synaptically to form the circuit. When the circuit is flooded with a neuromodulator such as histamine, histamine H2 receptor activation excites the cell body of each of the neurons to firing threshold and repetitive discharge. The neurons are connected synaptically with one another in a positive feed-forward configuration. Consequently, firing rates in all of the neurons of the circuit increase rapidly. Increased output from the circuit accomplishes rapid and coordinated activation of pools of motor neurons to effector systems. Rapid and coordinated activity of motor neurons is necessary to achieve rapid activation of the drive for secretion or other effector behavior simultaneously around the circumference and along the length of a segment of bowel. A consequence of a positive feed-forward driver circuit, such as that illustrated in Figure 24-8, is that excitation, once initiated in neurons of the circuit, will continue to escalate if left unchecked. Presynaptic inhibition functions to hold excitation within the entire circuit in check by suppressing transmission at the excitatory synapses that connect the neurons into the circuit. This essentially prevents disastrous “runaway” excitation in the circuit and explains the increase in amplitude of the histamine-evoked secretory cycles after blockade of presynaptic inhibition by a histamine H3 receptor antagonist (see Fig. 24-6B). The neuromodulatory action of histamine includes both excitation of the neuronal cell bodies

FIG. 24-8. Driver circuits are positive feed-forward synaptic circuits, the output of which drives the firing frequency in pools of enteric nervous system motor neurons. Neurons in the circuit have recurrent excitatory synaptic connections one with another, which results in positive feedback flow of synaptic excitation that leads to rapid buildup of firing within the entire population of driver neurons. Excitatory neuromodulatory substances released from neuronal, paracrine, or endocrine sources overlay the circuit and initiate firing in each neuronal constituent of the driver circuit. Presynaptic inhibitory receptors at the synapses in the driver circuit maintain a braking action that prevents “runaway” excitation in the circuit.

of the secretory driver circuit via histamine H2 receptors and presynaptic inhibition via histamine H3 presynaptic receptors (see Fig. 24-7). The H3 histamine receptor action applies a brake on circuit excitability. Application of a specific histamine H3 receptor blocker selectively removes the presynaptic braking action, resulting in increased neuronal excitation in the circuit and increased drive for each secretory cycle. Pattern Generator The ENS network responsible for driving cyclical secretion, when overlaid with histamine, also incorporates a pattern generator responsible for timing the cycles. However, the neural mechanism underlying the patterned timing and phasing of the rhythmic cycles is not understood. It is clearly a property of the circuit and a property that emerges from the H2- and H3-mediated actions of histamine at the level of single neural elements in the circuit (see Fig. 24-7). Operation of the pattern generator appears not to depend on presynaptic inhibition because the timing of the cycles does not change after blockade of histamine H3 receptors. Likewise, it does not depend on the concentration of the neuromodulator, with the exception of the need for a threshold concentration. The system behaves like a “switch” is present that activates the neural program, including the pattern generator, in an on/off manner. The neuromodulatory action of histamine to activate an enteric behavioral program is reminiscent of a similar neuromodulatory action of the intestinal hormone cholecystokinin.

678 / CHAPTER 24 An overlay of cholecystokinin on the ENS of the cat small intestine activates in all-or-none fashion a neural program for repetitive cycles of peristaltic propulsion (56). A similar repetitive patterning of activation of peristaltic reflex circuits (i.e., ascending circular muscle contraction and descending circular muscle inhibition) occurs when flat sheet intestinal preparations are tightly stretched in vitro (57–59).

a functioning unit necessary for the maintenance of fecal continence and normal defecation. The pelvic floor musculature exhibits physiologic and histologic properties similar to those of the tonic somatic muscles that maintain upright bodily posture against the forces of gravity. Weakening of the pelvic floor musculature (e.g., in advanced age) or traumatic damage to the musculature or its innervation (e.g., during childbirth) can underlie fecal incontinence and disordered defecation. Like other skeletal muscles, weakening of the pelvic floor musculature often can be corrected by isometric exercises that target this group of muscles. Histochemical and mechanical properties of the EAS, levator ani, and puborectalis muscles correspond to slowtwitch, tonic-type skeletal muscles in humans. The muscle properties in humans differ from quadrupedal animals (e.g., cat), where the pelvic floor muscles behave like fasttwitch skeletal muscle (60). Demands of an upright posture are postulated to have been the selective pressure for the evolutionary switch in the contractile and metabolic properties of the pelvic floor musculature in humans and other upright primates. The pelvic floor musculature is pictured as an inverted funnel consisting of the levator ani and external sphincter muscles forming a continuous sheet from the bottom margins of the pelvis to the anal verge, which is recognized as the transition zone between mucosal epithelium and stratified squamous epithelium of the perianal skin. The levator ani contract and restore the perineum to its normal position after defecation. Fibers of the puborectalis join behind the anorectum and pass around it on both sides to insert on the pubis.

INTEGRATED CONTROL OF THE ANAL CANAL AND PELVIC FLOOR Integrative neural control for defecation, release of flatus, and maintenance of fecal continence involves coordinated interactions between the ENS and CNS. The ENS organizes the motility component involving the smooth musculature of the colon and rectosigmoid region. Neural circuitry in the sacral spinal cord and supraspinal integrative centers has responsibility for control of the motility of the skeletal musculature of the pelvic floor.

Pelvic Floor Musculature The levator ani muscles are overlapping sheets of striated muscle that form the pelvic floor. They consist of the pubococcygeus, iliococcygeus, and ischiococcygeus (Fig. 24-9A). The levator ani contract and relax in concert with the puborectalis and the external anal sphincter (EAS) as components of

Pelvic floor musculature

Anorectal angle Coccyx

Pubic symphysis

Rectum Pubic symphysis

Urethra Perineum

Anorectal angle (~ 90°)

Puborectalis Pubococcygeus Iliococcygeus

A

Anus

Coccyx

B

Puborectalis muscle

Anus

FIG. 24-9. Integrative neural control of the musculature of the pelvic floor is responsible for the maintenance of fecal continence and normal defecation. (A) Pelvic floor muscle groups. The levator ani form the floor of the pelvis and are divided into pubococcygeus and iliococcygeus muscles. Both muscles arise from anterior regions of the pelvis. The iliococcygeus inserts into the coccyx. Divisions of the pubococcygeus from either side join to form the puborectalis. The puborectalis forms a loop around the rectum that becomes important in the maintenance of the anorectal angle and fecal continence. (B) Tonic contraction of the puborectalis muscle in conditions of rest closes the distal rectum and works in conjunction with the anal sphincters in the maintenance of continence. Contractile tension in the puborectalis is relaxed during defecation. Contraction of the puborectalis muscle forms an anorectal angle of approximately 90 degrees, which opens to a more obtuse angle of about 120 degrees when muscle tension is relaxed during defecation.

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 679 This forms a U-shaped sling that pulls the anorectal tube anteriorly, such that the long axis of the anal canal lies at nearly a right angle to that of the rectum (see Fig. 24-9B). Tonic contraction and “pull” by the puborectalis muscle narrows the anorectal tube from side to side at the bend of the angle, resulting in a mechanical barrier to the passage of feces and gas that is important in the mechanisms that ensure continence. The puborectalis sling and the upper margins of the internal and external sphincters form the anorectal ring, which marks the boundary of the anal canal and rectum. Surrounding the anal canal over a length of about 2 cm are the internal anal sphincter and EAS (Fig. 24-10). The external sphincter is skeletal muscle attached to the coccyx posteriorly and the perineum anteriorly. When contracted, it compresses the anus into a slit, thereby closing the orifice. The internal anal sphincter is a modified extension of the circular muscle coat of the rectum. It is composed of fatigue-resistant smooth muscle that, similar to other sphincteric muscles in the digestive tract, contracts tonically to sustain closure of the anal canal and is relaxed by inhibitory input from inhibitory enteric motor neurons.

Sensory Innervation Mechanoreceptors in the rectum detect distension and transmit the coded information to the neural networks of the large intestinal ENS and spinal cord on a moment-tomoment basis. Sensory innervation of the anal canal and

perianal skin differ from the rectum in that somatosensory nerves transmit multiple modalities of sensory information from these areas to interneuronal processing circuitry in sacral segments of the spinal cord and in the brain. Sensory receptors detect touch, pain, and temperature with high sensitivity in the anal canal and at the anal verge. Processing of information from these receptors by the spinal cord and higher brain centers determines the individual’s ability to discriminate consciously between the presence of gas, liquid, and solids in the anal canal. Mechanoreceptors in the muscles of the pelvic floor detect changes in the orientation of the anorectum as feces are propelled into the region and transmit the information to the CNS for processing that leads to conscious awareness of a threat to continence and an urge to defecate. Signals to the brain from mechanoreceptors in the pelvic floor musculature provide the first conscious warning of the arrival of stool in the rectum. The sensory mechanisms associated with the pelvic floor musculature account for the ability of an individual to experience the movement of feces in the anal canal and maintain fecal continence after surgical resection of the rectosigmoid region (e.g., surgical correction for Hirschsprung’s disease). Sensory mechanisms in the rectum differ from the anal canal and perianal skin in that detection of filling and distension is the principal stimulus parameter. The ability of patients to experience generally satisfactory defecation and continence after colectomy and surgical construction of an ileoanal pouch suggests that the presence of rectal sensory function is unnecessary for defecation or continence (61).

Distend rectum

Rectum

Internal anal sphincter

Rectal balloon

Internal anal sphincter

External anal sphincter

Anal balloon

A

B

Pressure recording Balloon distension

External anal sphincter

FIG. 24-10. Inflation of a balloon placed in the rectum evokes the rectoanal reflex. (A) The rectoanal reflex consists of simultaneous relaxation of intraluminal pressure in the internal anal sphincter and contraction (increased intraluminal pressure) in the external anal sphincter. (B) Method used to evoke the rectoanal reflex. Inflation of a balloon placed in the rectum distends the rectum. Two balloons, one placed in the lumen of the internal anal sphincter and a second in the external anal sphincter, are connected to pressure transducers and chart recorders for recording changes in intraluminal pressures evoked by distension of the rectum.

680 / CHAPTER 24 Rectoanal Reflex The rectoanal reflex reflects integration of the ENS and spinal control functions that is necessary for normal defecation. Tonic contraction of the internal anal sphincter, which is smooth muscle, and the puborectalis muscle, which is skeletal muscle, blocks the passage of feces into the anal canal and maintains continence with small volumes in the rectum. Rapid influx of feces and consequent distension of the rectum activates the rectoanal reflex, which results in relaxation of the internal sphincter and simultaneous contraction of the external sphincter (see Fig. 24-10A). Balloon distension in the rectum and recording of pressure changes in the lumen of the internal anal sphincter and EAS is a method used to study the rectoanal reflex (see Fig. 24-10B). Like other enteric reflexes, the rectoanal reflex involves stimulation of distension receptors in the rectal wall and processing of the sensory information by enteric neural networks in the ENS and spinal cord. Processing in the ENS leads to reflex excitation of inhibitory motor neurons to relax tension in the smooth muscle of the internal anal sphincter. Processing in the spinal cord leads to excitation of spinal motor neurons to contract the EAS. Conscious sensation of rectal fullness in the lower abdomen is experienced as the rectum fills and reflects CNS processing of information from the mechanoreceptors in the pelvic floor musculature. Relaxation of the internal anal sphincter allows contact of the rectal contents with the sensory receptors in the lining of the anal canal. Sensory signals that reach the brain from the anal canal serve as an early warning of the possibility of a breakdown of maintenance of continence. Continence in this situation is maintained by voluntary contraction and involuntary reflex contraction of the EAS and puborectalis muscle. Contraction of the external sphincter closes the anal canal, and contraction of the puborectalis sharpens the anorectal angle of the mechanical barrier. Operation of the large intestinal power propulsion program in diarrheal states (e.g., diarrhea-predominant irritable bowel syndrome or infectious enteritis) moves large volumes of liquid stool into the rectum with potential for overwhelming the mechanisms of continence and the embarrassment of soiling. Sensory detection of rectal distension by rapid influx of large volumes of liquid stool, transmission to the spinal cord, and supraspinal processing of the information underlie the sensations of fecal urgency in diarrheal states.

Spinal Motor Control Individual pools of motor neurons in the sacral spinal cord innervate the levator ani, puborectalis, and EAS. A pool of small motor neurons in the ventral horn of the sacral spinal cord at S2, named Onuf’s nucleus, project their axons in the pudendal nerve. Branches of the pudendal nerve directly innervate the EAS in humans. Branches of sacral nerves 3 and 4 that do not project in the pudendal nerve innervate the puborectalis (62,63). Nerves projecting from

one side of the sacrum innervate the puborectalis muscle only on the same side (64). Because of their separated innervation, interference with pudendal nerve conduction does not alter voluntary contraction of the puborectalis, but it does abolish voluntary and reflex contraction of the EAS. The EAS, puborectalis, and levator ani are each innervated by distinct pools of spinal motor neurons. Innervation by separate pools of motor neurons is consistent with each of the muscles operating as a functionally distinct entity. In this respect, the innervation of the pelvic floor musculature mimics the known cases where different muscles in different motor systems have separate spinal motor pools. Separate identities for the motor neuronal pools suggest that the EAS, puborectalis, and levator ani can be controlled independently by the integrative circuitry in the spinal cord. Individualized control allows the neural networks in the spinal cord to integrate the contractile activity of each muscle group into effective overall behavior of the pelvic floor. The EAS is stimulated to maintain tone by a low-frequency firing rate of 3 to 4 Hz in its motor innervation, which remains continuous in awake and sleeping humans (65). Muscle spindles, analogous to intrafusal fibers in other antigravity muscles, are present in the EAS and puborectalis muscle of animals and humans (66–68). Nevertheless, the extent to which the muscle spindles and set-point control of muscle length by spinal gamma motor neurons might be involved in the generation of the tonic motor input to the muscle is not understood. The tonic motor input to the EAS is abolished after bilateral transection of spinal dorsal roots S1 and S2 in animals. This effect of depriving the spinal cord of sensory input from the anal canal and pelvic floor suggests that a peripheral reflex consisting of dorsal root afferents and spinal motor neurons is responsible for the neurogenic tone in the sphincter. Such a reflex would be like somatomotor reflexes, where length detection by muscle spindles and monosynaptic connections to spinal motor neurons maintain tone in antigravity somatic muscles. Nevertheless, only small numbers of intrafusal fibers are found in the EAS, and no monosynaptically evoked responses can be recorded in the sacral ventral spinal roots after electrical stimulation of the sensory fibers in the pudendal nerves. This calls into question the involvement of muscle spindles in reflex control of the sphincter and raises questions as to whether mechanoreceptors in the anal canal or in perianal cutaneous regions are the sensory feedback component responsible for the tonic contractile behavior of the sphincter (60). Differences in reflex behavior of the EAS and levator ani in animal studies suggest that the two anatomically separate muscles are innervated with their own reflex connectivity. Reflex responses in the EAS are readily evoked by stimulation of cutaneous sensory receptors that have extensive receptive fields extending from the perianal region to the ankle in cats. Cutaneous receptive fields for reflex responses in the levator ani are much more restricted with reflex contraction more readily activated by rectal distension than by cutaneous stimulation (63). Filipini and Dubrovsky (63) suggest that reflex contractions of the levator ani were most important in the

INTEGRATIVE FUNCTIONS OF THE ENTERIC NERVOUS SYSTEM / 681 evacuation of stool. Reflex behavior of the EAS is considered to be important in the maintenance of continence, although not on a moment-to-moment basis, but during threatening situations when intrarectal pressure might become increased during events such as coughing, straining, lifting weights, or sudden movement to upright posture. Spinal reflexes therefore are postulated to have the more important role in neural control of the levator ani muscles. Supraspinal pathways come into play in addition to spinal reflexes for neural control of the EAS.

subgroup of chronically constipated patients (76). The association of paradoxical puborectalis contraction in patients with Parkinson’s disease and multiple sclerosis led to the suggestion that the paradoxical behavior of the pelvic floor musculature reflects defective transmission in descending suprasegmental pathways (77,78). Successful use of biofeedback techniques to train patients to avoid the paradoxical behavior of the puborectalis and EAS muscles in a subgroup of constipated patients is consistent with disordered descending control from the brain as an underlying cause (79).

Supraspinal Motor Control Descending spinal pathways transmit commands from the brain for voluntary contraction of the EAS and puborectalis muscle. Descending pathways provide monosynaptic excitatory synaptic input and polysynaptic inhibitory input to spinal motor neurons in Onuf’s nucleus that innervate the EAS. The ventromedial reticulospinal tract transmits the excitatory input, and the ventrolateral reticulospinal tract carries the inhibitory signals to the motor neurons in cats (69). Neurons in the nucleus gigantocellularis and nucleus raphe obscurus in the medullary reticular formation project in descending tracts that connect with the spinal motor innervation of the pelvic floor. Stimulation in the raphe magnus nucleus inhibits reflex responses of pudendal motor neurons evoked by electrical stimulation of sensory fibers in the central cut end of pudendal nerves (70). Lesions in the nucleus raphe obscurus of the medullary reticular formation lead to hyperreflexia in the EAS of rats (71). Electrical stimulation through the scalp in humans and animals activates rapidly conducting corticospinal pathways that contract the EAS, puborectalis, and levator ani muscles (63,72). Contraction of the EAS occurs in response to rectal distension in both healthy and paraplegic subjects. Nevertheless, healthy subjects have a significantly lower threshold for evoking the reflex than paraplegics. Enhancement of the reflex in healthy subjects is postulated to reflect a descending excitatory influence from the brain (73). Rectal distension in young children, with undeveloped voluntary control of the EAS, evokes relaxation in the sphincter. When the children advance to an age where they gain voluntary control of the sphincter, rectal distension evokes a brief contraction of the sphincter similar to what is found in adults. The changes in the distension reflex as the child matures are attributed to myelinization of the spinal pyramidal tracts (74). Suprasegmental input to the reflex circuitry in the sacral spinal cord determines both the amplitude of sensory-evoked spinal reflexes and the kind of reflex response that occurs. For example, voluntary straining evokes relaxation of the EAS and puborectalis sling. In contrast, increases in abdominal pressure during coughing or heavy lifting evoke an increase in the contractile activity of the two muscles (75). Voluntary straining to defecate normally evokes relaxation of tension in the puborectalis sling. Paradoxical contraction of the puborectalis muscle during voluntary straining to defecate is sometimes identified as a causative factor in a

INTEGRATIVE MECHANISMS FOR DEFECATION Defecation requires neural coordination of smooth muscles in the colon and rectosigmoid region and the skeletal muscles of the pelvic floor. Distension of the rectum by the mass influx of feces or gas evokes the urge to defecate or release flatus. Central processing of mechanosensory information from distension receptors in the rectum is the mechanism underlying this sensation. Local processing of the mechanosensory information in neural networks of the ENS activates the reflex for relaxation of the internal anal sphincter. At this stage of rectal distension, voluntary and involuntary contraction of the EAS and the puborectalis muscle prevent leakage. The decision to defecate at this stage is voluntary. When the decision is made, commands from the brain to the sacral cord “shut off ” the excitatory input to the external sphincter and levator ani muscles. Additional skeletal motor commands contract the abdominal muscles and diaphragm to increase intraabdominal pressure. Coordination of the skeletal muscle components of defecation results in a straightening of the anorectal angle, descent of the pelvic floor, and opening of the anal canal and anus. Programmed behavior of the smooth muscle during defecation includes shortening of the longitudinal muscle layer in the sigmoid colon and rectum, followed by strong contraction of the circular muscle coat (80). This behavior corresponds to the basic stereotyped pattern of peristaltic propulsion. It is the end point for intestinal peristalsis in that the circular muscle of the distal colon and rectum become the final propulsive segment, whereas the outside environment receives the forwardly propelled luminal contents. A voluntary decision to resist the urge to defecate is eventually accompanied by relaxation of the circular muscle of the rectum. This form of adaptive relaxation accommodates the increased volume in the rectum. As wall tension relaxes, the stimulus for the rectal distension receptors is removed, and the urge to defecate subsides. Receptive relaxation of the rectum is accompanied by a return of contractile tension in the internal anal sphincter, relaxation of tone in the EAS, increased pull by the puborectalis muscle sling, and sharpening of the anorectal angle. When this occurs, the feces remain in the rectum until the next mass movement further increases the rectal volume and stimulation of mechanoreceptors again signals the neural mechanisms for defecation.

682 / CHAPTER 24 EPILOGUE Trendelenburg (81) recognized the ENS as an independent integrative nervous system as early as the first quarter of the twentieth century, and J. N. Langley emphasized this in his landmark book on the autonomic nervous system published in 1921 (82). Langley was keenly aware that the few thousand efferent fibers in the vagal nerves were unlikely to contact each of the approximately 100 million neurons in the ENS. He reasoned that the neurons in the gut could be called neither sympathetic nor parasympathetic if all of them did not receive input from the CNS. Based on what has stood the test of time as sound reasoning, he called the intrinsic nervous system of the gut the “enteric nervous system” and identified it as a third division of the autonomic nervous system, which performs integrative functions independent of the sympathetic and parasympathetic divisions. Currently, in this century, there is little argument against reference to the ENS as the “brain-in-the-gut” (83) and a “second brain” (84). Action potential generation, synaptic transmission, multiple neurotransmitters, CNS-like glia, absence of connective tissue, isolation from blood vessels, and integrated microcircuits, all of which are properties of the CNS, are now well-known properties of the ENS (see the Structure-Function Relations with Other Autonomic Ganglia section in Chapter 23). Like in the CNS, emergence of complex behaviors of each of the specialized organs of the whole gut is a fundamental property of the neural networks of the brain-in-the-gut. Moreover, a case can be made for neural plasticity as another of the CNS-like properties of the ENS.

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postoperative continence in Hirschsprung’s disease. Z Kinderchir 1980;29:39–48. Katz PS, Harris-Warrick RM. Actions of identified neuromodulatory neurons in a simple motor system. Trends Neurosci 1990;13:367–373. Johnson BR, Peck JH, Harris-Warrick RM. Distributed amine modulation of graded chemical transmission in the pyloric network of the lobster stomatogastric ganglion. J Neurophysiol 1995;74:437–452. Hempel CM, Vincent P, Adams SR, Tsien RY, Silverston AI. Spatiotemporal dynamics of cyclic AMP signals in an intact neural circuit. Nature (Lond) 1996;384:166–169. Wang Y-Z, Cooke HJ. H2 receptors mediate cyclical chloride secretion in guinea pig distal colon. Am J Physiol 1990;258:G887–G893. Frieling T, Cooke HJ, Wood JD. Histamine receptors on submucous neurons in guinea pig colon. Am J Physiol 1993;264:G74–G80. Frieling T, Palmer JM, Cooke HJ, et al. Neuroimmune communication in the submucous plexus of guinea-pig colon after infection with Trichinella spiralis. Gastroenterology 1994;107:1602–1609. Xia Y, Fertel R, Wood J. Stimulation of formation of adenosine 3′,5′- phosphate by histamine in myenteric ganglia isolated from guinea-pig small intestine. Eur J Pharmacol 1996;316:81–85. Wood JD. Histamine signals in enteric neuroimmune interactions. Ann N Y Acad Sci 1992;664:275–283. Tamura K, Wood JD. Effects of prolonged exposure to histamine on guinea pig intestinal neurons. Dig Dis Sci 1992;37:1084–1088. Tamura K, Palmer JM, Wood JD. Presynaptic inhibition produced by histamine at nicotinic synapses in enteric ganglia. Neuroscience 1988; 25:171–179. Wood JD. Physiology of the enteric nervous system. In: Johnson LR, Alpers DH, Christensen J, Jacobson ED, Walsh JH, eds. Physiology of the gastrointestinal tract, 3rd ed. New York: Raven Press, 1994;423–482. Wood JD, Mayer CJ. Slow synaptic excitation mediated by serotonin in Auerbach’s plexus. Nature (Lond) 1979;276:836–837. Mawe GM, Branchek TA, Gershon MD. Peripheral neural serotonin receptors: identification and characterization with specific antagonists and agonists. Proc Natl Acad Sci U S A 1986;83:9799–9803. Monro RL, Bornstein JC, Bertrand PP. 5-HT7 receptors mediate slow synaptic transmission in myenteric AH neurons. Program No. 169.7. 2004 abstract viewer/itinerary planner. Washington, DC: Society for Neuroscience. North RA, Henderson G, Katayama Y, et al. Electrophysiological evidence for presynaptic inhibition of acetylcholine release by 5-hydroxytryptamine in the enteric nervous system. Neuroscience 1980;5:581–586. Pan H, Galligan JJ. 5-HT1A and 5-HT4 receptors mediate inhibition and facilitation of fast synaptic transmission in enteric neurons. Am J Physiol 1994;266:G230–G238. Weems WA, Seidel ER, Johnson LR. Induction in vitro of a specific pattern of jejunal propulsive behavior by cholecystokinin. Am J Physiol 1985;248:G470–G478. Spencer NJ, Smith TK. Simultaneous intracellular recordings from longitudinal and circular muscle during the peristaltic reflex in guineapig distal colon. J Physiol 2001;533:787–799. Spencer NJ, Hennig GW, Smith TK. A rhythmic motor pattern activated by circumferential stretch in guinea-pig distal colon. J Physiol 2002; 545:629–648. Spencer NJ, Smith TK. Mechanosensory S-neurons rather than AH-neurons appear to generate a rhythmic motor pattern in guinea-pig distal colon. J Physiol 2004;558:577–596. Krier J, Adams T, Meyer RA. Physiological, morphological, and histochemical properties of cat external anal sphincter. Am J Physiol 1988; 255:G772–G778.

61. Pezim ME, Pemberton JH, Dozois RR. Enteric continence and the ileal pouch-anal procedure. Semin Surg Oncol 1987;3:92–98. 62. Dubrovsky B, Filipini D. Neurobiological aspects of the pelvic floor muscles involved in defecation. Neurosci Biobehav Rev 1990;14: 157–168. 63. Filipini DL, Dubrovsky B. Pelvic floor muscles response to graded rectal distension and cutaneous stimulation. Dig Dis Sci 1991;36: 1761–1767. 64. Percy JP, Neill ME, Swash M, Parks AG. Electrophysiological study of motor nerve supply of pelvic floor. Lancet 1981;1:16–17. 65. Kwakami M. Electromyographic investigation on the human external sphincter muscles of anus. Jpn J Physiol 1954;4:196–204. 66. Walker LB. Neuromuscular spindles in the external anal sphincter of the cat. Anat Rec 1959;133:347 (Abstract). 67. Swash M. Neurology of the sphincters. Clin Exp Neurol 1987;23:1–14. 68. Borghi F, Di Molfetta L, Garavoglia M, Levi AC. Questions about the uncertain presence of muscle spindles in the human external anal sphincter. Panminerva Med 1991;33:170–172. 69. Mackel R. Segmental and descending control of the external urethral and anal sphincters in the cat. J Physiol (Lond) 1979;294: 105–122. 70. McMahon SB, Morrison JF, Spillane K. An electrophysiological study of somatic and visceral convergence in the reflex control of the external sphincters. J Physiol (Lond) 1982;328:379–387. 71. Holmes GM, Hermann GE, Rogers RC, Bresnahan JC, Beattie MS. Dissociation of the effects of nucleus raphe obscurus or rostral ventrolateral medulla lesions on eliminatory and sexual reflexes. Physiol Behav 2002;75:49–55. 72. Marsden CD, Merton PA, Morton HB. Direct electrical stimulation of corticospinal pathways through the intact scalp in human subjects. Adv Neurol 1983;39:387–391. 73. Frenckner B. Function of the anal sphincters in spinal man. Gut 1975; 16:638–644. 74. Molander ML, Frenckner B. Electrical activity of the external anal sphincter at different ages in childhood. Gut 1983;24:218–221. 75. Schuster MM. The riddle of the sphincters. Gastroenterology 1975;69: 249–262. 76. Yeh CY, Pikarsky A, Wexner SD, Baig MK, Jain A, Weiss EG, Nogueras JJ, Vernava AM 3rd. Electromyographic findings of paradoxical puborectalis contraction correlate poorly with cinedefecography. Tech Coloproctol 2003;7:77–81. 77. Chia YW, Gill KP, Jameson JS, Forti AD, Henry MM, Swash M, Shorvon PJ. Paradoxical puborectalis contraction is a feature of constipation in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry 1996;60:31–35. 78. Mathers SE, Kempster PA, Swash M, Lees AJ. Constipation and paradoxical puborectalis contraction in anismus and Parkinson’s disease: a dystonic phenomenon? J Neurol Neurosurg Psychiatry 1988; 51:1503–1507. 79. Sielezneff I, Sarles JC, Sastre B. [Anorectal asynchronism. Clinical, manometric and therapeutic data.] Presse Med 1994;23:1691–1694. 80. Kamm MA, van der Sijp JR, Lennard-Jones JE. Colorectal and anal motility during defaecation. Lancet 1992;339:820. 81. Trendelenburg P. Physiologische und Pharmakologische Versuche über die Peristaltik des Dünndarms. Deutsche Med Wchnschr 1917;43: 1225–1227. 82. Langley JN. The autonomic nervous system. Cambridge: W Heffer and Sons, 1921. 83. Wood JD. Intrinsic neural regulation of intestinal motility. Ann Rev Physiol 1981;43:31–49. 84. Gershon MD. The second brain. New York: Harper Collins, 1998.

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Extrinsic Sensory Afferent Nerves Innervating the Gastrointestinal Tract Michael J. Beyak, David C. E. Bulmer, Wen Jiang, C. Keating, Weifang Rong, and David Grundy Developmental Aspects, 685 Origins and Development, 685 Role of Axonal Guidance and Survival Molecules, 686 Anatomy of Extrinsic Afferent Fibers, 687 Peripheral Projections, 687 Central Projections, 690 Neurophysiology of Extrinsic Gastrointestinal Afferents, 692 Methodologic Considerations, 692 Ionic Determinants of Gastrointestinal Afferent Excitability, 693

Sensory Functions of Gastrointestinal Afferents, 696 Gastrointestinal Extrinsic Afferent Nerves: Integrative Physiology, 706 Regulation of Food Intake: Role of Vagal Afferents, 706 Gastrointestinal Afferents in Pathophysiology, 710 Conclusion, 716 Acknowledgments, 716 References, 716

Gastrointestinal (GI) tract gut functions are controlled and coordinated by the autonomic nervous system consisting of extrinsic (parasympathetic and sympathetic pathways) and intrinsic (enteric nervous system [ENS]) elements. The ENS comprises ganglionic nerve plexuses running between the muscle layers and in the submucosa. The extrinsic vagal and spinal innervation provide the anatomic connection between the central nervous system (CNS) and the gut wall and consists of efferent (sympathetic and parasympathetic), but also afferent, sensory nerves. These extrinsic sensory pathways provide the basis for spinal and brainstem reflex mechanisms that regulate digestive function, provide input to central autonomic circuits that regulate feeding and illness behavior, and cause both painful and nonpainful sensations.

This chapter focuses on the anatomy and physiology of the extrinsic afferent supply to the GI tract, starting with the origin and development of the extrinsic innervation of the gut, followed by an emphasis on the physiologic characterization of the peripheral afferent nerve terminals in the gut and the molecular mechanisms that endow these fibers with their particular response properties. We then discuss the role of these afferents in the modulation of GI functions such as food intake, nausea, vomiting, and alteration of afferent sensitivity in pathophysiologic conditions such as ischemia, intestinal anaphylaxis, visceral pain, and inflammation and their potential role in modulating responses to systemic inflammatory insults such as sepsis. Reviews in previous editions (1) are used as a starting point, and particular emphasis has been placed on advances made during the late 1990s and the beginning of this century.

M. J. Beyak: Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario, Canada, and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom. D. C. E. Bulmer, W. Jiang, C. Keating, W. Rong, and D. Grundy: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

DEVELOPMENTAL ASPECTS Origins and Development In the adult vertebrate, the extrinsic afferent innervation of the GI tract is derived from two sources: vagal and

685

686 / CHAPTER 25 spinal nerves. These primary sensory afferent fibers have their cell bodies located in the jugular/nodose or dorsal root ganglia (DRGs), respectively. Jugular/Nodose Ganglion Complex The jugular and nodose ganglion originate from the neural crest and the second epibranchial placodes, respectively (2). After gangliogenesis, these sensory neurons send projections centrally to the brainstem and peripherally to the visceral organs including the entire GI tract. The central processes terminate mainly in the nucleus tracti solitarii (NTS). The peripheral processes (axons) of these nodose neurons travel in the vagal nerves (cranial nerve X). The sensory cell bodies are located mainly in the nodose ganglia, but others in the jugular ganglia may also supply the GI tract. Data suggest that nodose and jugular sensory neurons supplying the cervical esophagus may be differentiated by chemosensitivity to adenosine triphosphate (ATP) and mechanical responses to distension, leading to speculation that they may subserve different physiologic functions (3), as is the case for the lung (4). Using DiI tracing in fixed mouse embryos, Sang and Young (5) and more recently Ratcliffe and colleagues (6) have detected vagal afferent fibers in the mouse esophagus as early as embryonic day 12 (E12) and in the stomach at E13. In subsequent embryonic days, both the density and the degree of specialization of afferent endings increase. For example, by E15, specialized intraganglionic laminar ending (IGLE)–like endings could be identified. Retrograde tracing from the esophagus revealed the presence of afferent terminals in the NTS as early as E13. By E16, vagal fibers have traveled as far as the jejunum but not the hindgut. Dorsal Root Ganglion Like the nodose ganglion, the DRGs consist of pseudounipolar neurons. These project centrally into the dorsal horn and peripherally run with the splanchnic and pelvic nerves to innervate the GI tract. In contrast with the sensory neurons in the nodose ganglion, a prominent feature of DRG neurons is their functionality and morphologic heterogeneity. These neurons have been classified into subgroups of large, medium, and small diameter that give rise to the myelinated and unmyelinated primary afferent nerve fibers that project to the periphery. The large neurons appear around E9.5 to E11.5, followed by small neurons around E10.5 to E13.5 (7). Neurogenins 1 and 2 (ngn1 and ngn2) are two neuronal determination genes encoding basic helix-loop-helix (bHLH) transcription factors that are required during different phases of DRG neurogenesis (8). ngn1 and ngn2 are coexpressed during much of the early phase, and both are required for the generation of TrkB and TrkC sensory neurons. However, ngn1 is expressed exclusively during the later phase, when small neurons are formed, and is required for most or all of the TrkA-expressing neurons that cause nociceptive afferents. ngn1 expression in the cervical DRG is first detected at E9

to E9.25, and TrkC is detected at E10. TrkA neurons could be detected at E12.5. That extrinsic DRG afferents innervating the adult gut are small, unmyelinated neurons and share many properties with cutaneous nociceptors (e.g., TrkA positivity, capsaicin sensitivity, expression of neuropeptides such as calcitonin gene–related peptide [CGRP]) may hold important clues as to their embryonic origin.

Role of Axonal Guidance and Survival Molecules The terminal distribution of afferents within the gut wall is determined by the interactions between signal molecules (cues) in the surrounding cells and the expression of receptors for these molecules on the growth cone, the tip of a growing axon. These signal molecules can regulate the proliferation and differentiation of neuronal crest precursors, promote axonal outgrowth and guide axons to their targets (guidance cues), match the number of peripheral neurons to their targets (survival factors), and refine the initial connections by regulating terminal branching and synaptogenesis (2,9–11). The neuronal guidance cues that are involved in neural crest cells (NCC) migration and axon navigation have been broadly classified as: (1) the classical neuronal guidance cues that function as either attractive or repulsive cues to guide migrating neural crest cells and axon growth during development; (2) neurotrophic factors; and (3) extracellular matrix and cell adhesion molecules (12). The classical neuronal guidance molecules are the semaphorins (Sema3A) (13), slits (14), ephrins (15), and dual functional netrins causing either attractive or repulsive effects (16). Sema3A signaling prevents the entry of the extrinsic axons to the distal hindgut. Ephrin signaling influences the trunk NCC that causes the sympathetic neurons, whereas the binding Slit to its receptor roundabout (Robo) prevents trunk level NCC from entering the gut. The netrin/DCC (deleted in colorectal cancer, a subset receptor for netrin) signaling mediates the centripetal migration of neural progenitors from myenteric to submucosal region of the mouse gut (17). Netrin/DCC signaling mechanism also functions in the longrange guidance of vagal sensory fibers to the gut, based on a preliminary study (6) that demonstrates that DCC-expressing vagal sensory axons are guided by netrins to targets in the gut. Neurotrophic factors are characterized by their functions not only in neural survival, but also in differentiation and the direction of axon (11,18,19). The neurotrophins and transforming growth factors (TGFs) are the two most important families of neurotrophic factors. The neurotrophin family contains at least five members: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6. These members signal through a receptor complex composed of two types of receptors: the nonselective p75 and more selective Trk subunits. NGF, BDNF, and NT-3 have high binding affinity to TrkA, TrkB, and TrkC, respectively. In comparison, the TGF family comprises glial cell line–derived neurotrophic factor (GDNF), transforming growth factor-β (TGF-β), neurturin (NTN),

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 687 and artemin, signaling through a receptor complex composed of a common receptor Ret tyrosine kinase and coreceptor subunit, GFR-α. GNDF, NTN, and artemin have high binding affinity to GFR-α family members GFR-α1, GFR-α2, and GFR-α3, respectively. Although there has been little study to date regarding the role of specific NGFs in targeting and survival of sensory axons projecting to the GI tract, a number of observations are relevant. The first is that NGF is required for survival of most nodose neurons early in development, although a small number of nodose neurons survive in NGF−/− mice. Interestingly, NGF dependence and tyrosine kinase A (trkA) expression are lost in later development and in the early postnatal period in most nodose neurons (20). In spinal afferents, NGF is crucial for survival of small, unmyelinated, peptidergic, “nociceptor-like” fibers (21). This is of particular interest in the GI tract, given that nearly all adult spinal afferents (~80%) contain CGRP and express the high-affinity NGF receptor trkA (22,23). Besides NGF, the mediators BDNF, NT-3, and NT-4 have been implicated as the key survival-promoting factors for the cranial sensory neurons during the early embryonic stages of target field innervation (20,24–26). For example, BDNF null mice have few nodose neurons, and loss of BDNF results in defective baroreceptor innervation, indicating an important role in targeting of visceral afferents (24). BDNF also is transported to the central terminals, suggesting a role in development of central terminal connections by this antegrade signaling mechanism. NT-4 is also likely important in visceral afferent development, especially in the development of specialized IGLE-like endings, because mice with NT-4 gene deletion have an almost complete loss of IGLEs (27). In the GI tract, few spinal afferents are isolectin B4 (IB4) positive and express the GDNF receptor, whereas the majority express TrkA (22,23). This observation likely provides a clue as to the targeting of extrinsic spinal sensory neurons to the GI tract and viscera. With regard to the GDNF family of neurotrophins, this pathway is clearly important in targeting of enteric neurons to the GI tract, and disruptions in this pathway are implicated in the genesis of Hirschsprung’s disease (28). In the nodose ganglion, GDNF deletion results in an almost complete loss of neurons in the nodose ganglion (29), and GDNF dependence of most nodose neurons is retained into adulthood. What role this family of growth factors has in targeting of vagal axons to the gut remains to be demonstrated.

ANATOMY OF EXTRINSIC AFFERENT FIBERS Peripheral Projections Vagal Afferent Fibers Vagal afferent fibers project distally from the inferior nodose and superior jugular ganglia, located bilaterally proximal to the carotid bifurcation, and innervate the digestive

NTS

SC

IML

NG

DRG RS

PVG

Vagal afferent fiber

spinal afferent fiber IFAN S LM MP

IPAN IPAN

CM SM M

FIG. 25-1. Schematic illustration of the different types of afferent neurons innervating the gastrointestinal tract. Vagal and spinal extrinsic primary afferent neurons possess cell bodies that lie outside the gut wall, in respective nodose/ jugular and dorsal root ganglia, and provide neuronal connection between the gut and central nervous system. Viscerofugal neuronal cell bodies lie within the myenteric ganglia and project outside the gut to synapse within the prevertebral ganglia (intestinofugal afferent neurons) or the spinal cord (rectospinal neurons). Intrinsic primary afferent neuronal cell bodies are found in the myenteric and submucosal plexuses and possess afferent fibers, which are contained within the gastrointestinal tract. CM, circular muscle; DRG, dorsal root ganglia; IFAN, intestinofugal afferent neuron; IML, intermediolateral column; IPAN intrinsic primary afferent neuron; LM, longitudinal muscle; M, mucosa; MP; myenteric plexus; NG, nodose ganglia; NTS, nucleus tracti solitarii; PVG, prevertebral ganglia; RS, rectospinal; S, serosa; SC, spinal cord; SM, submucosa.

tract from the pharynx to the proximal colon (Fig. 25-1). Sequentially, the vagus gives rise to pharyngeal, superior laryngeal, recurrent laryngeal, and esophageal branches, which innervate the pharynx, larynx, and esophagus (30,31). On penetrating the diaphragm, the vagus emerges as dorsal and ventral trunks, continuous with the right and left cervical vagi, respectively. The ventral trunk divides into three branches the hepatic, accessory celiac, and gastric branches, whereas the dorsal trunk divides into celiac and gastric branches (32, 33). The stomach is predominantly innervated by the gastric branches, with additional innervation to the fundus, antrum, and pylorus from the hepatic branch of the vagus (34–36).

688 / CHAPTER 25 The liver and biliary ducts are innervated by the hepatic branch, with a small contribution from the right vagus (37,38), whereas the duodenum is innervated by all branches of the subdiaphragmatic vagus, the greatest contribution provided by the gastric branches (39). The remaining small intestine and proximal colon are innervated by the celiac branches of the vagus (34,40,41). Vagal afferent fibers contribute most of the fibers present within the vagus nerve, outnumbering efferent fibers by a ratio of 9:1. Estimates of the number of afferent fibers in the subdiaphragmatic vagus range among species from 10,000 to 50,000, of which all but 2% are unmyelinated (1). Spinal Afferent Fibers The spinal afferents that innervate the GI tract can be subdivided into two populations, splanchnic and pelvic (42–44). These afferent fibers follow the path of sympathetic

Vagal

and pelvic parasympathetic efferent neurons that project to the gut wall and have their cell bodies in thoracolumbar and sacral DRGs, respectively. Because these afferents run in mixed-nerve bundles that contain the autonomic outflow from the CNS, they are often referred to as parasympathetic and sympathetic afferents. However, this terminology is a misnomer because parasympathetic and sympathetic are terms reserved exclusively for autonomic motor function. The correct terminology refers to them as vagal, pelvic, or splanchnic nerves, because these terms accurately describe the route the three kinds of afferents follow to the CNS. Aside from the visceral afferents in vagal, splanchnic, and pelvic nerves, somatic afferents that innervate the striated musculature of the pelvic floor project to the sacral spinal cord via the pudendal nerve. Spinal afferent fibers transverse paravertebral and prevertebral ganglia to reach their target organs, and pelvic spinal afferent fibers traverse the pelvic ganglia to innervate the distal colon and rectum (Fig. 25-2).

Spinal

Gut

To spinal cord

To NTS Pharynx

SCG

Pharyngeal Superior laryngeal

ICG SG

Recurrent laryngeal esophageal

T2 T3 T4 T5

Hepatic Gastric

Stomach

CG GSN SMG IMN

Celiac

IMG LCN

LSN

HGN

Colon

MPG PN

FIG. 25-2. Schematic representation of the vagal (left) and spinal (right) innervation of the gut. CG, celiac ganglia; GSN, greater splanchnic nerve; HGN, hypogastric nerve; ICG, intermediate cervical ganglia; IMG, inferior mesenteric ganglia; IMN, intermesenteric nerve; LCN, lumbar colonic nerve; LSN, lumbar splanchnic nerve; MPG, major pelvic ganglia; NTS, nucleus tracti solitarii; PN, pelvic nerve; SCG, superior cervical ganglia; SG, stellate ganglia; SMG, superior mesenteric ganglia.

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 689 Although spinal afferent fibers do not terminate within the autonomic ganglia they traverse, the presence of a collateral fiber innervation of the prevertebral ganglia has been demonstrated, suggesting that these spinal fibers may also participate in the generation of local axon reflexes (45,46). Spinal afferent fiber innervation of a particular organ within the digestive tract arises from a broad range of DRGs, although peak distributions are found (Fig. 25-3). As a result, a generalized, overlapping, viscerotopic distribution of spinal afferent fiber cell bodies exists. For example, an individual DRG may innervate many areas of the gut, although the greatest projection may be to one particular area of the gut. This situation is further complicated at the level of the spinal cord, where afferent fibers commonly give rise to collaterals,

Heart

Bladder

Cervical Dorsal esophagus root ganglia Thoracic esophagus C1 Lower C2 esophageal C3 sphincter Stomach C4 C5 Liver C6 Pancreas C7 Duodenum T1 T2 Ileum T3 Colon T4 T5 T6 T7 T8 T9 T10 T11 T12 L1 L2 L3 L4 L5 S1 S2 S3 S4

FIG. 25-3. Schematic illustration of the general viscerotropic distribution of the spinal afferent fiber innervation of the digestive tract within dorsal root ganglia (DRG). Figure illustrates the range (limits of open boxes) and peak distribution (shaded boxes) of DRG labeled after injection of tracer into specified organs from the following studies: esophagus (82,405–408), stomach (83,407,409–412), liver (413,414), pancreas (415–417), duodenum (407,418), ileum (175,419), and colon (22,23,50,59). DRG projecting to the heart (inferior cardiac nerve) (420–423) and bladder (424–429) are shown for comparison.

which course rostrocaudally over two to three spinal segments from their entry point (44,45,47–49). Pelvic spinal afferent fibers arise from a more restricted population of DRGs compared with splanchnic afferent fibers, which are located from L5-S3. However, some overlap occurs in the segmental distribution of lumbar splanchnic and pelvic afferent fibers in the spinal cord (48–50). In addition to the relatively poor viscerotopic distribution of spinal afferent fibers within the DRG, compared with somatic afferent fibers, visceral afferents account for less than 7% of cell bodies present within DRGs, factors that are believed to contribute to the poor localization of visceral versus somatic pain (1,45). In contrast with the vagus nerve, most nerve fibers present within splanchnic or pelvic nerves are efferent nerve fibers, with spinal afferents contributing to only 10% to 20% and 30% to 50% of total nerve fibers present, respectively. Furthermore, the greater afferent innervation of the GI tract is provided by the vagus nerve (see the earlier version of this chapter in the previous edition for a detailed description of afferent fiber numbers) (1). However, because of the greater proximal distribution of vagal afferent fibers with the gut, this ratio diminishes rostrocaudally along the GI tract such that, in the distal colon and rectum, the afferent innervation is predominantly spinal, via the pelvic nerves. Distribution and Morphology of Peripheral Afferent Terminals Using electrophysiologic and anatomic tracing techniques, investigators have demonstrated the presence of afferent fiber terminals within three main tissue layers of the GI tract, namely the mucosa, muscle (circular and longitudinal), and serosa/mesentery (see reviews by Berthoud and colleagues [42], Costa and colleagues [51], Grundy and Scratcherd [52], Powley and Phillips [53], and Sengupta and Gebhart [1]). Mucosa The existence of afferent fibers within the mucosal layer was originally suggested from electrophysiologic studies in which responses to light stroking of the mucosa or application of chemical mediators were lost on removal of the mucosa or treatment with local anesthetic (1,52). More recent studies using fiber tracing techniques have confirmed these early findings, demonstrating the presence of a network of vagal afferent fiber endings extending through the lamina propria, terminating around the crypts and throughout the villi, with some endings terminating at the basal lamina but not extending through the epithelial cells into the lumen (54–56). Mucosal vagal afferents have been described throughout the gut from the esophagus to the distal colon (30,35,54–57). Studies on spinal mucosal afferent fiber are more limited. Although their response to mucosal stroking has been documented (58–60), there is little direct evidence that they contribute to luminal sensing, particularly of nutrients.

690 / CHAPTER 25 Muscle Layers Vagal and spinal afferent fibers terminate within the muscle layers of the GI tract. Most spinal nerve fiber endings have been proposed to be bare nerve endings (61–64), although some reports have indicated the presence of specialized nerve endings within rectal afferent fibers (65,66). By contrast, most vagal afferent fiber endings to the muscle layer of the gut comprise two specialized endings, IGLEs and intramuscular arrays (IMAs), with only a small population of free nerve endings being reported in the intestines (38,56,67). IGLEs were first described by Nonidez in 1946 (68) and later characterized fully by Rodrigo in 1975 (69). They typically consist of a distinct cluster of terminal puncta arising from a single afferent fiber (although this parent axon commonly ramifies into several terminal endings, each consisting of an individual IGLE), which encapsulate a myenteric ganglion or pole of a ganglia. IGLEs are found in close apposition to the ganglia and surrounding muscle layers and are thought to be anchored to both by glial processes, an arrangement suited to the interpretation of shearing forces during stretch or contraction (53). IGLEs have long been speculated to function as mechanoreceptors within the gut, a hypothesis confirmed by an ingenious series of studies combining fast anterograde tracing techniques and electrophysiologic recordings (see Fig. 25-8) (70,71). The distribution of vagal IGLEs is greatest in the proximal gut, where IGLEs have been reported to innervate 100% and 80% of esophageal and gastric myenteric ganglia, respectively, and is weakest in the ileum and colon, where only 10% to 15% of myenteric ganglia have been reported to be innervated by IGLEs (56,57,72,73). Within individual organs, a general rostrocaudal decrease in the density of IGLEs occurs, whereas circumferentially, IGLE numbers are greatest near the point of nerve entry, for example, the mesenteric border, and the lesser curvature of the stomach. Additional areas of increased IGLE innervation are also reported, before and after the major sphincters of the gut, for example, the esophageal hiatus, the prepyloric area of the stomach, and proximal duodenum (56,67). In addition to vagal IGLEs, a population of rectal IGLEs (rIGLEs) has been described, which is supplied by pelvic afferent fibers (65,66). These rIGLEs resemble vagal IGLEs in many ways, although they are smaller, with less extensive branching and clustering of terminals. The second type of specialized vagal afferent endings found in GI smooth muscle, IMAs, were first distinguished by Berthoud and Powley in 1992 (35). IMAs typically consist of two or more parallel terminal processes originating from a single axon and interconnected by bridging elements that course in either circular or longitudinal smooth layers, where they have been found to form appositions with interstitial cells of Cajal (ICCs) (35,74,75). This arrangement of IMAs within the smooth muscle layers has lead to speculation that they function as a further type of vagal mechanosensor within the gut, possibly in conjunction with ICCs. However, data obtained by Zagorodnyuk and colleagues (71) failed to

find a correlation between labeled IMAs and areas of increased afferent fiber activity to von Frey hair probing, indicating that further investigation is need to confirm this hypothesis. IMAs are predominantly located within the stomach, particularly the proximal stomach, where a dense lattice pattern of IMAs is found, and the lower esophageal and pyloric sphincters (35,38,56,67). In the intestines, IMAs are sparse, with a slight clustering found at the junction of the proximal and midcolon segments. Serosa/Mesentery The afferent innervation of the serosa and mesentery is provided by spinal afferent fibers (1,52). Most afferent fiber endings in the serosa and mesentery are thought to be bare nerve endings. Electrophysiologic studies have indicated that in the colorectal area of the gut, the innervation of the mesentery is restricted to splanchnic afferent fibers compared with pelvic afferent fibers, whereas both splanchnic and pelvic fibers were found to innervate the serosa (58). The distribution of splanchnic afferent fiber endings innervating the serosa was found to be greatest near the mesenteric border of the colorectum, whereas serosal pelvic afferent fibers were located circumferentially throughout the colorectum. In addition, the presence of spinal afferent fibers with multiple receptive fields has been documented, some innervating several visceral organs (76,77).

Central Projections Vagal Afferent Fibers Projections from the GI tract to the nodose or jugular ganglia are predominantly ipsilateral, although as many as 20% of subdiaphragmatic afferent fibers may cross within abdominal and thoracic communicating branches (33,78,79). A crude topographic distribution of vagal afferent fiber cell bodies is found in the nodose ganglia: cell bodies projecting to the pharynx, larynx, and upper esophagus are located rostral to cell bodies projecting to the lower esophagus and stomach (80–83). Some studies have shown that distensionsensitive esophageal vagal afferent fibers originating from the nodose, but not the jugular ganglia, respond to application of ATP or α, β-methylene-ATP. Differences exist in the neurochemistry of vagal afferent fiber cell bodies located in the nodose ganglia compared with the jugular ganglia, suggesting that functionally different afferent fibers may arise from each ganglion (3,4,84,85). Central to the nodose/jugular ganglia, the principal site of termination of vagal afferent fibers within the CNS is the NTS, located within the dorsomedial brainstem, between the central canal and the floor of the fourth ventricle (86). Vagal afferent fibers also terminate in the closely associated area postrema, whereas few vagal afferent fibers also are found within the dorsal vagal motor nucleus and the spinal

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 691 trigeminal nuclei (87,88). In addition, a small population of vagal afferent fibers has been reported to project to the upper cervical (C1-4) spinal cord via a supraspinal route (89). Electrophysiologic studies have suggested that this projection may form the substrate of a vagal pain pathway responding to noxious distension of the thoracic esophagus and stomach (90,91). A viscerotopic distribution of vagal afferent fiber terminations within “organ-specific” subnuclei of the NTS exists, leading to the suggestion that reflex vagal input is primarily processed within these organ-specific subnuclei (Fig. 25-4) (92). For example, GI afferent fibers terminate strongly in the parvocellular (also referred to as the gelatinosa or subpostremal) subnuclei of the NTS (39,87,93–96), whereas dense labeling of pharyngeal and laryngeal afferent fibers are found in the intermediate and interstitial subnuclei, and esophageal afferent fiber labeling is observed primarily in the central subnucleus of the NTS (30,80,87,93). Cardiovascular and pulmonary afferent fibers principally terminate within the dorsolateral and ventral/ventrolateral subnuclei, respectively (92). This organ-specific distribution is not, however, mutually exclusive, and visceral afferents have been shown to distribute widely within other subnuclei of the NTS. In addition, the caudal commissural subnucleus of the NTS and its rostral continuation the medial subnucleus receive vagal afferent projections from all viscera, leading to the suggestion that these two areas are predominantly involved in the processing of general sensory input from the viscera. Vagal afferent input is relayed from the NTS via local medullary and ascending supramedullary projections (Fig. 25-5) (97–105). Local medullary connections are primarily involved in the generation of autonomic reflexes

terminating within the vagal preganglionic motor nuclei and caudal and ventral medulla. Ascending projections terminate within areas associated with the central control of food intake such as the medial, lateral, and paraventricular hypothalamus, and areas associated with generating the emotional and behavioral responses to visceral sensory input such as the periaqueductal gray (PAG), bed nucleus of the stria terminalis (BNST), paraventricular thalamic nucleus (PVT), and central nucleus of the amygdala (CNA). The major recipient of ascending projections from the NTS is the parabrachial nucleus (PBN), acting as a second major relay site for vagal afferent input within the brain. The PBN sends strong projections not only to areas directly innervated by the NTS such as the hypothalamus, PAG, and CNA, but also to areas not directly innervated by the NTS, such as the ventroposterior thalamus. The ventroposterior thalamus, in turn, projects to the insular cortex, an area strongly implicated in the affective response to visceral sensation (106–108). Some studies also have indicated that the somatosensory cortex, an area involved in the processing of the more cognitive and discriminatory aspects of pain, also receives vagal input (109,110). In addition, supramedullary structures, such as the PAG, CNA, and hypothalamus, also send descending projects back to the NTS, preganglionic nuclei, and other medullary areas involved in the generation of autonomic reflexes, forming the basis of higher center control of autonomic reflexes (92,99,111–113). Spinal Afferent Fibers Spinal afferent fibers project centrally through dorsal roots, and to a lesser extent ventral roots, to enter the spinal

Vagal

Spinal Gastrointestinal Cardiovascular DRG

Esophageal dm dl vl

ts i v

I c

pc m dvn

ap

Pharynx/larynx I

Pulmonary IV

All viscera

V VII

ts

II III

X

com dvn

FIG. 25-4. Schematic representation of the viscerotopic distribution of (left) vagal afferent fiber input from different visceral organs within the different subnuclei of the nucleus tracti solitarii and (right) spinal afferent fibers within the lamina of the dorsal horn. ap, area postrema; c, central subnucleus; com, commissural subnucleus; dl, dorsolateral subnucleus; dm, dorsomedial subnucleus; DRG, dorsal root ganglia; dvn, dorsal vagal motor nuclei; i, interstitial subnucleus; I, intermediate subnucleus; m, medial subnucleus; pc, parvocellular subnucleus; ts, solitary tract; v, ventral subnucleus; vl, ventrolateral subnucleus.

692 / CHAPTER 25

pvt

to BNST and PVN

lat hyp

vpm

vpl cna

m hyp

STT

SHT

pag

SMT pbn

SPT

na RVLRF

SRT

vmm gr cu nts

SST

nra CVLRF

DC

Vagus

Spinal

FIG. 25-5. Schematic representation of ascending vagal afferent inputs (left, arrows) and spinal afferent inputs from the spinal cord (right, circles) within the central nervous system. BNST, bed nucleus of the stria terminalis; cna, central nucleus of the amygdala; cu, cuneate nucleus; CVLRF, caudal ventrolateral reticular formation; DC, dorsal column; gr, gracile nucleus; lat hyp, lateral hypothalamus; m hyp, medial hypothalamus; na, nucleus ambiguous; nra, nucleus retroambiguous; nts, nucleus tracti solitarii; pag, periaqueductal gray; pbn, parabrachial nucleus; PVN, paraventricular hypothalamus; pvt, paraventricular thalamic nuclei; RVLRF, rostral ventrolateral reticular formation; SHT, spinohypothalamic tract; SMT, spinomesencephalic tract; SPT, spinoparabrachial tract; SRT, spinoreticular tract; SST, spinosolitary tract; STT, spinothalamic tract; vmm, ventromedial medulla; vmp, ventromedioposterial thalamus; vpl, ventroposterolateral thalamus.

cord via Lissauer’s tract. There they give off collaterals, which, in addition to traversing rostrocaudally along the spinal cord, course medially and laterally surrounding the dorsal horn to terminate mainly in lamina I and V (see Fig. 25-5) (47–49,114–116). In addition, a few collateral fibers continue to terminate in lamina VII, some in close proximity to preganglionic cell bodies, whereas others terminate in lamina X. A high degree of convergence occurs in the termination of somatic and visceral afferent fibers within the spinal cord, which is believed to form the basis of the somatic referral of visceral pain (47,117–125). Convergence of visceral afferent

fibers from the colon and rectum, with fibers from pelvic organs such as the bladder, vagina, and uterus, has also been reported within the lumbosacral spinal cord (126,127). Dorsal horn neurons project throughout the CNS via several predominantly contralateral ascending pathways located within the ventrolateral, lateral, and dorsolateral white matter of the spinal cord, namely the spinothalamic (109,128,129), spinohypothalamic (125,130,131), spinoreticular (132–134), spinomesencephalic/parabrachial (135–137), and spinosolitary tracts (138–140) (see Fig. 25-5). Furthermore, an additional postsynaptic ascending pathway also arises within the spinal cord, projecting via the dorsal column to the gracile and cuneate nuclei of the brainstem to the contralateral ventroposterolateral thalamus (141–143) (see Fig. 25-5). The spinothalamic and dorsal columns are the most important ascending pathways for the relay of visceral afferent fiber input from the spinal cord to the CNS (45,144). Although the dorsal column pathway was not originally thought to be involved in the signaling of nociceptive inputs (145), clinical reports, supported by experimental studies, have demonstrated that disruption of the midline spinal cord leads to relief of pain in patients with pelvic cancer or behavioral responses in animals, strongly implicating the dorsal column pathway in the transmission of visceral pain (143,146). Within the cerebral cortex, the somatosensory and limbic/ paralimbic cortices have been identified to respond to stimulation of the GI tract and are thought to be important in the processing of the cognitive and affective aspects of visceral pain, respectively (see Chapter 26). In addition to ascending pathways, the activity of neurons within the dorsal horn has been shown to be both facilitated and inhibited via a number of descending pathways, which travel ventral, ventrolateral, and dorsolateral to the spinal cord (147). These include a descending inhibitory pathway activated by vagal afferent input providing a mechanism via which vagal afferent fibers may influence spinal afferent input (148).

NEUROPHYSIOLOGY OF EXTRINSIC GASTROINTESTINAL AFFERENTS Methodologic Considerations Afferent Recording The electrical activity of extrinsic afferents to the GI tract have been recorded from perivascular nerve branches near the gut wall, from dorsal rootlets entering the spinal cord, or from branches of the vagal trunk in anesthetized animals and in isolated preparations. Perivascular nerve recording with wire or suction electrode is a useful technique for assessing the extrinsic GI afferents as a whole, but the limitation is that it is difficult to make conclusions on specificity of activation, receptive fields, or the thresholds to stimulation. A modification of this technique is to tease the nerve bundle until only a few fibers are recorded. If the action potentials generated by different fibers are sufficiently different in amplitude and waveform, it is possible to separate single

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 693 units electronically, thus allowing simultaneous assessment of the activity of several individual units. However, caution is necessary when using electronic discrimination, because as the number of viable units increases, the chance of action potentials from different units occurring simultaneously and thus summating increases. Another drawback of multiunit recording from perivascular nerve bundles is its inability to differentiate among vagal, spinal, and intestinofugal fibers, which potentially play different roles in GI function. Singleunit recording from dorsal rootlets or the vagal trunk or microelectrode recording from sensory ganglia can avoid some of the limitations of multiunit recordings, but they are more tedious, time consuming, and difficult to perform in the mouse, which is increasingly being used because of the potential for genetic manipulation. Dissociated Cells: Intracellular, Patch-Clamp Recording and Calcium Imaging In essence, fiber recording provides information on the discharge frequency of sensory neurons, but is it unable to provide information on the activity of ion channels or receptors that are responsible for the “generator potential” at the nerve terminals. Thus, an alternative approach to the study of impulse generation in visceral afferents in situ is to study neurons in culture by patch-clamp recording and calcium imaging. Using this approach, primary afferent neurons innervating the GI tract are labeled with fluorescent dyes injected into selected points of the gut wall and labeled neurons selected for subsequent study (149–151). Combining other cell biological and molecular techniques, this approach offers enormous potential, especially for investigating the biophysical and pharmacologic properties of ion channels on sensory neurons; but there are several issues that need to be considered. First, do the cell soma have the same sensory modalities

(ion channels, receptors) as the nerve endings? Second, does the culturing process (dissociation, incubation, adding NGFs, etc.) cause phenotypic changes in ion channels and receptors? Third, nerve endings in situ form elaborate connections with the extracellular matrix, and this has implications in sensory transduction. Nonetheless, these techniques have proved extremely valuable in the study of ion channels in GI afferents.

Ionic Determinants of Gastrointestinal Afferent Excitability Hodgkin and Huxley first demonstrated the critical role of sodium and potassium conductances in the initiation and termination of the action potential. The rapid upstroke of the action potential is mediated by regenerative opening of voltagegated sodium channels that are opened with a depolarizing stimulus, allowing influx of sodium ions. The action potential is terminated by the combination of time and voltagedependent inactivation of these channels and the opening of a voltage-sensitive outward (potassium) conductance. Using combined pharmacologic, electrophysiologic, and molecular techniques, researchers have made significant advances in the identification of the channels that underlie these conductances and how they may be influenced by disease states, with implications for the genesis and treatment of visceral pain. Voltage-Gated Sodium Channels in Extrinsic Gastrointestinal Afferents Broadly speaking, voltage-gated sodium channels in sensory afferents can be characterized based on their sensitivity to nanomolar concentrations of the puffer fish (fugu) toxin tetrodotoxin (TTX) (Fig. 25-6). TTX-sensitive (TTX-S) channels are blocked by low concentrations of TTX, whereas

TTX (1 µm)

Control 40 mV 20 ms

−52 mV

−54 mV

A Fast TTX-S

Slowt TTX-R

Persistent TTX-R

2 nA 1 nA 10 ms

100 ms

B FIG. 25-6. Sodium channels in dorsal root ganglion (DRG) neurons innervating the colon. (A) Most colonic DRG neurons have tetrodotoxin-resistant (TTX-R) action potentials. (B) Three voltage-gated Na+ currents can be distinguished based on TTX sensitivity and their inactivation kinetics. A fast inactivating TTX-sensitive (TTX-S) current, a slow inactivating TTX-R current, and an ultra slowly inactivating or persistent TTX-R current. (Reproduced from Beyak and colleagues [22], by permission.)

694 / CHAPTER 25 TTX-resistant (TTX-R) channels are blocked by concentrations of TTX 100- to 1000-fold greater (152,153). The α subunits of these channels determine most of their pharmacologic and biophysical properties, and nine subunits have been identified (154). In unselected DRG neurons, NaV1.7 and, to a lesser extent, NaV1.1, NaV1.2, NaV1.3, and NaV1.6 carry much of the TTX-S current, with NaV1.3 being particularly important in situations of nerve injury/axotomy (155). The molecular identity of TTX-S currents in visceral afferents is unknown; however, a variety of studies have recorded TTX-S currents from DRG and nodose cell bodies projecting to the stomach (156,157), ileum (158), and colon (22,159, 160). The TTX-S currents recorded in these studies have biophysical properties resembling those known for NaV1.7 (relatively low threshold for activation, fast inactivation, hyperpolarized inactivation curve, and slow repriming kinetics) (22,156–158). TTX-R currents have received a great amount of attention in recent literature, largely because of their preferential localization to sensory neurons, particularly small, unmyelinated, nociceptor-like fibers, as well as the relatively selective effect that inflammation and other disease states might have on these channels. The major TTX-R channels are NaV1.8 and NaV1.9 (152). NaV1.8 has a relatively high threshold for activation, slow inactivation kinetics, and rapid repriming kinetics (161). Gene knockout and modeling studies suggest a potential role for this channel in repetitive firing in response to a prolonged depolarizing stimulus, possibly by virtue of the channel’s rapid repriming kinetics (162). The second TTX-R channel NaV1.9 has unique properties. It has ultraslow or persistent inactivation kinetics, a hyperpolarized inactivation curve, and significant overlap between activation and inactivation curves (163,164). These properties suggest that this channel would likely be important in influencing overall membrane excitability and could amplify small depolarizing stimuli (165). Messenger RNA (mRNA) and immunocytochemical staining for NaV1.8 and NaV1.9

has been studied in bladder afferents. The former channel was present in most bladder afferents, whereas the latter was present in only a few (166). This observation seems also to be true in DRG neurons innervating the colon, ileum, and stomach, where NaV1.8-like currents are present in most DRGs innervating these organs (22,156–158,160,167), whereas only one study has systematically looked for persistent, NaV1.9like currents in GI afferents, and only a small proportion (~13%) of colonic DRGs had a persistent, low-threshold current (22). Because NaV1.9 is preferentially expressed in GDNF-sensitive (168), IB4-reactive neurons, perhaps it is not surprising that colonic afferents have little of this current, because most are IB4 negative (22,23). Thus, it seems likely that the major TTX-R current in GI afferents is carried by NaV1.8; however, evidence demonstrating either mRNA or immunohistochemically detectable NaV1.8 or NaV1.9 is lacking. Voltage-Gated K+ Channels in Extrinsic Gastrointestinal Afferents Voltage-gated potassium currents generally speaking serve to allow the membrane to accommodate a depolarizing stimulus, as well as to repolarize the membrane and limit repetitive firing (169). In sensory neuron cell bodies, the macroscopic Kv currents can be classified based on their inactivation kinetics. The first is a rapidly inactivating current, with a hyperpolarized threshold, and inactivation curve and is known as IA (Fig. 25-7). IA currents have been shown to be present in DRG neurons innervating the stomach (170), ileum (158), and colon (171), as well as the urinary bladder (172–174), and in nodose neurons innervating the stomach (170). The importance of this channel in determining membrane excitability is illustrated by that in both ileal (175) and bladder DRG neurons (158), blocking this current using the drug 4-aminopyridine (4-AP) results in a marked increase in the number of action

−100 mV

−60 mV

−100 – (−60)

Control

4-AP

Control – 4-AP

A

B FIG. 25-7. Whole-cell K+ currents in ileal dorsal root ganglion (DRG) neurons. Outward K+ currents can be distinguished based on (A) manipulating the holding potential, thus selectively inactivating the IA at more depolarized potentials, or (B) pharmacologically, using 4-aminopyridine (4-AP), revealing an inactivating (IA) current (right) and a sustained (IK) current (middle). (Reproduced from Stewart and colleagues [158], by permission.)

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 695 potentials fired in response to a depolarizing stimulus. Similarly, recordings from esophageal vagal single fibers demonstrate that 4-AP and α-dendrotoxins, both blockers of IA-like currents, increase basal firing rate and stretch-evoked firing, indicating an important role for these channels in regulating the excitability of the GI sensory nerve ending (176). The molecular determinants of the IA current are complex and are beyond the scope of this chapter; however, the macroscopic IA is likely a composite of several currents, with slight differences in activation properties and kinetics. The blockade of this current by α-dendrotoxins suggests the presence of Kv1.1, Kv1.2, and Kv1.6 subunits (177), which in heterologous systems cause an IA-like current. The second major macroscopic potassium current seen in GI extrinsic sensory neurons is a delayed rectifier or IK current. This current shows little inactivation over time and has a relatively depolarized threshold of activation (see Fig. 25-7). It is thought that this current contributes significantly to the repolarizing phase of the action potential and possibly the setting of the membrane potential (169). These currents have been demonstrated in DRG neurons innervating the ileum, stomach, colon, and bladder (158,170–172,174), as well as nodose neurons innervating the stomach (170). The Kv subunits causing this current in GI sensory afferents are unclear; however, based on studies in heterologous expression systems, Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1, Kv2.2, and Kv3.1 are prime candidates. Calcium-Activated Potassium Channels There exists a further class of potassium channels, some of which are weakly voltage sensitive but are primarily gated by increases in intracellular calcium concentration, which occur during membrane depolarization. The channels are classified by their single-channel conductance as small (SK), intermediate (IK), and large (BK). These channels may be important in regulating firing through increases in potassium conductance in response to calcium accumulation. They are also important in the slow postspike afterhyperpolarization (178,179). There has been no direct published evidence for the presence of these channels in GI afferent neurons; however, several indirect lines of evidence suggest a role. First, mesenteric afferent firing rate is increased by removal of extracellular calcium. Second, these channels are present in unselected vagal and DRG neurons (178, 179). Third, activation of BK channels in airway vagal afferents inhibits responses to chemical stimuli (180). However, the one study that has specifically examined the effects SK and BK channel inhibition on the firing of mechanosensitive afferents innervating the esophagus (176) failed to find any effect. In contrast, one report suggests that after infection mesenteric DRG neurons have a reduced afterhyperpolarization (181), which is thought to be caused by an IK-like potassium current. Clearly, further work in this area is needed to clarify the role of calcium-activated potassium channels in both vagal and spinal afferents innervating the GI tract.

Hyperpolarization-Activated Currents IH currents are activated at hyperpolarizing potentials and conduct cations (predominantly K+) into the cell. IH currents are mediated by channels of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family (182). On hyperpolarization of the membrane, these inwardly rectifying channels slowly open, returning the membrane toward its resting potential. This is evident on current-clamp recordings as a “sag” in the membrane potential at hyperpolarized voltages (Fig. 258). Currently, there has been no direct demonstration of HCN channels in GI nodose or DRG neurons; however, IH currents and HCN1, HCN3, and HCN4 subunits are present in nodose neurons, and they play a role in regulating aortic arch mechanoreceptor activity, because blockade of IH by external Cs+ increased somal excitability and reduced the threshold for discharge from these baroreceptor afferents innervating the aortic arch (183). In addition, preliminary experiments indicate the presence of an inward rectifier current in some colonic DRG neurons (Beyak and Vanner, unpublished observations, 2003) (see Fig. 25-8) and external Cs+, which blocks IH currents, causes an inhibition of spontaneous and stretchevoked firing in intestinal mesenteric afferents (Rong and colleagues, unpublished observations, 2004). Work in the guinea pig intestine suggests that this current may be an important mechanism of inflammation-induced alterations in neuronal firing of enteric neurons in inflammatory states (184), and this bears further investigation in extrinsic afferents. The physiologic effect of IH on neuronal firing is controversial and likely depends on the biophysical properties of the particular HCN channels present, as well as the state of the membrane potential. Being an inwardly rectifying current, IH should have a depolarizing influence on membrane potential. This is supported by the observation that Cs+ often hyperpolarizes membrane potential (183). However, this channel may also open partially at resting membrane potential, and therefore contributes to decreasing the membrane resistance, hence increasing the ability of the membrane to accommodate a depolarizing stimulus. Voltage-Gated Calcium Channels Voltage-gated calcium channels can be grouped based on their voltage sensitivity. High-voltage–activated channels include N-, P-, Q-, and L-type channels, and low-voltage– activated channels include T-type channels. Little work has been performed on GI or other visceral sensory neurons with respect to calcium channels; however, some findings bear particular mention. The first is that in bladder afferents, HVA channels are present, both L- and N-type, and the N-type channels are inhibited by nitric oxide donors (185). Second, in colon DRG afferents, high voltage activated (HVA) channels are present, and the N-, P-, and Q-like currents are inhibited by κ-opioids (186). Because N, P, and Q channels are important in calcium entry that allows synaptic release, these observations may be important for neurotransmission in the dorsal horn, as well as modulating transmitter release at the organ level via activation of axon-like reflexes.

696 / CHAPTER 25

−59 mV

3s

3s

0, −50, −100, −150, −200 pA

A

−60, −65, −70...−120 mV

B

FIG. 25-8. Hyperpolarization-activated currents in gut-projecting dorsal root ganglion (DRG) neurons. (A) Whole-cell patch-clamp recording from DRG neuron retrogradely labeled from the gut. Hyperpolarizing current command results in hyperpolarization, followed by a slow “sag” of the membrane potential toward rest (arrow), or inward rectification. Scale bar = 40 mV. This is followed by anode break excitation on repolarization of the membrane. (B) Voltage-clamp recording from same neuron using hyperpolarizing voltage commands demonstrate the onset of a slowly activating inward current. Scale bar = 200 pA.

The presence of low-voltage–activated T-type channels in visceral neurons has not been examined; however, the finding of an important role for these channels in mechanoreceptors (178,179,187) raises important questions about a potential role in GI mechanosensation, a phenomenon that has not been completely elucidated. Sensory Functions of Gastrointestinal Afferents It is now well accepted that extrinsic afferents convey a vast amount of information about the physical and chemical condition of the GI tract. However, the GI tract was originally thought to be a rather insensitive organ. Stimuli such as cutting, crushing, and burning, which cause pain if applied to the skin, are not perceived when applied to patients with open colostomies. One explanation for this is that visceral afferents have adequate stimuli that are different from those of somatic afferents. The issue of what constitutes an adequate visceral stimulus was addressed by Ruch (188): The adequate stimuli for visceral afferents are those arising from their environment and especially from their own activities and pathologic states. Such adequate stimuli include: (i) sudden distension against resistance; (ii) spasm or strong contraction, especially when accompanied by ischemia; (iii) chemical irritants; and (iv) mechanical stimulation, especially when the organ is hyperemic (stomach).

It is now clear that the GI tract is richly innervated by vagal and spinal afferents that respond to mechanical deformation and are sensitive to intraluminal nutrients, protons, and inflammatory mediators. Since the seminal work by Iggo (189) and Paintal (190,191), the activity of GI afferents in response to a variety of stimulations has been investigated

vigorously, and this has been examined in great detail by Sengupta and Gebhart in the previous edition of this textbook (1) and in other reviews (192,193). This section gives a rather concise review of the neurophysiology of GI afferents with more emphasis on the current understanding of the molecular mechanisms of afferent sensitivity. General Properties Most vagal and spinal fibers are C-fibers with a conduction velocity of slower than 2 m/sec. A smaller percentage of fibers are Aδ fibers with conduction velocity between 2 and 16 m/sec (1). Vagal and spinal GI afferents normally display some level of spontaneous discharge. The spontaneous activity persists in in vitro recordings (194), so it cannot be explained by the flow of intraluminal contents. This is in contrast with the urinary bladder afferents, which display a relatively low level of or no spontaneous activity normally but can develop spontaneous discharge after chemical stimulation (195–197). The spontaneous activity in small intestinal afferents is attenuated by 5-HT3 antagonist, granisetron, and by transient receptor potential vanilloid receptor 1 (TRPV1) antagonist, capsazepine, suggesting that tonic activation of 5-HT3 and TRPV1 contributes to the spontaneous activity (194,198). Mechanosensitivity In the literature, different forms of mechanical stimulation have been used to investigate the neurophysiologic properties of mechanoreceptors of the GI tract. Most commonly, balloon inflation or saline infusions are used. Distension can be done slowly (ramp distension) or rapidly as pressure pulses

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 697 sensitive to light stroking but are not easily accessible in tube preparations.

(phasic distensions). The mechanoreceptors have been classified by their pressure or volume thresholds to balloon or saline distension. However, notably, this approach is far from satisfactory because neither volume nor pressure per se can be considered as the actual mechanical force leading to activation of afferent terminals. Mechanical forces imposed on tissues induce stresses and strains in both circumferential and longitudinal directions and radial compressive stress and strain (199). For the study of mechanoreceptor kinematics, ideally the stimulus should be expressed in terms of magnitude and direction of stress, strain, and strain rate; however, this is impractical in most experiments. An alternative approach that allows measurement of circular or longitudinal tension, or both, has been adopted by several groups (58,60,70,76,200). A segment of gut (esophagus, stomach, colon, etc.) was opened up into a flat sheet, and the preparation was stretched with the tissue stretcher while monitoring circumferential tension. The receptive fields of mechanoreceptors can be pinpointed using calibrated von Frey hairs. The other significant advantage of the flat-sheet preparations is for characterizing mucosal receptors, which are not activated by distension, yet they are

Mechanosensitive Responses in Gastrointestinal Afferents Extrinsic afferents of the GI tract are composed of different functional populations with distinct stimulus–response properties. The activity of these different populations account for the typical biphasic responses to ramp distension seen in multiunit recordings of the mesenteric nerve in the rat and the mouse representing low- and high-threshold components (194,201). When single-unit activity was analyzed using spike sorting, the pressure–response profiles of single units could distinguish three functional types of afferent fibers to the mouse small intestine (194) (Fig. 25-9). The first type had a low threshold, and its activity reached a peak rapidly in the first few millimeters of Mercury increase in pressure. There was no further increase in discharge in these afferents with further increases in pressure up to 60 mm Hg. Indeed, in many cases, the activity decreased. These are typical of low-threshold, slowly adapting fibers described in many previous studies. The second type also had a low threshold

Rat

Mouse

Firing frequency

40 mmHg

75 50 25 0 500 250 0

−250 60

80 Hz

Intrajejunal pressure

IP Nerve activity Discharge rate (mmHg) (µV) (spikes/s)

Onset of ramp distension

1 min

0

C A ∆ Afferent discharge (spikes/s)

30 sec

4 3

100

B

Wide dynamic range fibers High threshold fibers Low threshold fibers

5

2 50

1 0

0

−1 0

20 40 Intrajejunal pressure (mmHg)

60

D

0

10 20 30 40 50 Intraluminal pressure (mmHg)

60

FIG. 25-9. Multiunit mesenteric afferent response to distension of the small intestine. (A) Example of biphasic multiunit response to jejunal distension in the rat in vivo. (B) The pressure–nerve response curve in control (open square) and in vagotomized (filled square) rat. *, significantly different (p < 0.05.) (C) Example of the multiunit mesenteric nerve to ramp distension in mouse jejunum preparation in vitro. (D) Pressure–response relations of three functional classes of fibers. IP, intrajejunal pressure. (A,B: Reproduced from Booth and colleagues [201], by permission; C,D: Reproduced from Rong and colleagues [194], by permission.)

698 / CHAPTER 25 for activation, but the discharge gradually increased further with the increase of pressure. These have been described as wide dynamic range fibers (a term originally used to describe a population of dorsal horn neurons). The third type had a threshold at more than 20 mm Hg, and it continues to encode into the noxious range of stimuli. These were classified as high-threshold fibers. The presence of low- and high-threshold fibers has been demonstrated by single-unit recordings from various regions of the GI tract (see Fig. 25-9) (202–205). It is generally accepted that although low-threshold fibers are of both vagal and spinal origin, high-threshold fibers are almost exclusively spinal afferents, a notion that is supported by a number of observations. The mesenteric afferent activity at low intraluminal pressure was diminished, whereas the level of activity at high distension pressure remained unchanged in vagotomized rat (201). In the stomach, all distensionsensitive vagal afferents are low-threshold fibers, whereas spinal afferents consist of both low- and high-threshold fibers (206,207). Vagal and spinal afferents likely convey information that is of a different nature, with vagal afferents conveying physiologic information, whereas spinal afferents can encode noxious events; however, there remains controversy regarding whether vagal afferents play a role in nociception as well. GI afferent fibers are also classified based on the location of their receptive fields as mucosal, muscle, and serosal or mesenteric receptors (Fig. 25-10). This approach of classification is preferred in many studies, because ultimately the position of the receptor endings within and outside the gut wall is largely responsible for determining receptor

Probing

characteristics (52). The electrophysiologic properties of these receptors have been investigated in detail by Blackshaw and coworkers (58,60,76,208,209). Mucosal Afferents. The mucosal afferents are sensitive to light stroking of the mucosa, generating a brief burst of impulses each time the stimulus passes over the receptive field. They are relatively insensitive to distension, contraction, or compression, except under circumstances when distortion of the mucosa occurs as a consequence (see Fig. 25-10). For example, friction between a luminal balloon and the mucosal surface during inflation and deflation of the balloon, or when a contraction passing over a stationary balloon evokes a brief burst of impulses (210). Mucosal afferents share some features with a population of afferents found in the urinary bladder (211,212) and colon (213), referred to as “silent nociceptors” (214). They normally show no resting activity, do not respond to distension but do respond to chemical stimuli, and may develop spontaneous firing and mechanosensitivity after exposure to chemical stimuli. These “silent” receptors are believed to be nociceptors. However, the location and the polymodal response nature of these fibers also suggest a key role in sensation of the presence and composition of luminal content (see detailed discussion in the following sections). Muscle Afferents. GI muscle afferents were initially described by Iggo (189,215,216) and Paintal (190,191). In these early studies, the typical response of a vagal afferent fiber to distension of the stomach consisted of an initial phasic component that lasted a few seconds, during which the discharge frequency reached 30 to 40 impulses per second, after which the discharge frequency adapted to

Stretch

Mucosal stroking

Serosal

Muscular

1 minute

Mucosal

FIG. 25-10. Classification of mucosal, muscle, and serosal afferents of the mouse colon. Colonic afferent terminals can be identified based on location of their receptive field and stimulus modality. Serosal endings respond only to probing of the serosal surface, muscular endings respond to stretch and probing, whereas mucosal endings respond to stroking of the mucosa with a von Frey hair, as well as probing. (Reproduced from Brierley and colleagues [58], by permission.)

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 699 a lower level that continued as long as the distension was maintained. Paintal adopted the term stretch receptor because he considered it unlikely that the receptors would be activated by an increase in intragastric pressure unaccompanied by stretching. Iggo introduced the term in-series tension receptor because the afferent discharge was also increased by contractions that occurred spontaneously in the goat stomach during distension or were evoked locally by electrical stimulation. Afferents that behave functionally as in-series tension receptors exist throughout the GI tract, although regional differences in response characteristics are seen. For example, the vagal mechanoreceptors supplying the corpus differ dramatically in their stimulus–response characteristics from those receptors of the antrum (217). Corpus mechanoreceptors display a low-frequency, irregular, spontaneous activity that increases dramatically when the volume of the stomach is increased within the physiologic range of gastric filling. In contrast, antral mechanoreceptors do not readily respond to increases in gastric volume, but instead generate a burst of impulses as each wave of contraction passes over the receptive field. That is, the corpus mechanoreceptors are sensitive to passive stretch, whereas antral mechanoreceptors respond preferentially to active contraction. One explanation for such regional difference in the response characteristics of muscle mechanoreceptors is that a single type of mechanoreceptors can encode different aspects of mechanical stimuli according to the mechanical responses of the muscle they innervate (217). Another explanation is that two classes of mechanoreceptors exist and have different regional distributions: one for detecting passive stretch (in-parallel receptors), and the other for active contraction (in-series receptors). This hypothesis is supported by the discovery of two morphologically distinct vagal mechanoreceptor endings, namely IGLEs and IMAs, within the smooth muscle layers of the gut. It has been hypothesized that the biophysical arrangement of IGLEs and IMAs allows them to transduce different forms of forces (53). IGLEs have been proposed to transduce shearing forces produced in association with changes of tension (both active and passive) and stretch in the muscle wall. Evidence supporting this view has been elaborated. Zagorodnyuk and coworkers (70,71,218) recorded vagal axons near an isolated segment of gut that was subjected to controlled circumferential stretch, mapped the receptive field of the unit using calibrated von Frey hairs, and filled its axon with an anterograde tracer that will identify its receptor endings (219) (Fig. 25-11). They demonstrated convincingly that the receptive “hot spots” correspond to the location of IGLEs. IMAs, being in parallel to smooth muscle fibers, have been proposed to transduce passive stretch, but direct electrophysiologic evidence for this remains lacking. Serosal Afferents. The third type of afferent is sensitive to probing with relatively strong forces, and because the threshold is typically lower when stimulated from the serosal side, they have been designated serosal receptors (60,220). These receptors were mainly described in the lower GI tract, but

they appear to exist throughout the gut (59,60,220,221), and the morphology of their endings has not yet been identified. Serosal afferents commonly have multiple receptive fields. Probing usually produces a short burst of impulses, although the mechanical threshold to probing and the frequency of the evoked response vary considerably. The response of serosal afferents to distension (or stretch) is also rather variable, often occurring only at the onset of distension (or stretch) consistent with the movement of the tissue. Bessou and Perl (222) describe the serosal mechanoreceptors in the small intestine as “movement receptors,” because movement of the mesentery evoked a maximal response, and distension or contraction of the adjacent viscus causing mesenteric distortion also evoked a response. Many of these serosal afferents also respond to chemical stimuli applied to the luminal side (60), suggesting multimodal chemoreceptors. Mechanisms of Mechanotransduction Our understanding of the molecular makeup of mechanosensors is still vague despite the widespread use of mechanosensation in biological systems. Sensory endings have close structural and functional interaction with the tissue they innervate, and the accessory elements (extracellular matrix) are part of the receptor units that are indispensable for normal signal transduction. There are also close interactions with other cell types such as epithelial endocrine cells, ICCs, and immune cells, which potentially can mediate or modulate mechanosensation. It has been proposed that cells transduce mechanical force by using two types of mechanisms: a physical mechanism that relies on mechanosensitive ion channels, and a chemical mechanism that involves mechanosensitive release of mediators from secondary cells in the gut wall. Mechanosensitive Ion Channels. Experiments in the 1980s by Corey and Hudspeth (223) in hair cells and by Sachs (224) in muscle showed that mechanical stimuli could directly activate ion channels. Mechanically induced current has been demonstrated in GI afferent neurons. Raybould and coworkers (225) demonstrated that 40% of gastric or colonic DRG neurons in culture responded to mechanical probing by an influx of calcium; the response was abolished by superfusion with gadolinium, a known blocker of mechanically induced currents in a variety of cell types. Su and coworkers (151) recorded a mechanosensitive potassium current in rat S1 DRG neurons supplying the colon. The current was blocked by tetraethylammonium, amiloride, and benzamil, but not by gadolinium. The molecular identity of the ion channels underlying the mechanosensitive currents remains unknown. However, progress in understanding mechanotransduction in other systems has provided important clues. Two families of potential mechanosensitive cation channels have been identified in genetic screens in the nematode Caenorhabditis elegans, in Drosophila melanogaster, and in zebra fish. These are the transient receptor potential (TRP) family of ion channels and degenerin/epithelial Na+ channels (DEG/ENaC)

700 / CHAPTER 25

Hot spot 1

Hot spot 2

B

Hot spot 3

C

A

von Frey hair/hot spot 3

von Frey hair/hot spot 1

Vagal unit

200 µV

1s 20 Hz

Rate

D

E

FIG. 25-11. Intraganglionic laminar endings (IGLEs) are the transduction sites of muscle mechanoreceptors. (A) Photomontage of preparation shows fluorescent biotinamide-labeled nerve fibers and dark carbon particles marking three hot spots. A filled IGLE was located close to each marked hot spot. (B, C) Confocal microscopic images at higher resolution show the characteristic branching lamellar structure of IGLEs near hot spots 1 and 3. Effects of applying the 0.4 mN von Frey hair to hot spots 1 and 3 are shown in D and E, respectively. This unit also was activated by circumferential stretch (not shown). Scale bars = 500 µm (A); 100 µm (B, C). (Reproduced from Zagorodnyuk and colleagues [70], by permission.)

(see reviews by Goodman and Schwarz [226] and Hamill and Martinac [227]). Both ion channel families have homologues in mammals. Transient Receptor Potential Channels. Mechanosensitive currents in sensory neurons share a number of properties with those mediated by members of the TRP channel family (228). These include inhibition by low micromolar concentrations of ruthenium red and by gadolinium, blockade by external Ca2+, and nonselective cation permeabilities. TRP-related channels have been found to play an essential role in a number of mechanosensory systems. In mammalian systems, TRPV4 is activated by hypotonicity in transfected cells and probably underlies similar currents in DRG neurons (229). TRPV4 null mutants appear to have some deficits in the responses to mechanical stimuli, as well as impaired osmosensitivity (229,230). Rat TRPV4 expressed in osm-9 mutant C. elegans restores osmotic and mechanical sensing behaviors (231). These studies clearly establish TRPV4 as a mechanotransducer in mammals. There is no functional evidence for the involvement of TRPV4 in mechanotransduction

in GI afferents. However, reverse transcriptase-polymerase chain reaction (RT-PCR) of single-nodose cell bodies, which had been labeled retrogradely from the upper gut, identified transcripts for TRPV4 together with several other TRP channels (232). Other TRP channels implicated in mechanosensitivity in mammals include TRPA1 and TRPN1, which may form heteromultimeric channels in hair cells in the inner ear (233). TRPA1 is also found in DRG neurons (234). There is also evidence to suggest that TRPV1 may play an important sensory role in the GI tract. TRPV1 (formerly known as VR1), was the first mammalian TRP channel to be cloned (235). It is activated by heat, protons, and vanilloid ligands such as capsaicin or endovanilloids such as anandamide and is also gated through the activation of protein kinases A and C (236–239). Although the channel is not believed to be gated mechanically, evidence linking mechanosensitivity and the TRPV1 receptor has been demonstrated in the attenuated mesenteric afferent sensitivity to jejunal distension seen in TRPV1

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 701 knockout mice (194) (Fig. 25-12). This suggests that TRPV1 may play a modulatory role, possibly via an effect on the overall excitability of the mechanosensitive nerve terminal. Acid-Sensing Ion Channels. Acid-sensing ion channels (ASICs) are nonselective cation channels belonging to the DEG/ENaC family (240). In mammals, the DEG/ENaC family comprises α, β, γ, and δ ENaC; ASIC1 (or BNaC2), ASIC2 (or BNC1 or MDEG) and ASIC3 (or DRASIC), and ASIC4 (or SPASIC); and BLINaC. Three lines of evidence suggest that DEG/ENaC ion channels may be mechanoreceptors (226). First, several DEG/ENaC ion channel proteins have been shown to be responsible for touch and stretch sensation in C. elegans. Second, DEG/ENaC proteins have been localized in mechanosensory structures. Third, mice lacking ASIC2 showed a reduced sensitivity in mechanosensitive neurons detecting light touch but not in other populations. Disrupting the ASIC3 gene increased sensitivity to light touch but reduced sensitivity of mechanoreceptors detecting a noxious pinch. ASIC channel blockers amiloride and benzamil are able to block mechanosensitive responses in a number of sensory systems. It has been proposed that members of DEG/ENaC family form a transduction complex on the cytoplasmic membrane of mechanoreceptors, and each individual subunit plays a different role in the transduction process. Few studies have investigated the potential role of ASICs in sensory transduction in the GI tract. Immunohistochemical studies have demonstrated the presence of these channels in the GI tract, although the cellular distribution of ASICs remains unclear (232,241). Zagorodnyuk and coworkers (218)

∆Afferent discharge (spikes/s)

80

Mechanosensitive Release of Transmitters Although the major focus of research on mechanosensitivity has been on mechanosensitive ion channels, it has become clear that mechanosensitive release of transmitters, most notably ATP, is as equally ubiquitous in eukaryotic cells (227). This putative mechanism of mechanotransduction is attractive for GI afferents because of both the close association between afferents and other cell types (e.g., mast cells, ICCs, enteric neurons, epithelial cells, enterochromaffin

Wild type TRPV1 knockout

60

40

20

0

A

have demonstrated that benzamil was able to block stretchinduced vagal muscle mechanoreceptor firing in guinea pigs, suggesting that ASICs may contribute to mechanotransduction, although this could be secondary to an effect on muscle tension. The role of ASICs in mechanosensitivity channels has been disputed because neither ASIC2 nor ASIC3 gene deletion affect mechanosensitive currents in DRGs (242). In addition, the mechanosensitive release of CGRP from the colon was unaltered in ASIC2 null mutant mice (243). In contrast, Page and colleagues (244) report that ASIC1 null mutant mice showed increased mechanosensitivity in all colonic and gastroesophageal mechanoreceptor subtypes. In addition, ASIC1 null mice showed almost double the gastric emptying time of wild-type mice. In contrast, loss of ASIC1 did not affect function in any of the five types of cutaneous mechanoreceptors, nor did it affect paw withdrawal responses or fecal output. It seems therefore that defining the role of ASICs in mechanotransduction must also take into account complex interactions between channel subunits.

0 10 20 30 40 50 60 Intraluminal pressure (mm Hg)

B

FIG. 25-12. Transient receptor potential vanilloid receptor 1 (TRPV1) plays a role in the mechanosensitivity of gastrointestinal afferents. (A) The pressure–response curve of multiunit activity from mesenteric nerve during ramp distensions in wild-type and TRPV1 knockout mice. (B) TRPV1 immunoreactivity in vagal afferent endings (A–D), nodose (E), and dorsal root ganglion (F) neurons supplying the rat gastrointestinal tract. IGLE, intraganglionic laminar ending; IMA, intramuscular array. (See Color Plate 15.) (A: Reproduced from Rong and colleagues [194], by permission; B: Reproduced from Ward and colleagues [268], by permission.)

702 / CHAPTER 25 [EC] cells) and the expression of a variety of transmitter receptors on the afferent terminals themselves. Burnstock (245) has hypothesized that stretch stimuli in tubular organs may release ATP from epithelial cells, which then activates sensory nerve terminals through interactions with P2 receptors. For example, epithelial cells in the urinary bladder release ATP on distension, and filling-induced bladder afferent discharge and micturition reflex can be blocked by P2 receptor antagonists or when the P2X3 receptor is knocked out. There have been several lines of evidence supporting the purinergic hypothesis for mechanotransduction in the GI tract. First, mechanosensitive release of ATP from the gut wall has been demonstrated (246). Second, P2 receptors have been identified on extrinsic (IGLEs) and intrinsic afferent neurons (23,218). Third, exogenous ATP has been shown to activate extrinsic and intrinsic afferent terminals (247–249). Fourth, P2 receptor antagonists were shown to reduce pelvic afferent response to colorectal distension in the rat (246). Mechanosensitive release of 5-HT from the gut wall also has been demonstrated. Balloon distension, puffs of nitrogen gas ejected from a pipette, and stroking the mucosa with a brush all evoked 5-HT release from EC cells in the mucosal epithelium (250) (see Ligand-Gated Ion Channels later in this chapter). However, although there is strong evidence for the involvement of 5-HT in mechanotransduction (and chemotransduction) for intrinsic afferents, that 5-HT participates in mechanotransduction in extrinsic mechanoreceptors still lacks electrophysiologic support. Granisetron at doses that completely blocked the response of vagal mucosal mechanoreceptors to 5-HT failed to affect the responses to both mechanical and chemical stimulation of the mucosa (251). The concept of mechanotransduction through mechanosensitive release of transmitters has been disputed by Zagorodnyuk and coworkers (218). Their major arguments are: (1) the onset of stretch-induced afferent discharge is fast (~5 msec), and thus is likely mediated by a direct mechanism involving mechanosensitive ion channels on afferent terminals; and (2) stretch-induced afferent discharge was not blocked by a P2 receptor antagonist (pyridoxal phosphate 6-azophenyl2′,4′-disulfonic acid tetrasodium [PPADS]) and, indeed, remained when the tissue was perfused with calcium-free solutions to block exocytotic release of transmitters. However, these investigators used flat-sheet preparations of the gut without the mucosal layer, which is an essential part of Burnstock’s purinergic hypothesis for mechanotransduction (245). Mechanoreceptors at different layers of the gut wall are specialized to encode different aspects of physical stimuli imposed on the gut tissue. It is likely that different transduction mechanisms may be used by different mechanoreceptors. The transduction in mucosal mechanoreceptors seems to be better explained by an indirect mechanism involving release of mediators than a direct mechanism involving mechanosensitive ion channels on nerve endings. Mucosal mechanoreceptors do not readily respond to distension (unless there is deformation of the mucosa as a result of overdistension), but they are sensitive to light stroking of the mucosa, suggesting

the transduction site might be on the luminal surface of epithelial cells. Chemical Sensitivity Extrinsic afferents of the GI tract, especially the mucosal afferents of the small intestine, are sensitive to a variety of chemical stimuli applied intraluminally. Accordingly, these afferents have been termed polymodal receptors (203). The responsiveness of GI afferents is a property that has been termed promiscuous chemosensitivity. Chemical mediators may be part of an integrated response such as inflammation or may be quite stimulus specific, such as cholecystokinin (CCK) released by the presence of fatty acids in the lumen. These actions are largely mediated via activation of both ligand-gated ion channels and other membrane-bound receptors such as G protein–coupled receptors (GPCRs). Ligand-Gated Ion Channels Receptor/ion channel complexes are present on both the vagal and spinal afferent fibers innervating the gut. These channels are gated by binding of various transmitters and, in some cases, protons, as well as mechanical stimuli. Among these are 5-HT3, P2X, N-methyl-D-aspartate (NMDA), TRP, ASIC, and nicotinic receptors. 5-HT3 Receptors. 5-Hydroxytryptamine (5-HT) can act on a number of GPCRs, as well as on ligand-gated channels known as 5-HT3 receptors (252). Immunohistochemical evidence suggests that 5-HT3 receptors are present on both DRG and nodose ganglion cells that innervate the GI tract (59,253). In the colon, 5-HT3 receptor ligands activate lumbosplanchnic afferents (59). Interestingly, these units had serosal receptive fields; thus, the source of endogenous 5-HT that may activate these endings is unclear, although 5-HT– mediated signaling may be important in inflammatory conditions (254). Furthermore, the 5-HT3 antagonist, alosetron, inhibited spinal cord c-fos expression in response to noxious colorectal distension, although these studies did not distinguish between a central and a peripheral action (255). 5-HT3 knockout mice have not been used to investigate GI sensory signaling, but these mutants have been shown to have altered somatic nociceptive signaling (256). Many aspects of ligand receptor interaction were established using isolated nodose neurons, which indicates that 5-HT activates a nonselective cationic current that is blocked by various 5-HT3 antagonists (257). 5-HT also has been shown to activate vagal endings innervating the gut wall. For example, in the stomach, 5-HT activated vagal single units, an effect that was blocked by 5-HT3 antagonists and by mucosal anesthesia (258). Similarly, in the intestine of the rat, 5-HT3 receptor activation stimulates afferent nerve activity; this also seems to be via an effect on mucosal afferents, and it is abolished by vagotomy. This aspect of 5-HT is discussed later in this chapter in the context of luminal sensing. P2X Receptors. Extracellular ATP is now recognized as an important signaling molecule in visceral afferents.

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 703 ATP can act on either P2Y receptors that are GPCRs or P2X receptors that are ligand-gated ion channels (259). Immunohistochemical evidence exists for P2X receptors (P2X3) on spinal afferents innervating the colon (23) and vagal endings (P2X2/3) (218,260,261) innervating the esophagus and stomach. ATP excites vagal endings innervating the stomach and esophagus through action on P2X receptors (209,218,262). Interestingly, this appears to be, at least in part, through activation of specialized IGLE-like endings, and antibodies to P2X2 subunits densely stain IGLEs (218,260, 261). In the intestine, P2X receptor activation also activates mesenteric afferents, and this appears to be through a direct mechanism (249). In the colon, ATP also has been implicated as having a role in mechanosensation (246) and in inflammation-induced visceral hypersensitivity (263). The P2X subunits mediating excitation have not yet been elucidated in GI afferents; however, experiments in isolated nodose and DRG neurons suggest that it is due to action on both P2X3 homomeric receptors and P2X2/3 heteromers (264). N-methyl-D-aspartate Receptors. Both morphologic and electrophysiologic evidence exists for the presence of NMDA receptors on GI primary afferents. Immunohistochemical studies have localized NMDA-R1 receptors to DRG cell bodies innervating the colon, and NMDA causes an increase in intracellular calcium in these cells (265). In addition, NMDA receptor antagonism inhibited visceral pain responses. In vagal afferents innervating the stomach, Sengupta and colleagues (266) showed that NMDA receptor activation excited gastric vagal afferents, and that blockade of these receptors inhibited responses to mechanical stimuli. However, these results have not been confirmed in all studies (218). The source of glutamate that activates these receptors is unclear; however, many visceral afferents express vesicular glutamate transporter-2 (VGLUT-2) (41), suggesting that under some conditions these endings may release glutamate, raising the possibility of an autocrine action. Transient Receptor Potential Vanilloid Channels. The TRP family of ion channels has been discussed earlier in this chapter in the context of mechanosensitivity. However, these channels clearly play a primary chemosensory role. They are gated by the pungent chili vanilloid, capsaicin, low pH (<6), and noxious heat. TRPV1 immunoreactivity has been demonstrated in cell bodies and nerve fibers of both vagal and spinal afferents innervating the GI tract (267,268), and many GI afferents respond to capsaicin. For example, in the ferret, approximately one-third of gastroesophageal vagal afferents respond to capsaicin (269). A similar proportion has been shown in the rat colon (76). Colonic (160) and ileal (175) DRGs respond to capsaicin with excitation and inward currents. In the small intestine, mesenteric afferents are activated by capsaicin (194), and this effect is lost in TRPV1-deficient animals. Extrinsic afferents of the upper GI tract are also generally sensitive to intraluminal acidity, although the response of individual afferents to acid may vary considerably according to the relative distance of their endings from the mucosal surface and the acid sensor they express. Intrajejunal infusion

of 20 mM HCl in Krebs (pH 4.8) evokes a pronounced increase in multiunit activity of the mesenteric nerve (194). Analysis of single-unit activity indicates that individual units responded to acid differently in terms of the latency, duration, and magnitude of the response. High-threshold fibers tend to reach peak activity faster than low-threshold and wide dynamic range fibers. Furthermore, the activity in lowthreshold fibers after acid infusion was much less compared with that seen in high-threshold or wide dynamic range fibers. There is increasing evidence linking TRPV1 and acid sensation. Responses to acid infusion in the jejunum are reduced in both TRPV1 knockout mice and in the presence of capsaicin (194) (Fig. 25-13). Experiments on isolated colonic DRG neurons also have demonstrated the key role for TRPV1 in acid-evoked currents and depolarization and have demonstrated that metabotropic 5-HT receptors can potentiate these effects (270). In humans, perfusion of the jejunum with capsaicin results in pain and cramping sensations, potentially implicating this channel in human visceral sensation (271). Experiments on somatic afferents have shown that TRPV1 mediates responses to inflammatory pain, and in the inflamed human gut, TRPV1 immunoreactivity is increased as it is in the esophagus (272–274). Thus, the TRPV1 channel may be important in both acid and nonacidrelated GI pain disorders. TRPV1 antagonist also attenuated acid-evoked duodenal hyperemia but failed to affect acidinduced gastric hyperemia (275–277). Thus, apparently other acid sensors are also involved in the transduction of acidity. Possible candidates include various TRPV4 members of the DEG/ENaC family of ion channels including the ASICs. Nonetheless, TRPV1 seems critically important as a polymodal chemotransducer in GI afferents. There has been little study of the role of other TRP channels in the GI tract; however, one study has examined the presence of TRPV2, TRPV4, TRPN1, and TRPM8 channels in GI vagal afferents, finding the mRNA for these channels, as well as that some of these vagal afferents respond to cooling and icillin, indicating that there are temperature-sensitive channels in upper GI tract afferents (278). In addition, TRPV4 channels have been implicated in mechanosensation (see earlier). Acid-Sensing Ion Channels. The presence of ASICs has been found in gut afferents (232), and that these channels are gated by acid suggests that they may have a role in GI chemosensation such as sensing of acid and ischemia in the gut. Support for the latter exists in cardiac afferents (279,280). These concepts await direct demonstration in the GI tract; however, it is likely that ASICs may play a chemosensory role in addition to acting as mechanosensors (see earlier). Nicotinic Receptors. Neuronal nicotinic receptors are pentameric channels formed as homomers (e.g., α7) or heteromers (e.g., α3β4). They are nonspecific cation channels and are important in fast synaptic transmission in the CNS and ENS. Hepatic branch vagal afferents have been shown to be activated by nicotine injected into the portal vein. However, these experiments were performed in vivo; therefore, a direct action on afferent terminals was not demonstrated directly. However, preliminary reports indicate that

704 / CHAPTER 25 HCI 20 mM

∆Discharge rate (imp/s)

60 40 20

Nerve activity (µV)

0 500

10

0 0

B 0

100

200

HCI 20 mM

1 min

4 3 2 1

A

500

600

20

10

0 0

0

300 400 Time (s)

HCI CPZ 10 µM + HCI

30

−250 5

∆Single unit activity (imp/s)

Wild type TRPV1 knockout

20

250

∆Discharge rate (imp/s)

Discharge rate (imp/s)

HCI 20 mM

100

200

300 400 Time (s)

500

600

C FIG. 25-13. Transient receptor potential vanilloid receptor 1 (TRPV1) contributes to the sensitivity of small intestinal afferent fibers to intraluminal acid in the mouse. (A) Example of the mesenteric nerve response to intraluminally applied HCl in a mouse jejunum preparation. The bottom trace illustrates the activity of several individual units with different response patterns. (B, C) The response in mesenteric nerve to intraluminal acid is attenuated in TRPV1 knockout mice and by the TRPV1 antagonist, capsazepine (CPZ). (Modified from Rong and colleagues [194], by permission.)

selective nicotinic agonists can activate gastric and mesenteric vagal afferents in vivo (intestine) and in vitro (stomach), an effect blocked by antagonists for receptors containing α3β4 subunits, and to a lesser extent in the intestine, by α7 antagonists (281,282). Furthermore, functional studies indicate the presence of these channels on IGLE-like mechanosensitive endings (281), raising the potential for communication between enteric neurons and vagal afferents, given the location of these endings in myenteric ganglia. Nicotine also is known to have inhibitory effects on food intake, and activation of vagal afferents may be one mechanism by which this takes place. Membrane Receptors Activating Intracellular Second Messenger Systems In addition to modulating afferent activity through ligandgated ion channels, chemical mediators can affect firing via an action on membrane receptors that subsequently activates a variety of intracellular signaling pathways. The most important of these groups is the GPCRs. Ligand binding to these receptors results in exchange of GDP for GTP bound to the α subunit, thereby dissociating it from the β subunit, leading to activation of downstream signaling pathways. GPCRs can be categorized broadly based on the cellular

second-messenger systems they activate: Gs activates adenylyl cyclase, Gi inhibits adenylyl cyclase, and Gq activates phospholipase C. G12 and G13 also have been described but serve unknown functions (283). There is substantial evidence implicating a role for GPCRs in regulating extrinsic afferent signaling in response to nutrient-derived stimuli and mechanosensitivity and in the development of symptoms associated with disease. These act in concert with ligandgated ion channels to “fine-tune” afferent sensitivity in a variety of conditions. Activation of G Protein–Coupled Receptor Signaling Pathways in Response to Luminal Stimuli. Several GPCR subtypes are involved in regulating afferent activity in response to luminal stimuli; these subtypes are shown in Table 25-1. A full description of the physiologic role of these receptors is covered later in this chapter (see Regulation of Food Intake: Role of Vagal Afferents). GPCRs involved in mediating nutrient signal information are activated by ligands released under physiologic conditions, and these signaling processes are specific in nature. In addition, these GPCR pathways appear to be susceptible to modulation, which ensures a degree of neuronal plasticity is maintained in the GI tract in response to food. Several other GPCR-mediated luminal-sensing mechanisms have been proposed to be present in mammals,

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 705 TABLE 25-1. G protein–coupled receptor signaling in response to luminal stimuli GPCR

Ligand

Expression

Role

References

CCKα (low affinity) OR-1 (high affinity)

CCK Orexin Ghrelin Anandamide

NPY

NPY/PYY

Vagus

Excitation—inhibits feeding Inhibits action of CCK, stimulates feeding Inhibition Stimulates feeding, effect dependent on vagus Inhibits gastric/duodenal motility via “ileal break”

301, 319, 430–432 325

GHSR Cannabinoid (CB1)

Vagus (stomach, jejunum) Vagus (coexpressed with CCK receptors) Vagus (stomach) Vagus

326 327 433, 434

CCK, cholecystokinin; GHSR, growth hormone-secretagogue receptor; GPRC, G protein–coupled receptor; NPY, neuropeptide Y; PYY, peptide YY.

A

5-HT

CAP

1 nA 10 s

these ligand–receptor interactions can directly activate or sensitize sensory afferent neurons, the consequences of which play an important role in the development of visceral hypersensitivity or pain (see reviews by Blackshaw and Gebhart [62], Grundy [290], Kirkup [291]). Furthermore, there is growing evidence that GPCRs can interact with voltage- and ligand-gated ion channels in GI afferents; for example, prostaglandin E2 (PGE2) augments TTX-R Na currents, and 5-HT (through an action on its subtype 2 and 4 receptors) potentiates TRPV1-mediated currents (Fig. 25-14). TRPV1 receptors are also enhanced by protease-activated receptor 2 (PAR2) activation, and it is known that PAR2 can activate GI afferents (292) (Fig. 25-15). Table 25-2 describes the major receptor subtypes involved in these processes. In addition to the receptors described in Tables 25-1 through 25-3, studies have revealed a large number of neurotransmitter-activated GPCRs with ligands and functions that are unknown (orphan receptors). Analysis of their expression using RT-PCR demonstrated that many of these receptors are expressed in the GI tract (293). Identifying the role of these orphan receptors will be an important feature of future research into GI physiology. Cytokine Receptor Superfamily. The family of receptors known as cytokine receptors also influence GI afferent function. These receptors are members of the immunoglobulin superfamily, and ligand binding results in receptor

B Normalized current

although their functional role remains unclear. Members of the bitter taste receptor (T2R/gustducin-α) family of GPCRs have been demonstrated to be present in the GI tract and enteroendocrine STC-1 cells (284), whereas the extracellular Ca2+ receptor is proposed to function as a receptor for aromatic amino acids and may contribute to sensory transduction in some regions of the gut (285,286). G Protein–Coupled Receptors and Modulation of Gastrointestinal Mechanosensitivity. GPCRs on afferent endings may also serve to modulate afferent responses to mechanical stimuli. This has been demonstrated for GABAB receptors, which attenuate responses of gastric vagal afferents to stretch (287). Similar effects have been found for galanin and mGluR agonists (288,289). Somatostatin also diminished intestinal responses to distention, and this effect appeared to be selective for spinal endings (201). These receptors may prove to be attractive targets for the treatment of visceral pain or for the modulation of GI vagal reflexes underlying disease conditions, such as transient lower esophageal sphincter relaxations in gastroesophageal reflux disease. Excitatory G Protein–Coupled Receptors Mediating Neuroimmune Responses. GPCRs expressed on sensory afferents are also receptive to mediators released from a variety of cell types (such as mast cells, macrophages, lymphocytes blood vessels, and neurons) during pathophysiologic conditions. The signal transduction processes initiated by

2

1 0 5-HT (−)

(+)

FIG. 25-14. Example of G protein–coupled receptor–mediated potentiation of ion (transient receptor potential vanilloid receptor 1) currents. (A) Whole-cell voltage-clamp recording from colon-projecting dorsal root ganglion neuron. Downward deflection is the inward current induced by 100 nM capsaicin (CAP; solid marker). In the presence of 5-hydroxytryptamine (5-HT; hatched line), current amplitude is significantly increased. (B) 5-HT results in a near doubling of normalized current amplitude. * indicates significant difference from control. (Reproduced from Sugiuar and colleagues [270], by permission.)

706 / CHAPTER 25 i

SLIGRL-NH2

JP (mm Hg)

10

ii LRGILS-NH2

0 AD (spikes s−1)

150

0 10 s

FIG. 25-15. Protease-activated receptor 2 (PAR2) stimulation excites intestinal mesenteric afferent nerves in vivo. Nerve recording showing effect of intravenous injection of the PAR2 agonist (SLIGRL; right) and the inactive reverse peptide (LRGILS; left) on mesenteric firing. SLIGRL caused a significant increase in firing, whereas the reverse peptide was without effect. AD, afferent discharge; JP, jejunal pressure. (Reproduced from Kirkup and colleagues [292], by permission.)

dimerization and subsequent activation of intracellular signaling pathways such as the c-Jun N-terminal kinase/signal transducer and activation of transcription 6 pathway (e.g., leptin receptor Ob-R) or the p53/mitogen-activated kinase/ nuclear factor-κB pathway (e.g., IL-1β). In particular, leptin and IL-1β have important effects on extrinsic GI afferents (see later); the interaction with these receptors and other GPCRs also highlights the complexity of these mechanisms in regulating afferent traffic from the GI tract.

GASTROINTESTINAL EXTRINSIC AFFERENT NERVES: INTEGRATIVE PHYSIOLOGY The extrinsic spinal and vagal innervation of the GI tract is critically involved in a variety of processes important in maintaining homeostasis of the organism, as well as maintaining integrity of the gut itself. These roles include reflex control of motility, secretion, blood flow, mucosal integrity, and gut immune function. These are integrated with behavioral responses including eating and illness behavior and are

associated with sensory experiences such as fullness, bloating, nausea, discomfort, and pain.

Regulation of Food Intake: Role of Vagal Afferents The regulatory systems controlling food intake are complex and involve neuroendocrine interactions among the gut, sensory afferent nerves, and hypothalamus, as well as higher centers in the CNS. The neuroendocrine control of food intake is reviewed elsewhere in this volume; however, we focus on the extrinsic afferent nerves innervating the gut and liver and highlight the potential role these may have in sensing nutrient intake and digestion and how this signaling may be perturbed in pathophysiologic states. Distention as a Satiating Stimulus The notion that distension of the stomach or intestine itself can result in satiation is intuitive. Experimental distension of the stomach in humans results in feelings of “fullness” before

TABLE 25-2. Excitatory G protein–coupled receptors mediating neuroimmune responses GPCR

Ligand

Expression

Role

References

A1 and A2B H1

Adenosine Histamine

Excitation Excitatory

EP1 and EP2

PGE2

Excitatory

435 203, 353, 369, 436, 437 159, 438–440

BK2

Bradykinin

Excitatory

201, 438, 440–442

PAR2

Trypsin, SLIGRL—NH2, mast cell tryptase

Mesenteric afferents Mesenteric afferents, vagal, colonic spinal Mesenteric afferents, colonic DRGs Mesenteric afferents, colonic spinal (splanchnic), vagal, esophageal, and spinal Mesenteric afferents

Excitatory, mediates neurogenic inflammation

292, 349

BK, bradykinin; DRG, dorsal root ganglion; EP, endoprostanoid; GPRC, G protein–coupled receptor; PAR2, protease-activated receptor 2; PGE2, prostaglandin E2.

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 707 TABLE 25-3. G protein–coupled receptors modulating mechanosensitive responses GPCR

Ligand

Expression

Role

References

µ, δ-Opioid κ-Opioid

Opioid agonists Opioid agonists

Excitatory Inhibitory

148, 443 391, 444, 445

SST-2

Somatostatin

Vagal (mesenteric) Vagal (gastric), colonic spinal (pelvic) Mesenteric afferents

201

5-HT (?2, 4)

5-HT

GALR-1 mGluR (type I and II) GABAB

Galanin Glutamate

Mesenteric, colonic spinal afferents, DRG Gastroesophageal vagal Gastroesophageal vagal

Inhibits basal vagal firing, spinal responses to distention and bradykinin Excitatory, potentiate responses to heat and protons (via TRPV1) Inhibitory Inhibits mechanosensitivity

GABA

Gastroesophageal vagal

Inhibitory, inhibits vagally evoked transient LES relaxations

59, 198, 270 288 289 287, 446, 447

5-HT, 5-hydroxytryptamine; DRG, dorsal root ganglion; GABA, γ-aminobutyric acid; GALR, galanin receptor; GPCR, G protein–coupled receptor; LES, lower esophageal sphincter; SST, spinosolitary tract; TRPV1, transient receptor potential vanilloid receptor 1.

discomfort and pain (294). In addition, consumption of nonnutritive drinks (e.g., water) induces sensations of fullness or satiety (295). Along these lines, endoscopic placement of a prosthetic intragastric balloon has been used to limit food intake in obese patients (296). Clearly, extrinsic primary afferents, especially vagal afferents, are finely tuned to detect slow low-pressure distension such as that which may occur during a meal (192,297). However, because pharmacologic antagonists to selectively block mechanosensitive afferents are unavailable, direct demonstration of this concept has been difficult. Experiments using animals with gene deficiencies that lead to abnormal development of mechanosensory endings, particularly IGLEs and IMAs, have provided important insights. Mice that have mutations in the c-kit pathway, a receptor tyrosine kinase, have been shown to be deficient in intramuscular ICCs and mast cells and, in addition, have a markedly reduced number of specialized mechanosensitive endings known as IMAs (see earlier) (75). Furthermore, these mice, as well as mice deficient in the steel factor, the c-kit ligand (27,75), eat smaller meals while maintaining normal overall caloric intake and body weight. Whether this is a consequence of altered sensory function or disordered motility has not been determined. A similar transgenic approach has been used to examine the role of IGLEs in the regulation of food intake. NT-4 knockout mice have an 80% to 90% reduction in IGLE number and have longer solid food meal duration, as well as larger liquid food meal size (27), perhaps indicating reduced satiety signaling. Similar to the IMA-deficient animals, body weight and overall caloric intake was maintained. In contrast, NT-4 knock-in mice, which have increased numbers of IGLEs, consumed smaller meals and ate liquid meals more slowly, indicating enhanced satiety. Furthermore, the dose–response curve for the inhibitory effect of CCK on food intake was shifted leftward, that is, CCK had a greater satiating effect at any given dose (298). These results highlight the importance of distension as a satiating stimulus, and they also provide insight into the

mechanism by which CCK enhances sensitivity to gastric distension (294). They also highlight the important dichotomy that although luminal factors may determine much of the size and pattern of meal intake, the overall need of the organism to maintain energy balance supersedes these factors by compensating in terms of frequency or timing of meals. Nutrient/Paracrine/Afferent Interaction In addition to mechanical stimulation, there is clear evidence that the presence of nutrients in the intestine stimulates the release of several mediators that can act either in a paracrine or endocrine fashion on afferent nerves. A prime example is fatty acids, particularly those with chain lengths of 12 or more carbon atoms, which cause the release of CCK from specialized enteroendocrine cells in the mucosa (299,300). CCK is a potent activator of intestinal vagal afferents (301), and in vivo experiments in rats have demonstrated that intraluminal fatty acids cause an activation of mesenteric afferents (302). The response to long-chain fatty acids is blocked by CCK1 receptor antagonism. In contrast, the response to shorter chain fatty acids (i.e., <10 carbon atoms) appears to occur via a CCK-independent mechanism (302), perhaps via a direct effect on the afferent terminal. In this respect, studies using a CCK-secreting cell line (STC cells) show that long-chain fatty acids release CCK directly (299,300), and this may represent a preabsorptive mechanism for signaling luminal content of nutrients. However, there is also evidence for postabsorptive signaling via CCK because apolipoprotein A-IV (ApoA-IV), which is released from enterocytes during chylomicron formation, also activates vagal afferents through a CCK-dependent mechanism (303). Products of protein digestion also release CCK (304–306). In this respect, protein hydrolysates activate intestinal vagal afferents via a CCKdependent mechanism (307). CCK causes a profound vagally mediated reflex inhibition of gastric emptying; therefore, CCK may not only inhibit food intake through a direct stimulation

708 / CHAPTER 25 on vagal afferents, but also secondary to inhibition of gastric emptying and increased gastric volume (308). CCK also has been shown to have a direct stimulating effect on gastric vagal afferents, indicating that vagal mechanoreceptors may signal both the quantity of gastric contents and composition of luminal nutrients via an endocrine action of CCK (308, 309). CCK may also have effects on other receptor families involved in the regulation of food intake. For example, Burdyga and colleagues (81) have demonstrated that CCK down-regulates the expression of cannabinoid 1 (CB1) and Orexin receptors, the activation of which are known to stimulate food intake. The CCKα receptor exists in two different affinity states (high and low) that can be differentiated by their sensitivity to CCK-JMV-180 (a high-affinity agonist and low-affinity antagonist) (310). Studies on isolated nodose neurons suggest that the low- and high-affinity receptors are linked to different calcium signal cascades (311). The paracrine-mediated action of CCK on intestinal vagal afferents leading to pancreatic secretion is via the high-affinity receptor (312), whereas it is the low-affinity receptor that features in satiety (313). Gastric vagal afferents are also inhibited by CCK-JMV, suggesting that the high-affinity receptor is predominantly expressed on vagal sensory neurons (314). The presence of carbohydrate in the lumen may also result in vagal afferent activation. Luminal glucose can release the neuromediator 5-HT from EC cells in the intestinal mucosa and from BON cells, which are a pancreatic neuroendocrine tumor cell line used as model of EC cells (315,316). Luminal glucose has been shown in Mei’s classical studies (317) to activate duodenal vagal afferents. More recent evidence suggests that this effect is partially sensitive to both 5-HT3 receptor antagonism and depletion of EC-like cell (but not neuronal) sources of 5-HT (318). Similar to CCK, 5-HT can cause a reflex inhibition of gastric emptying, possibly contributing to their suppressive effects on food intake (253). In addition, at least in the intestine, CCK and 5-HT appear to activate different populations of intestinal afferents (319), raising the possibility of nutrient-specific coding of vagal afferent input to the brainstem. Leptin is a peptide hormone that is released by adipocytes and also by the GI mucosa, and that is considered to play a role in long-term regulation of food intake. Mice lacking either leptin or the leptin receptor (Ob) are hyperphagic and massively obese (320). Leptin receptors are present on rat and human vagal afferents, raising the possibility that the actions of leptin on feeding are partially mediated via the vagus (321). The actions of leptin on vagal afferents currently are controversial and poorly understood. Leptin has been shown to inhibit and augment the firing of gastric and intestinal vagal afferents under certain circumstances. However, prior CCK administration reversed the inhibitory effects (322). Furthermore, some of the effects of leptin such as vagally mediated increases in pancreatic secretion appear to be mediated by CCK release (323). However, the relatively high concentrations of peptide needed to see these effects far exceed the picomolar concentrations seen in physiologic situations.

However, the observation that leptin levels are increased in intestinal inflammation may indicate a contributing factor in the anorexia and weight loss that often accompanies inflammatory bowel disease (IBD). Furthermore, the finding of autonomic innervation of abdominal fat (324) raises the possibility that leptin released from fat stores may also modulate autonomic afferent activity. A number of new mediators have been identified that serve not to terminate food intake, but rather to stimulate it. These hormones are collectively described as orexigenic and include Orexin itself and the growth hormone–releasing hormone, ghrelin. Orexin receptors have been described on vagal afferent cell bodies, using PCR and immunocytochemical techniques, and although Orexin alone had no effect on intestinal afferent nerve discharge, it was shown to suppress the responses to CCK (325). These studies also confirmed immunocytochemically that Orexin receptors, CCK-A receptors, and CART (cocaine and amphetamine regulatory transcript) often colocalized to the same neurons, supporting the notion that Orexin could modulate meal-related afferent signaling (Fig. 25-16). Ghrelin is released from the gastric mucosa and also has been demonstrated to have receptors and actions on vagal afferents neurons. Date and colleagues (326) showed that ghrelin inhibited gastric vagal afferent activity and that blockade of vagal afferent transmission either surgically or chemically (with capsaicin) inhibited the ghrelin-induced increase in food intake. These studies also provided evidence for the presence of ghrelin receptors on vagal afferent neurons. Thus, these studies show that inhibition of vagal afferent signaling may also be an important signal in the initiation of food intake. Fatty Acid Amides and Food Intake A group of compounds known as fatty acid amides are of interest in the regulation of food intake. The most studied, anandamide, is an endocannabinoid, that is, an endogenous compound that acts on CB receptors. This is of interest because CB receptor activation is known to increase food intake, and fasting increases intestinal anandamide levels by sevenfold (327). Furthermore, these hyperphagic effects of anandamide were prevented by chronic capsaicin treatment, which destroys a population of small unmyelinated sensory afferents, suggesting a role for extrinsic GI afferents. The effects of anandamide on intestinal afferent firing are unclear and await direct recording from intestinal afferents. Electrophysiologic data suggest that the CB diethanolamide may increase mesenteric afferent firing (328); however, this is hard to reconcile with a stimulatory effect on food intake. Perhaps the excitatory action is via activation of VR1 receptors, rather than CB1, as has been suggested for anandamide. In contrast, a related compound, oleylethanolamide (OEA), has been shown to have potent suppressive effects on food intake and weight gain (329–331), and intraperitoneal administration also results in activation of neurons in the NTS; both effects are blocked by chronic capsaicin treatment (332).

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 709

A

B

C

D

E

F

G

H

I

J

K

L

FIG. 25-16. The Orexin receptor OX-R1 in human nodose ganglia colocalizes with Ob-R, cholecystokinin (A) (CCK-A), cocaine and amphetamine regulatory transcript (CART), or calcitonin gene–related peptide (CGRP). (A, D, G, J) Individual neurons expressing OX-R1. Middle panels show the corresponding neurons exhibiting (B) Ob-R, (E) CCK-A, (H) CART, or (K) CGRP immunoreactivity. (C, F, I, L) Overlay of the relevant left and middle panels. Cells colocalizing both labels appear in yellow. Bars = 25 µm. (See Color Plate 16.) (Modified from Burdyga and colleagues [325], by permission.)

OEA levels are increased postprandially in the intestine and are inhibited by fasting. This compound has no effect on CB1 receptors, but it has been shown to activate both heterologously expressed and native (DRG) TRPV1 receptors (333). In addition to this action, much of the food-suppressive effects are lost in mice deficient in the peroxisome proliferator– activated receptor PPARα (334). Interestingly, this is a nuclear receptor, and whether OEA is transported to the nucleus

in the cell body of the afferent nerve to exert its effects or it releases another mediator from the intestinal epithelium is unknown. Hepatic Afferents as Nutrient Sensors The liver receives a rich vagal innervation, and many vagal afferents terminate within the portal vein (37), thus enabling

710 / CHAPTER 25 the detection of nutrients absorbed into the venous drainage of the GI tract. A number of nutrients administered into the portal vein can affect vagal afferent firing. In the case of glucose, Niijima (335) describes a decrease in hepatic vagal afferent discharge in response to intraportal D-glucose, but not L-glucose or other sugars. Experiments from the same group also have demonstrated either an excitatory or inhibitory effect of portal administration of amino acids, depending on the particular amino acid used (336); in particular, there appear to be glutamate sensors, which activate hepatoportal vagal afferents. The inhibitory effect of fat on food intake in diabetes also appears to be mediated via the hepatic branch of the vagus (337). Many of these studies used in vivo recordings from the hepatic branch of the vagus, which also innervates the distal stomach and proximal small intestine (37), and therefore secondary effects on gut motility or gut afferents could not be completely excluded. Nonetheless, these data suggest that liver afferents may be able to directly sense nutrient; however, the nature of the receptors involved are unknown, and the opposing effects of some of these nutrients do not correlate clearly with the expected role in excitation of vagal afferents as being a signal for satiation. It appears, therefore, that a variety of signals important in sensing and regulating food intake act via the afferent vagus nerve. This is perhaps not surprising because it would seem to be beneficial to have a mechanism for nerve-mediated signaling, to allow rapid communication of information regarding the presence of nutrients in the gut. Given the epidemic of obesity in the Western world, this area clearly requires further study, paying particular attention to how this signaling may be perturbed in obesity and diabetes, as well as designing pharmacologic targets for inhibition of food intake.

Gastrointestinal Afferents in Pathophysiology The GI mucosa forms a physical and functional barrier between the enteric microenvironment, containing a variety of antigens, as well as billions of bacteria, and functions as the body’s largest immune organ. Immune cells, including mast cells and macrophages, form an integral first line of defense against harmful antigenic material, and there is increasing evidence for a bidirectional communication between these cells and the extrinsic afferent innervation. Mediators released by these cells include amine transmitters (e.g., 5-HT, histamine), prostanoids, cytokines (e.g., IL1β and tumor necrosis factor-α [TNF-α]), as well as proteases, and these have been shown to be capable of exciting or sensitizing GI afferents. Illness Behavior: Systemic Inflammation, Cytokines, and the Vagus Nerve Activation of the immune system by pathogenic bacterial antigens leads to release of a cascade of cytokines that ultimately result in both local and systemic manifestations of inflammation and so-called illness, or sickness behavior.

Manifestations of the latter include fever, hyperalgesia, anorexia, and a reduction in exploratory behavior (338). The role of intestinal and hepatic sensory afferents in these responses is particularly attractive because: (1) many potentially harmful bacteria are contained in the gut lumen, and (2) breakdown of the luminal barrier has been shown to be a key component of the multiorgan dysfunction syndrome seen in critical illness. The result of this breakdown could be increased delivery of harmful bacterial products to resident macrophages in both the gut and liver, with the resultant activation of the cytokine cascade. The location of extrinsic afferent nerve terminals in the wall of the gut, as well as the portal vein (which ultimately receives all of the venous drainage from the gut), implies a potential role in sensing these events. The most commonly used model of systemic inflammation has been administration of the gram-negative bacterial cell wall product, lipopolysaccharide (LPS). LPS administration activates macrophages to release TNF and IL-1β, which, in turn, can activate intestinal and hepatic afferents (339,340). Furthermore, experiments using either surgical (e.g., vagotomy) or chemical deafferentation (e.g., chronic capsaicin treatment) attenuate early fever responses (338) and brainstem c-fos activation (341). In addition, vagotomy also attenuates some of the alterations in exploratory motor behavior induced by LPS administration (342). An intriguing finding concerns the paraganglia associated with the vagus nerve. These are collections of glomus-like cells, analogous to carotid body chemoreceptors. Studies have demonstrated the presence of IL-1 receptor on these cells, suggesting that they may transduce inflammatory signals, which ultimately are transmitted by vagal afferents (343). Vagus Nerve Anti-inflammatory Pathway Some work has also demonstrated a key role for the vagus nerve in down-regulating the systemic inflammatory response. Experiments have shown that vagotomy increases mortality in response to LPS injection, and vagal efferent stimulation attenuates both the systemic inflammatory response to LPS (e.g., TNF release by macrophages) and the mortality associated with it (344). Based on these observations, it has been concluded that the efferent vagus constitutes a critical anti-inflammatory pathway. Moreover, because a variety of cytokines can activate extrinsic intestinal and hepatic afferents and deafferentation with capsaicin exacerbates inflammation, there may be vagal reflex mechanisms that operate in local (organ) inflammation such as colitis (345). In addition, both CCK and leptin have been shown to have anti-inflammatory activity in colitis, an effect that is mediated via vagal afferents (346). There is also strong evidence for vagal afferents playing a role in the modulation of inflammatory responses throughout the body. Plasma extravasation during a neurogenic inflammatory response in the knee joint is subjected to feedback inhibition from the hypothalamic-pituitary-adrenal axis (347). The feedback inhibitory mechanism is stimulated by spinal nociceptive pathways, with vagal afferents supplying

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 711 the small and large intestines playing a modulatory role. Thus, after severing the coeliac branches of the vagus, the inhibition of bradykinin-induced plasma extravasation is potentiated. Presumably, therefore, ongoing vagal activity has an inhibitory input to this hypothalamic-pituitary-adrenal feedback loop, with a consequently greater inflammatory response with the vagus severed compared with when it is intact. Taken together, these data suggest the presence of a vagovagal anti-inflammatory reflex, which on the efferent side requires release of acetylcholine acting on nicotinic receptors; however, the afferent limb of this reflex is unclear. The whole question of how gut and liver afferents modulate both local and systemic inflammation requires further study. Role of Specialized Nonneuronal Cell Types in Afferent Sensitivity As discussed earlier, specialized nonneuronal cell types such as immune and EC cells serve important roles as transducers of a variety of stimuli, and because of their intimate association with sensory nerves, they deserve special mention here. The role of mast cells in mediating inflammatory responses in the gut has been documented extensively. Abundant evidence exists of signaling between mast cells and afferent terminals in which degranulation of mast cells in response to antigen or neurochemical stimulation results in the release of a “cocktail” of mediators including serotonin, histamine, cytokines, prostaglandins, NGF, leukotrienes, enzymes (such as mast cell tryptase), and platelet-activating factor. These mediators play a powerful role in activating or sensitizing afferent terminals through receptor-driven pathways or activation of ion channels. Mast Cells Mast cell degranulation can be stimulated by mediators such as substance P and CGRP released from afferent nerve terminals. This nerve–mast cell communication pathway functions as part of a “bidirectional” positive feedback loop, whereby mast cells release mediators that then excite sensory afferent neurons, which, in turn, further excite mast cell activity. One of the important mediators in this process is mast cell tryptase, which activates PAR2 present on sensory afferents. PAR2 plays a crucial role in sensitizing afferent neurons and also causes the release of substance P and CGRP (348–350). This system presumably acts to amplify the inflammatory response within the GI tract and leads to increased motility and secretion as part of the ENS response to mast cell degranulation. In addition, this is a likely mechanism contributing to visceral hypersensitivity and pain through the direct activation of spinal pathways and recruitment of “silent” nociceptive receptors. In addition, NGF, presumably derived from mast cells, also has been implicated in visceral hypersensitivity induced by neonatal stress (351). Preliminary experiments using mast cell–deficient mice and reconstitution of mast cells in these animals have

demonstrated the key role for mast cells in visceral pain responses in experimental pancreatitis (352). Anaphylaxis Intestinal anaphylaxis is a prototypical model of acute mast cell–mediated responses and can be readily induced in the laboratory in rodents sensitized to either ovalbumin or milk protein (β-lactoglobulin). In ovalbumin-sensitized rats, administration of the antigen results in both an increase in NTS c-fos activation and a prompt increase in mesenteric afferent nerve firing (Fig. 25-17), both of which are inhibited by 5-HT3 and histamine H1 receptor antagonism, as well as mast cell stabilization and luminal anesthesia (353–355). In addition, other mast cell mediators that may be implicated include proteases, acting through PAR2 receptors (292), and NGF (167), which can both acutely and chronically sensitize GI afferents. Given evidence that mast cells may be key mediators involved in sensitizing gut afferents after infection or in irritable bowel syndrome (IBS) (351,356,357), these observations may have particular relevance in the clinical setting. Enterochromaffin Cells The serotonin-containing EC cells are the most abundant enteroendocrine cells present in the gut; they are distributed throughout the GI tract, although the greatest concentrations are located in the small intestine and rectum. Release of 5-HT from the EC cells is Ca2+ dependent and is meditated by luminal (chemical or mechanical) or neuronal stimuli. Stimuli known to release 5-HT from EC cells include mucosal stroking, chemical stimulation with nutrients (e.g., glucose), toxins (e.g., cholera toxin, chemotherapeutics), and endogenous chemical stimuli such as adenosine. Evidence has demonstrated that EC cells also are influenced by GI inflammatory conditions. EC cell number is increased in clinical and experimental models of IBD (358,359). A further factor that may act to increase 5-HT levels is that expression of the serotonin reuptake transporter (SERT) is reduced in inflammation (358,359). Many of these changes such as increased EC cell number and alterations in SERT expression also occur in IBS, particularly in postinfectious IBS (360,361). These changes are potentially important because 5-HT has been shown to activate visceral afferents. For example, in the intestine, 5-HT results in mesenteric activation, which is directly through an action on 5-HT3 receptors and indirectly through an increase in motility, likely via a 5-HT2–dependent mechanism (198). This effect occurs through a direct action on vagal, but not spinal, afferents and cannot support a role for 5-HT in visceral pain signaling. In the colon, however, 5-HT can activate lumbosplanchnic (i.e., spinal) afferents via both 5-HT3– and non–5-HT3–dependent mechanisms (59). Thus, there is an experimental rationale for implicating 5-HT signaling in the pathogenesis of IBS and altered visceral sensitivity in the context of postinflammatory states. Indeed, 5-HT3 antagonists such as alosetron have proved efficacious

712 / CHAPTER 25

100 cm H2O

100 impulses s−1 BSA

EA

40 s

100 µV 50 ms

A

120 ∆ Afferent discharge (imp s−1)

Luminal EA Luminal BSA Doxantrazole + EA

90

Lidocaine + EA

60

30

0 0

60

120

B

180 240 300 Time course (sec)

360

420

480

FIG. 25-17. Effects of intestinal anaphylaxis on jejunal afferent nerve activity. (A) In rats sensitized to ovalbumin (EA), luminal administration of the bovine serum albumin (BSA) antigen results in a brief increase in afferent firing attributable to increase in intraluminal pressure. Ovalbumin (EA), in contrast, causes a prolonged increase in afferent firing. (B) Influence of mast cell stabilization and luminal anesthetic. The mast cell stabilizer, doxantrazole (triangles), and luminal lidocaine (diamonds) each attenuate the excitation induced by EA (open squares). These results indicate that mast cells release their contents to act primarily on luminal afferent endings. (Reproduced from Jiang and colleagues [353], by permission.)

in the treatment of IBS (362). Furthermore, the 5-HT4 agonist tegaserod also has been used in the treatment of constipation-predominant IBS (363). The mechanism by which this occurs is unclear, because studies have shown both inhibitory (364) and excitatory effects (270,365–367) of 5-HT4 agonism in visceral and somatic afferents. Given the prokinetic effects of tegaserod, it may be that this is not due to an effect only on visceral sensitivity, but perhaps alteration in motility.

Ischemia Intestinal ischemia elicits a profound activation of both vagal and spinal afferent fibers. Although most information available is on the response of spinal afferents to interruption of the intestinal blood flow (368–370), studies suggest that vagal afferent fibers may have a greater sensitivity to ischemia, perhaps as a consequence of their innervation of metabolically energetic tissue such as the intestinal mucosa (371).

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 713 Mechanistically, the release of a wide variety of inflammatory mediators such as histamine, 5-HT, bradykinin, prostaglandins, and IL-1β, and the buildup of local metabolites, particularly lactic acid and free radicals, contribute to the activation of afferent fibers during ischemia (148,368–370,372–377). One report suggests that a number of these mediators are derived via mast cell degranulation (378). More recently, using a novel in vitro preparation, investigators showed the absence of flow itself to be a potent stimulus in a population of mesenteric afferent fibers, providing evidence of an additional mechanism through which ischemia increases afferent nerve activity (379). Prolonged intestinal ischemia is fatal if surgical intervention does not occur swiftly. The activation of visceral afferents likely forms part of a protective axonal reflex hyperemia designed to maintain intestinal blood flow, and hence may play a protective role during periods of hypoperfusion (379–383). Physiologically, the reflex shunting of blood from the mesentery is part of the coordinated cardiovascular during exercise or on activation of the defense reaction during stressful stimuli. Electrical stimulation of areas involved in the generation of the defense reaction has been reported to reduce mesenteric blood flow by as much 80% (384). It is likely that ischemic afferents (particularly vagal afferent fiber) form part of a monitoring system designed to maintain sufficient blood flow during these periods of hypoperfusion. Vagal Afferents and Emesis The emetic response is an adaptive mechanism for the elimination of harmful contents from the GI tract. Detection of toxins by specialized cell types such as EC cells serves to protect the gut and the organism itself. 5-HT has been shown to be a major mediator of emetic and diarrheal responses to luminal toxins, and indeed emesis induced clinically, for example, as a side effect of chemotherapy and radiotherapy, appears to act through similar mechanisms. In the ferret, for example, intravenous cisplatin (a chemotherapeutic agent used in the treatment of some solid tumors) induces prodromal retching and emesis (385). Cisplatin-induced emesis can be attenuated by 5-HT3 receptor antagonism (386). In addition, both cisplatin-induced c-fos expression in the brainstem and mesenteric afferent activation are attenuated by 5-HT3 receptor blockers (387,388). Radiation-induced emesis also is significantly attenuated by 5-HT3 blockers (389). These experiments indicate the major mechanism through which chemotherapeutic agents such as cisplatin and radiation cause emesis is via release of intestinal 5-HT, which then subsequently acts on vagal afferents, stimulating the emetic reflex. These experiments are further supported by the clinical experience with 5-HT3 antagonists that have revolutionized the treatment of vomiting associated with the treatment of malignancy. They also further highlight the role of the EC cell as a luminal sensor, in this case causing a massive release of serotonin in response to a toxin.

Visceral Pain/Hypersensitivity Pain is defined by the International Association for the Study of Pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage.” Thus, the concept of pain includes not only the sensation of a potentially noxious stimulus, but also those cognitive and affective components that shape the conscious interpretation of the stimulus. Although the latter factors are clearly important (and perhaps the most important determinant of the degree of the impact on the individual and society), this chapter discusses those factors that affect the discharge of the nociceptive afferents that innervate the gut—the first link in the pain pathway. Pain arising from the GI tract normally is seen only in noxious mechanical or chemical stimulation of the organ. As discussed earlier, there is clear evidence for distinct populations of afferents that sense the complete range of mechanical stimuli: low-threshold afferents, which in the upper gut are predominately vagal; and wide dynamic range and high-threshold fibers, which are predominantly spinal fibers and mediate noxious sensation. These can be considered to be nociceptors in the classical Sherringtonian context in that they encode stimuli that are potentially tissue damaging. However, under certain conditions, particularly during inflammation and ischemia, nociceptors can become sensitized and can be activated by nonnoxious stimuli (analogous to allodynia) or increase their discharge to noxious stimuli (analogous to hyperalgesia). Furthermore, many of these alterations can result in changes that may persist after the inflammatory stimulus has gone, thus resulting in chronic visceral pain. The initial observations made by Ritchie (390) that patients with IBS perceived pain at lower thresholds of rectal distension than control subjects gave rise to the notion that visceral sensation was altered in this group of diseases. An abundance of research in subsequent years supports this idea. In the laboratory, the various models of visceral afferent sensitization and techniques used have been divided into the following categories: (1) studies on isolated cells (e.g., Ca2+ imaging, patch-clamp and intracellular recording); (2) electrophysiologic recordings of afferent nerve fibers (the relative strengths and weaknesses of these are discussed earlier in this chapter); and (3) studies on pseudo-affective responses and visceromotor reflexes. These techniques use a reflex response, which is interpreted to be a painful stimulus (e.g., abdominal contraction or change in blood pressure in response to colonic distension). First, it must be kept in mind that these responses are the result of activation of at least a three-neuron reflex arc (i.e., afferent neuron, interneuron, and motor neuron), with additional descending CNS inhibitory or facilitatory influences, and any changes seen may be due not only to an effect on afferents, but to an effect on any one (or all) of these components. Second, although these responses are widely used as surrogate markers for pain, many studies show that these reflexes are activated by subnoxious levels of distension, raising the

714 / CHAPTER 25 prospect that what may be activated is a physiologic reflex (e.g., defecation). Therefore, limitations of all of these techniques must be kept in mind when evaluating results of studies modeling visceral pain states. Because the topic of this chapter is extrinsic afferents, we focus here on acute and chronic mechanisms of peripheral afferent sensitization in the GI tract, with an emphasis on those studies that have used direct recordings from afferent cell bodies and nerve fibers. Role of the Vagus Nerve in Visceral Pain Although for years conventional wisdom suggested that the vagus nerve innervating the gut sensed primarily physiologic, nonnoxious stimuli and the spinal cord sensed stimuli into the noxious range, a number of recent investigations have implicated the vagus nerve as a sensor of noxious stimuli. The potential for vagal afferents supplying to stomach to encode noxious levels of stimulation has been described (207,391) and is important because this sensitivity implies a role in nociception rather than in physiologic control. Often, stimuli are such that would be considered to be supraphysiologic, applying both to the rate of distension and the magnitude. Rapid (phasic) distension or stretch give rise to afferent responses of greater magnitude than those to slow filling (218). At the onset of such distension wall tension is highest, exceeding that normally accounted under physiologic circumstances (207,217,297,392). This gives rise to a transient afferent response that declines rapidly together with wall tension as the stimulus is accommodated (71,176). This dynamic sensitivity of the endings reflects their ability to respond to changes in tension that exceed those encountered physiologically. Vagal afferents with low activation thresholds but responses that continue to encode levels of distension that when applied in humans give rise to pain have been described (207). Wide dynamic sensitivity, a term adopted from the literature on the sensitivity of dorsal horn neurons, describes this pattern of low-threshold but broad response profile, and although this term is not universally accepted in the field, it is a useful shorthand to distinguish between two populations of low-threshold afferents: those that saturate being low threshold, and those that continue to encode beyond the physiologic range being wide dynamic range. Vagal afferents with wide dynamic sensitivity have been described in the rat stomach (207,391). Thresholds were typically around 5 mm Hg but up to 18 mm Hg and varied dependent on the duration of the distension, implicating accommodation as a determining factor. Afferents responded in a graded fashion to pressures above threshold, an observation that might implicate vagal afferents in nociception. However, whether this sensitivity has any functional relevance is difficult to envisage because the stomach has its own pressure release valve in the lower esophageal sphincter (LOS). Levels of intragastric pressure are unlikely to exceed LOS pressure unless they arise ahead of a peristaltic antral contraction, propelling fluid toward and through the pylorus. Nevertheless, gastric distension to 80 mm Hg, a

level in excess of what might be likely encountered physiologically, stimulates c-Fos expression in the brainstem that is reduced but not abolished by vagotomy; these data are interpreted as supporting a role for vagal afferents in the processing of noxious visceral stimuli, perhaps by contributing to the affective-emotional component of visceral pain (140). Colonic distension also causes c-Fos not only in brainstem nuclei (NTS and rostral ventrolateral medulla), but in higher regions involved in pain processing including the periaqueductal gray, amygdala, hypothalamus, and thalamus (393). Interestingly, only the brainstem expression was lost after perivagal capsaicin. Thus, although activation of colonic vagal afferents is represented in the brainstem, it is spinal afferents that are the source of higher levels of brain activation. In addition, in interpreting these studies, it must be remembered that fibers with low thresholds for activation will still be activated by noxious stimuli. In early human studies (394), stimulation of the vagus below the level of the diaphragm did not cause pain, and vagal stimulation is increasingly being used in the treatment of intractable epilepsy, without reports of pain as a side effect. In contrast, the vagus nerve has clearly been shown to have an “antinociceptive” effect, acting through central reflex mechanisms (see reviews by Gebhart [147] and Janig and colleagues [347]). Mechanisms Underlying Chronic Visceral Hypersensitivity: Plasticity in Ion Channels and Receptors It is clear from the available evidence that the presence of a host of inflammatory mediators can stimulate or sensitize GI afferents. Many of these afferents, by activating cellular second messenger systems, then augment those ionic determinants of excitability discussed earlier; for example, PGE2 can increase TTX-R sodium currents (159) and inhibit potassium currents (395). These acute actions cannot completely explain the alterations in nerve activity seen in both clinical and experimental postinflammatory models. For example, up to a third of patients with IBS report an antecedent episode of gastroenteritis and have symptoms, despite no macroscopic or microscopic evidence of ongoing inflammation (396). Furthermore, in experimental models of bladder, ileal, or colonic inflammation, DRG afferents are hyperexcitable, even after removal from the “inflammatory milieu,” and this increased excitability persists in short-term cell culture (22,158,172,175) (Fig. 25-18). In addition, there is evidence that after the resolution of nematode infection in the mouse, there is long-term increased excitability of both nerve terminals (397) and DRG cell soma (181) of afferents innervating the gut. These observations suggest that longer term changes in either the activity or number of ion channels regulating neuronal excitability have occurred, and in recent years there has been evidence to support that this occurs in both visceral and somatic sensory afferents. Given the importance of sodium and potassium channels in the generation and regulation of action potentials, a number of studies have focused on inflammation-induced alterations in these families of channels.

EXTRINSIC SENSORY AFFERENT NERVES INNERVATING THE GASTROINTESTINAL TRACT / 715 FB control

Vm (mV)

40

100 ms

−60

600 pA

300 pA

FB-TNBS

Vm (mV)

40

−60 100 pA

50 pA

A 10 8 300 6 200

4

100

2

0

B

No. of spikes 2X rheobase

Rheobase (pA)

400

Unoperated control

FB control

FB-TNBS

0

FIG. 25-18. Effect of 2,4,6-trinitrobenzene sulfonic acid (TNBS) ileitis on dorsal root ganglion (DRG) excitability. Ileal projecting DRG neurons were labeled with Fast blue (FB) injected into the wall of the ileum. (A) Representative intracellular recordings from isolated FB–labeled DRG neurons. Note lower current threshold (rheobase) and increased number of action potentials at twice rheobase in TNBS ileitis animals (bottom) compared with control (top). (B) Summary of these findings. DRG neurons projecting to the inflamed ileum (FB-TNBS) have significantly decreased thresholds (solid bars) and fire more action potentials in response to a doubled threshold stimulus (hatched bars) compared with labeled neurons from control animals (FB control) or unoperated control animals. (Reproduced from Moore and colleagues [175], by permission.)

With regard to sodium channels, the effects of inflammation in the stomach, ileum, and colon have been examined. Using a model of acetic acid–induced ulceration, Bielefeldt and colleagues (156) have shown that, in gastric DRG neurons, the proportion of the total Na current made up by the TTX-R component is increased, and that the repriming kinetics of the TTX-S current are increased. In other studies using 2,4,6-trinitrobenzene sulfonic acid (TNBS) to cause inflammation that resembles IBD, ileitis and colitis cause an increase in the magnitude of TTX-R currents in DRG neurons innervating the inflamed gut (22,158), and colitis causes a reduction in threshold and acceleration of the repriming kinetics of this current (22,158). In experiments using mice deficient in the TTX-R NaV1.8 channel, sensitization of the colon by inflammatory stimuli is blunted, whereas responses to normally noxious stimuli are preserved (398). Similar to the approaches taken to study Na channels, investigators also have examined the influence of inflammation on potassium currents in GI DRG neurons. 4-AP, which blocks the IA current, mimics the changes in excitability seen in inflammation of the bladder (172) and intestine (175). Consistent with this observation, ileitis (158) and gastric ulcer (170) reduce the magnitude of this current and shift its availability curve in a hyperpolarizing direction. In contrast, the effects on the IK current are controversial. Stewart and colleagues (158) found that ileitis reduced the IK, whereas in experimental gastric ulcers (170) and experimental cystitis (172), the IK was unaffected. Thus, it may be that ion current changes are specific to the degree, type, or site of inflammation. The overall effect of inflammation is to augment those factors that favor increased excitability (i.e., depolarizing currents, increased membrane resistance) and reduce those that oppose it (e.g., hyperpolarizing currents) (Table 25-4). Although there are few studies on this topic, the available evidence suggests that changes in ligand-gated channels may also be important. For example, Page and colleagues (262) have shown that in vagal afferents innervating the inflamed esophagus, P2X receptor activation sensitizes mechanosensitive afferents, which does not occur in the control situation. Experimental colitis also up-regulates purinergic signaling in colonic sensory afferents (263). In addition, in human

TABLE 25-4. Summary of inflammation-induced changes in voltage-gated ionic currents in gastrointestinal sensory neurons Current

Change

Nodose/DRG (organ)

References

Potassium: IA

Decrease current amplitude; left shift of inactivation curve No change/decrease current amplitude Accelerate recovery/no change

DRG, nodose (stomach, ileum)

158, 170, 172

DRG, nodose (stomach, ileum)

158, 170, 172

DRG, nodose (stomach, ileum, colon)

22, 156–158

DRG, nodose (stomach, ileum, colon)

22, 156–158, 170

DRG (colon)

22

Potassium: IK Sodium: TTX-S fast (NaV1.7-like) Sodium: TTX-R (NaV1.8-like) Sodium: TTX-R persistent (NaV1.9-like)

Increase amplitude, accelerate recovery, reduce threshold No change

DRG, dorsal root ganglion; TTX-R, tetrodotoxin-resistant; TTX-S, tetrodotoxin-sensitive.

716 / CHAPTER 25 IBD, there appears to be an up-regulation of both VR1 and ASIC3 receptors (241,274). In somatic pain models, 5-HT3 (399), TRPV1 (400), and P2X (401) expression are increased. The mechanism of changes in ionic currents currently is unclear; however, there is compelling evidence for NGF as an important mediator. Neutralization of NGF can attenuate visceral hyperalgesia (402) and hyperexcitability, and administration of exogenous NGF can mimic some of the ion channel changes seen in inflammation (167). The importance of NGF is further supported by that NaV1.8 expression levels are NGF dependent and most colonic DRG neurons express the high-affinity NGF receptor trkA (22). In addition, NGF may also play a role in stress-induced alterations in visceral sensitivity. In neonatally stressed rats, colonic hypersensitivity and increased gut permeability are reduced by NGF neutralization. The implication is that stress causes an increase in gut permeability and possibly sensitization of afferent terminals that is mediated by NGF (351). In addition, there exists evidence in myenteric neurons (403) and in somatic DRGs (404) that cyclooxygenase products may play an important role, and this clearly warrants further investigation in extrinsic visceral afferents.

CONCLUSION The extrinsic sensory innervation of the GI tract, although sparse in comparison with its intrinsic counterpart, is the crucial link allowing detection of noxious and nonnoxious GI events by the CNS, thus permitting CNS control of critical GI functions and enabling conscious awareness of events in the GI tract. Recent advances have significantly enhanced our understanding of the molecular factors regulating excitability, as well as providing insight into how these mechanisms underlie complex phenomena such as mechanosensation, visceral pain, satiety, and regulation of food intake. Future work likely will focus on the mechanisms of alterations in excitability in disease states such as inflammation and obesity, as well as on the mechanisms of development and targeting of the extrinsic innervation to the gut. Expansion of our knowledge of the extrinsic afferent supply of the GI tract undoubtedly will impact on the understanding and treatment of human disorders such as the functional GI disorders, obesity, and both local (intestinal) and systemic inflammatory states.

ACKNOWLEDGMENTS M. J. Beyak was supported by the Canadian Association of Gastroenterology (CAG)/AstraZeneca/Canadian Institutes of Health Research (CIHR) Research Initiative Award. D. C. E. Bulmer and W. Rong were supported by GlaxoSmithKline. W. Jiang was supported by the Bardhan Trust. C. D. Keating was supported by the Biotechnology and Biological Sciences Research Council (BBSRC).

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CHAPTER

26

Processing of Gastrointestinal Sensory Signals in the Brain Anthony Hobson and Qasim Aziz Neuroanatomic Representation of Gastrointestinal Sensation, 727 Thalamus, 728 Lateral Thalamus, 728 Medial Thalamus, 729 Cerebral Cortex, 729 Primary Somatosensory Cortex (S1), 729 Secondary Somatosensory Cortex (S2), 729 The Insula, 730 Cingulate Cortex, 730 Other Brain Regions, 730

Functional Brain Imaging, 730 Functional Brain Imaging of Gastrointestinal Sensation, 732 The Thalamus, 732 Primary Somatosensory Cortex, 733 Secondary Somatosensory Cortex and Insula, 733 Anterior Cingulate Cortex, 734 An Integrated View of Gastrointestinal Sensory Processing in the Brain, 734 References, 735

The brain receives myriad sensory signals from gastrointestinal (GI) afferents that encode a wide range of peripheral activity. These signals predominantly help regulate our homeostatic balance and influence motility, secretion, and appetite (1). Most of these signals are subliminal, rarely reaching our consciousness; liminal sensations, however, such as satiety and fullness, together with supraliminal sensations such as pain complete this sensory spectrum. All of these sensations invoke activity in a complex array of cortical and subcortical structures via projections from both vagal and spinal visceral afferents (2–7). This chapter focuses predominantly on the processing of GI sensory signals in the brain that lead to the conscious perception of visceral sensations. Particular attention is paid to the neuroanatomic representation of GI sensation and an overview of advances in our knowledge gained from functional brain imaging studies in humans. Chapter 25 describes in detail the ascending tracts and brainstem terminations of

both vagal and spinal visceral afferents; therefore, this chapter concentrates predominantly on the thalamus and higher cortical regions.

NEUROANATOMIC REPRESENTATION OF GASTROINTESTINAL SENSATION Although it is convenient to consider distinct functional roles for vagal and spinal afferents innervating the GI tract, convergence of the two occurs at multiple supraspinal levels (4,8,9); therefore, their activity is inextricably linked. For instance, vagal afferents synapse bilaterally in the nucleus tracti solitarii (NTS), which also receives a wide range of input from spinal afferents, as well as extensive input from other brain regions (4). The NTS projects mainly to the parabrachial nucleus of the pons, which again receives input from multiple spinal dorsal horn laminae, several of which convey visceral information (3,4). The parabrachial nucleus then projects to higher structures including the hypothalamus, thalamus, insula, amygdala, prefrontal cortex, and many other cortical regions that process both vagal and spinal visceral information (3,4). Figure 26-1 shows a schematic of the spinal and brainstem visceral sensory pathways to the thalamus and cerebral cortex.

A. Hobson and Q. Aziz: University of Manchester, Hope Hospital, Salford M6 8HD, United Kingdom. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

727

728 / CHAPTER 26 Spinal visceral (Sympathetic) Forebrain Afferent system

Brainstem visceral (Parasympathetic) Forebrain Afferent system

Cortex Pain insular cortex

Thalamus Pain (Lateral) shell area

Spinal cord

Visoeral insular cortex

Visceral (Medical) shell area

Parabrachial complex

Pons

Medulla

Ventroposterlor thalamic complex

Cranial nerve

Thalamus The thalamus is the largest structure in the diencephalon and comprises a collection of small nuclei. There are six separate nuclear groups in each thalamic hemisphere, with two functional types: relay (specific) nuclei and diffuse projecting (nonspecific) nuclei. Relay nuclei convey information between specific subcortical and cortical regions. These nuclei mediate perception, voluntary movement, emotion, and language. Diffuse projecting nuclei receive many converging sources of input and project to multiple cortical regions (12).

Nucleus of the solitary tract

Lateral Thalamus

Chemosensory Mechanosensory Spinal root

Visceral organs

outside world. Furthermore, there is extensive convergence of visceral and somatic afferents within the spinal cord, resulting in noxious stimuli emanating from the GI tract activating viscerosomatic nociceptive afferents and their corresponding target thalamic nuclei (8,9,11). Therefore, when describing the functional properties of specific brain structures, we consider somatic and visceral responsiveness as part of an interlinked sensory system.

Pain and chemosensory temperature

FIG. 26-1. Schematic drawing illustrating the spinal and brainstem visceral sensory pathways to the thalamus and cerebral cortex. Note that the spinal afferent pathway crosses in the spinal cord; it projects bilaterally in the brainstem to the nucleus of the solitary tract and the parabrachial nucleus, but only ipsilateral projections are shown. Spinal visceral afferents relay in the lateral part of the ventroposterior thalamic complex, together with other spinothalamic afferents, especially nociceptive afferents. The pathway taken by visceral afferents conveyed in the facial, glossopharyngeal, and vagus nerves relays in the nucleus of the solitary tract and the parabrachial nucleus, then crosses to innervate the medial part of the contralateral ventroposterior thalamic complex. The brainstem visceral sensory pathway terminates in the anterior part of the insular cortex in comparison with the spinal pathway, which terminates more caudally in the insular area. (Reprinted from the Annual Review of Neuroscience, Volume 25 © 2002 by Annual Reviews, [4] by permission.)

Extensive integration of spinal and vagal afferents means that demarcation of vagally mediated homeostatic and liminal processes from spinally mediated sensation may not be the correct approach. Even in somatic pain research, the close link between the autonomic pathways and pain processing has culminated in several researchers considering pain as a much a “visceral modality” (4) and “homeostatic emotion” (10) as it is “interoceptive”—that is, pertaining to the integrity of the body rather than to its relationship with the

Within the lateral thalamus, neurons within the ventroposterolateral (VPL) and ventroposterior medial nuclei have been shown to respond to noxious stimuli. Nociceptive neurons in VPL respond maximally to intense mechanical stimuli, noxious heat, and ascending C-fiber volleys. The receptive fields of VPL neurons are restricted in size, display somatotopic organization, and almost all project to the primary somatosensory cortex (S1). Notably, approximately 85% of VPL neurons respond to both visceral and cutaneous stimuli. The visceral input to VPL, however, is not viscerotopic (13). The properties and projections of VPL neurons suggest a role in the sensory-discriminative aspects of pain processing (6). However, this has been challenged by Craig (10,14), who suggests that the properties of the thalamic area that make up the posterior part of the ventromedial nucleus (VMpo) may, in fact, be more suited to this function (see the next section). Two other nuclei in the lateral thalamus that contain nociceptive neurons are the ventroposterior inferior (VPI) and the medial part of the posterior complex (POm). These are organized somatotopically but have larger receptive fields than VPL neurons, and it is thought that these VPI neurons project to the secondary somatosensory cortex (S2). Neurons from POm have been shown to project to the insula in monkeys (6). After stimulation of VPL in a patient with angina pectoris, symptoms of chest pain were reproduced without concurrent changes in cardiovascular function (15). This suggests a role of VPL in visceral and referred pain; however, an alternative explanation is that changes in neuronal excitability within the thalamus, due to chronic pain, may result in enhanced excitability of passing thalamocortical axons from VMpo, and these may also have contributed to the generation of this response (10).

PROCESSING OF GASTROINTESTINAL SENSORY SIGNALS IN THE BRAIN / 729 Medial Thalamus Studies of neurons within the central lateral/central median nuclei of the medial thalamus in awake monkeys have shown that they have large, bilateral receptive fields, indicating that they do not contribute to the localization of pain. However, these neurons were able to distinguish among different pain intensities, suggesting that they may play a role in other aspects of sensory discrimination (16). Nociceptive neurons have also been found in the ventromedial posterior nuclei (VMpo), which respond to noxious heat, cold, and mechanical stimuli (17). These have small receptive fields and are organized somatotopically, projecting primarily to the insular cortex (17), as well as area 3a of S1 (ref). Studies by Vogt and colleagues (18) have also shown that there are projections from the medial thalamic nuclei to the anterior cingulate and prefrontal cortex, regions that process cognitive and emotional aspects of the pain response. VMpo also receives input from the vagus-NTS-parabrachial pathway, which then projects on to similar cortical areas (3) that receive spinal dorsal horn lamina I neurons. Lamina I neurons have been implicated in ongoing homeostatic regulation, with nociception being one aspect of this. The vagus performs a similar function for the GI tract, with the exception of encoding noxious stimuli. The vagus also has inputs to both the reticular nucleus, which projects to most other thalamic nuclei and influences synchronization of thalamocortical projections (3), and the intralaminar nucleus, which has widespread projections to the cerebral cortex and striatum (19,20). In summary, the thalamus plays an important role in relaying nociceptive and homeostatic information to higher brain regions and also to the descending pathways that facilitate the modulation of autonomic responses and pain. Although it has been postulated that projections from the lateral thalamic nuclei mediate sensory-discriminative aspects of pain processing, medial thalamic nuclei may mediate cognitive, affective, and motivational aspects of pain processing. This, however, may be an oversimplification, and current theories suggest the medial thalamus plays a role in both (4,6,10,14,21,22).

(see Functional Brain Imaging), this section concentrates mainly on data from animal and lesion studies.

Primary Somatosensory Cortex (S1) Lesion studies of S1 in humans have shown contradictory evidence for its role in pain perception. These studies have shown that lesions may cause hypoalgesia, hyperalgesia, or have no effect at all (25). The number and localization of nociceptive neurons within S1 may partly explain these conflicting results. S1 is involved predominantly in processing nonnoxious, somatosensory information, such as pressure and warmth, which gives us vital information about both our bodies and our external environment. Consequently, nociceptive neurons are greatly outnumbered by neurons that respond to tactile stimulation. In addition, nociceptive neurons project to distinct regions of S1, at the borders between the cytoarchitectonic areas 3b-1, and S1-2 (26). Therefore, it is highly probable that the location of the lesions in these studies may be important in determining their effect on pain perception. Animal studies have revealed abundant projections of nociceptive neurons to S1 from the VPL and VMpo nuclei in the thalamus. As mentioned earlier (see Lateral Thalamus and Medial Thalamus sections), these neurons are arranged somatotopically and have restricted receptive fields, and activity within these neurons is correlated with the duration and intensity of a noxious stimulus (25). These data suggest that nociceptive neurons in S1 encode the sensory discriminative aspects of pain processing. One study has shown the lateral aspects of S1 receive vagal inputs, and it has been suggested that this region is continuous with the intraoral trigeminal representation in S1 (21,22). Direct electrical stimulation of this region has been shown to produce visceral sensations such as nausea, but not pain (1). In the cat, the specific region of S1 that receives vagal inputs is area 3a, a region thought to be important in sensorimotor integration. As stated earlier, area 3a also receives nociceptive input from VMpo, and these findings have led to the proposal that pain and innocuous visceral sensation are different aspects of “a common homeostatic afferent system” (21,22).

Cerebral Cortex Secondary Somatosensory Cortex (S2) Despite abundant nociceptive projections from thalamic nuclei to many regions of the cerebral cortex, its role in pain processing remained in doubt for many years (23,24). Animal studies, and more recently human functional brain imaging studies, have indicated that the cerebral cortex plays an important role in both the sensory-discriminative and affective components of pain. The following sections summarize the role of the four main cortical regions that have consistently been shown to participate in pain processing. Because data from functional brain imaging studies are reviewed later in this chapter

S2 lies in the parietal operculum in the upper bank of the sylvian fissure (27). Processing of pain within S1 and S2 is in parallel, as opposed to the serial processing of tactile information (25). S2 receives nociceptive projections from the VPI thalamic nuclei (see Lateral Thalamus earlier in this chapter) that have mostly large and bilateral receptive fields. Projections from S2 enter the temporal lobe limbic structures via the insula. This is similar to the projections of tactile neurons, where the role of S2 is thought to include learning, memory, and object recognition. S2 is therefore thought to

730 / CHAPTER 26 be important in helping us to recognize the nature of a painful stimulus. Evidence to support this comes from lesion studies, which have shown that damage to S2 impairs the ability to identify the nature of noxious stimuli so that although the stimulus is still unpleasant, subjects cannot differentiate between mechanical or heat pain, for instance (25).

The Insula The insula has a complex role in pain processing that has not been adequately defined. It has two distinct regions: the granular posterior insular, which is mainly thought to process tactile, auditory, and visual somatosensory function; and the dysgranular anterior insula (AI), which is involved in olfactory, gustatory, and viscero-autonomic functions (28). Although most pain studies have shown nociceptive inputs to the AI, the presence of nociceptive inputs to the posterior insula also have been described. Although the insula receives projections from S2, studies have shown that it also receives direct projections from nociceptors within the VMpo in the medial thalamus (see Medial Thalamus earlier in this chapter). Insular nociceptive neurons have large receptive fields, respond to different sensory modalities, and are strongly activated by visceral stimuli. Although it is clear that the insula does not play an important role in sensory discrimination, lesion studies have shown that damage to the insula reduces pain affect and other affective pain reactions. It is therefore thought that the insula integrates information from direct thalamic projections with that received from S2 and possibly the area 3a of S1 (22) and relays this to limbic structures to modulate autonomic reactions and pain-related memory/learning process. The insular cortex also has been called the visceral sensory cortex because of its important role in the processing of gustatory, cardiac, and GI information (4). This is supported by studies that demonstrated that direct stimulation of the anterior portion of the insula produces visceral sensations such as nausea and fullness but not pain (29), whereas stimulation of the posterior portion produces somatic sensations including pain (29). Therefore, the AI, with its dense projections to limbic and subcortical structures, in addition to those from the vagus, may be considered an important part of a visceral sensory network.

Cingulate Cortex The cingulate cortex is an extensive area of the limbic system that overlies the corpus callosum and comprises anterior and posterior portions. Although highly interconnected, it is the anterior cingulate cortex (ACC) that has been predominantly implicated in pain processing (18,30). The ACC receives direct projections from the medial thalamic nuclei and has nociceptive neurons with large, bilateral receptive fields that do not display somatotopic organization. Lesion studies in patients after cingulotomy have shown that although

pain was still perceived, it was less distressing and there was less motivation to avoid the painful stimulus. Consequently, the ACC has a role in the affective-motivational aspects of pain processing. A subdivision of the ACC has been described as the “affective division” (31). This region comprises the subgenual ACC (Brodmann area 25), Brodmann area 33, and the rostral region of Brodmann area 24 (31). This division has extensive connections with the amygdala, periaqueductal gray, and mediodorsal and anterior thalamic nuclei, and it has been associated with autonomic function and emotional behavior (32). Lesions of this region significantly impair the ability of the autonomic system to respond to conditioned stimuli, abolish conditioned emotional vocalizations to painful stimuli, and result in reduced aggression, shyness, and emotional blunting (31). Further information regarding the functional properties of the ACC is given later in this chapter (see Secondary Somatosensory Cortex and Insula).

Other Brain Regions Many other brain regions participate in the processing of painful GI sensations including the dorsolateral prefrontal cortex, posterior parietal cortex, primary motor and supplementary motor cortices, hypothalamus, amygdala, hippocampus, cerebellum, and caudate nucleus (6,33,34) (Fig. 26-2). Evidence to help in understanding the role of these regions in GI pain processing is far from established, and further study is needed to ascertain their functional significance. In addition, studies that have examined the cortical effects of direct vagal stimulation have shown activation in a similar complex network of neural structures, once again underlining the close relation between vagal (nonpainful) and spinal cortical projections. The next section discusses the contribution of functional brain imaging on our understanding of the processing of GI sensory signals.

FUNCTIONAL BRAIN IMAGING Functional brain imaging has greatly enhanced the ability to noninvasively record brain activity in humans. The three main brain imaging techniques discussed in this section are functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and magnetoencephalography (MEG). PET relies on the principle of detecting positronemitting radionuclides that are injected intravenously into a subject positioned with his or her head in a specialized detection system. There are currently hundreds of radiolabeled compounds used in PET, each with its own characteristics and uses for imaging particular biochemical or physiologic processes (35). Although PET radiopharmaceuticals can be used to mark metabolic processes such as glucose uptake into active neural tissue and labeling of molecules that bind to specific receptors

PROCESSING OF GASTROINTESTINAL SENSORY SIGNALS IN THE BRAIN / 731

FIG. 26-2. Schematic of ascending pathways, subcortical structures, and cerebral cortical structures involved in processing pain. All of the regions shown have been activated in studies of gastrointestinal pain. ACC, anterior cingulate cortex; AMYG, amygdala; HT, hypothalamus; MDvc, ventrocaudal part of the medial dorsal nucleus; PAG, periaqueductal gray; PB, parabrachial nucleus of the dorsolateral pons; PCC, posterior cingulate cortex; PF, prefrontal cortex; PPC, posterior parietal complex; S1 and S2, first and second somatosensory cortical areas; SMA, supplementary motor area; VMpo, ventromedial part of the posterior nuclear complex; VPL, ventroposterior lateral nucleus. (Reproduced from Price DD. Psychological and neural mechanisms of the affective dimension of pain. Science 2000;228: 1769–1772, by permission.)

in the brain, the most commonly used in GI research is 15O, which is prepared as H215O-labeled water. This approach is used to create perfusion images that measure blood flow to the brain. Cerebral blood flow has been used as a surrogate measure of brain activation as increases in regional cerebral blood flow (rCBF) have been closely correlated to regional glucose consumption (6) and more latterly with neuronal activity (4). These studies have predominantly used block design paradigms that compare images taken during active task and rest periods. This type of paradigm forms the principle for the majority of functional brain imaging studies discussed in this chapter. fMRI produces images of active brain regions by detecting indirect effects of neural activity on local blood flow, volume, and oxygen saturation, giving detailed information regarding the functional neuroanatomy of the brain. The first fMRI images were produced using contrast agents to demonstrate an increase in blood volume in the visual cortex in response to a visual stimulus (9); however, it soon became clear that naturally occurring differences in the magnetic properties of blood constituents could be used to produce these images. The most commonly used fMRI technique relies on the detection of deoxygenated hemoglobin, because of its paramagnetic

properties within the blood, and identifies changes in its regional distribution during conditions that change neuronal activation (11). One limitation of PET and fMRI is that they do not have sufficient temporal resolution to map cortical activity as it dynamically changes, on a millisecond-by-millisecond basis. MEG is a noninvasive brain imaging tool that allows detection of cortical neuromagnetic activity. Although the spatial resolution of MEG is comparable with PET and fMRI, it also has millisecond temporal resolution. The MEG signal is thought to primarily reflect intracellular currents flowing in the apical dendrites of pyramidal cells oriented parallel to the skull surface. The neuromagnetic signal generated by a single cell is too small to be detected, and MEG typically requires approximately 1 million synapses to be simultaneously active to produce a detectable signal. Because there are more than 100,000 pyramidal cells per cubic millimeter of cortex, each containing thousands of synapses, MEG signals can be measured from relatively small regions of cortex (36). MEG does have some limitations in imaging deep structures such as the brainstem because of the distance of these structures from the MEG sensors (37).

732 / CHAPTER 26 Functional Brain Imaging of Gastrointestinal Sensation To date there have been approximately 40 studies that have used functional brain imaging for the study of GI sensation (for review see Derbyshire [7]). It has been demonstrated that even subliminal stimulation of the GI tract is capable of activating a widely distributed neural network (52). In addition, comparisons of somatic and visceral pain have shown that both activate a similar network of cortical and subcortical structures with subtle differences possibly accounting for the different psychophysiologic characteristics of the perceived sensations. Therefore, as in the previous section, we shall mainly review brain imaging data pertaining to the viscera; however, references to the somatic literature are given where it is appropriate to our understanding of GI sensory processing. Figure 26-3 shows a typical fMRI image depicting regions of cortical regions of activity commonly observed in studies of GI sensation.

The Thalamus To date, information regarding the brain processing of visceral sensation has been obtained after stimulation of the esophagus, stomach, rectum, bladder, and vagina, in addition to that acquired during dobutamine-induced chest pain (38–43). In healthy subjects, thalamic activity has been reported in only approximately 50% of studies, and this has been predominantly in response to noxious rather than innocuous visceral stimulation. Strigo and colleagues (44) have compared the brain processing of visceral and cutaneous pain in the human

brain using fMRI. In this study, esophageal distension and contact heat stimulation of the anterior chest wall were matched for subjective intensity and applied to healthy subjects in a counterbalanced order. Analysis revealed that although differences were seen in cortical regions such as the AI, a common pain neural network was activated encompassing the secondary somatosensory cortex, posterior parietal cortex, basal ganglia, and thalamus. The authors concluded that similar activations seen within these regions implicate their function in the identification of a stimulus as painful, rather than in the differentiation of the nature of painful stimuli (44). In a PET study that examined the effect of increasing intensity of gastric distension on cortical and subcortical activation, Ladabaum and colleagues (39) report significant thalamic activity only at the highest distension volumes, which produced a noxious stimulus. Peak activations were noted bilaterally in the ventroposterolateral nuclei and in the left dorsomedial nuclei, leading the authors to conclude that noxious gastric stimulation activates both the lateral and medial pain systems. The ability to noninvasively map the functional neuroanatomy of the visceral pain matrix has lead to several researchers embarking on clinical studies in patients with functional gastrointestinal disorders (FGDs), predominantly in patients with irritable bowel syndrome (IBS). Patients with IBS commonly report heightened perception of rectal distension (visceral hypersensitivity), and the aim of these studies has been to identify objective neural correlates of visceral hypersensitivity. Although the heterogeneity of the IBS population has meant that results from group imaging data have been inconsistent, several groups have shown increased

FIG. 26-3. This series of images depict increased neuronal blood flow, as determined by functional magnetic resonance imaging, in response to painful stimulation of the esophagus (increases in activity are shown as the white shaded areas and represent regions of statistically significant activity). Image 1 shows bilateral activation of the insula and thalamus, image 2 shows bilateral activation of S2 and lateral S1, image 3 shows activation of dorsolateral prefrontal cortex, image 4 shows activation of anterior cingulate cortex, and image 5 shows activation of the supplementary motor cortex. These are the regions most commonly reported in imaging studies of the gastrointestinal tract and are also activated in studies of somatic pain.

PROCESSING OF GASTROINTESTINAL SENSORY SIGNALS IN THE BRAIN / 733 thalamic activation in response to noxious rectal distension (45–47). The thalamic regions of interest identified in these studies encompassed the VPL and dorsomedial nuclei. The limited spatial resolution of the fMRI imaging techniques used in these studies meant that it was not possible to comment on activation of specific thalamic nuclei, and thus no comment could be made on the specific contribution of the thalamus to sensory and affective dimensions of pain. Future clinical studies using high-resolution imaging of the thalamus may be able to provide more information regarding the contribution of different thalamic regions to aberrant visceral pain processing in FGDs. In summary, painful GI stimuli produce bilateral activation of the thalamus incorporating the regions of the VPL and dorsomedial nuclei. Heightened activation of these regions has been associated with aberrant pain processing in patients with FGD. As the spatial resolution of FBI techniques improves, it will be possible to study the thalamus in greater detail, shedding further light on its role in GI sensory processing.

Primary Somatosensory Cortex Activation of S1 during experimental pain using PET and fMRI is also seen in only approximately 50% of studies (34) using both somatic and visceral stimuli. The unreliability of S1 activation has more to do with the stimulus characteristics used, which are incompatible with the response characteristics of S1 neurons (34). One of the major differences seen between these somatic and visceral studies is that although stimulation of a somatic structure, such as a limb, evokes neural activity in a discrete region of contralateral S1 (48), stimulation of a viscera, such as the esophagus, evokes bilateral activity that spreads diffusely from superior (trunk representation) to inferior (intraabdominal representation) S1 regions (7,49). This can be explained by that spinal afferent signals originating from the esophagus are divergent and may enter the spinal cord at several dermatomal levels (50), and this helps to explain the diffuse nature of the perceived esophageal sensation. fMRI has been used to study differences in the topographic representation of the anorectum and esophagus. In a study by Hobday and colleagues (40), rectal (visceral) stimulation resulted in bilateral activation of inferior S1, whereas anal canal (somatic) stimulation resulted in activation of S1 at a more superior level. A second study by Aziz and colleagues (51) demonstrates that unlike somatic sensation, which has a strong topographic representation in S1, visceral sensation is primarily represented in the secondary somatosensory cortex (S2), whereas representation in S1 was more diffuse. However, both of these studies do point to a crude viscerotopic organization of GI sensation in S1. One study has shown that S1 activity can be detected in response to rectal distension even at subliminal intensities (52). This study showed that neural activity occurred at distension levels that were not perceived by the subjects, and

this activity increased as the stimulus intensity reached liminal and supraliminal levels (52). Importantly, the locus of activity did not change, and this probably points to mediation of this response via both vagal and spinal afferents because both demonstrate similar dose–response curves to stimulation (53), with the exception that vagal afferents saturate at lower intensities in comparison with spinal afferents. Furthermore, both spinal and vagal afferents project to area 3a of S1, but only spinal afferents project to Brodmann areas 1 and 2 of S1, which also were activated in this study (52). Studies using MEG have confirmed that esophageal sensation is processed diffusely in S1, but the temporal information gained from this approach has shown that S1 activity occurs in parallel with that which occurs in S2 and posterior insula (54–57). This is identical to the processing of somatic pain and is in contrast with tactile processing, which occurs in series with S1 receiving sensory input before S2 (58).

Secondary Somatosensory Cortex and Insula The secondary somatosensory cortex and insula are the most commonly activated regions described in the somatic/ visceral pain literature (34,38,52,57,59–62). Activation in these regions is bilateral and encompasses a region comprising the depth of the sylvian fissure and the parietal/frontal operculi. Although difficult to discriminate when using group imaging, because of intraindividual variability, most studies depict two distinct loci within this region corresponding to the AI and posterior insula/S2. Several studies have shown that both these regions encode the intensity of stimulation as rCBF increases as the stimulus is intensified (59). It is clear that the AI is also modulated by changes in emotional state (63,64). A study showed increased activation of the AI when esophageal stimulation was delivered during a negative emotional context (65). Posterior insula/S2 does not appear to be modulated by attention or emotion, and its role appears to be in the recognition, learning, and memory of painful events (25). One study, rather surprisingly, revealed that the anterior portion of the insula was activated more intensely by thermal chest wall stimulation when compared with esophageal distension. Previous studies have shown bilateral activation of the AI to be the most consistent finding across all esophageal neuroimaging studies, as well as playing an important role in the emotional modulation of sensory processing (65). However, AI is also strongly activated by thermal stimulation (66), and the differences seen here may merely reflect that a greater number of somatic thermosensitive neurons are present in this region when compared with visceral-specific neurons. Both thermal and esophageal stimuli produced bilateral activation of the posterior insula, which is known to process somesthetic sensation, providing further evidence for the convergence of visceral and somatic pain. MEG data have shown that activity in the AI evoked by painful esophageal stimulation occurs approximately 20 msec later than that recorded in the posterior insula. This difference

734 / CHAPTER 26 in the temporal sequence of activation between these two insular regions may be partly explained by studies that have shown that the posterior/mid-insula region is part of a somesthetic network involved in processing painful and nonpainful somatic sensations (29,67), whereas the AI is part of a visceral sensorimotor network (29,67) (see The Insula section earlier in this chapter). Taken together, these findings suggest that although the posterior insula and AI have dense reciprocal connections (28), the significant latency difference of activation between these two regions probably reflects their respective roles in processing different aspects of the pain experience.

the cognitive and emotional aspects of pain or executive motor planning (35). Further evidence to support this comes from a PET study performed by Rainville and colleagues (74,75). This study used regression analysis of PET images to demonstrate that although ratings of pain intensity were correlated with activity in S1, ratings of pain unpleasantness were correlated with activity in the caudal ACC. Although the majority of data available to date focus on GI pain processing, it may also be possible to image brain activity involved in vagal processes, such as nutrient signaling (76). Functional brain imaging has considerable potential in helping us to study the central processing of GI sensory signals and provides us with an unparalleled opportunity to make fundamental advances in our understanding in this area.

Anterior Cingulate Cortex The ACC is also commonly activated during somatic and visceral pain research, with the most common activations occurring in the mid, rostral, and perigenual cingulate regions. Although not involved in spatial discrimination of pain stimuli, studies have shown that the cingulate cortex is functionally heterogeneous containing regions that process information regarding intensity, emotion, attention, and mood (34,68). In Buchel and colleagues’ study (68), increased rCBF in the dorsal anterior ACC was thought to be associated with cognitive processing as signal strength only increased between the conditions of no sensation and innocuous sensation, with no further increases seen with subsequently higher intensity stimulation levels. In contrast, in the dorsal posterior ACC, rCBF increased incrementally as stimulus intensity increased, indicating a role in sensory processing. Finally, a region in the ventroposterior ACC appeared to be specific for pain because this area did not distinguish between nonperceived and innocuous stimuli but did show incremental increases in signal strength at increasingly painful stimulation intensities (68). A study that has examined the intrastudy reproducibility of ACC activation to esophageal stimulation has shown that, when studying naive subjects, activity of dorsal ACC decreases when the data are acquired on a second occasion in comparison with the first study. This suggests that the ACC response habituates over time (69). This has important connotations for the interpretation of imaging data, especially when comparing activation patterns obtained from patients and trained healthy volunteers (70). Data from electroencephalography recordings after laser stimulation of the arm also have demonstrated that activation of the midposterior cingulate cortex occurs later than that of the somatosensory cortex, and that the locus of this activity moves anteriorly along the cingulate gyrus fading out in the prefrontal regions (71–73). These findings are similar to those described by Hobson and colleagues (57), who showed that after painful esophageal stimulation, activity was first seen within the mid-ACC (Brodmann areas 32 and 24) with later activity occurring in the perigenual and posterior areas. That the cingulate cortex is activated later than the somatosensory regions is consistent with its role in processing

AN INTEGRATED VIEW OF GASTROINTESTINAL SENSORY PROCESSING IN THE BRAIN It has long been accepted that somatic sensory and motor systems are finely regulated with discrete somatotopic representation throughout the CNS (6,25). Some work has demonstrated that the organization of GI/visceral sensory processing is equally complex with distinct viscerotopic representation at several CNS levels (4). In addition, there is extensive convergence of visceral and somatic afferents throughout the CNS (4,8,9,11,22). This complexity is required to regulate appropriate autonomic responses to a spectrum of sensory and emotional events that influence homeostatic function (2,4). Subtle differences in the cortical and subcortical representation of somatic and visceral sensations may explain some of the distinct psychophysical properties (see later) commonly attributed to sensations arising from the GI tract. To improve our understanding of these processes, we must consider GI sensory processing in the context of being part of a highly integrated sensory system. A prime example of the need to use this approach is in the study of the mechanisms of visceral pain. Pain arising from the GI tract has certain psychophysical properties that differentiate it from somatic pain in that it is poorly localized and induces strong autonomic reactions (77). However, the neuroanatomy of visceral pain as described both in this chapter and Chapter 25 means it cannot be considered as a wholly separate entity. Nociceptive signals arising from the GI tract are transmitted via primary afferents to the spinothalamic tract and postsynaptic dorsal column pathway (78). Here this relatively small afferent input converges with viscerosomatic nociceptive and nonnociceptive afferents (9) and is conveyed to several thalamic nuclei and onto cortical structures. Divergence of primary GI afferents results in the nociceptive signal entering the spinal cord across many dermatomal levels. If we take the esophagus as an example, noxious esophageal distension produces activity in neurons that project from C2 to T12 (50). In addition, rather than projecting specifically to contralateral S1, as seen with noxious stimulation applied to a limb, esophageal representation is diffuse, encompassing

PROCESSING OF GASTROINTESTINAL SENSORY SIGNALS IN THE BRAIN / 735 bilateral activation of S1 in the trunk and intraabdominal regions. This widespread activation of brain regions involved in the sensory discriminative aspects of pain certainly explains the diffuse nature of the sensation evoked by esophageal stimulation across the chest wall and into the back. Whereas direct stimulation of brain regions such as posterior insula induces localized somatic pain, the only GI sensations to be evoked in this way are vagally mediated liminal sensations, such as nausea and fullness (29). Because vagal afferents project to S1, S2, and insula cortices, this is not an unexpected finding (3) and helps, in part, to explain the strong autonomic reactions evoked by GI pain. However, the inability of stimulation of any brain region to evoke GI pain raises an important question whether “true visceral” pain as defined as “felt along the midline and accompanied by marked autonomic and emotional reactions” (79) merely reflects diffuse activation of the spinally mediated somatic pain network. This dichotomy is unhelpful and perhaps a more fruitful approach would be to consider the contribution that spinal, vagal, visceral, and somatic afferents make to the resultant pain experience generated within an integrated somatovisceral nociceptive system (4,10,80). In summary, we should no longer consider the processing of GI sensory signals in the brain as part of a restricted visceral network. A growing body of evidence suggests that sensory information arising from the viscera is part of a complex neurobehavioral system involved in homeostasis, pain, and emotional processing. The resurgence of interest in a field that dates back centuries, partly because of advances in functional brain imaging, provides us with an opportunity to greatly advance our understanding of the important role that GI sensory signaling plays.

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CHAPTER

27

Enteric Neural Regulation of Mucosal Secretion Helen Joan Cooke and Fedias Leontiou Christofi Reflex Modulation of Endogenous “Secretory Tone,” 738 Functional Neuoranatomy of Secretory Reflexes, 738 Enteric AH Neurons (Intrinsic Primary Afferent Neurons), 739 Interneurons, 740 Secretomotor Neurons, 740 Extrinsic Primary Afferent Neurons, 741 Sensory Enterochromaffin/BON Cells, 741 Chemosensitive Stimulation of 5-Hydroxytryptamine Release, 741 Mechanosensitivity of Enterochromaffin/BON Cell Model, 741 Endogenous Adenosine as a Physiologic Brake, 745 Excitatory Adenosine Receptors, 747

Secretory Reflexes, 748 Role for Adenosine Triphosphate in Sensory Signaling, 748 Mucosal Stroking Reflexes in Rodent Intestine, 749 Distention and Secretory Reflexes, 751 Secretory Reflexes in Humans, 752 Mediators of Synaptic Transmission in Secretory Reflexes, 754 Coordination of Secretion and Motility, 755 Cyclic Adenosine Monophosphate Signaling and Secretion, 756 Pathobiology of Reflex-Driven Intestinal Secretion, 758 Cholera Toxin–Induced Secretion, 758 Clostridium Difficile Enteritis, 759 Purines and Mucosal Disorders, 760 References, 761

The digestive tract is a unique organ because of its extensive surface area that is exposed to an enormous variety of stimuli including mechanical forces, diverse chemicals that are present in food, and toxins from enteropathic organisms. It must sort these signals and implement preprogrammed patterns that include coordinated movements of the musculature appropriately timed with stimulation of secretions, enabling smooth passage of the luminal contents and alterations in blood flow to provide metabolic nourishment to the cells. All of these functions operate in concert for appropriate digestive and interdigestive activities. This chapter focuses on the neurally evoked secretory component that is a consequence of chloride secretion into the intestinal lumen. This chapter examines the wiring of the neural circuitry that accommodates extremes in fluid secretion.

We give an overview of the circuits, the receptors that are present, and how these neural programs meet the minute-tominute need of the intestines. It was the ingenuity of Hubel (1) and his Iowa-endowed stamina that led to a major advance in methodology for studying enteric neural regulation of intestinal secretion. He adapted ordinary Ussing flux chambers, commonly used by epitheliologists to study ion transport, by adding stimulating electrodes to create an electrical field in the plane of the tissue while simultaneously recording a transepithelial shortcircuit current (Isc) indicative of ion transport. He demonstrated unequivocally that submucous neurons could regulate chloride secretion. His findings launched a whole new area of research on the enteric nervous system and its influence on epithelial cells. His contribution set the stage for all of us who have since jumped on the secretory bandwagon. There has been an explosion of articles on the enteric nervous system during the beginning of this century. Our focus is slanted to purines and their role in synaptic transmission, as well as their emergence as autocrine, neurocrine, and paracrine messengers (2,3). The complex interplay among these roles is discussed here and related to pathophysiology and relevance to intestinal secretory disorders.

H. J. Cooke: Department of Neuroscience, The Ohio State University, Columbus, Ohio 43210. F. L. Christofi: Departments of Anesthesiology and Physiology and Cell Biology, The Ohio State University, Columbus, Ohio 43210. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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738 / CHAPTER 27 REFLEX MODULATION OF ENDOGENOUS “SECRETORY TONE”

extrinsic and intrinsic afferent neurons, by releasing hormones or paracrine substances and immune mediators to modulate chloride secretory rates. “When awakened from its sleep, the lion can cause a tremendous roar.” This analogy characterizes the extremes of fluid and electrolyte secretion that can occur when the secretory reflexes are activated. Reflex-driven chloride secretion with its accompanying obligate of sodium ions and water is an important mechanism that flushes out the intestinal contents to protect the organism from massive invasion of microorganisms or foreign antigens (7). One of the clinical symptoms of excess chloride secretion is diarrhea.

Basal secretion is spontaneous fluid secretion that occurs in the resting state without any stimulation by exogenous compounds. Basal secretion is the net flux of ions and fluid into the intestinal lumen. “Secretory tone” was deduced from changes in basal Isc, a measure of chloride secretion. “Secretory tone” is dependent on the spontaneous release of endogenous neural or paracrine mediators (somatostatin, prostaglandins, adenosine triphosphate [ATP], adenosine) (3–6). Of particular interest are compounds that extend the life of ATP (ARL 67156) or nucleotidases that break down ATP (apyrase). They have opposing effects on Isc and implicate nucleotides as recently discovered modulators of “secretory tone” (5). Activation of the secretory reflex by exposing the gut to enteropathogenic microorganisms or to food antigens that are identified as “foreign” significantly enhances chloride secretion by direct actions on epithelial cells or neurons or indirect mechanisms through release of intermediary messengers. Thus, surveillance systems are in operation in the gut so that it may respond to threats to itself by activating

FUNCTIONAL NEUROANATOMY OF SECRETORY REFLEXES The secretory reflex is composed of various types of neurons with cell bodies that originate in either the submucous or myenteric ganglia within the gut wall (Fig. 27-1). The reflex contains intrinsic primary afferent neurons (IPANs) that carry information on the digestive state to the enteric ganglia, possibly interneurons that process the

Small intestine 5HT IPANS

Vasomotor

ACh,SP,Glu,CGRP

Secretomotor Non-Vasomotor

Myenteric CM Submucosal 5HT3?

VIP/cAMP

VPAC VIP

Epithelial

ACH

M3

EC

P2Y1 CHAT(−)

SP NK1

CHAT/NPY

5HT

NK1/ NK3

CHAT/CALRET

E

?

Axon reflex?

P2Y1

VIP/cAMP(−)

CHAT/CALB

5HT4

M3

VIP VPAC

ACH M3

Cl − Distortion

Cl Secretion Cl −

Cl −

FIG. 27-1. Generic intestinal secretory reflex based on guinea pig intestine. Neural reflex pathway regulating secretion includes submucous intrinsic primary afferent neurons (IPANs) that project to the mucosa, to submucous ganglia, and to myenteric ganglia. IPANs synapse with several types of cholinergic (ChAT) and vasoactive intestinal peptide (VIP) secretomotor neurons and release acetylcholine (ACh) at muscarinic receptors (M3) and VIP at VIP receptors (VPACRs) on epithelial cells, or arterioles. VIP neurons are VIP/cyclic adenosine monophosphate (cAMP) and VIP/cAMP(−) and may be further subdivided into VIP/cAMP/A2ARs and VIP/cAMP/A2AR(−). Cholinergic neurons are ChAT/calretinin (CALRET), ChAT/NPY, and ChAT(−). Mechanical distortion is one stimulus for activation of the reflex. Myenteric secretomotor neurons project to the mucosa as well. CGRP, calcitonin gene–related peptide; EC, enterochromaffin cell; 5-HT, 5-hydroxytryptamine; NK1, neurokinin-1; NPY, neuropeptide Y; SP, substance P; A2ARs, adenosine A2A receptors.

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 739 information, and secretomotor/vasomotor neurons that carry information to the effecter cells such as epithelial cells, arterioles, and glands (see Fig. 27-1). The neurotransmitters released target receptors on crypt epithelial cells to secrete chloride, other ions, and fluid. Our functional models are based primarily on guinea pigs, but this chapter addresses regional and species differences where possible.

Enteric AH Neurons (Intrinsic Primary Afferent Neurons) Most AH neurons are named for the long-lasting, after hyperpolarizing current after an action potential discharge and are often equated with IPANs. IPANs is a somewhat controversial classification because it implies that the first neuron in the reflex is sensory (see Chapter 23, AH- and S-type neurons). Until the controversy is resolved, we will continue to use the term IPANs, which is ingrained in the scientific vocabulary (2,7,8). Neurons that detect changes in the luminal environment or forces imposed on it are relatively few. The sensory aspects of these AH neurons have been characterized mostly in the guinea pig small intestine, with IPANs comprising about 15% of all submucous neurons and 25% to 30% of myenteric neurons. AH neurons have cell bodies located in either myenteric or submucous ganglia. Not all IPANs are AH neurons, as it has been reported in the colon that Dogiel I/S neurons might serve this role (9). Submucous IPANs may drive other IPANs via slow excitatory postsynaptic potentials (EPSPs), suggesting that these neurons may play an additional role as interneurons that simultaneously regulate directional spread of neural activity. In the guinea pig, stimulants such as pH (3.5 or 9–11), short-chain fatty acids, or 5-hydroxytryptamine (5-HT) and ATP depolarize AH cells and generate action potentials that are not a consequence of synaptic activity (10). Both myenteric and submucous IPANs have one sensory/primary afferent process projecting to the mucosa. Processes of submucous IPANs project to other IPANs, to submucous neurons, and to myenteric ganglia. They have Dogiel type II morphology with two or more long axonal processes that travel primarily circumferentially in the myenteric plexus, although in a minority of dendritic Dogiel type II neurons there may be a long descending axonal process (>10 cm) for long descending reflexes (7,8). In the submucous plexus, Dogiel type II neurons project in all directions with no apparent polarity (see Cyclic Adenosine Monophosphate Signaling and Secretory Reflexes later in this chapter). Electrical stimulation or receptor-mediated activation depolarizes AH cells and generates action potentials, followed by a characteristic long-lasting afterhyperpolarization of the membrane potential lasting longer than 1 to 10 seconds. During the AH potential the cell is refractory to firing action potentials (7,10,11). Modulation of the duration of the AH potential by transmitters, immune mediators, or paracrine mediators is therefore an effective mechanism for gating the excitability of these neurons.

There are four types of IPANs that have been identified. First, submucous, but not myenteric, neurons respond to distortion/deformation of the mucosa at sites less than 1 mm2 from the recording site. These may be modality specific because they have not been described for myenteric IPANs (8,11). The second type is submucous neurons that are sensitive to distention (3,12). The third type is myenteric neurons that are sensitive to chemicals such as acids or short-chain fatty acids, D-glucose, and 5-HT. The fourth type is myenteric neurons that react to onset of stretch or maintained stretch (13). In guinea pig small intestine, stretch (15% stretched), but not suction, increased the open probability of the largeconductance (BK) K+ channel. It is unclear if this is one of the components of mechanosensory signaling (10–13). Stretchsensitive activation of IPANs appears to occur primarily in myenteric plexus, although there is a report that stretch also can influence sensory transmission in the submucous plexus (2,3). A majority of IPANs are not sensory neurons per se, but they are activated by mediators released from sensory cells such as 5-HT from enterochromaffin cells (11,14). A unique feature of enteric IPANs that is not shared by other sensory neurons is that they receive strong slow excitatory synaptic inputs (slow EPSPs). Possible mediators of slow EPSPs are predicted on the basis of receptors on IPANs—tachykininergic (neurokinin-1/3[NK1/NK3]), serotoninergic (5-HT), purinergic (P2Y), calcitonin gene–related peptide (CGRP)— and for inflammatory mediators (bradykinin [B2], histamine [H2], prostaglandin E2 [PGE2]) (2,5,7,14–18). Less common (and region specific) are fast EPSPs possibly mediated through nicotinic, purinergic (P2X), and serotoninergic (5-HT3) or possibly other receptors (18–20). Immunohistochemically, IPANs are cholinergic with immunoreactivity for choline acetyltransferase (ChAT) and calbindin-D28 (CALB), a calcium-binding protein (2,7,16). About 80% of these neurons have substance P (SP). Their chemical code is ChAT/CALB/SP. One subset expresses glutamate, ChAT/SP/GLU (16,17); another subset of IPANs includes ChAT/CALB/CGRP (18). This chemical coding suggests that acetylcholine, SP, glutamate, and CGRP are putative transmitters in these IPANs that mediate synaptic transmission to secretomotor neurons or possibly to myenteric ganglia as well. In the guinea pig colon, CGRP has been shown to evoke an increase in Isc only when the myenteric plexus is intact (20). Because CGRP is reported to be present in submucous IPANs, release of CGRP may occur from processes of IPANs at synapses with myenteric neurons. There is a report that CGRP acts through myenteric neurons to facilitate 5-HT release by the action of tachykinins at NK2/NK3Rs (21). The chemical coding of IPANs in the guinea pig intestine is different from that in human and porcine intestines (22,23). Calbindin, found predominantly in IPANs in the guinea pig intestine, is found in porcine interneurons and Dogiel type II neurons that receive both fast nicotinic and muscarinic inputs. This emphasizes the differences among species and suggests that chemical coding must be done for each species and correlated with function to identify IPANs with any certainty.

740 / CHAPTER 27 Interneurons Once the IPANs have been activated, they release neurotransmitters at synapses with either interneurons (more rarely) or secretomotor/vasomotor neurons. Electrical recording from S/type 1 neurons, in response to application of pressure pulse of 5-HT, revealed fast synaptic transmission from cholinergic interneurons around the circumference and in the orad-to-caudad direction. The short projections of these neurons within a maximum of 3 mm may make it difficult to record slow synaptic transmission from IPANs (2,11,13). Myenteric 5-HT interneurons project to submucous ganglia where they release 5-HT at synapses with secretomotor neurons that express 5-HT3 receptors. In the guinea pig and human colons, there are submucous vasoactive intestinal polypeptide (VIP)–containing interneurons, which partially project to myenteric ganglia or to circular muscle, and therefore may be involved in coordination of secretion and motility (24,25). This is further reinforced by the finding of receptors for VIP (VPAC1 or VPAC2) on cell bodies of neurons in submucous ganglia.

Secretomotor Neurons Secretomotor or vasomotor neurons have S/type 1 electrophysiology and uniaxonal morphology and are subdivided into cholinergic (ChAT) or VIP-containing neurons. In the guinea pig ileum, the submucous ChAT neurons comprise about a third of the population, and VIP neurons comprise almost half (8). In the guinea pig small intestine, there are three types of ChAT neurons according to the following chemical coding: (1) S/type 1 cholinergic neurons coded as ChAT/calretinin (CALRET) are vasomotor and innervate the glands and arterioles; (2) ChAT neuropeptide Y (NPY) secretomotor neurons; and (3) ChAT−/secretomotor neurons lacking these markers (2,8). The mucosa is innervated by a small fraction of ascending cholinergic (13% of ascending neurons) secretomotor neurons and by a population of descending VIP secretomotor neurons (2,11,26–28). Most of the VIP neurons are secretomotor or vasomotor, but some are interneurons as alluded to earlier. These reflexes exhibit polarity with a pattern of innervation that amplifies the mucosal signal (26,27). The polarity is oral-to-anal such that cholinergic neurons provide converging and diverging inputs to secretomotor neurons in adjacent submucous ganglia (3). Secretomotor neurons are triggered by synaptic transmission from IPANs, causing release of acetylcholine or VIP that activate chloride-secreting crypt epithelial cells expressing muscarinic and VIP receptors (VPACR) (see Fig. 27-1). Both cyclic adenosine monophosphate (cAMP) and Ca+2 activate chloride ion channels that allow movement of chloride into the lumen (3,15). VPAC1R and VPAC2R are distributed (with or without coexpression) in crypt cells, or in cell somas and varicosities of submucous neurons, where they are thought to play a role in altering neurotransmitter release by presynaptic and postsynaptic mechanisms (3,29).

Cyclic AMP immunoreactivity and visualization with a new antiserum to acrolein-derivatized cAMP identified filamentous neurons with VIP immunoreactivity in response to the activator, forskolin (see Cyclic Adenosine Monophosphate Signaling and Secretory Reflexes later in this chapter). VIP neurons are subcategorized into cAMP-dependent (VIP/ cAMP) and cAMP-independent (VIP/cAMP−) categories (28). Because 100% of all VIP-immunoreactive neurons are also cGMP immunoreactive, it can be deduced that a majority of VIP secretomotor neurons are regulated by the activity of both adenylyl cyclase (AC) and guanylate cyclase (GC) (30). Negative cross talk between cAMP and cGMP pathways occurs, leading to cGMP suppression of AC/cAMP signaling in submucous neurons. This is evident from studies that an inhibitor of cGMP, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin1-one (ODQ), eliminates the inhibitory influence of cGMP and leads to a more robust cAMP response in submucous neurons. Cyclic GMP exerts an ongoing inhibitory influence on cAMP signaling. Negative cross talk between these second messengers, cAMP and cGMP, in the regulation of submucous neurons is a mechanism for dual regulation of excitability in submucous VIP neurons that is not shared by myenteric neurons. This technique for examining dynamic changes in intracellular free cAMP levels in submucous neurons should lead to a better understanding of the role of this second messenger in secretory reflexes. Cyclic AMP visualization revealed a subset of VIP neurons that contain functional adenosine A2ARs coupled to AC/cAMP. These are subclassified as VIP/cAMP/A2ARs or VIP/cAMP/A2AR−. Thus, the activity of subsets of VIP secretomotor neurons is differentially regulated and distinguished by AC/cAMP signaling, cAMP/cGMP negative cross talk, and A2AR activation. Previous studies in the central nervous system have shown that A2AR activation causes VIP neurotransmitter release in a subset of VIPimmunoreactive neurons (5,31). Comparing the distribution of cAMP-visualized neurons per ganglion per square centimeter indicates that neurons expressing A2ARs that generate cAMP in response to forskolin are found in subsets of ganglia of variable size (3–10 neurons/ganglion). A2AR activation or forskolin stimulation did not increase cAMP immunoreactivity in NPY secretomotor neurons. NPY neurons did not colocalize with cAMP immunoreactivity (NPY/A2AR−), further supporting the notion that A2ARs are coupled to AC only to specific functional groups of neurons within the submucous plexus (28,30). Furthermore, lack of dependence on cAMP signaling is a unique feature of NPY secretomotor neurons. There are two small populations (1% each) of myenteric secretomotor neurons that are ChAT or VIP neurons (2,25). A small population of VIP interneurons project from the submucous ganglia to the myenteric ganglia and may be important in coordinating secretion and motility (11,25). In the porcine model, one type of ChAT secretomotor neuron is characterized by dendritic and uniaxonal-like morphology and colocalizes with SP (22). In the guinea pig submucous plexus, SP is confined to cell bodies of IPANs. Other ChAT secretomotor neurons express somatostatin

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 741 in both plexuses. VIP neurons colocalize with VIP/galanin in the pig and ChAT/VIP in human colon (23). Important species differences do exist in the chemical coding of secretomotor neurons (22,23).

Extrinsic Primary Afferent Neurons Extrinsic primary afferent neurons (EPANs) are important in transmission of sensory signaling and in central and local reflexes. Their cell bodies are in the nodose ganglia and send information from the periphery to the central nervous system. Their central terminals are located in the nucleus tractus solitarius in the brainstem. Vagal afferents primarily innervate the more proximal regions of the gastrointestinal tract, and the spinal afferents innervate more distally (32,33). They convey nociceptive information from the gut to the central nervous system. They may participate in normal inflammatory processes in the gut by virtue of axon reflexes. Axon reflexes may occur when antidromic spread of action potentials invades collaterals of EPANs in proximity to enteric ganglia or immune cells, causing these collaterals to release SP or CGRP (see Fig. 27-1). Release of SP from EPANs can trigger mast cell degranulation and release of tryptase, which binds to protease-activated receptors known to release prostaglandins (PGE2). Vagal afferents with terminals in the serosal layer are activated by distortion but not by distension or muscle contraction. Spinal afferents are similar with the exception that they signal at a lower threshold and participate in physiologic functions and nociception. Afferent endings of submucous IPANs in the lamina propria of the mucosa are of particular interest to chloride secretion, because they respond to luminal chemicals and tactile stimulation (33). Therefore, there is a potential role for EPANs to augment chloride secretion by releasing transmitters that may augment the local activity initiated by the activation of a secretory reflex pathway.

SENSORY ENTEROCHROMAFFIN/BON CELLS In the rat, enteroendocrine and immune cells guard the intestine from microbial invasion or noxious agents. One of the endocrine cells most studied is the enterochromaffin cell, which releases 5-HT, a biogenic amine (14). 5-HT that is released locally acts as an autocrine mediator by stimulating receptors on enterochromaffin cells, as a paracrine messenger by affecting epithelial cells, and as a neurocrine mediator modulating neuronal activity (34). There has been limited progress in understanding the complex regulation of 5-HT release in tissue studies because of potential interference from mediators released from other cells as well. The human carcinoid BON cell is a unique model for studying properties of nontransformed enterochromaffin cells or for transformed cells such as carcinoid tumors (35). BON cells are derived from a metastasis of a pancreatic carcinoid tumor. These transformed cells (kindly donated by

Dr. Courtney Townsend, University of Texas) enabled us to examine the release of 5-HT in the setting of single cells that retained many of the mechanosensitive and chemosensitive properties of the nontransformed cells. BON cells retain their ability to respond to nutrients (glucose), mechanical stimulation (touch, pressure, shaking), or receptor activation (purine P1, P2, adenosine A1, A2A, A2B, A3R) (5,35–37). BON cells also serve as a model for carcinoid tumors, because injection of dispersed BON cells into nude mice forms carcinoid tumors with histology similar to that of the original tumor. BON cells can serve as dual models of 5-HT release in the gut (Fig. 27-2A) (38).

Chemosensitive Stimulation of 5-Hydroxytryptamine Release Protrusion of microvilli into the lumen raises the possibility that enterochromaffin cells may “taste” the luminal contents. Ingestion of a meal slows gastric emptying and secretion, whereas it stimulates biliary and pancreatic secretions (5,15). These processes require transduction of chemical and mechanical forces within the duodenum, processing in the central nervous system, and adjustments in intake and outflow to prevent overloading the digestive capacity of the intestine. In a human enterochromaffin cell model, BON cells (10–100 mM D-glucose) caused a graded increase in 5-HT release that was not caused by changes in osmolarity. D-glucose and the nonmetabolizable α-D-glucopyranoside stimulated 5-HT release that was inhibited by phloridzin, an inhibitor of D-glucose uptake. 5-HT was not released by the diffusive transport of fructose, a substrate for facilitated glucose transporter 2 (GLUT2) and GLUT5 proteins. BON cells express a sodium-dependent glucose transporter-1 (SGLT1)–like protein that is involved in chemotransduction of metabolizable and nonmetabolizable hexoses at high concentrations of D-glucose. It has similarities to SGLT1 and SGLT3, “a glucosensor” (39). The SGLT1-like protein in BON cells differs from that described in small intestinal myenteric neurons (40). The protein in BON cells has a 10- to 15-fold greater D-glucose threshold for 5-HT release than the 5 mmol/L threshold for depolarization of myenteric neurons (39,40). BON cells release 5-HT when triggered by either D-glucose or nonmetabolizable hexoses, whereas intracellular glucose metabolism was proposed to activate a subset of glucosensitive neurons. More recent findings indicate that SGLT3 is a potential glucosensor that binds but does not transport glucose in both myenteric and submucosal cholinergic neurons. Further studies are necessary to investigate the signaling pathways for SGLT3 in enterochromaffin cells or their model.

Mechanosensitivity of Enterochromaffin/BON Cell Model A comparison of enterochromaffin and BON cells indicates similarities in expression of receptors, ion transporters,

742 / CHAPTER 27 A BON cells serve as several models

BON Cells Innoculated 3 In vivo model for testing chemotherapeutic agents

2

EC: In vitro model

Immortalized

Transformed Carcinoid tumor: 1 In vitro model

BON Xenografts In nude-mice

5-HT

Enterochromaffin cell

Carcinoid tumor

B Enterochromaffin cell Ca+2

VMAT1

Voltage-sensitive L-& R type

5-HT VMAT2

Enterocyte

Ca+2 Sensitive +

K

EC receptors P2Y, P2X, *A?, N & M3, *a-& B-, adrenergic, VPAC, PAC1?, *SSTR, *H3, 5-HT3, 5-HT4, NT

Na+ Ca+2 Stores SERT-5-HT transporter

5-HT

C Human BON cells Contents or secretions Neuron specific enolase, Synaptophysin, Pancreastatin, Chromaogranin A, 5HT, Neurotensin, Atrial naturetic factor, VIP, PACAP, Prostaglands, Adenosine, APT

Stimuli Mechanical chemical

Voltage-sensitive L-type; R-type Ca2+

5-HT BON receptors *A1, *A3, A2a, A2b, P2Y1, P2X, M3, *a-& B-, VPAC, PAC1?, *SSTR, 5-HT: 5-HT3, 5-HT4, B1, B2, CCKB, D1, D2, D5, NT

VMAT1 VMAT2 Vesicles

Vesicles * Inhibitory

5-HT

FIG. 27-2. Similarities between enterochromaffin (ECs) and human BON cells. (A) BON cells serve as a model for nontransformed ECs and for in vitro and in vivo carcinoid tumors. Transformation (1) of ECs progresses to carcinoid tumors. Pancreatic carcinoid tumor cells that metastasized to the lymph nodes were immortalized (2) to BON cells. Inoculation (3) of nude mice with BON cells produces a BON cell xenograph that serves as an in vivo model of carcinoid tumors for testing therapeutic agents. (B) Enterochromaffin cells in tissue preparations. 5-Hydroxytryptamine (5-HT) is synthesized and transported into vesicles by vesicular monoamine transporters (VMAT1 and VMAT2) where it is stored. Exocytosis releases 5-HT into the bath where it acts as an autocrine, paracrine, or neurocrine messenger. 5-HT is released or modulated by neural, mechanical, and chemical stimuli that activate receptors on the cell surface. Voltage-sensitive Ca2+ channels (L-type) and Ca2+-activated K+ channels mediate changes in 5-HT release. (C) Human carcinoid BON cells. Asterisks denote inhibitory responses. ATP, adenosine triphosphate; NT, neurotensin; PAC1, type I pituitary adenylyl cyclase–activating polypeptide receptor; PACAP, pituitary adenylyl cyclase–activating polypeptide; SSTR, somatostatin receptor; A, adenosine; M3, muscarinic; (α,β), adrenergic; VPAC, VIP; B, bradykinin; CCK, cholecystokinin; D, dopamine; SSTR, somatostatin receptor; H3, histamine.

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 743 The mechanosensitive elements in BON and enterochromaffin cells remain unknown. Brush stroking that was used to elicit reflex pathways in tissue preparations could not be used as the mechanical stimulus to release 5-HT from cultured BON cells. Because forces may be continuously changing in the gut, a composite of several forces may more closely reflect the stimulus in vivo than a single force alone. Rotational shaking of cultured cells that simulated changes in hydrostatic pressure, shear forces, and centrifugal forces, all forces generated in the intestinal lumen, released 5-HT (Fig. 27-3). Turbulent shear stress is likely to occur in the small intestine and proximal colon where the luminal contents are fluid or in the distal colon when they become fluid during diarrheal states. Pressures of 75 mm Hg or greater or laminar shear stress of 1 to 2 dyne/cm2 applied to BON cells attached to a coverslip in a pressure chamber or a parallel-plate flow chamber (Flexcell, McKeespoet, PA) evoked 5-HT release (3).

Hydrostatic pressure Shear

Cells Symmetry axis

FIG. 27-3. Schematic representation of the mechanical forces that operate during rotational shaking in the human enterochromaffin/BON cell model. Rotational shaking is a composite of turbulent and laminar shear stress, hydrostatic pressure, and centrifugal forces. Dotted line indicates the static level of pressure. Rotational shaking decreases hydrostatic pressure at the axis of symmetry, whereas it increases near the wall of the chamber. These are some of the mechanical forces believed to be operating during the digestive or interdigestive states of the gut.

Gαq /Phospholipase C/Ca2+ The enterochromaffin/BON cell model has proved useful in the study of mechanosensitivity, signaling mechanisms, and receptors involved in the regulation of 5-HT release. Others have reported that balloon distention and puffs of nitrogen gas are mechanical stimuli that affect 5-HT release in intact mucosa/submucous preparations. Rotational shaking of tissue preparations or the enterochromaffin/BON cell model caused mobilization of intracellular Ca2+ via the Gαq-mediated pathway that includes phospholipase C (PLC) (Fig. 27-4). Blockade of PLC reduced the mechanically

ion channels, chemical mediators, or other mechanisms (see Figs. 27-2B and C). Administration of the Ca2+ ionophore, A23187, causes 5-HT release into the bath, yielding concentrations in the nanomolar range. This contrasts with receptormediated release of 5-HT that is a fraction of the total 5-HT capacity (37). The ability of A23187 to increase 5-HT release in the absence of mechanical stimulation indicates that influx of extracellular Ca2+ into the cell possibly through L-type Ca2+ channels can release 5-HT.

Peptide interference Gαq

U73122 blocks PLC

Mechanical stimulus

X Antisense uncouples R/Gαq

5-HT BAPTA-AM (Chelates Ca2+)

Ca2+

PLC

PIP2 IP3 Endoplasmic reticulum

5-HT

FIG. 27-4. Working model of how mechanical stimulation leads to activation of phospholipase C (PLC)/Ca2+-signaling pathway and 5-hydroxytryptamine (5-HT) release from human enterochromaffin/ BON cells. Mechanical stimulation releases adenosine triphosphate (ATP) or other nucleotides to activate Gαq/PLC/Ca2+. Pharmacologic studies targeting Gαq, receptor/Gαq interactions, antisense to Gαq C terminus, interfering peptides, PLC inhibitor (U73122), chelaters of intracellular free calcium (BAPTA-AM), or direct analysis of touch-induced increase in Fluo-4/AM-Ca2+ levels indicate that mechanical stimulation leads to autocrine activation of a receptor linked to Gαq/PLC/Ca2+, an increase in intracellular free Ca2+ levels, and eventual 5-HT release. IP3, inositol-1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; BAPTA-AM, 1,2-bis (2-aminophenoxy) ethaneN,N,N′,N′′-tetracetic acid-acetoxymethyl ester.

744 / CHAPTER 27 evoked release of 5-HT. Although these studies establish that mechanical stimuli evoked 5-HT release, and that the Gαq pathway is involved, there is still is a missing link. How is Gαq activated? Direct effects of the mechanical stimulus on the G protein cannot be completely ruled out, but it seems unlikely that this is the case. Evidence in favor of a purine nucleotide or metabolite of ATP as the ligand for the receptor coupled to Gαq in the mechanosensitive pathway is strong (see Fig. 27-4). In BON cells, peptides interfering with the interaction of Gαq-coupled receptors with Gαq or antisense probes that uncouple Gαq from its interactions with upstream events caused inhibition of 5-HT release and provided evidence for the activation of a receptor that triggered the Gαq signaling pathway. Figure 27-5 summarizes key enzymatic signaling pathways involved in the degradation of the high-energy phosphate ATP and formation of adenosine diphosphate (ADP), AMP, adenosine, and cAMP involved in autocrine regulation of 5-HT release (37,38,41–43). Results of a variety of experiments suggest that ATP is released by mechanical stimulation from BON cells. Nucleotides and their metabolites bind to receptors of a large class called P2Rs, which is subdivided into ionotropic P2XRs (P2X1-7) and metabotropic P2YRs (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12-15) (18,42,43). ATP or one of its metabolites is thought to activate P2YRs on BON cells linked to the Gαq/PLC/Ca2+ pathway, which is necessary for 5-HT release and activation of gut neural reflexes (see Fig. 27-5). Studies on cultured enterochromaffin/BON cells have been beneficial in identifying intracellular signaling pathways. The ability to identify subsets of BON cells that are linked

to various signaling pathways was facilitated by introducing touch/stretch experiments to single BON cells preloaded with Fluo-4/AM (Fig. 27-6). Touch/stretch could elicit Ca2+ responses in single enterochromaffin/BON cells by a smooth (fire polished) tip touch pipette guided by a motorized micromanipulator. Time-series Ca2+ analysis in laser confocal imaging studies, together with pharmacologic agonists and antagonists in BON cells, determined the receptors that trigger signaling pathways involved in 5-HT release. Both Ca2+-dependent and -independent signaling pathways have been found (44,45). Of particular interest is the P2Y1R coupled to the Ca2+ pathway. Messenger RNA and protein have been identified in the pathway. This receptor has been implicated in mechanotransduction and autocrine excitatory regulation of Ca2+-dependent 5-HT release from enterochromaffin cells in response to mechanical stimulation (37). All of these are important components of the mechanosensitive signaling pathway in human enterochromaffin/BON cells (3). Adenylyl Cyclase/Cyclic Adenosine Monophosphate Signaling in BON Cells In addition to receptors that signal through the Gαq/PLC/ Ca2+ pathway, some receptors on BON cells also use the AC/cAMP pathway. Therefore, the potential for interactions between receptors that have different signaling pathways is certainly a possibility (see Fig. 27-6D). P2Y1Rs coupled through Gαq /PLC/Ca2+ are excitatory, but the response may be attenuated by the state of activation of other inhibitory receptors such as P2Y12Rs (3,45). Ligands that bind to

ATP - - - ADENOSINE DEGRADATION ENERGY SOURCE SIGNALING MOLECULE ATP

cAMP

Adenylyl cyclase

ATPase

Adenylyl kinase

ADP P2YR

Phosphodiesterase

5′Nucleo -tidase

ATP

ADENOSINE Adenosine deaminase INOSINE

AMP

ADENOSINE

5′-AMP Adenosine kinase

Transporter

FIG. 27-5. Enzymatic signaling pathways in the degradation of high-energy phosphates adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP). ATP release is broken down to adenosine diphosphate (ADP), AMP, and finally adenosine that can activate cell-surface receptors. The nucleotides ATP and ADP can activate P2 receptors, and adenosine can activate P1 receptors. Adenosine is also formed from ATP inside the cell via AMP or cAMP intermediate steps. Adenosine is released from cells via a bidirectional transport mechanism that depends on the concentration gradient. ATP is unique in that it serves both the metabolic/energy demands of the cell and acts as an extracellular signaling molecule. During disease states, energy demand increases and the balance is disturbed between the availability of ATP as an energy source and that of ATP or its metabolite adenosine as signaling molecules.

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 745 Touch/pressure induced calcium response in the BON cell Micromanipulator

1 µm steps

Touch pipette

4

d

3

ro

15 - 50 µm

un

2

1

tip

5 µm

A

BON cell BON cell

7.5 µm Round tip

D. Purinergic autoregulation via mechanical

B

stimulation in human EC-derived BON cell 0s

9s

24 s ADP

ADP ATP

ATP 30 µM

Touch

?

−

AC

∆ in pixel intensity

C

?

P2Y12

P2Y1

ATP −

Gi

cAMP

Gαq ↑Ca2+

+ PLC

Cross-talk + 175 155 135 115 95 75 55 35

IP3

5-HT

0

15 30

45 60 75 90 105 120 135 150 Time (sec) BON Cell

5-HT

FIG. 27-6. Touch-induced Ca2+ responses in the enterochromaffin (EC)/BON cells to study regulation of Ca2+-dependent/mechanosensitive 5-hydroxytryptamine (5-HT) release. (A) (1) Cells were visualized under transmitted illumination, and the center of the cell was targeted for touch/mechanical stimulation with a blunt tip touch pipette guided by a motorized micromanipulator. (2) Side view of a BON cell before touch. (3) The cell was depressed in 1-µm increments to a maximum of 5 µm. (4) After touch, the cell returned to its original smooth shape with no signs of damage under transmitted illumination; cells completely recovered from an increase in intracellular free Ca2+ levels. (B) Pseudocolor images of a touch-evoked Fluo-4/AM Ca2+ response in a BON cell. (See Color Plate 17.) (C) Kinetics of the associated Ca2+ transient of cell in B. The Ca2+ response peaked in ~30 seconds and returned to baseline within 60 seconds. Ca2+ imaging was done using an LSM Zeiss 410 Confocal/REN 410 imaging system. Cells loaded with 5 µM fluo-4/AM for 1 hour were excited at 488nm using an Ar-Kr laser, and emissions were restricted to between 510 and 550 nm. (D) Autocrine regulation via mechanosensitive P2Y1 and putative P2Y12 purinoceptors in human enterochromaffin-derived BON cells. There was potential cross talk between Ca2+ and cyclic adenosine monophosphate (cAMP) second messenger. AC, adenylyl cyclase; IP3, inositol-1,4,5-trisphosphate; PLC, phospholipase C. Dual regulation of 5HT release occurs via excitatory P2Y1 and inhibitory P2Y12 purinoceptors.

P2Y1Rs may also bind to P2Y12Rs that are coupled to inhibition of cAMP. The net effect of release of nucleotides on secretion of 5-HT depends on actions at both P2Y1 and P2Y2 receptors (see Fig. 27-6D). P2Y1 and P2Y12R interactions likely occur in BON cells, and the response that predominates may depend on the relative coexpression of each P2 receptor subtype in subsets of BON cells.

Endogenous Adenosine as a Physiologic Brake ATP is present in high concentrations in cells, but it may enter the extracellular compartment by leakage from damaged cells, or it can be transported out of cells by

several mechanisms. In neurons, blood cells, and endocrine cells, ATP is copackaged in secretory granules with transmitters, hormones, or other mediators and is released by exocytosis. In epithelial cells, there is a 10,000-fold transmembrane gradient favoring the movement of ATP to the extracellular space; therefore, electrodiffusion via membrane channel proteins is another possibility (42). In BON cells, intracellular ATP is broken down to ADP, AMP, cAMP, and adenosine and can be released from cells (Fig. 27-5). Mechanical stimulation releases ATP and also increases extracellular cAMP associated with 5-HT release. Certain adenosine analogs or endogenous nucleotides modulate cAMP levels in parallel with Ca2+ levels involved in 5-HT release. The β-adrenergic receptor agonist, isoproterenol, gave a

746 / CHAPTER 27 A

B

p=0.02

C

p<0.001

10 p<0.05

0 BASE

0.5

1

[ADENOSINE DEAMINASE] UNITS/ml

5-HT 3.9 ± 0.1 (3) cAMP 8.4 ± 0.5 (3-5)

% Control

<.05 200 <.05 100

0

p=0.02

6 NS

4 2

Base MS NBTI 1µM

Base MS

Base MS NBTI 1µM

F

p<.0001

150 p<.001

125 p<.0001

5-HT 0.87 ± 0.6 (5-6)

p<.05

100

200

75 50 25

p < .05

p < .05

150 100 50 0

0

BASE +CSC AL

MS +CSC AL

50

0

IB MRS MRS+IB

p=0.05

0 Base MS

E

BASE

20 0

2

D

300

40

% CONTROL

0

p=0.04

60

nM 50 Co 0 ntr Ad CP e T M nM o l Ad nos + M RS CP en ine R 1 T o D S 19 + sin ea 11 1 IB e m 9 -M De in 1 EC am as A in e as e

5

80

Net increase in fluo4/Ca+2 pixel intensity

5HT Release pmole/w/30 min

15

8 5-HT Release (nM)

5-HT 1.0 ± 0.1 (4-16)

Endogenous adenosine Release (nM)

100

FIG. 27-7. Autocrine regulation of 5-hydroxytryptamine (5-HT) release by endogenous adenosine release from BON cells via inhibitory A1R/A3Rs and excitatory A2Rs. (A) The enzyme adenosine deaminase caused a dose-dependent increase in 5-HT release, indicating that release of endogenous adenosine provides an ongoing inhibition of 5-HT release. (B) Adenosine release during resting/unstimulated conditions (Base) and mechanical stimulation (MS) is augmented in the presence of the adenosine uptake inhibitor S-(p-nitrobenzyl)-6-thioinosine (1 µM NBTI). (C) In the same cells as A, NBTI blocked adenosine uptake and abolished mechanically evoked 5-HT. (D) Chemical stimulation with N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) decreased basal 5-HT and the A3R agonist cAMP levels. The A3R antagonist MRS1191 (10 nM) augmented 5-HT release but not cAMP levels (p < 0.05). The MRS1191 evoked 5-HT response was sensitive to IB-MECA inhibition. (E) Adenosine A1 and A3R inhibitory components of touch-evoked Ca2+ responses in enterochromaffin/BON cells. The A3R antagonist MRS1191 augmented the touch Ca2+ transient. Chronic exposure to the A1 antagonist CPT for 24 hours caused a significant augmentation in the touch Ca2+ response. The stimulatory effects of CPT and MRS1191 were additive on the touch Ca2+ response. Adenosine deaminase (5 U/ml)–induced augmentation is reduced by the A3R agonist IB-MECA (50 nM). (F) A2A/A2B antagonists [8-(3-chlorostyryl) caffeine] (CSC) and alloxazine (AL) inhibited mechanically evoked 5-HT release. Baseline values (pmol/well per 30 minutes) of 5-HT and/or cAMP are at the top of figures, with the number of experiments indicated in parentheses. MS, mechanical stimulation by rotational shaking at 80 g ; NS, not significant. (Modified from Kim and colleagues [36], by permission.)

concentration-dependent increase in intracellular Ca2+ monitored by Fluo-4/AM/fluorescence pixel intensity and an increase in cAMP in BON cells measured by enzymelinked immunosorbent assays. This was additional evidence of multiple signaling pathways regulating 5-HT release. Extracellular adenosine may also be generated by breakdown of extracellular ATP. Several sodium-dependent

electrogenic nucleoside transporters mediate the concentrative uptake of nucleosides back into the cells (43). Adenosine acts at A1, A2A, A2B, and A3 receptors (43,46). High-affinity A1 and A2A receptors are activated at nanomolar concentrations of adenosine, whereas the low-affinity A2B and A3 receptors are activated at submicromolar to micromolar concentrations. Depending on the relative and spatial

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 747 BON CELL Force

ATP ADENOSINE ATP ? Sensor

Internalization

A1

ATP

(−)

A1

ADP–AMP-ADO UTP-UDP-URIDINE

(+) A3

ADENOSINE

ADENOSINE A2a

A2b

5HT

FIG. 27-8. Working model of adenosinergic modulation of mechanically evoked 5-hydroxytryptamine (5-HT) release from enterochromaffin/BON cells. Endogenous adenosine release during rotational shaking or touch-evoked Ca2+ responses activates inhibitory A1 or A3 receptors and excitatory A2A or A2B receptors. A1Rs may internalize and stay desensitized for long periods. The mechanosensory signaling mechanisms remain unknown. Mechanosensitive release of endogenous adenosine acts in an autocrine manner to modulate 5-HT release. Adenosine (ADO) could be released as the precursor adenosine triphosphate (ATP) or as adenosine itself during mechanical activation. ADP, adenosine diphosphate; AMP, adenosine monophosphate; UTP, uridine tryphosphate; UDP, uridine diphosphate.

distribution of these receptors, adenosine may be stimulatory or inhibitory on Ca2+ responses or 5-HT release. The modulatory role of endogenous adenosine in regulation of 5-HT release from BON cells is illustrated with increasing concentrations of adenosine deaminase that catabolizes adenosine (see Fig. 27-7A). As endogenous adenosine was depleted by the enzyme, 5-HT release increased, illustrating that adenosine puts a brake on resting levels of 5-HT release (3,14,43). Additional proof comes from studies illustrating that mechanical simulation of BON cells can release sufficient adenosine to suppress 5-HT release. The apparent adenosine concentration of the buffer approximated 10 to 15 nmol/L for 1 million BON cells seeded and grown for 48 hours in 12-well culture plates (see Fig. 27-7). Mechanical stimulation caused adenosine levels to increase to 35 nmol/L. Greater concentrations of 100 nmol/L occurred if adenosine uptake was prevented with a nucleoside transport inhibitor, S-(p-nitrobenzyl)-6-thioinosine (NBTI) (see Fig. 27-7B). With this apparent level of adenosine, mechanically evoked 5-HT release was abolished (see Fig. 27-7C). These observations support the hypothesis that there is an inverse relation between endogenous adenosine and 5-HT release in the enterochromaffin/BON cell model (see Fig. 27-8). The adenosine receptor agonists that suppress 5-HT release are primarily A3Rs, because baseline 5-HT release increased with A3R antagonists and decreased with agonists (see Fig. 27-7D). Apparent concentrations of endogenous adenosine underestimate levels at receptor sites because of diffusion, dilutions, uptake, and lack of more sensitive methods to detect physiologic concentrations of adenosine. Although the A1R is a high-affinity receptor and is expected to be activated at

concentrations of adenosine in the nanomolar range, blockers of A1Rs did not consistently alter baseline 5-HT. This was thought to be due to high enough levels of endogenous adenosine that may have caused desensitization of A1Rs by internalization (see Fig. 27-8) (44). Once internalized, the kinetics of A1Rs trafficking require a long (24–48 hours) time to appear in the membrane, unlike the A3R or other G protein–coupled receptors that recycle back to the membrane within 60 minutes. Protection of the adenosine A1R with the competitive antagonist (8-cyclopentyl-1,3-dimethyl panthene) (CPT) prevents its internalization, and it revealed an additional A1R inhibitory component in touch/Ca2+ responses. Evidence that touch-activated Ca2+ was augmented with both A1 and A3R antagonists and adenosine deaminase indicates a role for these receptors in mechanosensory signaling (see Fig. 27-8). The antagonist MRS1191 increased 5-HT baseline without a parallel increase in cAMP. This suggests uncoupling of Ca2+ and cAMP in some pathways.

Excitatory Adenosine Receptors BON cells express all four adenosine receptors, A1, A2A, A2B, and A3, as do carcinoid tumors (see Fig. 27-8) (41). Unlike the adenosine inhibitory receptors that modulate “secretory tone,” the adenosine A2A and A2B receptors have a modest excitatory effect on resting 5-HT release revealed by antagonists (see Fig. 27-7F). If extracellular adenosine concentrations are sufficiently high to activate A3Rs, then A2Rs also should have been activated. The net effect of endogenous adenosine depends on number of

748 / CHAPTER 27 receptors and their distribution, as well as inactivation mechanisms for endogenous adenosine. Isoproterenol or 5′N-methylcarboxyaminoadenosine (MECA) caused parallel increases in both 5-HT and cAMP. Mechanical stimulation– evoked 5-HT release was abolished by A2A/A2BR antagonists (see Fig. 27-7, 27-8), supporting a stimulatory role of endogenous adenosine via A2Rs. Mechanically induced increase in 5-HT release was stimulated by A2A and A2B receptor agonists. Cross talk between cAMP and Ca2+ is suggested from purinergic studies and awaits further proof.

SECRETORY REFLEXES Modifications of Ussing chambers were necessary to apply a mechanical stimulus to elicit secretory reflexes (Fig. 27-9). To elicit a brushstroke, a flat sheet of mucosa was oriented in the vertical direction. The brush was inserted perpendicular to the tissue and rotated 360 degrees, or the chamber was rotated 90 degrees, the top was removed and a brush could be lowered onto the mucosal surface for a forward or backward movement.

Role for Adenosine Triphosphate in Sensory Signaling Brush stroking the mucosa causes reflex chloride secretion that involves release of 5-HT as a mechanism for

A

B Mucosal stroking - 360°

Mucosal Stroking– Forward Stroke 2 mm brush

rotation of brush

Isc

Mucosa PD

activation of IPANs. Mechanical stimulation activates the same pathways in BON cells and in IPANs. Both are linked to P2Y1Rs and to the GαqPLC/Ca+2 pathway leading to release of 5-HT release in the intestines in rodents (colon) and humans (small bowel from Roux-en-Y patients) (28). It is clear that the stimulation of P2 receptors exerts an excitatory tone on secretory pathways, which is attenuated by negative feedback mechanism triggered by activation of A1Rs (41). ATP is a new player in secretory reflexes evoked by stroking. Evidence for an endogenous nucleotide affecting chloride secretion comes from the finding that the secretion was altered by compounds that decrease ATP or increase ATP availability at P2Y1Rs. A purinergic-independent pathway exists as well. Apyrase or ARL67156 have opposing effects on stroking-evoked secretion. MRS2179, a specific antagonist at P2Y1Rs, significantly reduced stroking-induced secretion (Fig. 27-10B). The failure to abolish the response implies that there must also be present a purinergicindependent pathway (see Fig. 27-10). The potency profile of nucleotide analogs 2MeSADP = 2MeSATP >> ATP = ADP >> UTP = UDP is consistent with P2Y1Rs (see Fig. 27-10C). The effect of a P2Y1R agonist 2MeSADP on stimulating Isc responses was partially sensitive to blockade by the P2Y1R antagonist MRS2179 (see Fig. 27-10). These and other findings support a role for purinergic transmission in secretory reflexes induced by mucosal stroking.

360° brush

Isc

PD

Mucosa

C Distention – fluid removal CLAMP Isc Stopcock

PD Remove aliquot & replace

FIG. 27-9. Modified Ussing chambers used for mucosal stroking studies to evoke a reflex neurosecretory response. (A) A vertical configuration of the chamber permits a 2-mm brush to be placed on the mucosal surface. A single stroke represents the movement of the brush from one side to the other on the surface of the mucosa. (B) A complete 360-degree turn of the brush is another variation for stimulating reflexes. Stroke (1 second) is reproducible every 5 minutes for a 40-minute period. (C) Distention is accomplished by removing small volumes of fluid from the serosal side. Drugs are perfused separately on the serosal or mucosal surfaces of the mucosa/submucosa or whole-thickness colonic or small-bowel tissues. The short-circuit current (Isc) nulled out the spontaneous potential difference (PD) and was used as a measure of chloride secretion in the distal colon.

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 749 A

B ∗

Stroking-evoked Isc (% of control)

Stroking-evoked Isc µA/cm2

200 150 100

∗

50 0

MRS 2179

150 100 50 0

CON

APY

CON

0

ARL

D 400 300

2MeSADP 2MeSATP

200

ATP ADP

100 UDP

UTP

0 −100 −12 −11 −10 −9 −8 −7 −6 −5 −4 −3 Log [M]

Isc response to nucleotides (µA/cm2)

C

Nucleotide evoked Isc µA/cm2

200

5 10 15 20 25 30 35 40 Time-min

300 Control

200

100

0

-0-

-0-

Vehicle + 2MeSADP + MRS

FIG. 27-10. Mucosal stroking releases endogenous adenosine triphosphate (ATP)/nucleotides that activate excitatory neural P2Y1Rs, leading to an increase in short-circuit current (Isc)/chloride secretion in the guinea pig colon. (A) The ATPase/5′ nucleotidase inhibitor 6-N,N-diethyl-β,γ-dibromomethyleneD-adenosine-5′-triphosphate trisodium (10 µM ARL67156 or ARL) that prevents hydrolysis of ATPenhanced reflex evoked Isc (n = 6; *p < 0.05), whereas 10 U/ml apyrase (APY) that hydrolyzes ATP inhibited reflex-evoked Isc responses (n = 4). This indicates that endogenous ATP release contributes to the stroking response. (B) The selective P2Y1R MRS 2179 suppressed ~50% of the stroking response. (C) Concentration–response curves for nucleotides in the presence of 1 µM CPT (n = 4–23) antagonist, an antagonist at A1 receptors. The rank order of potencies of nucleotide agonists for stimulation of Isc was 2MeSADP = 2MeSATP >> ATP = ADP > UTP = UDP, which indicates a P2Y1R interaction. (D) The Isc response to the P2Y1R agonist 2MeSADP was nearly abolished by the P2Y1R antagonist MRS 2179 (10 µM; n = 4; p < 0.05). All experiments were done in the presence of 1 µM CPT to eliminate inhibitory influence of A1 receptor activation by endogenous adenosine (n = 4 each; *p < 0.05). (Modified from Cooke and colleagues [5], by permission.)

Mucosal Stroking Reflexes in Rodent Intestine The “secretory tone” in guinea pig colon depends on endogenous release of prostaglandins and nucleotides that increase chloride secretion in the face of its inhibition by adenosine acting on inhibitory A1Rs (3,45). When the submucous plexus is exposed to mechanical or chemical stimuli, secretory reflexes are triggered to increase chloride secretion greater than the resting levels. There are two reflex pathways for regulating chloride secretion in the guinea pig intestine. One is responsive to mucosal stroking, and the other is unresponsive to mechanical stimulation. Brush stroking induces 5-HT release and chloride secretion that is attenuated by antagonists at 5-HT1P, 5-HT4, NKI/NK3, P2Y1, CGRP, muscarinic M3, prostaglandins, EP2, P2Y1, and VPAC receptors (3,14–18). The implication of these studies is that brush stroking releases 5-HT, prostaglandins, tachykinins, acetylcholine, VIP, PGE2, ATP or ADP, adenosine, CGRP, and possibly others. Most of these components

are incorporated into a working model for secretory reflexes in guinea pig intestine (see Fig. 27-1). Further discussion is given later on nucleotides (i.e., ATP) because recent findings provide a more complete picture of the purinergic reflex. The mechanically evoked purinergic reflex in the guinea pig intestine starts in sensory enterochromaffin cells when a mechanical stimulus is the initiating event (Fig. 27-11). The mechanosensor is still unknown, but it may be an ion channel or some other element that converts forces on the cell membrane to a biological response such as release of ATP (3). Release of ATP or its metabolites such as ADP activate P2Y1Rs on the enterochromaffin cells, with the nucleotide acting as an autocrine mediator to bind to P2Y1Rs coupled to Gαq. The activation of P2Y1Rs and Gαq are followed by Ca2+ mobilization within the cells and exocytotic release of 5-HT. This sets the stage for 5-HT to activate 5-HT1PRs or 5HT4RS on the terminals of IPANs. Nucleotides may be released from the surrounding epithelial cells and may also activate IPANs, although this concept is difficult to prove.

750 / CHAPTER 27 Mechanical Activation of Purinergic Neural Circuitry in Guinea Pig Distal Colon

P2Y2R

NPY/ChAT ATP UTP

2

P2Y1R 3

CALB/IPAN *P2Y2R ChAT CALB/IPAN

1

*P2Y1R

ADP

Secretomotor

ATP

5HT4R 5HT1PR

5HT1PR

5HT

5HT

P2Y1R?

P2Y1R

EC

EC

Chemical stimulus?

Mechanical stimulation Stroking, brush

P2X1/3R +

Cl−

+ ACh

Cl−

FIG. 27-11. Working model of the role of P2YRs in the mucosal stroking reflex of the guinea pig distal colon. During mechanical activation by brush stroking the mucosa, 5-hydroxytryptamine (5-HT) activates the intrinsic primary afferent neuron (IPAN), releases endogenous nucleotides (adenosine triphosphate [ATP]), and activates P2Y1R on cholinergic secretomotor neurons, leading to an increase in short-circuit current (Isc)/chloride ion secretion (see synapse at 1). The preferred endogenous mediator at P2Y1Rs is adenosine diphosphate (ADP) that is formed by breakdown of ATP. A separate purinergic P2Y2R neural circuit pathway exists leading to chloride secretion that is not activated by stroking, in which P2Y2Rs are expressed in a subset (~30%) of IPANS (see cell body 2) and neuropeptide Y/choline acetyltransferase (NPY/ChAT) secretomotor neurons (see cell body 3). A tiny subset of such secretomotor neurons (<2%) express P2Y1Rs. The preferential agonists at P2Y2Rs are ATP and UTP. It is hypothesized that the P2Y2R neural circuit pathway may be activated by nonmechanical factors. ACh, acetylcholine; EC, enterochromaffin cell; CALB, calbindin. (Modified from Cooke and colleagues [5], by permission.)

IPANs may release SP, CGRP, glutamate, and acetylcholine at synapses with submucous cholinergic or VIP secretomotor/ vasomotor neurons, or with myenteric neurons. PGE2 released by mechanical stimulation selectively increases the excitability of VIP secretomotor neurons (45). Neural P2Y1Rs involved in mechanical reflexes are present on cholinergic secretomotor neurons that lack NPY (ChAT/NPY−/P2Y1R). The secretory response is attenuated by adenosine acting at presynaptic A1 or A2 receptors. Inputs from the sympathic nerves via norepinephrine inhibit VIP secretomotor neurons to prevent runaway secretion (not shown). The sympathetics and adenosine then gate the transmission to secretomotor neurons. Another mode of regulation includes excitatory purinergic input via P2Y1Rs from the myenteric plexus (47). The purinergic circuit derived from reflex or colocalization studies for P2YRs is shown in Figure 27-11 for guinea pig submucous plexus. Of the two distinct pathways depicted one pathway involves P2Y1Rs on secretomotor neurons and the other pathway involves P2Y2Rs on IPANS and secretomotor neurons. Mucosal stroking in the rat colon, like in the guinea pig, induces chloride secretion that is reduced, but not abolished, by P2Y1R antagonist. Other components of this response include release of SP, VIP, acetylcholine, ATP, prostaglandins, and possibly others. Chemical coding

indicates that 90% of rat submucous neurons express P2Y1Rs, whereas CALB-immunoreactive neurons, or putative IPANS, do not (Fig. 27-12). By extrapolation it appears that most secretomotor neurons (VIP/NPY) and interneurons express the P2Y1 and P2Y4 receptors. PY2Rs form baskets around putative IPANs or CALB-immunoreactive neurons (see Fig. 27-12). These findings implicate P2YRs as important mediators of synaptic transmission and purinergic reflexes in the rat colon in addition to guinea-pig colon (45). Touch or distention of the mucosa is another mechanical stimulus to study neural synaptic Ca2+ responses and purinergic reflexes in the intestine (Figs. 27-13 and 27-14). In a submucous ganglion, high K+ concentration, 2MeSADP, and SP caused an increase in Ca2+ in submucous neurons (48). Responses occurred together in the same neuron or in different neurons (see Fig. 27-13). Thus, subsets of submucous neurons can be defined by the types of receptors present. In the rat colon, of the neurons responding to high K+ concentrations, 17% displayed SP and 2MeSADP responses, 35% showed responses to SP alone, and 38% responded to 2MeSADP alone. Additional experiments are necessary to unravel the types of neurons and their characteristic chemical codes that will eventually define their specific role in reflex pathways.

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 751 A

Rat colon SMP

B

P2Y1R

Calretinin

C

D

P2Y1R

nNOS

E

F

P2Y2R

Calbindin

G

H

P2Y2R

ChAT

FIG. 27-12. P2Y2R immunoreactivity (IR) in the submucous plexus of the rat distal colon. (A, B) Strong coexpression of P2Y1R immunoreactivity (IR) in calretinin-positive neurons. (C, D) Coexpression of P2Y1R IR in a neuronal nitric oxide synthase (nNOS)–positive neuron. (E, F) P2Y1R-IR varicose fibers surround calbindin-IR cell bodies; E and F represent images from the same ganglion. Short, thick arrows represent P2Y1R IR in varicose fibers that appear as a basket of beads around an unlabeled calbindin cell body (asterisk). (G, H) P2Y2R-IR varicose fibers (short, thick arrows) form baskets or run close to the cell bodies of ChAT cholinergic neurons (asterisks). Scale bar = 30 µm. (Modified from Christofi and colleagues [68], by permission.)

IPANs are necessary for touch-evoked Ca2+ reflexes, because the touch-evoked response was blocked by tetrodotoxin, by severing the connections between the submucosa and mucosa, and by touching the ganglion near the recording site (45). Direct touch of the submucosa near the recording site does not appear to induce impulse generation in IPANs, because it bypasses the sensory afferent process. The entire touch-Ca2+ response was abolished by the P2Y1R antagonist, a finding that implies the touch response is entirely mediated by a purinergic pathway, whereas the purinergic component to chloride secretion in the stroking response in the rat colon was about 50%. This may reflect a second Ca2+-independent, nonpurinergic component to mucosal stroking in the rat. In support of this is the finding that the P2Y1R antagonist reduced chloride secretion in response to mucosal stroking, leaving a residual response that may act through another signaling pathway. Species differences do exist between the guinea

pig (see Fig. 27-11) and the rat (Fig. 27-15), but P2Y1Rs contribute to purinergic mechanosensory secretory reflexes in both species. Other P2YRs are implicated but await functional proof. Studies in the rat distal colon have identified other mediators and signaling pathways involved in descending pathways in the submucous plexus. Fiber tract electrical stimulation evoked synaptic Ca2+ responses that were abolished by tetrodotoxin and by a cAMP-dependent protein kinase inhibitor, GRTGRRNAI-NH2. Under PLC blockade, forskolin revealed a cAMP-dependent synaptic Ca2+ response or oscillations. The available data suggest that synaptic transmission in descending submucous reflex pathways involve a predominant protein kinase A (PKA) signaling pathway that facilitates synaptic transmission in some neurons that have a predominant PLC/Ca2+ signaling component.

Distention and Secretory Reflexes Stretching the gut wall by distention (see Fig. 27-9C) causes a neural reflex via extrinsic primary afferent neurons (EPANs) and IPANs that stimulates chloride secretion (49). Other stimuli, mucosal distortion or chemicals, release ATP that can act at P2XRs on EPANs. This may result in transmission of information to the central nervous system. The contribution of EPANs to capsaicin-sensitive, distentionevoked secretion in the guinea pig colon was nearly 50%, whereas the residual 54% was caused by IPAN stimulation (11,12,15,32). The capsaicin-activated vanilloid receptors (transient receptor potential vanilloid receptor 1 receptors [TRPV1Rs]) triggered by heat, acid, and possibly other chemicals colocalize with bradykinin B2Rs and purine receptors in EPANs in rat colon. In the guinea pig small intestine, the secretory response to capsaicin acting on TRPV1Rs is opposed by activation of cannabinoid receptors (cannabinoid-1 receptor [CB-1R]), which colocalize with TRPV1Rs on EPANs (33,34,49). Capsaicin-evoked secretion is inhibited by 50% by NK1R antagonist without any effect of NK3R or CGRP antagonists. Synaptic transmission to cholinergic but not to VIP secretomotor neurons occurs in the guinea pig ileum after capsaicin treatment. In contrast, in the distal colon, capsaicin-evoked secretion is abolished by NK1R and NK3R agonists and antagonists of muscarinic and VPACRs (12), indicating an involvement of both cholinergic and VIP secretomotor neurons. The rate of secretion can be further augmented by input from EPANs via an axon reflex. The finding that SP is present in IPANs and that NK1Rs were present on epithelial cells is evidence of the presence of the elements necessary for intrinsic axon reflexes within the submucosa, although functional proof of this concept awaits further investigation (3,15). A greater role of intrinsic axon reflexes is predicted to occur in inflammation, because the density of NK1Rs on epithelial cells may increase (15). Evidence has been presented that supports the presence of NK1Rs on secretomotor NPY-containing cholinergic neurons in the guinea pig ileum. In the guinea pig colon,

752 / CHAPTER 27 P2Y1 agonist Ca2+ responses rat distal colon submucous plexus

Basal

A

+

High K

B

% of high K+ responsive neurons

E

50

40

30 * *

*

20

10

50

100

D

SP

150

200

250

2M SA DP SP w/ 2M SP SA DP w/ oS SP P w/ o2 MS AD P

0

2MeSADP

2M SA DP

0

C

FIG. 27-13. Purinergic laser scanning confocal microscopy (LSM) Fluo-4/AM Ca2+ imaging in intact submucous plexus of rat distal colon. (A) Basal Ca2+ response. (B) Response to high K+ depolarization (75 mM). (C) The P2Y1R agonist 2MeSADP increased [Ca2+]i in three neurons (arrows). (D) Substance P (SP) increased intracellular free Ca2+ levels in six neurons and three of them also had Ca2+ responses to 2MeSADP; arrowheads designate cells with only SP responses. (See Color Plate 18.) (E) Average number of neurons/ganglion that responded to high K+, 2MeSADP, SP or to both SP and 2MeSADP. Drug exposure for high K+ was 100 to 120 seconds, and for 2MeSADP and SP was 120 to 200 seconds. At the end of each drug exposure, tissues were allowed 10 minutes to return to baseline before exposure to other drugs. Ca2+ images are pseudo-colored based on a gray scale ranging from 0 to 255 (see bottom left). Tissues were excited with an Ar-Kr laser at 488 nm, and emissions were collected by a photomultiplier tube after passing through a BP 505 to 550 filter Zeiss LSM (Carl Zeiss Microimaging Corporation, Thornwood, NY). Values are means ± standard error of the mean. The thickness of the optical slice was 4.7 µm. Scale bar = 30 µm. (Modified from Christofi and colleagues [68], by permission.)

NK1Rs may be present on VIP secretomotor neurons as well (3). There are many receptors on all components of secretory reflexes. Figure 27-16 is a generic composite from several species and regions of intestine, to illustrate the potential complexity of receptor-mediated signaling in the intestine. Not only are there different classes of receptors to consider, but also different regions of the gastrointestinal tract, as well as species.

Secretory Reflexes in Humans In the human jejunum, stroking the mucosa induces 5-HT release that activates a putative 5-HT4 receptor on afferent neurons to initiate a reflex secretory response (50). 5-HT1P, 5-HT3, or 5-HT2CRs are not involved in secretory reflexes to mucosal stroking. L-lysine in the diet acts as a 5-HT4R antagonist to modulate gut motor and secretory functions (51).

At concentrations normally found in the diet, this essential amino acid can act as an effective antagonist suppressing motility and secretion. This is important in that dietary nutrients can act in specific neuronal pathways as receptor antagonists affecting gut reflex function. Optical recording of synaptic Ca2+ signals can be used to monitor neuronal and synaptic activation in the human enteric nervous system ex vivo. Electrical stimulation of an interganglionic fiber tract with a titanium electrode or high K+ solutions elicited a synaptic Ca2+ transient response in Fluo-4/AM-loaded, microdissected submucosa of Roux-en-Y patients (28). Forskolin stimulation caused an increase in cAMP visualized using the acrolein-derivatized cAMP antibody and immunofluorescent labeling. Forskolin stimulation caused an increase in cAMP in Dogiel type II multipolar or Dogiel type I filamentous neurons. The p-site inhibitor 2′5′dideoxyadenosine or preabsorption of cAMP antiserum by cAMP prevented cAMP in human submucous neurons. Synaptic Ca2+ responses are sensitive to blockade

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 753 ‘Touch’ pipette

A

B

Pins

H

Mucosa SMP Perfusion

∆ Pixel fluorescence intensity to Fluo-4AM

Mucosa/SMP

Fluo-4 /AM second order neurons

Sylgard

Objective 40x oil SMP 1.3N.A

C Side view SMP

Sylgard

60 40

Control touch 10 µm MRS 2179 0.2 µm TTX

20 0 MRS 2179

−20

−40

No sylgard

Suction

E

D

30 µm

F

G

Touch+ 10 µm MRS 2179

Touch+ 0.2 µm TTX

∆ Pixel fluorescence intensity to Fluo-4AM

I Touch

Basal

50

Control touch 5 u/ml apyrase

40 30 20 10 0

2+

FIG. 27-14. Laser confocal imaging of touch-induced purinergic Ca signals in neurons of the intact microdissected submucous plexus of the rat distal colon. (A) Perfusion chamber used to record touch-induced Ca2+ responses in the neurons. It includes a recording chamber made of a 35-mm microwell dish with a 1-cm, number 0.0 cover-glass in the center of the bottom of the dish for visualizing the fluorescent neurons. A portion of the dish is embedded with a 2-mm-thick layer of Sylgard shaped around a half moon at one end of the dish. Perfusion with oxygenated buffer or drugs is exactly opposite to the suction. The microdissected mucosa-submucous plexus (M/SMP) preparation is stretched and fixed with many (15–20) micropins along the Sylgard layer. The remaining SMP without mucosa is stretched over the glass coverslip of the dish and secured with magnetic metal feet (not shown). A large ganglion of the intermediate layer of the submucous plexus is viewed with a 40× oil immersion objective (N.A. 1.3). (B) Inset shows Fluo-4/AM-loaded neurons representing second-order neurons that are visualized. (C) Side view of the tissue: The submucous plexus layer is located flat on the bottom of the dish, and the mucosa/submucosa lifts over the Sylgard ends at each side of the half moon. The tissue is touched for 20 seconds on the mucosal surface of the SMP portion that is stretched across the Sylgard with a fire-polished glass pipette to evoke a Ca2+ response in the neurons: the tissue is free to move/stretch downward during the touch, because it is located a distance of 2 mm above the glass bottom. (D–G) The P2Y1 antagonist MRS 2179 or tetrodotoxin (TTX) abolish the touch-induced Ca2+ fluorescence response; representative Ca2+ images from a single ganglion. (See Color Plate 19.) (H) Pooled data for the peak effects of MRS 2179 and TTX; averaged transients from n = 30 neurons from 3 to 4 separate ganglia. (I) Breaking down endogenous nucleotides reduces the touch-induced Ca2+ response in the neurons of the intact M-SMP. The touch response was determined in the presence of 5 units/ml apyrase exposed for 10 minutes. Apyrase reduced by 50% the control touch response (n = 4 neurons; p < 0.01). Values are means ± standard error of the mean. (Modified from Christofi and colleagues [68], by permission.)

by the P2Y1R antagonist MRS2179, and P2Y1Rs are colocalized on VIP-positive submucous neurons in the human gut. These preliminary findings suggest purinergic transmission associated with Gαq/PLC/Ca2+ pathways in putative VIP secretomotor or motoneurons, as well as Gs/AC/cAMP signaling pathways in the human submucous plexus.

Thus, P2Y1 purinergic transmission occurs in human, rat, and guinea pigs alike. Human submucous ChAT neurons make up 50% of all submucous neurons, and they have smooth cell bodies and filamentous dendrites (23,26). ChAT/VIP neurons in humans are presumably VIP secretomotor neurons, but their

754 / CHAPTER 27 Mechanical activation of purinergic neural circuitry in rat distal colon P2Y1R CALR/ChAT

P2Y2R

nNOS

P2Y1R

P2Y2R VIP secretomotor neuron

P2Y2R: CGRP/NPY/VIP/ACh or SP P2Y2R

ATP

P2Y2R ADP ATP UTP

UTP

P2Y2R CALB/IPAN

5HT1PR

VIP/NPY or SOM secretmotor neurons

P2Y1R, P2Y4R, SP-R

5HT P2Y1R EC

Cl−

Mechanical stimulation stroking/Touch

Cl−

Cl−

FIG. 27-15. Mechanical activation of purinergic neural circuitry in rat distal colon. Mucosal brush stroking or touch/stretch releases nucleotides that may include adenosine triphosphate (ATP), UTP, and adenosine diphosphate (ADP), which activate neural P2Y1, P2Y2, and P2Y4 receptors at presynaptic or postsynaptic sites of submucous neurons, leading to a net increase in short-circuit current (Isc)/chloride secretion. Intrinsic primary afferent neurons (IPANs) do not express P2YRs on their cell bodies, but they do receive strong synaptic inputs from extrinsic fibers (i.e., pelvic origin) with P2Y2Rs forming baskets of varicose fibers surrounding their cell bodies. A tiny subset of VIP secretomotor neurons expresses somatostatin (SOM). P2Y2, P2Y1, and P2Y4 receptors are coexpressed on more than 90% of neuropeptide Y/vasoactive intestinal peptide (NPY/VIP) putative secretomotor neurons that also express substance P (SP) receptors. P2Y1Rs are also expressed on putative SOM neurons. Activation of P2Y1Rs leads to an increase in intracellular free Ca2+ levels in the secretomotor neurons by ATP, UTP, or ADP. A nicotinic cholinergic synapse has not been ruled out. Nitric oxide synthase (NOS) neurons and CALR/choline acetyltransferase (ChAT) cholinergic neurons also express P2Y1Rs, but the functional role of these subsets of neurons is unknown. P2Y2Rs are discretely localized on some extrinsic fibers of SP, VIP, NPY, calcitonin gene–related peptide (CGRP), or cholinergic origin presumed to be involved in synaptic modulation of transmitter release. EC, enterochromaffin cell; 5-HT, 5-hydroxytryptamine; nNOS, neuronal nitric oxide synthase. (Modified from Christofi and colleagues [68], by permission.)

chemical coding differs from the guinea pig, where VIP and ChAT do not colocalize. Neurons with nitric oxide synthase were VIP immunoreactive. VIP-immunoreactive neurons project to the mucosa and within the submucous plexus (11). Data in humans imply that distention causes activation of IPANs, the output of which converges onto VIP and cholinergic secretomotor neurons via NK1 and NK3 receptors (26,27). SP is released from the varicose terminals of EPANs and binds to NK1Rs on secretomotor neurons to stimulate chloride secretion.

MEDIATORS OF SYNAPTIC TRANSMISSION IN SECRETORY REFLEXES Wood and Kirchgessner (16) reviewed slow excitatory metabotropic signal transmission in the enteric nervous system. In brief, application of focal electrical stimulation to

an interganglionic fiber tract or directly onto the surface of a ganglion leads to a slow EPSP response in both myenteric and submucous neurons. Both AH type 2 neurons with Dogiel type II multipolar morphology and S type 1 neurons with Dogiel type I uniaxonal morphology receive slow synaptic inputs. A slow EPSP is characterized by a slowly activating membrane depolarization and enhanced excitability that persists for several seconds to minutes and occasionally for hours (in pathophysiologic circumstances) after cessation of the stimulus and termination of the release of transmitter. Transmitters that mediate slow EPSPs activate metabotropic receptors that are coupled to ATP or guanosine triphosphate binding proteins and subsequent increase of intracellular free second messengers such as cAMP, cGMP, diacylglycerol, and inositol-1,4,5-trisphosphate (IP3). 5-HT (5-HT1P), ATP (P2Y1), acetylcholine (M1), SP (NK1/NK3), and possibly CGRP may function as metabotropic neurotransmitters in enteric neurons (8,16,18,19). ATP (or ADP) or a peptide CGRP

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 755 NEURAL RECEPTORS ON EPANS, IPANS OR SECRETOMOTOR NEURONS ILEAL EPANS

IPANS

COLON EPANS SP/CGRP

SP/CGRP

SECRETOMOTOR

A2 NC Grp-mGl5 IL-1B IL-6 B2 CB1 PAR2 P2Y1 P2X2 P2X3 NK3CGRP1 GM1 GM1 CTxB CDTxB

VIP

SP CHAT/NPY neurons NK1 (50%) NPY NK1 5HT3 NK3 B2 CB1 II/III mGlu P2Y1

VIP

NK1 5HT1P 5HT4 SST1/ SST2 B2 PAR2 P2Y2

ACH

PAR2 5HT3 TRV1

SECRETOMOTOR

CB1 TRV1 PAR2

VIP neurons

CHAT/SP CHAT/SP/GLU CHAT/CGRP

ChAT/NPY neurons NK1 SP NK1 NK3 NK3 VIP neurons NK1 VIP NK3 NPY P2Y1 NK1 P2Y2 NK3 IL1 PGE2 PGE2 A2A, B2 A2B CB1 P2Y1 P2Y4 5HT3 ACH VIP

EPITHELIUM Cl

Cl

FIG. 27-16. Neural receptors in the guinea pig ileum (left) and guinea pig colon (right). Based on the guinea pig, vasoactive intestinal peptide (VIP) and acetylcholine (ACh) are released by secretomotor neurons. CB1, cannabinoid-1; CGRP, calcitonin gene–related protein; ChAT, choline acetyltransferase; 5-HT, 5-hydroxytryptamine; NK, neurokinin; NPY, neuropeptide Y; PAR2, protease-activated receptor 2; PGE2, prostaglandin E2; SP, substance P; A2, adenosine; NC, nicotine; Grp1-mGlu5 and II/III mGlu, glutamate receptors, IL, interleukin; B2, bradykinin; P, purine; GM1, ganglioside; CTxB, cholera toxin B; CDTxB, Clostridium difficile; GLU, glutamate; TRV1, transient receptor vanilloid receptor (also called capsaicin receptor).

may serve as slow EPSP mediators in submucous neurons. Other putative mediators of slow EPSPs in submucous neurons include glutamate, adenosine, prostaglandins, interleukin-1β (IL-1β), IL-6, platelet-activating factor, histamine (H2), and protease-activated receptors. In enteric AH type 2 neurons, the available evidence suggests that mediators of slow EPSPs activate receptors coupled to AC to increase intraneuronal cAMP that activates PKA and ultimately closes Ca2+-dependent K+ channels associated with an increase in cell input resistance and excitation. In these neurons, postreceptor signaling also involves activation of the PLC signaling cascade, formation of IP3 or diacylglycerol, and phosphorylation of channel proteins via calmodulindependent protein kinases or PKC, leading to decreased cell input resistance and excitation. In S/type 1 neurons, the latter mechanisms predominate (8,16) and are involved in purinergic slow EPSPs mediated by P2Y1Rs. It is unknown if such purinergic slow EPSPs occur in secretory reflexes evoked by mechanical stimulation, mucosal stroking, distention, pressure reflexes, or chemical stimulation by ATP, 5-HT, or nutrients. Notably, CGRP8-37, a CGRP antagonist, had no effect on slow EPSPs in submucous neurons evoked by interganglionic fiber tract stimulation, but slow EPSPs were sensitive to blockade by a P2Y1R antagonist (22,48). In contrast, Pan and Gershon (17) report that submucous reflex-excitation and the consequent slow EPSPs in S/type 1 neurons could be blocked with CGRP antagonist, and these authors conclude that CGRP was involved. This difference is difficult to explain, although it raises the possibility that different sensory modalities may activate neural pathways for purinergic or CGRP neurotransmission.

Functional group 1 metabotropic glutamate receptors (mGluR) exist in the enteric nervous system that may play a role in secretion and motility. Group I receptors (mGlu1, mGlu5) activate PLC and undergo ligand-selective endocytosis after agonist activation or during enteric neural reflexes (16). Some data also exist for groups II and III mGluR in both neural plexuses, but their functional relevance in the submucous plexus remains unknown. Submucous primary afferent neurons are suggested to be the source of glutamate released to activate mGlu5Rs on secretomotor neurons. Furthermore, data showing that a group 1 mGluR antagonist 2-methyl-6-(phenylethynyl)-pyridine attenuates reflex-evoked slow EPSPs suggest that glutaminergic slow EPSPs involving mGlu5R may be involved in mechanosensory signaling and secretory reflexes (16). Ionotropic glutamate receptors also exist in the enteric nervous system that may have a potential role in learning and memory. Tetanic stimulation of interganglionic fiber tracts in the submucous plexus evokes a sustained potentiation of fast excitatory synaptic responses in some S-type neurons. A review by Wood and Kirchgessner (16) summarizes in detail the arrangement of glutaminergic neurons in IPANs and EPANs and secretomotor neurons and their pharmacologic properties.

COORDINATION OF SECRETION AND MOTILITY All aspects of digestive function are under the regulatory influence of extrinsic and intrinsic neurons. Because the initial hypothesis that intestinal propulsion was driven by local

756 / CHAPTER 27 neuronal reflexes within the intestine, much of the subsequent work focused on the myenteric plexus, a predominant player in driving peristalsis. However, more recent work has clearly demonstrated that autonomous reflexes also exist in the submucosal plexus (52). Even though the myenteric plexus strongly influences submucous functions in vitro, mucosalsubmucous preparations devoid of myenteric plexus still have intact neural reflex pathways regulating secretion. Furthermore, reflex-evoked dilation of arterioles and mucosal secretion from enterocytes are confined strictly within submucosal neurons. Endocrine cells or mast cells can act as sensory cells that are triggered to release mediators on mechanical or chemical stimulation. Neurons that are in proximity may be triggered by applied force or by chemicals to regulate the release of 5-HT from enterochromaffin cells. Mediators released from these sensory cells may affect enterochromaffin cells in an autocrine (direct) manner or adjacent epithelial cells in a paracrine (indirect) manner, or they may affect these cells indirectly through their actions on neurons in proximity (neurocrine). There has been limited progress in understanding the complex regulation of 5-HT release in tissue studies because of the potential interference from mediators released from other cells. By far, most of our information on sensory signaling is centered on 5-HT release and its activation of IPANs (2,11). Mucosal reflexes initiated by stroking or touch of the mucosa, balloon distention, and distortion of the mucosal surface cells and activation of enterochromaffin cells, alterations in luminal pH, nutrients, or nitrogen puffs on the mucosa all lead to secretion. Myenteric IPANs have their cell bodies in the myenteric plexus and a sensory process projecting to the mucosa. Intracellular recordings from myenteric AH/IPANs proved that mucosal stimulation causes excitation of these neurons that regulate motility. Submucous IPANs are presumed to respond to sensory stimulation, and this can be inferred from studies done in myenteric IPANs. It is not technically feasible to record mechanosensory responses in submucous IPANs because of their short afferent projections to the mucosal lining. Coordinated activity of neurons in submucous and myenteric plexuses is orchestrated, and appropriate secretion and vasomotor responses are triggered during normal or pathophysiologic circumstances. Few studies have explored the integrative activity of the enteric nervous system in the coordination of secretion and motility (53–55). Two complementary techniques are being used to study coordination. Secretion is monitored by Isc current analysis of chloride secretion responses of wholethickness colonic segments set up as flat sheets in Ussing chambers. Smooth muscle responses are monitored by either mini strain-gauge recordings of intestinal smooth muscle tension or sonomicrometric analysis of motion and change in muscle length (54). Dimaprit was used to activate histamine H2 receptors to identify neural circuits and transmitters involved in the coordination of secretion and muscle contraction. Dimaprit, acting on H2 receptors, was shown previously

to induce a cyclical secretory response (1.6–1.8 cycles per 5 minutes) that was coordinated with changes in tension (55). Cyclical secretion and large-amplitude contractions (LACs) were abolished by the H2 receptor blocker cimetidine, or with tetrodotoxin blockade. Secretion and muscle contraction become uncoupled after severing the neural connections between submucous and myenteric plexuses. The stereotypical secretory response is initiated in the submucous plexus because it persists after removing the myenteric plexus. These studies illustrate that a neural program activated by histamine via submucous neurons is important in flushing out the lumen, and they support the notion that the submucous plexus is capable of autonomous control of muscle contractions, and presumably motility, and coordination of secretory and motility functions. Mucosal stroking experiments provided proof that purinergic activation of P2Y1R contributes to the coordination response. Sonomicrometry uses tiny 1-mm diameter piezoelectric crystals to transmit and receive ultrasound waves and estimates motion with high spatial and temporal resolution. Isc was recorded simultaneously with muscle length by piezoelectric crystals fixed on the serosal surface 3 to 6 mm apart in the circular or longitudinal direction for monitoring responses in the two muscle layers, or alternatively, muscle tension was recorded by strain gauges sutured flush with the serosal surface. Our coordination studies give proof that sonomicrometry is an excellent complementary method to mini strain-gauge recordings of muscle responses. Data indicated that inhibitory adenosine A1Rs modulate the histamine H2–mediated stereotypic pattern of cyclical secretion and motility with either technique. In these studies, dimaprit caused cyclical increases in Isc of 170 ± 23 µA/cm2 at frequencies of 1.2 cycles per 5 minutes. Cyclical changes in Isc were coordinated with LACs measured by sonomicrometry as intercrystal distance of 221 ± 35 µm (equivalent to 66% of the peak carbachol response of 335 ± 56 µm; n = 4). In strain-gauge coordination studies, dimaprit caused LACs of 1.4 ± 0.4 g tension in association with large increases in Isc of 116 ± 51 µA/cm2. The A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) reduced or abolished cyclical Isc and LACs with either technique. Coordination of Isc and LACs was lost after CCPA. The adenosine A1R antagonist 8-cyclopentyltheophylline reversed the effects of the A1R agonist with both recording approaches.

CYCLIC ADENOSINE MONOPHOSPHATE SIGNALING AND SECRETION A new acrolein-derivatized cAMP antiserum was used to classify cAMP-dependent submucous neurons by visualization of their shapes and projections and by their cAMP immunoreactivity. Visualization is a powerful technique, because acrolein traps the intracellular free cAMP inside the cell and attaches to cellular protein components. In turn,

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 757 the acrolein-derivatized antiserum recognizes the proteinacrolein-cAMP complex. The use of a cAMP-dependent phosphodiesterase inhibitor prevents cAMP breakdown and permits the diffusion of cAMP throughout the cell. There is also a wide distribution of AC to all parts of the cell, and hence local cAMP production. These properties permit complete visualization of the neurons and their classification. Figure 27-17 shows shapes of neurons that display cAMP responses to forskolin. Forskolin is known to cause secretion in the intestine by multiple mechanisms including direct effects on epithelial cells (56) However, at low concentrations, forskolin acts mainly on the neurons to stimulate chloride secretion. Cholinergic neurons provide both diverging and converging inputs to VIP neurons, a mechanism to augment activation of VIP secretomotor neurons. Thus, AH neurons are likely to be good candidates for divergent patterns because of their multipolar morphology. In both neural plexuses, the majority of cAMP-visualized neurons displaying cAMP in

30 µm

A

D

B

E

C

F

FIG. 27-17. Cyclic adenosine monophosphate (cAMP) visualization of submucous neurons in intact microdissected submucosa in response to the adenylyl cyclase activator forskolin (0.1 µM). (A, B) Forskolin evokes an increase in intracellular AMP in Dogiel type II/multipolar neurons with smooth cell bodies or dendritic morphology. (C, D) Examples of Dogiel type I/uniaxonal neurons with filamentous dendrites visualized after stimulation with forskolin. (E) Dogiel type I neuron with simple morphology. (F) Dogiel type I responsive neuron with lamellar dendrites. Tissues were equilibrated for 45 minutes at 37°C in oxygenated Krebs buffer and stimulated for 20 minutes with 0.1 µM forskolin in the presence of the cAMP-dependent phosphodiesterase inhibitor Ro-20 1724 (10 µM). The adenosine A1R antagonist, CPT, increased the number of immunoreactive neurons. Tissues were then fixed in acrolein for 1 hour and processed for cAMP immunoreactivity using our newly developed acrolein-derivatized cAMP antiserum (raised in rabbit). Neurons are visualized using the avidin-biotin complex (ABC) method.

response to forskolin are uniaxonal Dogiel type I neurons with either filamentous or lamellar dendrites and small/ simple shapes, similar to morphology of secretomotor neurons. Based on cAMP visualization of cAMP immunoreactivity, multipolar neurons make up approximately 28% of all submucous neurons, whereas uniaxonal neurons make up about 72% of the remaining response. Some of the cAMP-immunoreactive neurons with filamentous morphology could be a subset of the VIP secretomotor neurons. The small shape or lamellar neurons expressing cAMP immunoreactivity could represent the secretomotor/vasomotor calretinin-immunoreactive neurons (57,58). The number of neurons displaying cAMP immunoreactivity was approximately threefold greater in the submucous plexus compared with the myenteric plexus (numbers of neurons per square centimeter) where such signaling is tightly regulated by phosphodiesterase, type PDE IV. The PDE IV inhibitors Ro-20 1724 or rolipram augment cAMP levels in submucous neurons by several fold more than in myenteric neurons. Basal levels of cAMP are tightly regulated by endogenous adenosine acting at inhibitory A1Rs, whereas A2ARs increase cAMP levels in submucous neurons. Prostaglandins, ATP, and SP stimulate cAMP in smaller subsets of neurons (Fig. 27-18). Pharmacologic agents acting through the AC/ cAMP or GC/cGMP signaling pathways increase intracellular free cAMP in submucous neurons with a potency profile as follows: ATP > forskolin + PDEI > forskolin > PDEI ≥ CGS 21680 (A2 agonist) > CPT (A1 antagonist) > PGE2 > SP > VIP, CGRP > Krebs (basal levels) As alluded to earlier, the AC/cAMP/PKA signaling pathway acts differently in the two nerve plexuses. This holds for cGMP where cGMP immunoreactivity occurs in a majority of submucous neurons in comparison with less than 2% of myenteric neurons (30). In contrast with myenteric neurons, cAMP-dependent submucous neurons do not have polarized projections and suggest a more fundamental role for cAMP signaling in neuronal circuits for widespread activation and spread of excitability in secretory circuits. Overall, differences exist between myenteric and submucous neurons in receptor coupling to AC/cAMP signaling (A1, A2a, prostaglandins, neuropeptides), polarity of cAMP-dependent neurons, PDE IV activity, numbers of neurons, and cross talk between cAMP and cGMP. The relation between cAMP immunoreactivity visualization and neuronal excitability remains unknown. However, mucosal stroking–evoked descending inhibition (relaxation), but not ascending excitation (contraction), is particularly sensitive to PKA inhibition, and this response was predictable from cAMP visualization studies, indicating that most cAMP-dependent neurons have descending projections. This suggests that cAMP responses detected by the acrolein-derivatized antiserum contribute to gut neural reflexes.

758 / CHAPTER 27 A

B p = 0.022, n = 3

600

400

200

0

p = 0.013

700 Number of cAMP ir Neurons/cm2

Number of cAMP ir Neurons/cm2

800

RO + F

600 500 400 300

p = 0.022

200 100 0

RO + CCPA

Krebs

RO

RO+CPT

C

Krebs

RO

CPT+RO

CPT+RO

FIG. 27-18. Receptors that are linked to the adenylyl cyclase/cyclic adenosine monophosphate (AC/cAMP) signaling pathway in submucous neurons are revealed by cAMP visualization with the acrolein-derivatized antiserum. (A) The cAMP response to forskolin in submucous neurons is sensitive to inhibition by the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1 µM). (B) The adenosine A1 R antagonist CPT alone augments the basal/R0-20 1724 response in submucous neurons. (C) Visualization of the effect of CPT on the AMP response in submucous neurons.

PATHOBIOLOGY OF REFLEX-DRIVEN INTESTINAL SECRETION Diarrhea is a continuing medical problem worldwide. Drugs that block or reduce secretion are needed. An understanding of the components of the reflex pathway is needed to target drugs. Cholera toxin or Clostridium difficile toxin are two examples used to illustrate the complex regulatory systems that modulate secretory diarrhea; however, there are many other examples (e.g., 59–66).

Cholera Toxin–Induced Secretion The pathways that mediate chloride secretion in response to cholera toxin are complex and involve multiple neural pathways and immune mediators (see Fig. 27-19A).

Submucous IPANs synapse with cholinergic or VIP secretomotor neurons, release acetylcholine and VIP that act on muscarinic or VIP (VPAC) receptors on epithelial cells, and stimulate secretion into the lumen. Cholera toxin–induced secretion is mediated by direct effects of the toxin A on epithelial cells, as well as effects of toxin B, which binds to ganglioside receptors GM1Rs on enterochromaffin cells and VIP neurons. Profuse intestinal secretion results from activation of secretory reflexes and increased excitability of VIP secretomotor neurons, as well as an increase in release of 5-HT from enterochromaffin cells (57,58). Not only is secretion mediated by activating submucous secretory reflexes, but secretion also occurs by activation of myenteric neurons. Effective and potent drugs that inhibit secretion are needed to prevent secretory diarrhea. Antagonists for 5-HT3, SP, and VPACRs reduce secretion, but some of these have been met with mixed results. The cystic fibrosis transmembrane

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 759 B Clostridium difficile toxin

A Cholera toxin IPANS

Myenteric

SP/CGRP

NE(-) NK1 α 6 NK3 VIP NK1 CHAT

α

VR1

VIP

V

5HT 2 GM1

V

GM1

5HT1P

7

VIP ACH 8 VPAC M3

4 9 Epithelium

5HT1P

5HT

VIP ACH VPAC M3 SP

Cholera

Tryptase PAR2

PGE2 HIS 5-HT NK1 PGE2 MC Mf IL-1b NFkB 3 IL-6

SP

Axon reflex

LTB4 PGE2 Others-TNB

2

NC EC

1 TxB

4

5HT3 Axon reflex

NFkB Epithelial

EC

5

V

Mf

CHAT

(+)

IL-1b IL-6

PAR2

4

NE(-)

PGE2

5HT2 C

3

7 NK1

(+)

SP/CGRP NK1 MC

VR1

Submucous

6

PGE2

EPANS

IPANS

EPANS

? ? Submucous

5

Sympathetic input

Sympathetic input

V

MYENTERIC

TxA

Focal infection

1 TxA

Cl Secretion

Cl Secretion

Cl. difficile

FIG. 27-19. Amplification of reflex-evoked intestinal secretion by extrinsic primary afferent neuron (EPANs) and by the secretory reflex. (A) Effect of cholera toxin on secretion. Cholera toxin has direct effects on epithelial and enterochromaffin cells (ECs) (1), and (2) toxin B (TxB) binds to GM1 ganglioside receptors on ECs and on vasoactive intestinal peptide (VIP) secretomotor neurons in the submucous plexus. (3) Activation of ECs releases 5-hydroxytryptamine (5-HT), which binds to 5-HT1P receptors and activates submucosal intrinsic primary afferent neuron (IPANs). (4) 5-HT acts on 5-HT receptors on immune cells. (5, 6, 7) Transmission from IPANS to VIP secretomotor neurons. (6) Prostaglandin E2 (PGE2) activates submucosal cholinergic secretomotor neurons. (7) VIP and acetylcholine (ACh) act on epithelial cells to stimulate chloride secretion. VPAC receptors are likely to be VPAC1. (8, 9) Secretion is through the submucous plexus, with a smaller component that is thought to be through the myenteric plexus. (B) (1) Clostridium difficile produces toxin A (TxA) (2). Once TxA is internalized in enterocytes, TxA activates nuclear factor-κB (NF-κB) and releases leukotriene (LTB4), PGE2, and tumor necrosis factor (TNF). After NF-κB activation, the proinflammatory mediators interleukins (ILs) are released from monocytes/macrophages MØ. (4) Stimulation of processes of EPANS occurs, followed by release of substance P (SP)/calcitonin gene–related protein (CGRP) that degranulates mast cells (MC) by activating neurokinin-1 (NK1) receptors. (5) SP-containing EPANs release SP on cholinergic secretomotor neurons. The mediators released from mast cells include histamine (HIS), 5-HT, PGE2, and tryptase. (6) Tryptase acts on proteaseactivated receptor 2 (PAR2) receptors on EPANs and on IPANs to stimulate further release of PGE2 (7). The sympathetic innervation with norepinephrine (NE) release and its binding to adrenergic receptors normally reduces secretion via VIP neurons. ILs suppress norepinephrine release. These cytokines act on sympathetics to lift the sympathetic brake that was caused by release of norepinephrine. TxA-induced release of proinflammatory mediators degranulate mast cells that amplify the secretory response to maximize secretion in defense of the host. ChAT, choline acetyltransferase.

conductance regulator (CFTR) chloride channel blocker, thiazolidine-type CFTR blocker, may be of potential therapeutic value in treatment of secretory diarrheas (59–61). Selective purinergic drugs, particularly those acting at A1, A3, and possibly P2Y1 receptors, remain to be tested.

Clostridium Difficile Enteritis In Clostridium difficile enteritis, toxin A activates nuclear factor-κB (NF-κB), which causes both epithelial cells

and macrophages to produce neutrophil attractants such as IL-6 (see Fig. 27-19B). Secretion induced by Clostridium difficile infection of intestinal loops initially is focal, but once EPANs are activated, the secretory response is further amplified to cause profuse secretion via the enteric nervous system. Axonal release of SP from EPANs near mast cells causes degranulation and amplifies the inflammatory response by releasing many mediators (62,63). In mast cell–deficient mice or when EPANs are exposed to capsaicin to prevent them from acting, inflammation is reduced in this model. The local inflammatory response

760 / CHAPTER 27 produced by the toxin is further amplified by an increase in receptor up-regulation of NK1Rs in epithelial cells and possibly other targets as well. Hyperexcitability of the neural circuits leads to amplification of the secretory response (64).

Purines and Mucosal Disorders Release of adenosine from crypt abscesses is believed to cause secretory diarrhea via putative A2b receptors (67). Patients with severe immunodeficiency disease have a deficiency of the enzyme adenosine deaminase and are unable to inactivate adenosine. These patients suffer from diarrhea, and in addition a failure to thrive, malabsorption, and recurrent infections caused by high plasma levels of adenosine acting to impair cellular immunity. Selective A2b receptor antagonists are not available for clinical applications, although they may hold potential therapeutic value. Adenosine is also an important endogenous modulator of inflammatory processes, exerting anti-inflammatory or proinflammatory effects, depending on the specific adenosine receptor subtype that is involved. Adenine (ATP, ADP) or uracil (UDP, UTP) nucleotides or nucleosides are important extracellular signaling molecules that act at cell-surface receptors. These prototypical purines influence sensory signaling and gut reflexes via putative A3R, A1R, P2Y1R, P2X, and perhaps others. In the enteric nervous system, and in particular the myenteric plexus, extracellular levels of endogenous adenosine are sufficient for ongoing suppression of neuronal excitability, fast EPSPS, as well as slow EPSPs, whereas inhibitory slow synaptic transmission is spared (66). These effects serve to complement the ability of adenosine to shut down excitatory neural activity in the gut microcircuits through its dual presynaptic and postsynaptic actions. In contrast, slow IPSPs are sensitive to adenosine in submucous neurons showing selectivity of action in the two neural plexuses. The therapeutic potential of purinergic receptors in intestinal secretor disorders remains to be established. The short half-life of adenosine and its potential side effects at other adenosine receptor subtypes limits its usefulness as a therapeutic drug. Recent efforts have focused on interventions that increase endogenous adenosine levels and enhance its protecting actions at its local cellular site of formation, whereas reducing systemic side effects. Purine nucleoside acadesine (AICA riboside [5-amino-1β-D-ribofuranosylimidazole-4-carboxamide]) is one such medication. After it is taken up by cells, it becomes incorporated in the de novo purine biosynthetic pathway by phosphorylation to 5-aminoimidazole-4carboxamide ribonucleotide (AICA ribotide, or AICAR); it can be metabolized further to inosine monophosphate (IMP). The adenosine-enhancing activity and protective actions of acadesine are believed to be a cause of the combined weak inhibitory action of AICAR on enzymes

that are reciprocally involved in the IMP to AMP conversion and of adenosine kinase and adenosine deaminase. It has been recognized that the anti-inflammatory effects of methotrexate and sulfasalazine used in the treatment of inflammatory bowel disease (IBD) and rheumatoid arthritis is a result of their ability to induce the accumulation of AICAR, thereby increasing local adenosine release at sites of inflammation. Adenosine A3 receptors are emerging as novel targets for treatment of inflammatory diseases including IBD and irritable bowel syndrome with predominant diarrhea that are associated with abnormal secretions. A3R agonist N(6)(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) was used to study gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)– induced colitis (68). The most exceptional cluster of genes that is sensitive to colitis in the RNU34 neurobio chip represents less than 10% of the gene pool. It illustrates two modes of colitis-related gene dysregulation. Oral IB-MECA prevented dysregulation in most genes, histopathology, gut injury, and weight loss and also blocked colitis-induced dysregulation of certain P2X or P2Y receptors but not adenosine receptor genes. The A3R agonist can prevent most gene dysregulation, histopathology, clinical signs of damage, and weight loss in TNBS-induced colitis (Fig. 27-20). Our studies did not delineate which receptors were involved. A3Rs are a more likely target for the protective effects of oral IB-MECA administration than A1Rs, although A2ARs have potential as well. Activation of A1Rs by IB-MECA would not be beneficial because chronic A1R activation could lead to rapid desensitization/internalization of A1Rs and disinhibition of enteric nerves, adding to neural dysfunction. IB-MECA is being tested in clinical trials for other inflammatory disorders such as rheumatoid arthritis and is apparently without toxicity. A clinical implication is the potential for testing the use of adenosine A3R agonists or other purinebased adenosine drugs (agonists or antagonists) in the prophylactic treatment of postoperative recurrence of Crohn’s disease after ileocolonic resection, which has been shown to be responsive to another purine drug, 6-mercaptopurine (6-MP). Purine-based drugs deserve thorough investigation as potential therapeutic targets for various purinergic receptors in intestinal diseases involving secretory diarrhea, mucosal injuries, and inflammation. Restoration of mucosal integrity and healing observed with IB-MECA by itself would restore normal function in IBD by restoring mucosal reflexes. In conclusion, the gut is an amazing organ that secretes fluid together with mucus and provides an insulating layer on top of epithelial cells. Chloride secretion is only one of the many elements in this barrier. We are beginning to discover that there are multiple reflex pathways that stimulate chloride secretion and contribute to this task. ATP or its metabolites now join the ranks of 5-HT, a known activator of mucosal reflexes.

ENTERIC NEURAL REGULATION OF MUCOSAL SECRETION / 761 A

B

C

Age-matched control

TNBS+IB-MECA

TNBS

M

M SM

SM

CM

M

SM CM

FIG. 27-20. N(6)-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) prevents cell and tissue injury in 2,4,6-trinitrobenzene sulfonic acid (TNBS)–induced colitis in rats. Colon sections from (A) age-matched control, (B) TNBS (C), and IB-MECA–treated TNBS rats. (B) TNBS administration caused areas of necrosis in mucosa (arrows) with evident regions of hemorrhage in both mucosa and submucosa (arrowhead) and infiltration of inflammatory cells (curved, hollow arrow). (C) Oral administration of IB-MECA (1.5 mg/kg BID for 6 days) nearly prevented the colitis. A few immune cells more than in age-matched control samples are still visible in submucosa (arrows) and circular muscle (CM) layer (arrowhead). M, mucosa; SM, submucosa. Hematoxylin and eosin staining. Scale bar = 50 µm.

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CHAPTER

28

Effect of Stress on Intestinal Mucosal Function Johan D. Söderholm and Mary H. Perdue Mucosal Barrier Function, 763 Specific Aspects of Barrier Function, 764 Other Components of Mucosal Barrier Function, 766 Stress Concepts, 766 Stress-Induced Changes in Mucosal Function in Humans, 767 Response to Physical Stress, 767 Response to Psychological Stress, 768 Stress-Induced Changes in Mucosal Function in Animals, 768

Animal Models, 768 Effects of Acute Stress, 769 Effects of Chronic Stress, 772 Effects of Early Life Stress, 774 Relevance for Human Intestinal Diseases, 775 Inflammatory Bowel Disease, 775 Food Allergy, 775 Irritable Bowel Syndrome, 776 Conclusion, 776 References, 776

The intestinal epithelium is the single layer of epithelial cells lining the gastrointestinal tract. It has conflicting functions, playing a major role in the digestion and absorption of nutrients, whereas at the same time limiting the uncontrolled uptake of material from the gut lumen. In constant contact with luminal contents on one side and various immune cells of the lamina propria on the other, the epithelium forms the most important barrier of the organism between the bodyproper and the external environment. Excessive passage of antigens or other noxious substances across a transiently or intrinsically leaky intestinal barrier can stimulate an inflammatory response. In a normal host, this reaction is downregulated when the triggering event has resolved, whereas in a susceptible host, chronic inflammation may develop. It has become increasingly recognized that stress has a major impact on intestinal physiology, can cause intestinal dysfunction, and can influence the course of gut disorders. For example, it has been established that early life stress and sustained stressful life events predispose humans to development of functional bowel disorders, such as irritable bowel syndrome

(IBS) (1). In addition, stress triggers relapses of chronic inflammatory bowel diseases (IBDs) (2). Our understanding of the complex brain–gut interactions involved in stress-related intestinal pathophysiology is, however, only beginning to evolve. This chapter focuses on stress-induced changes in mucosal functions (mainly epithelial secretion and barrier function) underlying mechanisms and clinical consequences.

MUCOSAL BARRIER FUNCTION The intestinal mucosa is continuously exposed to an immense load of antigens from ingested food, resident bacteria (1014 microbial cells of more than 400 different species), invading viruses, and so forth. In this milieu, the single cell layer of epithelium plays seemingly paradoxic roles, being the major site for digestion and absorption of nutrients, but also forming the most important barrier of the organism between the internal and external environments, with a surface area of approximately 300 m2. In addition, research conducted during the beginning of this century has shown that epithelial cells are involved in mucosal immunity (3). Normal epithelium allows small quantities of ingested antigens to cross and interact with immune cells, thus providing important surveillance of the luminal contents (4). However, excessive uptake of antigens and bacteria may overactivate the immune system and lead to local mucosal inflammation or even sepsis when bacteria are translocated to the circulation.

J. D. Söderholm: Department of Biomedicine and Surgery, Linköping University Hospital, Linköping, SE-581 85, Sweden. M. H. Perdue: Intestinal Disease Research Program, HSC-3N5C, McMaster University, Hamilton, Ontario, L8N3Z5, Canada. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

763

764 / CHAPTER 28 The ability to limit this uptake is denoted the barrier function of the intestinal mucosa. Mucosal barrier function includes physical diffusion barriers, regulated physiologic and enzymatic barriers, and immunologic barriers (Fig. 28-1). These components are under neuroimmunoendocrine control, and therefore may be affected by stress. Subepithelial myofibroblasts also are involved in regulation of epithelial function (5). The continuous layer of epithelial cells, interconnected by tight junctions, restricts both transcellular and paracellular permeation, thus constituting the principal part of the barrier. Active secretion of fluid and mucus containing secretory IgA also serves to

FIG. 28-1. Simplified schema of various components of the intestinal mucosal barrier. In the lumen (1), gastric acid, pancreatic juices, and biliary juices take part in barrier function by degradation of bacteria and antigens; pathogenic bacteria are kept under control by the normal flora. The microclimate of the epithelial cells is controlled by the unstirred water layer (2), the mucous layer and glycocalyx (3), and by secreted immunoglobulins, primarily IgA (4). Enterocytes constitute the major component of the barrier and are interconnected by junctional complexes, including tight junctions (5). Enterocytes can respond to a noxious stimulus with rapid secretion of chloride and water (6). The subepithelial basal lamina (7) contains large pores. In the lamina propria, a large number of cells interact with enterocytes to regulate barrier function, including subepithelial myofibroblasts (8), cells of acquired immunity including T and B cells (9), and cells of innate immunity including macrophages, mast cells, eosinophils, and neutrophils (10). In addition, the enteric nervous and endocrine systems (11) exert effects on epithelial function. The endothelium of the capillaries (12) represents the barrier from the mucosa to the circulation.

bind, dilute, and wash away noxious substances. The protective mechanisms of the bowel also include intestinal propulsive motility (6), which is coordinated with a structure–function relation. Thus, ongoing secretion from the relatively leaky crypts results in a situation where luminal contents can access only the relatively tight villous (small intestine) and surface (colon) epithelia (7,8).

Specific Aspects of Barrier Function Tight Junctions Adjacent enterocytes are joined to each other by junctional complexes; these complexes were first described by Farquhar and Palade (9). They consist mainly of tight and adherence junctions. Tight junctions are continuous cell–cell contacts near the apical end of the lateral cell interspaces, creating a highly regulated rate-limiting barrier to the diffusion of solutes, and also acting to separate specific lipid and protein components of the apical cell membrane from those in the basolateral cell membrane. Adherence junctions are another continuous circumferential contact zone positioned below the tight junctions and formed by adhesion molecules of the cadherin family and their cytoplasmic binding proteins α-, β-, and γ-catenins. In addition, the junctional complexes include desmosomes, which form spotlike dense adhesions between epithelial cells. Gap junctions are specialized regions for cell–cell communication mediating electrotonic coupling, allowing ionic currents to pass between cells. The details of tight junction structure and regulation are reviewed comprehensively in Chapter 63. In brief, the ability of epithelia to transport substances toward or away from the lumen depends on the polarity of the cells and on the tight junctions to prevent back diffusion through the intercellular space (gate function) (10). Tight junctions are selectively permeable to ions (depending on charge) and allow the passage of small molecules, but they are virtually impermeable to protein-sized molecules under normal conditions, thus constituting an effective barrier to macromolecules with antigenic potential. The tight junctions are formed by at least two types of transmembrane proteins: occludin and different variants of the 20 members of the claudin family (11). It is now thought that the varied tissue and cell-type distribution of the claudins explains the variable permeability observed among different tissues. These transmembrane proteins are connected to the cytoplasmic plaque proteins (e.g., zonula occludin 1 (ZO-1), ZO-2, ZO-3, cingulin, and 7H6), which are linked to the perijunctional actomyosin ring, the contraction of which regulates permeability (12). The tight junctions also are involved in maintaining the polarity of membrane proteins (e.g., ion channels and nutrient transporters) by preventing lateral movement of cell membrane constituents from the apical to the basolateral region in the outer cell membrane leaflet (fence function). The gate and fence functions of the tight junctions appear to be regulated in different ways (13).

EFFECT OF STRESS ON INTESTINAL MUCOSAL FUNCTION / 765 Transcellular Uptake of Protein Antigens Limited uptake of protein antigens by the intestinal mucosa is physiologic and crucial for the surveillance of luminal contents to generate appropriate down-regulated immune responses leading to oral tolerance (3,4). Antigen sampling is believed to occur primarily via the M cells in the follicleassociated epithelium (FAE) overlying lymphoid follicles and Peyer’s patches (14,15). The relative importance of uptake of soluble macromolecules via the comparatively infrequent M cells is, however, a point of discussion (16). Regular columnar epithelial cells are also capable of endocytosis, followed by transcytosis and/or processing and presentation of antigens via major histocompatibility complexes (MHCs) to immune cells (15). In general, three types of endocytotic uptake by the intestinal epithelium are recognized (4,17): highly specific, receptor-mediated uptake (e.g., uptake of immunoglobulins and growth factors from breast milk), adsorptive endocytosis (after binding of molecules to the cell membrane), and nonspecific fluid-phase endocytosis (of substances in the luminal fluid). The latter two types of endocytosis are relevant for the nonspecific uptake of dietary and bacterial antigens. The fate of the macromolecules after endocytosis is largely determined by membrane binding. Membrane-bound proteins can be sorted to vesicles that traverse the cell to the basolateral pole, whereas proteins in free solution are sorted in the tubulocisternae to vesicles that fuse with lysosomes. After a fluid-phase antigen is internalized in an apical membrane vesicle, the vesicle fuses with the early endocytotic compartment (18). This first step of endocytosis is actin dependent, whereas the following steps mainly are microtubule dependent. In the early endocytotic compartment, the antigen is sorted to different pathways: recycling to the apical membrane, delivery to lysosomes, or transcytosis. Most of what is internalized is recycled to the apical membrane. Proteins that remain inside the cell are transported to late endosomes that fuse with lysosomes, enabling degradation by lysosomal enzymes. In these compartments, which contain lysosome-associated membrane protein 1 (LAMP-1), the peptide fragments fuse with recycled MHC II molecules and are presented at the basolateral membrane. This targeting of the antigen to LAMP-1–positive late endosomes has been shown to regulate the immune response to endocytosed antigen in mice because the antigen-MHC II complex then interacts with a T-cell receptor (19). The class I–related CD1d molecules expressed at the surface of intestinal epithelial cells also appear to function as antigen-presenting molecules (15). Antigen presentation by enterocytes usually results in stimulation of CD8+ T cells, in contrast with the CD4+ cell stimulation produced by antigen-presenting cells in the lamina propria. Situations with enhanced antigen exposure or disrupted barrier may, however, result in stimulation of CD4+ T cells (15). Although the bulk of protein is transferred to the lysosomal pathway and degraded to peptide fragments and amino acids, even under normal conditions a small but significant amount of undegraded protein does cross the

epithelium (20,21). Whether this is the effect of an escape from lysosomal breakdown or a limited uptake via the paracellular route, or both, has not been fully resolved.

Specialized Antigen-Sampling Cells M Cells and Follicle-Associated Epithelium The biochemical features of the membranes facing the lumen in the FAE are different from the epithelium covering the surrounding villi. The FAE has less production of mucus, expresses smaller amounts of membrane-bound digestive enzymes, and has different glycosylation patterns in the brush-border glycocalyx (22). As a result, the epithelial cells of the FAE are more exposed to luminal contents. The FAE also lacks the underlying subepithelial myofibroblast sheath, and the basal lamina is more porous than regular villous epithelium (23). Moreover, 10% of the epithelial cells of the FAE are specialized epithelial M (microfold or membranous) cells (14). M cells lack brush borders, have a high endocytotic activity of adherent, as well as fluid-phase, macromolecules and particles, and have few lysosomes (24). Typically, the basolateral membrane is invaginated, forming a pocket for lymphocytes and dendritic cells. Thus, M cells are designed to deliver samples of luminal antigens by transepithelial vesicular transport to intraepithelial and subepithelial immune cells. Antigen uptake from the lumen is believed to be crucial for induction of protective mucosal immune responses, but it also provides a route of entry into the mucosa for various pathogens: bacteria, viruses, as well as protozoa (25). Many pathogens survive M cell transcytosis and go on to infect cells of the mucosa or the epithelium itself. The origin of M cells is not clearly understood. They may be a specialized cell lineage, but generally are thought to be a phenotypic conversion of enterocytes in the FAE, modulated by lymphocytes and cytokines (26). Caco-2 cells in culture can be converted to M-like cells by coculture with lymphocytes expressing B-cell markers (27). The number of M cells is increased rapidly by enteric infections (28,29). Dendritic Cells Dendritic cells are resident below and within intestinal epithelia along the entire intestinal tract (30) and are actively recruited to sites of mucosal inflammation. Dendritic cells sample invading microorganisms, and by presenting antigens and release of cytokines, they have an important immunoregulatory role. This is especially evident in Peyer’s patches, where immature dendritic cells in the subepithelial dome are in close contact with M cells and actively survey the antigens taken up from the intestinal lumen. Rescigno and colleagues (31) report that mucosal dendritic cells themselves are able to express tight junction proteins, interact with the epithelial cells, and send processes into the intestinal lumen to sample bacteria. Once dendritic cells have sampled antigen, they transform into mature cells and migrate out of the subepithelial dome area. Mature dendritic cells then present

766 / CHAPTER 28 phagocytosed antigens to lymphocytes found in the interfollicular regions or in the lymph nodes (32). In this manner, dendritic cells orchestrate the immune response. During an infection with pathogenic bacteria, recruited dendritic cells prime T cells to become effector cells, whereas tissueresident dendritic cells exposed to commensal flora induce the differentiation of regulatory T cells.

Other Components of Mucosal Barrier Function Ion and Fluid Transport Transport of fluid and ions across the epithelium can be divided into absorption (toward the bloodstream) and secretion (into the lumen). These functions are spatially distinct along the villus-crypt axis, with nutrient and fluid absorption occurring mainly in the upper part of the villi, and the crypt region being the site of electrolyte and water secretion (33). Epithelial transport functions are directly or indirectly dependent on the sodium/potassium pump mechanism, Na+/K+/ ATPase, located in the basolateral membrane of enterocytes. This energy-requiring ion pump creates an electrochemical gradient across the cell membrane due to the low intracellular Na+ concentration. This gradient facilitates the uptake into the cell of Na+, together with various nutrients via Na+-coupled cotransporters (e.g., for glucose, amino acids, vitamins). Water movement follows the osmotic gradient generated by the absorption of ions and nutrients. The Na+/K+/2Cl− entry ion transporter in the basolateral membrane of crypt cells creates an electrochemical gradient for Cl− (high intracellular concentration) that facilitates rapid secretion of Cl− and water through channels in the apical membrane when the cells are stimulated by secretagogues such as neurotransmitters or inflammatory mediators. This is an important “flushing” mechanism in innate defense that serves to wash away invading microorganisms, and it also is activated in response to stress. Mucous Secretion The major constituents of intestinal mucus are highmolecular-weight proteoglycans called mucins. Mucins consist of a central protein core with large numbers of oligosaccharides (accounting for up to 60–80% of the molecular mass) attached to specific regions of the core. The oligosaccharides are variable and complex, and the degree and type of glycosylation of mucins is central to their function. The patterns of glycosylation are tissue specific within the gastrointestinal tact. Mucins can be divided into two groups: secreted, gel-forming mucins (mainly in a form encoded by the MUC2 gene) and membrane-bound mucins (predominantly encoded by MUC4). Goblet cells of the surface epithelium store mucin in apically located granules. Mucin is secreted at a low baseline rate to maintain the mucous coat over the epithelium. In response to stimulation, such as by cholinergic nerves (34), goblet cells can substantially accelerate

their discharge of mucins. The discharge of mucins in response to stimulation occurs either by compound exocytosis, resulting in deep apical membrane cavitations, or by accelerated single-granule exocytosis (35). Colonic mucin release has been shown to be stimulated by signals from the enteric nervous system (acetylcholine, vasoactive intestinal polypeptide [VIP]), enteroendocrine cells (peptide YY, 5-hydroxytryptamine [5-HT]), and resident immune cells (mast cell mediators, interleukin-1β [IL-1β], nitric oxide [NO]) (36). Research has made clear that various types of stress have significant effects on several components of barrier function. This chapter reviews the most important findings in this area.

STRESS CONCEPTS The influence of psychosocial factors on gastrointestinal diseases has long been recognized by patients, but the effect of chronic life stress on intestinal diseases remains a controversial issue in the medical community (37). However, scientific studies are providing convincing evidence that stress has a major impact on intestinal physiology, importantly inducing mucosal barrier dysfunction. Stress is a normal component of life, and adequate responses to stressors are necessary for survival. However, there are large individual variations in coping with the environment and various situations (38). Selye (39) has defined stress as any threat to the homeostasis of an organism. This threat can be real (physical) or perceived (psychological) and caused by events in the environment or within the individual (e.g., by inflammation) (40). Stress also has been described as “the range of tensions of modern life” (41). Regardless of the type of threat, the principal stress responses triggered to maintain homeostasis are surprisingly similar. The normal integrated stress response is made up of different components, including the behavioral response (such as anxiety), autonomic responses (such as raised heart rate), and responses mediated by the hypothalamic-pituitaryadrenal (HPA) axis (such as cortisol release) (42) (Fig. 28-2). The response of the organism to stress is generated through a network of brain structures, mainly the paraventricular nucleus of the hypothalamus, amygdala, and periaqueductal gray. These structures receive input from visceral and somatic afferents, as well as from cortical structures, mainly the medial prefrontal cortex and the anterior cingulate and insular cortices (1,42). A critical mediator of the stress response is corticotropin-releasing hormone (CRH) and related peptides (urocortins I, II, and III). These peptides interact with three different G protein–coupled receptors (corticotropin-releasing factors 1-3 [CRF1-3] receptors), which are distributed in the brain, but also in viscera such as the intestine. The stress network has efferent connections to the pituitary, thereby mediating the neuroendocrine (HPA axis) response, and to the pontomedullary nuclei (PMN), generating the autonomic output to the body. Negative feedback loops from the adrenal (circulating glucocorticoids) and ascending monoaminergic projections (noradrenergic and serotonergic) regulate the

EFFECT OF STRESS ON INTESTINAL MUCOSAL FUNCTION / 767 Cortex Behavioral response

Amygdala PVN

PAG

CRH Pituitary ACTH

PMN −

−

Adr HPA-axis response

Autonomic response

Cortisol

NE, CRH, ACh Cytokines Visceral pathophysiology

FIG. 28-2. Overview of the neuroendocrine systems involved in the central and mucosal stress responses. The stress network has efferent connections to the pituitary gland, thereby mediating the hypothalamic-pituitary-adrenal (HPA) axis response, and to the pontomedullary nuclei (PMN), generating the autonomic output to the body. Negative feedback loops from the adrenal gland (cortisol) and ascending monoaminergic projections modulate the stress response. Cytokines released from stress-affected viscera can act as interoceptive stressors. ACh, acetylcholine; ACTH, adrenocorticotropic hormone; CRH, corticotropin-releasing hormone; NE, norepinephrine; PAG, periaqueductal gray; PVN, paraventricular nucleus.

stress response. Despite the integrated response to various stressors, there is large potential for variability in the peripheral response. In the hypothalamus, the major classes of visceromotor projections are separated from each other, and in the periphery, 12 different functional groups of sympathetic neurons have been identified that regulate visceral function. This is illustrated clinically by individual differences in stress symptoms (e.g., headache, heart palpitations, diarrhea). When a healthy individual encounters an acute challenge, physiologic systems are quickly turned on and off, matching the duration and severity of the stressor, a so-called adaptive response. An individual’s response to stress, however, is modulated by multiple factors; consequently, there are large variations in responses to stressors. The responses mainly depend on two principal factors: (1) how the individual perceives and interprets the situation (depending on one’s earlier experience of similar situations, and also on genetic predisposition and previous stress exposure); and (2) the physical condition of the individual’s body (because good physical condition results in better handling of strenuous events and mobilization of adequate adaptive stress responses) (38). The stress response, under certain circumstances, may become harmful and cause damage, a so-called maladaptive response. These maladaptive responses are also referred to

as pathologic stress or “wear and tear” on the body and occur via three types of responses: (1) overload—when the severity or the duration of the stressful experience exceeds an individual’s adaptive capacity; (2) failure to shut down— when the stress response persists between repetitive stressors, for example, hypertension in individuals with time-pressured work; and (3) inadequate response—for example, inadequate HPA activity in chronic inflammatory conditions (38). An overload situation may also turn into a failed shutdown or inadequate response. For example, early-life stress (such as maternal deprivation) may result in chronic hypersecretion of CRH (43). Chronic abuse and neglect has been associated with blunted and inadequate HPA axis responses to new stress (44). Moreover, exposure to a one-time life-threatening stress (rape, combat, natural disaster) may result in posttraumatic stress syndrome with alterations in memory, the HPA axis, and sympathetic responses (45). Obviously, in these cases, the individuals will be predisposed to disease in multiple organ systems, for example, the brain, the immune system, and the viscera, including the intestine. More frequently, maladaptive responses caused by chronic daily life stressors such as losses, financial problems, unemployment, and so forth have been associated with exacerbations of IBS symptoms (46) and increased numbers of relapses of ulcerative colitis (47). Stress-induced changes in mucosal function are believed to be an important mechanism involved in this association.

STRESS-INDUCED CHANGES IN MUCOSAL FUNCTION IN HUMANS A large body of evidence has shown that stress is involved in the pathogenesis of gastric stress ulcers in critically ill patients (48), as well as in peptic ulcer disease (49). Mechanisms include disturbances in the gastric mucosal barrier (50), mainly via changes in the prostaglandin-regulated secretion of mucus, and obstruction of back diffusion of acid through the tight junctions of the gastric surface epithelium. Historically, stress effects on the intestinal mucosa have received less attention, and there are few studies examining the influence of psychological stress on intestinal function in humans.

Response to Physical Stress A number of studies have documented the presence of increased intestinal permeability in patients suffering from trauma, thermal injury, sepsis after major surgery, or other critical illnesses (51,52). Moreover, a high incidence of bacteremia or endotoxemia, or both, was found in patients with hemorrhagic shock (53) and victims of thermal injury (54). Bacteria also were found in the mesenteric lymph nodes of patients undergoing intestinal surgery and in trauma patients (55,56). None of these studies found a correlation between the presence of bacterial translocation and the development of severe complications. In contrast, Doig and

768 / CHAPTER 28 colleagues (57) show that the degree of barrier dysfunction in patients admitted to intensive care was related to the development of multiple organ failure syndrome. This relation was not reproduced in patients with less severe illnesses who were undergoing major upper gastrointestinal surgery (58). There are a number of possible mechanisms for mucosal barrier dysfunction in patients exposed to various types of trauma or sepsis, for example, mucosal hypoxia and acidosis caused by hypoperfusion, oxidant stress and adenosine triphosphate depletion during reperfusion, and increased levels of proinflammatory cytokines. (51). Another possible mechanism for barrier dysfunction in patients who underwent surgery for severe trauma is extensive focal apoptosis of crypt epithelial cells and lamina propria lymphocytes of the intestine (59). The importance of the neuroendocrine stress response in that situation is shown by the positive clinical effects of epidural analgesia in the postoperative course after gastrointestinal surgery (60). Epidural analgesia, which blocks regional innervation to the intestine, may prevent the release of neurotransmitters from afferent pain fibers, block receptors, or interrupt the transmission of pain information in the dorsal horn of the spinal cord. In this manner, epidural analgesia attenuates the stress response, as shown by lower postoperative levels of adrenocorticotropic hormone (ACTH), cortisol, aldosterone, and glucose (61,62).

Response to Psychological Stress Studies using intestinal perfusion techniques in the human jejunum have revealed effects of acute stress on intestinal secretion. Barclay and Turnberg (63,64) found that psychological stress induced by dichotomous listening (63) or coldinduced hand pain (64) reduced mean net water absorption and transformed net sodium and chloride absorption to secretion. These stress effects were inhibited by intravenous atropine infusion, suggesting the involvement of cholinergic parasympathetic nerves. By extending this model, Santos and colleagues (65) found that jejunal water secretion induced by cold pain stress was accompanied by luminal release of the mast cell mediators, tryptase and histamine. These in vivo findings suggest that the central nervous system (CNS), mast cells, and the intestinal mucosa interact during stress in humans. Studies of human intestinal mucosa in Ussing chambers also support a role for neuroimmune interactions in the regulation of mucosal function. In noninflamed segments of surgically resected human bowel, anti-IgE stimulation of mast cells induced a secretory response that was inhibited by histamine receptor antagonists, cyclooxygenase inhibitors, and the neurotoxin, tetrodotoxin (66,67), suggesting that activation of mast cells releases mediators that stimulate intestinal ion transport directly through epithelial action and indirectly via nerves. Other neurotransmitters with the potential to mediate stress responses in human intestine are NO, substance P, and neurotensin (68–70). These studies provide important evidence that neuroimmune regulation of intestinal

ion transport occurs in humans. In contrast, no currently published studies have specifically examined the effect of stress on mucosal permeability in humans. Therefore, the mechanism by which psychological stress affects mucosal barrier function in human intestine remains to be elucidated (71,72). Preliminary data, however, show that regulation of barrier function in the human colon also involves CRH, cholinergic nerves, and mast cells (Wallon, Perdue, and Söderholm, unpublished results).

STRESS-INDUCED CHANGES IN MUCOSAL FUNCTION IN ANIMALS Animal Models As reviewed earlier, a large body of evidence proposes that severe physical stress (e.g., trauma, burns, and also major surgery) can cause gastrointestinal dysfunction and pathology (i.e., stress ulcers, bacterial translocation, and multiple organ dysfunction). In contrast, exposure to daily life stress is more relevant for general health, and epidemiologic studies have shown a link between chronic stress and a more severe course of IBS and IBD (47,73,74). Several animal models have been developed to study the possibility that life (psychological) stress promotes intestinal disease and the mechanisms involved. Models include rodents or other animals exposed to restraint stress, water avoidance/aversion stress, swimming stress, crowding stress, among others. In addition, models of social defeat and maternal deprivation, which had been used in psychological research, have been introduced into intestinal research. It is important to recognize that environmental stress results from the transport and handling of animals, and such stress has been shown to affect mucosal function (75,76). Well-controlled studies require that the animals are handled daily for 1 to 2 weeks before the study protocol starts. A widely used model for studies of intestinal mucosal function in rats or mice is acute (i.e., a single exposure) restraint or immobilization stress, where the animal is immobilized for periods from 30 to 240 minutes in an adjustable restraint device or by wrapping the upper and lower limbs with tape. Variations include cold restraint stress (CRS), where the restrained animal is placed in a cold room (usually 8°C), or water immersion restraint stress, where the restrained animal is immersed vertically in 20°C water to the level of the xiphoid process. Another common model is the so-called water avoidance stress (WAS), where the animal is placed on a small platform surrounded by water at room temperature for 20 to 60 minutes. Because this model induces minimal physical stress, it is believed to be a good model of psychological stress. The latter models are based on the natural aversion of rodents to water. Rodents exposed to chronic mild stress are used in psychiatric research as models of depression. Such psychological models have been used in studies of intestinal function. In chronic models, animals are exposed to stress over time

EFFECT OF STRESS ON INTESTINAL MUCOSAL FUNCTION / 769 (e.g., isolation, overcrowding, or repeated daily exposure to WAS). In addition, among environmental factors that modulate an individual’s response to stress, psychological trauma in early life is of special importance. Maternal deprivation/ separation is a commonly used animal model of neonatal stress: Rat pups are separated from the dam for a period of 2 to 4 hours daily during the first 2 to 3 weeks of life. These models have been used in studies of gut pathophysiology (77–79).

Effects of Acute Stress Ion Secretion Secretion of ions and water in response to bacteria and noxious substances is one aspect of epithelial host defense. In vivo techniques for assessing secretion involve measurements of net water flux in intestinal loops. As mentioned earlier, experimental acute stress in humans stimulates water secretion in the jejunum (63–65), and a similar secretory response was found in the ileum and colon of rats exposed to restraint stress (80). Ion secretion can also be studied in Ussing chambers by monitoring increases in short-circuit current (Isc), as an indication of active, luminally directed anion secretion. Ussing chambers also are used to examine changes in bidirectional fluxes of radioactive isotopes (e.g., chloride and sodium), to determine the ionic driving forces for the water movement. An alternative approach involves identifying the effect of exclusion of a specific ion in the circulating buffer. In rat studies, an increased baseline Isc was found in isolated jejunal segments after acute restraint stress (81). Substitution of Cl− in the buffers eliminated the abnormality, suggesting that net Cl− secretion was provoked by stress. In contrast, a reduced Isc response to electrical transmural nerve stimulation was found in small intestinal tissues from stressed rats, suggesting an impaired responsiveness of the enteric nerves or epithelial cells. However, the epithelial response to bethanechol or VIP was unimpaired, implicating stress-induced nerve changes, for example, depletion of neurotransmitters. These findings were later verified and shown to involve cholinergic pathways (82). Similar findings regarding Isc were also found in the rat colon (83,84). Thus, acute stress in rats increases baseline chloride secretion in the jejunum and colon by a mechanism involving cholinergic nerve stimulation, which implies that stress induces increased baseline activity of enteric neurons. Mucous Secretion Castagliuolo and colleagues (85,86) demonstrated the effects of restraint stress on colonic mucin secretion in a series of experiments. Thirty minutes of immobilization stress caused a significant increase in mucin release from colonic mucosal explants and goblet cell depletion by histologic evaluation, which was coupled with increased colonic mucosal levels of the mast cell mediators, rat mast cell protease II (RMCPII),

prostaglandin E2, and cyclooxygenase-2 messenger RNA (mRNA). The findings of stress-activated mucin release have been corroborated by studies documenting goblet cell activation by environmental stress in rats (76). In addition, a study in rats showed altered colonic mucin glycoprotein and mucus-containing goblet cells after acute stress (87). Barrier Function In an early report of stress-induced permeability changes, Saunders and colleagues (81) showed that rats subjected to acute restraint stress had overt barrier dysfunction despite normal light microscopic results. In this study, jejunal electrical conductance and permeability to the inert marker molecules mannitol and 51chromium-ethylene-diamine tetraacetic acid (51Cr-EDTA) were increased significantly. Subsequent experiments showed enhanced gut permeability in stressed Wistar–Kyoto rats (with low cholinesterase activity) compared with the parent Wistar strain (82), and the possible involvement of cholinergic nerves was further emphasized by atropine inhibiting the stress response. To determine if the stress-induced epithelial barrier defect extended to macromolecules with antigenic potential, Kiliaan and colleagues (88) studied transport of a model protein, horseradish peroxidase (HRP; molecular weight, 45 kDa), in isolated jejunal segments. HRP was used both for flux studies (by assessing enzymatic activity), and to determine passage routes because its reaction product can be visualized by electron microscopy. Wistar–Kyoto rats exposed to CRS for 2 hours showed increased permeability to HRP and to 51Cr-EDTA. Electron microscopy showed an increased size and number of HRPcontaining endosomes in the enterocytes, as well as HRP within the tight junctions in the jejunum of stressed rats. Stress-induced increases in colonic permeability also have been reported (83,84,87). Santos and Saunders and colleagues showed that isolated colonic segments from Wistar–Kyoto rats exposed to acute stress had increased conductance, as well as increased permeability to HRP and the bacterial chemotactic peptide f-MLP (83). These experiments implicated a role for CRH in the colonic epithelial pathophysiology. Pfeiffer and colleagues (87) demonstrated increased paracellular permeability induced by CRS, which involved cholinergic nerves and mast cells, whereas glucocorticoids were of minor importance. With in vivo techniques, Meddings and Swain (75) confirmed that acute stress reduces mucosal barrier function. Using both restraint stress (RS) and swimming with a floatation device as stress models, the authors identified signs of increased paracellular permeability throughout the gastrointestinal tract by assessing fractional absorption of sucrose, lactulose/mannitol, and sucralose, reflecting the gastroduodenum, small bowel, and colon, respectively. It has been demonstrated that acute stress also affects barrier function in the FAE of rats. One hour of WAS significantly increased transmucosal flux of the protein antigen, HRP, whereas paracellular permeability was less affected (89). Thus, compared with villous epithelium, antigen transport across the FAE appears to occur to a greater extent, both under normal and adverse conditions.

770 / CHAPTER 28 Adaptation to acute stress is also possible. Wistar–Kyoto rats exposed to continuous WAS for 60 minutes demonstrated significantly increased Isc, conductance, and HRP flux, whereas rats subjected to stress 3 × 20 minutes with a 5-minute rest (in their home cage) between each stress period showed rapid adaptation, having values similar to those in nonstressed control animals (90). Mechanisms Involved in the Effects of Acute Stress Studies of intestinal mucosal function in stressed rats have demonstrated the ability of various physical and psychological stressors to modulate small-bowel and colonic permeability, both by increasing transcellular endocytosis of macromolecules and by facilitating paracellular passage of ions and molecules of various sizes. Mucosal mast cells play a key role in the mechanism, possibly activated via neurons or other cells releasing CRH. Acetylcholine is a key neurotransmitter, although whether it is released in response to mast cells or CRH has yet to be clarified. Various other factors may affect the magnitude of the stress response. A simplified paradigm of the mechanisms thought to be involved in stress-related barrier dysfunction is shown in Figure 28-3. Acetylcholine Barclay and Turnberg (63) show that stress-induced secretion in the human jejunum was inhibited by intravenous atropine infusion, suggesting a cholinergic parasympathetic

nervous mechanism. Studies in rodents confirmed these findings: Jejunal chloride secretion after restraint stress was inhibited by pretreating the rats with atropine or atropine methyl nitrate (which does not cross the blood–brain barrier), but not with hexamethonium (82), with similar findings regarding changes in jejunal permeability to 51Cr-EDTA. In addition, the magnitude of the intestinal response to stress was inversely correlated with mucosal cholinesterase activity in two rat strains (response in Wistar–Kyoto > Wistar). The increase in transcellular endosomal uptake and flux of the macromolecule HRP also was inhibited by atropine (83,88). Taken together, these results suggest that acetylcholine is a mediator of stress-induced transport abnormalities in the jejunum and colon (i.e., chloride secretion) and increased permeability via muscarinic receptors located in the gastrointestinal tract. Castagliuolo and colleagues (91) also showed the importance of cholinergic mechanisms in the stress-induced intestinal response: Atropine inhibited colonic mucin and RMCPII release after restraint stress (82). Mast Cells Several studies from various groups have highlighted the importance of mast cells in stress-related changes in intestinal mucosal function. Mast cell involvement in mucosal stress changes were first observed by an increased release of RMCPII during acute restraint stress in rats (85,91). The stress-induced increase in mucin release was inhibited by pharmacologic stabilization of mast cells with lodoxamide (91)

Bacterial products

Antigens

Epithelial cells Intestinal mucosa Granulocyte

Mast T cell

B cell

cell

CRH

CRH Nerve fibers

ACh Modulators: Adrenergic nerves opioid pathways

FIG. 28-3. Schema of potential mechanisms involved in stress-induced effects on intestinal mucosal function. Stress triggers mucosal nerves to release corticotropin-releasing hormone (CRH) that activates mast cells. (CRH may also be derived from immune cells.) Other released neurotransmitters such as acetylcholine (ACh) may act directly or indirectly on the epithelium. Mast cell mediators (e.g., tumor necrosis factor-α and tryptase) disrupt barrier function to antigens and bacteria. These mediators and others stimulate epithelial ion secretion and mucous secretion. (Modified from Söderholm and Perdue [72], by permission.)

EFFECT OF STRESS ON INTESTINAL MUCOSAL FUNCTION / 771 or doxantrazole (83). Using intestinal perfusion in humans, Santos and colleagues (65) report that cold pain stress induced water secretion in the jejunum, combined with luminal release of the mast cell mediators tryptase and histamine. Moreover, studies of mucosal ultrastructure demonstrated mucosal mast cell activation in combination with various signs of barrier disturbance during stress (76,88,92). These results suggest that signals from the CNS have the ability to modulate intestinal mast cell activity. It has been shown that stress-induced activation of colonic mucosal mast cells involves central release of IL-1, and secondarily secretion of CRH from the hypothalamus (93). More compelling evidence for a role of mast cells in stress-induced mucosal dysfunction has been obtained by studies using mast cell–deficient (Wv/ Wv) mice and (Ws/Ws) rats. Castagliuolo and colleagues (86) report that RS-induced mucin and prostaglandin release in the colon did not occur in mast cell–deficient mice, in contrast with normal animals. However, mast cell–deficient mice that had their mast cells reconstituted by injection of precursors had the same colonic response to stress as normal mice. In a study of intestinal barrier function during chronic stress (5 days of WAS) in mast cell–deficient and normal rats, Santos and colleagues (94) showed that the stress-induced increase in jejunal Isc and permeability to macromolecules were absent in mast cell–deficient (Ws/Ws) rats. In addition, in +/+ rats subjected to 5-day stress, there was an increased number of activated mast cells in the jejunal mucosa, and the increase in macromolecular permeability lasted for at least 3 days after the end of the stress period. These studies were the first to provide direct evidence that intestinal barrier dysfunction after stress is dependent on mast cells, and they further underline the importance of mast cells in the regulation of intestinal transport. Mast cell–mediated regulation of barrier function in the small intestine and colon has subsequently been confirmed by other stress studies in mast cell–deficient rats (95,96). Several mast cell–released factors may mediate mucosal dysfunction. For example, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and IL-4 have well-recognized effects on intestinal barrier function (97–99), and proteases such as tryptase may affect permeability via activation of proteaseactivated receptors (PARs) (100,101). In a short report, Alptekin and colleagues (102) demonstrate increased lipid peroxidation and reduced glutathione levels in intestinal tissue after water immersion of rats, suggesting that exposure to reactive oxygen species might be another possible mechanism for increased epithelial permeability during stress. Corticotropin-Releasing Hormone Mast cells often are found close to neurons and are activated by neuropeptides, such as substance P and CRH. The mediators of intestinal mucosa mast cell activation during stress have not been fully elucidated. CRH has been implicated in various stress-induced abnormalities, including gastrointestinal abnormalities. In the human colon, CRH immunoreactivity and mRNA has been found in epithelial cells of

the crypts (103). These cells also were positive for 5-HT, and it was concluded that CRH probably is produced by enterochromaffin cells. Central, as well as peripheral, injections of CRH and urocortin-1 mimicked stress responses in the colon (104). Peripheral (intravenous or intraperitoneal) injection of CRH mimicked stress-induced changes in colonic function regarding mucin release (91), ion secretion, and permeability (paracellular and transcellular) (83,84). These effects were associated with RMCPII secretion and activation of mast cells and were inhibited by doxantrazole. Moreover, restraint stress–induced epithelial changes were inhibited by the CRH antagonist, α-helical CRH (83,84). There are two main subtypes of CRH receptors, CRF1 and CRF2. In the rat colon, the CRF1 receptor has been localized to colonic crypt stem cells and goblet cells, as well as in scattered cells of the surface epithelium (105). In addition, CRF1 receptor immunoreactivity was found in the myenteric and submucosal nerve plexuses of the colonic wall. The CRF2 receptor was less abundant and was mainly found close to the luminal surface of the crypts and in submucosal blood vessels. Selective CRF1 receptor antagonists were shown to inhibit stress effects on intestinal motor function, whereas blocking of the CRF2 receptor had no effect on motility (106). The relative importance of the specific CRH receptors in mucosal function is unclear. However, CRH injected intraperitoneally caused mucosal changes that were inhibited by the muscarinic antagonists, atropine and pirenzepine, the ganglion blocker, hexamethonium, and the adrenergic blocker, bretylium (83,87,91), implicating a role for intestinal nerves. In another study, Castagliuolo and colleagues (85) suggest the neuropeptide neurotensin as a candidate for transmitting the CRH-induced effects within the colonic wall. CRH-induced effects are not inhibited by blocking steroid synthesis (83,91,107) or adrenalectomy (87), suggesting that the HPA axis is not important in the CRH-mediated gut changes. Taken together, the findings in these studies suggest that CRH is important for stress-induced changes in colonic epithelial function, and that its effects are mediated by mucosal receptors, via enteric nerves, and involve activation of mast cells. Glucocorticoids In contrast with the findings discussed earlier, Meddings and Swain (75) report that glucocorticoids are important for the increase in the in vivo intestinal permeability seen in rats exposed to environmental stress of various degrees. Stressinduced permeability changes were absent in adrenalectomized and glucocorticoid antagonist–treated animals and were mimicked by dexamethasone. Increased permeability by high-dose dexamethasone also has been reported previously (108). Thus, the role for glucocorticoids in intestinal barrier function in relation to stress needs further evaluation. Opioids A role for opioids in the stress-induced increase in colonic permeability was found in a study of adaptation to acute

772 / CHAPTER 28 stress in Wistar–Kyoto rats (90). As shown previously, 60 minutes of WAS significantly increased ion secretion and tissue conductance, as well as macromolecular permeability. In contrast, when rats were allowed two 5-minute periods of rest during the 60 minutes, the secretory and permeability parameters were maintained at control levels. That is, stressinduced changes in colonic mucosal function were subject to adaptation. This adaptive response was inhibited by intraperitoneal injection of either naloxone or methylnaloxone, suggesting that the adaptive effects were mediated by peripheral opioid receptors. Another mechanism involved in mucosal adaptive responses could be the activation of the early immediate genes, c-fos and nerve growth factor (NGF)–induced gene (NGFI-A), which were shown to be induced in rat gastric and duodenal mucosa in response to the water immersion restraint stress (109).

Effects of Chronic Stress Long-term perceived stress in humans is more important than acutely stressful life events for the risk for exacerbation of ulcerative colitis (47,73). Similarly, two animal studies showed that subjecting rodents to stress reduced their threshold for induction of inflammation in hapten-induced colitis (107,110), corroborating clinical observations. Qiu and colleagues (110) have shown that chronic stress–induced reactivation of colitis in mice (that had recovered from hapteninduced colitis 8 weeks earlier) required CD4+ T cells and involved changes in mucosal barrier function (reduction of mucus and increased permeability of the colon). There are now many studies that have described changes in gut function in situations of chronic stress. Clearly, there are important consequences of such changes (see later for further discussion). Barrier Function and Host Defense Studies examining the effects of chronic stress on intestinal function used 1-hour sessions of WAS on 5 or 10 consecutive days. Stressed rats reduced their food intake and lost weight during a 5-day period of exposure to stress (94). Epithelial chloride secretion (Isc), ionic permeability (conductance), and macromolecular permeability (HRP flux) were increased in the jejunum and colon (95,96). Although mast cell–deficient (Ws/Ws) rats exposed to stress also lost weight, they were normal with respect to epithelial function. Moreover, epithelial cells in stressed control rats demonstrated mitochondrial swelling, whereas those in Ws/Ws rats were unchanged in this respect. The epithelial barrier defect and mitochondrial damage were associated with mucosal mast cell hyperplasia; a large proportion of mast cells was activated and in contact with depleted nerve varicosities (Fig. 28-4). A longer period of repetitive WAS stress (10 days) was shown to induce significant pathology in the intestinal mucosa (96). Mucosal barrier dysfunction in the ileum and colon was associated with ultrastructural changes in epithelial cells, namely

enlarged mitochondria and presence of autophagosomes (an early indication of apoptosis). In addition, commensal bacteria were identified adhering and penetrating into enterocytes (Fig. 28-5). Moreover, infiltration and activation of inflammatory cells and increased myeloperoxidase activity were found in the mucosa. In contrast, exposure of Ws/Ws rats to a similar stress protocol did not alter epithelial function, mucosal morphology or bacterial interactions with the gut surface, further highlighting the importance of mast cells in stress-induced mucosal changes. Although it was shown earlier that severe sleep deprivation in rats causes systemic invasion of enteric bacteria (111), Söderholm and colleagues (96) suggest in their study that relatively mild ongoing psychological stress also impairs epithelial defense to bacteria, thereby facilitating intestinal inflammation. Moreover, ultrastructural assessment of intestinal changes after environmental stress (76) (low vs high personnel activity in housing rooms) demonstrated pathologic changes both in epithelial cells and in the capillary endothelium. Additional studies in mice exposed to repeated mixed restraint and acoustic stress sessions showed that increased permeability of the colon, including bacterial translocation, was preceded by mucosal overexpression of IFN-γ (mRNA and protein) (112). The stress-induced permeability increase was mediated partly by myosin light chain kinase and was abolished in IFN-γ knockout and severe combined immunodeficiency mice. These findings substantiate the importance of neuroimmune interactions in chronic stress–induced mucosal changes. Chronic stress also affects intestinal mucous release and production. Reduced mucin contents and a decreased number of goblet cells were observed after repeated exposure to restraint (87) or water avoidance (96) stress. These changes involved cholinergic nerves and mast cells, whereas the adrenal gland was of less importance. The stress-induced decrease in mucin was associated with a greater number of adhering bacteria (96), and it enhanced susceptibility to hapten-induced colitis (87,110) and chemotherapy-induced epithelial damage (113). Velin and colleagues (89) report another aspect of stressinduced bacterial transepithelial transport. Uptake of Escherichia coli K-12, as studied by confocal microscopy and in vitro in Ussing chambers, was increased 30-fold across FAE in rats exposed to chronic stress compared with control rats. Moreover, the stress-induced increase in permeability to protein antigens was more pronounced in the FAE than in regular villous epithelium. Therefore, an increased antigen exposure in the Peyer’s patches might contribute to the initiation of proinflammatory responses in the intestinal mucosa in chronic stress. Further studies are needed to elucidate the mechanisms of stress-induced effects on FAE. Green and colleagues’ study (114) of porcine intestine in Ussing chambers provides evidence that bacterial uptake in FAE also is regulated by nerves. Intracellular internalization of pathogenic strains of Salmonella and E. coli in vivo was increased by mucosal exposure to norepinephrine and also by the neuronal conduction

EFFECT OF STRESS ON INTESTINAL MUCOSAL FUNCTION / 773

A

C

B FIG. 28-4. Transmission electron photomicrographs of mucosal mast cells in rat intestine. (A) A nonactivated mast cell (MC) in the mucosa of a nonstressed rat showing numerous homogeneous electron-dense granules. (B) An activated mucosal mast cell (MC) from a stressed rat. The mast cell shows so-called piecemeal-type degranulation, indicated by loss of intragranular density without fusion of intergranular membranes, retained oval granules in the cytoplasm, and absence of extruded granules contents or granule membranes. Piecemeal-type degranulation is the predominant pattern observed in activated mast cells in stressed animals. Frequently, the activated mast cells are in close proximity to emptied nerve terminals (N). (C) A mast cell (MC) in the mucosa of a stressed rat, showing enlarged granules with solubilization of intragranular contents and fusion of intergranular membranes indicative of a mixed anaphylactic-piecemeal degranulation pattern. Original magnification × 3000. (Reproduced from Santos and colleagues [95], by permission.)

blocker, saxitoxin, which strongly supports a neuronal component in the regulation of bacterial uptake in the FAE. Intestinal Flora Accumulating evidence exists that stress alters the microenvironment for resident bacteria and changes the conditions for bacteria–enterocyte contacts. Neonatal stress (maternal separation) in Rhesus monkeys decreased the number of Lactobacilli in the fecal flora and made the animals more susceptible to opportunistic bacterial infections (115). Mimicking stress with a high-dose dexamethasone (as also used in Meddings and Swain’s study [75]), Spitz and colleagues (108) report an impaired colonic IgA secretion, increased permeability to the bacterial chemoattractant f-MLP, and enhanced bacterial adherence to the mucosa. Gut-derived sepsis occurs when a microbe with particular virulence factors encounters certain conditions within the host; that is, it results from host–pathogen interactions that take place

within the gut lumen itself (116,117). For example, Wu and colleagues (118) show that in mice exposed to surgical stress (hepatic resection), expression of the barrier-disrupting virulence factor, PA-I lectin/adhesin, was increased in luminal Pseudomonas aeruginosa. Similar events, including increased invasiveness of commensal bacteria, occur in response to chronic psychological stress (96). Resident bacteria appear to gain the ability to take advantage of a weakened host. Such changes may involve catecholamine signaling from the stressed host, because norepinephrine and dopamine increased the adherence of E. coli to murine cecal mucosa in Ussing chambers (119). Moreover, the mucosal/epithelial response of the stressed host to commensal microbes might change, initiating nuclear factor-κB signaling and production of IL-8 (120). Some approaches to remedy this situation might be the use of probiotic bacteria to interfere with bacteria– epithelia interactions (121), or high-molecular-weight polyethylene glycols that can act as a surrogate mucin in a stressed (and thereby mucus-deprived) host (122).

774 / CHAPTER 28

A

B

C

FIG. 28-5. Effect of chronic stress on bacterial attachment and internalization into the epithelium. (A, B) Scanning electron photomicrographs of the luminal aspect of ileal enterocytes in rats. (A) In sections from nonstressed rats, the luminal bacteria were washed away during processing. (B) In many sections from stressed rats, luminal bacteria were attached to the epithelial cells and were not washed away. (C) Transmission electron photomicrograph showing the apical region of ileal enterocytes in a stressed rat. Two bacteria are adhering/internalizing into the enterocyte, with disappearance of microvilli and pronounced condensation of the epithelial cytoskeleton at the adherence sites. Scale bars = 10 µm (A, B); 1 µm (C). (Reproduced from Söderholm and colleagues [96], by permission.)

Effects of Early Life Stress Early life experience plays an important role in stress responsiveness throughout adult life (123). Neonatal stress in animals can result in increased release of CRH, altered expression of glucocorticoid receptors, as well as changes in the norepinephrine and γ-aminobutyric acid systems of the CNS (124,125). Moreover, it has been shown in humans that adverse early life events are associated with changes in the HPA axis and hyperresponsiveness to stress in later life (44). Coutinho and colleagues (77) report that rats exposed to neonatal maternal separation showed visceral hyperalgesia and increased colonic motility in response to stress at 2 months of age. In the first study of effects on mucosal function, Söderholm and colleagues (78) found that adult Sprague–Dawley rats (a nonstress-susceptible rat strain) that had been exposed to neonatal maternal deprivation demonstrated an increased baseline ion secretion and reacted to mild acute stress (30-minute WAS) with significantly increased conductance and transepithelial transport of macromolecules in colonic mucosa, whereas mild stress had minimal effects in control animals. The enhanced mucosal

susceptibility to stress involved peripherally located CRH receptors. Long-term vulnerability of the mucosal barrier in rats subjected to neonatal stress was verified in two studies by Barreau and colleagues (126,127). In 12-week-old Wistar rats, previous maternal separation caused increased colonic paracellular permeability in vivo, together with translocation of enteric bacteria to lymph nodes and increased myeloperoxidase activity in colonic mucosa. Moreover, an exaggerated immune response to intracolonic infusion of the hapten 2,4,6-trinitrobenzene sulfonic acid (TNBS) was demonstrated. In a follow-up study, it was shown that the neonatal stressinduced effects on colonic barrier were abolished by daily treatment with anti-NGF antibodies or mast cell stabilizers and were mimicked by injections of NGF in the neonatal period. From these three studies it can be concluded that neonatal stress in the form of maternal deprivation in rats induces long-term phenotypic changes in mucosal function by mechanisms that appear to involve increased release of NGF and CRH and also include activation of mast cells. From a study in Rhesus monkeys, it was suggested that maternal separation may also affect the intestinal flora; Bailey and Coe (115) show that separation around the time of weaning

EFFECT OF STRESS ON INTESTINAL MUCOSAL FUNCTION / 775 decreased the number of Lactobacilli in the feces and increased the susceptibility to opportunistic bacterial infections.

RELEVANCE FOR HUMAN INTESTINAL DISEASES Current evidence suggests that the stress-induced gut mucosal response is primarily mediated by neuroimmune interactions between the autonomic and enteric nervous systems and the intestinal immune system. As can be concluded from the studies reviewed in this chapter, chronic stress has a pronounced effect on host defense against luminal bacteria, has consequences for the development of intestinal inflammation in animal models, and increases the risk for exacerbation of intestinal diseases (1). In addition, many studies have identified that chronic stress is associated with a decrease in systemic immune function. Thus, from a clinical perspective, models of chronic stress are more relevant than models of acute stress. In contrast, some studies in humans indicate that acute stress may influence the symptoms of gastrointestinal disorders by altering motor function or visceral perception (128,129), or by inducing marked alterations in ion and water secretion (63–65). Following are some examples of the potential relevance of stress-induced mucosal pathophysiology with respect to human intestinal diseases.

Inflammatory Bowel Disease The current paradigm for the pathogenesis of IBDs, including Crohn’s disease and ulcerative colitis, is that IBD represents the outcome of the interaction between the predisposition of the host (due to genetic and environmental factors) and an inappropriate immune response to bacterial flora in the intestinal lumen. From the information presented on stressinduced gut mucosal pathophysiology, it is clear that chronic stress may be implicated in the development of disease. However, this is a controversial concept. Although many patients with IBD and their physicians are convinced that there is an association, the scientific evidence for an epidemiologic connection between IBD and stress is conflicting. Two recent well-designed studies have shown, however, that persistent stress and life events increase the risk for exacerbations of ulcerative colitis (47,73). It also has been suggested that a stress-susceptible patient subgroup may be identified from those individuals without the perinuclear antineutrophil cytoplasmic antibody (130). As the biological consequences resulting from psychological stress are being identified and the mechanisms are evolving, stress is becoming a more or less accepted risk factor in ulcerative colitis (131). An association between stress and Crohn’s disease also has been suggested, but the epidemiologic evidence is less clear (74). Structural and functional alterations of enteric nerves have been reported in both ulcerative colitis and Crohn’s disease (132). In addition, changes in CRH, mast cells, and intestinal barrier function have been identified in both stress models and IBD,

implying that these factors may transduce the impact of a stressful environment from the CNS to the gut mucosa. Kawahito and colleagues (103) have demonstrated production of CRH in enterochromaffin cells of normal human colonic epithelium. Subsequently, increased expression of CRH was identified in macrophages and epithelial cells in the colon of patients with ulcerative colitis (133), as well as in colitis in rats (134). However, Wlk and colleagues (135) have presented evidence that local production of CRH was proinflammatory, because CRH antagonists had an antiinflammatory effect in Clostridium difficile infection in mice. Intestinal inflammation also increased CRH production in the paraventricular nucleus of the hypothalamus, but there it appeared to have protective properties with blunting of inflammatory responses (107,136). Therefore, the role of CRH in IBD is unresolved. Compared with observations in control intestine, IBD mucosa demonstrates increased numbers and degranulation of mast cells (137), and increased levels of histamine are found in the gut lumen of patients with IBD (138,139). Crowe and colleagues (140) suggest that prior activation of mast cells may account for the reduced epithelial secretory response to anti-IgE found in IBD mucosa. Mast cells are also a major source of TNF-α in the intestine of Crohn’s disease (CD) patients (141,142). Mucosal barrier dysfunction is a feature of intestinal inflammation in humans (71), and it has been implicated as a pathogenic factor in IBD (143–145). The importance of TNF-α in the mucosal barrier disturbance has been highlighted in recent years. Lamina propria–derived monocytes/ macrophages from patients with Crohn’s disease spontaneously secreted TNF-α, inducing changes in baseline Isc and permeability when cocultured with T84 human colonic monolayers (146). Moreover, increased paracellular and transcellular permeability in Crohn’s disease mucosa was associated with enhanced expression of TNF-α (147–149). In addition, mast cell tryptase has been implicated as a mediator of disruption of the integrity of the intestinal barrier via activation of PAR2 receptors in the intestinal mucosa (100,101). It must be kept in mind, however, that although physical stressors (e.g., surgery and trauma) have been shown to increase intestinal permeability in humans, there are currently no published studies examining the influence of psychological stress on intestinal barrier function in humans. This is an important area for future research.

Food Allergy Patients with food allergies experience symptoms that may be mediated by mast cell activation, resulting in changes in ion secretion and barrier function (150,151). There is evidence that the increased intestinal permeability seen in people with food allergies (71,152,153) involves TNF-α (154). Studies of normal human intestine in Ussing chambers (66) showed that mast cell–induced chloride secretion was inhibited by the nerve conductivity blocker, tetrodotoxin. Santos and

776 / CHAPTER 28 colleagues (65) demonstrated that either cold pain stress or luminal antigen challenge in healthy volunteers or patients with food allergies, respectively, resulted in release of mast cell mediators (histamine, tryptase, and prostaglandin D2). These findings strongly suggest neural involvement in mucosal anaphylaxis in humans. Although effects of stress on the systemic immune response have been clearly demonstrated (155), it is unclear if or how stress modulates the mucosal immune response to oral antigens.

Irritable Bowel Syndrome IBS is a functional disorder characterized by abdominal pain and alterations in bowel habits in the absence of demonstrable pathology by endoscopy, radiology, histology, and so forth. In patients with IBS, a relation between stress and symptom severity is well described, together with abnormal responses to acute stress in the autonomic nervous system, the HPA axis, and visceral pain perception (see Mayer [1] for review). Exposure to stressful life events increases the risk for development of IBS after bacterial gastroenteritis (156). In this patient group, immune activation and increased permeability persisted several months after the infection was cleared (157). An increased number of intestinal mucosal mast cells have been demonstrated in patients with IBS (158,159), and this was related to changes in anorectal function (160) and abdominal pain (161). Fukudo and colleagues (162) report that patients with IBS also had increased colonic motility and ACTH responses to intravenous injection of CRH. Posserud and colleagues (129) have shown an exaggerated neuroendocrine response (greater CRH and ACTH concentrations in serum) after acute stress in these patients. It is important to determine if patients with IBS also exhibit exaggerated mucosal responses to stress.

CONCLUSION In conclusion, an extensive literature indicates that various types of psychological and physical stress have an impact on several components of intestinal mucosal function, mainly ion secretion, mucous secretion, and barrier function. With respect to the mucosal changes induced by acute stress, cholinergic nerves and mast cells play a crucial role, possibly activated by local release of CRH, whereas the magnitude of the stress response can be modulated by adrenergic and opioid pathways. The stress–response pattern in the mucosa of different individuals is determined by genetic factors (as shown by enhanced susceptibility in rat strains with decreased levels of cholinesterase and reduced susceptibility in mast cell–deficient rodents), and not less importantly by environmental factors. This has been demonstrated by the lifelong stress hyperresponsiveness in animals exposed to neonatal trauma. It has become evident that, compared with acute stress, models of chronic stress better represent exposure of humans

to daily life stress. Although some similar mechanisms are involved in both types of stress-induced mucosal barrier function, there seems to be a discrepancy between acute and chronic stress with respect to neuroimmune interactions. Although nerve–mast cell interactions dominate in acute stress, there is evidence that CD4+ T cells also are involved in chronic stress. Moreover, chronic stress has more pronounced effects on the defense against luminal bacteria. Consequently, chronic stress affects the course of experimental colitis and may even trigger mucosal inflammation by a mast cell–dependent breakdown of the barrier to the normal flora. Clinical studies have substantiated the postulate that chronic stress affects the course of inflammation, with an increased number of exacerbations in patients with IBD exposed to ongoing stress. What direction should this line of research take in the future? A key question is whether stress-induced intestinal permeability changes are relevant for human diseases. Various mucosal changes resulting from stress in rodents also are present in the human intestine and may be involved in intestinal diseases. However, although it is clear that physical stress (e.g., trauma and surgery) causes barrier dysfunction in patients, there is a paucity of information related to the impact of psychological stress on intestinal permeability in humans. Another challenge is to identify critical pathophysiologic targets that may lead to novel therapeutic modalities. More information related to stress-induced cellular changes in enterocytes is needed, including the signals involved in endocytotic uptake and tight junction permeability. Additional studies should further characterize mast cell products affecting enterocyte function, the cellular origin of CRH in the mucosa, and the critical receptor subtype. Most animals and humans encounter psychological stress of various degrees during life without developing overt gastrointestinal diseases. In rodents, the opioid pathway appears to be involved in defensive and adaptive mechanisms, but little is known about the pathways involved in human adaptation. There is a growing body of evidence that stress modulates intestinal mucosal function. However, when dealing with stress-related symptoms and disease flare-ups in the clinical situation, mucosal dysfunction, which may initiate or promote pathogenic immune reactions, or both, needs to be put in context with stress-induced motility changes and immune dysfunction. Much work is needed to define the role and understand the clinical relevance of each of these elements in the search for new therapies for stress-induced intestinal pathophysiology. Meanwhile, it should be remembered that stress-related changes in disease activity are not just in the patient’s mind, but in the mind-gut axis!

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CHAPTER

29

Effect of Stress on Gastrointestinal Motility Michèle Gué Effects of Stress on Gastrointestinal Motility, 782 Healthy Subjects, 782 Patients with Irritable Bowel Syndrome, 782 Experimental Animals, 783 Central Corticotropin-Releasing Factor Receptors and Stress-Related Gastrointestinal Motor Disturbances, 783 Stress Inhibits Gastric Motility via CorticotropinReleasing Factor 2 Receptor Activation, 784 Stress Stimulates Colonic Motility via Corticotropin-Releasing Factor 1 Receptors, 784

Autonomic Dysfunction, 784 Peripheral Corticotropin-Releasing Factor Receptors Involved in Stress-Induced Colonic Motor Alterations, 785 Effects of Stress on Visceral Perception, 785 Role of Corticotropin-Releasing Factor Receptors in Stress-Induced Visceral Hyperalgesia, 786 Sex Difference in Stress-Induced Visceral Hypersensitivity, 786 Conclusion, 786 References, 787

Alteration of gastrointestinal (GI) motor functions is an important part of the visceral responses to stress. Stress is widely believed to play a major role in functional GI disorders, especially in the onset and modulation of irritable bowel syndrome (IBS). IBS is the most common GI disorder characterized by altered bowel habits associated with abdominal pain or discomfort. This stress-related disorder involves dysregulation of the nervous system, altered GI motility, and increased visceral sensitivity. Inhibition of gastric emptying and stimulation of colonic motor functions are the commonly encountered patterns induced by stressors, both in animals and in humans. Activation of brain corticotropin-releasing factor (CRF) receptors mediates stress-induced inhibition of gastric motility and stimulation of colonic motor function through different CRF receptor subtypes. Convergent studies have established the role of the brain CRF1 receptors in the colonic motility, visceral hypersensitivity, and anxiogenic responses to stress, whereas CRF2 receptors may be important in reduction of gastric emptying and in dampening stress sensitivity.

Pioneer clinical observations pointed out the GI link to the brain as early as the beginning of the nineteenth century. Beaumont (1) observed in a fistulous subject that “fear, anger or whatever depresses or disturbs the nervous system” was correlated with suppression of gastric functions such as gastric secretion, delay in gastric emptying, and digestion. One century later, Cannon (2) and Selye (3) brought experimental evidence that the “fight or flight” response is associated with a reduced gastric acidity and motor activity and often is accompanied by gastric ulcers. Stress-induced alterations in intestinal motility have been recognized since 1902, when Cannon (4) noted changes in contour and flow of intestinal contents in cats confronted by a growling dog, and in 1934, Hall (5) established the use of a defecation score as an index of emotionality in rats exposed to unfamiliar surroundings. The development of quantitative techniques to measure GI motility in experimental animals and humans has documented well the stress-induced alterations of the GI motility. Many forms of physical or psychological stress might alter GI functions in humans and might contribute to various functional GI disorders such as IBS. IBS is a pathology defined as a chronic functional bowel disorder, where functional means “not explained by structural or biochemical abnormalities” characterized by continuous or remittent abdominal pain and is associated with altered bowel habits; diarrhea, constipation, or both; and bloating (6,7). IBS symptoms are multidetermined and are generated from

M. Gué: Department of Physiology, Université Paul Sabatier, INSERM U388, Laboratoire de Pharmacologie Moléculaire et Physiopathologie Rénale, Toulouse, France. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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782 / CHAPTER 29 dysregulation at various levels of the brain-gut axis. IBS is manifested by GI motor reactivity to various stimuli and low sensation and pain thresholds. Experimental animal models have contributed evidence that the central nervous system (CNS) response to stress modulates the autonomic nervous system outflow, activates the hypothalamic-pituitary-adrenal (HPA) axis, and alters pain modulatory mechanisms; these effects can be associated with changes in GI motility and visceral sensitivity. The link between IBS and psychosocial factors, particularly psychological factors, is evident based on (1) physiologic studies showing stress-induced changes in GI functions, (2) a temporal relation between life events and GI disorders, (3) epidemiologic studies, and (4) the relief of IBS symptoms by psychological interventions or drugs that influence emotional well-being (8). Despite the early recognition that various stressors as emotional, chemical, or physical stimuli can affect GI functions, it took more than 50 years to elucidate the neurohumoral mechanisms underlying such alterations. In 1981, Vale and coworkers (9) characterized the structure of the CRF as a novel 41-amino-acid hypothalamic peptide that stimulates the synthesis and release of adrenocorticotrophic hormone and β-endorphin from the pituitary gland (9) and established its major role in modulating responses to stress (10). Furthermore, the localization of CRF-like immunoreactivity in brain areas involved in the regulation of visceral (11) opened a new field of investigation to evaluate the involvement of neuronal CRF in stress-induced GI disorders. Since then, activation of CRF signaling pathways in the brain has been shown to reproduce the overall endocrine, autonomic, visceral, and behavioral responses to stress (12–15). For more two decades, CRF family peptides (9) have been recognized as modulators of gastrocolonic motility based on the use of nonselective members of the CRF peptide family. However, recent years have witnessed in rapid succession the identification of potent selective CRF1 and CRF2 receptor ligands. These developments (16,17) are useful to define the functional role of specific CRF receptor subtypes in the regulation of GI motor function and visceral sensitivity. This chapter reviews evidence for the involvement of stress-induced alterations in GI motility and visceral sensitivity in the genesis of IBS and the contribution of experimental animal models for the characterization of neurobiological mechanisms that are involved in stress-induced changes in GI functional disorders.

EFFECTS OF STRESS ON GASTROINTESTINAL MOTILITY Healthy Subjects Several lines of evidence suggest a modulatory or causative role of the CNS in the pathophysiology of IBS, and that symptoms of IBS are at least partially related to abnormal GI motility (18,19). Several studies using acute stress performed in healthy control subjects demonstrated that stress induces differing motor effects in the upper and lower

regions of GI tract. Among stress paradigms used (e.g., cold pain, labyrinthine stimulation, psychological stress), all result in inhibition of gastric emptying and the postprandial phasic motor activity in the gastric antrum (20–23), as well as reduction in the incidence and frequency of fasting migrating motor complexes (24,25). In contrast, acute physical stress (e.g., hand immersion in cold water) and psychological stress (e.g., dichotomous listening and mental arithmetic) stimulate contractile activity and propulsive colonic motor function in control subjects (26–29). In summary, experimental studies clearly demonstrate that acute or short-term stress applied to healthy control subjects induces inhibition of gastric emptying and stimulation of colonic transit. These observations led to the conclusion that these alterations might play a pathologic role in dyspeptic symptoms and alterations in stool frequency and consistency in patients with stress-related functional GI disorders. In agreement with this hypothesis, long-term studies in healthy volunteers showed that chronic stress causes symptoms consistent with the diagnosis of a functional GI disorder (30). Despite that studies on the effects of stress on GI motility in healthy volunteers need to be investigated further for a better understanding of the pathophysiologic mechanisms involved, some technical difficulties, as well as ethical considerations, have been limiting. Consequently, several experimental animal models have been developed to address the unanswered questions.

Patients with Irritable Bowel Syndrome Although stressful experiences produce GI symptoms in healthy volunteers, patients with IBS are particularly susceptible, with a greater reactivity to stress. Psychological stress is widely believed to play a major role in IBS by precipitating exacerbation of symptoms. Indeed, patients with IBS report more lifetime and daily stressful events, including severe abuse history, than medical comparison groups or healthy control subjects (18,31). In patients with IBS, stress scores are correlated with the number of disability days and clinical visits for bowel symptoms (8,32). It is clearly demonstrated in experimental studies that psychological and physical stress applied in both healthy volunteers and experimental animals induces GI motility disorders and, in turn, may cause abdominal symptoms and altered stool habits. About 25% to 75% of patients with IBS have disturbances in GI motility (7). Regarding gastric motility, anger stress used as a strong emotional stressor specifically inhibits antral motility in patients with IBS but not in healthy control subjects (33). In addition, studies in patients with functional dyspepsia also showed an inhibition of propulsive gastric motor activity in response to stress (34). Consequently, it can be suggested that stress-related inhibition of gastric motility might be one of the principal pathophysiologic mechanisms involved in the genesis of dyspeptic symptoms in the various functional GI disorders.

EFFECT OF STRESS ON GASTROINTESTINAL MOTILITY / 783 The literature almost unanimously reports an increased colonic motor response to various psychological (e.g., strobe test, fear, mental arithmetic) and physical (e.g., ice water and cold pressure tests) stressors, as determined by different parameters of colonic motor activity (e.g., spike and 2–4 cpm slow-wave activity, contractile and motor activity) in patients with IBS compared with healthy control subjects (27–29,35). In these studies, it is well demonstrated that stress induces a greater colonic motor response in patients with IBS than in control subjects, which is correlated with electroencephalographic power spectral changes, suggesting an exaggerated responsiveness to stress in these patients (35). Many studies have identified abnormal patterns of contractile activity in the colon of patients with IBS. Patients with diarrheapredominant IBS have a greater number of fast colonic contractions and propagated contractions with subsequent accelerated transit (36). Patients with abdominal pain have more “cluster” contractions, which are groups of brief propagated intestinal contractions of higher amplitude than in healthy control subjects (37). A model of IBS connected with enhanced stress responsiveness has been proposed based on animal experimental data (38,39).

greater sensitivity to the effects of stress on intestinal transit were observed in the late afternoon in rats (44). Interestingly, a short session of inescapable foot shocks induces long-term sensitization of the colonic contractile response to a novel stressor applied 2 weeks later (32). We can summarize that in experimental animals the most consistent pattern of GI motor alterations induced by various psychological and physical stressors is that of delaying gastric emptying and accelerating colonic motility and transit. However, studies on the modulation of the gut motor response to acute stress are relatively scarce, and the underlying mechanisms remain poorly characterized. In addition, all of these studies on stress-induced GI motility disturbances have been performed with a short-term stress, whereas long-term exposure to stressors have received little attention (45). All of these considerations pointed out the limitations of the animal studies of stress-related GI motor alterations. Nevertheless, the close parallels between the effects of acute stress on GI motility in humans and laboratory animals suggests that the central and peripheral pathways mediating the stress effects on GI motility delineated in experimental animals are similar in humans.

Experimental Animals

CENTRAL CORTICOTROPIN-RELEASING FACTOR RECEPTORS AND STRESS-RELATED GASTROINTESTINAL MOTOR DISTURBANCES

Acute stress applied to laboratory animals induced differential motor effects depending on the level of the GI tract. In fasted conscious dogs, acoustic stress produced by exposure to loud noise inhibits gastric migrating motor complexes without affecting intestinal motility (40). In fed animals (mice, rats, guinea pigs, dogs, monkeys), various stressors such as water avoidance, radiation, handling, acoustic stimulation, hemorrhage, abdominal or cranial surgery, tail shock, trunk clamping, wrap restraint, swimming, or anesthetic exposure results in a slowing of gastric emptying (41–43). Acute exposure to wrap restraint, cold water swim, or ether also slows down intestinal transit in rats (44). In accordance with the observations in humans, colonic transit, colonic motility, and defecation are stimulated by the various stressors used such as conditioned fear to inescapable foot shocks and water avoidance, restraint or open field stress, tail shock, and loud noise (43–47). Although the above gut motor changes represent the most characteristic patterns induced by various stressors, differential responses are also encountered after specific stimuli, for instance, acute exposure to cold, which is used to study gastric erosion formation induced by stress and activates both gastric and colonic motility and transit in rats (41,48). GI motor responsiveness to acute stress is affected by intrinsic factors such as the animal species, strain, sex, estrus cycles, circadian pattern, and previous stress experiences. For example, Fisher rats are more sensitive than Sprague– Dawley rats to acute wrap restraint-induced inhibition of small intestinal transit (44). Female rats are more sensitive than male rats to the intestinal inhibitory effect of chronic wrap restraint (44); furthermore, circadian variations with a

Activation of CRF receptors is a key signaling mechanism involved in the integration of behavioral, immune, autonomic, and visceral responses to stress including alterations of GI motor functions (12,14). In particular, CRF or urocortin (Ucn) injected centrally or peripherally inhibits the propulsive motor activity of the stomach, whereas stimulating that of the colon in rats, mice, and dogs, and mimics the effects of stress (46–49). Water avoidance and restraint stresses that are largely used as models of acute psychological/physical stress induce a rapid increase in CRF gene transcription in the parvocellular region of the paraventricular nucleus (PVN) of the hypothalamus, which is associated with activation of the HPA axis (50,51). The implication of the brain CRF signaling pathways in the stress-related GI motor activation has been studied intensively using CRF and CRF-related ligands, as well as receptor antagonists (14). In humans, intravenous injection of CRF increases colonic motility, and the contractile response is greater in patients with IBS than in healthy subjects (35). CRF and CRF-related peptides, Ucns, interact with two cloned G protein–coupled receptors, CRF1 and CRF2, that are expressed from distinct genes (52). Results from studies using CRF1 receptor knockout mice and selective CRF1 antagonists in rodents and monkeys established that CRF1 receptors located in the CNS are involved in centrally injected CRF and stress-related stimulation of colonic motor function (46,47).

784 / CHAPTER 29 Stress Inhibits Gastric Motility via Corticotropin-Releasing Factor 2 Receptor Activation Stressors inhibit gastric motor function in a manner that is reversible by central pretreatment with nonselective CRF receptor antagonists (47,48). Furthermore, central injection of nonselective receptor agonists delays gastric emptying (47). Through vagal mechanisms, central infusion of nonselective CRF receptors has been shown to reduce antral gastric motility, to inhibit high-amplitude contractions, and to shift duodenal activity from fasted to fed motor pattern (47,53,54). Such CRF- and Ucn1-induced gastric inhibition was reversed by nonselective CRF receptor antagonists but not by a selective CRF1 receptor antagonist, NBI-12914. These results suggest that brain CRF2 receptors mediate stress-induced inhibition of gastric motor function. In addition to the central CRF2 receptor activation for stress-induced gastric motility inhibition, peripheral CRF2 receptor activation delays gastric emptying. In that way, intravenous or intraperitoneal administration of agonists with high or selective CRF2 receptor affinity has been shown to delay gastric emptying more potently than does CRF (47,48,55). Conversely, peripheral pretreatment with specific CRF2, but not CRF1, antagonists blocks the Ucn-induced gastric motor inhibition (47,48,56). Similar to its central injection, peripheral administration of Ucn has been shown to reduce antral gastric motility in fed rats and shifts gastric patterns from a fasted to a fed state (53). In addition, peripheral administration of selective CRF2, but not CRF1, receptor antagonists attenuates stressinduced gastric motor inhibition. These observations suggest that stress releases CRF-like peptides that inhibit gastric motility through brain and gut CRF2 receptors; putative nervous structures include centrally the PVN and dorsal vagal complex via an undefined subset of its descending autonomic efferents (56,57), as well as the enteric nervous system (53).

receptor antagonist, α-helical CRF9-41, D-Phe12 CRF12-41, or astressin in rats or mice (44,58,59). Objective evidence indicates that the CRF1 receptor subtype is involved in the colonic motor response to central CRF. The icv injection of CRF1 receptor–specific agonists, ovine CRF and rat/human CRF, as well as the CRF1/CRF2 receptor agonist Ucn1, increased colonic propulsive activity, whereas the receptor agonists, Ucn2 and Ucn3, injected icv had no effect on colonic motility in mice (52,62). Furthermore, the icv injection of selective CRF1 receptor antagonists, NBI-35965 or NBI27914, inhibited icv CRF-induced stimulation of colonic transit and defecation, whereas icv astressin2-B had no effect (59,62). Studies with CRF1 receptor knockout mice and selective CRF1 receptor antagonists confirmed the importance of brain CRF1 signaling pathways in the colonic motor response to various stressors. α-Helical CRF9-41, D-Phe12 CRF12-41, and astressin injected icv into the PVN or LC/ Barrington nucleus complex inhibited the stimulation of colonic transit. Moreover, selective CRF1 receptor antagonists such as CP-154,526, NBI-35965, NBI-27914, and antalarmin injected icv or peripherally blunted the acceleration of colonic transit induced by restraint and defecation induced by water avoidance stress (WAS) restraint, or social stress (63). In mice, the icv injection of the CRF1 antagonist, NBI-35965, abolished the defecatory response to restraint stress, whereas the CRF2 receptor antagonist, astressin2-B, had no effect (62). CRF1 receptors in the PVN are up-regulated by central CRF and acute stress (64,65), and CRF1 receptor antagonists blocked LC activation induced by icv CRF or stress (66). All of these studies provide evidence for CRF1 receptor–mediated action at these brain structures to stimulate colonic motility. The available data strongly suggest that hypersecretion of cerebral CRF could be involved in the pathophysiology of the stress-related exacerbations of symptoms in IBS and other functional GI disorders. Consequently, CRF1 receptor antagonists might be potential future therapy in IBS, particularly in patients with anxiety, depression, and chronic stress (67) (Fig. 29-1).

Stress Stimulates Colonic Motility via Corticotropin-Releasing Factor 1 Receptors

AUTONOMIC DYSFUNCTION

The implication of the brain CRF signaling pathways in stress-related colonic motor activation has been studied intensively using CRF and CRF-related ligands, as well as receptor antagonists (14). CRF or Ucn1 administered intracerebroventricularly (icv) mimics the colonic response to stress as shown by the increased colonic motility, decreased colonic transit time, and induction of defecation in rats, mice, and Mongolian gerbils (14,44,58,59). The colonic response to icv CRF is centrally mediated (58). Brain structures involved in the CRF response have been localized in specific hypothalamic (PVN) and pontine areas (locus ceruleus [LC], sub-LC/ Barrington nucleus complex), which are also sites involved in CRF-induced behaviors that are symptomatic of anxiety and depression (60,61). The central action of CRF is receptor mediated as shown by blockade of icv CRF-induced colonic motor activation by the nonspecific CRF1/CRF2

Central CRF and stress stimulate colonic motor function through the autonomic nervous system. Convergent studies show that central CRF-induced sustained stimulation of colonic motor activity is mediated neurally through the activation of parasympathetic cholinergic pathways and occurs independently of the activation of the HPA axis in rats. Transneuronal labeling showed that the PVN and LC/sub-LC/ Barrington complex send direct projections to the intermediolateral column at the S1 segment of the rat sacral spinal cord that provides the parasympathetic innervation of the colon (68,69). Pharmacologic and surgical treatment showed that the CRF-induced colonic motor stimulation was unchanged after hypophysectomy, adrenalectomy, and noradrenergic blockade but reversed by vagotomy, ganglionic blockade, and atropine (58,70–72). Similarly, restraint stress–induced colonic motor stimulation involved the same pathways as

EFFECT OF STRESS ON GASTROINTESTINAL MOTILITY / 785 Nevertheless, several experimental data provide experimental evidence that both central and peripheral receptors are involved in the stress-induced colonic alterations (14,79).

Stress

CRF

Urocortin 2 Urocortin 3

Urocortin 1

CRF1

Colonic motility Defecation Visceral pain IBS

CRF2

Gastric motility Gastric emptying Visceral pain

FIG. 29-1. Schematic representation of roles of corticotropinreleasing factor (CRF) receptors and their endogenous ligands in gastrointestinal (GI) responses to stress. CRF1 receptors are thought to activate GI motility and visceral pain, and then to be involved in symptoms of irritable bowel syndrome (IBS). CRF2 receptors are thought to delay gastric emptying, inhibit gastric motility, and modulate visceral pain.

those involved with icv CRF injection. The colonic responses to restraint stress were not changed by hypophysectomy, adrenalectomy, naloxone, indomethacin, or bretylium, but they were abolished by vagotomy, ganglionic blockade, and atropine (58,73,74). In summary, peripheral autonomic pathways convey acute stress and stimulation of colonic motor function, whereas associated activation of the pituitaryadrenal axis does not play a role.

PERIPHERAL CORTICOTROPIN-RELEASING FACTOR RECEPTORS INVOLVED IN STRESS-INDUCED COLONIC MOTOR ALTERATIONS Activation of colonic signaling pathways is implicated in the colonic responses to acute stress in laboratory animals. CRF or Ucn injected peripherally inhibits the propulsive motor activity of the stomach, whereas stimulating that of the colon in rat, mice, and dogs mimics the effects of stress (46,49,75,76). Restraint stress–induced increase of colonic motility and stimulation of colonic transit were prevented or blunted by peripheral injection of α-helical CRF9-41 or astressin, but were not affected by the injection of selective CRF2 antagonists (74,76,77). Several CRF1 antagonists administered peripherally also inhibit the stress-related increase in colonic motility. Furthermore, in humans, intravenous injection of CRF increases colonic motility, and the contractile response is greater in patients with IBS than in healthy subjects (78). The mechanisms whereby stress recruits the peripheral pool of CRF to activate CRF1 receptors in stress-related colonic motor responses remain unknown.

EFFECTS OF STRESS ON VISCERAL PERCEPTION In addition to altered bowel movements, IBS is also characterized by abdominal pain (80). Evidence suggestive of alterations in stress-induced modulation of viscerosomatic sensitivity comes from human and animal studies. Such observations have led to the hypothesis that an excessive colonic response or “hypersensitivity” to stress is as important as motor changes in the cause of functional GI disorders (81). Acute stress is known to be associated with heightened sensitivity in the upper and lower gut (82,83). Furthermore, it has long been recognized that patients with IBS are hypersensitive to colonic distension (84); in contrast, patients with IBS have normal or even increased tolerance to somatic stimuli. The use of psychological laboratory stressors in healthy volunteers suggests a stress-induced increase in colonic or rectosigmoid sensitivity to distension (85), and another study supports the hypothesis of stress-induced altered modulation of visceral perception in patients with IBS (82). Hypotheses for the cause of IBS have ranged widely from the original view that the disorder reflects a primary disturbance of the gut mucosa to an emerging conception of the syndrome as emanating from complex disordered interactions between the digestive tract and its innervations (86,87). Several studies strongly support a modulatory or causative role of the CNS in the pathophysiology of IBS. Murray and colleagues (88) report that an acute stress alters gut-efferent autonomic innervation in both control subjects and patients with IBS, with a significant delay in normalization for IBS. In addition, only patients with IBS show heightened visceral sensation, suggestive of involvement of a different regulatory mechanism, either central, peripheral, or both (88). Furthermore, a majority of patients associate stressful life events with initiation or exacerbation of their symptoms (89); emotional state caused by stress or a psychiatric disorder such as anxiety or depression modifies intestinal reactivity (90). Behavioral studies, including relaxation (91) and hypnosis (92), and low-dose tricyclic antidepressant have ameliorated symptoms in patients with IBS (93). Symptomatic improvement in patients with IBS after hypnotherapy may be partly because of changes in visceral sensitivity (94,95). In animal studies, an increased frequency of abdominal wall contractions is an established pseudo-affective parameter to semiquantitatively determine visceral pain (96). In a previous study, we demonstrated that a partial restraint stress applied 2 hours before colorectal distension induced an increase in abdominal cramps, suggesting that stress sensitizes the animals to the distension stimulus by inducing visceral hypersensitivity (97). In that study, we also determined the effect of stressor on somatic pain tolerance, as determined by the tail-flick paradigm. It was reported that stressed rats show a delay in the tail-flick response up to 45 minutes after the end of the

786 / CHAPTER 29 stress, an observation that agrees with the well-described phenomenon of stress-induced hypoalgesia (97). Taken together, the results show that, in rats, stress induces visceral hypersensitivity associated with somatic hyposensitivity.

ROLE OF CORTICOTROPIN-RELEASING FACTOR RECEPTORS IN STRESS-INDUCED VISCERAL HYPERALGESIA Some data suggest that CRF acting at both central and peripheral levels of the nervous system might be implicated in stress-related visceral hypersensitivity. Indeed, strains of rats with high anxiety levels displayed enhanced visceral sensitivity to colorectal distension (98,99). Furthermore, acute and chronic stress enhances visceral sensitivity to colorectal distension in rats (99,100). As an initial report (97), we reported that the stress-induced visceral hyperalgesia was blocked by icv injection of the nonselective CRF antagonist, α-helical CRF9-41. In addition, CRF, injected icv, dose dependently mimicked the stress-induced enhancement of abdominal contractions in response to rectal distension, suggesting that central CRF plays a physiologic role in the stress-related visceral hypersensitization. In accordance with this work, Million and colleagues (101) report that adults rats, previously submitted to neonatal maternal separation, are hypersensitive to colorectal distention after a water avoidance stress session. Now, neonatal maternal deprivation is known to increase brain CRF levels in adult rats (102,103), and peripheral injection of a selective CRF1 antagonist, NBI-35965, that crosses the blood–brain barrier prevented water avoidance stress-induced hyperalgesia to colorectal distention in adults that had been neonatally stressed (101). Likewise, antalarmin injected intraperitoneally prevented both the icv CRF-induced hypersensitivity to colorectal distention and the visceral hypersensitivity observed in a high-anxiety strain of rats (104). In humans, it has been reported that stress increased colonic sensations during short-term colonic distension (38,105). Peripheral injection of ovine CRF, a preferential CRF1 receptor agonist mimicked the effect of stress by lowering the stooling threshold and the sensation of discomfort to distention of the colon (106). Furthermore, a clinical investigation reported that α-helical CRF9-41 administered intravenously reduced the higher symptoms (i.e., motility and visceral perception) in patients with IBS subjected to electrical stimulation compared with healthy subjects (107). These findings are consistent with a hyperalgesic effect of CRF mediated through CRF1 receptors. In contrast, recent studies indicate that the activation of peripheral CRF2 receptors presents an antinociceptive modulatory effect. Indeed, intravenous injection of humanUcn2 (h-Ucn2) reduced visceral hyperalgesia during colorectal distension (108,109), and administration of h-Ucn2 induced an antinociceptive effect in a model of duodenal distension-induced visceral pain in rats (110). These effects of h-Ucn2 were blocked by peripheral administration of CRF2 antagonists, astressin2-B and antisauvagine-30 (108,110), and strongly support the hypothesis that Ucn2 exerts

antinociceptive effects through the activation of peripheral CRF2 receptors (111). Consequently, it is suggested that stress-induced endogenous release of CRF or Ucn might modulate colonic motility and visceral pain through a simultaneous action on CRF1 and CRF2 receptors, depending on the peptides released and their relative affinity for CRF receptors (see Fig. 29-1).

SEX DIFFERENCE IN STRESS-INDUCED VISCERAL HYPERSENSITIVITY Sex appears to play an important role in IBS. Among patients who report symptoms of IBS, there is a female preponderance, which some reviews have reported to be as high as 80% (18,112,113). The severity of abdominal pain in women with IBS has been reported to vary with stress and ovarian hormone status, and these women commonly report worsening symptoms in relation to the menstrual cycle (114, 115). Furthermore, pharmacologic ovariectomy of female patients with IBS by administration of the long-acting, gonadotropin-releasing hormone analogue, leuprolide acetate, is effective in controlling the symptoms of abdominal pain (116,117). This clinical observation suggests that female individuals might be more predisposed to the mechanism(s) that causes this syndrome than male individuals. Despite the attention to female patients with IBS, sex differences in gut sensitivity have not been explored in animals, because all current studies on the effect of stress on colonic motility or sensitivity in rats have been performed in male animals (100). One study (118) shows that female rats are more sensitive than male rats because they present a greater number of abdominal cramps for the same volume of rectal distension, and restraint stress enhances this sex difference. Indeed, after stress, female rats exhibit an exacerbated visceral response for a small volume of rectal distension, suggesting that stress induces a visceral allodynia in female rats (Fig. 29-2). Ovariectomy significantly decreased the response of abdominal cramps to rectal distension both in no-stressed and in stressed female rats, suggesting that sexual hormones are involved in such responses (118). Treatment of ovariectomized female rats with gonadal steroid hormones, especially with progesterone, modulates the visceral sensitivity and restore the number of abdominal cramps compared with intact female rats, whereas they have no effect on stress-related visceral hypersensitivity (118). Taken together, the results of the animal and human studies indicate that evidence of a physiologic component is based on sex differences in visceral sensitivity, autonomic nervous system, stress reactivity, and specific effect of estrogen and progesterone on gut function (113). However, the link between these components and GI motility and sensitivity needs to be clarified.

CONCLUSION The connections between the brain and the gut are among the deepest and most phylogenetically ancient of all neural

EFFECT OF STRESS ON GASTROINTESTINAL MOTILITY / 787 45

45

Males

Abdominal contractions/5 min

40 35

Control (sham)

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30 25

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20

15

15

10

10

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0 0.4

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FIG. 29-2. Sex differences in partial restraint stress (PRS)–induced rectal hypersensitivity to distension in rats. Response of the abdominal striated muscle, expressed in number of contractions per 5 minutes, to increasing volume of rectal distension, after sham stress (control) or PRS. Note that female rats are more responsive than male rats to distension both after sham PRS or PRS session. Asterisks indicate significantly different (p < 0.05) from corresponding sham PRS values; ‡ significantly different (p < 0.05) from corresponding male values. Dunnett’s test for multiple comparisons after analysis of variance.

control systems. The design of experiments to study this system was an intellectual and technical challenge. However, recent studies have demonstrated a major role for the brain in the control of gut motility and visceral perception, not only in the physiologic state, but also during pathophysiologic situations such as stress and functional gut disorders. The most consistent pattern of GI motility induced by various stressors is that of delaying gastric emptying and stimulation of colonic motor function, which is mimicked by central administration of CRF. Findings related to the differential CRF receptor subtypes indicate that the inhibition of gastric emptying by CRF is mediated by interaction with CRF2 receptors, whereas CRF1 receptors are involved in the colonic and anxiogenic responses to stress. Furthermore, findings indicate that CRF1 receptors also are implicated in stressrelated visceral hypersensitivity in rodents and humans. Newly developed, selective, peripherally acting CRF1 receptor antagonists are expected to become useful tools to delineate the respective involvement of central and peripheral CRF1 receptors in the colonic motor and hypersensitive responses to stress; finally, they may provide insight into their role in functional bowel diseases and as a potential therapeutic target for the treatment of IBS.

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CHAPTER

30

Central Corticotropin-Releasing Factor and the Hypothalamic-Pituitary-Adrenal Axis in Gastrointestinal Physiology Yvette Taché and Mulugeta Million Historical Considerations, 791 Chemistry of the Hypothalamic-Pituitary-Adrenal Axis, 792 Corticotropin-Releasing Factor–Related Peptides, 792 Corticotropin-Releasing Factor Receptors, 793 Corticotropin-Releasing Factor Receptor Antagonists, 794 Corticotropin-Releasing Factor Binding Protein, 795 Brain Distribution of Corticotropin-Releasing Factor Ligands and Receptors, 795 Brain and Spinal Corticotropin-Releasing Factor and Urocortin Expression, 796 Brain Corticotropin-Releasing Factor Receptor Expression, 796 Central Action of Corticotropin-Releasing Factor Peptides to Influence Gastric Function, 797 Inhibition of Gastric Acid Secretion, 797 Stimulation of Gastric Bicarbonate Secretion, 798

Inhibition of Gastric Emptying, 798 Alteration of Gastric Motility, 800 Central Actions of Corticotropin-Releasing Factor Peptides to Influence Small Intestinal Function, 802 Stimulation of Duodenal Bicarbonate Secretion, 802 Alterations of Jejunoileal Absorption, 803 Slowing of Small Intestinal Transit, 803 Alterations of Small Intestinal Motility, 804 Anti-inflammatory Action, 804 Central Actions of Corticotropin-Releasing Factor Peptides to Influence Colonic Function, 804 Stimulation of Colonic Propulsive Motor Function, 804 Increased Colonic Permeability, 807 Inflammation, 807 Colonic Hypersensitivity, 808 Conclusion, 809 Acknowledgments, 809 References, 809

HISTORICAL CONSIDERATIONS

the role of the pituitary gland in the adrenal hypertrophic response to various stressful stimuli (2) (Fig. 30-1). Thereafter, the concept of hypothalamic neurohumoral control of the pituitary secretions came from the seminal contributions of Geoffrey Harris (3,4), who in the 1950s stated that stressinduced adrenocorticotropin (ACTH) secretion is “under neural control via the hypothalamus and the hypophyseal portal vessels of the pituitary stalk.” However, it was not until 1955 that the concept was validated simultaneously and independently by Guillemin and Rosenberg (5) and Saffran and Schally (6). They demonstrated the existence of hypothalamic factor(s) that elicited ACTH release from the pituitary in intact rats, which was named corticotropinreleasing factor (CRF) in line with its ability to stimulate ACTH release and in keeping with that its molecular structure was not yet characterized (5,6). Although CRF was one

Hans Selye pioneered the concept of biological stress, borrowing the term from physics where stress stands for the interaction between a deforming force and the resistance to it. His initial reports in 1936 positioned the adrenal cortex and the gut at the center of the stress syndrome (1) and established Y. Taché and M. Million: CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, Los Angeles, California 90073. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

791

792 / CHAPTER 30

FIG. 30-1. Dr. Hans Selye (University of Montreal, Montreal, Quebec, Canada) lecturing on hypothalamic-pituitary-adrenal (HPA) axis.

of the first hypothalamic-releasing factors to be named, the elucidation of its structure lingered for nearly three decades. However, its search paved the way for the isolation and structural identification of several other hypothalamic-releasing factors in the 1970s (7). In 1981, Vale and colleagues (8) succeeded in purifying and isolating CRF from ovine hypothalami as a 41-amino-acid (aa) peptide. Since then, considerable advances have been made in understanding mechanisms regulating the hypothalamic-pituitary-adrenal (HPA) axis under basal and stress conditions (9). With the previous recognition that circulating levels of ACTH and glucocorticoids are increased by various physical (internal or external) or perceived perturbations representing a threat to homeostasis, the HPA axis became de facto the endocrine hallmark of the adaptive response to stress (10) and positioned CRF as the prime hypothalamic mediator (8). Within the hypothalamus, a discrete population of hypophysiotrophic neurons in the parvocellular portion of the paraventricular nucleus (pPVN) synthesizes CRF and coexpresses arginine vasopressin (AVP). These peptides are released into the hypophyseal-portal circulation from axon terminals located in the Zona externa of the median eminence. CRF binds to specific CRF receptor subtype 1 (CRF1) receptors located on membranes in the anterior pituitary corticotrope cells to stimulate ACTH secretion, whereas AVP interacts with V1b pituitary receptors to potentiate the ACTH release (11,12). Studies using CRF1−/− and V1b−/− receptor mice clearly showed that under stress conditions, both the hypothalamic CRF/CRF1 and AVP/V1b systems are required and play a crucial role in regulating the pituitary ACTH response (12,13). The CRF-CRF1–mediated increase in circulating

levels of ACTH stimulates the release and synthesis of corticosteroid hormones (corticosterone in rats, cortisol in humans) from the adrenal cortex (11), which completes the HPA activation occurring during stress. The physiologic importance of CRF expanded quickly, far beyond the classical action of mounting the ACTH response to various stressors. Experimental studies established that CRF injected into the brain had stress-inducing properties generating the overall behavioral, autonomic, immune, and visceral responses that were largely independent from the pituitary-adrenal stimulation (14–19). This provided the framework for CRF to fulfill the insightful concept advanced by Selye in the 1950s regarding the existence of a “first mediator” that triggers the adaptive bodily response to stress (10). Of relevance to the gastrointestinal physiology were early reports establishing that CRF acts in the brain and the periphery to inhibit gastric emptying and acid secretion, whereas stimulating colonic motor function in rats (20–22). The effects of exogenous administration of CRF mimicked the early experimental observations of Walter Cannon showing the inhibition of gastric emptying and acid secretion in cats stressed by facing a barking dog (23). The central injection of the peptide CRF antagonist, α-helical CRF9-41, further indicated that brain CRF receptors were involved in restraint and surgical stress–induced alterations of gut secretory and motor functions (22,24). In addition, experiments in hypophysectomized rats demonstrated that the central CRF actions to alter gut function were mediated by autonomic nervous system (ANS)–dependent pathways and unrelated to the activation of hypothalamic-pituitary axis (20,25). These findings paved the way to a number of investigations delineating central and peripheral sites of CRF actions and autonomic effectors, together with physiologic relevance of CRF signaling pathways in the adaptive response of the gut to stress (26,27). This chapter first reviews existing knowledge on the expanding members of mammalian CRF-related peptides, their pharmacologic characteristics on cloned CRF1 and CRF2 receptors, and the development of selective CRF1 and CRF2 receptor antagonists. CRF receptor–mediated central actions of exogenous CRF and related peptides on gastric, small intestinal, and colonic function and autonomic/neurocrine pathways mediating their effects on the gut are reviewed. Evidence that activation of central CRF receptors plays a role in stress-related alterations of gut function, development of visceral hypersensitivity, and modulation of experimentally induced intestinal inflammation in experimental animals also is addressed.

CHEMISTRY OF THE HYPOTHALAMICPITUITARY-ADRENAL AXIS Corticotropin-Releasing Factor–Related Peptides CRF is a well-conserved 41-aa peptide with an identical primary structure in humans, primates, dogs, horses, and rodents, whereas ovine CRF (oCRF) differs by 7 aa, together

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 793 with other artiodactylous ruminants that underwent several residue changes (28). In the 1980s, two novel nonmammalian CRF-like peptides were discovered in nonmammalian vertebrates, namely sauvagine, a 40-aa peptide isolated from the serous gland of amphibian Phyllomedusa sauvagei (29), and urotensin-I, a 41-aa residue peptide isolated from the neurosecretory system (urophysis) of teleosts (30,31). These peptides originally were considered as CRF orthologs in the frog and in fish, in line with their 50% homology with CRF. However, the cloning of CRF peptides in these species (32,33) encouraged the quest for possible additional CRF family members in mammals. In 1995, urocortin 1 (Ucn 1, also known as urocortin) (34) was characterized from rat midbrain as a 40-aa peptide with 45% sequence identity with rat and human CRF (r/hCRF) (Table 30-1), 44% to 63% with urotensin-I sequenced from various fish species (28,35), and 35% with sauvagine (28). The structure of Ucn 1 is highly conserved across mammalian species with rat, mice, and sheep primary sequences sharing 100% identity, and 95% with that of human Ucn 1 (hUcn 1) (35–37). However, the mismatches between brain distribution of CRF ligands (CRF and Ucn 1) and CRF receptors (38) kindled the search for additional endogenous CRF receptor ligands. Screening in human and mouse genome databases for sequence homology with the CRF/Ucn 1 family of peptides, including urotensin-I and sauvagine, culminated in the simultaneous cloning of two novel putative urocortin isoforms by two independent groups (39,40). This resulted in nomenclature divergences, which have been addressed (34). Mouse urocortin 2 (mUcn 2) is a 38-aa peptide that shares 34% homology with r/hCRF (see Table 30-1) and 42% with rUcn 1 (41). The hUcn 2 counterparts (34) share 76% homology with mUcn 2 (40,41) and originally was named urocortinrelated peptide (URP) (41) and stresscopin-related peptide (SRP) (40). Mouse urocortin 3 (mUcn 3) and hUcn 3 (39), also named human stresscopin (40), are more distantly related to r/hCRF (see Table 30-1), r/hUcn 1, and mUcn 1 (18% and 21% homology, respectively) (39). Because Ucn 2 and 3 were identified by molecular cloning strategies, the exact size of mature peptides has yet TABLE 30-1. Members of the corticotropin-releasing factor family of peptides in humans and rodents Peptides r/hCRF rUcn 1 hUcn 1 hUcn 2, (hURP) mUcn 2 hSRP hUcn 3 mUcn 3 SCP

Length (aa) 41 40 40 38 38 43 38 38 40

Identity (%) References 100 45 45 34 34 31 32 26 26

259 36 260 41 41 40 39, 61 39 40

aa, amino acids; CRF, corticotropin-releasing factor; h, human; m, mouse; r, rat; Ucn, urocortin; URP, urocortinrelated peptide; SCP, stresscopin; SRP, stresscopin-related peptide.

to be established, particularly because hUcn 2 (URP) lacks the consensus proteolytic cleavage site allowing for C-terminal processing (39). The predicted 38-aa mature hUcn 2 and 3 peptides have been assigned based on the structures of precursor genes (39,41), although Hsu and Hsueh (40) postulated the existence of N-terminally extended peptides, stresscopin-related peptide and stresscopin, composed of 43 and 40 aa, respectively (see Table 30-1). Less-conserved, extended N terminus between mouse and hUcn 2 and 3 and other pharmacologic considerations currently favor the 38-, rather than the 43/40-aa, versions of Ucn 2 and 3, respectively (34). The evaluation of the vertebrate genome reflects four groups of paralogous chromosomal regions in humans and mice. This is indicative that the vertebrate genome has duplicated twice by either regional duplication or by tetraploidization, causing paralogous genes constituting the many gene families and superfamilies (42). Phylogenetic profiling of the four CRF family members issued from four unique CRF-related genes indicates that CRF/Ucn 1 and Ucn 2/3 represent two parallel branches of functional paralogs in vertebrates (43).

Corticotropin-Releasing Factor Receptors CRF ligands interact with CRF1 or CRF2 receptors, or both, cloned from two distinct genes that have 70% identity at the aa level within species homologues (14,34). Both CRF1 and CRF2 belong to the class B1 subfamily of the 7-transmembrane domain receptors that signal largely, but not exclusively, by coupling to Gs-adenylate cyclase (44). Both CRF receptors have been cloned in a variety of species from amphibians to rodents to humans (45,46), and exist as a family of related proteins produced from multiple alternatively spliced forms of messenger RNA (mRNA) (47) (Table 30-2). Corticotropin-Releasing Factor 1 Receptors: Molecular Structures and Variants The human and rat genomic structure of CRF1 receptor contains 14 and 13 exons (GenBank accession numbers AF039510-AF3523 and U53486-U53498), respectively (46). An increasing number of CRF1 variants have been isolated and characterized mainly in human, rodent, and hamster skin and human myometrium and placenta (47–50). In human skin, 8 alternative splice variants have been reported, CRF1α (lacking exon 6, the most prevalent form), CRF1β (with a 29-aa insert into the first intracellular loop), CRF1c (40-aa deletion from the N-terminal domain), CRF1d (lacking exon 6- and 14-aa deletion in the 7-transmembrane domain), and CRF1e-h (47). In rodent skin, three to four isoforms have been identified (CRF1α, CRF1c-f) (47) (see Table 30-2). The expression of these CRF1 alternative splice variants is tissue specific (skin vs myometrium), is linked with the functional activity of the tissues (pregnant vs nonpregnant myometrium), and could be influenced by environmental factors (ultraviolet light), particularly in the

794 / CHAPTER 30 TABLE 30-2. Mammalian corticotropin-releasing factor receptors and variants CRF receptor subtypes

Variants

Tissue

References

CRF1 CRF1 CRF1 CRF1 CRF2 CRF2 CRF2 CRF2 CRF2

α, β, c, d, e, f, g, h α, d, e, j, h, f, k, m, n α, c, e, f β α, d α, β β1, β2 γ α, soluble α α-tr

Human skin Hamster skin Mouse skin Human myometrium Human, rodent brain/heart Human skeletal muscle Human brain Brain mice Rat brain

47, 48 47 49, 58 58 55 56, 52,

51 50

60 59

CRF, corticotropin-releasing factor.

skin (47,50,51). However, similar information is still to be obtained in the brain and the gut. Functional characterization of these isoforms indicate that only CRF1α is coupled directly to adenylate cyclase, whereas the majority of other isoforms are lacking ligand binding or signaling domains, or both (47,51). However, soluble isoforms may play a modulatory role as shown with CRF1e decreasing and CRF1h amplifying the CRF1α-coupled cyclic adenosine monophosphate (cAMP) production induced by Ucn 1 in transfected cercopithecus aethiops (COS) cells (51). Corticotropin-Releasing Factor 2 Receptors: Molecular Structures and Variants CRF2 receptors exist in three functional splice variants in humans, 2α, 2β, and 2γ, and two in rodents, 2α and 2β, which are structurally distinct in their N-terminal extracellular domains (52–55) (see Table 30-2). Genomic sequence from pig, bovine, cat, rat, and mice failed to identify the CRF2γ receptors, suggesting that this variant is found exclusively in humans (55,56). Rodent CRF2 isoforms are derived from the alternative splicing of exons 1 to 3 in their mRNA precursors (57). In the final translated proteins, the 34-aa N-terminal extracellular region of the CRF2α (involved in ligand–receptor interaction) is replaced by 61 aa in CRF2β and 20 aa in CRF2γ, whereas the C-terminus is common to all CRF2 receptor splice variants (34). Sequence comparison revealed high CRF2α homology between rats and mice (94%) and mice and humans (92%) (56). Presence of CRF2α in amphibian species suggests an earliest occurrence of this splice variant during vertebrate evolution, whereas CRF2β is less conserved and appears to be evolutionary younger and found only in mammals (61). CRF2β1 and CRF2β2, resulting from aberrant splicing, have been reported in the human skeletal muscle (58) (see Table 30-2). In rat brain, CRF2α-tr contains only three transmembrane domains because of the presence of a premature stop codon leading to unspliced introns 6 and 7 (52,59). Chen and colleagues (60) report a novel mouse CRF2α splice variant that includes the first extracellular domain of the CRF2α receptor and acts as a soluble binding protein (sCRF2α) that is capable of modulating CRF family ligand activity.

Corticotropin-Releasing Factor Receptor Pharmacology Radioreceptor and functional assays have demonstrated that CRF1 and CRF2 receptors differ considerably in their binding characteristics to several natural CRF ligands (34,61). However, there are no interspecies differences between rat and human CRF1 and CRF2 receptor pharmacologic profiles (62). CRF1 shows no appreciable binding to Ucn 2 and 3, but binds with high affinity to Ucn 1, CRF, and sauvagine (63). In contrast, CRF2 receptor binds to Ucn 1, 2, and 3 with a greater affinity than CRF, making this receptor subtype highly selective for urocortin signaling (35,41,56,64). Although CRF is 100-fold more potent at the CRF1 than the CRF2 receptor (65), there are no natural agonists with similar selectivity for CRF1 as Ucn 2 and Ucn 3 for CRF2 receptors (34). A search for developing selective CRF1 agonist resulted in the synthesis of cortagine, a chimeric peptide [Glu21, Ala40] [sauvagine1-12] × [h/rCRF14-30] × [saugavine30-40] that displays high affinity to the rCRF1 (2.6 nM), low affinity to the mCRF2β (540 nM), and no affinity to the CRF binding protein (CRF-BP) (66). Binding characteristics of CRF receptor variants, CRF2β, CRF2α, and CRF2γ isoforms, are almost identical with high affinity for Ucn 1, 2 and 3 and low affinity for r/hCRF and ovine CRF (39,55,62,67). Interestingly, the mouse sCRF2α receptor displays low affinity for Ucn 2 and 3, whereas binding Ucn 1 (Ki 6.6 nM), and to a lesser extent CRF (23 nM), and inhibits the cAMP and extracellular signal–regulated kinase 1/2-p42,44 responses to Ucn 1 and CRF (60). In contrast, rat CRF2α-tr binds with low affinity to CRF (Kd 12.7 nM) and does not bind to Ucn 1 (59).

Corticotropin-Releasing Factor Receptor Antagonists Key to the assessment of the role of CRF receptors in the stress response was the development of specific CRF receptor antagonists (Table 30-3). Earlier studies relied on the use of nonselective CRF1/CRF2 receptor peptide antagonists, mainly α-helical CRF9-41 (68), D-Phe12 CRF12-41 (69), followed by the potent and long-acting antagonists, astressin and astressin-B (70). These peptides are competitive antagonists

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 795 TABLE 30-3. Selective corticotropin-releasing factor receptor subtypes 1 and 2 antagonists Name

References

Nonpeptide, CRF1 antagonists with brain penetration on peripheral administration CP-154,526 NBI 30775/R121919 DMP695, DMP696, DMP904 Antalarmin SSR125543A NBI 27914, NBI 35965, NBI 30545 SN003 R278995/CRA0450 CRA1000, CRA1001

78, 261, 262 81, 263 264–266 267 268 77, 79, 80, 269 270 271 272

Peptide-selective CRF2 receptor antagonists Antisauvagine-30 Astressin2-B K41498

72, 85 71 273

CRF, corticotropin-releasing factor.

that bind equally to the α, β, and γ variants of CRF2 receptors and sCRF2α (55,56,60,67). Notably, α-helical CRF9-41 also acts as a partial agonist (65). Selective peptide CRF2 receptor antagonists, namely antisauvagine-30, K41498, [D-Phe11,His12,Nle17] sauvagine11-40, and the more potent, longer acting analog, astressin2-B, have been developed and characterized (71,72). Pharmacologic studies established that antisauvagine-30 and astressin2-B act as competitive antagonists at CRF2 receptors (73). In addition, 123I-K31440, Ac-[D-Tyr11, His12,Nle17]sauvagine11-40 has been developed as a potential single-photon emission computed tomography ligand (74). As a whole, these peptide antagonists generally display a poor penetrance to the brain. For instance, we showed that an intravenous (iv) injection of astressin, at a dose that blocked iv CRF-induced delayed gastric emptying, did not influence intracisternal (ic) CRFinduced gastric emptying inhibition (75). Likewise, α-helical CRF9-41 infused iv did not influence intracerebroventricular (icv) CRF-induced inhibition of pentagastrin-stimulated acid secretion, whereas injected icv, the antagonist blocked the icv CRF action (76). In contrast, all CRF1 antagonists have been designed as small hydrophobic molecules that cross the blood–brain barrier, including the first developed CP-154,526 (77,78) and several others listed in Table 30-3. The newly developed CRF1 antagonists, NBI 30775 (also labeled R 121919), NBI 30545, and NBI 35965, have the distinct advantage of being water soluble (79–81) compared with all previously developed CRF1 receptor antagonists that are not water soluble. These compounds have been instrumental in establishing the role of brain CRF-CRF1 signaling pathways in stress-related activation of the HPA axis and anxiogenic behavior (14).

Ucn 1 with an affinity equal to or greater than that of CRF1 receptors, whereas oCRF displays low affinity, and Ucn 2 and 3 have no appreciable affinity to the CRF-BP (35,39,83,84). The aa residues 6 to 33 of r/hCRF are sufficient to bind with high affinity to the CRF-BP, which differs from the binding requirement on CRF1 receptors (82). The peptide CRF antagonists, α-helical CRF9-41, have high affinity to the CRF-BP, whereas astressin does not (85,86). In vitro studies have established that the CRF-BP functions to sequester CRF and Ucn 1, limiting the availability of free ligands for CRF-mediated actions (83,84). Interestingly, studies indicate that the CRF-BP is expressed within hypothalamic circuitries modulating the neuroendocrine and autonomic responses to stress in rats (87). In particular, the majority of CRF-BP–immunoreactive cell bodies within the PVN are located in the subdivisions (dorsal cap) known to project to the spinal cord and brainstem structures (88) and positioned in close association with CRF- and Ucn 1–immunoreactive varicosities (87), as well as regions that contain CRF1 receptor gene expression (89). These data provide neuroanatomic support for the modulation of CRF and Ucn 1 availability at CRF1 receptors. Such a possibility is further supported by studies in CRF-BP–deficient mice that display anxiogenic behavior related to the increased “free” CRF availability in the absence of CRF-BP (90). However, CRF-BP–deficient or CRF-BP–overexpressing female mice have an unchanged basal and stress HPA axis response that has been related to adaptive compensatory mechanisms including the increase in hypothalamic AVP secretion (90,91).

Corticotropin-Releasing Factor Binding Protein

BRAIN DISTRIBUTION OF CORTICOTROPINRELEASING FACTOR LIGANDS AND RECEPTORS

The action of CRF and CRF-like peptides can be modulated by the CRF-BP, a 37-kDa protein that consists of 7 exons and 6 introns (82). The CRF-BP binds r/hCRF and

The molecular characterization of CRF, Ucns, and CRF1 and CRF2 receptor variants paved the way to establishing their localization in the central nervous system using

796 / CHAPTER 30 immunohistochemistry, in situ hybridization, and polymerase chain reaction methods in rodents (92). Available evidence indicates that CRF and Ucns have a distinct pattern of distribution, and that Ucn 1 may serve as the major CRF2 ligand within the lower brain, whereas Ucn 2 and 3 serve as the major CRF2 ligand in the forebrain (34).

Brain and Spinal Corticotropin-Releasing Factor and Urocortin Expression Central Nervous System Distribution of CorticotropinReleasing Factor Detailed organization of the CRF-immunoreactive cells and fibers in the rat brain has been described extensively (93–95). CRF mRNA and protein are abundantly expressed throughout the brain with major sites of expression in the PVN, cerebral cortex, amygdalar-hippocampal complex, and pontine Barrington’s nucleus. The PVN is the major site of CRF-containing cell bodies within the pPVN, which project to the medial eminence. In addition, the pPVN contains a discrete group of neurons in the dorsal and ventral parts, sending direct projections to the brainstem and spinal cord nuclei, respectively, that regulate autonomic outflow to the viscera (96,97). CRF neurons in the central amygdala project to the PVN and parabrachial nucleus, and those in the bed nucleus of stria terminalis to the dorsal vagal complex (DVC). CRF-containing neurons in the Barrington’s nuclei contribute CRF innervation to the locus ceruleus (LC) and sacral spinal cord (93,98). Central Nervous System Distribution of Urocortins There is little neuroanatomic overlap between the distribution of CRF and Ucn in the rat brain (99). The brain distribution of Ucn 1 is limited, although broadly expressed in the periphery (35,100,101). The most prominent site of Ucn 1 gene expression and immunoreactivity is found in the Edinger–Westphal nucleus (EWN) in various species (38,100,102). Ucn 1 immunoreactivity also is detected in the lateral superior olive, olfactory bulb, supraoptic nucleus (SON), ventromedial hypothalamus (VMH) and magnocellular parts of the PVN, lateral hypothalamic area, and cranial nerve motor nuclei (facial, hypoglossal, and ambigual nuclei) (38,102). Ucn 1–immunoreactive fibers project to brain nuclei that contain CRF2 receptors, mainly the lateral septum, dorsal raphe, interpeduncular nucleus, and nucleus tractus solitarius (NTS) and area postrema (38). However, several brain regions rich in CRF2 receptors do not contain Ucn 1–immunoreactive fibers, particularly in the VMH and the amygdala (38,103). Ucn 2 gene expression is localized in the PVN, SON, and arcuate nucleus of the hypothalamus (ARC) and LC, as well as in several cranial nerve motor nuclei (trigeminal, facial, and hypoglossal nuclei) and the ventral horn of the spinal cord (41). The neuroanatomic distribution of Ucn 2–immunoreactive protein has not been well characterized because of the lack of specific Ucn 2 antibody that does not

cross-react with other CRF family members. One report showed immunoreactive Ucn 2 neurons to be present in both the parvocellular and magnocellular PVN (104). Ucn 3 mRNA is detected in the PVN mainly in the dorsal and ventral aspects, the amygdala (basomedial nucleus) and basomedial nucleus of the stria terminalis (99). Dense Ucn 3–immunoreactive fibers innervate the lateral septum, the amygdala except for the central nucleus, the dorsal aspect of the VMH, and the dorsal raphe, as well as the area postrema (39,103,104). Functional Significance The abundant and widespread distribution of CRF is consistent with its physiologic role in regulating behavior, endocrine, and autonomic functions in response to stress (92,94). Ucn 1 distribution provides anatomic support for a role in the modulation of autonomic outflow and sensorimotor integration that may involve the integration of afferent signals from visceral origin (105). Dense Ucn 1 fibers ramify with the intermediolateral column of thoracic and lumbar spinal cord most likely originating from EWN projections, where the highest expression of Ucn 1 is found (106,107). The NTS and area postrema (38) that project to core groups of nuclei pivotal to the influence of autonomic outflow (108–110) are densely innervated by Ucn 1 fibers. Various immunologic, painful, chemical, and psychological stresses activate Ucn 1–containing neurons and up-regulate Ucn 1 gene expression within the EWN independently from the glucocorticoid increase (111–113). Interestingly, the activation of Ucn 1–containing neurons in the EWN displays a relatively long onset and duration (111,112). These observations led to the postulation that central Ucn 1 may be involved in some aspects of poststress–related adaptation (112). Likewise, Ucn 2 gene expression is up-regulated by restraint stress in the pPVN within 2 hours, and the response is adrenal dependent and site specific because Ucn 2 mRNA was not altered in the LC (114). Urocortin 3 mRNA is expressed in stress-related brain regions, including PVN and amygdala, and is up-regulated by restraint stress selectively in the PVN independently from glucocorticoids (99). However, Ucn 2 or 3 injected icv or in vitro does not stimulate the pituitary adrenal secretion and therefore is not involved in initiating the endocrine response to stress but appears to attenuate the behavioral anxiogenic response (40,99,115). The presence of Ucn 1 and 3 terminals in moderate density in the periaqueductal gray and laminae I and V of the dorsal horn, an area directly involved in the processing of pain signals (116), suggests a possible role in pain transduction mechanisms.

Brain Corticotropin-Releasing Factor Receptor Expression In the rat brain, CRF1 expression is high in the forebrain, subcortical limbic structures in the septal region, and amygdala, whereas in the hypothalamus, the expression is low, but

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 797 its expression is largely up-regulated during stress or by CRF (117–119). In keeping with the activation of the HPA axis by CRF, the CRF1 receptor is expressed densely in the anterior and intermediate lobe of the pituitary (120). In rodents, CRF2 expression is confined to subfornical structures with high expression in the lateral septum, amygdala (with the exception of central nuclei), and hypothalamus (including high levels in the VMH and SON) (121). In the hindbrain, the dorsal raphe, area postrema, NTS, and chorionic plexus express CRF2 receptor (121). A close association has been found between the Ucn 3–immunoreactive terminal fields and expression of CRF2 receptor in most of the brain examined (103). Notably, in the rat brain, CRF2α is the main variant expressed, whereas in nonneuronal tissues, either in the brain (cerebral arterioles) or periphery, only the CRF2β variant is expressed (122). In contrast, in humans, CRF2β and CRF2γ are expressed mainly in the brain neurons, and CRF2α is found in both the periphery and in the brain (55,58).

CENTRAL ACTION OF CORTICOTROPINRELEASING FACTOR PEPTIDES TO INFLUENCE GASTRIC FUNCTION Inhibition of Gastric Acid Secretion Corticotropin-Releasing Factor Acts in Hypothalamic/Pontine Nuclei to Inhibit Gastric Acid Secretion Several reports have established that CRF injected into the cerebrospinal fluid (CSF) either into the lateral brain ventricle (icv) or into the cisterna magna (ic) at nanomole doses induced a dose-related rapid onset and sustained inhibition of gastric acid secretion in conscious or anesthetized rats. Central CRF inhibits vagally stimulated acid secretion induced by either central injection of thyrotropin-releasing hormone (TRH), 2-deoxy-D-glucose, pylorus ligation, or gastric distention, as well as the acid response to pentagastrin, whereas not altering that of histamine (20,123,124). Similarly, in conscious dogs, the injection of CRF into the third ventricle inhibited 2-deoxy-D-glucose–, a peptone meal–, and pentagastrin iv infusion–stimulated, but not histaminestimulated, acid secretion (125,126). In healthy human volunteers, CRF applied intranasally, a route established to maximize delivery of peptides or drugs into the brain (127,128), resulted in an increase in gastric pH associated with a change in mood, whereas circulating cortisol levels were not altered, indicative of a central action to inhibit acid secretion (129). The CSF injection of CRF-induced inhibition of acid secretion is mediated centrally through interaction with CRF receptor and is not related to leakage of the peptide into the periphery (130), where CRF also inhibits acid secretion (19). Sauvagine injected icv suppresses gastric acid secretion with a greater potency than icv CRF, suggesting a CRF2 receptor– mediated effect in pylorus-ligated rats (131). However, the

CRF receptor subtype involved in CRF action needs to be further ascertained using selective CRF2 agonists and antagonists. Brain-responsive sites have been identified in the hypothalamus, namely the VMH, PVN, and lateral hypothalamic nuclei in which microinjection of CRF induced a doserelated suppression of acid secretion in conscious pylorusligated rats (124,132,133) (see Fig. 30-4). The hypothalamic ARC cell bodies activated by microinjection of kainic acid inhibit pentagastrin-stimulated acid secretion, and the response is blocked by astressin microinjected into the PVN (134). These data indicate that endogenous CRF signaling pathways in the PVN induced by ARC activation inhibit acid secretion consistent with the effect of exogenous injection of the peptide in the PVN (132). In contrast, the caudate putamen or dorsomedial frontal cortex did not influence acid secretion when microinjected with oCRF under the same conditions showing brain site specificity (132,133). Bilateral microinjections of r/hCRF into the pontine area, namely the LC/subceruleus (SC), also suppressed the acid response to iv infusion of pentagastrin in urethane anesthetized rats, whereas CRF microinjected into sites within the vicinity, but outside the LC/SC, had no effect (135). Pharmacologic studies indicate that the synthesis of cyclooxygenase products and naloxone-sensitive opiate system are part of the brain downstream effector mechanisms contributing to CRF inhibitory actions in rats and dogs (126,136). Central Corticotropin-Releasing Factor–Induced Inhibition of Gastric Acid Secretion Is Mediated by the Autonomic Nervous System The peripheral pathways through which central CRF inhibits gastric acid secretion involve the ANS (see Fig. 30-4). In rats, central CRF and more potently sauvagine inhibit gastric vagal efferent discharges (137) and stimulate sympathetic outflow as shown by the increase in circulating levels of epinephrine and norepinephrine in rats and dogs (125,138). Ganglionic blockade with chlorisondamine, spinal cord transection at the fifth cervical level, noradrenergic blockade with bretylium, α2-adrenergic blockade with yohimbine, and acute adrenalectomy abolished the inhibitory effect of ic, icv, or LC/SC injection of CRF on gastric acid secretion (20,76,135). Notably, the vagus nerve also contributes to the inhibitory effect of ic, but not icv or LC/SC, injection of CRF (20,76,135). The additional involvement of vagal pathways may be related to a more direct access of CRF when injected into the cisterna magna to modulate neurons in the DVC (139). These data combined with the unaltered gastric inhibitory response to central injection of CRF and sauvagine in hypophysectomized rats and to peripheral injection of naloxone (20,76) clearly established that the central action of CRF is neurally mediated and unrelated to the activation of the HPA axis (ACTH, β-endorphin). Gastric mechanisms are still to be established, but they do not involve prostaglandin synthesis or the suppression of gastrin release that is unchanged after icv injection of CRF (20,126). CRF injected icv induced

798 / CHAPTER 30 a vagal cholinergic-dependent increase in circulating levels of somatostatin (140). However, the role of somatostatin is still to be established, because icv CRF induced a sympatheticdependent, not vagal-dependent, gastric acid inhibition, and the effects of somatostatin receptor antagonists have not been tested under these conditions. In the dog, icv injection of CRF-induced inhibition of pentagastrin-stimulated gastric acid secretion was prevented by ganglionic blockade with hexamethonium, whereas vagotomy or adrenalectomy had no effect, suggesting a primary role of the gastric sympathetic noradrenergic nervous system. Peripheral mechanisms involving AVP also are supported by the increased plasma levels of AVP by icv CRF and the dampening of the CRF effect after peripheral injection of a vasopressin receptor antagonist (125,126,141). Circulating gastrin and β-endorphin do not play a role as reported in rats (126,141). Brain Corticotropin-Releasing Factor Signaling Pathways Contribute to Stress and Anorexigenic Brain Peptide–Induced Inhibition of Gastric Acid Secretion Early clinical and experimental observations demonstrated that emotional stimuli inhibit gastric acid secretion. Beaumont (142) noticed that when his fistulous subject experienced “fear, anger or whatever depresses or disturbs the nervous system,” gastric secretion was inhibited. Cannon (23) showed that flight-or-fight response in cats resulted in decreased gastric acid secretion. The role of CRF signaling pathways in stress-related inhibition of acid secretion is supported by pharmacologic evidence that ic injection of CRF antagonist, α-helical CRF9-41, completely prevented brain surgery– induced reduction of gastric acid secretion in pylorusligated rats (24). Likewise, partial restraint stress–induced decreased gastric acid secretion is prevented by icv injection of α-helical CRF9-41, whereas the antagonist injected peripherally had no effect (138). The lack of change in basal acid secretion in rats injected into the CSF with the CRF receptor antagonist indicates that brain CRF receptors are not involved in basal acid secretion regulation but do play a role under stress-related conditions (24,138). In addition to stress, activation of central CRF signaling pathways is part of the brain mechanisms through which some anorexigenic brain peptides inhibit gastric acid secretion. In particular, cocaine- and amphetamine-regulated transcript (CART) peptide ic injection-induced inhibition of gastric acid secretion in pylorus-ligated rats was completely blocked by prior ic injection of α-helical CRF9-41 (143). Likewise, the effects of neuromedine U injected icv to decrease gastric secretory volume and acid secretion were prevented by anti-CRF IgG injected icv (144). In contrast, gastrin-releasing peptide (GRP) icv injection-induced inhibition of gastric acid secretion in pylorus-ligated rats was not influenced by icv injection of α-helical CRF9-41 (138). Central injection of interleukin-1β (IL-1β) in pylorusligated rats or peripheral administration of endotoxininduced inhibition of stimulated acid secretion by gastric

distention in urethane-anesthetized rats also are not altered by ic injection of α-helical CRF9-41 (145,146). Stimulation of Gastric Bicarbonate Secretion Corticotropin-Releasing Factor Acts in the Hypothalamic Nuclei to Increase Bicarbonate Secretion First evidence of brain regulation of gastric bicarbonate secretion came from studies showing that microinjection of oCRF into hypothalamic nuclei such as the PVN, lateral hypothalamus, and VMH increased the gastric volume of secretion, whereas total acid output and acid concentration were inhibited in conscious rats (132) (see Fig. 30-4). Such a response is peptide and site specific because bombesin, calcitonin gene–related peptide, or calcitonin microinjected into these hypothalamic nuclei and oCRF or sauvagine injected ic inhibit both gastric secretory volume and acid concentration (20,131,147). Further studies established that oCRF microinjected into the VMH stimulated gastric concentration and output of bicarbonate and potassium secretion, and the responses were not secondary to the inhibition of acid secretion in conscious pylorus-ligated rats (133). Therefore, the net acute effect of central CRF is to protect the gastric mucosa as shown by the inhibition of gastric erosions induced by cold restraint (133). Inhibition of Gastric Emptying Corticotropin-Releasing Factor and Related Peptides Injected into the Brain Inhibit Gastric Emptying in Various Species The central action of CRF to inhibit gastric transit has been well documented under various experimental conditions (148). Initial reports showed that CRF injected icv or ic exerts a potent and long-acting inhibition of gastric emptying of a nonnutrient viscous solution in conscious male or female rats (22,25,149). Other studies established that CRF injected icv into the fourth ventricle or cisterna magna inhibits gastric emptying of saline, glucose, acid, and a peptone liquid meal delivered intragastrically, as well as gastric emptying of a physiologic meal (ingestion of Purina chow) in conscious rats (150–152). Similarly, in mice, icv injection of CRF results in a potent inhibition of gastric emptying of nonnutrient liquid and an ingested solid Purina chow meal (153,154). In dogs, icv infusion of CRF for 15 minutes delayed the total gastric emptying time of a solid nutrient meal for 6 to 8 hours (155). In addition to CRF, all other nonmammalian and mammalian CRF-related peptides, namely sauvagine, urotensin I, Ucn 1, Ucn 2, and less potently, Ucn 3, injected icv or ic inhibit gastric emptying of liquid or solid meal, whereas the CRF analog, oCRF9-33OH that displays high affinity to the CRF-BP but does not bind to CRF receptors (82), had no effect in rats and mice (154,156–158).

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 799

The CRF1/CRF2 peptide antagonists, α-helical CRF9-41, D-Phe12 CRF12-41, astressin, or astressin-B, injected ic or icv blocked central CRF, urotensin I, and sauvagineinduced delayed gastric emptying in rats, mice, and dogs (149–151,153–156,162–167). Convergent evidence indicates that CRF2 receptor subtype mediates the delayed gastric emptying induced by central injection of CRF and related peptides in rodents. First, the rank order of potency of ic or icv CRF-related peptides (sauvagine > urotensin I > r/hCRF) is consistent with the CRF2-mediated event (156,157), as well as the increasing ic astressin/agonist ratios (3:1, 10:1, and 16:1) required for full reversal of ic r/hCRF, urotensin I, or sauvagine, respectively (156). Second, selective CRF2 agonist, Ucn 2, and to a lesser extent Ucn 3, injected icv in mice delayed gastric emptying of a solid meal (154). Third, the inhibition of gastric emptying of a solid meal induced by ic Ucn 1 in rats and icv CRF, Ucn 1, and Ucn 2 in mice is prevented by astressin2-B (154,156,158) (Fig. 30-2). Lastly, selective CRF1 antagonists, NBI 35965 and NBI 27914, had no effect when injected ic or peripherally at doses that prevent icv CRF- or Ucn 1–induced stimulation of colonic motor function assessed simultaneously in rats or mice (154,156,158,167) (see Fig. 30-2). The Autonomic Nervous System Mediates Central Injection of Corticotropin-Releasing Factor–Induced Inhibition of Gastric Transit Consistent sets of observations established that the inhibition of gastric emptying induced by ic or icv injection of CRF in rats involves the ANS and is not related to activation of the HPA axis (see Fig. 30-4). First, neither hypophysectomy nor acute adrenalectomy altered icv CRF action in rats (25,149,168). Second, ACTH injected iv did not mimic the icv CRF inhibitory action in rats and dogs (169), and a CRF1 antagonist known to prevent central CRF-induced HPA activation did not prevent the inhibition of gastric emptying (156). Lastly, ganglionic blockade with chlorisondamine abolished icv CRF-induced delayed gastric emptying in rats and mice (153,156).

15

30

12 9

20 6 10

Bead latency (min)

Gastric emptying (% in 2 h)

40

3

A rUcn 1

B

40

15

30

12 9

20 6 10

Bead latency (min)

Corticotropin-Releasing Factor 2 Receptor Mediates Central Injection of Corticotropin-Releasing Factor and Related Peptide-Induced Inhibition of Gastric Emptying

r/hCRF

Gastric emptying (% in 2 h)

The effect of CRF injected into the CSF is mediated centrally and is not related to leakage into the periphery, where CRF is also a potent inhibitor of gastric emptying in rodents and dogs (22,149,159). This was established first by the unaltered action of ic or icv CRF by peripheral injection of CRF receptor antagonist or CRF antibody at doses that blocked peripheral CRF-induced delayed gastric emptying in rats and mice (149,153). Brain sites of action of CRF to inhibit gastric emptying have been localized in selective hypothalamic (PVN) or spinal (L5-L6 levels) sites, whereas CRF microinjected into the lateral hypothalamus and LC/SC did not influence gastric transit in rats and mice (153,160,161) (see Fig. 30-4).

3

Vehicle

NBI 35965

Astressin2B

FIG. 30-2. Inhibition of gastric emptying of a solid meal by intracerebroventricular (icv) corticotropin-releasing factor (CRF; 0.5 µg) (A) and urocortin 1 (Ucn 1) (0.5 µg) (B) in conscious mice: reversal by the selective CRF2 antagonist, astressin2-B (10 µg) injected icv, whereas the selective CRF1 antagonist, NBI 35965 (50 µg) injected icv, had no effect. In contrast, NBI 35965 prevented icv CRF- and Ucn 1–induced reduction of distal colonic transit time, whereas astressin2-B had no effect in the same mice. Dashed lines represent the mean gastric emptying (41.2 ± 5.0%) and bead latency time (11.8 ± 1.2 minutes) in animals treated with vehicle or antagonists + vehicle. *p < 0.05 versus vehicletreated group (analysis of variance). (Modified from Martinez and colleagues [154], by permission.)

Vagal or sympathetic pathways, or both, have been reported to be involved in central CRF-induced inhibition of gastric emptying depending on the site of peptide injection and the nutrient and nonnutrient nature of the meal. Vagotomy blocked icv CRF-induced delay of total gastric emptying time of nutrient meal in dogs (155) and icv or ic CRF and ic sauvagine-induced inhibition of gastric emptying of liquid nonnutrient solution in rats (149,160,168). In contrast, ganglionectomy and propanolol were reported to block ic CRF-induced delayed gastric emptying of solid nutrient meal in rats, showing a mediation through sympathetic pathways and β-adrenergic receptors (152). Similarly, Lenz and colleagues (25) report that icv CRF-induced delayed gastric emptying of a liquid nonnutrient meal was blocked by bretylium, whereas vagotomy had no effect. Possible involvement of capsaicin-sensitive afferent fibers also was reported after icv CRF-induced decreased gastric emptying of an intragastric glucose solution (151).

800 / CHAPTER 30 Other studies investigated the peripheral transmitters through which ANS mediates gastric motor response and found a possible role of gastric somatostatin. Fourth ventricle injection of CRF induces a vagal cholinergic release of somatostatin into the circulation, and somatostatin antagonists block the CRF action (140,170). Other studies ruled out a role of prostaglandin (164) and naloxone to alter the effects of CRF injected icv in mice (153) and ic in rats (149), although Lenz and colleagues (25) report a blockade of icv CRF induced delayed gastric emptying in rats by naloxone.

There is also evidence that some peptides acting centrally to inhibit gastric emptying recruit CRF signaling pathways. For instance, fourth ventricle injection of CART-induced delayed gastric emptying of an intragastric glucose solution is inhibited by fourth ventricle injection of α-helical CRF941 in rats (175). However, neither ic adrenomedullin nor icv GRP-induced delayed gastric emptying are influenced by ic D-Phe12 CRF12-41 and icv α-helical CRF9-41, respectively, at doses sufficient to block the ic or icv CRF inhibitory action (138,176).

Brain Corticotropin-Releasing Factor Receptors Mediate Inhibition of Gastric Transit Induced by Various Stressors and Specific Brain Peptides

Alteration of Gastric Motility

All CRF receptor antagonists injected into the CSF did not alter basal gastric emptying of a liquid nonnutrient or solid nutrient meal in rats, mice, and dogs, suggesting that central CRF signaling pathways are not involved in basal and postprandial regulation of gastric emptying (153,155,171). However, under conditions of stress, ic or icv injection of CRF receptor antagonists, D-Phe12 CRF12-41, astressin, or astressin-B, completely prevents the delayed gastric emptying induced by either psychological/physical (swim stress, restraint), visceral (abdominal surgery, trephination, peritoneal irritation with intraperitoneal 0.6% acetic acid), immunologic (iv IL-1β), or chemical (ether) stressors (Table 30-4). These antagonist actions are mediated centrally as shown by the lack of reversal of peritoneal irritation; iv IL-1β or partial restraint induced delayed gastric emptying by peripheral injection of CRF receptor antagonists at doses efficient when injected into the central nervous system (138,172,173). The CRF receptor subtype involved has not been investigated for all stressors. Astressin2-B injected ic prevents restraint stress (152), whereas selective CRF1 antagonists blocked postoperative gastric ileus, and CRF1 knockout mice did not develop postoperative gastric ileus after abdominal surgery (174). These data provide new insights to the underlying mechanisms of stress-related upper gastrointestinal motility disorders.

Central Corticotropin-Releasing Factor Alters the Pattern of Gastric Motility in Various Species Consistent with the central action of CRF and related peptides to inhibit gastric emptying, CRF and Ucn 1 injected into the CSF alter gastric contractility pattern and propagation of motor events in various species. In sheep, icv injection of CRF induced a centrally mediated and long-lasting inhibition of postprandial antral motility (177). In fasted dogs, earlier reports by Buéno and colleagues (169) showed that icv injection of CRF induced an immediate and sustained disruption of the interdigestive pattern of gastric motility resulting in the abolition of the cyclic activity front in the antrum that was replaced by irregular contractions of small amplitude (178,179). However, in dogs fed with a solid meal immediately after icv infusion of CRF, a dose that delayed the total gastric emptying time, no significant effect on the amplitude and duration of the gastric pyloric contraction was observed, whereas the mean distance of propagation of gastric contractions together with their frequency were increased (155). The delayed emptying results from inhibition of gastric propagative contractions and alterations in duodenal propulsion (155). Fasted urethane-anesthetized rats responded to CRF or Ucn 1 injected ic by a suppression of high-amplitude gastric contractions evoked by central vagal stimulants (ic TRH analog or 2-deoxy-D-glucose) (Fig. 30-3), whereas those induced by the peripheral stimulant, carbachol, were not modified (180,181). Further studies

TABLE 30-4. Stress-induced delayed gastric emptying: Blockade by central injection of peptide corticotropin-releasing factor receptor subtypes 1 and 2 antagonists Stressors

↓GE (%)

CRF antagonist

Ether 70–140 seconds Partial restraint Restraint Restraint Peritoneal irritation Swim × 20 minutes Trephination Abdominal surgery Abdominal surgery Abdominal surgery Interleukin-1β ic Interleukin-1β iv

32–56 25–30 30 50 50 22 81 83 60 60 60 50

α-Helical CRF9-41 α-Helical CRF9-41 α-Helical CRF9-41 Astressin2-B α-Helical CRF9-41 α-Helical CRF9-41 α-Helical CRF9-41 α-Helical CRF9-41 [DPhe12] CRF12-41 Astressin [DPhe12] CRF12-41 [DPhe12] CRF12-41

Route

Dose (µg/rat)

ic icv PVN ic ic icv ic ic ic ic ic ic

50 40 50/site 10 40 30 100 50 10 3 20 20

Reversal (%) 100 100 100 100 100 100 79 87 100 100 52 100

References 163 138 160 152 172 151 163 163 166 162 164 173

CRF, corticotropin-releasing factor; GE, gastric emptying; ic, intracisternal; icv, intracerebroventricular; PVN, paraventricular nucleus.

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 801

AUC of corpus contractions (mV min−1)

2

Vehicle or RX-77368 30 ng, ic

Vehicle (ic) + Vehicle (ic)

1.8 1.6

Vehicle (lc) + RX-77368 (ic) rUch (1 µg ic) + RX-77368 (ic) Vehicle or rUcn (ic)

rUch (3 µg ic) + RX-77368 (ic) rUch (10 µg ic) + RX-77368 (ic)

1.4 1.2 1 0.8 0.6 0.4 0.2 0 −30 −20 −10 0

A ic Vehicle

B ic Vehicle

10 20 30 40 50 60 70 80 90 100 110 120 Minutes

ic Vehicle

RX-77368 30 ng, ic

0.3 mV 10 min

C

D

E

rUcn 1 µg, ic

RX-77368 30 ng, ic

rUcn 3 µg, ic

RX-77368 30 ng, ic

rUcn 10 µg, ic

RX-77368 30 ng, ic

F FIG. 30-3. Urocortin 1 (Ucn 1) injected intracisternally induced a dose-related inhibition of vagally stimulated gastric contractions induced by intracisternal (ic) RX 77368 in fasted urethaneanesthetized rats. Animals were acutely implanted with strain gauge on circular smooth muscles of the greater gastric curvature. (A) Mean ± standard error of the mean of area under the curve (min−1) of gastric contractions. (B–F) Representative trace with various treatments. (Reproduced from Chen and colleagues [158], by permission.)

in conscious rats showed that icv CRF changed the fasted pattern of gastric motility to a fed-like motor pattern with irregular contractions of high frequency, whereas in fed rats, icv CRF maintained the fed pattern with a reduced amplitude of pressure wave in the antrum (182). Corticotropin-Releasing Factor 2 Receptor Mediates Central Injection of Corticotropin-Releasing Factor and Urocortin 1–Induced Inhibition of Gastric Motility Intracisternal Ucn 1–induced inhibition of vagally stimulated gastric motility in fasted rats is prevented by ic injection of astressin2-B, whereas NBI 27914 had no effect (158). These data indicate that central action of Ucn 1 to

alter gastric contractility involves CRF2 receptors in line with results on the inhibition of gastric emptying (158). Central Corticotropin-Releasing Factor–Induced Alterations of Gastric Motility Is Mediated Vagally The iv injection of cortisol and ACTH does not reproduce the disruption of antral motility in fasted dogs, ruling out a mediation through the stimulation of the HPA axis (169). Several reports have shown that the neuronal pathways through which central CRF alter gastric motility in dogs and rats is mediated vagally (155,179,182,183). For instance, icv CRF induced a rapid abolition of antral cyclic migrating myoelectrical complexes (MMCs) in fasted dogs and triggered fed patterns in fasted rats. These effects were abolished

802 / CHAPTER 30 by vagotomy but were unaltered by sympathectomy or adrenergic blockade using propanolol and phentolamine (179,182) (see Fig. 30-4). Consistent with vagal-dependent mechanisms, the DVC is a responsive site that inhibits gastric motility stimulated by central vagal activation using TRH into the DVC (181) or iv infusion of bethanecol (183). Peripheral mechanisms through which central CRF disrupts gastric MMC in fasted dogs may be related to the inhibition of cyclic variation of plasma motilin (178). In rats, inhibition of vagal cholinergic input, as well as activation of nonadrenergic, noncholinergic input play a role in the central inhibitory action of CRF on gastric motility (181,183).

CENTRAL ACTIONS OF CORTICOTROPINRELEASING FACTOR PEPTIDES TO INFLUENCE SMALL INTESTINAL FUNCTION Stimulation of Duodenal Bicarbonate Secretion Central Corticotropin-Releasing Factor Stimulates Duodenal Bicarbonate Secretion in Rats CRF injected icv is one of the most potent peptides to stimulate duodenal bicarbonate secretion compared with other stimulants (icv injection of TRH or somatostatin analog) and a number of inactive peptides (icv bombesin, neurotensin, calcitonin, vasoactive intestinal peptide, gastrin, growth hormone–releasing factor, gonadotropin-releasing hormone, and β-endorphin) in conscious rats (123,184). CRF injected icv also stimulates duodenal bicarbonate secretion and transmembrane potential in anesthetized rats (185). The central

effects of CRF action are CRF receptor mediated, although the receptor subtypes remain to be established. Activation of Hypothalamic-Pituitary-β-Endorphin Axis Mediates Duodenal Bicarbonate Response to Central Corticotropin-Releasing Factor Central injection of CRF-induced stimulation of duodenal bicarbonate secretion is not mediated by the ANS because it is unaltered by chlorisondamine, vagotomy, or blockade of sympathetic nervous system with bretylium and by adrenalectomy (184). Evidence indicates the involvement of the hypothalamic-pituitary axis releasing β-endorphin. Hypophysectomy and naloxone prevent the duodenal bicarbonate response (184), and β-endorphin, unlike ACTH, injected iv at doses reproducing the circulating levels induced by icv CRF, stimulates duodenal bicarbonate secretion (184). Lastly, icv and iv CRF display a similar potency to stimulate duodenal bicarbonate secretion consistent with the activation of the hypothalamicpituitary axis by CRF injected centrally or peripherally (184). These data provided the first evidence that central CRF influences gut secretory function through the hypothalamic-pituitary-intestinal axis (Fig. 30-5). Brain Corticotropin-Releasing Factor Receptors Are Involved in Stress-Related Duodenal Bicarbonate Secretion The icv injection of α-helical CRF9-41 abolished both icv CRF and partial restraint stress–induced threefold increase in bicarbonate secretion in conscious rats (184). These data suggest that activation of brain CRF receptors during stress

Central CRF: Effects on the stomach

Motor alterations

Secretion Effects

Brain sites

Acid

- ic - icv - LC/SC

Pathways

- Hypothalamus (PVN, VMH, LH) Bicarbonate

- Hypothalamus (PVN, VMH, LH)

- Vagal

CRF receptors

Effects

- CRF2 ?

Sympathetic - α2-adrenergic - NE Does not involve - HPA - Not known

GE

Brain sites - ic - icv - PVN - Spinal L6-S1

Pathways

CRF receptors

- Vagal Sympathetic - β-adrenergic - NE

- CRF2 - CRF1 ?

Does not involve - HPA

Alter motility

- ic - icv

- Vagal - CRF2 Does not involve - HPA or - Sympathetic activity

FIG. 30-4. Summary representation of central corticotropin-releasing factor (CRF)–induced inhibition of gastric acid secretion and motility, and stimulation of bicarbonate secretion: brain CRF receptor subtypes involved, brain sites of action, and neuronal pathways. Note that alterations of gastric secretory or motor function are not related to the stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and are mediated mainly by CRF2 receptor–induced autonomic changes. GE, gastric emptying; LC/SC, locus ceruleus/subceruleus; LH, lateral hypothalamus; NE, norepinephrine; PVN, paraventricular nucleus; VMH, ventromedial hypothalamus.

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 803 Central CRF: Effects on the intestine

Secretion & absorption Effects

Bicarbonate secretion

Brain sites

- ic - icv

Ileal absorption - icv water chloride sodium

Pathways

Motor alterations & inflammation CRF receptors

- HP-β-endorphin - Dose not involve ANS

- Sympathetic - Does not involve HPA or Vagus

- Not known

Effects

Brain sites

Transit

- icv

Alter motility

- icv

Pathways

CRF receptors

- Vagal - Does not involve HPA or Sympathetic system

- Not known

- Vagus - Does not involve HPA

- Not known

- Not known - ic Inflammation - icv

- HPA

- CRF1 ?

FIG. 30-5. Summary representation of central corticotropin-releasing factor (CRF)–induced alterations of small intestinal secretion, motility, and modulation of experimental inflammation. Note that central CRF-induced stimulation of duodenal bicarbonate secretion is mediated by hypothalamicpituitary-β-endorphin and the anti-inflammatory response by the hypothalamic-pituitary-adrenal (HPA) axis, whereas inhibition of transit and propagative motility is mediated vagally. Brain sites and CRF receptor subtypes of CRF actions remain to be established. ANS, autonomic nervous system; ic, intracisternal; icv, intracerebroventricular.

results in alterations of duodenal secretory function that enhances the resistance of the duodenal mucosa (184), similar to that observed in the gastric mucosa (133). Alterations of Jejunoileal Absorption Central Corticotropin-Releasing Factor Stimulates Ileal Water Absorption through Sympathetic Pathways CRF injected icv induces a CRF receptor–mediated stimulation of ileal water, chloride, and sodium absorption in conscious rats (186). The enhanced ileal water transport is not secondary to the activation of HPA axis because the response is not altered by hypophysectomy, adrenalectomy, naloxone, and vasopressin and is mediated by the activation of sympathetic noradrenergic outflow to the gut (186) (see Fig. 30-5). The absorptive action of icv CRF is blocked by chlorisondamine, bretylium, and phentolamine, whereas vagotomy had no effect (186). In addition, the increased ileal water transport was reproduced by peripheral administration of norepinephrine (186), whereas the glucocorticoid, dexamethasone, exerts an inhibitory effect on jejunal water absorption (187). These data are consistent with norepinephrine acting on α receptors, being the peripheral effector in the ileal response to icv CRF (186). Brain Corticotropin-Releasing Factor Receptors Are Involved in Stress-Related Alteration of Absorption Activation of brain CRF receptors is involved in restraintinduced stimulation of ileal water absorption lasting for 60 minutes in rats as shown by the use of icv α-helical

CRF9-41 before stress (186). However, under conditions of chronic, intermittent, water avoidance stress (WAS) for 1 hour per day for 5 days, there was an increase in the baseline jejunal epithelial ion secretion indicated by the short-circuit current (Isc) in rats (188). Likewise, anxiety-prone Wistar–Kyoto rats exposed to cold restraint stress showed a significant increase in jejunal secretion as evidenced by increased Isc across isolated rat jejunal tissue compared with control tissues (189,190). The role of CRF signaling pathway under these chronic conditions of stress-induced increased secretion in jejunal segment has not been investigated. Chronic stress has been shown to induce a rapid inhibition of glucose absorption in the loop of fed rats (187). This change results from the increased corticosterone that inhibits GLUT2 trafficking to the jejunal brush-border membrane, indicative of a mediation through activation of the HPA axis (187). Such inhibition of intestinal delivery of glucose has been viewed in the context of not antagonizing cortisol to mobilize energy stores in response to chronic stress (187). Slowing of Small Intestinal Transit Central Corticotropin-Releasing Factor Delays Small Intestinal Transit through Vagal Pathways Central CRF delays small intestinal transit in rats, although the response has been little studied compared with that of the upper or lower gut (22,25,138). Dose–response studies showed that icv injection of CRF results in a more modest inhibition of small intestinal transit than gastric emptying in conscious rats (22,25,138). The response to icv CRF is not related to the activation of the HPA axis because slowing of

804 / CHAPTER 30 intestinal transit was not altered by hypophysectomy or adrenalectomy (25). In addition, the iv administration of ACTH and β-endorphin, at doses that reproduced levels occurring during restraint, did not influence small intestinal propulsion (191). The blockade by chlorisondamine, vagotomy, and naloxone, unlike noradrenergic inhibition (25), indicates a role for vagal efferent pathways and central opioid receptors as observed for the inhibition of gastric emptying (see Figs. 30-4 and 30-5). The less prominent vagally mediated action of central CRF on intestinal versus gastric transit may reflect the lesser vagal innervation of the small intestine compared with the stomach (139). Do Central Corticotropin-Releasing Factor Receptors Play a Role in Stress-Related Slowing of Small Intestinal Transit? The action of icv CRF on small intestinal transit is receptor mediated, although the subtype involved remains to be established (22,138). Conflicting data have been reported regarding the role of brain CRF receptors in the inhibition of small intestinal transit induced by stress. An earlier report by Williams and colleagues (22) shows that α-helical CRF9-41, injected icv at doses that suppressed colonic transit response, did not influence wrap restraint–induced reduction of small intestinal transit in female rats. In contrast, Lenz and colleagues (138) report a blockade of partial restraint–induced small intestinal transit delay by icv α-helical CRF9-41 in male rats. Whether sex differences or modalities of the partial restraint may account for these divergent results needs to be assessed further. Alterations of Small Intestinal Motility Central Corticotropin-Releasing Factor Alters Pattern of Intestinal Motility in Various Species through Vagal Pathways A number of studies have demonstrated that CRF ligands injected into the brain alter the pattern of small intestinal motility, although the responses varied with the fed or fasted state and species under study. CRF infused icv for 15 minutes after a nutrient meal in dogs increased the frequency of proximal duodenal contractions, whereas the amplitude and durations were not altered (155). However, in fed rats, icv Ucn 1 increased the amplitude of pressure waves in the duodenum (182). By contrast, in fed sheep, icv CRF induced a longlasting inhibition of duodenal motor activity (177). Early studies in fasted dogs have shown that oCRF, injected icv in doses as low as 20 to 100 ng/kg, inhibits the occurrence of MMC not only in the duodenum, but also in the proximal jejunum (169,179). Similarly, other studies have shown that Ucn 1, injected icv, disrupted the fasted pattern of duodenal motor activity and replaced it with a fed pattern (182). In rats and dogs, the central CRF actions on duodenal motility involve the activation of CRF receptors, as shown by the blockade of the effect with icv α-helical CRF9-41 and

vagal-dependent mechanisms, whereas the stimulation of the HPA axis does not play a role (155,169,182). Collectively, these studies indicate that central CRF induces vagally mediated alterations of small intestinal coordinated movements leading to a decreased transit (see Fig. 30-5). Do Central Corticotropin-Releasing Factor Receptors Play a Role in Stress-Related Alterations of Small Intestinal Motility? Clinical and experimental studies established that stress influences small intestinal motility. For instance, in humans, acute psychological stress suppresses duodenal phase II migrating motor complex (192). In rats, restraint abolished the recurrence of MMCs and replaced it with a continuous and irregular activity in the jejunum and ileum (193). However, whether central administration of CRF antagonists prevents the alterations of intestinal motility pattern in response to stress remains to be investigated.

Anti-inflammatory Action Brain CRF is well known to exert anti-inflammatory effects through the release of ACTH and glucocorticoids (194). Clinical practice clearly showed that glucocorticoids are the mainstay of treatments against intestinal inflammation (195). Experimental evidence shows that blockade of glucocorticoid receptors enhanced small intestinal inflammation induced by Clostridium difficile toxin A in rats (196). Conversely, CRF injected ic also abrogated both the increased expression of ICAM-1 and leukocyte recruitment in lipopolysaccharide-induced mesenteric inflammation in rats (197). Furthermore, the same study showed that blockade of endogenous glucocorticoid receptors, but not blockade of peripheral CRF receptors with astressin, reversed the inhibitory action of CRF on leukocyte–endothelial cell interactions during endotoxemia-induced mesenteric inflammation (197). These data suggest the anti-inflammatory effects of cerebral CRF to be mediated through HPA axis activation (see Fig. 30-5).

CENTRAL ACTIONS OF CORTICOTROPINRELEASING FACTOR PEPTIDES TO INFLUENCE COLONIC FUNCTION Stimulation of Colonic Propulsive Motor Function Corticotropin-Releasing Factor and Urocortin 1 Act in the Brain to Stimulate Colonic Motor Function in Various Species Numerous reports have established that CRF injected icv, although inhibiting gastric and intestinal transit, stimulates colonic transit and induces rapid onset of defecation in freely moving female and male Sprague–Dawley rats, male Wistar

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 805 rats, and male mice and gerbils (22,25,154,161,162,198–200), but not in anesthetized Sprague–Dawley rats (201). Likewise, CRF injected into the cisterna magna also results in stimulation of defecation in Fischer or Lewis rats (202), whereas Ucn 1 injected icv stimulates colonic transit in mice (154). Consistent with an acceleration of propulsive colonic motor function, icv CRF induces a rapid occurrence of colonic spike burst activity in fasted or fed male Sprague–Dawley (203,204) and Wistar rats (205). Brain nuclei responsive to CRF leading to stimulation of colonic motor function have been localized in specific hypothalamic (PVN) and pontine areas (LC, SC/Barrington nucleus complex) (160,161,206,207), which are also sites involved in central CRF-induced anxiety and depression (208,209). CRF microinjected into the PVN and LC/ Barrington nucleus complex increased tonic and phasic motor activity, decreased colonic transit time, and induced watery fecal output (160,207,210). Brain nuclei specificity was established by the inactivity of CRF microinjected into the lateral, ventromedial or anterior hypothalamus, central amygdala, nucleus stria terminalis, medial preoptic area, or zona incerta under similar conditions in freely moving male rats (160,207,210). Corticotropin-Releasing Factor 1 Receptor Mediates Central Injection of Corticotropin-Releasing Factor and Urocortin 1–Induced Stimulation of Colonic Motor Function Several initial studies have established that icv α-helical CRF9-41 blocked icv CRF-induced stimulation of colonic transit and increased the frequency of contractions in freely moving rats (22,138,204). α-Helical CRF9-41 and astressin injected icv also reduce the icv CRF-induced defecation (22,167,199). Studies have established that brain CRF1 receptors are involved in the colonic response to icv CRF in rats. This is supported by the rank order of potency of icv oCRF, r/hCRF, and Ucn 1 > Ucn 2 > Ucn 3 to induce defecation in mice consistent with a CRF1-mediated effect (154). In addition, icv injection of NBI 35965 and NBI 27914 antagonized icv CRF- and Ucn 1–induced stimulation of colonic transit, whereas icv astressin2-B had no effect in mice or rats (154,167). Parasympathetic Nervous System Mediates Central Injection of Corticotropin-Releasing Factor–Induced Stimulation of Colonic Motor Function Pharmacologic and surgical approaches established that icv CRF-induced stimulation of colonic motor function (transit and increased frequency of spike-burst activity) does not involve the activation of the HPA axis because the colonic responses were not altered in hypophysectomized and adrenalectomized rats or by pretreatment with naloxone (25,204). However, the stimulation of colonic transit, phasic and tonic contractions, and defecation induced by CRF injected icv or into the PVN were prevented by chlorisondamine and

atropine, attenuated by vagotomy, and unchanged by bretylium (25,160,206). Likewise, endogenous release of CRF in the PVN induced by activation of ARC cell bodies stimulates colonic transit, and the response is partly abrogated by vagotomy (211). As the vagus contributes innervation mainly to the proximal colon, activation of sacral parasympathetic outflow involved in the regulation of distal colonic motor function (139) also mediates the central action of CRF to stimulate colonic transit (160). Effector mechanisms within the colon that activate colonic transit involve parasympathetic mediated activation of 5-HT3 receptors because icv CRF-induced defecation is blocked by peripheral administration of atropine and the 5-HT3 antagonists, ramosteron and azasetron, whereas icv injection of these antagonists had no effects (199). These data suggest the activation of the peripheral serotonin pools from either enterochromaffin cells or enteric neurons that need further characterization. Activation of Corticotropin-Releasing Factor 1 Signaling Pathways in the Brain Mediates Various Stressors and Specific Brain Peptide–Induced Stimulation of Colonic Motor Function Studies in humans have shown that various stressors, including dichotomous listening, intermittent hand immersion in cold water, fear, anxiety, and stressful interviews, increased colonic motility in healthy subjects (212–216). Primates exposed to a social stressor, for example, an intruder paradigm, manifested stress responses including urination and defecation (15). In rodents, stressors as diverse as openfield test, conditioned fear, loud sound, restraint, cold exposure, fear conditioning, water avoidance, inescapable foot or tail shocks, and central injection of IL-1 activate propulsive colonic motor function as assessed by the shortening of transit time, stimulation of colonic myoelectric activity or motion, as well as induction of diarrhea (22,154,204, 217–219) (Fig. 30-6E). Convergent evidence indicates that central CRF peptides and receptors play an important role in the colonic motor response to stress. This is clearly established by the mimicry of the stress response by centrally administered CRF receptor agonists and the blockade of the colonic stress response by CRF receptor antagonists (220). The icv injection of α-helical CRF9-41 abolishes wrap restraint– and partial body restraint–induced stimulation of colonic transit in female and male rats (22,138). α-Helical CRF9-41, D-Phe12 CRF12-41, and astressin injected icv attenuated the defecatory response to wrap restraint in female and male Sprague–Dawley, female Lewis, and female Fischer rats (22,162,198,202). α-Helical CRF9-41 injected icv also antagonized the increased frequency of colonic spike-bursts induced by conditioned fear stress (204). The use of selective CRF1 receptor antagonists injected icv or peripherally and CRF1 receptor knockout mice established the importance of the brain CRF1 signaling pathways in the colonic motor response to various stressors in rodents

806 / CHAPTER 30

A

B

C

D

Plasma CORT (ng/ml)

500

E

No stress WAS (30 min)

400 300 200 100 0

10

No stress

WAS

Fecal pellet (Nb/h)

8 6 #

4 2 0

F

20

0

20

NBI 35965 (mg/kg, sc)

FIG. 30-6. Water avoidance in rats is a stress model that activates neurons, and corticotropin-releasing factor (CRF) gene transcription in the paraventricular nucleus (PVN) resulting in CRF1 receptor–mediated hypothalamic-pituitary-adrenal (HPA) stimulation (increase in adrenocorticotropin [ACTH] and corticosterone [CORT]) and activation of colonic motor response (defecation) in conscious rats. Fos expression, a marker of neuronal activation in the paraventricular nucleus of the hypothalamus in non stressed (A) and following 1-hour exposure to water avoidance stress (WAS) (B). Dark-field micrograph showing the induction of CRF heteronuclear RNA signal in the parvocellular portion of the paraventricular nucleus (pPVN) in nonstressed (C) and rats exposed to WAS for 30 minutes (D). Increase in plasma corticosterone levels induced by 30 minutes of WAS; *p < 0.005 compared with nonstress (E). Stimulation of colonic motor function assessed by defecation score induced by 60 minutes WAS and the inhibitory action of CRF1 antagonist pretreatment (F). mPVN, magnocellular portion of the paraventricular nucleus; sc, subcutaneous. (A: Modified from Bonaz and Taché [198], by permission; B, C: Modified from Kresse and colleagues [228], by permission; D: Modified from Million and colleagues [79], by permission.)

and monkeys (221) (Table 30-5). In particular, CP-154,526, CRA 1000, NBI 27914, NBI 35965, and antalarmin injected icv or peripherally blunt the acceleration of colonic transit induced by restraint and defecation in response to WAS (see Fig. 30-6E), restraint, social stress, and the diarrhea elicited by morphine withdrawal. In mice, restraint stress–induced defecation was abolished by icv NBI 35965, whereas the astressin2-B injected icv at doses that blocked CRF2-mediated action on gastric emptying had no effect (154). In an openfield test, CRF1 knockout mice had significantly less defecation than wild-type mice (222), providing additional support for the involvement of CRF1 signaling pathways in the colonic motor response to stress. In addition to stress, central CRF receptors are recruited as part of brain pathways through which centrally acting neuropeptides or cytokines stimulate colonic motility. In particular, icv α-helical CRF9-41 abolished IL-1β–induced increases in spike-burst frequency in the colon, indicative that the brain CRF receptors are involved in immune-related alterations of distal colonic motor function (223). The icv injection of α-helical CRF9-41 or astressin prevents the stimulation of colonic transit induced by neuropeptide Y (NPY) microinjected into the PVN or by icv CART (224,225), whereas not altering icv GRP-induced stimulation of colonic transit (138). These data indicate that selective peptides such as CART, as well as NPY, known to activate CRF-containing neurons in the PVN (226,227), recruit brain CRF signaling pathways that impact the regulation of colonic motility. Notably, all the centrally administered CRF receptor antagonists by themselves do not affect basal or postprandial colonic transit and motility or defecation events, indicating that central CRF receptors do not play a role in the regulation of colonic motor function under nonstress conditions (220). Neurofunctional studies support the PVN and LC/ Barrington’s complex as sites of action for CRF to stimulate colonic motor function. These brain sites are activated in response to WAS, as shown by Fos expression as a marker of neuronal activation (198) (see Fig. 30-6B) compared with nonstressed rats that have low basal Fos (see Fig. 30-6A). WAS induces a rapid CRF gene transcription in the PVN (see Fig. 30-5D) and an increase in plasma corticosterone (see Fig. 30-5E) compared with the absence of CRF gene transcription under resting conditions (see Fig. 30-5C) (228). The icv injection of α-helical CRF9-41 reduced by 44% and 60% Fos expression induced by WAS selectively in these hypothalamic and pontine nuclei, respectively, together with the 60% reduction in the defecation score (198). In addition, α-helical CRF9-41 microinjected into the PVN prevents partial restraint– and WAS-induced stimulation of colonic transit and defecation (160,206). Likewise, Lewis rats that have a blunted hypothalamic CRF response to stress exposure (229) displayed a reduced activation of neurons in the PVN and sacral parasympathetic nucleus, as shown by Fos expression, that are correlated with an attenuated colonic motor response (202). One study shows that the activation of the ARC by kainate stimulates colonic transit, which is

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 807 TABLE 30-5. Stress-induced stimulation of colonic motor function: Blockade by nonpeptide corticotropin-releasing factor subtype 1 antagonists CRF antagonists

Dose (route)

Stress/Species

Colonic inhibition

CP-154,526 CP-154,526 CP-154,526 CRA 1000 NBI 27914 NBI 35965 NBI 35965 Antalarmin CRF1 knockout

20 mg/kg (sc) 20 mg/kg (sc) 30 mg/kg (sc) 20 mg/kg (sc) 100 µg/rat (icv) 50 µg/mice (icv) 20 mg/kg (sc) 20 mg/kg (po) —

Partial restraint/female rats WAS/male rats Morphine withdrawal/male rats Morphine withdrawal/male mice WAS/male rats Restraint/mice WAS/male rats Social intruder/male monkeys Open-field test/female mice

Transit (55%) Defecation (55%) Diarrhea ∼(50%) Diarrhea ∼(50%) Defecation (67%) Defecation (100%) Defecation (53%) Defecation (40%) Defecation (p = 0.0008)

Reference 274 275 276 277 167 154 79 15 222

CRF, corticotropin-releasing factor; icv, intracerebroventricular; po, by mouth; sc, subcutaneous; WAS, water avoidance stress.

blocked by astressin, injected into the PVN but not into extra PVN sites (211). Tracing studies showing that a proportion of CRF-immunoreactive neurons in the Barrington’s nucleus and PVN are linked transsynaptically to visceral organs such as the colon provide further anatomic support for the role of pontine and PVN CRF signaling pathways in the regulation of pelvic organ functions (230,231).

Increased Colonic Permeability Given the antigenic load that the gut has in its lumen, keeping the integrity of the epithelium is critical to prevent entrance of rogue organisms and agents into the body. Gastrointestinal epithelium is equipped with tight junction and transcellular and paracellular transport systems to assure not only barrier functions against toxic or infectious agents, but also to create concentration gradients that are essential for normal functioning (232). Epithelial barrier function could be compromised in the face of direct physical, chemical, or neurohumoral assaults to the epithelium. There is now growing evidence that stress directly impacts the barrier function of the gut, as described in detail in Chapter 28. However, the role of central CRF signaling pathways remains to be characterized. Inflammation Activation of Brain Corticotropin-Releasing Factor Receptors Dampen Experimental Colitis Several studies have shown the anti-inflammatory role of central CRF on the colon. Repeated icv injections of CRF inhibit the development of chronic trinitrobenzensulphonic acid (TNBS) colitis in female Fischer and Lewis rats (233). Conversely, blockade of brain CRF receptors by icv α-helical CRF9-41 or astressin worsened stress-induced aggravation of colitis (233,234), suggesting that central CRF exerts an anti-inflammatory effect in the colon in Wistar, Lewis, and Fischer rats. The protective role of endogenous brain CRF was also revealed in Lewis rats, which display a decreased hypothalamic CRF response to stress compared with the Fischer

strain (229). Repeated stress worsened the development of colitis in both strains, but the level of inflammation was greater in the Lewis rats, which displayed lower corticosterone response to repeated WAS and restraint stress (233). Some studies further extend these observations by showing that chronic stress enhanced colitis induced by dextran sulfate consumption only in a subgroup of rats that displayed low corticosterone levels (235). In addition, adrenalectomy, without implantation of corticosterone pellet, is lethal to rats with colitis (236). Therefore, centrally mediated CRF-mediated activation of the HPA axis and the related hypersecretion of glucocorticoids provide a major anti-inflammatory mechanism to modulate colitis. Colitis Modulates Hypothalamic Corticotropin-Releasing Factor Signaling Pathways The alterations of the HPA axis under conditions of experimental colitis have been demonstrated (228,236). In particular, it was established that TNB-induced colitis results in CRF gene expression in the hypothalamic magnocellular neurons of the PVN, whereas dampening the acute stressinduced CRF gene transcription, in the pPVN, and plasma corticosterone response (228). TNBS also induces c-fos mRNA expression in brain nuclei involved in the autonomic, behavioral, and neuroendocrine responses to a stimulus (PVN, amygdala, LC, parabrachial nucleus, NTS) and in circumventricular organs (lamina terminalis, subfornical organ, area postrema) (237). CRF pathways, particularly in the PVN, were activated in this model as evidenced by a robust signal of c-fos and CRF1 receptor transcripts in the PVN (237). In addition, numerous CRF neurons expressed c-fos or CRF1 receptor transcripts in the PVN of TNBStreated animals (237). Taken together, the brain CRF signaling pathway not only modulates, through its HPA axis and the sympathetic-adrenalmedullary axis, the intestinal inflammatory response to stress (238), but it also plays a role in the perception and processing of systemic stress stimuli to and from the intestinal tract. Thus, brain CRF signaling occupies a strategic place to modulate the intestinal response to a variety of stress stimuli, including systemic inflammation to eventually bring homeostasis.

808 / CHAPTER 30 5000

An initial report by Gué and colleagues (239) established that icv injection of CRF mimicked the stress-induced colonic hyperalgesia in male Wistar rats submitted to colorectal distention. This represents a centrally mediated action because peripheral administration of CRF did not reproduce the colonic hypersensitivity (240). The role of central CRF receptors in the hyperalgesic response was further shown by the fact that both stress- or icv CRF–induced colonic sensitization to colorectal distention were blocked by icv injection of α-helical CRF9-41 (239). Convergent evidence indicates that central CRF1 receptor mediates the development of colonic hypersensitivity to stress or repeated colorectal distention. Intracerebroventricular CRF-induced colonic hypersensitivity to tonic colorectal distention is blocked by antalarmin in rats (241). When adult male Long Evans rats subjected to neonatal maternal separation were exposed to acute WAS, they experienced development of colonic hypersensitivity to the poststress second set of phasic colorectal distention, and the response was blocked by prior oral treatment with NBI 35965 (79). In male Wistar rats, acute WAS induced a delayed colonic hypersensitivity to phasic colorectal distention that was prevented by pretreatment with CP-154,526 before the stress exposure (242). Similarly, the colonic hypersensitivity induced by two sets of repeated tonic colorectal distention in female rats (Fig. 30-7A) was abolished by antalarmin (240) (see Fig. 30-7B). Likewise, in the anxiety-prone Wistar–Kyoto rats, intracolonic instillation of acetic acid–induced colonic hyperalgesia to a second set of tonic colorectal distention was inhibited by antalarmin (241). Both CP-154,526 and antalarmin are CRF antagonists that readily cross the blood–brain barrier. Mechanisms of Corticotropin-Releasing Factor Antagonist-Induced Blockade of Colonic Hypersensitivity Central CRF1 receptors have been implicated in the activation of the sympathetic nervous system (243). However, such a mechanism does not play a role in stress-related colonic hypersensitivity because sympathectomy did not affect the delayed hypersensitivity induced by WAS in Wistar rats (242). CRF1 receptor antagonists may exert a beneficial effect by preventing the activation of LC neurons induced by colorectal distention. Electrophysiologic studies in anesthetized rats showed that [DPhe12] CRF12-41, administered icv or microinfused into the LC, ic astressin, and iv NBI 35965 prevented LC neuronal activation in response to central injection of CRF and colorectal distention (244,245). In the periphery, mast cell stabilizer doxantrazole prevents icv CRF and stress-induced colonic hypersensitivity to a second set of colorectal distention (239). Central CRF may

4000 500 sec

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A 600 % difference of AUC (2nd vs 1st distention)

Central Corticotropin-Releasing Factor 1 Receptors Are Involved in Stress-Related Hypersensitivity to Colorectal Distention

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FIG. 30-7. Corticotropin-releasing factor 1 receptor (CRF1) antagonist, antalarmin, prevents colonic hypersensitivity induced by repeated tonic colorectal distentions in female rats. (A) Representative illustration of abdominal contraction traces from rats with acute strain gauge on the external abdominal muscles. Hypersensitivity is observed at the second set of two 3-minute On/Off distentions at 30, 40, and 60 mm Hg pressure performed 30 minutes after the first set. (B) Antalarmin inhibits colonic hypersensitivity to repeated colorectal distention. AUC, area under the curve; sc, subcutaneous. *p < 0.05 vs. vehicle. (Modified from Million and colleagues [240], by permission.)

contribute to colonic hypersensitivity by activating colonic mast cells. The icv injection of CRF induced a rapid increase in the release of rat mast cell protease II, prostaglandin E2, and mast cell histamine levels in the colon (246,247), and α-helical CRF9-41 injected icv prevents both icv CRF and stress-induced enhancement of colonic mast cell content of histamine (247). Relevance of the Central Corticotropin-Releasing Factor Signaling Pathways in Irritable Bowel Syndrome There has long been clinical suspicion that stress plays a role in functional diseases such as irritable bowel syndrome (IBS) symptom generation and exacerbation (248). The clinical relevance of the stress effect on functional diseases is exemplified by the studies that showed that psychological factors including life events and hypochondria measures are clear predictors of the development of IBS after gastroenteritis (249). Patients with IBS also display an exaggerated colonic motor response to mental stress (212,213). Ford and colleagues (250) showed that mental stress increased levels of sensations of gas and pain during sigmoid distentions in

CENTRAL CORTICOTROPIN-RELEASING FACTOR / 809 healthy volunteers. Stressful life events often are reported to be associated with onset or exacerbation of IBS symptoms (251). In addition, enhanced colonic mechanosensation in the absence of colonic injury is the hallmark of IBS (252,253). Existing preclinical evidence (see earlier) that CRF1 receptor antagonists prevent stress and repeated colorectal distention–related colonic hypersensitivity may suggest that activation of brain CRF1 receptors are part of the underlying mechanisms of IBS symptoms (220).

CONCLUSION The past decade has witnessed rapid advances in our knowledge of brain-gut axis as it relates to the underlying mechanisms of stress-related alteration of gut function (16,171). The characterization of CRF, Ucn 1, 2, and 3 (see Table 30-1), as well as the cloning of CRF1 and CRF2 receptors and their variants (see Table 30-2), and the development of selective CRF receptor subtype antagonists (see Table 30-3) have intensified investigations of the relative roles of receptors and ligands in the various aspects of the stress response (14,34,61). It is clear that CRF1 or CRF2 receptors, or both, are involved in circuits that are called on to coordinate the stress responses through endocrine, autonomic, behavioral, and immune arms (14,254). The gastrointestinal tract, by virtue of its key position as an interface between the internal milieu and external environment, is profoundly affected by stress stimuli and is endowed with an intricate physical and neurochemical regulatory system. As detailed in this chapter, activation of central CRF signaling contributes to the prominent gastrointestinal secretory and motor and hyperalgesic responses by engaging discrete brain regions, such as the hypothalamus, the pontine nuclei LC/SC, as well as the ANS and HPA axis (see Figs. 30-4, 30-5, and 30-8). This large line of evidence provides new insight, at the molecular levels, on the early seminal reports by William Beaumont, Walter Cannon, and Steward Wolf (23,142,255,256) that brain alters gut function under stress conditions. Of interest are preclinical observations showing that activation of brain CRF1 pathways reproduces some symptoms in IBS as it relates to the induction of colonic hypersensitivity to colorectal distention, enhanced bowel movements and diarrhea (220) (see Fig. 30-8), and comorbidity with anxiety and depression (257,258), as well as the potential role of brain CRF signaling in the early phase of postoperative gastric ileus (174,220).

ACKNOWLEDGMENTS This work was supported by the National Institute of Arthritis, Metabolism and Digestive Diseases grants R01 DK-33061, R01 DK-57236, and DK57238-01A1S1; Center grant DK-41301 (Animal Core); grant P50 AR-049550; and VA Merit and Senior Scientist Awards. The authors thank Drs. J. Rivier (Salk Institute, La Jolla, CA), D. Grigoriadis (Neurocrine Biosciences Inc., La Jolla, CA), and E. D. Pagani

Central CRF: Effects on the large intestine

Motor alteration, inflammation & visceral pain Effects

Stimulates -Transit -Defecation -Diarrhea -Motility

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FIG. 30-8. Summary representation of the effects of central corticotropin-releasing factor (CRF) on colonic motor function and visceral hypersensitivity and experimental colitis through action of CRF1 receptors. Note that central CRF stimulates colonic motor response and induces hypersensitivity to colorectal distention, and inhibits colitis induced by trinitrobenzensulphonic acid (TNBS) primarily through the activation of CRF1 receptors. HPA, hypothalamic-pituitaryadrenal; ic, intracisternal; icv, intracerebroventricular; LC, locus ceruleus; PVN, paraventricular nucleus.

(Center Research Division, Pfizer Inc., Croton, CT) for the generous supply of different CRF agonists and antagonists used in these studies. Miss Teresa Olivas’s help in the preparation of the manuscript is gratefully acknowledged.

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CHAPTER

31

Neural Regulation of Gastrointestinal Blood Flow Peter Holzer Physiologic Relevance of the Gastrointestinal Circulation, 817 Organizational Characteristics of the Gastrointestinal Circulation, 817 Innervation of Gastrointestinal Blood Vessels, 819 Multiplicity of Gastrointestinal Vasomotor Neurons, 819 Origin, Distribution, and Chemical Coding of Gastrointestinal Vasomotor Neurons, 819 Functional Implications of Vasomotor Neurons in the Regulation of Gastrointestinal Blood Flow, 820 Sympathetic Vasoconstrictor Neurons, 820 Parasympathetic Vasodilator Pathways, 822 Enteric Vasodilator Neurons, 823 Primary Afferent Vasodilator Neurons, 825

Interactive Control of Gastrointestinal Circulation, 830 Interactions between Enteric and Sympathetic Vasomotor Neurons, 830 Interactions between Primary Afferent and Sympathetic Vasomotor Neurons, 831 Interactions between Primary Afferent and Enteric Vasodilator Neurons, 831 Influence of Local and Paracrine Mediators on Gastrointestinal Vasomotor Neurons, 831 Summary, 832 Acknowledgments, 832 References, 832

PHYSIOLOGIC RELEVANCE OF THE GASTROINTESTINAL CIRCULATION

The mucosal barrier in the lower esophagus, stomach, and duodenum prevents back-diffusion of luminal acid into and damage to the mucosa. This defensive process depends to a large extent on the blood-borne transport of bicarbonate from the parietal cells to the mucosal surface, where alkali is needed to neutralize the acid that intrudes the gel-like mucous layer lining the surface epithelium (4–8). In light of these circulatory demands, it is not surprising that the GI mucosa is among the tissues that are most susceptible to ischemic insults. Intestinal hypoperfusion, together with intestinal motor stasis, in critically ill patients causes a sequence of bacterial overgrowth, intestinal barrier disruption, bacterial translocation, sepsis, and multiple organ dysfunction or failure (2,9). Recognition of these gut-driven complications and the association of ischemic colitis with irritable bowel syndrome (10) have led to a renewed interest in GI circulation and its regulation (see Chapter 41).

Adequate blood flow is critical to the physiologic function of the gastrointestinal (GI) tract, and inappropriate perfusion of the gut is quickly followed by functional disturbance and structural deterioration of the mucosa (see Chapter 65). The mucosal lining of the alimentary canal is one of the most highly perfused tissues in the body, receiving up to 80% of the total resting blood flow to the gut (1,2). This rate of vascular perfusion is necessary to meet the high oxygen utilization associated with GI secretion and absorption (2,3). Further roles of the GI circulation are to deliver nutrients and hormones to the gut, to distribute hormones released from GI endocrine cells and absorbed nutrients, and to remove metabolic products and other waste material. In addition, adequate blood perfusion is of paramount relevance to maintenance of the GI mucosal barrier, which hinders transepithelial migration of toxic chemicals, antigens, and pathogenic microorganisms (2).

ORGANIZATIONAL CHARACTERISTICS OF THE GASTROINTESTINAL CIRCULATION

P. Holzer: Department of Experimental and Clinical Pharmacology, Medical University of Graz, A-8010 Graz, Austria. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

The blood supply of the mammalian GI tract is delivered by three branches of the aorta. The stomach and liver are

817

818 / CHAPTER 31 supplied by the celiac artery, the small intestine and proximal colon by the superior mesenteric artery, and the distal colon by the inferior mesenteric artery (2). Total blood flow through these arteries is in the range of 25% of the cardiac output in the interdigestive state (2). It has long been known that food intake, as well as mechanical or chemical stimulation of the GI mucosa, leads to a reflex increase in blood flow, the hyperemia figuring most prominently in the submucosal arterioles (2–4,11,12). These observations reflect the organizational principles of GI blood flow regulation. Apart from giving off some arterioles to the external muscle layers, the mesenteric arteries feed primarily into a network of arterioles in the GI submucosa. This submucosal arteriolar plexus issues microvessels perpendicularly through the mucosa and other microvessels that run into the external muscle layer (1,5). The extensive capillary networks in the mucosa and muscle drain into venules that, in general, follow the pattern of arterioles. Because of this arrangement, about three fourths of the blood delivered to the GI tract is fed into the submucosalmucosal arteriolar circuit (4,11,13). Consequently, GI mucosal blood flow is largely determined at the level of the submucosal arterioles, which constitute the final resistance vessels of the mesenteric circulation (1,4,5,14) and represent the most important target for blood flow regulation, although the feed arteries are also relevant (3,15). Another aspect of regulation, capillary recruitment, is relevant to

postprandial hyperemia. Whereas in the interdigestive state only a fourth of the mucosal capillaries is perfused to a normal extent, many capillaries open after ingestion of a meal to contribute to postprandial hyperemia (2). A third possibility of local blood flow regulation, namely the opening of arteriovenous anastomoses, does not apply to the stomach (4,5). In the intestine, however, there is an extensive network of intramural and extramural collateral blood vessels so that occlusion of a major mesenteric artery does not result in the expected reduction in intestinal blood flow (16,17). The GI vascular system is regulated by an elaborate system of metabolic, endocrine, and neural control mechanisms to adjust GI blood flow to a wide range of physiologic needs (2,3,12,15,18). The GI resistance vessels are densely innervated by primary afferent and sympathetic efferent neurons and, in addition, receive inputs from parasympathetic efferent and enteric neurons (Fig. 31-1). It has long been known that neurogenic constriction of GI blood vessels results from activation of sympathetic pathways (3,19,20). In contrast, the mechanisms whereby GI blood flow is increased during digestive activity or in the face of pending injury remained little understood, and it was only in the past 15 years that the complex mechanisms of neurogenic vasodilatation in the GI tract have been elucidated (3,21). The chief aim of this chapter is to review the function and relevance of the multiple vasomotor pathways that regulate GI blood flow and their mutual interactions.

DRG Primary afferent

Sympathetic

Parasympathetic

ACh PVG

LM ACh

MP ATP/NE − CGRP +

CM ACh +

Arteriole

VIP +

Enteric SM MU

FIG. 31-1. Diagrammatic overview of the multiple innervation of gastrointestinal resistance vessels. Plus signs denote vasodilatation; minus signs denote vasoconstriction. ACh, acetylcholine; ATP, adenosine triphosphate; CGRP, calcitonin gene-related peptide; CM, circular muscle; DRG, dorsal root ganglion; LM, longitudinal muscle; MP, myenteric plexus; MU, mucosa; NE, norepinephrine; PVG, prevertebral ganglion; SM, submucosa; VIP, vasoactive intestinal polypeptide.

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 819 INNERVATION OF GASTROINTESTINAL BLOOD VESSELS Multiplicity of Gastrointestinal Vasomotor Neurons Unlike the blood vessels of other organ systems, the vasculature supplying the digestive tract is under the control of as many as four different divisions of the nervous system (see Fig. 31-1). Neurogenic constriction of GI blood vessels results solely from activation of sympathetic pathways (3,19,20), whereas neurogenic vasodilatation in the GI tract is governed by three different classes of neurons: enteric neurons (1) that also relay regulatory input from efferent neurons of the parasympathetic nervous system (2), and extrinsic primary sensory neurons (3). These afferent neurons originate from dorsal root ganglia and can regulate vessel diameter indirectly via autonomic reflexes and directly through release of vasoactive neuropeptides from their perivascular plexus of fibers (21). Origin, Distribution, and Chemical Coding of Gastrointestinal Vasomotor Neurons

have synapse-like specializations, except that varicosities may come as close as less than 100 nm to vascular smooth muscle cells (29). Because only the outer layer of the vascular smooth muscle layer is in contact with noradrenergic axons, effective sympathetic neuromuscular transmission relies on electrical coupling among the muscle cells (30). Although the principal transmitter of preganglionic sympathetic neurons is acetylcholine, norepinephrine (noradrenaline) is the main transmitter of postganglionic sympathetic nerve fibers. However, there is now unequivocal evidence (3,31) that sympathetic constriction of the splanchnic circulation is comediated by adenosine triphosphate (ATP) and norepinephrine (see Fig. 31-1). Immunohistochemical investigations have shown that sympathetic effector neurons can be identified by their organotopic “chemical coding,” that is, the combination of coexisting messenger molecules that varies with different innervation targets (32). Thus, noradrenergic neurons supplying GI blood vessels contain neuropeptide Y (NPY) but not somatostatin, whereas those innervating enteric ganglia may contain somatostatin but do not express NPY (23,32–35).

Sympathetic Neurons

Parasympathetic Neurons

The preganglionic fibers of the sympathetic pathways to the GI tract originate from the thoracic and lumbar levels of the spinal cord. Although part of the ganglionic transmission takes place in the paravertebral ganglia of the sympathetic chain, only a minority of the postganglionic sympathetic neurons supplying the digestive tract is derived from the paravertebral ganglia (22,23). Most preganglionic fibers project to the prevertebral ganglia (celiac ganglion, superior mesenteric ganglion, and inferior mesenteric ganglion), where they synapse on postganglionic neurons (19,22,23). Typically, the efferent fibers of sympathetic pathways reach the prevertebral ganglia via the thoracic and lumbar splanchnic nerves. In the foregut, however, some postganglionic sympathetic nerve fibers originating from sympathetic chain ganglia, notably the superior cervical and stellate ganglia, reach the stomach and small intestine via the vagus nerves (24,25). The organotopic projections of postganglionic neurons from the prevertebral ganglia reach the alimentary canal as discrete nerves or as paravascular nerves using the track taken by the arteries as a conduit. Tyrosine hydroxylase–positive noradrenergic axons of extrinsic sympathetic origin form a dense plexus of fibers around arteries and arterioles in the GI tract of rat, guinea pig, rabbit, and cat (26–28). In contrast, capillaries appear to lack an innervation by noradrenergic axons, and intramural venules and veins receive only a sparse supply by sympathetic nerve fibers, whereas extramural capacitance veins are surrounded by an appreciable number of noradrenergic axons (27). Within the vessel wall, the plexus of noradrenergic fibers is situated in the vascular adventitia and at the border between adventitia and media (27,28). The terminal axons exhibit swellings or varicosities that are the sites of transmitter release. The sympathetic neuromuscular junctions usually do not

The parasympathetic innervation of the GI tract is provided by the vagus and pelvic nerves. Whereas the preganglionic nerve fibers in the pelvic nerves originate in the sacral spinal cord, those running in the vagus nerves emanate from the vagal nuclei (dorsal vagal motor nucleus and nucleus ambiguus) of the brainstem and project to the enteric ganglia of the GI tract, where they impinge on enteric neurons within the myenteric plexus (32,36,37). Thus, in the gut, there are no distinct “postganglionic parasympathetic” neurons, and the effects that parasympathetic pathways have on GI functions including blood flow are relayed by the enteric nervous system (see Fig. 31-1). The principal transmitter of the preganglionic parasympathetic neurons is acetylcholine, which is also the major mediator of the postganglionic parasympathetic pathways in the enteric nervous system. However, the chemical coding of enteric neurons is highly elaborate (32,38–42), and the enteric vasodilator neurons that receive input from preganglionic parasympathetic nerve fibers have been little characterized. Vasoactive intestinal polypeptide (VIP) may be one vasodilator messenger of these pathways, given that vagal efferent fibers make contact with myenteric neurons containing VIP (36). Nitric oxide (NO) synthase and gastrin-releasing peptide also have been found in enteric neurons receiving vagal efferent input (36), but it is unknown whether these neurons project to GI blood vessels. Enteric Neurons The intrinsic innervation of the GI tract originates from the two ganglionated plexuses of the enteric nervous system, the myenteric and submucosal plexuses (32). Parasympathetic and sympathetic neurons are in close contact with enteric

820 / CHAPTER 31 neurons, which, in the stomach, also form synaptic connections with vagal afferent neurons (43). Enteric neurons innervate all layers of the digestive system including the vasculature, and their distinct projections to the GI effector tissues have been identified with neuroanatomic lesion and tracing techniques combined with functional studies (3,32,38–42,44). These studies have shown that arteries and arterioles in the GI submucosa and mucosa are innervated by enteric neurons (28,44,45) that originate in the submucosal, but not myenteric, plexus (see Fig. 31-1). One population of these neurons is cholinergic, as revealed by the presence of choline acetyltransferase and the vesicular acetylcholine transporter (28,44–47). The other, noncholinergic population stains for VIP, may also contain galanin and dynorphin (28,44,48–52), and exhibits a distribution in the arteriolar vessel wall similar to that of sympathetic neurons (48,53). Further markers of enteric vasomotor neurons include gastrin-releasing peptide, enkephalin, and somatostatin (51,52). Unlike arterioles, venules and veins receive only a loose innervation by VIP-immunoreactive nerve fibers. Enteric vasomotor neurons in the guinea pig colon also express substance P, whereas this neuropeptide is largely absent from arteriolar projections of submucosal enteric neurons in the guinea pig small intestine (47,54,55). Apart from their chemical coding, enteric vasomotor neurons can be differentiated by their morphology and projection targets (44,56) (see Chapter 21). Many of these neurons appear to project both to submucosal arterioles and the mucosal epithelium, and thus appear to subserve both a vasodilator and secretomotor function. Although the axons of cholinergic secretomotor/vasodilator neurons pass through many submucosal ganglia, those of noncholinergic secretomotor/vasodilator neurons run only short distances within the plexus (44). Myenteric neurons expressing VIP also innervate extramural arteries supplying the GI tract (35,48,50,57). However, these intestinofugal neurons project mainly to the prevertebral ganglia, and there make synaptic contact only with those sympathetic neurons that do not innervate splanchnic blood vessels (33,34,57). Primary Afferent Neurons The splanchnic arterial system is richly supplied by primary sensory neurons that originate from dorsal root ganglia (see Fig. 31-1; see Chapter 25). These spinal afferents are pseudounipolar neurons that give off centrally and peripherally directed processes. The short central processes project into the dorsal horn of the spinal cord, whereas the long peripheral processes supply both somatic and visceral tissues in a segmental organotopic manner (58–61). On their way to the GI tract, the peripheral fibers of visceral afferents take the course of sympathetic nerve trunks and pass through the prevertebral ganglia, where they give off collaterals to form axodendritic and axosomatic synapses with the sympathetic ganglion cells (57,59,62–65). Within the abdomen, the major targets of sensory vasomotor neurons are the mesenteric arteries and intramural arterioles (66), whereas the venous

system is sparsely innervated (67–69). Although the network of varicose afferent nerve fibers surrounding the mesenteric resistance vessels become looser toward the gut wall, the submucosal and mucosal arterioles retain a prominent perivascular plexus of sensory nerve fibers (59,62,67,69–75). Like sympathetic and enteric axons, the afferent nerve fibers are situated in the adventitia and at the border between adventitia and media but hardly penetrate into the muscle layer. Analysis of the chemical coding has shown that practically all spinal afferent neurons projecting to the abdominal viscera of the rat and guinea pig synthesize calcitonin gene–related peptide (CGRP), and a majority of them contains substance P (59,75–79); other peptides such as galanin and dynorphin might also occur. Regarding the rodent stomach, CGRP can be considered as a quantitative and exclusive marker of spinal afferent nerve endings, because the expression of this peptide in other neurons is negligibly small (59,62,73–75). The occurrence of CGRP and substance P by sensory neurons supplying the splanchnic vasculature of other species has been less well examined but appears to be subject to species differences (80,81). CGRP occurs in two isoforms, CGRP-α and -β (77), and most of the CGRP synthesized by primary afferent neurons of the rat is CGRP-α (75,82–84). Neurogenic changes in splanchnic blood flow comprise sympathetic vasoconstriction and cholinergic, as well as nonadrenergic, noncholinergic (NANC), vasodilatation. Apart from noncholinergic enteric vasodilator neurons, CGRP-containing nerve fibers originating from the dorsal root ganglia have been identified as the most important group of NANC vasodilator neurons in the GI vasculature (3,18,21,66,76,85). Theoretically, vagal afferents could also belong to this class of neurons, given that the GI tract is supplied by many afferent neurons that run in the vagus nerves and originate from the nodose ganglia. Besides other targets, these vagal afferent nerve fibers innervate the rat portal vein (86) and submucosal blood vessels in the rat stomach (87,88). The chemical coding of these neurons, however, is largely unknown, because less than 10% of the vagal afferent nerve fibers to the rat, mouse, and guinea pig stomach contain CGRP and substance P (59,75,88).

FUNCTIONAL IMPLICATIONS OF VASOMOTOR NEURONS IN THE REGULATION OF GASTROINTESTINAL BLOOD FLOW Sympathetic Vasoconstrictor Neurons Effects of Nerve Stimulation The available evidence indicates that neurogenic vasoconstriction in the splanchnic circulation results solely from activation of the sympathetic nervous system, and that the sympathetic nerve fibers supplying the GI vasculature are vasoconstrictor fibers (3). Electrical stimulation of sympathetic pathways to the stomach decreases total gastric

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 821 blood flow, blood flow in the celiac and gastroepiploic arteries, and blood flow in the gastric mucosa, the latter being reduced more significantly than total gastric blood flow (4,5,15,89,90). Blood flow to the small and large intestines is similarly reduced by sympathetic nerve stimulation. This is because of constriction of the mesenteric arteries and intramural blood vessels, particularly the submucosal arterioles within the GI wall (3,31,91–96). The reduction of rat mesenteric blood flow caused by cigarette smoke (97) is probably due to nicotine-mediated stimulation of sympathetic constrictor neurons, because nicotine does not constrict the isolated and perfused mesenteric vascular bed (98). Apart from increasing the resistance of the arterial system, sympathetic nerve stimulation also constricts the larger veins of the GI tract, thus decreasing the volume of blood contained in the splanchnic vasculature (12,32,92). The decrease in GI blood flow wanes after 1 to 3 minutes of prolonged sympathetic nerve stimulation to reach a new steady-state level, a phenomenon known as “autoregulatory escape” (91,93,94,99). When stimulation is stopped, flow may increase to a level greater than that recorded before stimulation, a process referred to as “post-nerve stimulation hyperemia” (95). The escape from sympathetic constriction of submucosal arterioles in the rat gastric submucosa is not caused by opening of arteriovenous shunts, but rather results from relaxation of the initially constricted vessels (91). Further studies have indicated that autoregulatory escape in the stomach (99) and intestine (94) and post-nerve stimulation hyperemia (95) are active processes that involve sensory vasodilator nerves. Transmitter Mechanisms Norepinephrine and Adenosine Triphosphate as Principal Cotransmitters Although traditionally norepinephrine has been considered to be the main transmitter of sympathetic vasomotor neurons, sympathetic constriction of splanchnic resistance

vessels is comediated by the “sympathetic triad” of ATP, norepinephrine, and NPY (Table 31-1). ATP and norepinephrine are potent vasoconstrictors that act directly on vascular smooth muscle, whereas NPY has comparatively little direct effect on the muscle but facilitates the action of norepinephrine and ATP (96,100,101). The cotransmitter role of ATP and norepinephrine (see Fig. 31-1) has been demonstrated in the mesenteric arterial bed of the rat, rabbit, cat, and dog (102–105). Electrical stimulation of the perivascular sympathetic nerve supply leads to a biphasic response that consists of an initial rapid contraction followed by a prolonged, sustained vasoconstriction. Pharmacologic analysis has revealed that, typically, ATP acting via ionotropic P2X purinoceptors is the dominant mediator of the initial fast vasoconstrictor response, particularly if the sympathetic nerve supply is stimulated at low frequencies, whereas norepinephrine acting via a1-adrenoceptors is responsible for the delayed and maintained phase of constriction (100–107). The relative contribution that ATP and norepinephrine make to sympathetic vasoconstriction varies along the tree of the mesenteric arterial bed. Whereas sympathetic constriction of large mesenteric arteries is mediated by both ATP and norepinephrine, that of small arteries and intramural arterioles is predominantly brought about by ATP (31,102–108). Specifically, ATP seems to be the sole mediator of sympathetic constriction in small arteries of the rabbit jejunum (107,108) and in submucosal arterioles of the guinea pig small intestine (31). This inference is based on the findings that neither adrenoceptor antagonists nor depletion of norepinephrine stores by reserpine reduces sympathetic vasoconstriction, whereas both the neurogenic and ATP-induced vasoconstrictor responses are blocked by P2 purinoceptor antagonists and by desensitization to the P2X purinoceptorselective agonist α,β-methylene ATP (31,107,108). The role of ATP and norepinephrine in sympathetic vasoconstriction is consistent with their ability to contract mesenteric arteries, as well as intramural arteries and arterioles of

TABLE 31-1. Overview of the vasomotor neurons and their transmitters that control splanchnic blood flow Vasomotor neurons

Transmitters (receptors)

References

Sympathetic vasoconstrictor neurons

Adenosine triphosphate (P2X purinoceptors)a Norepinephrine (α1-adrenoceptors)a Neuropeptide Y (Y1 receptors) Acetylcholine (M3 receptors)a Vasoactive intestinal polypeptidea Substance P and neurokinin A (tachykinin NK1 receptors) Galanin Dynorphin Calcitonin gene–related peptide (CGRP1 receptors)

31, 100–108 31, 100–108 139 46, 47, 55, 152, 173 144, 152 47, 178

Enteric vasodilator neurons

Primary afferent vasodilator neuronsa

Substance P and neurokinin A (NK1 receptors) Nitric oxide aMajor

vasoactive transmitters of the splanchnic vasomotor neurons.

152 152 85, 195, 199, 200, 205, 209, 215, 219, 246, 252, 253, 267–273 205, 225, 273 194, 255

822 / CHAPTER 31 the rat, guinea pig, rabbit, and dog GI tract, without affecting the diameter of venules (31,102,105,106,108–111). Norepinephrine mimics the effect of sympathetic nerve stimulation to attenuate blood flow in the stomach of the dog, cat, and rat (4,5,112–114). The reduction of gastric blood flow caused by norepinephrine and sympathetic nerve stimulation is blocked by α-adrenoceptor antagonists (112,114,115) and most likely mediated by α1-adrenoceptors, because stimulation of α2-adrenoceptors by clonidine causes vasodilatation in the rat gastric mucosa (116). The lack of involvement of norepinephrine in the sympathetic constriction of small arteries in the rabbit jejunum and submucosal arterioles in the guinea pig intestine contrasts with the ability of norepinephrine to elicit strong constriction of these vessels via activation of α1-adrenoceptors (31,107–109). Because nerve stimulation does release norepinephrine from sympathetic axons surrounding small arteries and submucosal arterioles (31,102), it is thought that P2X purinoceptors are clustered at synapse-like junctions, whereas α1-adrenoceptors lie outside these specialized junctions, and therefore are not accessible to synaptically released norepinephrine (3,20,108). It has occasionally been observed that blockade of α-adrenoceptors reverses the constrictor action of sympathetic nerve stimulation and norepinephrine in the splanchnic vasculature to a slight dilator action (32). Because this type of vasodilatation is abolished by β-adrenoceptor antagonists, it follows that β-adrenoceptors mediating vasodilatation are present in the splanchnic circulation. This inference is in keeping with the ability of isoproterenol, a selective β-adrenoceptor agonist, to enhance blood flow in the gastric mucosa (117–119) and superior mesenteric artery (120) of the rat. The epinephrine-evoked increase in blood flow through the human superior mesenteric artery is likewise brought about by stimulation of β-adrenoceptors (121). Under physiologic circumstances, though, β-adrenoceptor–mediated vasodilatation in response to sympathetic nerve stimulation is not observed because it is overridden by vasoconstriction caused by α-adrenoceptor and P2X purinoceptor activation. In certain sympathetic neurons, dopamine, a precursor of norepinephrine, is considered to be a transmitter in its own right (122). Whether this applies to sympathetic vasomotor neurons innervating the splanchnic vasculature is unknown. Dopamine, which is used in critical care medicine to improve cardiac output and regional perfusion (123,124), exerts a species-dependent mixture of vascular effects because of its affinity for various monoamine receptors. The rabbit left gastric artery contracts in response to dopamine; once this constrictor effect is blocked by an α-adrenoceptor antagonist, dopamine dilates the artery via activation of dopamine receptors (125,126). Dopamine increases mucosal blood flow in the rabbit stomach (125,126) but decreases it in the rat stomach (116,127) and canine gut (128) through activation of α1-adrenoceptors. Under pathologic conditions, dopamine has been found to reduce gastric blood flow in septic patients (129,130) and to attenuate intestinal oxygenation in hemorrhagic pigs (131). Thus, dopamine does not

appear to have any beneficial action on GI circulation in critically ill patients (116,123,124,132). Neuropeptide Y NPY is a 36-amino-acid peptide that is coreleased with norepinephrine from sympathetic vasoconstrictor fibers (133). This peptide is a comparatively weak constrictor of the rat and pig mesenteric arterial bed (134,135) and of the rabbit gastroepiploic artery (136). Accordingly, NPY reduces rat gastric mucosal blood flow via a mechanism independent of α-adrenoceptors (113) and causes vasoconstriction in the feline colon (137). A prominent action of NPY is to potentiate the constrictor effect of norepinephrine and ATP on the rat mesenteric arterial bed (134,138,139), rabbit gastroepiploic artery (136,140), and rat portal vein (141). Both the vasoconstrictor response to NPY and its action to augment norepinephrine- and ATP-induced mesenteric vasoconstriction are mediated by postjunctional NPY Y1 receptors (134,135,138,139). Analysis of the response to sympathetic nerve stimulation in the rat mesenteric arterial bed with the Y1 receptor antagonist BIBP-3226 indicates that about 30% of the sympathetic vasoconstriction depends on NPY (139). Autoregulatory Inhibition of Sympathetic Neurotransmitter Release The sympathetic neuroeffector junctions in the splanchnic vascular bed are subject to feedback inhibition of sympathetic neurotransmitter release. This autoregulatory mechanism involves prejunctional α2-adrenoceptors targeted by norepinephrine (31,102,106,108), prejunctional adenosine receptors targeted by adenosine, a breakdown product of ATP (106), and prejunctional NPY Y2 receptors targeted by NPY (134,141,142). Consequently, the further release of both norepinephrine and ATP is blunted (31,107,108). Attenuation of the sympathetic vasoconstrictor tone is likely to contribute to the effect of the α2-adrenoceptor agonist clonidine to enhance blood flow in the rat gastric mucosa (116). In the small arteries and arterioles in which ATP is the sole mediator of sympathetic vasoconstriction, the only role of coreleased norepinephrine is to exert a negative feedback control of sympathetic neuroeffector transmission via prejunctional α2-adrenoceptors (3,31,107,108).

Parasympathetic Vasodilator Pathways In assessing the impact of parasympathetic efferent pathways on the splanchnic circulation, it is important to consider that parasympathetic vasomotor effects are relayed by enteric vasodilator neurons (32,36,37) and primary afferent vasodilator neurons (143). Electrical stimulation of the vagus nerve leads to an increase in blood flow through the stomach of the dog (91,116,144), cat, (145) and rat (146,147). As shown by videomicroscopic analysis, submucosal

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 823 arterioles in the gastric corpus dilate in response to vagal nerve stimulation (146), whereas they constrict after acute vagotomy (148). Total gastric blood flow (149) and gastric mucosal blood flow (150,151) are likewise reduced immediately after vagotomy. These observations suggest that vagal efferent neurons exert a tonic vasodilator influence on the gastric circulation. Cholinergic vasodilator mechanisms play only a minor role in the gastric vasodilator responses to electrical stimulation of the distal end of the severed vagus nerve in the dog and rat (144,146,147). Thus, gastric vasodilatation caused by short stimulation of the vagus nerve in the rat remains unchanged by atropine (146), whereas that caused by prolonged vagal nerve stimulation is attenuated (147). Although acetylcholine can dilate submucosal and mucosal arterioles in the rat stomach (111,146) and the majority of enteric vasodilator neurons in the guinea pig intestine is cholinergic (46,152), it follows that electrically evoked vagal output to the stomach activates primarily a noncholinergic vasodilator mechanism (see Table 31-1). These vagally driven noncholinergic vasodilator systems are likely to comprise noncholinergic enteric vasodilator neurons (152), primary afferent vasodilator neurons of dorsal root ganglion origin (143), and vagal afferents causing gastric vasodilatation (147). The possibility that vagal efferent impulses stimulate noncholinergic enteric vasodilator neurons has been corroborated in the dog, in which the gastric vasodilatation caused by electrical stimulation of the vagus nerve is inhibited by hexamethonium, an antagonist of nicotinic acetylcholine receptors blocking transmission between preganglionic vagal efferents and enteric neurons (144). However, the gastric vasodilatation is not blocked by atropine (144), which indicates that noncholinergic enteric vasodilator neurons are responsible for the vascular response to vagal nerve stimulation. It is likely that these neurons use VIP as their transmitter, because vagal efferent fibers make contact with myenteric neurons containing VIP (36), VIP is released in the dog stomach after vagal nerve stimulation (144,153), VIP is a potent vasodilator in the dog and rat stomach (144,153–155), and VIP is a mediator of noncholinergic enteric vasodilator neurons in the guinea pig gut (152). Local administration of thyrotropin-releasing hormone (TRH) into the brainstem excites preganglionic efferent neurons of the vagus nerve (156) and, unlike electrical stimulation of the cut vagus nerve, provides an experimental model to study the physiologic GI effects mediated by the parasympathetic nervous system. Importantly, the gastric vasodilator mechanisms recruited by electrical nerve stimulation differ profoundly from those evoked by central TRH, and in the case of TRH depend on the peptide dose administered. Stimulation of preganglionic vagal efferents by intracisternal application of the stable TRH analog RX-77368 (30 ng) elicits gastric vasodilatation that is predominantly mediated by cholinergic vasodilator neurons because it is blocked by atropine (157–159) but left unaltered by a VIP receptor antagonist (155). Acetylcholine, in turn, increases

gastric mucosal blood flow via stimulation of NO formation in the endothelium (159–161). Although the cholinergic vasodilatation in the stomach is elicited by an intracisternal dose of RX-77368 (30 ng) that also induces gastric acid secretion, the increase of mucosal blood flow occurs independently of the secretory response (157,159,161). Whereas a high prosecretory dose of RX-77368 (30 ng) elicits cholinergic hyperemia in the stomach, intracisternal administration of this TRH analog at a dose (1.5 ng) that is too low to evoke gastric acid secretion enhances gastric mucosal blood flow by a noncholinergic mechanism (143). Thus, different doses of intracisternal RX-77368 recruit different populations of preganglionic vagal efferents that activate different vasodilator systems in the stomach. Specifically, intracisternal application of RX-77368 (1.5 ng) leads to activation of primary afferent neurons running in the splanchnic nerves (162), which are likely to mediate the noncholinergic increase in gastric mucosal blood flow (143,155), because the hyperemia is blocked by defunctionalization of primary afferent vasodilator neurons by capsaicin or antagonism of their vasodilator transmitter by a CGRP receptor antagonist (143,158). It remains to be explored whether activation of mast cells (163) provides a link between the TRH-evoked stimulation of preganglionic parasympathetic efferents (156) and the stimulation of primary afferent vasodilator neurons releasing CGRP in the stomach (143,162). The possibility that vagal afferent neurons play a vasodilator role has been envisaged from the finding that the noncholinergic increase of gastric mucosal blood flow evoked by prolonged electrical stimulation of the vagus nerve is absent after capsaicin-induced defunctionalization of afferent neurons in the vagus nerve (147). Because 80% to 90% of the axons in the vagus nerve are afferent nerve fibers (164), it is highly probable that electrical stimulation of the cut vagus nerve causes antidromic activation of vagal afferents, which evoke gastric mucosal hyperemia by a noncholinergic mechanism that has not yet been elucidated. Enteric Vasodilator Neurons Effects of Nerve Stimulation Combined application of neurophysiologic and videomicroscopic techniques has disclosed that enteric neurons of the submucosal plexus regulate the diameter of submucosal arterioles in the guinea pig small and large intestines (3). These enteric neurons are exclusively vasodilator neurons (3,165), because the only vascular effect that is elicited by electrical stimulation of the submucosal plexus is cholinergic and noncholinergic dilatation of nearby submucosal arterioles (3,46,47,152,165). The arteriolar dilatation is mediated by submucosal enteric neurons, because it is blocked by tetrodotoxin but left unaltered by extrinsic denervation of the gut and removal of the myenteric plexus (47). Stimulation of vagal efferent neurons is likewise able to activate enteric

824 / CHAPTER 31 vasodilator neurons in the rat and dog stomach; these neurons mediate gastric hyperemia by both cholinergic and noncholinergic mechanisms (144,157–159). The vasodilator neurons of the submucosal plexus are efferent neurons of enteric reflex pathways that are involved in the control of digestion (Fig. 31-2). GI vasodilatation is linked to the regulation of motility, secretion, and absorption (165,166), and the enteric nervous system with its topographic and functional organization is poised to coordinate these elements of digestive activity (39–42,44,56,165,167,168). Ingestion of a meal has long been known to increase GI mucosal blood flow, a response in which metabolic, paracrine, endocrine, and neural factors are involved (1,2,11,12). However, the neural regulation of postprandial hyperemia remains incompletely understood (2,3). Although one study holds that luminal stimulation with a bile-oleate mixture induces mesenteric vasodilatation via activation of primary afferent vasodilator neurons (169), other animal studies have shaped the concept that postprandial hyperemia is largely independent of the extrinsic and intrinsic innervation of the gut (2,3,170). In contrast, the postprandial increase of splanchnic blood flow in humans is inhibited by atropine, which suggests that enteric vasodilator neurons contribute to postprandial hyperemia (171). This contention receives strong support from the demonstration of intramural vasodilator reflexes triggered by mechanical stimulation of the mucosa (3,168,172). In isolated preparations of the guinea pig small intestine consisting of submucosal plexus and mucosa, it has been found that mucosal stroking causes relaxation of

precontracted submucosal arterioles partly through a neural reflex (172). The reflex is independent of extrinsic neurons because it persists after chronic extrinsic denervation of the gut, but it is interrupted by hexamethonium, which indicates that it is relayed by ganglionic transmission via nicotinic acetylcholine receptors (172). The afferent reflex arc seems to be constituted by intrinsic primary afferent neurons (IPANs) with nerve endings in the mucosa. Their stimulation by stroking involves 5-hydroxytryptamine (5-HT) released from enterochromaffin cells because the vasodilator reflex is attenuated by the 5-HT3/5-HT4 receptor antagonist tropisetron (172). Conveyed to the submucosal plexus, the afferent input is transmitted to the efferent reflex limb, which is made of cholinergic vasodilator neurons, as the reflex dilatation of submucosal arterioles is blocked by atropine (172). Further analysis has shown that the enteric vasodilator neurons in the submucosal plexus receive polysynaptic inputs from the myenteric plexus (165,167,168). Whereas vasodilator responses relayed solely in the submucosal plexus are confined to relatively short distances (<5 mm), those relayed by the myenteric plexus extend over several centimeters in an orad and aborad direction (165,167,168,172). These long vasodilator reflexes coordinated by the myenteric plexus can be evoked both by mucosal stroking and by distension of the intestinal wall (see Fig. 31-2); these stimuli activate different populations of IPANs because only the vasodilator response to mucosal stroking is blunted by the 5-HT3/5-HT4 receptor antagonist tropisetron (165,168,172). The afferent input is signaled to the myenteric plexus,

LM

IPAN

IPAN

MP CM ACh +

IPAN

IPAN

VIP +

ACh + Arteriole

VIP + SM

5-HT3R

5-HT4R

5-HT4R

MU

FIG. 31-2. Diagrammatic overview of vasodilator reflex pathways in the enteric nervous system. The afferent arc of these pathways is constituted by intrinsic primary afferent neurons (IPANs), and the final efferent arc is constituted by submucosal vasodilator neurons that release acetylcholine (ACh) or vasoactive intestinal polypeptide (VIP) as the principal vasorelaxant transmitter. Short-distance reflex pathways are relayed solely in the submucosal plexus, whereas polysynaptic long-distance reflex pathways run in the myenteric plexus. Submucosal IPANs innervating the mucosa (MU) bear 5-hydroxytryptamine subtype 4 receptors (5-HT4R), whereas myenteric IPANs innervating the mucosa express 5-HT3 receptors (5-HT3R). Plus signs denote vasodilatation. CM, circular muscle; LM, longitudinal muscle; MP, myenteric plexus; SM, submucosa. (This graph is based on a scheme proposed by Vanner and MacNaughton [165].)

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 825 where the neural impulses are transmitted in an orad and aborad direction before they are sent to the submucosal plexus to activate submucosal vasodilator neurons (165,167,168). The polysynaptic transmission within the myenteric and submucosal plexuses involves hexamethonium-sensitive nicotinic acetylcholine receptors, whereas the dilatation of submucosal arterioles is brought about by atropine-sensitive muscarinic acetylcholine receptors (167,168). It is tempting to hypothesize that these longdistance vasodilator reflexes are particularly relevant to digestive regulation because they may integrate blood flow, motility, and secretion in a temporally and spatially coordinated manner. Transmitter Mechanisms A large component of the arteriolar dilatation that in the guinea pig ileum is elicited by stimulation of submucosal enteric neurons is cholinergic (46,173), which is in keeping with the chemical coding of submucosal vasodilator neurons (28,44–47). Further analysis has revealed that dilatation in 48% of the submucosal arterioles is solely caused by acetylcholine, in 31% of the arterioles is mediated in part by acetylcholine, and in 21% of the arterioles is brought about by a transmitter other than acetylcholine (152). The predominant mediator role of acetylcholine is in keeping with its ability to dilate submucosal arterioles in the rat stomach (111,160,174) and guinea pig intestine (47,152,173,175). Acetylcholine-induced vasodilatation is brought about by M3 muscarinic acetylcholine receptors, which stimulate the formation of NO in the arteriolar endothelium (160,173,175). In this context, it is notable that inflammatory bowel disease is associated with a loss of NO-mediated vasodilatation (176), which entails that the function of cholinergic enteric vasodilator neurons is likewise impaired. The noncholinergic dilatation of arterioles that stimulation of submucosal enteric neurons elicits in the guinea pig small intestine is most likely caused by VIP (see Fig. 31-2 and Table 31-1), although galanin and dynorphin could also be involved (152). This transmitter spectrum is in keeping with the chemical coding of submucosal enteric neurons in the guinea pig small intestine, which besides cholinergic neurons (28,44–47), include a sizable population of noncholinergic neurons staining for VIP, galanin, and dynorphin (28,44,48–52,177). The role of VIP as principal mediator of noncholinergic enteric vasodilator neurons is mirrored by the high activity of the peptide to dilate vessels in the small intestine of the guinea pig (47,152) and stomach of the dog and rat (144,153–155). Whether galanin, dynorphin, and related peptides participate in the hyperemic response to stimulation of noncholinergic enteric vasodilator neurons has not yet been proved. Because galanin and dynorphin are able to dilate submucosal arterioles in the guinea pig ileum (152), it is conceivable that they play a neurotransmitter role. The neurogenic vasodilatation elicited by submucosal plexus stimulation in the guinea pig colon likewise involves a cholinergic and noncholinergic component (47,55).

Unlike in the small intestine, however, the noncholinergic dilatation of submucosal arterioles in the colon appears to be mediated by substance P and VIP (47). Both peptides are able to dilate submucosal arterioles in the colon, and the noncholinergic vasodilatation elicited by enteric nerve stimulation is reduced by a substance P receptor antagonist (47). Substance P acting via tachykinin NK1 receptors also dilates submucosal arterioles in the guinea pig ileum (177,178) and enhances blood flow in the stomach and intestine of several species including human (179–181). Despite this pharmacologic activity, substance P does not appear to participate in the enteric nerve–mediated dilatation of submucosal arterioles in the normal guinea pig ileum. When the ileum is deprived of its extrinsic innervation, however, myenteric neurons expressing substance P begin to sprout and to form a prominent plexus of fibers around submucosal arterioles (54,55,178). Under these conditions, substance P plays a vasodilator transmitter function as shown by the ability of a substance P receptor antagonist to suppress the noncholinergic neurogenic dilatation of submucosal arterioles in extrinsically denervated segments of the guinea pig ileum (178).

Primary Afferent Vasodilator Neurons Afferent Nerve–Mediated Vasodilatation in the Gastrointestinal Tract Stimulation by Electrical Impulses and Capsaicin The splanchnic resistance vessels receive not only a dense supply by sympathetic nerve fibers, but also by primary afferents that originate in the dorsal root ganglia (see Fig. 31-1). There is now unequivocal evidence that sensory nerve fibers are responsible for the NANC dilatation of mesenteric arteries (66). Experimentally, the type of the neuroeffector transmission in the inferior mesenteric artery of the guinea pig is governed by the frequency at which the periarterial nerve plexus is stimulated (182,183). Although high-frequency (10–20 Hz) stimulation leads to noradrenergic depolarization and constriction, low-frequency (2–5 Hz) stimulation elicits slow inhibitory junction potentials and relaxation of precontracted vessels (184). The hyperpolarization remains unaltered by blockade of sympathetic nerve transmission and muscarinic acetylcholine receptors (184). Electrical stimulation of isolated rat mesenteric arteries causes a similar dilatation of the vessels precontracted by α-adrenoceptor agonists or endothelin, especially when sympathetic nerve transmission has been blocked (76,85,185,186). Instrumental for the functional and neurochemical analysis of the NANC vasodilator system was the availability of a neuropharmacologic tool, capsaicin, with which the activity of primary afferent neurons can be selectively manipulated. By binding to the transient receptor potential ion channel of vanilloid type 1 (TRPV1), capsaicin potently stimulates sensory neurons that express TRPV1 (187). Capsaicin-induced excitation is soon followed by desensitization and, with greater doses of the drug, long-lasting defunctionalization of sensory

826 / CHAPTER 31 neurons (188,189). Importantly, enteric, parasympathetic, and sympathetic efferent neurons do not appear to be affected directly by capsaicin (187–189). Many studies have proved that the NANC vasodilator fibers in the mesenteric arteries of the dog, rat, guinea pig, and rabbit are susceptible to the neuropharmacologic actions of capsaicin (85,180,184,191–195). Stimulation of afferent nerve fibers by capsaicin mimics not only the hyperpolarizing response to NANC nerve stimulation (184), but also the dilatation caused by electrical stimulation of isolated mesenteric, hepatic, and splenic arteries of the rat and guinea pig (184,191,192,196). Blood flow in the superior mesenteric artery of the rat and dog is similarly augmented by periarterial administration of capsaicin (190,193). Conversely, defunctionalization of primary afferent neurons by pretreatment with capsaicin inhibits the dilator response to both capsaicin application and electrical nerve stimulation in the isolated mesenteric and hepatic arterial beds of the rat and guinea pig (85,184,191,192,196,197). By the use of capsaicin it has been possible to demonstrate that primary afferent vasodilator neurons influence the diameter of intramural resistance vessels throughout the GI tract, and that their stimulation augments blood flow in the GI mucosa (3,21,66,198). Topical application of capsaicin causes dilatation of submucosal and mucosal arterioles in the rat stomach, whereas venules are not affected (199,200). Serosal arterioles in the rat jejunum (201) and submucosal arterioles in the guinea pig duodenum and ileum (202–204) are likewise dilated by capsaicin, an effect that is gone after extrinsic denervation of the gut. Further evidence that the capsaicin-induced arteriolar dilatation is mediated by primary afferent neurons comes from the observation that, unlike the response to enteric nerve stimulation, the dilator effect of capsaicin is insensitive to atropine but blocked by a CGRP and substance P receptor antagonist (205). The action of capsaicin to dilate GI resistance vessels is matched by its ability to enhance blood flow through the rabbit esophagus (206,207), rat gastric mucosa and left gastric artery (99,208–217), rat duodenum and superior mesenteric artery (218,219), rat jejunum (217), dog ileum (220), and rat colon (221). In several instances, it has been shown that the hyperemic response to capsaicin is absent in rats that had been pretreated with a dose of capsaicin that defunctionalizes primary afferent neurons (99,208,210,212,217,221). Stimulation by Pathophysiologic Stimuli Ever since capsaicin-sensitive afferent neurons were envisaged as NANC vasodilator neurons, the question arose as to how these neurons, the main function of which is to send sensory information to the central nervous system, influence tissue blood flow. It is now established that perivascular sensory nerve fibers control, when stimulated, vascular functions by release of transmitter substances from their peripheral axons in the tissue (222,223). Because capsaicin-sensitive afferent neurons are by and large nociceptive neurons that respond to pending or actual tissue

damage (188,189), the concept has emerged that primary afferent vasodilator neurons operate predominantly under pathophysiologic conditions (3,21,66,198). The noxious stimuli that in the GI tract activate primary afferent vasodilator neurons comprise aggressive secretory products such as hydrochloric acid and bile salts, as well as several proinflammatory messengers including prostaglandins and bradykinin. Sensory vasodilator neurons play an important role in the hyperemia that acid challenge evokes in the mucosa of the esophagus, stomach (Fig. 31-3), and duodenum (216,218, 224,225). When the mucosal barrier in the rat stomach is disrupted with ethanol, hypertonic saline, or taurocholate, acid back-diffusion from the lumen to the lamina propria

DRG SN

CG

LM

MP CM NO + NO + Arteriole NO

CGRP + SM

H+

MU

FIG. 31-3. Diagrammatic overview of the possible neural pathways whereby mucosal acid back-diffusion (H+) increases blood flow in the rat stomach by a peripheral axon reflex-like circuit. H+ excites primary afferent neurons that originate in the dorsal root ganglia (DRG) and, at the level of the splanchnic nerve (SN), arborize into several branches that innervate mucosa (MU), submucosal arterioles, myenteric plexus (MP), and celiac ganglion (CG). The reflex hyperemia is mediated by calcitonin gene-related peptide (CGRP) and nitric oxide (NO) formed most likely in the endothelium. Hypothetically, NO released from primary afferent or myenteric neurons also could be involved. Plus signs denote vasodilatation. CM, circular muscle; LM, longitudinal muscle; SM, submucosa.

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 827 enhances blood flow in the left gastric artery and mucosa; this response is mediated to a large degree by capsaicinsensitive afferent neurons (216,224,226–228). The increase of gastric mucosal blood flow accompanying pentagastrinevoked gastric acid secretion is mediated partly by acidinduced stimulation of primary afferent vasodilator neurons (229). Similarly, the increase in mesenteric and mucosal blood flow after exposure of the rat duodenum to excess acid is blunted by capsaicin-induced defunctionalization of sensory neurons (218,219). It has not been fully elucidated how backfluxing acid activates primary afferent vasodilator neurons in the stomach and duodenum. Primary afferent neurons express a multitude of acid sensors including TRPV1 (230), which mediates the GI vasodilator effect of capsaicin (219,231,232). By the use of the TRPV1 blocker capsazepine, it has been found that this ion channel appears to participate in the acid-induced hyperemia in the rat duodenum (219) but not stomach (232). Primary afferent vasodilator neurons can be activated or sensitized by several proinflammatory stimuli. The role of prostaglandins in this process has been controversial (21,198,210,211,219,233–238), but emerging evidence indicates that prostaglandins released by acidified ethanol or hypertonic saline can sensitize capsaicin-sensitive afferent neurons in the rat and mouse stomach via activation of prostaglandin EP2 and IP receptors, respectively (239,240). Bradykinin appears to have a similar action, given that the bradykinin B2 receptor antagonist Hoe-140 attenuates the gastric vasodilator response to acid back-diffusion after barrier disruption with ethanol or hypertonic saline (241,242) and the mesenteric hyperemia evoked by intraduodenal capsaicin administration or acid challenge (243). The sensitizing effects of prostaglandins and bradykinin may be partially related to the property of TRPV1 to become more active when phosphorylated by protein kinase A or C, which are downstream messengers of prostaglandin and bradykinin receptor activation, respectively (187). Histamine released from mast cells is a further factor that contributes to the stimulation of primary afferent vasodilator neurons by gastric acid back-diffusion after barrier disruption with hypertonic saline (244,245). Other stimuli that activate sensory vasodilator neurons include deoxycholate in the rabbit esophagus (246), epidermal growth factor in the rat stomach (247,248), bile-oleate in the rat jejunum (169), and acetic acid in the rat colon (221). Nerve Circuitry of Afferent Nerve–Mediated Vasodilatation Nerve-selective ablation of capsaicin-sensitive afferent nerve fibers has revealed that the mucosal hyperemic response to local capsaicin administration and acid backdiffusion in the rat stomach is mediated by primary afferent vasodilator neurons that originate in the dorsal root ganglia (209,249). Because acute acid challenge of the rat gastric mucosa is not signaled to the spinal cord (250), it has to be inferred that the acid-induced increase of mucosal blood flow is relayed entirely by a peripheral reflex circuit (21,198). This concept is consistent with the observation

that the gastric mucosal hyperemic response to both acid back-diffusion and intraluminal capsaicin takes place in the absence of any systemic changes in blood pressure and heart rate (208,216,224,251). The existence of a peripheral reflex arrangement can be deduced from the inhibitory action of tetrodotoxin (224), but the neural pathway whereby acid challenge of the superficial lamina propria is signaled to resistance vessels in the submucosa is not fully understood (see Fig. 31-3). One explanation is that the gastric hyperemic response to acid back-diffusion results from an axon reflex between mucosal and submucosal collaterals of afferent neurons (224,252). This conjecture is supported by the observation that the acid-evoked hyperemia is mediated by CGRP (252,253), which is the major peptide transmitter of primary afferent vasodilator neurons (3,18,21,66,198). Evidence for an axon reflex-type innervation has been found in submucosal arterioles of the guinea pig ileum in which focal application of capsaicin evokes vasodilatation that spreads beyond the application site (203). Further analysis of the neural circuitry underlying the gastric vasodilator response to mucosal acid back-diffusion has challenged the axon reflex concept. Although noradrenergic sympathetic neurons (251) and cholinergic enteric neurons (224) are not involved, the acid-induced hyperemia is inhibited by transection of the splanchnic nerves, removal of the celiac/superior mesenteric ganglion complex, and blockade of nicotinic acetylcholine receptors by hexamethonium (216,251). It follows that the gastric vasodilator response to acid back-diffusion depends on intact pathways through the celiac ganglion and cholinergic transmission either in the prevertebral or enteric ganglia (see Fig. 31-3). These findings may be accommodated in a peripheral axon reflex-like circuit in which primary afferent neurons arborize at the level of the splanchnic nerve and issue branches that supply the gastric mucosa and resistance vessels, respectively (see Fig. 31-3). In addition, these branches interact with sympathetic neurons in the celiac ganglion or with neurons in the myenteric plexus, or both (21,198). The second possibility is envisaged from the finding that mucosal acid challenge stimulates nitrergic neurons in the myenteric plexus (254). Like the hyperemia (216,224), acid-induced stimulation of nitrergic myenteric neurons is reduced by defunctionalization of capsaicin-sensitive afferent neurons (254). Although NO is a mediator of the acid-induced hyperemia (234), it has not yet been proved that the acid-stimulated nitrergic neurons in the myenteric plexus participate in vasodilator reflex pathways. It needs to be taken into account that primary afferent neurons innervating the splanchnic vasculature may themselves be able to release NO (194,255).

Transmitter Mechanisms Calcitonin Gene–Related Peptide The major vasorelaxant transmitter of primary afferent vasodilator neurons in the rat and guinea pig gut is CGRP (see Figs. 31-1 and 31-3 and Table 31-1). This 37-amino-acid

828 / CHAPTER 31 peptide is expressed by almost all dorsal root ganglion afferents that innervate the viscera of small rodents and form a plexus of fibers around extramural and intramural resistance vessels of the gut (59,75,77,79). Stimulation of afferent nerve fibers in the splanchnic vasculature by electrical impulses, capsaicin, or acid challenge leads to a calciumdependent release of CGRP, a response that is blunted after chemical ablation of sensory neurons (192,256–266). Direct pharmacologic evidence for a transmitter role of CGRP has come from studies using immunoneutralization of CGRP or CGRP receptor blockade with CGRP8-37, a fragment of human CGRP-α that is an antagonist at CGRP1 receptors (267). Specifically, the dilatation of rat and rabbit mesenteric arteries evoked by periarterial nerve stimulation is inhibited either by a CGRP antibody (85) or CGRP8-37 (195,268–270). Likewise, CGRP8-37 depresses the capsaicininduced dilatation of mucosal and submucosal arterioles in the rat stomach and guinea pig ileum (199,200,205,271), the hyperemia in the rabbit esophagus evoked by deoxycholate (246), the hyperemia in the rat gastric mucosa evoked by capsaicin and acid back-diffusion (209,215,252,253,272), and the capsaicin- and acid-induced increase of blood flow in the rat duodenum and superior mesenteric artery (219,273). Notably, the data obtained with CGRP8-37 are not totally conclusive because adrenomedullin and amylin, two peptides with structural homology to CGRP, also have some affinity to CGRP1 receptors, and their vasodilator responses are attenuated by CGRP8-37 (198,267). Although amylin can be expressed in sensory neurons (274), there is no information whether amylin and adrenomedullin are transmitters of primary afferent vasodilator neurons. The role of CGRP as a transmitter of primary afferent vasodilator neurons in the gut fits with the high activity with which this peptide relaxes precontracted mesenteric arteries of rats, guinea pigs, rabbits, and humans (69,76,85,191, 192,197,275–278). Other extramural vessels that are relaxed by CGRP include the left gastric, gastroepiploic, splenic, and hepatic arteries of the rat, guinea pig, and rabbit (69,196,276,279). Within the gut, CGRP dilates mucosal and submucosal arterioles in the rat stomach and guinea pig ileum (199,200,202,205,271), whereas venules are not affected (199,200). Accordingly, blood flow in the rabbit esophagus, rat left gastric artery and gastric mucosa, rabbit stomach and duodenum, as well as rat and canine superior mesenteric artery is augmented by CGRP (154,209,246,253,273, 280–282). In the dog, blood vessels of the intestine seem to be more sensitive to CGRP than those of the stomach (282). In analyzing the dilator action of CGRP, differences between extramural and intramural resistance vessels of the gut have been found. Like the nerve stimulation-induced NANC response, the CGRP-induced dilatation of the left gastric, hepatic, and superior mesenteric arteries is independent of the endothelium (186,196,276,279). Similarly, the hyperemia caused by intravascular CGRP and luminal acid back-diffusion in the rat left gastric artery is independent of NO (253,280), an important vasodilator formed in

endothelial cells. In contrast, the relaxant action of CGRP on precontracted arteries of the human stomach requires the presence of the endothelium (283), much as the capsaicin- and CGRP-induced dilatation of submucosal arterioles in the rat stomach depends on the formation of NO (199,271). The increase of gastric mucosal blood flow induced by topical capsaicin, luminal acid back-diffusion, and intravascular CGRP is also suppressed after blockade of NO synthesis (211,213,234,253,272,280,284,285). The similarities in the pharmacology of nerve stimulationand CGRP-induced vasodilatation and the presence of CGRP receptor binding sites on the endothelium of GI resistance vessels (75,286) make a strong case for CGRP being the major transmitter of primary afferent vasodilator neurons. Whereas NO formed in the endothelium mediates most of the dilator action of CGRP on intramural vessels, CGRP acts directly on vascular smooth muscle to dilate extramural arteries of the GI tract. This concept is not necessarily at variance with the observations that the gastric mucosal hyperemic reaction to CGRP depends only partly on NO (280), and that the CGRP-induced dilatation of gastric submucosal arterioles is less susceptible to NO synthase inhibition than the capsaicin-induced dilatation (271). That endogenously released CGRP may activate receptors different from those targeted by exogenously administered CGRP needs to be considered to explain these discrepancies. Because of their complex structure, the precise distribution of vascular CGRP receptors in the GI vasculature has not yet been explored. Functional CGRP receptors are composed of the calcitonin receptor–like receptor (CRLR), the receptorassociated membrane protein-1, and the receptor component protein that adds in coupling the receptor to guanosine triphosphate–binding protein Gs (287). CRLR has been localized to vessels of the human intestine (288), and from the potent vasodilator activity of CGRP in the GI tract it can be inferred that functional CGRP1 receptors are present on the muscle and endothelium of GI resistance vessels. In addition, there is functional evidence that primary afferent vasodilator neurons in rat mesenteric resistance vessels bear CGRP1 autoreceptors, which regulate transmitter release via a negative feedback mechanism (289). Substance P and Neurokinin A Many dorsal root ganglion afferents innervating the splanchnic vasculature of rodents contain the tachykinins substance P and neurokinin A (59,75–79). With capsaicin as a probe, it has been possible to show that these peptides are released from primary afferent neurons in the rat, guinea pig, and porcine stomach (259,290–294), and from the vascularly perfused guinea pig small intestine (295). However, the evidence for substance P as a transmitter of primary afferent vasodilator neurons in the rat GI tract is limited (see Table 31-1). Thus, tachykinins have been ruled out as mediators of the NANC dilatation of rat mesenteric arteries (270), and both substance P and neurokinin A are

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 829 inactive in dilating the rat mesenteric arterial bed (68,76, 186,270) and mucosal arterioles in the rat stomach (200). Likewise, tachykinin receptor agonists are unable to enhance blood flow in the mucosa of the rabbit esophagus and rat stomach (154,214,246,253,296,297). Finally, tachykinin NK1 and NK2 receptor antagonists fail to inhibit the mucosal hyperemia that in the rat stomach is evoked by intragastric capsaicin or luminal acid back-diffusion (210,253,296–298). Notwithstanding these negative findings, there is some evidence that tachykinins participate in the vascular responses to stimulation of primary afferent vasodilator neurons in the rat intestine and in the GI tract of other species. For instance, the acid-induced increase of esophageal blood flow in the opossum is inhibited by combined blockade of NK1 and NK2 receptors with CP-99,994 and SR-48,968, respectively, although substance P per se is without effect on blood flow in this tissue (225). Despite the low efficacy of tachykinins to increase blood flow in the rat superior mesenteric artery, the mesenteric hyperemia that is elicited by duodenal acid challenge is attenuated by the tachykinin NK1 receptor antagonist CP-96,345 (273,299). Mesenteric arteries isolated from guinea pigs (300), rabbits (301), and humans (278) are relaxed by substance P via activation of tachykinin NK1 receptors and release of NO from the endothelium, and tachykinins have been found to increase intestinal blood flow in many species including dogs, pigs, and humans (179,180,181,302,303). However, it has not yet been systematically explored whether tachykinins are transmitters of primary afferent vasodilator neurons in the intestine. Only in the guinea pig ileum has such a role been substantiated, given that the capsaicin-induced dilatation of submucosal arterioles is reduced by the tachykinin NK1 receptor antagonist CP-96,345, which fits with the vasodilator activity of substance P in this vascular bed (47,177,178,202,205). Because the vasodilator response to capsaicin is suppressed only by a combination of CGRP8-37 and CP-96,345, it appears as if CGRP and substance P comediate the vasodilator response to sensory nerve stimulation in the guinea pig small intestine (205). Substance P and neurokinin A also have been implicated in the GI hyperemia evoked by extrinsic nerve stimulation in the dog and cat (302,303). Thus, splanchnic nerve stimulation under blockade of ganglionic transmission causes both cholinergic and noncholinergic vasodilatation in the canine stomach, the noncholinergic component being inhibited by the tachykinin receptor antagonist spantide (303). Stimulation of the pelvic nerve releases substance P and neurokinin A in the feline colon, where these tachykinins may mediate the accompanying NANC vasodilatation (302). It is unclear, however, whether the tachykinins involved in the vascular effects of splanchnic and pelvic nerve stimulation are released from primary afferent or enteric vasodilator neurons. In the rat stomach, substance P and neurokinin A not only lack a vasodilator activity, but are, in fact, able to reduce blood flow. Thus, tachykinins constrict venules in the gastric

mucosa (200), reduce vascular perfusion of the isolated stomach (304), and attenuate gastric mucosal blood flow through activation of NK1 receptors (296,297). Furthermore, tachykinins inhibit the gastric vasodilator response to stimulation of primary afferent vasodilator neurons by capsaicin (214) and mucosal acid back-diffusion (297,305). This antihyperemic effect of tachykinins is mediated by NK2 receptors (297) and represents a negative feedback system that is triggered by acid-induced release of tachykinins during mucosal acid back-diffusion (298). Substance P appears to inhibit sensory nerve–mediated gastric hyperemia through liberation of mast cell proteases that may degrade CGRP, and thus prevent its action as a vasodilator transmitter (214,305), whereas neurokinin A acts by another mechanism that has not yet been elucidated (297). Tachykinins also have been found to constrict mesenteric veins of the rat via activation of tachykinin NK1 receptors and to contribute to the nonadrenergic mesenteric venoconstriction that is triggered by perivascular nerve stimulation (270). This implication of tachykinins is unlikely to reflect an action of primary afferent neurons, because the nonadrenergic venoconstriction remains unaltered after capsaicin pretreatment (270). The portal vein of the rat (306), rabbit (307), and dog (308) is likewise constricted by tachykinins, the response of the rat portal vein being brought about by stimulation of tachykinin NK3 receptors (306). Pathophysiologic Implications Although capsaicin-induced defunctionalization of primary afferent vasodilator neurons has been reported to alter basal hemodynamics in the rat intestine (193), mucosal blood flow in the rat stomach remains unaltered by chemical ablation of these neurons (21). Sensory vasodilator neurons are hence thought to be of minor importance in the regulation of GI blood flow under physiologic conditions (3,21,198), but to make an important contribution to the changes in vascular function that are seen under pathophysiologic circumstances such as inflammation and pending tissue damage. This mode of activation is consistent with the nociceptive function that enables primary afferent vasodilator neurons to react to noxious stimuli (21,198,309). In this capacity, sensory vasodilator neurons operate as a local emergency system, a role that is exemplified by the vasodilator response to acid challenge in the esophagus, stomach, and duodenum (21). Given that adequate blood flow is of paramount relevance to the maintenance of mucosal integrity, afferent nerve–mediated hyperemia supports a wide range of processes that either strengthen the resistance or aid the repair of the GI mucosa (21,198,235,237,238). Consequently, the GI mucosa becomes easily vulnerable when the function of primary afferent vasodilator neurons or their effector targets is compromised (21). This is true in aged rats (310–312); in rats chronically pretreated with nicotine, taurocholate, or monochloramine (313–315); and in experimental uremia (316), cirrhosis (317,318), and diabetes (319).

830 / CHAPTER 31 Primary afferent vasodilator neurons are known not only to regulate the diameter of resistance vessels, but also to control the permeability of venules (222,223). Whereas the former function is brought about by CGRP, the latter function is mediated by substance P, which acts on endothelial NK1 receptors to increase venular permeability for plasma proteins and leukocytes (222,223). Arteriolar dilatation and an increase of venular permeability are typical of neurogenic inflammation, a reaction that is mediated by release of CGRP and substance P from perivascular sensory nerve fibers. There is increasing evidence that substance P released from afferent nerve endings contributes to experimental inflammation in the gut (179,181,320,321).

INTERACTIVE CONTROL OF GASTROINTESTINAL CIRCULATION Appropriate regulation of the tone in GI resistance vessels depends on a balance between the opposing inputs of vasoconstrictor and vasodilator neurons (66,96). This is particularly true for the splanchnic vascular bed, which, unlike other vascular beds, is under the control of four different vasomotor pathways (see Fig. 31-1). Neuroeffector transmission is fine-tuned by a number of mechanisms (Fig. 31-4). First, transmitter release from sympathetic and probably enteric and primary afferent vasomotor neurons is subject to autoregulatory feedback inhibition. Second, there is a mutual interaction between the sympathetic vasoconstrictor and primary afferent as well as enteric vasodilator neurons.

Specifically, primary afferent and enteric vasodilator neurons counterbalance sympathetic vasoconstriction both via prejunctional inhibition of sympathetic neurotransmitter release and postjunctional antagonism of the sympathetic vasoconstrictor response (3,66,96,322,323). Third, neuroeffector transmission from the vasomotor neurons is under the influence of local, paracrine, and endocrine messengers.

Interactions between Enteric and Sympathetic Vasomotor Neurons Both exogenous and endogenous acetylcholine, released by vagal nerve stimulation, inhibits noradrenergic transmission in the canine gastric artery by a prejunctional site of action (115). Vasoconstriction elicited by stimulation of sympathetic nerve fibers in the submucosal arterioles of the guinea pig small intestine also is attenuated by acetylcholine (322). This effect consists of prejunctional and postjunctional components, the prejunctional effect being attributed to inhibition of ATP release (322). Given that acetylcholine is a major transmitter of enteric vasodilator neurons, it would appear that sympathetic vasoconstrictor tone in the gut is controlled by the enteric nervous system (see Fig. 31-4). This inference is supported by the observation that electrical stimulation of enteric vasodilator neurons reduces the constriction of submucosal arterioles elicited by subsequent sympathetic nerve activation, the inhibitory effect being brought about via a prejunctional site of action (324).

Primary afferent vasodilator

α2R − Y2 R − CGRP α2R − ATP

−

ACh NE NPY

Sympathetic vasoconstrictor

Y2R −

Arteriole −

VIP Y2R −

Enteric vasodilator

−

FIG. 31-4. Diagrammatic overview of reciprocal interactions among sympathetic vasoconstrictor, enteric vasodilator, and primary afferent vasodilator neurons in the control of gastrointestinal resistance vessels. Minus signs denote inhibition of transmission at prejunctional or postjunctional level, as indicated. ACh, acetylcholine; α2R, α2-adrenoceptor; ATP, adenosine triphosphate; CGRP, calcitonin gene–related peptide; NE, norepinephrine; NPY, neuropeptide Y; VIP, vasoactive intestinal polypeptide; Y2R, NPY Y2 receptor.

NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 831 neurons (333), whereas that of norepinephrine is mediated by α2-adrenoceptors (185). Accordingly, clonidine reduces the mucosal vasodilator response to acid back-diffusion in the rat stomach, a response that is prevented by the α2-adrenoceptor antagonist idazoxan (116).

Other neural factors that inhibit transmitter release from sympathetic vasoconstrictor neurons in the rabbit mesenteric and ileocolic arteries include opioid peptides acting via δ- and κ-, but not µ-, opioid receptors (325–327). Cannabinoids inhibit sympathetic neurotransmission in the rat mesenteric arterial bed both via prejunctional cannabinoid CB1 receptors and a postjunctional site of action (328). Furthermore, sympathetic vasoconstriction in the rat mesentery is blunted by γ-aminobutyric acid (GABA) acting via GABAB receptors (329). Because opioid peptides, endocannabinoids, and GABA are expressed in the enteric nervous system, it is conceivable that their inhibitory influence on sympathetic vasoconstriction reflects, at least in part, a regulatory input of enteric neurons on sympathetic vasoconstrictor neurons. Some of the opioid peptide-positive fibers in the mesenteric nerves of the guinea pig are indeed axons of enteric neurons (57). Sympathetic neurons, in turn, may control enteric vasodilator neurons because a NPY Y2 receptor agonist inhibits, via a prejunctional site of action, the effect of enteric neuron stimulation to dilate submucosal arterioles in the guinea pig small intestine (142).

Transmitter release from primary afferent vasodilator neurons in the rat mesenteric artery is inhibited by opioid peptides acting via µ-opioid receptors (334). The mucosal hyperemia in response to gastric acid back-diffusion is likewise attenuated by morphine (224). It is conceivable that these inhibitory effects of opioid receptor agonists reflect an inhibitory control of enteric neurons over sensory nerve– mediated vasodilatation, given that opioid peptides are known to inhibit transmitter release from sensory nerve endings (223) and a likely source of opioid peptides in the splanchnic vasculature of the guinea pig is enteric neurons (57).

Interactions between Primary Afferent and Sympathetic Vasomotor Neurons

Influence of Local and Paracrine Mediators on Gastrointestinal Vasomotor Neurons

Capsaicin-induced defunctionalization of sensory neurons enhances sympathetic constriction of the rat mesenteric arterial bed, which indicates that a loss of the vasodilator input from CGRP-releasing afferent neurons allows sympathetic vasoconstriction to take place unopposed (94,96,186,196,330,331). Sympathetic constriction of submucosal arterioles in the guinea pig ileum is likewise amplified by capsaicin pretreatment (323). The finding that chemical ablation of capsaicin-sensitive afferent neurons attenuates the autoregulatory escape from sympathetic vasoconstriction and post-nerve stimulation hyperemia in the rat stomach and intestine (94,95,99) implies that sympathetic vasoconstriction is normally counteracted by afferent neuron–mediated vasodilation (see Fig. 31-4). This inference is borne out by the finding that capsaicin-induced stimulation of sensory neurons attenuates sympathetic vasoconstriction in small and large mesenteric arteries of the rat (330,332). Because CGRP inhibits the mesenteric vasoconstrictor response to both sympathetic nerve stimulation and norepinephrine but fails to alter the stimulus-induced release of norepinephrine from sympathetic nerve fibers (85,330), it is thought that primary afferent vasodilator neurons oppose sympathetic vasoconstriction in mesenteric arteries mainly at a postjunctional level (185,323). Conversely, sympathetic neurons interact with primary afferent vasodilator neurons predominantly at a prejunctional level. Thus, norepinephrine and NPY inhibit sensory nerve–mediated vasodilatation by depressing the release of CGRP and other NANC vasodilator transmitters (76,185, 197,269). The prejunctional effect of NPY is likely to involve Y2 receptors, which are expressed by primary afferent

A number of local and paracrine messengers that activate primary afferent vasodilator neurons are able to inhibit the activity of sympathetic vasoconstrictor neurons. For instance, prostaglandins activate or sensitize capsaicin-sensitive primary afferent neurons (239,240) but inhibit the stimulationevoked release of NPY and norepinephrine in a differential manner. Whereas prostaglandin I2 blunts the release of both norepinephrine and NPY, prostaglandin E1 attenuates the release of norepinephrine but not NPY, and prostaglandin E2 preferentially counteracts the release of NPY (335). Another paracrine messenger with an influence on sympathetic neurotransmission is histamine, which dilates submucosal arterioles in the guinea pig ileum in part through histamine H3 receptor–mediated inhibition of sympathetic neurotransmitter release (336). In addition, histamine can stimulate primary afferent vasodilator neurons (245). The vascular endothelium releases a number of factors that control not only the activity of vascular smooth muscle, but also that of vasomotor neurons. One of these messengers is NO, which in the GI tract can be synthesized in endothelial cells, enteric neurons, and perivascular nerve fibers (337,338), which appear to be axons of primary afferent neurons, at least in the inferior mesenteric artery of the guinea pig (194,255). Whatever the source, there is evidence for an antagonistic interaction of NO with sympathetic vasoconstrictor neurons. Thus, inhibition of NO synthase augments sympathetic vasoconstriction in isolated strips of the monkey mesenteric artery (339). In the rat mesenteric vasculature, NO causes vasodilatation not only by stimulating guanylate cyclase in vascular smooth muscle, but also by chemically decreasing the biological activity of

Interactions between Primary Afferent and Enteric Vasodilator Neurons

832 / CHAPTER 31 norepinephrine, but not ATP and NPY (340). Through this influence, NO can indirectly modulate the negative feedback inhibition of sympathetic neurotransmitter release via α2adrenoceptor stimulation (341). Furthermore, NO can interact with primary afferent vasodilator neurons. Whereas in the rat mesenteric arterial bed, endothelium-derived NO counteracts the sensory neuron–mediated NANC relaxation (342), the ability of NO synthase inhibitors to cause gastric vasoconstriction is amplified after capsaicin-induced ablation of afferent neurons (343). A further mediator released from the endothelium is endothelin, which blunts sympathetic vasoconstriction in the rat mesentery (344). This vascular effect is related to inhibition of the stimulation-evoked release of NPY, but not norepinephrine, via activation of prejunctional endothelin ETB receptors (344).

SUMMARY More than any other vascular circuit, the splanchnic circulation is governed by an elaborate network of vasodilator and vasoconstrictor neurons, and this neural system is just one component of GI vascular control that, in addition, comprises metabolic, paracrine, and endocrine factors. As is reflected by this complex regulation, GI blood flow is of paramount relevance to the function and integrity of the gut. Work in the past 10 years has significantly advanced our understanding of the multiple innervation of GI resistance vessels and its functional characteristics. Particular progress has been made in the elucidation of enteric and primary afferent vasodilator neurons and in the identification of the transmitter mechanisms whereby sympathetic, enteric, and primary afferent vasomotor neurons control splanchnic resistance vessels. The work ahead needs to address, among other issues, the functional genomics of the splanchnic vascular system and its dynamics in health and disease. There is increasing awareness that inappropriate perfusion of the alimentary canal not only impairs GI function, but also contributes to systemic illness including sepsis and multiple organ dysfunction (2,9).

ACKNOWLEDGMENTS The author greatly appreciates the artistry of Evelin Painsipp in preparing the figures. This work is supported by the Austrian Scientific Research Funds (FWF grant L25B05), the Jubilee Funds of the Austrian National Bank (grant 9858), and the Zukunftsfonds Steiermark (grant 262). This chapter is dedicated to Paul H. Guth, a pioneer in the study of gastric blood flow.

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NEURAL REGULATION OF GASTROINTESTINAL BLOOD FLOW / 839 322. Kotecha N. Mechanisms underlying ACh induced modulation of neurogenic and applied ATP constrictions in the submucosal arterioles of the guinea-pig small intestine. Br J Pharmacol 1999;126:1625–1633. 323. Coffa PF, Kotecha N. Modulation of sympathetic nerve activity by perivascular sensory nerves in the arterioles of the guinea-pig small intestine. J Auton Nerv Syst 1999;77:125–132. 324. Kotecha N, Neild TO. Actions of vasodilator nerves on arteriolar smooth muscle and neurotransmitter release from sympathetic nerves in the guinea-pig small intestine. J Physiol 1995;489:849–855. 325. von Kügelgen I, Illes P, Wolf D, Starke K. Presynaptic inhibitory opioid δ- and κ-receptors in a branch of the rabbit ileocolic artery. Eur J Pharmacol 1985;118:97–105. 326. Illes P, Ramme D, Starke K. Presynaptic opioid δ-receptors in the rabbit mesenteric artery. J Physiol 1986;379:217–228. 327. Nguyen K, Barrios V, Duckles SP. Prejunctional effects of opioids in the perfused mesentery of the rat and rabbit: interactions with α2-adrenoceptors. Life Sci 1991;48:931–938. 328. Ralevic V, Kendall DA. Cannabinoids inhibit pre- and postjunctionally sympathetic neurotransmission in rat mesenteric arteries. Eur J Pharmacol 2002;444:171–181. 329. Li YJ, Duckles SP. GABA agonists and omega conotoxin GVIA modulate responses to nerve activation of the perfused rat mesentery. Life Sci 1991;48:2331–2339. 330. Kawasaki H, Nuki C, Saito A, Takasaki K. Role of calcitonin generelated peptide-containing nerves in the vascular adrenergic neurotransmission. J Pharmacol Exp Ther 1990;252:403–409. 331. Ralevic V, Karoon P, Burnstock G. Long-term sensory denervation by neonatal capsaicin treatment augments sympathetic neurotransmission in rat mesenteric arteries by increasing levels of norepinephrine and selectively enhancing postjunctional actions. J Pharmacol Exp Ther 1995;274:64–71. 332. Ahluwalia A, Vallance P. Interaction between sympathetic and sensory nerves in rat small arteries: involvement of nitric oxide. Am J Physiol 1996;271:H969–H976. 333. Zhang X, Shi T, Holmberg K, Landry M, Huang W, Xiao H, Ju G, Hökfelt T. Expression and regulation of the neuropeptide Y Y2 receptor

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32

Neural Control of the Gallbladder and Sphincter of Oddi Gary M. Mawe, Gino T. P. Saccone, and María J. Pozo Gallbladder, 841 Properties of Gallbladder Ganglia and Neurons, 841 Neuronal Mechanisms Associated with Gallbladder Emptying, 843 Neuronal Mechanisms Associated with Gallbladder Filling, 844 Interprandial Gallbladder Motility, 845

Sphincter of Oddi, 845 Properties of Sphincter of Oddi Ganglia and Neurons, 845 Neuronal Mechanisms Associated with Decreases in Sphincter of Oddi Resistance, 846 Interprandial Sphincter of Oddi Activity, 846 References, 847

Approximately 80 years ago, Ivy and Oldberg (1) demonstrated that an extract from the duodenal mucosa contained a compound that promoted gallbladder emptying via a hormonal mechanism, and they named the yet to be identified compound cholecystokinin (CCK), for its stimulatory effect on the gallbladder. Despite that biliary disease is an extremely common ailment, resulting in hundreds of thousands of surgeries each year, only recently have we begun to unravel the mechanisms by which CCK and other neuroactive compounds contribute to the regulation of gallbladder and sphincter of Oddi (SO) function. This chapter provides an overview of the properties of gallbladder and SO function, with an emphasis on the neurophysiologic aspects of biliary motility.

GALLBLADDER Properties of Gallbladder Ganglia and Neurons The wall of the gallbladder consists of three layers: mucosa, muscularis, and serosa. A ganglionated plexus that lies at the interface between the muscularis and serosal layers provides the local innervation of the muscular and epithelial tissues of the organ, and in some species, ganglia are also sparsely distributed in the lamina propria of the mucosal layer. The main ganglionated plexus is composed of an array of small, irregularly shaped ganglia that are arranged in no discernible pattern (Fig. 32-1A and B). Extrinsic nerves, which include sensory, vagal preganglionic, and sympathetic postganglionic fibers, travel in perivascular and paravascular nerve bundles that are connected to the ganglionated plexus. The muscularis and mucosal layers also contain nerve fibers that represent a mixture of projections from intrinsic neurons, sympathetic postganglionic fibers, and sensory fibers. The neurophysiologic properties of gallbladder neurons have been determined by intracellular recording techniques in whole-mount preparations of guinea pig (2), opossum (3), and human (4) gallbladders. Unlike in the enteric nervous system, where neurons are divided into subgroups by their electrical properties, gallbladder neurons can be described as a single population. Gallbladder neurons rarely exhibit

G. M. Mawe: Department of Anatomy and Neurobiology, The University of Vermont, Burlington, Vermont 05405. G. T. P. Saccone: Department of General and Digestive Surgery, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. M. J. Pozo: Department of Physiology, Nursing School, University of Extremadura, Cáceres, Spain.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

841

842 / CHAPTER 32 spontaneous activity, and they are rapidly accommodating; that is, they fire only one to a few action potentials at the onset of a prolonged depolarizing current pulse, regardless of the amplitude or duration of the current pulse. These properties suggest that gallbladder neurons only fire action potentials when they are activated by neurotransmitters, hormones, or inflammatory mediators. Therefore, gallbladder neurons only release their neurotransmitters onto target tissues in response to incoming commands. Gallbladder neurons receive two types of synaptic inputs, both of which are excitatory (2). One type is the fast excitatory postsynaptic potential (EPSP) (Fig. 32-1C), which is mediated primarily by the release of acetylcholine from vagal preganglionic nerve endings and often is associated with action potential generation. The other is the slow EPSP, which typically is manifested as a prolonged depolarization that is not associated with neuronal firing (Fig. 32-1D and E).

A

Therefore, the vagal preganglionic axons represent the principal driving force for generating neuronal output from gallbladder ganglia to the smooth muscle. As described later, the efficacy of ganglionic transmission can be modulated by physiologic signals that act presynaptically on vagal terminals or postsynaptically on gallbladder neurons. Immunocytochemical and histochemical studies have demonstrated that a variety of neuroactive compounds are expressed by gallbladder neurons. In all species that have been studied to date, including human, dog, guinea pig, opossum, and Australian possum, all gallbladder neurons are immunoreactive for choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine, indicating that all gallbladder neurons are cholinergic. In addition, a number of other compounds and associated synthetic enzymes have been detected in these neurons, including tachykinins, neuropeptide Y (NPY), nitric oxide synthase (NOS), vasoactive

B

Human gallbladder cholinesterase

100 µm

10 mV 10 msec

Human gallbladder NADPH-diaphorase

100 µm

Substance P

D

10 mV Single pulse Fiber tract stimulations

C

20 Hz Fiber tract stimulation

20 sec

E FIG. 32-1. The structure and function of gallbladder ganglia are relatively simple, as compared with ganglia of the gut. (A) Photomicrograph of two ganglia in a whole-mount preparation of a human gallbladder that was histochemically stained for cholinesterase. One of the ganglia consists of a single neuron, and the other contains about 12 neurons. Ganglia are interconnected by axon bundles that are contiguous with nerves that travel along blood vessels. (B) Photomicrograph of a gallbladder neuron in a whole-mount preparation of a human gallbladder that was histochemically stained for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, which labels nitrergic neurons. Gallbladder neurons are relatively simple in structure, consisting of a single axon and a small number of short processes. (C) Synaptic responses of a guinea pig gallbladder neuron evoked by stimulation of nerves traveling along the cystic duct. All gallbladder neurons respond to cystic nerve stimulation, and these synaptic inputs are eliminated by vagotomy, indicating that all gallbladder neurons receive direct input from the central nervous system. (D, E) Brief administration of substance P and highfrequency stimulation of interganglionic fiber bundles evoke prolonged depolarization of guinea pig gallbladder neurons. These responses, and similar responses evoked by capsaicin administration, are inhibited by application of a neurokinin-3 receptor antagonist, indicating that extrinsic sensory fibers provide slow excitatory synaptic input to gallbladder neurons.

NEURAL CONTROL OF THE GALLBLADDER AND SPHINCTER OF ODDI / 843 the threshold for a direct action of CCK on gallbladder muscle strips. Furthermore, meal-induced gallbladder contractions and contractions induced by physiologic concentrations of CCK in vivo are significantly attenuated by neural blockade in several species, including human (11–20). Also, hexamethonium, which blocks the vagal preganglionic input to gallbladder neurons, inhibits CCK- and meal-induced gallbladder contractions (11,17,18). These data indicate that CCK can act on nerves to promote gallbladder motility. Therefore, a neural mechanism is involved in the prokinetic effects of postprandial CCK release. Results of electrophysiologic studies indicate that gallbladder neurons do not respond directly to CCK, but this peptide does have a potent presynaptic excitatory effect on nerve terminals in gallbladder ganglia (3,21,22). CCK increases the amplitude of the cholinergic fast EPSPs in gallbladder ganglia and can convert subthreshold fast EPSPs to responses that are accompanied by one or more action potentials (3,21). The concentration–effect relation of the presynaptic action of CCK in gallbladder ganglia indicates that CCK can act physiologically at this site. The action of CCK in gallbladder ganglia peaks at 1.0 nM and has an EC50 of 33 pM. Furthermore, in the presence of 10 pM CCK, which approximates the postprandial serum concentration, the peptide increases synaptic currents by about 20%. The nerve terminals that are sensitive to CCK are vagal preganglionic nerve terminals because these synaptic inputs are eliminated by vagotomy (22). It is likely that postprandial gallbladder contractions are also promoted by CCK, released from enteroendocrine cells,

intestinal peptide (VIP), pituitary adenylate cyclase– activating peptide (PACAP), orphanin FQ (OFQ), cocaineand amphetamine-regulated transcript peptide (CART), galanin (GAL), and somatostatin (SOM). The distribution of these compounds among gallbladder neuronal subpopulations is species specific. (See Balemba and colleagues [5], and references therein, for a detailed overview.) Neuronal Mechanisms Associated with Gallbladder Emptying Hormonal Cholecystokinin Postprandial gallbladder contraction is triggered by gastric emptying leading to the release of CCK from enteroendocrine cells in the epithelial lining of the duodenum. Although the concept that CCK acts as a hormone to cause gallbladder emptying is solidly established, it is also likely that CCK acts at several sites to promote functional gallbladder motility (Fig. 32-2). The most direct means of gallbladder contraction is for hormonal CCK to act on receptors located on gallbladder smooth muscle, which expresses the CCKA but not the CCKB receptor (6). It is likely that the low-affinity CCKA receptor is present in gallbladder smooth muscle, because the median effective concentration (EC50) for the CCK-induced gallbladder contraction is in the 10 to 50 nM range (7–9). However, it is unclear whether CCK receptors on gallbladder smooth muscle are a normal physiologic site of action for CCK, because postprandial concentrations of CCK in the serum are in the 10 to 20 pM range (10,11), which is below

Sensory nerve SP/+

CGRP/− Sympathetic nerve

CGRP/+

−

SP/+

ACh/SP

+

Vagal efferent nerve

Symbol key CCK Norepinephrine Alpha-2 receptor CCK-A receptor CGRP Substance P

ACh/NO/ VIP/PACAP

Hormonal CCK

+/−

+

FIG. 32-2. Schematic diagram demonstrating the mechanisms by which intrinsic and extrinsic nerves can modulate gallbladder motility. Note that within gallbladder ganglia, vagus nerve terminals are targets of modulatory activity via presynaptic facilitation and inhibition. ACh, acetylcholine; CCK, cholecystokinin; CGRP, calcitonin gene–related peptide; NO, nitric oxide; PACAP, pituitary adenylate cyclase–activating peptide; SP, substance P; VIP, vasoactive intestinal peptide.

844 / CHAPTER 32 acting via a paracrine mechanism on vagal afferent nerve fibers in the lamina propria of the duodenum. Subdiaphragmatic vagal afferent fibers are sensitive to CCK, and postprandial physiologic responses, such as increased gastric motility and pancreatic secretion, have been attributed to CCK-mediated increases in vagal afferent activity (23,24). After a meal, CCK stimulates vagal afferent nerve fibers, which act in the vagal motor complex to increase the rate of firing of vagal preganglionic neurons. Furthermore, CCK in gallbladder ganglia, as described earlier, can act to increase the amount of acetylcholine released from the vagal motor terminals each time they are activated. Acetylcholine As described earlier, all gallbladder neurons are thought to be cholinergic because they all are immunoreactive for the biosynthetic enzyme, ChAT. It is likely that release of acetylcholine represents the principal means of neuronally mediated contraction of gallbladder smooth muscle. Electric field stimulation of nerves in gallbladder muscle strips elicits a contraction that is significantly diminished, if not blocked completely, by the muscarinic receptor antagonist, atropine (25–28). The contractile effect of acetylcholine is mediated primarily through the activation of M3 receptors (29,30) and involves the activation of the phospholipase 3-inositol triphosphate pathway (31,32). Tachykinins Tachykinin-containing nerves are widely distributed in the gallbladder, including extrinsic afferent nerves coexpressing calcitonin gene–related peptide (CGRP), as well as gallbladder neurons and their axons, and it is likely that tachykinins can elicit action in gallbladder ganglia and in the muscularis. In gallbladder ganglia, tachykinins depolarize gallbladder neurons and increase their excitability (see Figs. 32-1D and 32-2). Furthermore, antagonism of the neurokinin receptor that mediates the substance P (SP) response, the neurokinin3 (NK3) receptor, attenuates capsaicin-induced depolarizations and slow EPSPs. These results indicate that SP is released in gallbladder ganglia, and that it is likely to mediate long-lasting excitatory synaptic events. These results suggest that the tachykinin/CGRP-immunoreactive sensory fibers could act as the afferent limb of a local axon reflex circuit within the wall of the gallbladder. It is possible that in response to inflammation or increased intraluminal pressure, tachykinins and CGRP may be released within ganglia by sensory fibers and act directly on intrinsic neurons to facilitate ganglionic transmission. Physiologic or pathophysiologic modifications of the peptidergic content of the afferent innervation could result in changes in the modulation of efferent nerves or smooth muscle contractility. Tachykinins also produce a direct, concentrationdependent contraction of the isolated gallbladder muscle strips, with a rank-order potency of neurokinin A (NKA) > neurokinin B (NKB) > SP (33–35), which is a characteristic

of NK2 receptors. In the gallbladder, the binding of tachykinins to NK2 receptors is linked to protein kinase C activation (34). That tachykinins are coexpressed with acetylcholine in gallbladder neurons indicates that these compounds may act together to promote gallbladder emptying on vagal stimulation. Because most of the neurally mediated contractile response of gallbladder muscle strips is blocked by atropine, it is unclear how much of a role tachykinins play in neurally mediated gallbladder contractions, and therefore gallbladder emptying.

Neuronal Mechanisms Associated with Gallbladder Filling Several neuroactive compounds are candidates for mediating gallbladder relaxation and resultant gallbladder filling. These include CGRP, norepinephrine, VIP, PACAP, and nitric oxide (NO), all of which are capable of inducing gallbladder smooth muscle relaxation. However, it is difficult to resolve which of these compounds act physiologically to promote gallbladder filling, because many of their effects on the gallbladder have been observed at supraphysiologic concentrations. The following sections describe several potential mechanisms that could contribute to this process. Potential Inhibitory Actions of Gallbladder Neurons Subsets of gallbladder neurons are VIP- or PACAPimmunoreactive (36–39) and also immunoreactive for NOS (36–38,40). VIP is released by nerves in the gallbladder in response to electrical stimulation of the vagus nerves (41,42). VIP and PACAP cause relaxation of resting or precontracted gallbladder muscle strips from several species including human (43–46), probably through the activation of adenylyl cyclase and increased cyclic adenosine monophosphate (cAMP) levels. A broad range of intracellular processes are affected by cAMP to decrease gallbladder smooth muscle excitability and induce relaxation, including smooth muscle hyperpolarization, resulting from activation of KATP and BK conductances (46a, 46b), reduction of [Ca2+]i caused by the inhibition of both Ca2+ influx and Ca2+ release from SR, and desensitization of the contractile machinery to Ca2+ (46a). Evidence for nitrergic relaxation of gallbladder smooth muscle includes the finding that inhibition of NOS increases gallbladder tone, increases agonist-induced contractions, and abolishes electrically induced nonadrenergic, noncholinergic relaxation (47,48). Furthermore, NOS inhibition reduces stimulation-induced neurogenic relaxations, and NO donors have an inhibitory effect on gallbladder tone (47–50), which is mediated through guanylate cyclase activation. Carbon monoxide (CO) released from intrinsic gallbladder neurons may also play a role in gallbladder filling. Gallbladder neurons express the synthetic enzyme, heme oxygenase 2; CO relaxes gallbladder muscle strips; and inhibition of heme oxygenase 2 diminishes nonadrenergic, noncholinergic relaxations. Despite these findings, it is

NEURAL CONTROL OF THE GALLBLADDER AND SPHINCTER OF ODDI / 845 difficult to conceive how gallbladder neurons could provide an unambiguous inhibitory signal to gallbladder smooth muscle because all gallbladder neurons express ChAT, and are therefore likely to be cholinergic and excitatory (51). Activation of these neurons would lead to release of acetylcholine plus other compounds such as NO or VIP, or both, thus providing the gallbladder smooth muscle with a mixed signal. Inhibitory Actions of Sensory Nerves in the Gallbladder The neuroactive peptide, CGRP, which is present in extrinsic sensory fibers, can decrease tension in gallbladder muscle strips and cause a hyperpolarization of gallbladder smooth muscle cells (see Fig. 32-2) (52). However, as described earlier for inhibitory actions of intrinsic gallbladder neurons, it is unlikely that CGRP released from gallbladder afferent fibers contributes to physiologic relaxation of the gallbladder, because tachykinins are costored with CGRP in these nerve fibers (40,51,53), and tachykinins have an excitatory effect on gallbladder smooth muscle (33–35,54). Inhibitory Actions of Sympathetic Nerves in the Gallbladder Gallbladder relaxation could be mediated by the release of norepinephrine from sympathetic nerves (see Fig. 32-2). Stimulation of the splanchnic nerves leads to a decrease in gallbladder tone (55–57), and it is likely that this effect is mediated both through the direct effects of sympathetic nerves on gallbladder smooth muscle and via the inhibition of vagal input to gallbladder neurons. Electric field stimulation–induced relaxation of cat gallbladder muscle strips can be completely blocked by the β-adrenoreceptor antagonist, propranolol, whereas the relaxation is unaltered by VIP receptor antagonists or by NOS inhibition (46). In addition to directly relaxing gallbladder smooth muscle, sympathetic nerves may facilitate gallbladder filling by decreasing vagal tone in the organ. Stimulation of sympathetic nerves at low intensities results in a decrease in the excitatory response to vagal stimulation (57). Furthermore, application of exogenous norepinephrine, or release of norepinephrine from sympathetic nerves, causes an activation of α2 adrenoreceptors located on vagal terminals in gallbladder ganglia and a suppression of vagal synaptic input to gallbladder neurons (22,58).

Interprandial Gallbladder Motility Between meals, the gallbladder undergoes periods of increased pressure during phase II of the migrating myoelectric complex (MMC). The purpose of this interprandial gallbladder motor response, which occurs in concert with increased antral and duodenal motor activity, is to be associated with clearing cellular and undigested debris from the intestines, and also with helping maintain the enterohepatic

circulation of bile salts (59). Increases in gallbladder motility associated with phase II of the MMC, as well as those induced by motilin, are attenuated by atropine or hexamethonium, indicating that this response is neurally mediated (60). It is not yet clear whether this involves a vagal reflex or if it is mediated by the neural circuitry that exists between the gut and the gallbladder. Acute inhibition of vagal reflex activity leads to a decrease in MMC-related gallbladder emptying (61); however, MMC-related contractile activity is relatively normal after chronic vagotomy (20,62,63).

SPHINCTER OF ODDI Properties of Sphincter of Oddi Ganglia and Neurons The majority of studies of SO neurobiology and motility have involved the guinea pig and Australian possum models. Therefore, most of the following information is related to ganglia in the SO region in these species. Because of its vast interspecies variation in morphology and function (64), the SO has been the subject of much controversy, and one must be cautious when extrapolating data from experimental animals to the human condition. Ganglia in the guinea pig SO are similar in shape and density to the ganglia of the myenteric plexus of the duodenum, and interganglionic nerve bundles can be traced between the myenteric plexus of the duodenum and that of the SO (65,66). Most of these neurons have a Dogiel type I morphology, consisting of a single long process and several short processes that measure less than the diameter of the cell body. The processes of SO neurons typically leave the ganglionated plexus to innervate the smooth muscle of the SO; therefore, most SO neurons are thought to be motor neurons. In the Australian possum SO, numerous nerve cell bodies are observed within two plexuses consisting of ganglia connected by internodal strands (67). Examination of wholemount preparations revealed a proximal-to-distal gradient of innervation. NOS-labeled nerve cell bodies are abundant, representing approximately 50% of the neurons. Most nerve cell bodies are monoaxonal with a mainly smooth soma and few short dendrites; however, some filamentous neurons with fine dendritic processes also are present (68). The electrical properties of SO neurons have only been investigated in the guinea pig, in which neurons are classified as Tonic or Phasic cells based on their active electrical properties (65). Tonic cells frequently exhibiting spontaneous activity are quite excitable and generate action potentials throughout a depolarizing current pulse. Phasic cells do not exhibit spontaneous activity, are relatively inexcitable, and generate action potentials only at the onset of a depolarizing current pulse. Three types of synaptic responses can be detected in guinea pig SO ganglia. Fast EPSPs can be detected in most neurons and slow EPSPs in about 25% of the cells, whereas inhibitory postsynaptic potentials (IPSPs) are detected in about 10% of the neurons (65,69,70). Fast EPSPs are mediated by nicotinic receptors; slow EPSPs are mediated, at

846 / CHAPTER 32 least in part, by the release of tachykinins from extrinsic sensory fibers and involve the activation of NK3 receptors (71). Release of norepinephrine from sympathetic postganglionic nerves elicits IPSPs and inhibits fast EPSPs through a presynaptic action (69). As in the gallbladder, numerous neuroactive compounds are present in SO nerves. For example, among the species that have been investigated, there is evidence for the following neuroactive compounds: acetylcholine, NO, VIP, tachykinins, CGRP, NPY, GAL, OFQ, SOM, enkephalin (ENK), serotonin (5-hydroxytryptamine [5-HT]), peptide histidine isoleucine (PHI), gastrin-releasing peptide (GRP), and bombesin. (See Mawe and colleagues [72] for a detailed description of the species-specific distributions of these compounds.) The most thorough investigation of neurochemical content and distribution in the SO has been conducted in the guinea pig, in which two major subpopulations of neurons are observed: those that are immunoreactive for ChAT, and those that are immunoreactive for NOS (66,73). The cholinergic (ChAT-positive) neurons represent about two thirds of the population, are also immunoreactive for tachykinins or ENK, or both, and are thought to be excitatory motor neurons. The nitrergic neurons are also immunoreactive for either VIP or NPY and are thought to be inhibitory motor neurons. Nerve fibers, thought to be extrinsic afferent axons, are immunoreactive for tachykinins and CGRP and are abundant in the ganglionated plexus of the guinea pig SO (66). Also, tyrosine hydroxylase–immunoreactive, sympathetic, postganglionic nerves are abundant in guinea pig SO ganglia and nerve bundles (69). There is no relation between the electrical properties of SO neurons (Tonic vs Phasic) and their chemical coding patterns (cholinergic vs nitrergic) (74). The general neurochemical content of nerves in the Australian SO ganglia and nerve bundles are similar to those reported in the guinea pig SO (75–77). There appears to be a greater number of NOS-positive cell bodies compared with the guinea pig SO and a greater number of neurons immunoreactive for both ChAT and NOS (68).

Neuronal Mechanisms Associated with Decreases in Sphincter of Oddi Resistance After a meal, when CCK is released from enterochromaffin cells, changes in SO motor patterns allow bile and pancreatic secretions to flow into the duodenal lumen. As described earlier, considerable interspecies variation exists in the SO anatomy and function; in some species, the SO acts as a resistor, whereas in others, it functions as a pump. In human, cat, and dog, bile flow is associated with a decrease in SO tone (16,78–82). In contrast, in guinea pig, rabbit, and North American opossum, the SO contracts rhythmically, resulting in a pumping action that propels fluids from the ducts into the duodenum (7,83–85). Studies in the Australian possum suggest that the SO can act as either a pump or a resistor, depending on common bile duct and duodenal pressures (82). However, regardless of the species, or the type of response that is involved, the physiologic effect of

CCK is to decrease resistance, and therefore promote the flow of bile. The CCK-induced increase in bile flow through the SO is inhibited by muscarinic blockade with atropine or by complete neural blockade with tetrodotoxin, or by both, indicating that the effect of CCK on SO tone involves a neural mechanism (16,17,83–85). Furthermore, VIP antiserum and NOS inhibitors have both been shown to attenuate the inhibitory actions of CCK on SO tone (86,87), suggesting that CCK can decrease SO smooth muscle tone through an indirect effect on SO neurons. Unlike gallbladder neurons, which do not respond directly to CCK, CCK causes a prolonged depolarization and bursts of action potentials in guinea pig SO neurons (88). However, the concentration– effect relation of this response is out of the physiologic range for hormonal CCK (10,11). Another possibility is that CCK, released at high concentrations from enterochromaffin cells, activates a neural circuit that influences SO ganglionic transmission. Several lines of evidence support the existence of a neural circuit between the mucosa of the duodenum and SO ganglia, and suggest that CCK can activate this circuit (Fig. 32-3). Retrograde axonal tracing studies have confirmed the existence of a direct neural projection from the myenteric plexus of the duodenum to the SO in the guinea pig (89) and the Australian possum (67). In the guinea pig, the duodenum–SO projection neurons are cholinergic (89), and neurons in SO ganglia receive nicotinic excitatory synaptic input from the duodenal myenteric plexus (90). Experiments involving electrical recordings from duodenal neurons that were retrogradely labeled from the SO have demonstrated that all of these neurons express CCK receptors, and many of these neurons are intrinsic primary afferent neurons (89). Intrinsic primary afferent neurons in the intestines are known to send projections to the lamina propria, where they may be activated by local release of CCK from enterochromaffin cells (91–93). These data support the view that CCK released from duodenal enterochromaffin cells could initiate changes in SO resistance by way of a local neural circuit. However, it remains unclear whether this neural circuit is actually involved in postprandial changes in SO tone. Other potential functions for this circuit include the mediation of changes in SO tone that accompany interprandial bile flow during phase II of the MMC (see later). Alternatively, it could provide a means of increasing SO tone when luminal pressure in the duodenum is increased, thus inhibiting the flow of duodenal contents into the biliary and pancreatic duct system.

Interprandial Sphincter of Oddi Activity Between meals, SO resistance is maintained by phasic contractions of the smooth muscle that are superimposed on a basal level of tonic pressure that is adequate to route most of the hepatic bile to the gallbladder. During the fasting state, bile flowing toward the small bowel is diverted into the gallbladder by the resistance at the SO by basal and phasic contractions. Thus, intraductal pressures are increased, and

NEURAL CONTROL OF THE GALLBLADDER AND SPHINCTER OF ODDI / 847 Duodenum Myenteric ganglion

Sphincter of Oddi SO ganglion

ACh NO + ACh Epithelium CCK

+

Smooth muscle

FIG. 32-3. Schematic diagram demonstrating the neural circuitry between ganglia of the duodenum and the sphincter of Oddi (SO). Neurons in the myenteric ganglia of the duodenum project to the SO. Some of these neurons are intrinsic sensory neurons that express cholecystokinin (CCK) receptors. Stimulation of the duodenal mucosa evokes fast excitatory synaptic input to SO neurons. ACh, acetylcholine; NO, nitric oxide.

bile flow is diverted to the region of least resistance, the cystic duct and gallbladder. High pressures in the biliary system also trigger relaxation and compliance of the gallbladder. Resistance at the SO and increased compliance of the gallbladder allows for the diversion of most of the bile away from the gut and into the gallbladder. There are, however, interprandial changes in SO activity that correspond to phase II of the small intestine MMC. The interprandial SO activity associated with the MMC involves a decrease in SO resistance that accompanies increased gallbladder motor activity and bile flow. However, the MMC-related flow of bile does not depend on increased gallbladder motility because dogs that have undergone cholecystectomy also exhibit cyclic bile flow that corresponds to the MMC (94). Changes in SO activity that accompany the MMC have been documented in many species, including human (60,94,95). As was the case for the gallbladder, the mechanisms that underlie the MMC-related changes in SO activity are not entirely clear; however, it appears likely that the duodenum–SO neural circuitry is involved. After truncal vagotomy, MMCrelated SO activity is normal in the opossum. Furthermore, relocation of the duodenal papilla in the opossum, which leaves the blood supply and extrinsic nerves intact but eliminates myoneural continuity between the duodenum and the SO, leads to a marked reduction in electrical activity of the SO during phases II and III of the MMC (96). Also, surgical relocation of the duodenal papilla reduces the responsiveness of the SO to motilin (97). Together, these data support the concept that an intrinsic neural circuit mediates the interprandial SO motor response, but because some cyclic activity remains after translocation of the SO, it is likely that vagal reflex activity or humoral factors, or both, also are involved.

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82.

83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93.

94. 95.

96. 97.

differently to neural and cholecystokinin octapeptide stimulation in vitro. Dig Surg 2000;17:241–249. Grivell MB, Woods CM, Grivell AR, Neild TO, Craig AG, Toouli J, Saccone GT. The possum sphincter of Oddi pumps or resists flow depending on common bile duct pressure: a multilumen manometry study. J Physiol 2004;558:611–622. Cox KL, Von Schrenck T, Moran TH, Gardner JD, Jensen RT. Characterization of cholecystokinin receptors on the sphincter of Oddi. Am J Physiol 1990;259:G873–G881. Hanyu N, Dodds WJ, Layman RD, Hogan WJ. Cholecystokinininduced contraction of opossum sphincter of Oddi. Dig Dis Sci 1990;35: 567–576. Vogalis F, Bywater RAR, Taylor GS. Propulsive activity of the isolated choledochoduodenal junction of the guinea pig. J Gastroenterol Motil 1989;1:115–121. Wiley JW, O’Dorisio TM, Owyang C. Vasoactive intestinal polypeptide mediates cholecystokinin-induced relaxation of the sphincter of Oddi. J Clin Invest 1988;81:1920–1924. Pauletzki JG, Sharkey KA, Davison JS, Bomzon A, Shaffer EA. Involvement of L-arginine-nitric oxide pathways in neural relaxation of the sphincter of Oddi. Eur J Parmacol 1993;232:263–270. Gokin AP, Hillsley K, Mawe GM. Cholecystokinin (CCK) depolarizes guinea pig sphincter of Oddi neurons by activating CCK-A receptors. Am J Physiol 1997;272:G1365–G1371. Kennedy AL, Mawe GM. Duodenal sensory neurons project to sphincter of Oddi ganglia in guinea pig. J Neurosci 1998;18:8065–8073. Mawe GM, Kennedy AL. Duodenal neurons provide nicotinic fast synaptic input to sphincter of Oddi neurons in guinea pig. Am J Physiol 1999;277:G226–G234. Clerc N, Furness JB, Li ZS, Bornstein JC, Kunze WAA. Morphological and immunohistochemical identification of neurons and their targets in the guinea-pig duodenum. Neuroscience 1998;86:679–694. Song Z-M, Brookes SHJ, Costa M. All calbindin-immunoreactive myenteric neurons project to the mucosa of guinea-pig small intestine. Neurosci Lett 1994;180:219–222. Furness JB, Trussell DC, Pompola S, Bornstein JC, Smith TK. Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections. Cell Tissue Res 1990;260: 261–272. Scott RB, Strasberg SM, El-Sharkawy TY, Diamant NE. Regulation of the fasting enterohepatic circulation of bile acids by the migrating myoelectric complex in dogs. J Clin Invest 1983;71:644–654. Marzio L, Neri M, Capone F, di Felice F, de Angelis C, Mezzetti A, Cuccurullo F. Gallbladder contraction and its relationship to interdigestive duodenal motor activity in normal human subjects. Dig Dis Sci 1988;33:540–544. Tanaka M, Senninger N, Runkel N, Herfarth C. Sphincter of Oddi cyclic motility. Effect of translocation of the papilla in opossums. Gastroenterology 1990;98:347–352. Tanaka M, Senninger N, Runkel N, Herfarth C. Hormonal control of opossum sphincter of Oddi motility: role of myoneural continuity to duodenum. J Surg Res 1992;53:91–97.

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33

Brainstem Control of Gastric Function Richard C. Rogers, Gerlinda E. Hermann, and R. Alberto Travagli Efferent Autonomic Overlay, 852 Visceral Afferent Inputs to Brainstem Reflex Control Circuits, 854 Reflex Actions Triggered by Visceral Afferent Inputs, 855 Motility, 855 Secretion, 856 Components and Characteristics of Vago-Vagal Gastric Control Reflexes, 856 Vagal Efferent Neurons in the Dorsal Motor Nucleus of the Vagus Nerve, 856 Solitary Nucleus, 857 Details of Specific Vago-Vagal Reflexes, 858 Modulation of Brainstem Gastric Reflex Control Circuits by Descending Central Nervous System Pathways and Circulating Agents, 860 Appropriation of Gastric Vago-Vagal Reflexes: TRHergic Inputs to the Dorsal Vagal Complex from the Raphe Nuclei, 861 Reflex Facilitation: Oxytocin Inputs to the Dorsal Vagal Complex from the Paraventricular Nucleus, 861

Selective Efferent Activation: Corticotrophin-Releasing Factor Activation of the Vagal Nonadrenergic, Noncholinergic Path, 862 Short-Term Receptor Trafficking and Plasticity: A New Concept for Vago-Vagal Reflex Control, 863 Circulating Factors Controlling Brainstem Vago-Vagal Reflex Mechanisms, 864 The Dorsal Vagal Complex and Chemosensitivity, 864 Tumor Necrosis Factor-α and Gastric Stasis, 866 Tumor Necrosis Factor-α Potentiation of Vago-Vagal Reflex Responsiveness, 866 Mechanism of Tumor Necrosis Factor Action in the Brainstem, 866 Constitutive Expression of Tumor Necrosis Factor Receptors on Brainstem Afferents, 867 Summary, 869 Acknowledgments, 869 References, 869

dependent functionally on extrinsic (i.e., descending) nervous input (5,6). Interest in this brainstem integrative circuitry has increased because of the observations that factors modulating upper GI function during changes in digestive status, activity, stress, or disease may do so via direct action on brainstem gastric reflex control circuits. This realization has accelerated mechanistic physiologic and pharmacologic work on this circuitry over the last 10 years. These efforts comprise much of the advance in our knowledge of brainstem control of gastric function since the publication of the previous edition of this treatise. This review first summarizes what is known about the essential structure and function of brainstem reflex control of the stomach. Next, we focus on the mechanistic details of gastric reflex control circuitry, especially on how extrinsic, descending central nervous system (CNS) pathways (neural) and circulating (humoral) factors acting in the brainstem can alter the behavior of the stomach by affecting the activity of gastric reflex control circuits.

Every investigation that is guided by the principals of Nature fixes its ultimate aim on gratifying the stomach. Athenaeus of Naucratis, “Banquit for the Sophists,” A.D. 200

The smooth, coordinated control of digestive processes by the upper gastrointestinal (GI) tract is the province of the caudal brainstem. The stomach possesses an intrinsic nervous plexus, like the rest of the gut (1,2). Whereas the intestine is capable of considerable independent and integrated neural control (3,4), the stomach and esophagus are completely R. C. Rogers, G. E. Hermann, and R. A. Travagli: Department of Neuroscience, Pennington Biomedical Research Center, Baton Rouge, Louisiana 70808. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

851

852 / CHAPTER 33 Galen (7) began to understand the relationship between brainstem and stomach with his dissection studies of animals in the second century. Originally described as the “sixth” cranial nerve pair by this pioneer in anatomic study, the large plexus of nerves entering and leaving the brainstem near the junction with the spinal cord is the combination of cranial nerve pairs IX, X, and XI. Galen clearly describes the contacts of this wandering nerve: “For from it (the vagus), not only heart and lungs, liver, stomach, mesentery, and intestines, but also all the muscles of the larynx and certain others receive branches.” The intimate anatomic relationship between vagus and stomach is reflected in an alternate name for this nerve used in anatomic texts through the 19th century: the pneumogastric nerve (8). A rich body of literature describes the intervening 1800 years of work on the innervation and control of the stomach. Likewise, the descriptive anatomy of the CNS components of brainstem gastric reflex circuitry has been researched actively since the time of Cajal (9). No attempt is made here to exhaustively review the historical descriptive literature concerning brainstem–gastric interactions through 1994. Instead, there are many excellent reviews in the literature (e.g., 2,10–16).

EFFERENT AUTONOMIC OVERLAY CNS control over the stomach is mediated by parasympathetic and sympathetic pathways that either originate in or are controlled by neural circuits in the brainstem. The parasympathetic motor supply to the stomach is provided by the vagus nerve. The cell bodies of origin for the great majority of gastric-projecting parasympathetic efferents are located in the dorsal motor nucleus of the vagus nerve (DMV). This locus for parasympathetic control has been appreciated for at least a century (9) and has been confirmed in numerous modern studies using retrograde tracing of nerve fibers from stomach to brainstem (17–19). A few neurons of the nucleus ambiguus (NAmb) also project to the stomach, though the role for these neurons in gastric control is uncertain. GI-projecting NAmb neurons are engaged principally in the vagal control of the esophagus and lower esophageal sphincter, as well as cardiorespiratory structures (4,20,21), as described in detail in Chapter 36. The DMV, a paired structure adjacent to the central canal in the dorsal medulla, consists of approximately 20,000 efferent neurons (Fig. 33-1) (13,14,22). These primary vagal efferents branch and diverge widely throughout the digestive tract innervating, as Galen observed, virtually every digestive organ in the abdomen from the verge of the esophagus to the transverse colon. However, vagal innervation of the stomach is the densest (23). Extrinsic neural inputs to the stomach act on smooth muscle and the related interstitial cells of Cajal via the enteric plexus (24). Descending vagal input to glandular tissue also passes through the intermediate of the enteric plexus (11,25). Powley and his colleagues at Purdue University (26,27) have

AP NST

Solitary tract vagal afferents

DMV

Vagal efferent root

NA

FIG. 33-1. Peroxidase tracing study demonstrates the components of vago-vagal neurocircuitry in the medulla of the rat. Visceral afferents in the vagus nerve collect in the medulla as the solitary tract and terminate in the nucleus of the solitary tract (NST) and area postrema (AP). The neurons of the NST and AP process these data and send projections to vagal efferents in the dorsal motor nucleus of the vagus nerve (DMV), completing the basic reflex loop. The AP and the medial-most part of the NST are outside the blood–brain diffusion barrier. This characteristic explains how circulating factors can regulate gastric functions by modulating vagal projections. NA, nucleus ambiguus.

approached the anatomic details of the vagal-enteric plexus connection. Using anterograde Phaseolus vulgaris tracing methods, these investigators demonstrated an extremely dense network of varicose efferent vagal axons entering the stomach at the hilus that fan out throughout the entire gastric myenteric plexus. Individual vagal fibers collateralize extensively, forming a variety of pericellular arborizations and terminal complexes made up of both en passant and end swellings. Single axons frequently innervate subsets of neurons within the ganglia. Most myenteric neurons are contacted by varicosities of more than one vagal fiber. The termination patterns of vagal preganglionic fibers in the duodenal and cecal myenteric plexuses resemble that seen in the stomach in many aspects, but the density is less in the intestine than in the stomach. Thus, the enteric plexus of the stomach is under the coordinated control of a substantial portion of the DMV (26). Vagal preganglionic fibers also innervate the submucosal ganglia, though this input is sparse. The potency of vagal control over the stomach has been known since ancient times. Rufus of Ephesus, a generation before Galen, observed that cutting the vagus nerve stopped

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 853 digestive motions of the stomach (referenced by Holle [10]). Before this, Marcus Tullius Cicero (~60 B.C.) (referenced by Holle [10]) made the following observation: “In alvo multa sunt mirabiliter effecta quae constat fere e nervis” (“a number of interesting effects on the stomach happen because of nerves”). Whereas Beaumont (28) observed that cognitive and emotional states were strongly and acutely expressed in the motor and secretory behavior of the stomach, Tiedemann (1831; referenced by Holle [10]) concluded that these changes in secretion of gastric juice and acid are, at least partially, under the control of the pneumogastric (vagus) nerve. Pavlov (29) determined that the vagus nerve formed the critical connection between brain and stomach responsible for conditioned changes in secretomotor function. He also postulated that the vagus nerve contains both excitatory and inhibitory gastric control fibers, a concept that is now well accepted. Studies from the last century to the present have shown that section of the vagus nerve can have conflicting effects on the behavior of the stomach. In general, secretory functions of the stomach are reduced dramatically, whereas motility effects are mixed. Specifically, the reservoir function of the fundus is reduced as a consequence of an increase in tone, whereas antral motility is depressed (11,30–33). This loss of vagal input transforms the stomach into a noncompliant sack incapable of properly performing its milling, transit regulating, and reservoir functions (5). Consequently, solids are retained and fluids are dumped from the vagotomized stomach. Regulation of gastric emptying and filling mechanisms are reviewed in detail in Chapter 37. The conflicting effects of vagotomy (e.g., increased fundic tone and reduced antral motility) suggested to Pavlov that the vagus nerve controlled gastric motility through both inhibitory and excitatory pathways. This differential function of vagal input to the stomach is reinforced by results obtained from vagal stimulation experiments. In the antrum, stimulation of the vagus nerve leads to an increase in tone and contractility, whereas in the fundus, the result is often a profound relaxation (11). The anatomic and pharmacologic basis for these observations is fairly clear. More than 95% of vagal preganglionic neurons express choline acetyltransferase, the marker for neurons producing acetylcholine as a transmitter (34). However, it is also the case that some preganglionic neurons in the DMV coexpress markers for other neurotransmitter synthesis pathways, including nitric oxide synthase (NOS) and catecholamines (35–38). The physiologic significance of these other potential transmitter synthesis pathways in the DMV is unclear. However, anatomically, these markers for alternate transmitter production tend to predict the location of neurons involved in the inhibitory effect of vagal input to the stomach (36,38). For example, NOS-containing neurons are found in the extreme caudal and rostrolateral portions of the DMV. There is independent evidence that nonadrenergic, noncholinergic (NANC) neurons could be concentrated in the same area. This is not to say that there is a connection between NOS in these DMV neurons and NANC gastroinhibition, but the correlation is suggestive.

Most data demonstrate that blockade of nicotinic receptors eliminates all effects of vagal input to the stomach. Schemann and Grundy (39) used a novel intact vagus in vitro gastric myenteric plexus preparation to demonstrate that postganglionic neurons in the enteric plexus receive nicotinic cholinergic input exclusively. All myenteric plexus neurons responsive to electric vagal stimulation produced only excitatory postsynaptic potentials, and these were blocked with nicotinic cholinergic antagonists. Divergent postganglionic effects of vagal stimulation on target tissues of the stomach are mediated by excitatory and inhibitory enteric neurons. The principal excitatory neurons are cholinergic and act on muscarinic receptors in gastric smooth muscle and on parietal cells. Activated muscarinic receptors depolarize smooth muscle (and the interstitial cells of Cajal) to augment smooth muscle–generated pacemaker activity that drives peristalsis and tone (24). Enteric control of GI motility is reviewed in Chapter 24. Likewise, neural control of gastric secretion is dominated by vagal inputs to the enteric plexus that primarily activate secretion through muscarinic inputs to parietal cells (see review by Hirshowitz [25]). Although vagal efferents primarily control gastric secretion through cholinergic enteric neurons, precise regulation is the sum of interactions between cholinergic and peptidergic neurons and the local hormonal milieu (13,14,25,40). Details of enteric control over gastric secretion are reviewed in Chapter 27. Direct vagal inhibitory action on the stomach is restricted to control of gastric motility and tone (11) and is not seen in gastric secretion (25). A number of functional studies from the 1950s to the present implicate a NANC inhibitory path between the nicotinic vagal efferents and gastric smooth muscle. The two most likely candidates mediating this connection are nitric oxide (NO) and vasoactive inhibitory peptide, although other mediators (e.g., adenosine, 5-hydroxytryptamine [5-HT]) also have been implicated (30,31,41–48). Activation of this vagal NANC path produces a profound relaxation of the proximal stomach and depresses motility in the antrum. Together, these excitatory and inhibitory vagal efferent control mechanisms provide the brainstem with the tools to exert an exquisite degree of control over gastric motility (2,49). Whereas sympathetic inputs to the intestine produce a profound inhibition of secretomotor function as a consequence of actions within the enteric plexus, as well as on smooth muscle and secretory epithelia itself (11,50), the gastric effects of sympathetic input are more subtle. Sympathetic input to the stomach originates from preganglionic neurons in the intermediolateral column (IML) of the T6 through T9 divisions of the spinal cord. These cholinergic neurons control postganglionic adrenergic neurons in the celiac ganglion, and these neurons, in turn, provide the stomach with most of its sympathetic supply (11). Sympathetic input to the stomach is directed primarily to the intermural ganglia and blood vessels, very little direct input to smooth muscle is visible, and its physiologic significance is unclear (2,11). Sympathetic regulation of motility primarily involves

854 / CHAPTER 33 inhibitory presynaptic control of vagal cholinergic input to postganglionic neurons in the enteric plexus, as well as from plexus neurons to smooth muscle. The magnitude of inhibitory sympathetic effects on motility is directly proportional to the level of background vagal efferent input. In contrast, sympathetic input to parietal cells and onto the arteriolar supply to secretory epithelia exerts a substantial and physiologically significant inhibitory effect (51).

VISCERAL AFFERENT INPUTS TO BRAINSTEM REFLEX CONTROL CIRCUITS Together, the parasympathetic and sympathetic pathways provide the brainstem with the means to regulate the function of the stomach with a high degree of precision. At the most essential level, these efferent inputs to the stomach are regulated by an array of visceral sensory inputs that arrive primarily, although not exclusively, from afferent fibers in the vagus nerve. Vagal afferent input is sorted out by neurons in the nucleus of the solitary tract (NST) that, in turn, regulate the activity of inhibitory and excitatory vagal projections to the stomach (2,14,49,52,53). By analogy with brainstem cardiovascular regulation, it is likely that the NST also makes connections with reticular efferent neurons, such as the rostroventrolateral reticular area, that project to the thoracic IML and regulate sympathetic input to the stomach. In addition, spinosolitary pathways carry visceral afferent information that traverses the dorsal roots. However, compared with “vago-vagal” brainstem reflex control of the stomach, almost nothing is known about brainstem–spinal interactions in the regulation of gastric function. Vagal afferents carry a large volume of information about the physiologic status of the gut directly to brainstem circuits regulating gastric function. The cell bodies of origin for this afferent pathway are contained in the nodose ganglion. These neurons are the cranial equivalent of dorsal root ganglion cells in that they are bipolar, connecting gut directly with brainstem with no intervening synapse (49,54–56). Visceral afferents in the vagus nerve are of two essential types: “mucosal” and “muscular” receptors. This classification scheme originated as a result of early neurophysiologic studies on the sensitivities of different visceral afferent units in mixed visceral nerves rather than as a precise anatomic description of the sensors themselves (11). From neurophysiological data, mucosal afferents were presumed to be present below the mucosal epithelium throughout the GI tract (33). This afferent category has been identified in the esophagus, stomach, and small intestine and is particularly highly concentrated in the antrum and pylorus (57). Anterograde dye tracing studies of the nodose ganglion reveal vagal afferent terminals ramifying widely through the submucosa, and these are likely to be mucosal-type afferents (40,58–60). The mucosal-type sensor was originally described as a rapidly adapting mechanosensor. Indeed, even current neurophysiologic studies identify this afferent by mechanical

probing of the exposed mucosa via gentle brush stroking or von Frey hair stimulations. Such sensors are typically silent at rest but are exceedingly sensitive. Light touch (25–50 mg pressure) is sufficient for crisp bursts of activity of 5 to 10 spikes per second (61). This sensitivity profile has led to the suggestion that these afferents are responsible for “grading” the size of chyme particles contacting the antral mucosa during the “milling” process (62,63). These particle size data can be used by brainstem reflex control mechanisms to regulate transit or retropulsion necessary for trituration of solids. Although mucosal afferents are not sensitive to tension per se, the sensitivity of the receptor to touch can be modulated by ongoing changes in the contractile state of the musculature or, potentially, by chemical agents released by nearby enteric neurons (57). Indeed, mucosal afferents also possess sensitivity not just to neurotransmitters, but also to pH, nutrient concentrations, osmolarity, and temperature (2). These afferents are also sensitive to circulating and paracrine released hormones. Cholecystokinin (CCK), leptin, interleukin-1 (IL-1), neurokinins, and serotonin (5-HT) head a long list of agents that can act on vagal afferents to dramatically regulate gastric motility, with CCK probably the most widely recognized and studied example. CCK effects to inhibit gastric motility and secretion, as well as food intake, are most likely the result of the peptide’s activation of these multifunction sensors (2,14,64,65). Most chemosensitive mucosal vagal afferents terminate in the small intestine, though some reports suggest that mixed-mode (chemosensitive and mechanosensitive) mucosal afferents also terminate in gastric mucosa (61,66). The muscle afferents have also been classified by neurophysiologic criteria. Unlike mucosal afferents, GI muscle afferents respond to distension with a graded, nonadapting response. These sensors are essentially tension receptors with the capability to follow stresses generated within muscle layers to a high degree of fidelity (2,57,67). Histologically, two types of tension receptors have been tentatively identified. The intraganglionic laminar endings form a network of fine processes around myenteric ganglia. It is likely that these sensors are positioned such that shear forces generated around the ganglia by adjacent muscle layers activate these afferents. Intramuscular afferents parallel the circular and longitudinal smooth muscle fibers and may be responsible for in-series rather than shear tension reception. Like mucosal receptors, muscular GI afferents may be chemosensitive, particularly to monoamines and cholecystokinin (2,68,69). Spinal visceral afferents of the upper GI tract are essentially serosal in nature. They have multiple, widely divergent, receptive fields and generally exhibit higher thresholds for mechanical activation than vagal afferents. These afferents also can be chemosensitive, especially to substances released from adjacent tissues in reaction to ischemia and inflammation such as bradykinin and substance P. These physiologic characteristics suggest that these afferents are involved in spinal reflex inhibition of GI activity that occurs after excessive distension, ischemia, or inflammation (16,57,70,71).

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 855 REFLEX ACTIONS TRIGGERED BY VISCERAL AFFERENT INPUTS A detailed discussion of the extrinsic regulation of gastric emptying and gastric acid secretion are contained in Chapters 37 and 49. The essential mechanisms are reviewed here to provide a context for the following section on brainstem reflex circuitry and its modulation by extrinsic factors.

Motility Activation of visceral afferent inputs to the brainstem (see earlier description) typically evokes a reduction in gastric tone or motility. One of these reflexes was first described nearly a century ago by Cannon and Leib (72), who observed a relaxation of the fundus and corpus in response to swallowinduced esophageal distension (Fig. 33-2). This “receptive relaxation” allows the fundic reservoir to expand to receive ingesta, thereby keeping intragastric pressures low (73). Studies have shown that receptive relaxation essentially disappears after acute vagotomy, but some adaptive gastric reservoir function returns after several weeks. This illustrates a latent ability of the gastric enteric plexus and intrinsic smooth muscle mechanisms to reassume a measure of control over gastric emptying (74,75). Earlier studies of receptive relaxation implicated vagal inhibitory (i.e., NANC) pathways as the dominant efferent factor in the reflex (76). However, more recent neurophysiologic studies implicate a combination of afferent-mediated withdrawal of cholinergic vagal input in conjunction with

2 gm

Esophageal distension [160ul]

2 gm

Esophageal distension [160ul]

FIG. 33-2. Demonstration of the esophageal-gastric vagovagal reflex. This reflex normally functions to increase the compliance of the stomach to accommodate ingesta in the esophagus. Gastric motility is monitored via a strain gauge secured to the fundic portion of the stomach. (Top) In vagally intact preparation, distension of the esophagus activates vagal afferent projections to the nucleus of the solitary tract (i.e., in the pars centralis), which then evokes an increase in gastric compliance (i.e., relaxation in tone) via regulation of the activity of relevant vagal projections to the stomach. (Bottom) Vagotomy or destruction of vagal afferent fibers in the medulla completely eliminates the reflex. (Modified from Rogers and colleagues [73], by permission.)

activation of the NANC pathway (73,77) to mediate this response. The potential for this dual effect of visceral afferents on vagal efferent outflow offers a mechanism for incrementation or amplification of these descending effects on a number of “vago-vagal” gastric control reflexes. However, with the exception of the receptive relaxation reflex, there is a mismatch in neurophysiologic versus pharmacologic data regarding the pathway(s) responsible for the vagal efferent effect. For example, the following stimuli all produce a significant reduction in proximal gastric motility and tone: esophageal distension, antral mucosal contact and distension, intestinal distension and intestinal nutrients, intestinal acid and irritant stimulation, and the action of CCK and other hormones (2,11,63,73,77). All of these reflexes are eliminated by vagotomy. Pharmacologic evidence suggests that virtually all of these reflexes depend on activation of a vagal inhibitory NANC pathway, whereas neurophysiologic evidence suggests that withdrawal of cholinergic excitatory inputs to the stomach may be primarily responsible (73,77,78). This disparity may be explained by a peculiarity in the organization of CNS reflex control circuits in the medulla. Given that more than 90% of DMV neurons are “gastroexcitatory,” neurophysiologic methods favor the probability of finding DMV neurons involved in descending cholinergic excitation of the stomach (79). However, the relatively smaller number of DMV neurons coupled to the NANC path produces a proportionally large inhibition of the stomach (12,36). The result is that it is easier to demonstrate a potential reflex cholinergic withdrawal (i.e., reduction in excitation) using neurophysiologic tools, whereas a pharmacologic demonstration of a brief NANC inhibition (i.e., activation of an inhibitory pathway) is easier to observe than the withdrawal of a tonic agonist effect. Antral motility is also regulated mainly by vago-vagal reflexes (2,11). Fundic distension can provoke an increase in antral motility that is vagally dependent (80,81). This is possibly the only known example of an excitatory vagal gastro-gastric motility reflex. Intestino-antral reflexes are similar to intestino-fundic reflexes in that they are powerfully inhibitory and largely eliminated by vagotomy (63). As before, pharmacologic studies implicate activation of vagal NANC pathways in mediating these reflexes. Pyloric motility control is a complex mix of intramural, splanchnic, extramural plexus, and vagal control. As such, this combination resembles the autonomic control of the intestine more than the stomach, which is subject to unambiguously dominant vago-vagal control (11,63). Maintenance of the tonic closure of the pylorus appears not to involve vagal mechanisms. However, relaxation of the pylorus, and therefore regulation of transpyloric flux, does involve potent, if not exclusive, vagal control. Earlier studies (82,83) showed that vagal afferent stimulation causes a reduction in transpyloric flow, whereas vagal efferent stimulation caused mixed excitatory and inhibitory effects on the antrum/pylorus. These results suggested that vagal afferent fibers can regulate pyloric flux by the familiar mechanism of co-control over

856 / CHAPTER 33 opposing vagal efferent pathways (63). More recent studies emphasize the dominance of vagal reflex modulation of NANC mechanisms (84). Thus, afferent information may be differentially processed and, ultimately, recruit different subpopulations of efferents to evoke the appropriate response.

Secretion Stimulation of vagal afferents can activate vagal efferent outflow to provoke gastric secretion. Electric vagal afferent stimulation (85), as well as gastric distension (86,87), activates vagal efferent projections to the stomach and increases gastric acid secretion via direct action of acetylcholine on parietal cells, as well as on enteric neurons that control gastrin secretion. Gastrin, in turn, activates acid secretory mechanisms through a hormonal mechanism (see Chapters 4 and 49). In addition, gastric secretion can be driven to quite high levels as a consequence of central commands regulating the excitability of vagal efferent neurons in the DMV. These “cephalic phase” events are discussed in the next section. Although physiologically important, vagal mechanisms are probably secondary in impact to gastro-gastric (enteric) and hormonal mechanisms of secretion control (25). Intestinal feedback has potent effects to inhibit gastric acid secretory mechanisms. For example, the intestinal hormone, secretin, which is released in response to duodenal acidification, causes inhibition of gastric secretion via a vago-vagal reflex mechanism. The details of this mechanism and sites of action are not yet known (88). Intestinal nutrients, especially fats, can also cause significant inhibition of gastric secretion, as well as motility (25,63,89), through action on vagal afferent projections. Many of the ultimate actions of vagal afferents on gastric secretory functions involve local axon reflex interactions with the enteric plexus as opposed to “long loop” vago-vagal connections involving the brainstem (16). In this case, chemoactivation of one branch of a visceral afferent may cause the release of neurotransmitters (typically peptides) from adjacent branches of the same nerve fiber. These “sensory-efferent” local axon reflexes play a protective role for the GI mucosa and may be modulated by descending vagal efferent projections, but the mechanistic details are currently unavailable (16). Gastric vago-vagal reflex regulatory patterns are not static or stereotypic and are subject to considerable modulation by other neurohumoral mechanisms (49,52,63). The abovecited paracrine-neurocrine actions of gut hormones directly on vagal afferents within the GI tract are well-known examples of humoral modulation of vagal reflex-regulated functions. However, it has become clear that CNS pathways, nutrients, gut hormones, and cytokine products of immune activation can have a profound impact on vago-vagal control through actions directly within brainstem circuits. Emerging evidence suggests that vagal afferents, and the neural circuits they regulate within the CNS, could be also affected by circulating agents (49,53,90–93).

COMPONENTS AND CHARACTERISTICS OF VAGO-VAGAL GASTRIC CONTROL REFLEXES Vagal Efferent Neurons in the Dorsal Motor Nucleus of the Vagus Nerve Retrograde tracing experiments have shown that there is a mediolateral organization within the rat DMV. That is, its medial portion contains the somata of neurons that contribute axons to the vagal gastric branches (anterior and posterior), the lateral portion of the DMV contains the somata that send axons to the vagal celiac branches (celiac and accessory celiac), and the hepatic branch originates from scattered somata of the medial portion of the left DMV (17–19,94–98). This mediolateral columnar organization of the DMV is not maintained, however, when considering the target organs of the vagal branches themselves. In fact, individual organs can be innervated by multiple branches. For example, gastric and hepatic branches innervate the stomach, whereas the duodenum is innervated by gastric and celiac branches (23,99). Although the DMV may not be rigidly organized organotropically, there is evidence to suggest that vagal innervation of the stomach is segregated within the DMV by function. The two descending vagal pathways described in the previous section (i.e., the NANC and the postganglionic cholinergic) appear to be localized in different regions of the DMV. That is, putative NANC neurons are probably located in the caudal medial and anterolateral divisions of the DMV, whereas the cholinergic excitatory neurons are located in the more rostral and medial divisions (36,100). One of the remarkable properties of vagal efferent neurons is that most of them maintain a spontaneous, slow (1–2 Hz), pacemaker-like activity (79,101–103). Although the expression of this activity is permitted by sources of afferent control, it is not generated by synaptic input (102). Rather, this activity is a fundamental property of vagal efferent projections to the GI tract. One immediate implication is that the stomach, at rest, is the recipient of vagal efferent outflow that is continuously sculpted by afferent input (i.e., vagal, descending CNS, and humoral) impinging on the ongoing activity of the DMV neurons. Furthermore, the slow pacemaker activity of DMV neurons implies that afferent inputs altering the membrane potential by even a few millivolts, in either direction, can result in dramatic changes of the vagal motor output (104,105) (Fig. 33-3). There appear to be several intrinsic morphologic and physiologic properties that distinguish between DMV neurons projecting to the stomach versus other visceral targets. Morphologic diversity has been reported in both the rat and the human DMV (78,95,97,98,101,106). The properties of DMV neurons are well matched to the physiology of the stomach and its intramural innervation such that minute changes in DMV activity can have dramatic effects on gastric function. In a relatively recent study, the morphologic properties of DMV neurons were analyzed with respect to their GI projections and their electrophysiologic

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 857

1 mV

changes in input from either the cholinergic excitatory branch or the NANC inhibitory branch of the DMV. Therefore, slight changes in DMV activity in either division translate into dramatic effects on gastric function. The power of this extrinsic innervation can be appreciated by the modulation of GI motor function seen after vagotomy, where fundic compliance is lost and antral motility is depressed (5,30,31,63,114). 3 sec

FIG. 33-3. In vivo electrophysiologic recording made from a single dorsal motor nucleus of the vagus nerve (DMV) neuron illustrating the characteristic pacemaker activity of these neurons. Distension of the esophagus by inflating a 160 µl balloon in the thoracic esophagus (recorded in lower trace) halts the spontaneous activity of the DMV cell. This neuron is probably responsible for controlling cholinergic tone of the stomach. Withdrawal of cholinergic tone is one mechanism by which visceral afferents can cause gastric relaxation, that is, vago-vagal reflex. (Modified from McCann and Rogers [79], by permission.)

properties (101). Morphologically, gastric-projecting neurons are smaller and tend to have a less complex dendritic arborization than intestinal-projecting neurons. These morphologic differences appear to converge with a diversity in the electrophysiologic properties of DMV neurons (101,105,107–110). For example, it has been demonstrated that DMV neurons can be distinguished based on their electrophysiologic and morphologic properties, which, in turn, can be correlated with the target organs they innervate (101). Specifically, smaller gastric-projecting DMV neurons are more apt to change membrane potential in response to synaptic inputs than DMV neurons that project elsewhere. This “size principle,” which is widely accepted for spinal motoneuron activation and recruitment (111), states that neurons with the smallest cell bodies have the lowest threshold for synaptic activation, and therefore can be activated by weaker synaptic inputs. The smaller sized gastricprojecting DMV neurons have membrane properties such as a higher input resistance or the presence of Ih that makes them prone to respond to a greater range of synaptic inputs than the larger intestinal-projecting DMV neurons. These intrinsic properties of DMV neurons suggest that morphologic and electrophysiologic differences in neuronal groups may reflect underlying functional differences in the GI-projecting DMV. The resting membrane potential of gastric smooth muscle is non-uniform with a gradient from approximately −48 mV in the proximal stomach to −70 mV in the distal stomach (112). The threshold for smooth muscle contraction is around −50 mV (113), and the myenteric plexus of the stomach essentially serves as a “follower” of vagal efferent input (39). That is, there is a high degree of fidelity for activation of gastric myenteric neurons by vagal efferent inputs (2). As a result, the proximal stomach is highly sensitive to minute

Solitary Nucleus The general hypothesis of a vago-vagal reflex connecting vagal afferent input, brainstem interneurons, and vagal efferent projections to the stomach was formulated years ago (2,6,11,52,53,115). While this early model is certainly an oversimplification, it still explains many of the salient features of brainstem reflex control of the stomach. In this model, reflex action is initiated by stimulation of vagal afferent mechanosensory or chemosensory pathways. This increase in afferent traffic enters the brainstem via the solitary tract and terminates primarily in the medial divisions of the NST. These neurons make connections with efferent DMV neurons, completing the loop (Fig. 33-4). Afferent input to the NST is organized in a distinct viscerotopic manner: the intestine is represented in the subnuclei commissuralis and medialis, the stomach in the subnuclei medialis and gelatinosus, and the esophagus in the subnucleus centralis (56,116,117). This viscerotopic organization of afferent information, together with the knowledge that the DMV may be organized in a more functional manner, led to the hypothesis that vago-vagal reflex functions were organized in a sort of functional matrix (118). Contributions of vagal afferent input from different regions of the gut can lead to different patterns of gut-directed vagal efferent outflow. Accordingly, afferents serving one gut region would synapse on a subset of NST neurons that, in turn, terminate on an appropriate collection of DMV neurons, completing the reflex. Medial NST neurons (i.e., the cells that receive vagal afferent input from the gut) possess long mediolaterally oriented dendrites (~600 µm) that extend across the terminal zones of all GI afferent inputs (2,119). A priori, such an arrangement tends to favor a convergence of vagal afferent input of different modalities and different visceral loci onto select populations of NST neurons. Selected NST neurons would provide dominant local synaptic control over the appropriate DMV projections to the stomach (53,118) and determine a specific pattern of gastric control (2,6,115). In general, stimulation of the proximal GI (i.e., esophagus, fundic stomach) vagal afferents produces a mixed activation of some gastric-projecting vagal fibers and inhibition of others. Activation of vagal afferents from more distal sites in the antrum and intestine produce inhibition of practically all vagal outflow to the stomach (see reviews 2, 49, 52, 53, 78, 92). In combination with other physiologic studies, we see that vagal afferent input integrated by the NST ultimately evokes gastroinhibition by either the withdrawal of

858 / CHAPTER 33 Vago-vagal reflex circuit

NST (+) glut

(+) glut

Afferents

gaba, NE (−)

(+) glut, NE

5 sec DMN

Cholinergic 5 sec “NANC” NO, VIP, Adensine? 5 sec

FIG. 33-4. Schematic model of a basic vago-vagal reflex circuit (left) with neurophysiologic recordings of nucleus of the solitary tract (NST) and dorsal motor nucleus (DMN) neurons (right). Visceral afferents are activated by a variety of potential stimuli including distension, mucosal contact, duodenal acidification, paracrine hormone action (e.g., cholecystokinin [CCK]), and so forth. Afferents activated by these stimuli excite select NST neurons using glutamate as a neurotransmitter (top right panel is an example of NST activation due to esophageal distension). The NST is composed of a variety of neuronal phenotypes that can have different effects on downstream targets such as DMN. For example, one class of NST neuron inhibits a select population of DMN neurons that controls tonic cholinergic input to the stomach (bottom right panel is an example of DMN neuron inhibited by gastric distension). Another class of NST neuron activates a subpopulation of DMN neurons that activates inhibitory inputs (i.e., “nonadrenergic, noncholinergic,” or NANC) to the stomach (middle right panel is an example of DMN neuron activated by gastric distension). Activation of these parallel branches of the reflex can dramatically reduce gastric tone and motility. NE, norepinephrine; NO, nitric oxide; VIP, vasoactive inhibitory peptide.

cholinergic tone (i.e., generated by the pacemaker activity of DMV neurons) or the activation of the vagal NANC pathway, or a combination of both, as described previously (see Fig. 33-4). Since NST neurons do not appear to possess pacemaker activity, their inputs onto DMV neurons have to be “driven” by synaptic activity, either from the afferent vagus nerve, from other CNS areas, or via circulating hormones (120,121). Afferent vagal fibers enter the brainstem in the solitary tract and activate NST neurons via the release of excitatory neurotransmitters, mainly glutamate (52,53,122–125). It has been appreciated for some time that localized glutamate injections directly into the medial NST can evoke rapid, large, and vagally mediated gastric relaxations similar to those evoked by stimulation of vagal afferents (12). Vagal afferent fibers within the CNS also possess receptors for a variety

of transmitters and hormone-like agents that can modulate reflex function (126–129). There are numerous phenotypes of neurons in the NST that can potentially contribute input to the DMV (130) and invoke powerful effects on vagally mediated gastric function. However, indirect evidence suggests that the NST primarily controls the DMV through GABAergic (79,102,131), glutamatergic (102,125,131,132), and catecholaminergic inputs (77). Discrete microinjection of γ-aminobutyric acid (GABA) antagonists, such as bicuculline, into the DMV dramatically increases motility and secretion (133), presumably by eliminating tonic inhibition on DMV neurons. Microinjections of glutamate or catecholamine agonists into the DMV also evoke significant GI effects (77,131,134). Direct evidence of discrete synaptic inputs from NST onto DMV neurons has been provided by several investigators (73,102,135–137). Stimulation of the NST subnucleus commissuralis induced noradrenergic α2-mediated inhibitory potentials in approximately 10% of the DMV neurons (136). Electrical stimulation of portions of the NST evoked both inhibitory GABA-mediated or excitatory glutamate-mediated currents in DMV neurons, or both (102,138,139). In addition, there appears to be an excitatory monosynaptic pathway from NST to DMV, which uses ionotropic glutamate transmission (137). Thus, as described previously, vagally mediated GI motor and secretory functions are finely tuned by these inputs from the NST.

Details of Specific Vago-Vagal Reflexes While the general scheme of vago-vagal reflex control of the stomach is accepted, there are only a few examples in which the connectional and neuropharmacologic details of a specific circuit approaches completeness. The esophageal distension-gastric relaxation or receptive relaxation reflex described by Cannon and Leib (72) is a possible exception. Vagal distension–sensitive afferents in the esophagus are the essential sensors activating the reflex (33). Esophageal vagal afferents are unique in that they terminate in a highly circumscribed region of the NST, specifically the pars centralis portion (NSTc) (15,56,73,77,140–143). In vivo electrophysiologic recordings made from this region of the brainstem reveal that localized esophageal distension produces a crisp activation of NSTc neurons; no inhibitory responses are observed (73). These NST neurons project (Fig. 33-5) to virtually all parts of the DMV, the NAmb, other subdivisions of the NST, as well as subjacent areas involved in control of the oral cavity and pharynx such as the reticular formation and the area postrema (144,145). Neurophysiologic studies also showed that esophageal distension tends to activate those DMV neurons in areas containing presumptive NANC preganglionic efferents, whereas DMV neurons located in areas likely to control cholinergic excitation of the stomach were inhibited by esophageal distension (73).

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 859

eso. dist 5 sec

DMV ST

NSTc 1 mm anterior to calamus

DMV neuron

NSTc afferents 10 µ

FIG. 33-5. Top panel: Neurons in the nucleus of the solitary tract, pars centralis (NSTc), are specifically activated by esophageal distension (eso dist). Middle Panel: Neurobiocytin injections into the NSTc area responding to this stimulus demonstrated that neurons in this subdivision project throughout the dorsal motor nucleus of the vagus nerve (DMV). Bottom panel: Camera lucida drawing of dense preterminal arborizations of neurobiocytin-filled NSTc projections that surround individual DMV cells. (Modified from Rogers and colleagues [73], by permission).

NE 1 µM

50 mV

0.5 mV

(i.e., onto the area postrema and solitary nucleus) and is nearly eliminated by the combination of the two drugs. In contrast, antagonism of NOS, GABA, or β-adrenoceptor actions did not affect the reflex. In vitro neurophysiologic studies (96,136) on gastric DMV neurons have shown that these neurons may be either activated or inhibited by norepinephrine via α1 or α2 adrenoceptors, respectively (Fig. 33-6). Parallel studies (147,148) show that brainstem reflex regulation of gastric function may be dependent on noradrenergic neurons in the NST. Although the esophageal-gastric reflex does not appear to be dependent on nitrergic neurons in the NSTc, it does appear that esophageo-esophageal interactions that regulate swallowing are dependent on this subset of neurons. Therefore, esophageal distension may initiate gastric relaxation through a brainstem reflex containing noradrenergic neurons. However, NOS neurons in the NSTc and their connections with vagal efferent neurons in the NAmb are probably involved in the feedback regulation of esophageal peristalsis during voluntary swallowing (149) (see also Chapters 35 and 36 regarding swallowing mechanisms). The precise circuits underlying other brainstem vago-vagal reflexes are not as well understood. The gastric accommodation reflex causes a fundic relaxation in response to antral mucosal contact or distension of the antrum. Either stimulus

A

10 µM B

The NSTc is unusual in that it is composed of two clearly defined neuronal phenotypes; NOS-containing neurons form a dense “core” of this subnucleus, which, in turn, is surrounded by a thin “shell” of neurons that contain tyrosine hydroxylase (TH; i.e., presumptive norepinephrine) (77). These NOS-NSTc neurons are thought to play a role in regulating the parasympathetic control of digestion. Direct injection of the NO precursor, arginine, into the DMV produced tonic gastric relaxation, whereas the NOS inhibitor, NG-nitro-L-arginine methyl ester, increased gastric tone. Both of these effects are mediated vagally (36). The DMV receives a dense input of NOS-immunoreactive (NOS-IR) fibers; however, it is unclear whether these terminals originate from neurons in the NSTc (77,146). Surprisingly, the NOS neurons in the NSTc are probably not involved in producing the receptive relaxation reflex. Repetitive distension of the esophagus primarily activates the TH-IR neurons in the NSTc, while NOS-IR–positive neurons were unresponsive (77). The relaxation reflex is partially blocked by either an α1 or an α2 adrenoceptor antagonist application onto the floor of the fourth ventricle

100 µM

C 1 µM D 10 µM E 100 µM F

FIG. 33-6. There are two subpopulations of dorsal motor nucleus of the vagus nerve (DMV) neurons: those that are excited by norepinephrine (NE), and those that are inhibited. (A–C) Dose-dependent increase in excitation produced by NE. Excitatory responses were blocked by prazosin, an α1 adrenoceptor antagonist (not shown). (D–F) The reaction of a different DMV neuron to NE. Inhibitory responses to NE were blocked by yohimbine, an α2 antagonist (not shown). (Modified from Martinez-Pena y Valenzuela, and colleagues [96], by permission.)

860 / CHAPTER 33 causes a brisk activation of neurons located in the medial portion of the NST (mNST), though the responsive cells are not confined to a specific subnucleus, as in the case of the receptive relaxation reflex (79). Antral stimulation activates NST neurons via vagal afferents and requires intact glutamate neurotransmission (150,151). This same stimulation inhibits virtually all DMV neurons examined (79). These data would suggest that antral control over fundic compliance involves a primary withdrawal of vagal cholinergic excitatory “tone.” However, pharmacologic studies have shown that fundic compliance may be regulated primarily by the activation of a vagal NANC pathway (2,30,31). As discussed previously, this disconnect of physiologic and pharmacologic observations is probably the result of differences in the relative sensitivity of different experimental approaches. For example, histologic studies of phenotypes and projection patterns of DMV neurons have shown that most DMV neurons provide input to cholinergic enteric plexus neurons (152). A much smaller number of DMV neurons are likely to provide input to putative NANC inhibitory enteric neurons (99). Thus, there is a higher probability or bias in favor of electrophysiologically identifying and recording from the former rather than the latter subpopulation of DMV neurons (78,79). In contrast, pharmacologic proof that a signal has been withdrawn (i.e., tonic cholinergic input to the stomach) is much harder to obtain than the demonstration that an effect is caused by the de novo release of an agonist (i.e., as happens when the NANC path has been activated). Even less information is available concerning the potential mechanism behind the fundic-antral excitation reflex observed by Andrews and colleagues (80). The presumption is that activation of fundic distension-sensitive vagal afferents causes the activation of antral-innervating DMV neurons through a reflex circuit. Although no specific investigations on this brainstem reflex have been performed, data from Rinaman (153) and Blackshaw and Grundy (154,155) provide a potential circuit by which vagal afferents may directly activate DMV neurons that could, in turn, activate cholinergic enteric projections to the stomach. Note that this evidence is not reflex specific; it may also explain why some vagal afferent stimulation can cause an increase in gastric acid secretion through direct activation of efferent vagal cholinergic projections (85–87). While some types of vagal afferent stimulation can cause an increase in acid secretion, recall that most vagal afferent stimulation that impacts acid secretion is inhibitory. Duodenal acidification and distension activates NST and inhibits DMV neurons (156). Since duodenal distension and acidification depresses gastric motility, as well as secretion, this circuit may regulate either function. In addition, although the preceding examples have shown that actions of agonists within the dorsal vagal complex (DVC) often result in a tandem increase (or decrease) of both gastric motility and gastric acid secretion, it must not be assumed that such a relation is mandatory. Indeed, the action of the neurotransmitter/hormone oxytocin within the DVC is to cause gastric

hypomotility (157), while augmenting gastric acid secretion (158). Thus, specific chemicals may encode the “appropriate” gastric responses that this circuitry may control.

Modulation of Brainstem Gastric Reflex Control Circuits by Descending Central Nervous System Pathways and Circulating Agents The connection between, sensory perception, emotion or mental activity, and gastric function was well appreciated in ancient times, even if the mechanisms were not understood. Hippocrates (~630 B.C.) in his “Nature of Man” identified a particular emotional state (“choleric,” i.e., irritable, temperamental) with gastric trouble. The 11th century physician, Avicenna, who wrote copious advice about diet and digestion, sagely observed that “Mental excitement or emotion…hinder digestion” (159). Beaumont’s early 19th century observations on Alexis St. Martin’s shotgun blast–induced chronic gastric fistula firmly established a link between mental state and the secretory and motile activities of the stomach. This link was strengthened by Pavlov (29), Cannon (160), and Wolf and Wolff (161) in classic works on the connection between brain and gut that spanned the early part of the 20th century. Knowledge of even the most essential details of CNSautonomic control of the upper GI tract has been obtained only recently. The delay in progress was due, fundamentally, to the difficulty in tracing the diffuse, unmyelinated projections to and from the dorsal medullary circuitry responsible for vagal control of the stomach. The application of modern retrograde tracing methods to the medial NST and DMV has revealed an enormous number of potential sources of CNS input to both nuclei. The details of these projections are cataloged in a review by Blessing (13). Essentially, the NST and DMV regions containing vago-vagal reflex control circuits receive input from the spinal cord, ventrolateral medulla, raphe nuclei, several components of the reticular formation, spinal trigeminal nucleus, A5 (pontine) and A6 (locus coeruleus) adrenergic areas, parabrachial and Kölliker Fuse nuclei, and dorsal tegmental regions including the periaqueductal gray. From the forebrain, the NST and DMV receive descending input from a large and interconnected complex of neurons in the paraventricular and lateral hypothalamic areas, central nucleus of the amygdala, and bed nucleus of the stria terminalis (13). Significant cortical projections to the NST and DMV come from the medial prefrontal and insular regions. Within each one of these potential sources of descending projections are embedded numerous phenotypes of potential modulatory neurons. These regions projecting to the NST and DMV are richly interconnected. Indeed, the NST maintains reciprocal connections with practically all of the areas mentioned above (162,163). To say that the CNS has infinite latitude in the control of gastric vago-vagal reflex circuits would not be much of an exaggeration. At this time, there are relatively few wellunderstood effects of specific CNS pathways on vagal reflex

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 861 circuits in the brainstem. However, we begin to see patterns of control emerge that may help to explain how the brain regulates gastric function in response to such disparate challenges as cold exposure, the anticipation of feeding (i.e., the “cephalic” phase of digestion), the effects of stress, fear, or pain, and metabolic status.

Appropriation of Gastric Vago-Vagal Reflexes: TRHergic Inputs to the Dorsal Vagal Complex from the Raphe Nuclei One of the best-described examples of CNS modulation of gastric vagal reflex control originates in the medullary raphe nuclei, specifically the raphe obscurrus and pallidus. These midline structures are found at approximately the same rostrocaudal level as the solitary and dorsal motor nuclei. Raphe inputs to the DMV and NST were among the first to be described (164). The early work of Tache and colleagues (165), as well as our own efforts, quickly determined that the principal peptide neurotransmitter contained by medullary raphe neurons—that is, thyrotropin-releasing hormone (TRH)—could act in the DVC to produce a dramatic and lengthy increase in gastric motility and secretion by activating cholinergic vagal projections to the stomach (12,157,158). The physiologic role of this increased gastric activity may parallel the role of TRH and the raphe to respond to cold stress (49,166). Here, TRH functions at several critical junctures to regulate energy utilization and heat generation. In addition to the well-known role of TRH to regulate metabolic heat production through thyrotropin, raphe-TRH projections to the spinal cord can activate sympathetic inputs to the brown adipose tissue to induce thermogenesis. Shivering behavior can be initiated by TRH input to ventral horn neurons of the spinal cord. TRH action in the DVC may also assist thermogenesis by augmenting gastric motility, which is itself a thermogenic activity (49). TRH action to drive motility and secretion may also anticipate the increase in feeding behavior that occurs with exposure to cold and may represent an indirect component of the anticipatory or “cephalic phase” control over gastric function (167). Early in vivo neurophysiologic studies helped illustrate how TRH could exert such a potent effect to excite this pathway. TRH was found to activate DMV neurons and inhibit NST neurons that were part of the vago-vagal reflex pathway (168,169). This dichotomy of TRH effects on the DMV and the NST is reflected in the morphology of TRHcontaining raphe terminals. Ultrastructural analysis showed that TRH terminals on NST neurons were symmetric (characteristic of inhibitory effects), whereas TRH terminals on DMV neurons were asymmetric (characteristic of excitatory effects) (153). This anatomic and physiologic arrangement helps to explain why TRH can produce such a potent and long-lasting vagally mediated increase in gastric motility and secretion. TRH augmentation of efferent parasympathetic activity (109) vigorously stimulates gastric motility and acid secretory responses. Normally, these events would

be duly recorded by gastric and intestinal afferents with the result that vagal afferent inputs to the brainstem would act to terminate the primary TRH effect through vago-vagal reflex pathways. However, by inhibiting NST neurons while activating DMV neurons, TRH essentially commandeers the brainstem circuitry normally involved in the negative feedback regulation of gastric function to, instead, promote a vigorous and long-term increase in vagal efferent input to the stomach. Raphe projections to the DVC contain not only TRH, but also serotonin (5-HT). The two agonists are co-released from raphe terminals (170) and there is evidence for an interesting synergy of action between them. While application of 5-HT, alone, onto the DVC has minimal or no primary effect on gastric motility or acid secretion, the indolamine can synergize with TRH to produce larger effects than those elicited by TRH, alone (171,172). Furthermore, once “primed” by TRH, the DVC can respond to 5-HT to produce increases in motility and secretion (171) (Fig. 33-7). The mechanism of the synergy was thought to involve TRH-induced modulation of transduction mechanisms sensitive to 5-HT (171). This view was confirmed by the work of Browning and Travagli (104) (Fig. 33-8). These investigators first observed that the synergistic effect of 5-HT and TRH was not caused by an interaction at the level of the DMV membrane (173). In subsequent work (135), they observed that part of the TRH-mediated excitation of DMV neurons was caused by a reduction in the inhibitory influence of NST input onto DMV neurons. The result for the DMV neuron was withdrawal of inhibitory (GABA) inputs. Furthermore, TRH increased cyclic adenosine monophosphate (cAMP) in NST neurons that, in turn, unmasked a population of essentially silent 5-HT1A receptors. Ordinarily, 5-HT has no significant effect on NST neurons. However, with the unmasking of these 5-HT receptors, serotonin is now able to further reduce the activity of the NST neurons (see Fig. 33-8). These results suggest that activation of presynaptic TRH receptors initiates an intracellular signaling cascade that increases the levels of cAMP sufficiently to unmask previously silent 5-HT1A receptors on presynaptic nerve terminals within the DVC. Thus, this combination of the disinhibition of cholinergic vagal efferent activity obtained by inhibition of the GABAergic NST-DMV synapse in conjunction with the direct activation of the DMV neurons by TRH resulted in dramatic and long-term changes gastric acid secretion and motility (172,174,175) (see Fig. 33-7).

Reflex Facilitation: Oxytocin Inputs to the Dorsal Vagal Complex from the Paraventricular Nucleus Potentially anorexic or nauseagenic stimuli (e.g., high circulating levels of CCK) can cause gastric stasis via direct action on vagal afferent-brainstem reflex mechanisms. However, these circulating stimuli also strongly activate oxytocinergic neurons in the paraventricular nucleus (PVN) (176–178).

862 / CHAPTER 33 Baseline 5HT (1) 5HT (2) 5HT (3)

TRH (4) 5HT (5) 5HT (6) 5HT (7)

FIG. 33-7. The effects of serotonin (5-hydroxytryptamine [5-HT]) and thyrotropin-releasing hormone (TRH) on gastric motility patterns were monitored via a strain gauge secured to the corpus portion of the stomach of an anesthetized rat. Microinjection of 5-HT (8 pmol in 4 nl) into the DMV produced a small increase in motility and tone, an effect that was lost with repeated injections (5-HT (1) to (3)). TRH (1 nmol in 1 µl) applied to the surface of the dorsal medulla evoked a large increase in gastric motility and tone (and secretion). After gastric motility returned to baseline levels following the TRH application, subsequent 5-HT injections (5-HT (5) to (7)) are now capable of eliciting a significant increase in gastric motility. This observation provided evidence that one agonist could “gate” the actions of others, perhaps by regulating receptor function through convergent transduction mechanisms. Subsequently, this has been proved to be the case (see Fig. 33-8). (Modified from McCann and colleagues [171], by permission.)

Oxytocinergic projections from the PVN to the DVC were among the first to be identified phenotypically (179). PVN projections to the DVC are strongly excitatory, and oxytocin activates both NST and DMV neurons (180). Oxytocin injected into the DVC produces a potent gastric relaxation that can be mimicked by electrical stimulation of the PVN and oxytocin antagonists abolish the effects of PVN stimulation on gastric function (85,157,158,181,182). It is likely that PVN oxytocin pathways produce their effects on gastric function in the same general way as TRH (183, 184); but in this case, PVN oxytocin increases the sensitivity of vago-vagal reflexes regulating gastric secretion and motility. Recall that the resultant effects on gastric function caused by action of oxytocin within the DVC is gastric hypomotility (157) while augmenting gastric acid secretion (158). In contrast, lesioning the PVN sharply diminished the sensitivity of brainstem vago-vagal reflex mechanisms to afferent stimulation (85).

Selective Efferent Activation: Corticotrophin-Releasing Factor Activation of the Vagal Nonadrenergic, Noncholinergic Path A wide variety of stressors including trauma, surgery, pain, and restraint (185–188) can cause a vagally mediated gastric stasis. Corticotrophin-releasing factor (CRF) is implicated in many of the behavioral and physiologic responses to threatening (stressful) circumstances (189,190). CRF-containing projections from the PVN of the hypothalamus and

Barrington’s nucleus terminate within the NST and DMV (191–193), and these DVC structures express receptors for the peptide (194). CRF injections into the DVC can cause a significant gastric stasis and CRF antagonists applied centrally block stress-induced gastric relaxation. Neurophysiologic studies show that CRF action is specifically excitatory on DMV neurons due to interaction with CRF2 receptors (195). The inhibitory effects of CRF on gastric motility are achieved by specific activation of DMV neurons that are part of the preganglionic, NANC (inhibitory) vagal pathway. Microinjection of CRF into the DMV continued to evoke gastric inhibition even if peripheral cholinergic muscarinic receptors were saturated by bethanechol; however, pretreatment with systemic NOS inhibitors abolished these gastric effects. These results provide direct proof of the existence of vagal NANC pathways and reinforce the concept of the fundamental role of brainstem circuits in the descending CNS control of GI functions, as well as delineate the selective vagal pathways used by CRF activation. Many parallel examples of CNS modulation have come to light (196). Some potentially important influences over DVC circuits include: opioids (132), neuropeptide Y (NPY) (197), cannabinoids (198), vanilloid (199), leptin (200), melanocortin (201), dopamine (202), and norepinephrine (96). A detailed examination of the effects of opiates on vago-vagal release mechanisms has revealed another novel and potentially powerful means by which vago-vagal reflexes can be modulated by means of receptor trafficking and short-term plasticity.

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 863 Glutamate (+) TRH and 5HT (−)

NST

TRH or 5HT (−) NST

Afferents

GABA (−)

GABA (−)

DMV TRH (+)

Stomach

A Vagal afferent 5HT

4

1a NST

5

cAMP/PKA 3

2 −

K+ (out)

TRH

6 +

GABA

B

DMV

Control 5HT

7

1

Control TRH

Control TRH + 5HT

FIG. 33-8. (A) Schematic illustrating the effects of thyrotropin-releasing hormone (TRH) and 5-hydroxytryptamine (5-HT) to modulate gastric vago-vagal reflexes. Two subpopulations of nucleus of the solitary tract (NST) neurons are shown that are activated by vagal afferent input from the gut. When activated, these NST cells inhibit the dorsal motor nucleus of the vagus nerve (DMV). Roughly half of these NST neurons are inhibited by TRH or 5-HT; the other half is inhibited by the combination of TRH plus 5-HT. This disinhibition of DMV neurons (by inhibiting NST neurons), in combination with the direct activation of DMV neurons by TRH, is responsible for the potent and prolonged effects of TRH plus 5-HT to regulate gastric function. (B) Modes of action of TRH and 5-HT to regulate vagovagal circuitry. Schematic indicates that TRH potently and directly activates DMV neurons (1), while inhibiting NST neurons (2–4). TRH and 5-HT effects synergize in some NST neurons as a consequence of TRH modulation of the sensitivity of 5-HT1a receptors. The convergence of 5-HT and TRH effects within the NST reinforces the disinhibition of the same DMV neurons that are directly activated by TRH. Ordinarily, TRH or 5-HT alone has limited effects on these NST neurons, but together these agonists inhibit the NST by invoking an outward (hyperpolarizing) current (5). This causes a reduction in the inhibitory postsynaptic currents (IPSCs) generated in the DMV by the NST (6,7) (see recordings in C). Taken together, the result is that TRH can command a prolonged increase in gastric motility by activating the DMV (directly) and, at the same time, blunting the feedback consequences at the NST. (C) Under normal circumstances (i.e., no drug), stimulation of the NST evokes a large outward current (i.e., inhibitory) in DMV neurons as seen in these in vitro electrophysiologic recordings of DMV neurons. When this in vitro preparation is exposed to either 5-HT or TRH alone under these stimulation conditions, there is no effect on the inhibitory (i.e., outward) current. However, when both 5-HT and TRH are applied in combination, there is a reduction in the magnitude of the inhibitory synaptic input as a consequence of electrical stimulation of the NST. cAMP, cyclic adenosine monophosphate; GABA, γ-aminobutyric acid; PKA, protein kinase A. (Modified from Browning and Travagli [104], by permission.)

C

Short-Term Receptor Trafficking and Plasticity: A New Concept for Vago-Vagal Reflex Control Another variant for modulation of reflex control is the possibility of agonist-activated receptor trafficking. This option might explain how particular aspects of vago-vagal reflex control can be rapidly switched on and off by a number of signal molecules. As an example, Browning and colleagues (132) showed that opioid peptides modulate only glutamatergic currents between NST and DMV, leaving the GABAergic currents unaffected. This electrophysiologic observation was paralleled by immunohistochemical data that showed the presence of µ-opioid receptors (MOR) on glutamate- but not GABA-containing profiles apposing DMV neurons. In subsequent work, Browning and colleagues (203) demonstrated that TRH increased levels of cAMP in NST neurons (see the previous section) that

induced a fast (within 5 minutes), but short-lasting (1 hour), translocation of MOR from intracellular stores to the membrane of GABAergic NST neurons (Fig. 33-9). This effect takes place on a time scale unlikely to involve de novo receptor synthesis and provides a means for a rapid and situation-specific up-regulation of opioid effects on reflex sensitivity. Interestingly, this phenomenon is not limited to the modulation of GABA currents by opioids only, but it may be a common means by which other neurotransmitters (especially the pancreatic polypeptides) (197) regulate NST influences over DMV neurons. In summary, a growing number of neurotransmitters appear to regulate the insertion of pre-assembled receptors into the membrane of NST neurons. This means that one agonist can “permit” another to regulate information flow through the DVC. One can almost imagine “logic circuits” in the NST with such equations as “IF” (NPY) “AND” (opiates),

864 / CHAPTER 33 ME

Control

NTS

Golgi complex MOR GABA

Increase cAMP

DMV

100 pA

NTS

50 ms Control

Circulating Factors Controlling Brainstem Vago-Vagal Reflex Mechanisms

ME

DMV

A

the stomach. Gastric transit and motility are greatest during feeding, a time when the esophageal-gastric relaxation reflex should be functioning to depress motility, while enlarging the fundic reservoir (206–208). Somehow, the receptive relaxation reflex is “suspended” during, and just after, the act of feeding (52).

B

FIG. 33-9. Modulating cyclic adenosine monophosphate (cAMP) levels in the brainstem induces receptor trafficking in vago-vagal reflex circuit elements. (A) Upper panel: Representative traces of electrically evoked inhibitory postsynaptic currents (IPSCs) in a gastric-projecting dorsal motor nucleus of the vagus nerve (DMV) neuron voltageclamped at −50 mV. Perfusion with 10 µM Met-enkephalin (ME) does not affect the amplitude of IPSCs evoked by electrical stimulation of the nucleus of the solitary tract (NTS). Lower panel: After 5-minute exposure to 10 µM forskolin to elevate cAMP levels, perfusion with 10 µM ME reduces the amplitude of the IPSCs. Holding potential = −50 mV. Each trace represents the average of 3 IPSCs evoked 20 seconds apart. (B) Proposed scheme of the synaptic connections between NTS and DMV that are modulated by µ-opioid receptors. Upper panel: Under control conditions, that is, when the levels of cAMP are low, the terminals of GABAergic neurons in the NTS store µ-opioid receptors (MOR) in internal compartments associated with the Golgi complex. In this situation, µ-opioid agonists such as ME cannot modulate the release of γ-aminobutyric acid (GABA) onto DMV neurons. Lower panel: Conditions that increase cAMP levels within the GABAergic terminal provoke the release of the MOR complex from the Golgi complex that are then transported to the membrane apposing DMV neurons. In this situation, µ-opioid agonists such as ME can modulate the release of GABA onto DMV neurons. This scheme has been verified in parallel immunocytochemical studies (203). (Modified from Browning and colleagues [132], by permission.)

“THEN” (augment gastric motility and secretion).” It is not too much of a stretch of the analogy to superimpose another level of analysis on the circuit: “IF you anticipate eating (i.e., CNS-NPY release [204]) AND you’re happy about it (i.e., feeding related opiate release [205]), THEN prepare the stomach for a meal.” Although this analysis might appear dangerously anthropomorphic, consider the other “logical” possibilities. If feeding is anticipated, but some other conflicting stimulus (e.g., stress) prevents opiate release, then digestion may not be activated. Likewise, opiate release without an anticipation to feed may not activate digestion. This sort of permissive mechanism could also explain one of the persistent paradoxes in vagal reflex control of

The case has already been made for the potent and physiologically significant effects of CCK to activate gastric and duodenal vagal afferents. There is still some controversy about whether CCK action is primarily paracrine or endocrine. However, the realization that gut hormones could signal changes in vagal reflex functions by acting on afferent fibers has fueled speculation that other circulating or paracrine factors could regulate digestive functions through action on these circuits as well. Indeed, a number of receptors have been identified on vagal afferents and their activation may lead to increases (5-HT3, CCK1, vanilloid receptor 1 [VR1], neurokinin-1, apolipoprotein A4, IL-1) or decreases (ghrelin, leptin, κ opioid, GABAB) in afferent activity (209). These effects on afferent excitability may be reflected in changes in gastric reflex function (52). The recognition that vagal afferents are essentially polysensory (i.e., sensitive to nutrients, secreta, transmitters, hormones, and distension) has helped explain the breadth of factors that can influence gastric function through afferentdriven vagal reflexes. However, the brainstem possesses its own independent and equally complex chemosensitivity. This capacity often is reflected in profound changes in gastric function seen in normal and disease states.

The Dorsal Vagal Complex and Chemosensitivity The term dorsal vagal complex (DVC) is most accurately used to describe the combination of the DMV, NST, and area postrema. The area postrema has been singled out as a unique site for the detection of circulating factors that may be important to the regulation of autonomic and behavioral functions. However, the distinction between area postrema and NST has blurred. Like the area postrema, the NST possesses large, fenestrated capillaries and has been shown in numerous studies to be outside the blood–brain diffusion barrier (210,211). Like the NST, the area postrema receives vagal afferent input. The area postrema makes essentially the same afferent connections as the NST and many of the neuronal phenotypes found in the NST also are found in the area postrema (19,212–217). To further the complication, dendrites from vagal motoneurons in the DMV penetrate the NST and occasionally the boundary with the area postrema (214). Thus, cellular components of the area postrema, NST, and DMV together (as the DVC) receive vagal afferent inputs and are equally likely to be under the simultaneous

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 865 influence of circulating signal molecules. Numerous chemical influences on the excitability of NST, DMV, and area postrema neurons have been catalogued. Much of this work has been done to investigate the phenomena of emesis and taste aversion learning (216,218–222). These topics are relevant to gastric control because gastric relaxation is a consistent prodrome for emesis (218) and agents causing taste aversion are commonly associated with gastric relaxation (223,224). These function-specific chemospecificities are described in Chapters 35–37 concerning emetic mechanisms and 34 concerning the regulation of feeding behavior. Under physiologic conditions (i.e., excluding the effects of toxins and drugs), vago-vagal reflexes that control the stomach are engaged by three classes of circulating molecules: nutrients, hormones, and cytokines. An example of each is reviewed in the following sections. Glucose Among some of the most common circulating signal molecules, glucose has received relatively little attention in relation to its influence on vago-vagal reflexes. This is particularly surprising given that CNS glucose homeostasis is clearly a factor controlling gastric function via the vagus nerve. Microinjections of glucose into the NST or DMV of anesthetized rats decrease gastric motility and intragastric pressure. These effects appear to be more related to the excitatory actions of glucose on NST (225) than inhibitory actions on DMV, as suggested previously (226,227). Sensitivity to glucose by the DVC is a critical component of the dramatic CNS-mediated increase in gastric acid secretion and motility that occurs with hypoglycemia (25). Neurons in both NST and DMV are affected by glucose; they are distinguished as gluco-responsive (i.e., glucoexcitatory) and gluco-sensitive (i.e., gluco-inhibitory) neurons. NST neurons are always excited (i.e., gluco-responsive) by increased glucose levels likely via blockade of KATP channels resulting in depolarization (228–232). Inhibition of DMV neurons by hyperglycemic versus excitation by hypoglycemic conditions have been shown to be the result of indirect effects, that is, because of a direct effect of glucose on GABAergic NST neurons impinging onto DMV. These in vitro observations support the in vivo data that NST neurons are tonically inhibiting the activity of DMV neurons (131,225). Thus, it is hypothesized that acute hyperglycemia affects the central components of vago-vagal reflexes by inducing a decrease in gastric motility and an increase in intragastric pressure via disinhibition of preganglionic vagal neurons. Dorsal Vagal Complex Detection of Pancreatic Polypeptide and Peptide YY The gut hormones pancreatic polypeptide (PP) and peptide YY (PYY) exert powerful control over digestive functions (49,92,233). PP, a hormone first identified as a contaminant of bovine insulin, is produced exclusively by

δ cells of the pancreas. Intravenous PP inhibits vagally stimulated pancreatic exocrine secretion, while increasing gastric motility and acid secretion (92,234,235). These two apparently conflicting effects (stimulation of gastric digestion and inhibition of pancreatic secretion) make physiologic sense when considering the secretory capacity of the exocrine pancreas. Taylor (236) has estimated that the pancreas is capable of releasing 8 to 10 times the amount of digestive enzymes necessary to digest a meal. PP augments the gastric phase of digestion while preventing the pancreas from digesting itself and the proximal duodenum. PP levels increase sharply at the onset of feeding, and the release of PP is largely under vagal reflex control (237). Curiously, PP receptors are absent from the digestive tract, and the effects of PP appear to be entirely dependent on intact vagal connections with the brainstem (92,234,235). This paradox led to the prediction that PP receptors are found only in physiologically significant concentrations within the DVC. These predictions were borne out by a combination of careful transport and radioligand binding studies (91). The application of PP directly to DVC circuitry completely and potently replicates the circulating effects of the peptide (235). Neurophysiologic studies further show that vagal efferent neurons involved in excitatory control of the stomach are themselves activated by PP (238). A significant population of DMV neurons not related to gastric reflex control is inhibited by PP and these may provide excitatory input to the pancreas (239). PYY may exert control over gastric vagal reflex circuitry using the same general mechanism. PYY is released primarily by endocrine cells in the ileum in response to fatty acids in the gut lumen (236). PYY effects are “enterogastrone”like given that this peptide causes a significant depression in gastric transit (240,241). PYY emerges as the critical component of the “ileal brake” mechanism by which gastric transit is inhibited by the presence of fatty acid in the ileum. As is the case for PP, PYY receptors are concentrated in the DVC (90) and PYY can gain access to the DVC through fenestrated capillaries. Low doses of PYY cause a Y2 receptor–mediated inhibition of gastric transit that is reflected in the receptor-specific inhibition of gastroexcitatory vagal efferent neurons in the DMV (197,242,243). This Y2 receptor specificity is significant, given the fate of PYY released by the ileum. A nearly ubiquitous diaminopeptidase (DAP IV; [244]) rapidly converts PYY to a shortened form of the PYY molecule, PYY 3-36. PYY 3-36 is a specific Y2 receptor agonist. In all likelihood, PYY 3-36 depresses gastric motility by acting specifically on Y2 receptors in the DMV (49,92,242,243,245). In addition to direct effects pancreatic polypeptides may have on DMV neurons, these peptides also inhibit glutamatergic synaptic transmission between the NST and the DMV (197). Pharmacologic evidence suggests that different “Y” receptors are involved. Specifically, activation of Y1, Y2, and another pancreatic peptides class receptor, possibly the putative Y3 receptor, inhibits excitatory synaptic transmission from the NST to GI-projecting DMV neurons.

866 / CHAPTER 33 The effects of the putative Y3 receptor activation are mediated by release of norepinephrine and its subsequent interaction with α2 adrenoceptors. However, evidence indicates that Y1-3 modulation of vagal circuits undergoes the same type of short-term plasticity in the modulation of GABAergic currents seen with opioids. That is, the state of activation of the circuit, which is reflected in cAMP levels, determines the type of modulatory response produced by these peptides. Immune Activation, Cytokine Detection by the Dorsal Vagal Complex, and Gastric Vago-Vagal Reflex Control Activation of the immune system in response to illness or injury initiates a cascade of cytokine release meant to recruit immune effector cells and, effectively, isolate the challenge. The physiologic and behavioral repertoire that follows infection has been referred to as “sickness behavior” and is characterized by ancient hallmarks of disease: fever, fatigue, listlessness, hypersensitivity to touch or pain, significant changes in sleep patterns, loss of appetite, gastric stasis, and emesis (246–248). The production of cytokines by the immune system in response to infection or injury obviously plays a critical role in coordinating the host’s defense against this challenge. This process is triggered by proinflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α], IL-1, IL-6) that are produced by peripheral phagocytic cells. The acute physiologic reactions by the host, which include the “sickness behaviors,” serve to eliminate the challenge and restore homeostasis via local and systemic immunologic, neuroendocrine, metabolic, and behavioral reactions (249–252). TNF-α is one of the early proinflammatory cytokines to be released by activated macrophages and lymphocytes. Cancer or exposure to radiation evokes production of TNF-α by nonimmune cell types as well (253). Systemic injection of TNF-α can mimic many of the autonomic signs associated with illness such as increased body temperature (252,254–256), increased plasma corticosterone levels (257), gastric hypomotility (258), suppressed appetite, as well as nausea and vomiting (255,256,259,260). CNS binding sites for TNF-α demonstrated a particularly high density in the brainstem (261) and TNF-α was shown to be able to cross the blood–brain barrier via a specific and saturable transport system (262). Subsequently, Nadeau and Rivest (263) reported the constitutive expression of messenger RNA for the TNF receptor type 1 (TNFR1) on circumventricular organs, although the residence of TNFRs on elements of the DVC had not been demonstrated. It seemed reasonable to expect that if TNF-α is responsible for changes in gastric function during illness or pathologic conditions, then perhaps one of its sites of action is within the DVC. α and Gastric Stasis Tumor Necrosis Factor-α Microinjections of TNF-α directly into the DVC produce a dose-related suppression of gastric motility (Fig. 33-10A

and B). In these studies, strain gauges were secured to the antral portion of the stomach of anesthetized rats. Basal gastric motility and tone is minimal under anesthesia. Therefore, maximal gastric motility was evoked via vagal, cholinergic stimulation (i.e., application of 0.2 nmol TRH onto the floor of the fourth ventricle). Against this vagally stimulated gastric motility, gastric stasis could be evoked rapidly by unilateral injections of TNF-α directly into the DVC. This suppression of gastric motility effect had a threshold in the subfemtomolar range (0.02 fmol in 20 nl volume) and was dependent on intact vagal pathways (264). α Potentiation of Vago-Vagal Tumor Necrosis Factor-α Reflex Responsiveness Subsequent electrophysiologic studies showed that nanoinjection of TNF-α into the DVC produces an activation of identified NST neurons (265) and an inhibition of identified DMV neurons (266). According to the basic vago-vagal reflex circuit, these neurophysiologic observations are consistent with the physiologic observations of gastroinhibition. Of particular interest was the observation that sensory neurons exposed to TNF-α displayed a hypersensitivity to natural stimulation that outlasted the exposure to the TNF-α. That is, those neurons responsive to gastric distension were identified as gastric-NST; when exposed briefly to small amounts of TNF-α (0.03 fmol in 3 nl; 5-second exposure), these neurons were hyper-responsive to subsequent exposures to identical magnitudes of gastric distension (see Fig. 33-10C to F) (265). This prolonged potentiation of the NST response to stimulation after exposure to TNF-α may provide a basis by which increased circulating levels of TNF-α (e.g., during illness) may alter sensory activation. In the case of gastricrelated NST, this prolonged activation would, ultimately, evoke gastric stasis.

Mechanism of Tumor Necrosis Factor Action in the Brainstem Systemic, endogenous production of TNF-α (and other cytokines) provoked by the systemic administration of lipopolysaccharide (LPS) (267) was sufficient to elicit gastric stasis (268). Without the availability of a specific antagonist for TNF-α, the TNF-α–specific adsorbing construct (e.g., etanercept) was used to suppress the effects of TNF-α. In this case, application of the TNF adsorbent to the fourth ventricular surface blocked induction of gastric stasis (269) that was normally elicited by systemic LPS challenge. Secondly, up-regulation of the immediate early gene product, c-Fos, serves as a marker for neuronal activation (215). Systemic exposure to LPS will induce cFos activation in NST neurons; this up-regulation of c-Fos also can be blocked by application of the TNF adsorbent to the fourth ventricular surface (270). Together, these data verified that peripherally generated, endogenous TNF-α

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can gain access to neurons of the DVC and inhibit gastric motility. Activation of NST neurons directly by TNF-α was demonstrable using immunohistologic techniques to stain for c-Fos generation (271). The majority of afferents to the NST are glutamatergic (272,273); therefore, it was hypothesized that one means of activation of NST neurons by TNF-α might be via its interaction with glutamatergic vagal afferents. Co-injection of glutamate antagonists (e.g., NBQX or MK-801) with TNF-α directly into the DVC suppressed TNF-α–induced c-Fos generation in NST neurons. These data suggested that TNF-α might act to alter vago-vagal reflex functions through presynaptic modulation of glutamate release from afferents (271).

FIG. 33-10. (A, B) Polygraph records strain gauge activity monitoring gastric motility. Basal gastric motility and tone of a food-deprived and anesthetized animal are minimal. Therefore, gastric motility was maximally stimulated by application of the peptide thyrotropin-releasing hormone (TRH; 0.2 nmol; i.e., vagal, cholinergic stimulation) directly onto the exposed brainstem (TRH intracerebroventricularly [icv]). (A) When stimulated gastric motility has plateaued (approximately 5–10 minutes after TRH application); unilateral microinjection of 20 nl phosphate-buffered saline (PBS) was made into the left dorsal vagal complex (DVC). No change in motility was observed. (B) When tumor necrosis factor-α (TNF-α) (10−9 M = 0.02 fmol in 20 nl) was microinjected into the DVC, gastric motility was rapidly suppressed for prolonged periods (in excess of 2 hours in this example). Unilateral vagotomy on the side of the TNF injection blocks the effect of TNF (data not shown; refer to original article by Hermann and Rogers [264]). (C–F) Effects of gastric distension, PBS, or TNF-α microinjection on a single neuron of the nucleus of the solitary tract (NST). (C) (Upper left trace) Raw oscillograph record of the identification of a gastric distension-related neuron in the NST. Distension of antrum (bottom trace) produces a brisk increase the firing rate of the NST neuron; phase locked to the stimulus. (Upper right trace) Integrated rate-meter record of the same event. Inset shows 20 superimposed NST spikes; profile of identified NST neuron responding to gastric distension alone. (D) Integrated record of gastric motility. Control injection of PBS (3 nl delivered at arrow) has no effect on neuron’s firing rate; same neuron as recorded in C. Inset shows neuron’s signature spike profile. (E) TNF (0.03 fmol) microinjected onto same NST neuron causes activation of identified neuron. (F) Response to gastric distension of same NST neuron depicted in C, now 30 minutes after exposure to and recovery from the TNF injection seen in E. Note the greatly potentiated response to gastric distension that significantly outlasts the stimulus. Inset shows superimposed, individual NST spikes identifying this neuron as the same as that seen in C. These data show that the gastroparesis caused by TNF action in the central nervous system is probably due to excitation of NST neurons activated by gastric vagal afferent input. Activation of NST neurons that form the afferent limb of gastric vago-vagal control reflexes produces gastroinhibition. The primary effect of TNF is amplified by a long-term potentiation of responsiveness to vagal afferent input. Scale bars for insets = 200 µV/2.5 msec. (Modified from Hermann and Rogers [264] and Emch and colleagues [265], by permission.)

Constitutive Expression of Tumor Necrosis Factor Receptors on Brainstem Afferents There are two cell-surface receptor types for TNF-α: TNFR1 (also referred to as p55, p60) and TNF receptor type 2 (TNFR2; also referred to as p75, p80). The TNFR1 receptor possesses the domain responsible for the cascade of cellular signaling molecules ultimately leading to apoptosis (274–279). The TNFR2 receptor may contribute to the initiation of this cascade via unmasking or activating the dormant apoptotic machinery associated with TNFR1 (280). TNFR2 receptors also exist in a soluble form (sTNFR2) and are thought to function as a ligand-passing molecule (281), thus acting as a TNF-α transport molecule subserving

868 / CHAPTER 33 presented as fine filamentous strands of dark reaction product as they diverge to enter the medial solitary nucleus located dorsal to the DMV at the level of the area postrema. Adjacent histologic sections from the same animal indicated that only TNFR1-IR, and not TNFR2-IR, is constitutively expressed in the fibers of the solitary tract as well as spinal trigeminal and area postrema afferents. Curiously, TNFR1-IR was not observed in the peripheral vagal trunks, even though it was observed in the nodose ganglion and the centripetal vagal fibers leaving the nodose and proceeding onto the brainstem (see Hermann and colleagues [129]). Thus, TNF-α may alter vago-vagal reflex control of the gut by acting on vagal afferents within the NST and area postrema. Furthermore, binding of TNF-α to presynaptic vagal afferents may produce the sort of longterm potentiation to otherwise innocuous visceral afferent input similar to our neurophysiologic results (265). Grassi and colleagues (286) have described a model where TNF-α alters presynaptic glutamate release in

TNFR1 activation. In vitro studies have shown that interaction with TNF-α can induce shedding of the TNFR2 receptor into the medium (282). In the capacity of a TNF-α transport molecule, one can hypothesize a role for the sTNFR2 receptor to prolong the inflammatory response by the intermittent delivery and release of TNF-α to TNFR1. There also have been reports that the TNFR2 receptor may play an important signaling role in chronic inflammatory conditions such as Crohn’s disease (283). Although there have been reports of high density of binding sites for TNF-α within the brainstem (261) and constitutive expression of messenger RNA for the TNFR1 on circumventricular organs (263,284), the immunohistochemical demonstration of TNFRs on vagal afferents (Fig. 33-11A to D) (129) required the employment of a heat-induced antigen retrieval technique (285). Dense TNFR1-IR is constitutively present on central (but not peripheral) vagal afferents in the solitary tract and nucleus as well as on afferents entering the spinal trigeminal nucleus. Fibers in cross section

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FIG. 33-11. Immunocytochemical demonstration of the p55 tumor necrosis factor (TNF) receptor on vagal afferents within the nucleus of the solitary tract (NST) and area postrema (AP). (A) Vagal afferents entering the medulla (X aff) are immunopositive for the TNF-p55 receptor. Note that trigeminal afferents projecting to the spinal trigeminal nucleus (SN V) also are labeled. Since these afferents are implicated in pain transmission, this finding may explain the hyperalgesia and allodynia that occurs in disease processes in which TNF is generated by an activated immune system. (B) Higher magnification of p55-labeled vagal afferents in the solitary tract. (C) p55-immunoreactive vagal afferent fibers ramify widely in the medial NST (mNST), the location of neurons that form the afferent limb of gastric vago-vagal reflexes. (D) p55-labaled vagal afferents also enter the area postrema, where they are completely outside the blood–brain barrier. (E) Cartoon diagram showing the possible loci at which TNF could affect vago-vagal reflex function. Most data now suggest that TNF probably modulates reflex function by regulating glutamate release from vagal afferent terminals in the NST and AP (271). AMPA, α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid; GABA, γ-aminobutyric acid; ICP, inferior cerebellar peduncle; NMDA, N-methyl-D-aspartate. (Modified from Hermann and colleagues [129], by permission.)

BRAINSTEM CONTROL OF GASTRIC FUNCTION / 869 cultured hippocampal neurons by increasing calcium ion availability to the presynaptic transmitter release mechanism. Thus, TNF-α, acting at TNFR1 receptors on the presynaptic afferents, indirectly increases glutamate release. Our observations of TNFR1-IR on neurons and fibers of the nodose ganglion tempt us to suggest that a similar mechanism may exist for circulating TNF-α to act directly on these neurons and, ultimately, to potentiate transmitter release onto NST neurons. The constitutive presence of these receptors on these afferents helps explain why inflammatory processes that generate TNF-α may disrupt GI functions.

SUMMARY The DVC is the focal point of an enormous convergence of sensory afferents, descending CNS pathways, and circulating signal molecules. The static performance of the circuits in the brainstem produces timely and critical reflex control over the stomach. However, it is now apparent that the vagal reflex control circuits are not static entities functioning as simple relays between brain and gut. These brainstem circuits achieve exquisite coordination of digestive processes with ongoing and anticipated changes in behavior as a consequence of the diversity of descending CNS input. In addition, circulating factors can cause rapid changes in brain–gut control through their direct action on brainstem vagal reflex control circuits. A multitude of possible modulatory mechanisms exist within these circuits to guarantee speed, precision, and flexibility in the control of digestive processes.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grants DK-52142, DK-55530, and DK-56373. Support was also provided by NSF grant 00456291.

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CHAPTER

34

Neural and Hormonal Controls of Food Intake and Satiety Timothy H. Moran Food Intake and Energy Balance, 877 Metabolic Signals and Their Mediation, 877 Hypothalamic Systems Involved in Feeding Control, 878 Within-Meal Feedback Signaling, 881 Gut Neural Signaling, 882 Within-Meal Gut Peptide Signaling, 883

Across-Meal Peptide Signaling, 885 Interactions between Gut Peptide and Hypothalamic Signaling, 887 Summary, 888 References, 888

FOOD INTAKE AND ENERGY BALANCE

METABOLIC SIGNALS AND THEIR MEDIATION

The body has multiple redundant controls for the maintenance of energy balance. The developing obesity epidemic highlights the primacy of systems that stimulate food intake. In the face of caloric abundance and a reduced need for regular physical exertion, mean body weights are increasing, and the prevalence of disorders secondary to excess weight gain are increasing at an alarming rate. Despite what seems to be an intractable problem, our knowledge of physiologic systems that either drive or limit food intake has greatly increased during the turn of this century. This increasing knowledge holds the promise for the eventual development of rational therapies for facilitating weight loss and weight maintenance. This review focuses on two interrelated regulatory systems involved in feeding control. These are systems that mediate responses to the following signals: (1) signals related to metabolic state, especially those that indicate the degree of stored or available energy; and (2) signals that arise from the gastrointestinal tract before, during, and after meals, the stimulation of which is modulated by the nutrient content of what is consumed. The interaction between these systems plays a major role in determining the overall regulation of energy balance.

A role for signals related to the availability of energy stores in the control of food intake has long been postulated. Depletion/repletion models tied to carbohydrate availability (1) and the extent of fat stores (2) dominated the early years of feeding research. Although none of these individual models was sufficient to explain the multiple variations in food intake that occur throughout the life cycle, there is considerable evidence for food intake controls that depend on monitoring fuel availability and utilization. Thus, administration of metabolic inhibitors that act on differing metabolic pathways can be shown to stimulate food intake. For example, treatment with either 2-deoxy-D-methyl glucose, an inhibitor of glucose utilization (3), or methyl palmoxirate, producing a blockade of fatty acid oxidation (4), stimulate food intake in satiated animals. Further support for a role for monitoring energy availability in food intake control comes from experiments showing a relation between circulating glucose and meal initiation in both rats and humans. Campfield and colleagues (5,6) have demonstrated that transient declines and partial restorations in blood glucose levels reliably predict meal initiation. Demonstrations that experimentally induced declines in blood glucose can result in meal initiation have led to the suggestion that the relation may be more than correlational (7). Early studies of mouse genetic obesity models suggested the importance of circulating factors in overall body weight control. Having identified two different mutations in mice that led to obesity, Coleman (8) conducted parabiosis experiments

T. H. Moran: Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

877

878 / CHAPTER 34 involving obese ob/ob, obese db/db, and normal mice in which the blood supply between two mice in a parabiotic pair was shared. The results showed that when paired with db/db mice, ob/ob mice became hypoglycemic, lost weight, and eventually died—a similar response to that seen in normal mice combined with db/db mice. In contrast, when combined with normal mice, ob/ob mice gained less weight than they otherwise would but were fully viable. The results led Coleman to conclude that the ob/ob mouse lacked a circulating satiety factor that in its absence results in hyperphagia and obesity, whereas the db/db mouse produced the factor but lacked the ability to appropriately respond to it. The positional cloning of leptin as the product of the ob gene (9) and subsequent identification of the leptin receptor as the protein product of the db gene (10,11) has provided the basis for understanding Coleman’s observations. Leptin not only normalizes food intake and body weight in ob/ob mice, but also reduces food intake in normal mice and rats (12). Leptin is produced in white adipose tissue and circulates in proportion with the adipose mass, increasing in plasma as animals and humans become obese (13) and decreasing in times of food deprivation (14). Thus, leptin provides a signal of the availability of the body’s energy stores. Leptin receptors are members of the cytokine receptor superfamily. Multiple leptin receptor isoforms that arise from differential splicing have been identified. The predominant, short form (Ob-Ra) is widely expressed in multiple tissues including the choroid plexus and brain microvasculature (15). These brain binding sites have been proposed to function as part of a saturable transport system allowing circulating leptin to gain access into the brain (16). The long form of the leptin receptor (Ob-Rb) is highly expressed within hypothalamic nuclei with identified roles in energy balance. Greatest concentrations of the long form of the leptin receptor are found within the arcuate, paraventricular, and dorsomedial hypothalamic nuclei, as well as within the lateral hypothalamus (17). The action of leptin at these hypothalamic sites results in the activation or inactivation of hypothalamic pathways containing various anorexigenic and orexigenic peptides (18) (see later). Interactions between leptin and Ob-Rb result in activation of Janus-activated kinase-2 (JAK2) and signal transducer and activator of transcription-3 (STAT3) to regulate gene transcription (19). Interaction of leptin with its receptor has also been demonstrated to activate phosphatidylinositol 3-kinase (PI3K), providing an additional mechanism for the signaling effects of leptin (20). The ability of leptin to inhibit food intake can be shown to depend on both of these signaling pathways (20,21). Although leptin currently is viewed as the major adiposity factor important for the long-term control of energy balance, similar actions have been shown for insulin. Insulin is also secreted in proportion to the body mass (22), mechanisms for the transport of insulin into brain have been demonstrated (23), and, as discussed later, insulin interacts with the same hypothalamic circuits as leptin to affect overall energy balance (24). Increased leptin and insulin levels with increased adiposity would be expected to provide a significant negative

feedback signal to reduce food intake and body weight, yet the incidence of obesity continues to increase, providing an argument against the potency of such signals. Developed insensitivity to leptin and insulin has been postulated to explain the lack of responsivity of obese animals and humans to the negative feedback signal of prolonged increases of plasma leptin and insulin levels. Leptin insensitivity has been shown to be a two-stage process with initially reduced feeding inhibitory responses to peripheral, but not central, leptin administration and eventual loss of response to central leptin with further obesity. Thus, a down-regulation of leptin transport may precede a down-regulation of central leptin receptors against a background of high circulating leptin levels (25,26). Similar mechanisms for insulin insensitivity have been proposed (27).

Hypothalamic Systems Involved in Feeding Control An overall role for the brain in body weight control was originally suggested from patient data demonstrating that tumors compressing the base of the brain resulted in obesity. Froelich (28) suggested that obesity in response to such tumors was the result of altered pituitary function; however, based on autopsy results showing compression of the base of the brain, Erdheim (29) proposed that the obesity resulted from a neural lesion. The classic experiments of Hetherington and Ranson (30) resolved this controversy. Using the Horsely–Clarke stereotaxic device, they demonstrated that bilateral lesions of the medial hypothalamus resulted in hyperphagia and obesity in experimental animals. Work from Anand and Brobeck (31) suggested that the food intake and body weight could have multiple hypothalamic controls from experiments demonstrating that lesions of the lateral hypothalamus produced profound anorexia and weight loss. Subsequent work demonstrated that stimulation of these hypothalamic regions had the opposite effects. Medial hypothalamic stimulation inhibited food intake, whereas stimulation of the lateral hypothalamus produced food intake. Results such as these led Stellar (32) to propose the classic dual-center hypothesis for the role of the hypothalamus in food intake. The ventromedial region was viewed as a satiety center important for inhibiting food intake, whereas the lateral hypothalamus was viewed as a feeding stimulatory center. The identification of leptin and its brain sites of action allowed rapid advances in our understanding of the hypothalamic influences on food intake and shifted the focus from nuclei-specific influences to the identification of orexigenic and anorexigenic peptide systems that respond to circulating signals that vary with overall metabolic state. A major site for feeding control is the hypothalamic arcuate nucleus. As shown in Figure 34-1, the arcuate contains two distinct neuronal populations that both express leptin and insulin receptors (18,33), and leptin and insulin have specific effects on these two neuronal populations. The first is a population that expresses the propeptide proopiomelanocortin (POMC). POMC is a precursor peptide that is

NEURAL AND HORMONAL CONTROLS OF FOOD INTAKE AND SATIETY / 879 Arcuate nucleus peptide interactions

Y1/Y5

MC4

2nd Order neurons

Orexigenic

Anorexigenic POMC

NPY/AgRP GABA ObRb IR ObRb

IR

FIG. 34-1. Schematic of signaling within the hypothalamic arcuate nucleus. Neuropeptide Y/agouti-related protein (NPY/AgRP) and proopiomelanocortin (POMC)-containing neurons contain both leptin (Ob-Rb) and insulin (IR) receptors. Neurons containing the orexigenic peptides NPY/AgRP are inhibited by leptin and insulin, and neurons containing POMC, the precursor of the anorexigenic peptide αmelanocyte-stimulating hormone (α-MSH), are activated by these adiposity signals. The activity of second-order neurons outside the arcuate is modulated by NPY or the balance between the melanocortin agonist α-MSH and antagonist AgRP. GABA, γ-aminobutyric acid; MC4, melanocortin 4.

processed into multiple peptides including a-melanocytestimulating hormone (a-MSH). a-MSH is an anorexigenic peptide in that it inhibits food intake after its exogenous administration (34). Leptin activates POMC-containing neurons both by altering gene expression (33,35) and by direct depolarization (36), resulting in increased POMC messenger RNA (mRNA) expression and the release of a-MSH. Leptin also interacts with a second population of arcuate neurons that contain the orexigenic peptides neuropeptide Y (NPY) and the endogenous melanocortin antagonist agouti-related protein (AgRP). Leptin reduces NPY and AgRP mRNA expression (37) and hyperpolarizes these neurons, thus reducing the release of these orexigenic stimuli (36). Leptin inhibition of these neurons also results in the reduction of a GABAergic inhibitory input onto POMC-containing neurons (36). Thus, increased leptin levels at times of metabolic excess directly activate an arcuate anorexigenic pathway and reduce activity in arcuate orexigenic pathways. Low leptin levels, occurring at times of nutrient deficit, result in a reduction of inhibitory influences on NPY/AgRP neurons, a lack of activation of POMC-containing neurons, and an overall increase in orexigenic signaling. The evidence for important roles for NPY and the melanocortins in energy balance derives from a number of sources. Intracerebroventricular (icv) or direct hypothalamic injection of NPY potently stimulates feeding in a variety of paradigms (38–40), and repeated or chronic NPY administration results in obesity (40,41). Hypothalamic NPY gene

expression and release increase in response to food deprivation (42) or exercise (43) and decrease in response to overconsumption of a highly palatable high-energy diet (44). Although chronic treatment with NPY results in obesity, absence of NPY or its receptors does not result in the absence of food intake or metabolic wasting. Murine knockout models that do not express NPY or NPY receptors are viable (45–47). Rather than negating a role for NPY in food intake control and energy balance, these results should be interpreted to suggest that there are multiple redundant systems available for stimulating food intake and the absence of one is not sufficient to significantly block this critical behavior. This interpretation is supported by data demonstrating that although the absence of NPY signaling does not affect food intake and body weight in wild-type mice, it does modulate the phenotypes produced by leptin signaling deficits (48). Cell bodies of neurons expressing NPY are also found in the dorsomedial hypothalamic nuclei (49). These neurons do not express leptin receptors (50) and NPY expression in these neurons is not increased by short term food deprivation but is elevated in response to chronic food restriction (51), chronic exercise (52,53) and the metabolic demands of lactation (54). A significant increase in dorsomedial hypothalamic nuclei NPY signaling does to appear to underlie the obesity of Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking cholecystokinin-A (CCK-A) receptors (55). Thus, there are multiple hypothalamic systems responding to energy demands with increases in NPY signaling. The orexigenic effects of NPY are thought to be mediated through interactions with both the Y1 and Y5 receptor subtypes. Both of these receptors are expressed in hypothalamic areas involved in feeding control (56), and compounds with specific activity at these receptors affect food intake (57). As noted earlier, knockout of NPY receptors has little effect on basal food intake. However, Y1-deficient mice have been reported to have a reduced refeeding response to food deprivation (47), and Y5 knockouts have a reduced feeding response to exogenous NPY administration that can be eliminated by the administration of a Y1 receptor antagonist (46). Recent data demonstrating the ability of chronic NPY infusion to induce sustained increases in food intake and obesity in both Y1 and Y5 knockouts further suggest redundancies in these receptor networks (58). Melanocortins are the other arcuate peptides that play significant roles in mediating the energy balance effects of leptin and insulin. Melanocortins can have both feeding stimulatory and feeding inhibitory actions. POMC, with hypothalamic expression limited to the arcuate nucleus, is the precursor for a variety of peptides. Among these is the anorexigenic melanocortin agonist peptide a-MSH. Central administration of a-MSH or synthetic melanocortin agonists potently inhibit food intake (59,60). Arcuate POMC neurons contain both leptin and insulin receptors, and administration of both peptides has been demonstrated to increase POMC gene expression (35,61). POMC mRNA expression also has been demonstrated to decrease with food deprivation (62) and increase with overfeeding (Hagan et al., 1999), suggesting

880 / CHAPTER 34 a regulatory role for this peptide in feeding control. Important roles for melanocortin signaling in energy balance are demonstrated in experiments examining the effects of POMC (63) or melanocortin receptor (64) knockouts. Such interruptions in melanocortin signaling result in obese phenotypes, and mutations in POMC and the melanocortin 4 receptor also are associated with human obesity (64). A unique aspect of melanocortin signaling is the existence of an endogenous melanocortin antagonist, AgRP. AgRP is colocalized with NPY in the medial arcuate nucleus, and its expression is up-regulated by fasting and downregulated by leptin and insulin (65). AgRP or synthetic melanocortin antagonists increase food intake when administered centrally and their effects are long lasting (59). The feeding inhibitory actions of central melanocortins are mediated primarily through interactions with melanocortin 4 receptors. Melanocortin 4 receptor knockouts are hyperphagic and obese and do not reduce their food intake in response to administration of the melanocortin 3/4 agonist MT11 (66). Melanocortin signaling is thought to represent the balance between a-MSH and AgRP signaling at melanocortin 4 receptors with increased food intake in the presence of increased AgRP and decreased food intake in the presence of increased a-MSH. Arcuate NPY/AgRP- and POMC-containing neurons have primary projections to both the paraventricular nucleus (PVN) (67,68) and to the lateral hypothalamus (33), two nuclei involved in autonomic regulation. The PVN appears to play a major role in anorexigenic signaling. Lesions of the PVN result in hyperphagia and obesity (69), suggesting that this nucleus plays a major role in limiting food intake and body weight. The PVN contains multiple neuronal populations and is a major hypothalamic site with projections to the dorsal vagal complex (70), the brain site receiving neural input from the gastrointestinal tract. NPY and melanocortin receptors are found on multiple neuronal cell types within PVN, and some of the peptides expressed by these neurons may mediate the downstream actions of decreased NPY and increased melanocortin signaling (Fig. 34-2). Candidate peptides for this action include oxytocin, corticotropinreleasing factor, and gastrin-releasing peptide (GRP). All of these peptides reduce food intake after their exogenous central administration (71–73), and alterations in melanocortin or NPY signaling have been shown to alter the expression of these PVN peptides (74–76). The specific actions through which these peptides may affect intake are discussed later in this chapter (e.g., see Interactions between Gut Peptide and Hypothalamic Signaling). In contrast with the feeding inhibitory action of PVN stimulation, the other major hypothalamic projection site for arcuate neurons, the lateral hypothalamus, appears to play a role in stimulating or maintaining food intake (Fig. 34-3). Lateral hypothalamus lesions result in aphagia and profound weight loss (77), and lateral hypothalamus electrical or chemical stimulation increases intake in sated animals (78). The lateral hypothalamus is the site for neurons containing two additional peptides that have been implicated in feeding

PVN GRP MCH Anorexigenic outputs

OXY Orexigenic outputs

CRH

ORX

LH

POMC

NPY/AGRP ARC

FIG. 34-2. Schematic of paraventricular nucleus (PVN) and LN sites that mediate the downstream actions of alterations in arcuate signaling. Anorexigenic outputs are mediated in the PVN through alterations in corticotropin-releasing hormone (CRH), oxytocin (OXY), and gastrin-releasing peptide (GRP) signaling. Orexigenic outputs are mediated in the lateral hypothalamus (LH) through alterations in orexin (ORX) and melanin-concentrating hormone (MCH) signaling. NPY/AgRP; neuropeptide Y/agouti-related protein; POMC, proopiomelanocortin.

Mode of action of peripheral signals Amylin GLP-1 Direct access AP Arcuate nucleus

Direct access

NTS

Ghrelin? PYY(3-36)? Vagal afferent inputs

CCK Gastric distention Ghrelin? Glucagon PP PYY(3-36)?

FIG. 34-3. Schematic of actions of peripheral signals in feeding control. Vagal modes of action have been demonstrated for multiple signals. Direct action dependent on access to the area postrema (AP) or arcuate nucleus have been demonstrated or proposed for other peptides. CCK, cholecystokinin; GLP-1, glucagon-like peptide-1; NTS, nucleus of the solitary tract; PP, pancreatic polypeptide; PYY(3-36), peptide YY3-36.

NEURAL AND HORMONAL CONTROLS OF FOOD INTAKE AND SATIETY / 881 control, orexin, (79) and melanin-concentrating hormone (MCH) (80). Both of these peptides are orexigenic and have been proposed to be downstream targets of leptin signaling (81,82). Orexin-containing neurons are found in the perifornical area of the hypothalamus and project from there throughout the hypothalamus (83). Prepro-orexin expression is increased in response to deprivation (84), and administration of an orexin A antagonist has been demonstrated to inhibit food intake, suggesting a role for endogenous orexin A in food intake control (85). MCH expression is increased in obesity, and MCH levels are modulated by fasting (80). MCH neurons in the lateral hypothalamus are a distinct population from those expressing hypocretin/orexin, although, like orexin neurons, they are innervated by arcuate nucleus NPY/AgRP- and POMC-containing fibers (86). NPY has been demonstrated to activate orexin and MCH neurons (87), and leptin has been demonstrated to inhibit orexin and MCH mRNA expression (87,88). Lateral hypothalamus neurons directly innervate autonomic centers in the hindbrain including the dorsal vagal complex (83). As well as responding to circulating hormones such as leptin and insulin, hypothalamic neurons are responsive to alterations in local nutrient concentrations. Hypothalamic neurons have long been known to alter their activity in response to changes in glucose concentration (89). Two classes of responses have been noted. Neurons that increase their activity in response to increased glucose have been termed glucose-responsive or glucose-stimulated neurons, whereas those that increase their activity in response to decreases in glucose concentration have been called glucosesensitive or glucose-inhibited neurons (90). There are now reasons to think that hypothalamic neurons also respond to local changes in the availability of fatty acids. Intraventricular administration of oleic acid inhibits food intake and reduces hepatic glucose production (91). Because similar effects are not found with medium-chain fatty acids, suggesting that long-chain fatty acids provide a signal for nutrient availability and, in the presence of increased concentrations as produced by local oleic acid infusion, a signal of nutrient abundance (92). Results with compounds that inhibit fatty acid synthesis are consistent with the idea of neuronal nutrient signal. Administration of the fatty acid synthase inhibitor C75 results in decreases in food intake and body weight (93), and does so by altering patterns of hypothalamic gene expression, reducing NPY and AgRP signaling, and increasing POMC expression relative to levels in pair fed animals (94,95). C75 also results in reductions in the level of the phosphorylated subunit of adenosine monophosphate–activated protein kinase (pAMPK) within the basal hypothalamus, an action that appears to be secondary to increases of hypothalamic adenosine triphosphate levels (96). A similar action has been demonstrated for leptin. Leptin decreases pAMPK in both the arcuate and PVN, and such an action is necessary for the feeding inhibitory effects of leptin (97). Constitutively active AMPK expression in the hypothalamus increases food intake and prevents the anorexic actions of leptin. Together, these data suggest that AMPK acts as a physiologic energy sensor

within the hypothalamus, and that alterations in AMPK may be a common final readout of a variety of treatments that act within the basal hypothalamus to affect energy balance. Although the hypothalamus has been the main focus of study for anorexigenic and orexigenic peptides, a number of these also have ingestive effects when delivered to the dorsal hindbrain. Thus, fourth cerebroventricular administration of NPY (98) or a melanocortin antagonist (99) potently increases food intake, whereas a melanocortin agonist (99), cocaineand amphetamine-regulated transcript (CART) (100), and urocortin (101) inhibit food intake when administered at this site. These hindbrain actions suggest that the central feeding regulatory system is a distributed system. How these hindbrain and hypothalamic systems interact with one another remains to be determined.

WITHIN-MEAL FEEDBACK SIGNALING In many species, including human, food intake occurs in distinct bouts or meals. Meal initiation is determined by a variety of factors, especially food availability. During a meal, ingested nutrients contact a variety of receptors within the oral cavity and gastrointestinal tract, resulting in neural and hormonal signals that contribute to the determination of meal size. Meal size can be highly variable, and alterations in meal size appear to be a major determinant of overall food intake. Taste plays an important role in determining meal size. Palatable or good-tasting substances are consumed more rapidly and in greater amounts than unpalatable foods. A number of experimental paradigms have been developed to isolate the influences of taste on ingestion. The most widely used is sham feeding. In this paradigm, animals are equipped with a fistula that can be opened or closed depending on the test condition. The fistula can be located in the esophagus or, more commonly, in the stomach. When the fistula is opened, ingested liquids drain from the fistula, minimizing postingestive feedback. For example, with a gastric fistula, although nutrients contact the mouth esophagus and stomach, they do not collect in the stomach or pass into the upper intestine eliminating signals derived from gastric distention or nutrient absorption. Pavlov (102) first reported the effect of sham feeding on ingestion. He demonstrated that dogs with open esophageal fistulas did not experience satiety but would continue to eat for hours. The sham feeding paradigm has clearly demonstrated the important role of orosensory stimuli in ingestion. Increasing the concentration of saccharide solutions or oil emulsions increases the amount consumed in a linear fashion over an extensive range of concentrations (103–105). Such data have led to the view that palatability provides a positive feedback signal maintaining ingestion once it is initiated. Analyses of patterns of sham feeding have not only demonstrated effects of palatability on ingestion, but also have revealed pregastric contributions to satiety. Sham feeding does eventually stop, and a number of processes have been

882 / CHAPTER 34 proposed to contribute to the cessation of sham feeding including oral metering (106), habituation (107), and sensoryspecific satiety (decreasing pleasantness of a specific food as more is ingested) (108). The amount sham fed also depends on the experience of the animal with the sham feeding paradigm. Although the first time rats sham feed, they double their intake compared with the fistula closed condition, continued experience with sham feeding significantly increases intake over the next three or four tests. These data have been interpreted to demonstrate the presence of a conditioned inhibition on food intake that is caused by an association of the oral stimulation with postingestive negative feedback. Only with continued experience with sham feeding is this conditioned inhibition on intake gradually overcome (109,110). In normal ingestion, consumed nutrients contact multiple mechanosensitive and chemosensitive receptors that provide feedback information to the brain important to the controls of meal size. The potential range of feedback mechanism that could be operating during a meal is dependent on the distribution of ingested nutrients during that meal. Traditional measures of rates of gastric emptying had led to the view that a relatively small portion of ingested nutrients passed through the pylorus during a meal, leading to the conclusion that a considerable portion of the within-meal feedback signaling arose from the stomach and was related to gastric distention. Kaplan and colleagues (111) have demonstrated, in the rat, that when the stomach is filled at rates mimicking normal ingestion rates, gastric emptying is much more rapid during than after the period that the stomach is filled, and that emptying occurs at a constant rate for the duration of the filling period and is not affected by nutrient concentration. Similar results were found whether the meal was infused into the stomach or ingested by the rat (112). These data demonstrate that a significant portion of ingested nutrients (as much as 30% in the rat) enters the duodenum, contacts duodenal receptors, and is available for absorption. Similar dynamics of gastric emptying during fill also have been demonstrated in rhesus monkeys (113). Thus, in addition to the stomach, a significant proportion of the upper intestine provides potential sites for feedback signal generation during a meal that could be important for meal termination.

Gut Neural Signaling The presence of nutrients within the digestive tract elicits both neural and chemical signals that can provide information about the quantity and quality of the ingested nutrients. The gastrointestinal tract receives both spinal and vagal innervation, providing the potential for extensive direct neural feedback signaling between the gut and brain. Spinal afferents arise from gastrointestinal mucosa and submucosal layers, smooth muscle and vasculature. Although the traditional view of the role of spinal afferent signaling has been that they are activated only in response to noxious or harmful stimuli (114), some work has suggested their involvement

in coordination of gastrointestinal motility and in gut-brain signaling. Chemical or surgical disruption results in alterations in gastric emptying (115) and diminishes the feeding inhibitory effect of duodenal nutrient infusions (116). The vagus is the major neuroanatomic link between the gastrointestinal tract and brain. Vagal afferent fibers with cell bodies in the nodose ganglion arise from the digestive organs and project to the nucleus of the solitary tract (NTS) with a distribution approximating a viscerotopic representation of the alimentary canal within the NTS (117). In the rat, afferent fibers constitute the majority of vagal fibers, accounting for greater that 75% of subdiaphragmatic vagal nerve fibers (118). The subdiaphragmatic vagus has two major trunks that travel along the esophagus. In the rat, the ventral trunk bifurcates into the left gastric, hepatic, and accessory celiac branches. The dorsal trunk bifurcates into the right gastric and celiac branches. The nomenclature implies a specificity of innervation. However, branches often innervate additional organs. For example, the hepatic branch provides the major innervation to the proximal duodenum, and some duodenal innervation also derives from the gastric branches (119). The terminations and response properties of vagal afferents depend, in part, on the target organ from which they arise. Within the gastric and intestinal musculature, two distinct mechanosensitive vagal afferent terminations have been identified. Intramuscular arrays (IMAs) are found in both the longitudinal and circular muscles and consist of an array of free endings lying in parallel with the muscle fibers (120). IMAs primarily are located in the stomach, concentrated in the pyloric and esophageal sphincters. IMAs have been proposed to serve as stretch receptors responding to passive stretch (121). The other primary termination, intraganglionic laminar endings (IGLEs), is localized to the myenteric plexus and consists of a series of arborizing endings within the myenteric ganglia (122). IGLEs are widely distributed throughout the esophagus, stomach, and intestine, and these endings have been proposed to be tension receptors responding to local muscle contraction (121). The location of IGLEs has led to the proposal that they also have the ability to respond to local chemical stimulation arising from myenteric neurons or paracrine release from the gastrointestinal secretory cells (123,124). Polymorphic individual vagal afferent fibers have been identified that have collateral terminations consisting of both IMAs and IGLEs (120). Mechanosensitive gastric vagal afferents increase their firing in response to increasing gastric load volume. Slowly adapting mechanoreceptive fibers increase their response rate with increasing gastric volume (125). The fibers remain active while load volume is retained, and they show an off response during which activity briefly decreases to less than baseline levels when the load volume is removed. Individual afferents are differentially tuned such that there are differences in their dynamic range (126). Some afferents reach their maximal activity at small intragastric volumes, whereas others do not begin to respond until a significant gastric load is present. Gastric mechanoreceptive vagal afferents do not respond directly to the chemical character of the

NEURAL AND HORMONAL CONTROLS OF FOOD INTAKE AND SATIETY / 883 gastric load. Response rate is similarly increased by nutrient and nonnutrient load volumes that are restricted to the stomach by a pyloric noose (127). Consistent with this gastric load–related feedback signaling, gastric distention has been demonstrated in multiple paradigms to inhibit food intake (128,129), and this inhibition is significantly attenuated after vagotomy (130). Vagal afferent terminations also have been identified within the duodenal mucosa (131). Although fine afferent fibers have been localized within the intestinal villa, the terminations do not appear to penetrate into the duodenal lumen, leading to the view that any direct nutrient stimulation would be before rather than after absorption. Alternatively, nutrient absorption could stimulate local chemical release and activate vagal afferents secondary to such release. Duodenal vagal afferents are activated by both intraluminal load volume and nutrient character. Duodenal, slowly adapting, mechanoreceptive fibers have been identified. Similar to gastric mechanoreceptive fibers, activity increases with increases in load volume. Duodenal vagal afferents are also directly responsive to nutrient character. For example, both intestinal casein (132) and lipid infusions (133) have been shown to result in increases in vagal afferent activity. Although gastric vagal activity is not directly responsive to intragastric nutrient character, gastric afferent responsivity can be altered by duodenal nutrient (134). Thus, gastric vagal afferent activity is modulated in the presence of duodenal nutrients. As noted earlier, these nutrient-induced alterations in vagal afferent activity may reflect the actions of duodenal nutrient–induced release of GI peptides. For example, the brain-gut peptide CCK is released by the duodenal presence of nutrient digestion products. Local arterial CCK administration results in increases in vagal gastric mechanoreceptive afferent activity similar to those produced by intragastric load or intraduodenal load (135,136). CCK also plays a role in the response of duodenal afferents to nutrients. Administration of a CCK antagonist blocks the increase in vagal afferent activity produced by intraduodenal casein (132). CCK-induced changes in vagal afferent activity appear to result from a direct action of the peptide on the vagal afferent fibers. Vagal afferents contain and transport CCK receptors (137), and CCK can be demonstrated to activate cultured vagal neurons as indicated by CCK-induced alterations in cytosolic calcium (138). Elimination of aspects of vagal afferent- or peptideinduced feedback can result in significant alterations in the way that rats pattern their food intake. Surgical vagal deafferentation results in alterations in meal patterns in rats maintained on a liquid diet, in that such rats consume larger, less frequent meals than sham-operated control animals (139). Meal frequency is reduced in response to these meal size increases such that overall food intake is unchanged. Similar alterations in meal size have been reported in response to capsaicin-induced chemical vagal deafferentation. These rats consume larger meals on a novel diet (140) or with calorically dilute sucrose access (141). Although these data demonstrate a role for vagal afferent feedback in the controls

of meal size, the particular signal(s) that is being blocked has yet to be identified.

Within-Meal Gut Peptide Signaling A number of peripherally acting peptides with roles in the controls of food intake have been identified. The best characterized of these is the brain-gut peptide CCK. Exogenously administered, CCK was originally demonstrated to decrease food intake in rats in 1973 (142). This feeding inhibitory action of CCK and CCK agonists has been demonstrated in a variety of species including nonhuman primates and human (143,144). Exogenously administered CCK reduces meal size and results in an earlier appearance of a behavioral satiety sequence (145). A role for CCK in the control of the size of individual meals was first suggested by experiments examining the feeding effects of repeated, mealcontingent, CCK administration. CCK consistently reduced meal size without producing significant changes in overall daily food intake (146). Such actions are consistent with the dynamics of nutrient-induced CCK release. Plasma CCK increases rapidly after meal initiation, peaking within 10 to 15 minutes and gradually returning to baseline levels. The half-life of CCK in circulation is on the order of a few minutes. Thus, CCK can serve as a signal for terminating an individual meal without affecting a subsequent meal or overall food intake. The satiety actions of CCK depend on interactions with multiple receptor sites. Important populations of CCK-A receptors, the receptor subtype through which the satiety actions of CCK are mediated, are found on vagal afferent fibers and on circular muscle cells within the pyloric sphincter. Surgical or chemical disruption of subdiaphragmatic vagal afferent innervation significantly affects the ability of CCK to inhibit food intake (147–149). The nature of this disruption is a rightward shift in the CCK satiety dose– response curve. Low doses of CCK that inhibit food intake in intact rats are ineffective for reducing intake after vagal afferent lesions. Higher doses inhibit intake but to a smaller degree. In contrast, surgical removal of the pyloric sphincter does not affect the ability of low doses of CCK to inhibit intake but truncates the dose–effect curve such that the additional suppression that normally accompanies higher CCK doses is eliminated (150). Results such as these have led to the proposal that CCK satiety is determined by multiple factors and is, in part, secondary to its local gastrointestinal actions. A role for endogenous CCK in satiety is supported by data demonstrating that CCK antagonists with specificity for the CCK-A receptor increase food intake (151,152). In the primate, the effect is dose related with a maximum increase of around 40% in daily 4-hour food intake. This increase is almost completely accounted for by an increase in the size of the first meal (152). Alterations in meal patterns also are evident in rats or mice lacking CCK-A receptors. OLETF rats have been demonstrated to have a ~6kb deletion in the

884 / CHAPTER 34 CCK-A receptor gene spanning the promotor region and the first and second exons. This deletion prevents protein expression, resulting in a CCK-A receptor knockout rat (153). OLETF rats are obese and hyperphagic. Characterization of their spontaneous solid food intake has revealed a 35% increase in daily food intake resulting from an 80% increase in meal size combined with an insufficient decrease in meal frequency (154). CCK-A receptor knockout mice also exhibit alterations in the size of their spontaneous meals. Meals size is increased, although not to the same degree as in OLETF rats, and CCK-A receptor knockout mice compensate for the increase in meal size by decreasing the number of neals consumed (50). Satiety actions also have been demonstrated for the mammalian bombesin-like peptides, GRP and neuromedin B (NMB). These peptides reduce food intake after peripheral exogenous administration (155–157). Bombesin is more potent and has a greater maximal effect on intake than either mammalian peptide, an effect that can be best explained by its high affinity for both GRP and NMB receptors. Thus, bombesin activates both mammalian pathways and produces an effect similar in magnitude to combined GRP and NMB administration (158). Both vagal and spinal afferents contribute to the mediation of the satiety actions of abdominal bombesin-like peptides. Either combined vagotomy, dorsal rhizotomy and cord section, or neonatal capsaicin administration is necessary to abolish the effects of bombesin on food intake (159,160). Bombesin-like peptides also inhibit food intake after central administration, and the site of action for this effect is within the caudal hindbrain (161,162). There does appear to be a relation between the central and peripheral actions of these peptides because central antagonist administration can block the effect of peripherally administered peptides (73). Such results suggest the possibility that peripherally administered bombesin-like peptides may exert some of their actions through a central site. A role for endogenous mammalian bombesin-like peptides in satiety is supported by data demonstrating increases in short-term food intake after antagonist administration. Central GRP (163,164) and NMB receptor antagonists (165) have been demonstrated to increase food intake in a variety of feeding paradigms. These data provide further support for a central site of action as being important for the feeding effects of bombesin-like peptides and are consistent with a role for endogenous bombesin-like peptides in the controls of meal size. As discussed later in this chapter, data suggest actions of GRP in integrating hypothalamic and satiety signaling. Satiety actions for the pancreatic peptides glucagon and amylin also have been demonstrated. Eating rapidly elicits an increase in pancreatic glucagon secretion (166), and because such an increase is also stimulated by sham feeding, this appears to be a cephalic-phase response (167). Glucagon is cleared rapidly from the circulation by the liver (166), and the liver appears to be the site of the satiety action of glucagon (168) based on data demonstrating that hepatic-portal infusion of glucagon at meal onset elicits dose-related reductions in

meal size (169). The satiety action of glucagon requires the presence of other forms of consequences of ingestion, because glucagon alone does not affect sham feeding (170), but it can increase the degree of feeding suppression produced by CCK in a sham feeding paradigm (171). A role for endogenous glucagon in the control of meal size is supported by data demonstrating the ability hepatic portal infusions of glucagon antibody to increase meal size (172). The ability of glucagons to suppress food intake has been demonstrated in human subjects (173). Amylin inhibits feeding in a dose-dependent and behavior-specific manner after either peripheral or central administration (174). Amylin is obligatorily cosecreted with insulin by pancreatic b cells (175). Thus, amylin levels increase rapidly with meal onset and remain increased for a significant period during and after meals. The site of action for amylin is within the area postrema, a hindbrain structure lacking a complete blood–brain barrier. The area postrema contains a dense concentration of amylin receptors, and area postrema lesions block the feeding inhibitory actions of peripherally administered amylin (176). Amylin inhibits sham feeding, but higher doses are required than those required for inhibiting real feeding, suggesting that amylininduced signals normally combine with other inhibitory feedback to inhibit food intake (177). A physiologic role for endogenous amylin in feeding controls is supported by experiments demonstrating increases in food intake in response to peripheral (178) or central administration of amylin antagonists (179,180). All of these peptides that have proposed roles in contributing to meal termination share the property that their signaling, whether direct peptide or through vagal afferent activation, results in the modulation of neural activity within the dorsal hindbrain as demonstrated by the induction of c-Fos. CCK (181), bombesin-like peptides (182), and amylin (183) have all been demonstrated to induce c-Fos in the NTS, the site of vagal afferent termination, and within the area postrema, a hindbrain site with an incomplete blood–brain barrier. Considerable integration of within-meal feedback signaling takes place within the NTS. Experiments in which nutrient exposure was either limited to oral, gastric, duodenal, or combinations of these sites have revealed both negative and positive interactions. For example, gastric nutrient exposure reduces the degree of NTS c-Fos activation induced by oral exposure during sham feeding (184), and combined gastric and duodenal exposure results in greater activation than either exposure alone (185). Signal integration at this hindbrain site is consistent with the sufficiency of the hindbrain for mediating many aspects of within-meal feedback signaling. Although decerebrate rats will not spontaneously consume food, they are capable of metering their intake if nutrients are infused into their mouths by either swallowing or allowing the food to drip out. Using such oral feeding procedures, Grill and colleagues have demonstrated the sufficiency of the hindbrain for mediating sophisticated within-meal feedback on controls (186–188).

NEURAL AND HORMONAL CONTROLS OF FOOD INTAKE AND SATIETY / 885 Across-Meal Peptide Signaling Some work has demonstrated the ability of a number of additional gut peptides to inhibit food intake after their peripheral administration, and the dynamics of their nutrientstimulated release and feeding effects are consistent with actions that may extend beyond the individual meal. Thus, peptides such as glucagon-like peptide-1 (GLP-1), pancreatic polypeptide (PP), peptide YY3-36 (PYY3-36), and ghrelin have actions in feeding that do not depend solely on terminating the individual meal that modulated their release. GLP-1 is a posttranslational product of proglucagon that is released by ileal endocrine cells on ingestion of nutrients. Circulating levels of GLP-1 increase within minutes of food ingestion and remain increased for more than 3 hours (189). Carbohydrate (190) and long-chain unsaturated fatty acids (191) are particularly good secretagogues. The rapid increase in plasma GLP-1 after meal initiation makes it unlikely that this is a local response to nutrients in the ileum. Duodenal nutrients likely stimulate endocrine and neural mediators that activate GLP-1 secretion (192,193). Consistent with this view, pharmacologic or surgical vagotomy significantly attenuates meal-stimulated increases in GLP-1 secretion (194). GLP-1 functions as a gut hormone that helps regulate blood glucose and feeding behavior in mammals (195). Exogenous administration of GLP-1 reduces blood glucose in both normal and diabetic subjects (196) by stimulating insulin secretion and synthesis in response to changes in blood glucose (197) and by suppressing glucagon secretion (198). GLP-1 also is involved in the ileal brake. Gastric inhibitory and intestinal motility actions of ileal nutrients are partially mediated through GLP-1 release (199,200). In human subjects, peripheral GLP-1 infusions have been reported to reduce food intake in lean, obese, and type II diabetic subjects (201–204). However, peripheral bolus injections of GLP-1 into the rat have little (205) or no (206) effect on food intake. The short half-life of peripheral GLP-1 provides a potential explanation for the inability of exogenous GLP-1 to elicit an effect on food intake in the rat. In vivo studies in rats have demonstrated that dipeptidyl peptidase IV, an enzyme present in the capillaries surrounding intestinal cells that catabolize GLP-1, cleaved 50% of a bolus GLP-1 infusion within 2 minutes (207). GLP-1 analogs that are resistant to degradation have been shown to reduce food intake and body weight in lean and obese rats (208), and recent data demonstrate that prolonged intravenous GLP-1 infusions do reduce food intake (209). The mechanisms underlying the feeding inhibitory actions of intestinally derived GLP-1 are not understood, although a number of possibilities have been suggested. In human studies, exogenous GLP-1 infusions that produce physiologic postprandial plasma levels result in significant reduction in size of a test meal similar to those found with CCK (210), suggesting a similar mediation. The possibility of such vagal mediation is further suggested from data demonstrating that vagal afferent denervation abolishes the

actions of central and peripheral GLP-1 on gastric emptying (211). Whether such afferent denervation also affects the feeding inhibitory actions of GLP-1 has not been determined. A second possible mode of action for peripheral GLP-1 to exert its effects is via specific carriers or endothelial leaks that allow peripheral GLP-1 access to central binding sites. Evidence supporting this idea comes from data demonstrating that when radiolabeled GLP-1 is injected peripherally, it can be identified in the area postrema and in other circumventricular organs (212). These data suggest that peripheral GLP-1 gains access to the brain and may bind to brainstem sites from which efferent projections are directed to major hypothalamic nuclei involved in energy homeostasis (213). Consistent with such mediation are data showing the ability of centrally administered GLP-1 to reduce food intake (214). Although the site of action for central GLP-1 to inhibit feeding appears to be within the dorsal hindbrain (215), GLP-1 administration does activate hypothalamic arcuate neurons, and an intact arcuate is necessary for the feeding suppression to occur (216). Dependence on the arcuate suggests that at least some aspects of GLP-1–induced inhibition on intake depend on alterations in hypothalamic signaling and affect intake beyond a single meal. In fact, repeated administration of GLP-1 analogs can result in long-term reduction in food intake and reductions in body weight (208). Effects of peripheral GLP-1 or GLP-1 analogs on hypothalamic gene expression have yet to be identified, thus how GLP-1 may affect food intake beyond a single meal situation is not yet understood. PP is a member of the NPY and PYY family of peptides. PP has a high affinity for the Y4 receptor subtype. PP is produced in endocrine cells in the pancreas and released into the circulation in response to food intake. PP exerts a variety of regulatory actions in the gastrointestinal tract including inhibition of pancreatic exocrine secretion, gall bladder contraction, modulation of gastric acid secretion, and stimulation of gastrointestinal motility. The release of PP has multiple components, with an early cephalic peak followed by a prolonged plateau (217). A role for PP in food intake was first suggested from studies in mice demonstrating that twice daily peripheral PP injections into obese mice reduced food intake and body weight (218). Some work has suggested a role for PP in both food intake and energy expenditure (219) and identified PP-induced decreases in the expression of NPY, orexin, and ghrelin as potential mechanisms for these actions. A vagal mediation has been suggested based on experiments demonstrating PP-induced increases in vagal afferent activity and the blockade of the feeding inhibitory actions by vagotomy (219). Exogenous PP administration has also been shown to result in reductions in food intake in human subjects (220). How the actions of PP may combine with those of other peptides that affect food intake has yet to be determined. However, an action in limiting the release of the orexigenic peptide ghrelin has been proposed (219). PYY is a gut peptide secreted from I cells in the distal intestine. PYY has long been identified as a peptide involved in the

886 / CHAPTER 34 ileal brake (221,222). PYY inhibits gastric emptying and mediates the effects of ileal fat on gastric motility. PYY3-36 is an alternate form of PYY found throughout the gastrointestinal tract with the greatest concentrations found at distal small and large intestinal sites (223). We have shown that PYY3-36 has similar effects on gastric emptying (224), but whether it also plays a direct role in the gastric inhibitory effects of ileal nutrients has not been assessed directly. PYY3-36 has been shown to be released into the plasma in response to a meal (223), although little work has been done on the dynamics of PYY3-36 release, and most of the statements about the pattern of release of PYY3-36 have been based on information inferred from data that measured plasma levels of PYY. The temporal pattern of PYY release is different from that of other gastrointestinal peptides with suggested roles in feeding control. Plasma levels of PYY gradually increase after meal initiation, reaching a peak at about 60 minutes, and high levels are maintained for a number of hours after a meal (225). Such a pattern of release suggests the potential for a role in the control of food intake across meals rather than in meal termination. Batterham and colleagues (226) have reported significant suppression of food intake in rodents in response to peripheral bolus PYY3-36 administration and significant decreases in food intake in human subjects in response to PYY3-36 infusions. An action of peripheral PYY3-36 through arcuate nucleus Y2 receptors was proposed. The Y2 receptor is a presynaptic inhibitory receptor, and PYY3-36 has been demonstrated to have relative specificity for this NPY receptor subtype (227). Consistent with this proposal, PYY3-36 was shown to inhibit food intake when injected into the arcuate nucleus intact but not in Y2−/− mice, and PYY3-36 increased activity in POMC neurons through decreasing the frequency of inhibitory postsynaptic currents, indicative of a decrease in GABA release. Because arcuate NPY neurons exert a tonic GABAergic inhibitory influence on arcuate POMC neurons, this action of PYY3-36 was interpreted to support a feeding inhibitory action of PYY336 through inhibition of arcuate NPY neurons, resulting in increased activity in arcuate POMC neurons. Some work has demonstrated feeding inhibitory actions of PYY3-36 on mice lacking either the melanocortin 4 receptor (228) or POMC (229), demonstrating that increased melanocortin activity is not necessary for the feeding inhibitory action of PYY3-36. An important aspect of the initial report was the finding that repeated PYY3-36 administration resulted in reductions in body weight. Such a finding is consistent with actions that depend on alteration in hypothalamic signaling. Data suggest a vagal mediation. Koda and colleagues (230) have demonstrated that the vagus nerve contains Y2 receptors and that intravenous PYY3-36 administration activates gastric vagal afferent fibers. Furthermore, they have shown that vagotomy blocks the ability of PYY3-36 to inhibit food intake and to activate arcuate NPY neurons. Batterham and colleagues (226) also demonstrated the ability of PYY3-36 to inhibit food intake in human subjects, and this finding has been extended to obese subjects (231).

PYY3-36 also reduces food intake in nonhuman primates (224). The feeding inhibitory action appears to be specific to a reduction in the size of spontaneous meals throughout the daily 6-hour feeding period (224). Although the original findings of a feeding inhibitory action of exogenous PYY3-36 have been replicated and extended by some investigators (230,232,233), others have not able to demonstrate the feeding inhibitory actions of PYY3-36 in rodents. A report combining results from multiple laboratories presented negative data on the ability of PYY3-36 to inhibit food intake and reduce body weight in rodents across a range of doses and testing paradigms (234). The reasons for the differences in results are unclear. However, in light of such disagreement, the overall role for PYY3-36 in feeding control remains uncertain. Additional information on its patterns of meal-induced release, its relative feeding contribution in relation to other nutrient-induced peptide signals, and the role of the endogenous peptide to feeding control would significantly advance our understanding of the actions of this peptide in feeding control. Ghrelin was first identified in 1999 as the endogenous ligand for the growth hormone stimulating receptor (GHSR) (235). A novel role for ghrelin in feeding control was suggested from data demonstrating that peripheral and central ghrelin administration potently stimulates food intake and decreases energy expenditure in rodent models (236,237). A physiologic role for ghrelin in energy balance is supported by data from experiments demonstrating the ability of ghrelin antagonists to reduce food intake (238) and by demonstrations of decreased body weight and fat mass in ghrelin knockout mice exposed to a high-fat diet (239). Ghrelin is secreted by endocrine cells within the stomach and circulates in plasma. Fasting results in increases in circulating ghrelin (240), and humans have been shown to display preprandial increases and postprandial declines in circulating ghrelin levels (241). Meal-induced declines in plasma ghrelin levels appear to depend on postgastric nutrient factors (242), although the specific site and mechanism underlying these declines remains to be determined. Importantly, ghrelin levels after gastric bypass surgery have been reported to be chronically suppressed, and this suppression has been proposed to play a role in the decreased appetite and weight that accompany bypass surgery (243). This finding suggests that lower intestinal nutrient contact may be important in producing declines in plasma ghrelin levels. Notably, there is some disagreement about the ability of gastric bypass to affect plasma ghrelin levels, and the effect may depend on the specifics of the bypass surgical procedure (244). As with PYY3-36, multiple sites and mechanisms of ghrelin action have been proposed. A vagal site is suggested from data demonstrating that the vagus nerve contains GHSR, ghrelin decreases activity in gastric vagal afferent fibers, and vagotomy blocks the ability of peripheral ghrelin to activate arcuate NPY neurons and stimulate food intake (245). A direct hypothalamic action for peripheral ghrelin also has been proposed. Such an action depends on peripherally circulating ghrelin gaining access to the arcuate nucleus, a possibility

NEURAL AND HORMONAL CONTROLS OF FOOD INTAKE AND SATIETY / 887 based on the view that the arcuate does not have a complete blood–brain barrier. Findings that are taken to support such a site of action include the following demonstrations: (1) arcuate NPY neurons contain GHSRs; (2) peripheral ghrelin induces c-fos in NPY neurons (246); (3) ghrelin fails to induce food intake in NPY knockout mice (247); and (4) ghrelin activates NPY neurons in slice preparations (248). However, the ability of ghrelin to activate NPY-containing neurons could depend on activation of an ascending pathway. Fourth and lateral ventricular ghrelin reduces food intake comparably (249), and both result in increased arcuate NPY mRNA expression. Furthermore, icv anti–ghrelin antibody decreases food intake, whereas direct arcuate injection of the antibody does not (250). Ghrelin-containing neurons have been found in the medial hypothalamus (248), suggesting the additional possibility that GHSR on NPY neurons may be normally activated by a distinct central ghrelin pathway. Figure 34-3 presents a schematic of the major proposed modes of action for gut peptides in feeding control. Feedback actions dependent on vagal afferent transmission have been demonstrated or proposed for multiple gut peptides involved in both meal termination and across meal signaling. Other peptides have proposed actions dependent on direct hindbrain access to sites receiving ascending neural inputs. In contrast, other peptides have been proposed to have direct access to hypothalamic sites and actions dependent on modulating hypothalamic signaling.

INTERACTIONS BETWEEN GUT PEPTIDE AND HYPOTHALAMIC SIGNALING As detailed earlier, some of the effects of gut peptides have been proposed to depend on alterations in hypothalamic signaling, whether this is through direct contact or is secondary to activation of ascending pathways. There is also evidence for interactions between hypothalamic peptide signaling and the processing of ascending within meal satiety signals. A number of the clearest demonstrations of interactions involve the adiposity signal leptin. As noted earlier, leptin circulates in direct relation to the degree of adiposity, serving as a feedback signal for the overall regulation of energy balance. Both peripheral and central leptin administration reduce food intake, and a number of experiments have demonstrated that the effects of leptin on feeding are specific to reducing meal size without changing meal frequency (251–253). How does a signal that is critically involved in regulating hypothalamic pathways involved in energy balance result in reductions in the size of individual meals? Leptin may result in reductions in meal size through its direct actions on taste sensitivity. Leptin specifically reduces chorda tympani and glossopharyngeal sensitivity to sweet stimuli without altering responses to other tastants (254). This appears to be a direct effect at the level of the taste bud because leptin hyperpolarizes the taste cell. Leptin may also decrease meal size by altering the reinforcing effects of ingestion. Leptin has been

shown to reduce the rewarding efficacy of electrical brain stimulation (255). Such an action may lead to earlier meal termination because the ability of the ingested nutrients to maintain feeding may be reduced. The ability of leptin to limit meal size also appears to depend, in part, on its interactions with within-meal feedback signaling. For example, central administration of leptin at doses that are subthreshold for inhibiting feeding when administered alone enhance the satiating potential of peripheral CCK or an intragastric preload (185,256). This action of leptin appears to depend on its ability to enhance the degree of NTS neural activation produced by these peripheral manipulations. That is, leptin enhances the dorsal hindbrain representation of ascending vagal afferent feedback signals arising from CCK or gastric preload-induced gastrointestinal stimulation. Reducing leptin levels through food deprivation has the opposite result—the satiating potency of CCK is reduced (257,258). This effect may be mediated through enhanced NPY signaling because NPY administration has the opposite effect to that of leptin on both the behavioral and neural activation potency of CCK. NPY administration attenuates the ability of CCK to reduce food intake and to induce c-fos in the NTS (258). Such actions of alterations in leptin or its downstream signaling appear to depend on altered responsivity of individual NTS neurons to ascending vagal afferent signaling (259). Central leptin enhances the degree of electrophysiologic activity to a range of gastric load volumes, whereas central NPY administration has the opposite effect. After NPY administration, the activation produced by a given intragastric volume is significantly reduced. Such actions of leptin or NPY could depend on local hindbrain actions. The dorsal hindbrain contains both leptin and NPY receptors (260–262), and hindbrain injection of these peptides alters feeding in the predicted directions (98,261). Alternatively, the modulation of hindbrain responsivity to within meal signals could depend on activation of a descending pathway from the hypothalamus to the dorsal hindbrain. Data support such an interpretation. Adenoviral replacement of functional leptin receptors within the arcuate nucleus of Koletsky rats (Koletsky rats lack all forms of the leptin receptor at all sites) was sufficient to normalize behavioral and neural activation responses to CCK (263). These data demonstrate that forebrain signaling induced by leptin limits meal size by regulating hindbrain responses to within-meal satiety signaling and support the model proposed in Figure 34-4. A number of neuropeptide systems are candidates for mediating this hypothalamic to hindbrain modulation (see Fig. 34-4). Work by Blevins and colleagues (74,264) has provided support for a role for PVN oxytocin-containing neurons in mediating the action of leptin in modulating responses to CCK. Oxytocin-containing neurons are localized to the parvocellular subdivision of the PVN (265). These neurons project to the NTS, and the PVN is the source for NTS oxytocin-containing fibers (266,267). Blevins and colleagues (74) have demonstrated that leptin increases c-fos expression in PVN oxytocin-containing neurons, and that

888 / CHAPTER 34 Negative energy balance ARC

PVN

Ob-R Leptin

NPY AgRP

OXY GRP CRH

POMC

MCH ORX

NTS ___________

+ + + + + + + + ++

Leptin Ob-R LH Positive energy balance

Gastrointestinal afferent signals

FIG. 34-4. Schematic of hypothalamic influences on nucleus of the solitary tract (NTS) responses to ascending satiety signals. Leptin modulates NTS responses to gastrointestinal afferent signaling. Proposed mechanisms include descending hypothalamic to hindbrain pathways arising in the paraventricular nucleus (PVN) and lateral hypothalamus (LH). Increases in corticotrophin-releasing hormone (CRH), oxytocin (OXY), and gastrin-releasing peptide (GRP) signaling increase NTS neural responses to satiety signals. Increases in orexin (ORX) and melanin-concentrating hormone (MCH) signaling may reduce responses to gastrointestinal inhibitory feedback. POMC, proopiomelanocortin

regions of the NTS activated by CCK are densely innervated by oxytocin-containing fibers. They have further shown that ventricular administration of an oxytocin antagonist attenuated the effect of leptin on food intake and resulted in a significant decrease in the potentiating effect of leptin on CCK activation of NTS neuronal c-fos expression (264). Furthermore, administration of an oxytocin antagonist in the fourth ventricle increased food intake. Oxytocin has long been known to affect gastrointestinal function (268), and local hindbrain administration of oxytocin has been shown to activate gastric-related NTS and dorsal motor nucleus (DMX) neurons (269). Whether oxytocin modulates the NTS electrophysiologic response to gastric distention or other satiety signals has not been assessed directly. GRP-expressing neurons localized to the magnocellular layer of the PVN are another potential candidate. These neurons project to the dorsal vagal complex (270,271). Administration of bombesin peptides in the fourth ventricle inhibits food intake (161), and administration of a GRP antagonist in the fourth ventricle increases intake (163), demonstrating a role for the endogenous hindbrain peptide in feeding modulation. Electrophysiologic studies from Zhang and colleagues (272) have shown that NTS neurons that are responsive to gastric distention also respond to local application of GRP, and that activation of NTS neurons induced by electrical stimulation of the PVN can be modulated by local administration of GRP antagonists. It is not known whether local application of GRP has the ability to modulate responses to gastric distention or whether GRP can affect the activity of CCK-responsive NTS neurons. Finally, a discrete population of parvocellular PVN neurons expresses corticotropin-releasing hormone (CRH) (265),

and PVN CRH neurons project to the NTS (273). Intracerebroventricular administration of CRH can be shown to inhibit food intake and to activate c-fos expression within the caudal brainstem (72,274), and icv administration of CRH antagonists blocks the anorexic actions of both leptin (275) and the melanocortin agonist MT-11 (76), suggesting that there is also a role for CRH signaling in these pathways. Thus, multiple pathways may underlie hypothalamic to hindbrain signaling mediating the interactions between energy balance and satiety.

SUMMARY The body contains multiple systems for regulating overall energy balance. These systems derive from and control different aspects of ingestive behavior and its consequences. Although adiposity and satiety signaling have different primary sites of mediation within the brain, these are interacting systems that together control energy intake.

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75. 76.

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CHAPTER

35

Pharyngeal Motor Function Reza Shaker Pharyngeal Motor Function during Deglutition, 895 Pressure Profile of the Pharynx, 896 Pharyngeal Pressure Phenomenon in Relation to Swallowed Material, 898 Deglutitive Laryngeal Motor Function, 900 Nasopharyngeal Motor Function during Swallowing and Belching, 901 Upper Esophageal Sphincter and Its Pressure Phenomena, 902 Opening of the Upper Esophageal Sphincter, 903 Upper Esophageal Sphincter, Pharyngolaryngeal, and Esophageal Reflexes, 904 Esophagoglottal Closure Reflex, 904

Pharyngeal (Secondary) Swallow, 905 Cerebral Cortical Representation of Pharyngeal/Reflexive and Volitional Swallow in Humans, 906 Pharyngo-Upper Esophageal Sphincter Contractile Reflex, 907 Pharyngoglottal Adduction Reflex, 908 Laryngo-Upper Esophageal Sphincter Contractile Reflex, 908 Pharyngeal Inhibitory Reflexes, 908 Mechanisms of Airway Protection during Belching, 909 References, 910

The pharynx has a complex muscular structure and is suspended from the base of the skull and styloid process. It lies anterior to the cervical spine and is attached to the mandible and the hyoid bone anteriorly, although arbitrarily, it is divided into three anatomic segments—that is, nasopharynx (from the skull base to the soft palate), oropharynx (from the soft palate to the pharyngoepiglottic fold), and hypopharynx (from the pharyngoepiglottic fold to the lower boundary of the pharynx, i.e., the upper esophageal sphincter [UES]). Whereas the nasal and oral cavities connect to the nasopharynx and oropharynx, with the tongue forming the anterior wall of the oropharynx, the laryngeal structures, namely the arytenoids, cricoid, thyroid, and epiglottis, form the anterior wall of the hypopharynx. Motor function of the pharynx contributes to swallowing, retrograde transit through the esophagopharyngeal axis, namely eructation, respiration, and phonation. This chapter does not focus on pharyngeal function related to respiration

or phonation. The pharynx also is involved in a number of reflexes that affect the larynx, esophagus, and its sphincters. These reflexes will also be discussed. During both deglutition and eructation there exists a close functional relation between the pharynx and the larynx, thus discussion of pharyngeal motor function without that of the larynx would not be complete. Therefore, this chapter also discusses laryngeal motor function as it relates to deglutition and eructation. Because the main component of the UES (i.e., the cricopharyngeus [CP] muscle) is considered part of the pharyngeal muscular structure, its function is closely tied to that of the pharynx and larynx during both anterograde and retrograde transit. This chapter also discusses the UES.

PHARYNGEAL MOTOR FUNCTION DURING DEGLUTITION For descriptive purposes, swallowing could be divided into four consecutive phases: (1) preparatory; (2) oral; (3) pharyngeal; and (4) esophageal (1,2). These phases merely represent the anatomic regions traversed by the swallowed food. The term oropharyngeal swallowing in this chapter is used to refer to oral/pharyngeal phases of swallowing. During the preparatory phase, for the most part, the food bolus remains in the oral

R. Shaker: Division of Gastroenterology and Hepatology, Digestive Disease Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

895

896 / CHAPTER 35 cavity, undergoing physical and some chemical changes. Through mastication and mixing of the bolus with saliva, it develops suitable physical qualities for transit through the aerodigestive tract. During this phase, the bolus is sized, shaped, and positioned on the dorsum of the tongue for initiation of the oral phase of swallowing. During the oral phase, sequential squeezing of the tongue against the hard and soft palates generates a peristaltic pressure wave (3) that propels the bolus from the oral cavity into the pharynx. During the pharyngeal phase, the pharynx, UES, and larynx (4) become elevated, and three of the four routes to the pharynx—namely, the nasal cavity, oral cavity, and larynx (5–7)—become sealed off, whereas the fourth route, the UES, opens and the bolus is transported into the esophagus by rapid, forceful posterior tongue movement (thrust) continued from the oral phase, as well as the peristaltic contraction of the pharyngeal constrictors against the soft palate, base of the tongue, and larynx. During the pharyngeal phase of swallowing, the nasopharynx is sealed by contraction of the superior pharyngeal constrictor, elevation of the soft palate, and its contact with the posterior pharyngeal wall. The oral cavity is closed by elevation of the tongue base and its contact with the hard and soft palates (8). During this phase, important biomechanical events involving intrinsic glottic, as well as suprahyoid and infrahyoid muscles, take place that result in closure of the airway (9,10), as well as in the opening of the UES (11,12). These events include adduction of the true vocal cords and arytenoids (first tier of airway closure), followed by vertical approximation of the adducted arytenoids to the base of the epiglottis (second tier of closure), then descent of the epiglottis covering the closed glottis, thereby closing the laryngeal vestibule (third tier of closure). Beginning with the vocal cords closure or shortly afterward, the entire larynx is pulled upward and forward by the contraction of the suprahyoid muscle group. This displacement results in the positioning of the closed larynx under the tongue base away from the path of the swallowed material, thereby providing additional protection against aspiration and helping to close the laryngeal vestibule (13–15). During oropharyngeal swallowing, the UES transiently relaxes and subsequently is pulled upward and forward by the contraction of the same suprahyoid muscles that displace the larynx. This traction results in forced opening of the UES that also is modified by the bolus size (11,12). Figure 35-1 shows the temporal relation of the events that take place during the oropharyngeal phase of swallowing. As observed under normal conditions (10), oropharyngeal swallowing begins with the closure of the vocal cords, signifying the activation of airway protection, and ends when the cords return to their resting positions. During this time, the respiration is reflexively inhibited (16–20). All other swallowing events, including pharyngeal peristalsis, starts and ends while the cords are fully closed (10). During the esophageal phase of swallowing, the bolus is transported further into the esophagus and stomach. From a functional point of view, events that take place during oropharyngeal swallowing contribute to the following

Pharyngeal peristalsis

UESO PT-O OT-O

SH-O TB-O SM-O

Vestibular closure

Vocal cord adduction

Superior hyoid movement 0

0.5

1.0

1.5

2.0

2.5

FIG. 35-1. Relation of deglutitive vocal cord kinetics to other events of the oropharyngeal phase of swallowing during 5-ml barium swallows. Bolus transit through the pharynx and across the upper esophageal sphincter (UES) begins and ends while the vocal cords are at maximal adduction. OT-O, onset of bolus movement from the mouth; PT-O, arrival of bolus into pharynx; SH-O, onset of superior hyoid movement; SM-O, onset of submental myoelectrical activity; TB-O, onset of tongue base movement; UESO, UES opening. (Modified from Shaker and colleagues [10].)

two functions: transit of the bolus and protection of the airway. The transit and protective aspects of oropharyngeal swallowing are highly coordinated, and studies have shown that oropharyngeal transit occurs during the full activation of the protective aspect of swallowing (10). A successful oropharyngeal swallow requires the effective and coordinated actions of the anatomic elements involved in these two functions. Table 35-1 presents the head and neck muscles that contribute to the pharyngeal phase of swallowing and their innervations. Therefore, normal oropharyngeal swallowing is defined as complete transit of the ingested material from the mouth into the esophagus without compromising the airway. Oropharyngeal dysphagia may develop when the efficacy or coordination, or both, of either transport or protective aspect of oropharyngeal swallowing are compromised. Contraction of the pharyngeal muscles, including the constrictors and posterior tongue thrust, generate a peristaltic pressure wave that results in transit of the swallowed bolus out of the pharynx and into the esophagus.

PRESSURE PROFILE OF THE PHARYNX The pharynx is both axially and radially asymmetric in its dimensions, and as such its pressure profile during deglutition varies depending on the orientation and location of the recording sensor (21–23). Figure 35-2 shows the endoscopic view of the pharynx from the skull base to the UES. To overcome the problem of asymmetry, pressure recordings have been made from posterior orientation and from a given distance proximal to the UES high-pressure zone (24). This approach makes it possible to measure the parameters of the pharyngeal peristalsis at comparable sites for the study of

PHARYNGEAL MOTOR FUNCTION / 897 TABLE 35-1. Muscles of the neck participating in deglutitive pharyngeal motor function Muscles

Neural control

Palatal muscles Tensor veli palatini Levator veli palatini Uvular Palatoglossus

V Pharyngeal plexus X and XI Pharyngeal plexus X Pharyngeal plexus XI

Palatopharyngeus Salpingopharyngeus Superior pharyngeal constrictor Middle pharyngeal constrictor Inferior pharyngeal constrictor

Laryngeal muscles Thyroarytenoid Transverse and oblique arytenoid Lateral and posterior cricoarytenoid Thyroepiglottic Lingual muscles Superior and inferior longitudinal Transverse Verticalis Hyoglossus Genioglossus Styloglossus Suprahyoid and infrahyoid muscles Mylohyoid and anterior belly of digastric Posterior belly of digastric Stylohyoid Geniohyoid Thyrohyoid Sternohyoid Omohyoid Sternothyroid

IX X (recurrent and external branch of superior laryngeal nerve [SLN]) Pharyngeal plexus Pharyngeal plexus Pharyngeal plexus (XI)

A

B

C

D

Pharyngeal plexus (XI) Pharyngeal plexus (XI) (and recurrent laryngeal nerve [RLN], ext. branch of SLN) X (RLN) X (RLN)

FIG. 35-2. Endoscopic views of the pharynx, descending from the skull base (A) to the upper esophageal sphincter (D). B, Pharyngeal cavity proximal to uvula. C, Pharyngeal cavity at uvula. (See Color Plate 20.)

X (RLN) X (RLN) XII XII XII XII XII XII

200 Wave amplitude (mm Hg)

Pharyngeal muscles Stylopharyngeus Cricopharyngeus

100

0

P1

P2

P3

Young

P4

P1

P2

P3

P4

Young

V VII VII XII XII Ansi cervicalis Ansi cervicalis Ansi cervicalis

the effect of bolus variables such as volume, consistency, and temperature on the pharyngeal peristalsis. Optimal recording of pharyngeal peristalsis requires instrumentation with a flat frequency response of up to 50 Hz (25). Infused catheter systems satisfy the requirement for accurate recording of esophageal peristaltic waveforms that have a frequency content of less than 5 Hz (26,27) but substantially misrecord pharyngeal waveforms (28). Thus, intraluminal transducer probes are the only instrumentation available for accurate recording of pharyngeal peristaltic amplitude. As seen in Figures 35-3 and 35-4, the amplitude of peristaltic pressure wave increases and its duration decreases as

FIG. 35-3. Differences in the amplitude of pharyngeal peristalsis between young and elderly healthy subjects during a 5-ml water swallow. The amplitude of hypopharyngeal pressure is significantly higher in elderly versus young individuals. P1 is 6.0 cm above the upper esophageal sphincter (UES); P2 is 4.5 cm above the UES; P3 is 3.0 cm above the UES; and P4 is 1.5 cm above the UES. *p < 0.05, for P4 versus P1; †p < 0.05, for P4 in elderly versus P4 in young individuals. (Modified from Shaker and colleagues [24].)

the peristaltic wave propagates from the proximal pharynx (6 cm above the UES) to the hypopharynx (1.5 cm above the UES). The amplitude of hypopharyngeal pressure is significantly greater and its duration significantly longer in healthy elderly individuals compared with healthy young individuals (24). The reason for this difference appears to be a compensation for the alteration in the UES opening mechanism in the elderly (20,30) (see later). This speculation is supported by a finding of increased intrabolus pressure in the hypopharynx of elderly compared with young people (Table 35-2) (p < 0.05). Abnormalities of pharyngeal peristalsis are seen

898 / CHAPTER 35 and further increase with mashed potato swallows was not appreciable.

Wave duration (sec)

1.0

PHARYNGEAL PRESSURE PHENOMENON IN RELATION TO SWALLOWED MATERIAL

0.5

0.0

P1

P2

P3

P4

P1

Young

P2

P3

Transit of a bolus can be studied from two perspectives: (1) dynamics, namely, studies of forces that induce bolus movement, as described earlier; and (2) kinematics, namely, studies of the motion of the bolus itself. Whereas ample data (3,21,23,24,31,32) are available on the “forces” that induce bolus movement, information regarding the “motion” of the bolus, particularly solid bolus movement, is limited. Available information on bolus kinematics generally pertains to liquid bolus (33). Studies (34) have shown that solid bolus kinematics is similar to the kinematics of the head of a liquid bolus. From a kinematic point of view, the head of a liquid bolus behaves differently from the tail of the bolus, and the velocity characteristics of the tail, but not the head, of a liquid bolus are similar to those of pharyngeal peristalsis, and both have constant velocity. During swallowing, associated with the anterior movement of the larynx and UES opening before the arrival of the bolus, a pressure gradient develops in the area of the hypopharynx, UES, and proximal esophagus. In this region, the more distal sites exhibit a lower pressure compared with the proximal sites. Frequently, these pressures are subatmospheric. The duration and magnitude of these recorded subatmospheric pressures are significantly different from those induced by transducer “ringing” (34). Viewed from a dynamic perspective, at any instant during the pharyngeal phase of swallowing, three distinct dynamic pressure zones exist within the anatomic region extending from the hypopharynx to the proximal esophagus (34): (1) the peristaltic zone, which corresponds fluoroscopically to the region of luminal closure; (2) the bolus zone, which corresponds fluoroscopically to the open lumen that is occupied by the bolus; and (3) most distally, the prebolus zone, which corresponds fluoroscopically to the open lumen that is occupied by air or liquid mist. The pressure phenomena in the pharyngeal peristaltic and bolus zones have been well studied (21,24,32). Limited available studies indicate that within the prebolus zone there exists an incrementally decreasing

P4

Elderly

FIG. 35-4. Differences in the duration of pharyngeal peristalsis between young and elderly healthy subjects during a 5-ml water swallow as in Figure 35-3. The duration of hypopharyngeal pressure is significantly longer in elderly versus young subjects. P1 is 6.0 cm above the upper esophageal sphincter (UES); P2 is 4.5 cm above the UES; P3 is 3.0 cm above the UES; and P4 is 1.5 cm above the UES. *p < 0.05, for P4 versus P1 in young subjects; †p < 0.05, for P4 in elderly versus P4 in young subjects. (Modified form Shaker and colleagues [24].)

in disorders that affect pharyngeal muscles such as muscular dystrophy or pharyngeal neural control such as those seen after cerebrovascular accidents (CVAs). Figure 35-5 shows an example of absence of pharyngeal peristalsis recorded in a patient with Kearns–Sayre syndrome induced by cytochrome c oxidase deficiency. In both young and elderly people, bolus volume and temperature do not alter the parameters of the pharyngeal peristaltic pressure wave. Swallowing of mashed potato, in contrast, significantly increases its amplitude and duration. These findings suggest the existence of a consistency, but not volumeresponsive, modulatory mechanism between the pharynx and the brainstem. These findings are similar to the earlier reported effect of mashed potato on the lingual peristalsis (3). Amplitude of the hypopharyngeal peristaltic pressure wave during swallowing of mashed potato has been reported to be similar among young and elderly people, whereas it was found to be significantly greater in the elderly for dry and water swallows. An explanation for this phenomenon could be that, in elderly adults, the peristaltic amplitude has already approached its physiologic limit for water swallows

TABLE 35-2. Effect of Aging and Position on Intrabolus Pressure (mmHg) in the Hypopharynx Bolus variables Volunteer position Upright Supine

WS5 Young

WS10 Elderly

Young

Elderly

WS20 Young

Elderly

MP5 Young

Elderly

MP10 Young

4.8 ± 1.4 8.5 ± 5* 5.7 ± 1.3 11.2 ± 3.2* 7.7 ± 1.3 14.4 ± 3.9* 10.4 ± 2.4 18.8 ± 2.9* 16.8 ± 3.1 13.3 ± 2.1† 19.7 ± 1.5*† 17.6 ± 1.7† 21.2 ± 3.3*† Not tested 17.0 ± 3.0† 29.0 ± 4.2*† 23.6 ± 3.8†

Elderly 34.5 ± 6.7* 42.8 ± 7.6*†

Values are means ± SE; n = 14 young and 12 elderly volunteers. P < 0.05 in *elderly volunteers compared with young and in supine position compared with upright. WS5, 5-ml water swallow; WS10, 10-ml water swallow; WS20 water swallow. MP5, 5-ml mashed potato swallow; MP10, 10-ml mashed potato swallow. [From: Shaker R, Ren J, Podvrsan B, Dodds WJ, Hogan WJ, Kern M, Hoffmann R, Hintz J. Effect of aging and bolus variables on pharyngeal and upper esophageal sphincter motor function. AJP:Gastrointest Liver Physiol 264(27):G427–G432, 1993.] †

PHARYNGEAL MOTOR FUNCTION / 899 200

mmHg SW

mmHg 100 Kearns–Sayres 0 syndrome mmHg 100

0 200 mmHg 0 200 mmHg

SW

SW

0 mmHg 100

0 200 mmHg

0 100 mmHg

UES

0 200 mmHg

0

UES

100 0 100 mmHg

mmHg

0 100

0

mmHg

0

0

5 Time in seconds

10

5 Time in seconds

0

10

FIG. 35-5. Example of pharyngeal manometric recordings during a 5-ml water swallow in a patient with Kearns–Sayre syndrome (KSS) and an age-matched healthy control subject. Recordings are obtained using six intraluminal pressure transducers spaced 1.5 cm apart. During resting, the most distal transducer is within the upper esophageal sphincter (UES); during swallowing, because of orad excursion of the UES, the transducer located immediately above the most distal transducer becomes located within the sphincter. This phenomenon is heralded by a hump (arrow) followed by a pressure decline because of UES relaxation. With this arrangement, during swallowing, the remaining four transducers record pharyngeal pressure activity from a span of 4.5 cm proximal to the UES. As seen, the patient with KSS generates negligible pharyngeal pressure compared with the control subject (note differences in scale). SW, swallow. (Modified from Shaker and colleagues [32a].)

50 Solid pelllet Liquid bolus head Liquid bolus tail

40

Velocity (cm/s)

pressure distribution across the UES such that the proximal esophagus exhibits a lower pressure compared with the distal pharynx. These pressures frequently reach subatmospheric range. These pressure declines are associated temporally with deglutitive anterior displacement of the cricolaryngohyoid complex (34). From a kinematic perspective, during swallowing and associated with anterosuperior laryngeal movement and UES opening when the epiglottis has reached its tilted position covering the glottis, there exist two zones of bolus acceleration located, respectively, at the supraepiglottic and infraepiglottic regions. The velocity and acceleration of the bolus in the supraepiglottic and infraepiglottic zones are similar. However, these zones are separated by a region of bolus deceleration (Fig. 35-6) located between the edge of the tilted epiglottis and the posterior pharyngeal wall: the pharyngoepiglottal space. It has been reported that the instances of maximum bolus acceleration across the UES are closely associated with maximum pressure differences between the pressure site immediately distal to the bolus position and the pressure in the proximal esophagus (34). It is speculated that this pressure decline provides a preferential path to the bolus away from the airway by facilitating its transport across the UES and into the esophagus. In this context, this pressure decrease, in addition to its possible role in bolus transport, may act as an airway-protective mechanism.

30

20

10

0 0.0

0.5

1.0

1.5

2.0

2.5

FIG. 35-6. Composite time history of average velocity of solid pellet, liquid barium bolus head, and tail traversing pharynx. As seen, velocity and acceleration of barium pellet and liquid barium bolus head were similar. However, both were significantly different from those of liquid barium bolus tail. (Modified from Kern and colleagues [34].)

900 / CHAPTER 35 Intrabolus pressure has been found to be a reliable indicator of the magnitude of resistance to bolus flow (35,36). In patients with Zenker’s diverticulum, intrabolus pressure has been found to be significantly increased compared with that in age-matched control groups, and it is reported to decrease after myotomy of the CP muscle in these patients (37). Intrabolus hypopharyngeal pressure has been found to be significantly greater in subjects with CP bar compared agematched control subjects (38). In addition, the intrabolus hypopharyngeal pressure also has been found to be significantly greater in asymptomatic healthy elderly compared with young people (24,30,39). Overall study of bolus kinematics induced by the pharyngeal phase of swallowing dynamics suggests (34) the existence of two distinct modes of transport for solid barium pellet, as well as liquid barium boluses. One mode of transport is characterized by rapid acceleration of material across the base of the tongue and through the UES. This mode is associated temporally with the transsphincteric pressure decline, as well as with the forces generated by rapid posterior movement of the tongue base, seen on fluoroscopy. The second mode of transport is characterized by a slower clearance of material from the oropharynx associated with the oropharyngeal peristaltic pressure wave. These modes of transport appear to be secondary to the structural and kinematic events that occur in the pharynx and pharyngesophageal segment. Barclay (1), in 1927, postulated from high-speed cineradiographic recordings of passage of food through the pharynx the presence of a short-duration “negative” pressure in the pharynx “sucking the food down.” He reported a subatmospheric pressure of 14 to 16 inches of water (26–36 mm Hg) in the pharyngoesophageal segment during swallowing. Although the phenomenon of subatmospheric (“negative”) pressures has been reported in some later studies (1,40–42), this phenomenon has not been discussed in other studies. The phenomenon of “negative” pressures is further complicated by that in a few published reports, the duration of some of these “negative” pressure excursions, although not reported directly as such, when measured from the presented figures occur in time intervals that are consistent with the solid-state strain gauge “ringing” effects on the catheter. According to the findings of one study (34), both physiologic and “ringing”induced negative pressure excursions may occur during swallowing. Differences in recording sites and techniques may explain the discrepancies reported previously.

DEGLUTITIVE LARYNGEAL MOTOR FUNCTION Discussion of pharyngeal motor function during deglutition is not complete without discussing laryngeal swallowrelated motor function that helps in transit of the swallowed food through the pharynx without invasion of the airway. Closure of the vocal cords is an integral part of various functions involving the pharynx. These include swallowing

(10), coughing (43), straining and Valsalva maneuver (44), belching (45), and several airway protective reflexes such as esophagoglottal closure (46) and pharyngoglottal closure (47,49) reflexes. Duration of vocal cord closure during these events has been measured by direct videoendoscopic techniques (10,45,46) and differ depending on the function; likewise, the closure pressure that the cords generate during these functions varies depending on the performed function. During swallowing, vocal cord closure pressure together with two additional closure mechanisms, namely, aryepiglottal adduction and epiglottal descent, provide a sealed barrier against entry of the swallowed food in transit through the pharynx (10,49). The vocal cord closure is the most distal barrier, occurring first and lasting the longest of the abovementioned deglutitive airway closure mechanisms (10,49). During coughing, vocal cord closure helps to generate a high subglottic pressure. Subsequently, their sudden opening is associated with an accelerated expiratory flow (43). Studies of vocal cord closure pressure during a number of voluntary functions and tasks such as swallowing, coughing, straining, and phonation indicate that although not perceived by humans, the magnitude of contraction of striated muscles involved in closure of the vocal cords varies depending on the performed tasks. These varying contractions result in generation of different closure pressures commensurate to the respective task and function (Fig. 35-7) (50). Vocal cord closure is the result of contraction of adductor and tensor muscles of the larynx. The adductor muscles include the lateral cricoarytenoid and interarytenoid muscles. Whereas contraction of the lateral cricoarytenoid results in median rotation of the vocalis processes of the arytenoid cartilages, completely adducting the anterior portion of the cords and closing the anterior part of the tracheal inlet, the contraction of the interarytenoid muscle results in median adduction of the arytenoids, whereby adducting the posterior part of the cords and closing the posterior part of the tracheal inlet. The tensor muscles, including vocalis and thyroarytenoid muscles and their contractions during the tested tasks, most probably provide the tensed contact surface that helped in generation of reported pressures. A combination of various degrees of contraction of these adductor and tensor muscles, and possibly the cricothyroid muscle, is suggested to be responsible for the production of differing task-dependent intercordal pressures. The sphincteric function of the vocal cords and their resistance to ingress of swallowed material is an integral part of airway protection during swallowing. Studies using cadavers have reported that artificially adducted vocal cords can prevent the ingress of air with pressures as high as 140 mm Hg (51). Among the three tiers of laryngeal closure during swallowing, closure of the cords is temporally the first and anatomically the most caudad tier. Duration of complete vocal cord closure during swallowing has been reported to be about 1.7 seconds (10,49). Vocal cord closure in the majority of instances is followed by laryngeal elevation (10,49). In a minority of instances, the cords start their movement toward complete closure during the upward and forward excursion

PHARYNGEAL MOTOR FUNCTION / 901 tasks. This may be because of a combination of significantly lesser degrees of adductor and tensor muscle contractions. Nonetheless, because videoendoscopic images indicate wavelike contact of cords with each other and with the transducers during phonation and pressing against the pressure sensor, it is likely that the lack of intense contraction of tensor muscles resulting in the absence of a firm surface against the strain gauge may have contributed to the low pressure reported after the initial pressure spike during phonation (10).

mmHg 400

# ∗

Vocal cords pressure

∗

∗

300

200

100

0 Strain

Swallow

Phonation

∆

100 Intratracheal pressure

Cough

80 60

∆∆

40 20 0 Swallow

−20 Strain

Phase I Phase II Cough

Phonation

FIG. 35-7. Comparison of intercordal and intratracheal pressure during straining, swallowing, coughing, and phonation. Intercordal pressure during straining, coughing, and swallowing was significantly greater than that of phonation (*p < 0.05). Straining produced pressures significantly greater than coughing (#p < 0.05). Intratracheal pressure during coughing induced a pressure significantly greater than all other studied events (∆p < 0.05). Intratracheal pressure during straining was significantly greater than those of phonation and swallowing (∆∆p < 0.01). For all studied events, intercordal pressures were significantly greater than intratracheal pressures. (Modified from Shaker and colleagues [50].)

of the larynx (10). For this reason, even with endoscopic control, and with available pressure-recording devices, it is impossible to measure the pressure of the cords during the entire period of their closure. Therefore, the reported deglutitive closure pressures represent only a brief period of their closure. Because of the short period of vocal cord closure during swallowing and coughing, only extremely sensitive recording devices with high rise rate such as strain-gauge transducers could be used to study this phenomenon. In addition to upward movement, because of subsequent approximation of arytenoids to the base of the epiglottis and epiglottal descent during swallowing, measurement of pure deglutitive vocal cord pressure, except for the brief initial phase, currently is virtually impossible. The vocal cords produce a closure pressure significantly greater than that of the intratracheal pressure during coughing. The intercordal pressure during phonation after an initial spikelike pressure increase is minimal compared with other

Nasopharyngeal Motor Function during Swallowing and Belching The mechanism of nasopharyngeal closure (NPC) is different during swallowing and belching. During swallowing, NPC has two tiers of closure: palatal elevation and proximal pharyngeal muscle adduction; during belching, it consists of only palatal elevation. NPC is tightly coordinated with oralpharyngeal bolus transit during swallowing and with biomechanical events during belching. Similar to deglutitive glottal closure, the duration of NPC during swallowing is not modified by bolus volume. Velopharyngeal closure during swallowing and speech has been studied using various radiographic techniques and has identified different patterns of closure during these functions. Recent studies using videoendoscopy have shown that the mechanism of deglutitive NPC, in addition to palatal elevation and its contact with the posterior pharyngeal wall, includes the adduction of the most proximal part of the lateral pharyngeal wall, identifying a second function for these muscles, in addition to their known role in generating pharyngeal peristalsis and bolus transport. The onset of NPC during both swallowing and belching identified endoscopically precedes its onset seen fluoroscopically. This discrepancy is probably caused by the lack of adequate visualization of the various components of the proximal pharynx that are involved in NPC by videofluoroscopy in lateral projection (Fig. 35-8). NPC during swallowing begins from the proximal pharynx and extends caudad. This pattern of closure is opposite to the pattern of deglutitive laryngeal closure, which begins from the most caudad layer and extends orad. During belching, there is no adduction of the proximal pharyngeal muscles, and NPC is primarily caused by palatal elevation and velopharyngeal contact. During belching, participation of the nasopharynx includes elevation of the soft palate. This suggests the contraction of the levator veli palatini and possibly other palatal muscles such as tensor palatini, which are innervated by branches of the vagus and mandibular nerves, respectively (45). The onset of UES relaxation during swallowing precedes the onset of NPC. UES relaxation occurs after the onset of vocal cord closure (52), indirectly suggesting that NPC also occurs after the onset of deglutitive vocal cord closure. Studies (53) indicate that the arrival of the barium bolus head into the pharynx occurs ~0.2 seconds after the complete

902 / CHAPTER 35 NPC-O NPC-C

NPO-O NPO-C Endoscopy

VPC-O VPC-C

VPO-O VPO-C

USER-O BA-PX (Manometry)

Fluoroscopy

UES opening Hyoid bone movement Vestibular closure

VC-O VC-C 0

VO-O

VO-C

1.0 Time in seconds

2.0

FIG. 35-8. An example of temporal relation of deglutitive (10-ml barium swallow) nasopharyngeal closure (NPC) with other swallowing events. As shown, endoscopically identifiable onset of NPC occurs earlier (0.1 ± 0.02 seconds) than fluoroscopically seen onset of velopharyngeal closure, and upper esophageal sphincter (UES) opening occurs while nasopharynx is closed. BA-PX, arrival of barium at the pharynx; NPC-C, complete NPC; NPC-O, onset of NPC; NPO-C, complete nasopharyngeal opening; NPO-O, onset of nasopharyngeal opening; UESR-O, onset of UES relaxation; VC-C, complete vestibular closure; VC-O, onset of vestibular closure; VO-C, complete vestibular opening; VO-O, onset of vestibular opening; VPC-C, complete velopharyngeal closure; VPC-O, onset of velopharyngeal closure; VPO-C, complete velopharyngeal opening; VPO-O, onset of velopharyngeal opening. (Modified from Dua and colleagues [53].)

closure of the nasopharynx. It is conceivable that nasal regurgitation may occur in individuals in whom this coordination is disturbed. Duration of NPC is not influenced by the volume of the swallowed bolus, suggesting that this part of the swallowing cascade is stereotypically programmed and is not under a modulatory sensory feedback control. Interestingly, similar findings have been reported on the duration of the deglutitive glottal closure (2). However, studies indicate that certain aspects of swallowing, such as duration of UES opening (54), duration of hyoid bone movement (55), amplitude of pharyngeal peristalsis (10,56), and velocity of lingual peristalsis (3), are modifiable by volume or consistency of swallowed boluses. These findings suggest that although the airwayprotective aspect of oral-pharyngeal swallowing is stereotypically controlled, its bolus transit function is subject to modification by sensory feedback from the swallowed bolus.

UPPER ESOPHAGEAL SPHINCTER AND ITS PRESSURE PHENOMENA The UES provides a high-pressure zone between the pharynx and esophagus. Its major component, the CP muscle, attaches to the posterior aspect of the lamina of the cricoid cartilage like a c-clamp. Muscle fibers from inferior pharyngeal constrictors (IPCs) and proximal esophagus contribute

to the genesis of the UES high-pressure zone. This sphincter provides the most proximal physical barrier of the gastrointestinal tract against pharyngeal and laryngeal reflux of gastric content, whereas maintaining a basal pressure preventing entry of air into the esophagus during inspiration. The UES contracts in response to segmental distention of the esophagus (57) and may provide additional protection against pharyngeal regurgitation during gastroesophageal reflux (GER). However, this contractile response may not be present in all events (45). The UES, in contrast, relaxes either completely or partially in the majority of instances in response to abrupt generalized distention of the esophagus. In the remainder, however, it may contract or remain unaffected (24). The response of the UES to intraesophageal pH has been the subject of controversy. Although some studies report an increase in UES pressure caused by acidification of the esophageal lumen (58,59), others have not found such a response (60,61). UES resting pressure decreases during sleep (62). Whereas stress enhances the UES resting pressure (63), it is not uncommon to find the UES resting pressure during rest and relaxation to be difficult to detect manometrically. Resting UES pressure has been the subject of multiple studies. Some of these studies were done using a water-perfused catheter with side holes, some with strain gauges, and some with sleeve sensors. Pelemans and Vantrappen (64), using a pneumohydraulic system, found decreased resting UES pressure in subjects older than 74 years. Weihrauch and colleagues (65) found that mean UES pressure in younger subjects (20–49 years old) was significantly greater than that in older subjects (50–80 years old). Measurements of resting UES pressure were performed with the rapid pull-through technique. Cook and colleagues (66) did not find significant age-related changes in resting UES pressure measured with a sleeve device; however, in their study, the age range of study subjects was between only 21 and 55 years. Similarly, Wilson and colleagues (67) did not find age-related changes in a study of 50 healthy subjects aged 17 to 62 years. In contrast, Fulp and colleagues (68) were able to show that healthy elderly subjects older than 62 years had lower resting UES pressures than younger control subjects. Shaker and colleagues (24) studied the effect of aging (age, ≥70 years) on the resting UES pressure and its response to esophageal air and balloon distention. The results of this study indicate that the resting UES pressure is significantly reduced in elderly compared with young people, whereas its response to esophageal segmental and generalized distention is preserved (24). The major component of the UES, the cricopharyngeal muscle, is a striated muscle, and it is conceivable that with aging it becomes subjected to loss of muscle fiber. Then again, it is also conceivable that excitation impulses that maintain the cricopharyngeal tone in the awake state are decreased in elderly people. The presence of the UES contraction response to esophageal balloon distention suggests that the protective role of the UES against pharyngeal reflux of gastric acid is preserved in elderly people. The UES responses to esophageal distention by both air and balloon are variable (24). One possible explanation for this phenomenon might be the differences in the sensitivity and

PHARYNGEAL MOTOR FUNCTION / 903 concentration of recruited esophageal stretch receptors during different trials of esophageal distention by air and balloon. In most species, the CP attaches to the posterior lamina of the cricoid cartilage and forms a c-clamp shape muscular band producing maximum tension in anteroposterior compared with lateral directions (69–71). Two sets of muscle fibers have been identified in the human CP: the horizontally oriented fibers, that is, pars fundiforms, and an oblique band of fibers, that is, pars obliqua. These latter fibers extend from the lateral aspect of the cricoid cartilage to the posterior midline raphe, where they blend superiorly with the thyropharyngeus (TP) (72). Unlike the TP, the pars fundiforms of the CP has no median raphe. In most animal species, the CP, although distinct from the TP, contains a median raphe (73,74). The CP is composed of variable-sized striated muscle fibers of small-to-average diameter (25–35 µm), which contrary to most other striated muscles are not oriented in strict parallel fashion (72,75,76). The CP contains about 40% of endomysial connective tissue, much of which is elastic, but has no muscle spindles (75–77). It has been suggested that, unlike most other striated muscles that insert on the skeletal framework, the muscle fibers of the CP insert onto the connective tissue framework, thereby forming a muscular network (77). CP muscle fibers include both the slow-twitch type I (72,76,77) and the fast-twitch type II (72,75,77,78) fibers. This arrangement provides an anatomic basis for the various functions of the CP or UES, namely, maintaining constant basal tone and rapid relaxation and contraction during swallowing, belching, vomiting, and other reflexes. Comparison of the UES intraluminal high-pressure zone to the anatomic components of the pharyngoesophageal segment (11,69–71,79) shows an increased pressure encompassing the proximal cervical esophagus, CP, and IPC (TP in animals). Experimental evidence suggests contribution of each of these muscular elements to the UES high-pressure zone. Comparison (80) of the manometric location of the UES high-pressure zone with the radiographic location of the pharyngoesophageal junction, in most studies, has shown that the peak UES pressure corresponds with the IPC in humans (11,71,81) and animals (69,70). Although some investigators have reported that the electromyographic activity of both the TP (IPC) and the CP fluctuated with changes in UES pressure and both relaxed during swallowing (70), the CP has been considered the primary muscular component of the UES by most investigators because of the finding that the peak UES pressure does indeed correspond with the CP (79). The assumption of CP being the main component of the UES is supported by the fact that most studies in humans or animals report that the CP, and not the IPC (TP) (69), exhibits a continual basal tone (69,82–87), relaxes during swallowing (69,82–86,88), and exhibits fluctuations in its activity with changes in UES pressure (69,86,89). However, experimental evidence suggests that the UES is more complex than comprising only one muscle (90). Considering all of the studies, it appears that the muscle(s) contributing to the UES, also called the pharyngoesophageal segment high-pressure zone, is called into action depending

on its physiologic function. For example: basal tone is produced by the CP and IPC (TP) and possibly the infracricoid esophagus (69,70,83–88); active relaxation during swallowing (69,83–89) and belching (91) occurs primarily on the CP; changes in activity with respiration (70,87,88,92) occur in both the CP and the IPC; UES contraction and relaxation during retching and vomiting, in contrast, occur by the simultaneous action of the CP, IPC, and proximal cervical esophagus (83,91); UES contraction in response to esophageal or pharyngeal stimulation (i.e., the esophago-UES and pharyngoUES reflexes; see later) occurs with the CP (87) rather than the IPC; and contraction of the UES during coughing and sneezing occurs in both the CP and the IPC (93). Therefore, the pharyngoesophageal high-pressure zone commonly referred to as the UES may comprise the contribution of the CP, IPC, and proximal cervical esophagus depending on the physiologic state, but the CP is the only muscle that participates in all physiologic states of the UES (90).

OPENING OF THE UPPER ESOPHAGEAL SPHINCTER Opening of the UES during swallowing is determined by the combined effect of relaxation and distensibility of the CP muscle and retraction of the cricoid cartilage caused by contraction of the suprahyoid muscle group involved in UES opening. UES opening also is influenced by the distending force of the oncoming bolus traversing through the pharyngoesophageal junction (11,12,70,94,95). The relative contribution of these individual factors to UES opening, as well as the relative significance of malfunction of these factors to disordered UES opening, has not been quantified. Among the above-mentioned factors, only the suprahyoid muscle group appears to be accessible to external noninvasive manipulation, such as an exercise. The cross-sectional area of the UES opening during deglutition is reduced significantly in elderly compared with young people (30,96). This reduction is associated with a significant change in biomechanical events that accompany UES deglutitive opening. Among these events, the magnitude of anterior movement of the larynx, a correlate to the function of the UES opening muscles, is reduced, whereas the hypopharyngeal intrabolus pressure, an indicator of resistance to pharyngeal outflow, is increased in elderly people (24,30,96). Studies using a simple isotonic/isometric neck exercise aimed at suprahyoid UES opening muscles indicate that, in the elderly, deglutitive UES opening can be augmented significantly by exercise (97a). This augmentation has been reported to be associated with a significant increase in the magnitude of maximum anterior excursion of the larynx and a significant decrease in hypopharyngeal intrabolus pressure. The finding of a significant decrease in the hypopharyngeal intrabolus pressure indicates that the observed augmentation in biomechanical events, such as anterior laryngeal excursion, anteroposterior diameter, and cross-sectional area of the UES, indeed does result in physiologic consequences, that is, a decrease in pharyngeal outflow resistance.

904 / CHAPTER 35 A study of the effect of this simple isotonic and isokinetic head raising exercise on the UES deglutitive opening and functional outcome of swallowing in a small group of patients with dysphagia and aspiration due to abnormal UES opening has reported a significant improvement in some deglutitive biomechanical measurements in these patients, most notably a significant increase in anterior laryngeal movement, UES maximum anteroposterior opening, a significant decrease in postswallow pyriform sinus residue, as well as a resolution of postdeglutitive aspiration. These biomechanical changes were found to be accompanied by a significant improvement in the functional outcome measures associated with resumption of oral feeding in all patients (97). Volitional control of UES opening muscles has been reported and is used in Mendelsohn’s maneuver to prolong the superior and anterior excursion of the larynx, and thus improve swallowing efficiency (98). This prolongation results in a longer duration of the UES opening during swallowing (99).

UPPER ESOPHAGEAL SPHINCTER, PHARYNGOLARYNGEAL, AND ESOPHAGEAL REFLEXES The UES is one of the components of the airway-protective mechanisms against entry of gastroesophageal refluxate into the aerodigestive tract (24,29). However, because the magnitude of UES pressure is quite variable and it decreases significantly during sleep (62) and periods of calmness, it is conceivable that the pressure of gastroesophageal refluxate may overcome the UES if it occurs during periods of low UES pressure. Hence, UES function during GER events has been of interest and the subject of several previous studies. Although some of these studies report an increase in UES pressure after experimental esophageal acidification (60,100,101), others have not observed a similar response (58,70) after gastroesophageal acid reflux events. For these reasons, the UES function during gastroesophageal acid reflux events remains incompletely understood. This difficulty also stems from the fact that GER results in intraluminal pressure increase, pH changes, or both. The UES response to intraluminal pressure increase and distention is variable depending on the intensity and nature of the stimulus. In addition, the UES response to esophageal acidification has been controversial. Differences in recording techniques also might have added to these difficulties. Earlier reports generally used the station pull-through technique for recording the UES pressure, whereas more recent studies have used a modified sleeve device for this purpose. In 1957, Creamer and Schlegel (102) described the contractile response of the UES to esophageal balloon distention and water infusion. This contractile response was found by Car and Roman (82) to involve the CP muscle. Freiman and colleagues (100) showed that bilateral cold block of the cervical vagus completely abolished the UES response to HCl infusion and partially blocked its response to balloon distention in dogs.

Using the pull-through technique, Wallin and colleagues (101) reported an increase in UES pressure after 1 minute of intraesophageal acid infusion in humans. However, this increase was not maintained 4 minutes later. Gerhardt and colleagues (58), using the pull-through technique, demonstrated that the UES pressure augmentation occurs in response to volume distention, and that this response is further enhanced by HCl and is not dependent on osmolarity of the infusate. Using a sleeve sensor, Vakil and colleagues (60) monitored the UES pressure continuously during acid reflux events and compared the mean UES pressure between a period of 135 seconds before and after reflux events and did not find a significant reflux-induced UES pressure augmentation. This study corroborated an earlier report by Kahrilas and coworkers (62), who also did not find a UES pressure increase in response to GER during sleep. In the latter study, UES pressure during 28 reflux events, defined as intraluminal esophageal pH less than 4.0 for a minimum of 15 seconds, was compared with similar periods before and after these events. Average duration of acid exposure time during these reflux events was reported as 152 ± 137 seconds. Therefore, a sustained long-duration UES response was not documented in these studies. The most critical time for the UES-protective function during a reflux event begins with the onset of the entry of refluxate into the esophagus until its clearance from the esophagus by a secondary or primary peristalsis, namely, during the time of increased intraesophageal pressure because of a reflux event. In a study of healthy control subjects and patients with reflux esophagitis, Torrico and coworkers (103) evaluated UES pressure change at the onset of a reflux event. A total of 321 reflux events were identified by the development of abrupt reflux-induced intraesophageal pressure increase (IPI); 285 events occurred in patients and 36 in control subjects. In control subjects and patients, 33 of 36 and 252 of 285 IPI events, respectively, were associated with a pH reduction. Among patients and control subjects, 99% and 100%, respectively, of all IPI events regardless of a pH decline, were associated with an abrupt increase in UES pressure (34 ± 2 and 27 ± 6 mm Hg, respectively). The average percentage of maximum UES pressure increase over prereflux values ranged between 66% and 96% (control subjects) and 34% and 122% (patients). IPIs induced by both acidic and nonacidic reflux events evoke strong UES contractile responses. These findings suggest the existence of a strong positive relation between UES tone and IPIs induced by GER events, shown previously by experimental distention. Although both acidic and nonacidic reflux events induce UES contraction, intraluminal pH level decreases to less than 4 appear to augment this contractile response.

ESOPHAGOGLOTTAL CLOSURE REFLEX Large-volume GER episodes may cause an instantaneous increase in intraesophageal pressure that might overcome the

PHARYNGEAL MOTOR FUNCTION / 905 UES. This circumstance potentially leaves the upper airway vulnerable to aspiration. Studies have documented the existence of an esophagoglottal closure reflex in humans (46), as well as in the feline species (104). Stimulation of this reflex results in adduction of the vocal cords and closure of the introitus to the trachea. To evoke this reflex, esophageal distention may involve the entire body of the esophagus, such as the distention caused by air insufflation, or it may be regional, such as the distention caused by a short balloon. In either case, the distention must be abrupt to evoke the reflex. The prime candidates for the sensory signal for this reflex are the stretch receptors present in the body of the esophagus. The afferent nerves undoubtedly run in the vagus nerve, carrying the sensory impulse to the brainstem because vagotomy abolishes this reflex (104). The efferent fibers are likely vagal motor fibers to the larynx that traverse the recurrent laryngeal nerve and stimulate the adductor muscles of the glottis. The target muscles are thought to include all or some of the glottal adductors (104). These muscles include thyroarytenoid that is also an isometric tensor of the cords, cricothyroid (also an isotonic tensor), lateral cricoarytenoid, and, finally, interarytenoid muscle that closes the posterior gap in the glottis. Except for the cricothyroid muscle, which is innervated by the external division of the superior laryngeal nerve, all adductor muscles are innervated by the recurrent laryngeal nerve. Because the posterior glottal gap becomes closed on direct viewing when this reflex is triggered, it appears that in addition to the lateral cricoarytenoid muscles, the interarytenoid muscle is also involved. Because the glottis, in response to esophageal distention, shortens and narrows, it is also possible that the thyroarytenoid muscles have a strong participation in this reflex. Reflex connections between the digestive tract and the respiratory system have been reported previously (105). Such studies generally focus on a reflex connection between acid-sensitive receptors in the esophagus and pulmonary bronchi, such that refluxed acid may induce bronchiolar spasm. Coordination of the digestive and respiratory systems during swallowing is well established (10,106). The esophagoglottal closure reflex is an example of close coordination between digestive and respiratory systems during retrograde esophageal transit. The physiologic role of the esophagoglottal closure reflex could be postulated to be one of the airway-protective mechanisms during retrograde esophageal and pharyngeal transit, such as those occurring during belching, GER, regurgitation, and possibly vomiting. Studies have documented that this reflex is evoked during spontaneous GER episodes (107). Other studies have shown that this reflex is absent in about half of the individuals older than 70 years. The function of this reflex in patients with esophagitis or supraesophageal complications of GERD has not yet been evaluated. However, a study using a feline model demonstrated that acute experimental esophagitis either completely abolishes this reflex or results in significant reduction in its frequency of activation (108).

PHARYNGEAL (SECONDARY) SWALLOW Mechanical stimulation of the pharyngeal wall in animals (109) and injection of water into the pharynx in humans (110,111) trigger swallowing; which is called pharyngeal reflexive swallow. This additional stimulus to the initiation of swallowing may play a role in airway protection from pharyngeal reflux of gastric contents, as well as inadvertent spillage of oral contents into the pharynx, during the preparatory phase of swallowing. Studies (112) have characterized the pharyngeal swallow and determined the threshold volume of liquid required to trigger this type of swallowing in young and elderly volunteers. These studies (110–113) have shown that the swallowing mechanism in humans can be readily activated by water stimulation of the pharynx. They also showed that swallows triggered by direct stimulation of the pharynx are different from volitional or primary swallows by not inducing sequential contact of the proximal tongue with the hard palate that is known to occur during primary swallows. Therefore, pharyngeal reflexive swallow does not result in transit of the oral bolus while it clears the pharynx. In this regard, the pharyngeal swallow could be compared with secondary esophageal peristalsis, which usually spares the activation of the peristaltic wave from areas proximal to the point of stimulation (114–116). For this reason, the term secondary swallow is used interchangeably with pharyngeal swallow. Except for lingual peristalsis and transit of oral bolus, the rest of the deglutitive biomechanical events during both types of swallows were found to be similar (Table 35-3). A significantly larger volume of liquid is required to trigger a pharyngeal swallow in elderly people (112). From a functional point of view, therefore, pharyngeal swallows may help to prevent aspiration by two mechanisms: (1) activating the swallow-induced glottal closure (this, in turn, seals off the airway and prevents possible aspiration of material that may either fall into the pharynx inadvertently during the preparatory phase of swallowing or enter the pharynx during large-volume GER episodes); and (2) clearing the pharynx of materials that enter it during reflux from the esophagus. TABLE 35-3. Comparison of the biomechanical events during primary and pharyngeal swallow Biochemical events Lingual peristalsis Tongue base movement Oral volume clearance Pharyngeal volume clearance Hyoid bone movement Velopharyngeal contact Vocal cord closure Aryepiglottal approximation Epiglottal descent Laryngeal elevation Vestibular closure

Primary swallow + + + + + + + + + + +

Source: Shaker and colleagues (112).

Pharyngeal swallow − + − + + + + + + + +

906 / CHAPTER 35 The demonstrated need for larger volumes to trigger pharyngeal swallow in elderly patients could potentially have clinical implications. In the interval between the entry of subthreshold volume of gastric content into the pharynx and occurrence of a primary swallow, the material may be inhaled into the airway. This possibility is further enhanced by the fact that the rate of spontaneous swallowing in elderly people is lower than in young people (117,118). Several areas within the oropharyngeal cavity have been shown to be sensitive for elicitation of the swallowing reflex. Stimulation of the anterior faucial pillars, the tongue, and the epiglottis, as well as the larynx and the posterior pharyngeal wall (16,119), have been shown to trigger swallowing. It has been shown that the spontaneous (primary) swallow occurs infrequently during stable sleep (117,118,120). The stimulation of pharyngeal swallow during sleep has not been studied. However, its nocturnal activation can potentially play an important role in preventing the contact of refluxed gastric content with laryngeal structures and its subsequent aspiration.

CEREBRAL CORTICAL REPRESENTATION OF PHARYNGEAL/REFLEXIVE AND VOLITIONAL SWALLOW IN HUMANS The central neural control mechanism of swallowing is not completely understood; however, a large body of evidence indicates the existence of two levels of control at the brainstem (121–124) and the cerebral cortex (125–132). Current understanding of the cerebral cortical involvement in swallowing is still developing, but clinical evidence derived from patients with CVA (133–138) and, more recently, functional studies of the human brain (125,126,129,132) indicate cortical involvement in swallowing to be multifocal and bilaterally represented. Most commonly cited of these foci include those corresponding to areas in the sensorimotor cortex, prefrontal cortex, anterior cingulate, insular (125,126,129,132), and parietooccipital regions (126,132). The nature of involvement of these foci in various aspects of volitional swallowing such as intent, urge, planning, previous experience, as well as sensorimotor control has not been systematically investigated. In addition to volition, swallowing can be initiated reflexively by direct injection of minute amounts of water into the pharynx. These swallows are irrepressible and occur despite the intention to avoid swallowing (112). Comparison of the topography of the cerebral cortical regions that are associated with pharyngeal reflexive and volitional swallows indicates significant differences between cortical representation of volitional and reflexive swallows in humans. Imaging studies (125,126,129,132) have shown that multiple regions of the human cerebral cortex are involved with volitional swallow. These not only include the sensorimotor cortex, but also involve areas such as the prefrontal cortex, anterior cingulate gyrus, insular cortex, as well as areas corresponding to cuneus and precuneus regions. These latter areas are believed to be involved in a number of functions not necessarily directly related to swallowing.

The cingulate gyrus, for example, is important in conscious feeling and in the processing of stimuli linked to emotion. The activation of pharyngoesophageal sensory pathways conceivably could be conveyed to the cingulate gyrus either directly or after a relay in the hypothalamus and amygdala. Similarly, the prefrontal cortex is involved in the generalized arousal associated with emotional experiences (139,140). Studies have shown that the insular cortex receives projections from the thalamic nuclei, and its neurons are important for processing information about the internal state of the body’s homeostasis. Insular lesions affect the ability to exhibit emotion in response to various stimuli, particularly painful ones (141). The precuneus region is part of the posterior association area of the brain. It integrates information emanating from more than one sensory modality (141,142). It is clear from these findings (125,126,143) that these areas are also involved in deglutition. The functional importance of this multifocal cortical involvement in swallowing is evidenced clinically by development of swallowing disorders after CVAs involving a wide range of cortical regions (133–138), and not directly related to the sensorimotor aspect of swallowing. Contrary to the multifocal cortical representation of the volitional swallow, cerebral cortical representation of pharyngeal reflexive swallow is reported to be focused on the sensorimotor cortex (Fig. 35-9). Reflexive/pharyngeal swallow can occur in response to direct pharyngeal stimulation either by inadvertent premature spill of oral bolus or reflux of gastric content into the pharynx (112). Pharyngeal reflexive swallow is irrepressible and can be induced experimentally by injection of minute amounts of water into the pharynx directed posteriorly. Studies comparing the biomechanical events during volitional and pharyngeal reflexive swallows have shown that except for the absence of lingual peristalsis and transfer of oral bolus into the pharynx, all other biomechanical deglutitive events during reflexive pharyngeal swallow are similar to those of volitional swallows (112). For this reason, the activation of nonsensorimotor cortical regions observed in volitional swallowing probably represents the volitional aspects of the swallow such as intent, urge, decision making, memory, as well as information processing related to deglutition. This notion is partially supported by reports of activation of cingulate gyrus and insular cortex in healthy volunteers experiencing a swallowing urge (144). Within the sensorimotor cortex, activated voxel volumes during volitional swallow were reported to be significantly larger on the right compared with the left hemisphere (125,126,143). In contrast, for reflexive swallow, there seemed to be a significantly larger left hemispheric volume of activated voxels compared with the right hemisphere (143). The functional significance of this finding currently is unknown. Studies of the relation between brain injury and associated deglutitive deficits report both anterior and posterior cortical lesions, as well as brainstem strokes in isolation or in combination with cortical lesions, to be associated with dysphagic symptoms such as aspiration (133,145,146), diminished deglutitive hyolaryngeal mechanics (146), poor

PHARYNGEAL MOTOR FUNCTION / 907 Cortical regions of activity Volitional swallowing – Young subject

Reflexive swallowing – Young subject

left

left

right

right

A

B

FIG. 35-9. A representative example of cerebral cortical functional magnetic resonance imaging (fMRI) activity during volitional swallowing (A) and reflexive swallowing (B). There were substantial differences in cortical activity regions between volitional and reflexive swallow. Although the former was represented in the primary sensorimotor area, the prefrontal, cingulated, insula, and parietooccipital regions, the later was represented in the primary sensorimotor cortex. (See Color Plate 21.) (Modified from Kern and colleagues [143].)

timing and coordination of deglutitive events (144,147,148), and sensory discrimination. These findings suggest the involvement of multiple cortical regions in volitional swallowing as reported by imaging studies. Some CVA patients without detectable brainstem involvement exhibit a deficit in pharyngeal reflexive swallow; the cerebral cortical representation of reflexive pharyngeal swallow in the sensorimotor region offers an explanation for this finding.

PHARYNGO-UPPER ESOPHAGEAL SPHINCTER CONTRACTILE REFLEX Pharyngeal mechanical stimulation in cats (87) and water stimulation in humans (149) have been shown to induce an increase in the resting tone of the UES: the pharyngo-UES contractile reflex. It is speculated that this reflex functions as an airway-protective mechanism whereby retrograde entry of small volumes of liquid into the pharynx from the stomach can result in augmentation of UES tone, reducing the chance of further regurgitation into the pharynx. Contrary to rapid water stimulation that results in abrupt UES pressure increase, during slow, continuous water injection into the pharynx, the UES pressure increases gradually but precipitously before the occurrence of the pharyngeal swallow. In the young group in the supine position during slow continuous injection, the UES pressure was reported to increase from a basal pressure of 50 ± 7 mm Hg to a pressure of 94 ± 9 mm Hg, which is a 76% ± 16% pressure increase (p < 0.05). In the elderly, although a similar trend was reported, the pressure difference was not statistically significant. Similar findings were reported for the upright position.

The afferent limb of the pharyngo-UES contractile reflex is thought to be mediated by the glossopharyngeal nerves. In animal studies, cutting the glossopharyngeal nerves blocked the pharyngo-UES contractile reflex but did not block the esophago-UES contractile reflex or the responses of the TP or CP muscles during swallowing (87). The efferent limb of the pharyngo-UES contractile reflex is mediated by the pharyngoesophageal nerves (somatomotor nerves) that branch from the vagal trunk just rostral to the nodose ganglion (74). Transection of this nerve eliminated basal tone of the CP muscle and blocked the CP muscle response to all reflex stimuli, pharyngo-UES contractile reflex, esophago-UES contractile reflex, and swallowing, but did not block the response of the TP muscle during swallowing. Transection of the vagus nerves at the cervical level (i.e., below the nodose ganglion) had no effect on the pharyngo-UES contractile reflex but blocked the esophago-UES contractile reflex, which indicates that the recurrent laryngeal nerve (that branches from the vagal trunk in the thoracic cavity) serves no role in this reflex (87). It is possible, however, that activation of the pharyngeal receptors may affect laryngeal muscle activity that may be mediated through the recurrent laryngeal nerves (150). A study evaluated the effect of volume, temperature, and anesthesia on the pharyngo-UES contractile reflex in humans (149). Pharyngeal water injection at a threshold volume of 0.1 ± 0 ml invariably resulted in a significant increase in the UES pressure in all subjects. This pressure increase was not affected by volumes and temperatures. The threshold volume for UES pressure increase was significantly less than that for triggering a pharyngeal swallow. Results were found to be similar for slow continuous injection.

908 / CHAPTER 35 Application of a topical anesthetic to the pharyngeal mucosa completely abolished this reflex (149). A study (151) of patients with posterior laryngitis who were believed to have suffered from reflux of gastric content into the pharynx and larynx reported that the threshold volume required to evoke the pharyngo-UES contractile reflex was significantly greater than that of the control subjects. However, after stimulation of the pharyngo-UES contractile reflex, the maximum postinjection pressure in patients was similar to that of the control subjects, suggesting differences in the afferent, but not the efferent, arm of the reflex in the patient group.

PHARYNGOGLOTTAL ADDUCTION REFLEX Studies have shown that injection of minute amounts of water into the pharynx results in brief closure of the vocal cords (48,152). Gradual entry of liquid into the pharynx induces partial adduction, whereas rapid injection causes complete closure of the cords. It is postulated that this adduction response is a part of the complex mechanisms that protect the airway from retrograde aspiration. The threshold volume that stimulates this glottal adduction is reported to be significantly smaller than that required to trigger an irrepressible pharyngeal swallow but similar to that required to induce a pharyngo-UES contractile reflex (48,152). Evidence suggests that a significantly larger volume of liquid is needed to trigger a pharyngoglottal reflex in elderly compared with young people (48,152).

LARYNGO-UPPER ESOPHAGEAL SPHINCTER CONTRACTILE REFLEX Studies (153) using an air stimulation technique have shown that afferent signals originating from the larynx induce contraction of the UES: laryngo-UES contractile reflex. Frequency elicitation of this reflex is significantly less in elderly compared with young people, whereas the magnitude of UES pressure change remains unchanged, indicating a deleterious effect of aging on the afferent arm of this reflex. The laryngo-UES contractile reflex is reported to be altered in some patients with UES dysphagia (153). The afferent arm of this reflex in humans includes the laryngeal mechanoreceptor and internal division of the superior laryngeal nerve (155), a branch of the vagus nerve. The efferent arm is undoubtedly the vagus nerve (156,157), including superior laryngeal nerve and recurrent laryngeal nerve, although glossopharyngeal nerve cannot be excluded because it serves branches into the pharyngeal plexus. The central pathway for this reflex is probably different from those of deglutition because the contractile response to the stimulation of this reflex is the opposite of the relaxation response of the UES to volitional, subconscious, and reflexive or pharyngeal swallow. This reflex is different from the pharyngo-UES contractile reflex that is triggered by stimulation of mechanoreceptors in the posterior pharyngeal wall

(89,149), whereas the effector organ and efferent arc are the same for both reflexes; the sensory field and afferent arc are different in that the pharyngo-UES contractile reflex is mediated via the glossopharyngeal (89) nerve with possible contribution from the superior laryngeal nerve. Earlier studies have shown that air stimulation of the larynx also has an inhibitory effect on the lower esophageal sphincter (LES) (158). It is conceivable that activation of the laryngo-UES contractile reflex may counteract the possible consequences of the inhibitory effect of laryngeal stimulation on the LES, namely, pharyngeal reflux of gastric content by increasing the UES pressure. In addition, although highly speculative at this stage, this reflex may be activated during pharyngeal reflux events by contact of regurgitated material with the laryngeal mucosa and by inducing an augmentation of UES resting pressure, possibly preventing further entry of refluxate into the pharynx and larynx. It is conceivable that deterioration of this reflex in elderly people may negatively affect airway protection against aspiration in this age group, especially during nighttime reflux events in the supine position, when the airway is most vulnerable. In summary, there are currently at least nine reflex mechanisms identified that either augment the UES pressure barrier, close the airway, or clear the pharynx and esophagus from gas, liquid, or solid volumes. These reflexes include: (1) pharyngo-UES contractile reflex; (2) pharyngoglottal adduction reflex; (3) pharyngeal/reflexive swallow; (4) laryngo-UES contractile reflex; (5) laryngeal adductor reflex; (6) esophagoglottal closure reflex; (7) esophago-UES contractile reflex; (8) esophago-UES relaxation reflex; and (9) secondary esophageal peristalsis. Except for secondary esophageal peristalsis, the remaining eight reflexes involve the pharynx, UES, and larynx either as the afferent site or effector organ. These reflexes may be activated as the result of pressure events associated with antegrade, as well as retrograde, transit of material through the pharyngoesophageal axis. Available evidence indicates that one or several of these reflexes may be stimulated in response to a single stimulus, such as stimulation of the pharyngo-UES and pharyngoglottal closure reflexes as a consequence of stimulation of the pharyngeal mucosa or stimulation of secondary peristalsis, esophago-UES contractile reflex, and esophagoglottal closure reflex in response to esophageal distention. Aging appears to adversely affect these reflexes. Emerging evidence suggests the impairment of these reflexes in some disorders involving the aerodigestive tract, such as posterior acid laryngitis or dysphagia with predeglutitive aspiration.

PHARYNGEAL INHIBITORY REFLEXES In addition to stimulatory reflexes emanating from the pharynx, as described earlier, pharyngeal stimulation may exert an inhibitory effect on esophageal peristalsis, as well as the LES. Studies (159) have shown that minute amounts of liquid injected either abruptly or slowly into the pharynx induce LES relaxation. The duration and magnitude of LES

PHARYNGEAL MOTOR FUNCTION / 909 relaxation induced by pharyngeal water injection are different from those of the normal swallow. Neither the duration nor the magnitude of this relaxation is volume dependent. It also has been shown (160) that sensory impulses initiated from the pharynx by water injection inhibit the progression of primary esophageal peristalsis. This, in turn, inhibits esophageal bolus transit. This inhibitory effect is capable of overcoming the facilitating effect that the presence of a bolus induces on the swallowing apparatus, and the threshold volume required to inhibit the progression of peristalsis induced by swallowing liquid boluses is significantly greater than that induced by the dry swallows; however, this threshold is not dependent on bolus volume or consistency (161). In a substantial minority of young and elderly healthy volunteers, pharyngeal water stimulation has been shown to result in gastroesophageal acid reflux in the postprandial period (162).

Glottal function during belching consists of the following sequences: (1) vocal cord adduction concurrent with adduction of arytenoids, resulting in full closure of the introitus to the trachea before the UES relaxes or opens; (2) anterior/ caudad movement of the glottis; and (3) opening of the UES that is either slitlike or triangular (45). During some belches, the adducted arytenoids approximate the base of the epiglottis, whereby closing the supraglottic portion of the laryngeal vestibule. Supraglottic closure of the laryngeal vestibule, when present, is associated with an increase in intragastric pressure (45). Glottal closure and its precedence to the UES relaxation and opening during belching is an important protective function against aspiration of food particles, acid mist, and gastric content that may be regurgitated into the pharynx during belching and could be aspirated into the trachea. Glottal closure during belching in humans is in sharp contrast to the glottal function in ruminants, in which the glottis remains open during belching, resulting in tracheal reflux of gastric gas (154). The time interval between the onset of vocal cord adduction and their complete closure, as well as the time interval between the onset of their abduction and their return to resting open position during belching, is fixed and indicates that the closure and opening of the cords during belching is preprogrammed by a stereotyped neural program. In contrast, the duration of complete cord closure during belching is modified according to the volume of belched air (45). Based on these observations, it has been suggested (45) that the neural pathway during belching invokes both those controlling the larynx and the UES functions, in that the neural pathway for glottal closure during belching begins with the vagus nerve and the stretch receptors in the esophageal wall that carries the signal to the brainstem. The efferent vagal fibers, through the recurrent laryngeal nerve, carry the signal to the glottal adductor muscles—interarytenoid, lateral cricoarytenoid, and thyroarytenoid. Coupled with this activity, the signals carried by the vagus to the brainstem

MECHANISMS OF AIRWAY PROTECTION DURING BELCHING Belching is defined as voiding of gas from the stomach and esophagus through the mouth. However, it is known that distention of the esophagus by air may also initiate an esophageal belching that does not involve gas reflux from the stomach. Ventilation of gastric or esophageal gas across the UES into the pharynx may be accompanied by entry of food particles, acid mist, and so forth into the hypopharynx and may predispose the airway to aspiration. Studies indicate that the glottis is actively involved in the belch reflex by activation of its closure mechanism (45). They also indicate the existence of a close coordination between the UES and glottal function during belching. These studies also show that the glottal closure mechanism is activated and the vocal cords become closed before UES relaxation and its subsequent opening during belching. The existence of the same coordination during regurgitation and vomiting has been speculated but not yet studied.

TABLE 35-4. Function of various elements involved in belching and swallowing Hyoid movement Events Swallowing Esophageal belch without increase in intragastric pressure Esophageal belch with increase in intragastric pressure Gastric belch without increase in intragastric pressure Gastric belch with increase in intragastric pressure

Vocal cord adduction

Vestibular supraglottic

+ +

+ −

+ −

+ −

+ −

+ +

+ ±

− ±

+

+

−

−

−

+

±

±

+

−

−

−

−

+

±

±

+

+

−

−

−

+

±

±

Source: Shaker and colleagues [45].

Closure Epiglottal subepiglottic descent

Laryngeal elevation

Anterior Superior

Inferior

910 / CHAPTER 35 from the esophageal wall stretch receptors stimulate suprahyoid and infrahyoid muscles through the 10th, 5th, and 12th and ansi cervicalis, resulting in UES relaxation and opening. Table 35-4 shows a comparison of the functions of the various elements involved in belching and swallowing.

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912 / CHAPTER 35 104. Shaker R, Ren J, Medda B, Lang I, Cowles V, Jaradeh S. Identification and characterization of the esophagoglottal closure reflex in a feline model. Am J Physiol Gastrointest Liver Physiol 1994;266:G147–G153. 105. Wright RA, Miller SA, Corsello BF. Acid-induced esophago-bronchialcardiac reflexes in humans. Gastroenterology 1990;99:71–73. 106. Sasaki CT, Isaacson G. Functional anatomy of the larynx. Otolaryngol Clin North Am 1988;21:595–612. 107. Shaker R, Ren J, Hogan WJ, Liu J, Podvrsan B, Sui Z. Glottal function during postprandial gastroesophageal reflux. Gastroenterology 1993;104:A581. 108. Ren J, Shaker R, Medda B, Bonnevier J, Kern M, Dunn B. Effect of acute esophagitis on the esophagoglottal closure reflex in a feline model. Gastroenterology 1995;108:A677. 109. Paterson WC, Rattan S, Goyal RK. Experimental induction of isolated lower esophageal sphincter relaxation in anesthetized opossums. J Clin Invest 1986;77:1187–1193. 110. Nishino T, Takizawa K. Yokokawa N, Hiraga K. Depression of the swallowing reflex during sedation and/or relative analgesia produced by inhalation of 50% nitrous oxide in oxygen. Anesthesiology 1987;67:995–998. 111. Nishino T. Swallowing as a protective reflex for the upper respiratory tract. Anesthesiology 1993;79:588–601. 112. Shaker R, Ren J, Zamir Z, Sarna S, Liu J, Sui Z. Effect of aging, position, and temperature on the threshold volume triggering pharyngeal swallows. Gastroenterology 1994;107:396–402. 113. Ebihara T, Sekizawa K, Nakazawa H, Sasaki H. Capsaicin and swallowing reflex. Lancet 1993;341:432. 114. Patterson WG, Hynna-Liepert TT, Selucky M. Comparison of primary and secondary esophageal peristalsis in humans: effect of atropine. Am J Physiol 1991;260:G52–G57. 115. Christensen J, Lund GF. Esophageal responses to distension and electrical stimulation. J Clin Invest 1969;4I:408–419. 116. Paterson WG. Neuromuscular mechanisms of esophageal responses at and proximal to a distending balloon, Am J Physiol Gastrointest Liver Physiol 1991;260:G148–G155. 117. Lichter I, Muir RC. The pattern of swallowing during sleep. Electroencephalogr Clin Neurophysiol 1975;38:427–432. 118. Lear CS, Flanagan JB, Moorrees CF. The frequency of deglutition in man. Arch Oral Biol 1965;10:83–100. 119. Pommerenke W. A study of the sensory areas eliciting the swallowing reflex. Am J Physiol 1928;84:36–41. 120. Dent J, Dodds WJ, Friedman RH, et al. Mechanism of gastroesophageal reflux in recumbent asymptomatic human subjects. J Clin Invest 1980;65:256–267. 121. Carpenter DO. Central nervous system mechanisms in deglutition and emesis. In: Handbook of physiology. The gastrointestinal system. Motility and circulation. Schultz SG, Woods JD (eds). Bethesda, MD: American Physiological Society, 1989;685–714. 122. Jean A. Brainstem organization of the swallowing network. Brain Behav Evol 1984;25:109–116. 123. Miller AJ. Deglutition. Physiol Rev 1982;62:129–184. 124. Sessle B, Henry JL. Neural mechanisms of swallowing: neurophysiological and neurochemical studies on brain stem neurons in the solitary tract region. Dysphagia 1989;4:61–75. 125. Hamdy S, Mikulis DJ, Crawley A, Zue S, Lau H, Henry S, Diamant NE. Cortical activation during human volitional swallowing: an eventrelated fMRI study. Am J Physiol Gastrointest Liver Physiol 1999;277:G219–G225. 126. Hamdy S, Rothwell JC, Brooks DJ, Bailey D, Aziz Q, Thompson DG. Identification of the cerebral loci processing human swallowing with H215O PET activation. J Neurophysiol 1999;81:1917–1926. 127. Martin RE, Sessle BJ. The role of the cerebral cortex in swallowing. Dysphagia 1993;8:195–202. 128. Miller AJ, Bowman JP. Precentral cortical modulation of mastication and swallowing. J Dent Res 1977;56:1154 (Abstract). 129. Mosier K, Patel R, Liu W-C, Kalnin A, Maldjian J, Baredes S. Cortical representation of swallowing in normal adults: functional implications. Laryngoscope 1999;109:1417–1423. 130. Sumi T. Reticular ascending activation of frontal cortical neurons in rabbits, with specific reference to the regulation of deglutition. Brain Res 1972;46:43–54. 131. Sumi T. Some properties of cortically evoked swallowing and chewing in rabbits. Brain Res 1969;15:107–120. 132. Zald DH, Pardo JV. The functional neuroanatomy of voluntary swallowing. Ann Neurol 1999;46:281–286. 133. Alberts M, Horner J, Gray L, Brazer SR. Aspiration after stroke: lesion analysis by brain MRI. Dysphagia 1992;7:170–173.

134. Gordon C, Hewer R, Wade D. Dysphagia in acute stroke. Br Med J 1987;195:411–414. 135. Horner J, Massey E, Riski J, Lathrop D, Chase K. Aspiration following stroke: clinical correlates and outcome. Neurology 1988;38:1359–1362. 136. Meadow J. Dysphagia in unilateral cerebral lesions. J Neurol Neurosurg Psychiatry 1973;36:853–860. 137. Robbins J. Swallowing after cortical CVA: right vs left hemisphere differences. ASHA 1985;27:123 (Abstract). 138. Veis S, Logemann J. Swallowing disorders in persons with cerebrovascular accident. Arch Phys Med Rehabil 1985;66:372–375. 139. Aggleton JP. The contribution of the amygdale to normal and abnormal emotional states. Trends Neurosci 1993;16:328–333. 140. LeDoux JE. Brain mechanisms of emotion and emotional learning. Curr Opin Neurobiol 1992;2:191–197. 141. Talbot JD, Marrett S, Evans AC, Meyer E, Bushnell MC, Duncan GH. Multiple representation of pain in human cerebral cortex. Science 1991;251:1355–1358. 142. Pandya DN, Seltzer B. Association areas of the cerebral cortex. Trends Neurosci 1982;5:386–390. 143. Kern MK, Jaradeh S, Arndorfer RC, Shaker R. Cerebral cortical representation of reflexive and volitional swallowing in humans. Am J Physiol Gastrointest Liver Physiol 2001;280:G354–G360. 144. Kern MK, Arndorfer RC, Hyde JS, Shaker R. Cortical representation of swallow urge in young healthy volunteers. Neurogastroenterol Motil 1999;11:269 (Abstract). 145. Field LH, Weiss CJ. Dysphagia with head injury. Brain Inj 1989;3: 19–26. 146. Logemann JA, Shanahan T, Rademaker AW, Kahrilas PJ, Lazar R, Halper A. Oropharyngeal swallowing after stroke in the left basal ganglion/internal capsule. Dysphagia 1993;8:230–234. 147. Robbins J, Levine RL. Swallowing after unilateral stroke of the cerebral cortex: preliminary experience. Dysphagia 1988;3:11–17. 148. Robbins J, Levine RL, Maser A, Rosenbek JC, Kempster GB. Swallowing after stroke of the cerebral cortex. Arch Phys Med Rehabil 1993;74:1295–1300. 149. Shaker R, Ren J, Xie P, Lang IM, Bardan E, Sui Z. Characterization of the pharyngo-UES contractile reflex in humans. Am J Physiol 1997;273:G854–G858. 150. Barlett D. Upper airway motor systems. In: Schultz SG, ed. Handbook of physiology. The respiratory system. Control of breathing. Bethesda, MD: American Physiological Society, 1986;363–394. 151. Ulualp SO, Toohill RJ, Kern M, Shaker R. Pharyngo-UES contractile reflex in patients with posterior laryngitis. Laryngoscope 1998;108:1351–1357. 152. Shaker R, Ren J, Bardan E, Easterling C, Dua K, Xie P, Kern M. Pharyngoglottal closure reflex: Characterization in healthy young, elderly and dysphagic patients with predeglutitive aspiration. Gerontology 2003;49:12–20. 153. Kawamura O, Easterling C, Aslam M, Rittmann T, Hofmann C, Shaker R. Laryngo-upper esophageal sphincter contractile reflex in humans deteriorates with age. Gastroenterology 2004;127:57–64. 154. Dougherty RW, Hill KJ, Campeti FL, McClure RC, Habel RE. Studies of pharyngeal and laryngeal activity during eructation in ruminants. Am J Vet Res 1962;23:213–219. 155. Sasaki CT, Weaver EM. Physiology of the larynx. Am J Med 1997; 103:9S–18S. 156. Sasaki CT. Understanding the motor innervation of the human cricopharyngeus muscle. Am J Med 2000;108:38S–39S. 157. Lang IM, Shaker R. An overview of the upper esophageal sphincter. Curr Gastroenterol Rep 2000;2:185–190. 158. Noordzij JP, Mittal RK, Arora T, Pehlivanov N, Liu J, Relbel JF, Levine PA. The effect of mechanoreceptor stimulation of the laryngopharynx on the oesophago-gastric junction. Neurogastroenterol Motil 2000;12:353–359. 159. Trifan A, Shaker R, Ren J, Mittal RI, Saeian K, Dua K, Kusano M. Inhibition of resting lower esophageal sphincter pressure by pharyngeal water stimulation in humans. Gastroenterology 1995;108:441–446. 160. Trifan A, Ren J, Arndorfer R, Hofmann C, Bardan E, Shaker R. Inhibition of progressing primary esophageal peristalsis by pharyngeal water stimulation in humans. Gastroenterology 1996;110:419–423. 161. Bardan E, Xie P, Ren J, Dua K, Shaker R. Effect of pharyngeal water stimulation on esophageal peristalsis and bolus transport. Am J Physiol 1997;272:G265–G271. 162. Xie P, Ren J, Bardan E, Mittal RK, Sui Z, Shaker R. Frequency of gastroesophageal reflux events induced by pharyngeal water stimulation in young and elderly subjects. Am J Physiol Gastrointest Liver Physiol 1997;272:G233–G237.

CHAPTER

36

Motor Function of the Esophagus Ray E. Clouse and Nicholas E. Diamant Overview, 913 Neuromuscular Anatomy, 913 Gross Anatomy, 913 Upper Esophageal Sphincter, 914 Esophageal Body, 914 Lower Esophageal Sphincter, 914 Innervation, 914 Extrinsic Parasympathetic Innervation, 914

Extrinsic Sympathetic Innervation, 915 Intramural Innervation, 915 Coordinated Motor Events, 916 Upper Esophageal Sphincter, 916 Esophageal Body, 917 Lower Esophageal Sphincter, 921 Deglutitive Inhibition and Refractoriness, 923 References, 923

OVERVIEW

through the posterior pharynx and esophagus. Redundancy of control mechanisms in the involuntary component, particularly in the smooth muscle region of the esophagus, also adds complexity to the physiology underlying esophageal motor function. Presumably, some of this redundancy allows for successful motor function despite impairments that might be imposed by disease.

Esophageal motor functions result from a variety of interacting control mechanisms. These mechanisms link esophageal function to oropharyngeal aspects of swallowing (see Chapter 35), gastric motor function, other thoracic organs, and the central nervous system. This chapter describes motor physiology of the upper esophageal sphincter (UES) in relation to the esophageal body, the esophageal body itself, and the lower esophageal sphincter (LES). Functions of these regions include: (1) facilitation of aborad food transport into the stomach once it leaves the oropharynx; and (2) prevention of orad escape of gastric contents into the esophagus and pharynx. Physiology of this motor behavior is made more complex by the transition from striated muscle and its dominant central control (UES and cervical esophagus) to smooth muscle and its prominent local control mechanisms, influenced by central modulation. The swallowing process also combines voluntary and involuntary processes acting in concert: the oropharyngeal phase initially is voluntary but becomes involuntary as the motor sequence proceeds

NEUROMUSCULAR ANATOMY Gross Anatomy The esophagus begins proximally at the pharyngoesophageal junction (level of the UES), traverses the posterior mediastinum, and ends at the gastroesophageal junction (level of the LES). Anatomic esophageal length ranges from 18 to 26 cm in the adult, varies with age and presence of hiatal herniation, and has some correlation with body height (1,2). The cervical esophagus includes the proximal 4 to 5 cm from the pharyngoesophageal junction to the suprasternal notch. The thoracic esophagus continues beyond this point, coursing behind the trachea and its bifurcation. The esophagus remains anterior to the aorta as it enters the hiatus, an elliptical diaphragmatic opening. Only 0.5 to 2.5 cm of esophagus is present in the abdomen before it merges with gastric structures (3). The hiatus is rimmed by diaphragmatic crura, and at times, the median arcuate ligament. The anatomic relation of the esophagus to the crura is maintained by the collagen and elastic fibers of the phrenoesophageal membrane.

R. E. Clouse: Departments of Medicine and Psychiatry, Washington University, St. Louis, Missouri 63110. N. E. Diamant: Departments of Medicine and Physiology, University of Toronto, Toronto Western Hospital, Toronto, Ontario, Canada. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

913

914 / CHAPTER 36

Vagus nerves

Recurrent laryngeal nerves

Right recurrent laryngeal nerve

the inferior pharyngeal constrictor (8–10). Muscles within the esophageal body below the cricopharyngeus are not major contributors to sphincter pressure but may explain some of its distal aspect (11). The thyrohyoid and geniohyoid muscles are involved in active sphincter opening with swallows (12).

Esophageal Body Left recurrent laryngeal nerve

Thoracic chain Anterior esophageal plexus

Anterior vagal trunk Posterior vagal trunk

FIG. 36-1. Extrinsic innervation of the esophagus. (Reproduced from Boyce and Boyce [4], by permission.)

This membrane inserts into the circumference of the esophagus, both above and below the diaphragm. With aging, fat separates these fibers, and the anatomic relation of the distal esophagus to the crura is less well maintained (4). The cervical esophagus is innervated by the recurrent laryngeal nerves as they return proximally from the vagus nerves (Fig. 36-1). The two vagal nerves (left and right) join sympathetic fibers to create an esophageal plexus (5,6). At some point above the diaphragm, the anterior and posterior vagal trunks emerge from this plexus, traverse the diaphragmatic hiatus, and divide below the diaphragm to innervate abdominal organs. Sympathetic innervation is provided by sympathetic nerves in the plexus and along the vascular supply by fibers from the superior cervical ganglion, thoracic ganglia, and celiac ganglion.

Upper Esophageal Sphincter The UES is defined in physiologic terms, but its anatomic correlates are quite evident. The 1-cm region of maximal pressure within the sphincter corresponds to the location of the cricopharyngeus muscle (7). Other muscles and local structures also are involved in producing the 2- to 4-cm-wide high-pressure zone surrounding the region represented by the cricopharyngeus, such as the cartilaginous hypopharynx and

The muscularis propria of the esophageal body is composed of an outer longitudinal muscle layer and an inner circular muscle layer. Muscle fibers of the muscularis mucosae are also oriented in a longitudinal fashion and are of smooth muscle type throughout the length of the esophagus. Muscle type within the two layers of the muscularis propria changes from striated in the proximal esophagus to smooth muscle distally. The esophageal body averages 18 to 21 cm in length as measured manometrically (13,14). Approximately 4 cm of the proximal circular muscle layer is entirely striated muscle; at 4.7 cm from the cricopharyngeus, the circular muscle layer is a mixture of both muscle types in equal proportion (15). Further transition to smooth muscle occurs over an additional 4 to 8 cm such that the distal 10 to 14 cm of the circular muscle layer are entirely smooth muscle. Transition locations within the longitudinal muscle layer vary to a small degree from these values.

Lower Esophageal Sphincter A thickened segment within the circular muscle layer having different ultrastructural characteristics from smooth muscle in the esophageal body represents the anatomic equivalent of the LES (16,17). The thickened segment is 2.5 to 3.5 cm in length and angles upward from the lesser to greater gastric curvatures. Besides some completely circular rings, muscle fibers extending from the thickened region onto the greater and lesser curves of the stomach can be identified. Sling fibers form long oblique loops, do not completely encompass the distal esophagus, and straddle the greater curvature of the stomach (18). Clasp fibers form short, semicircular rings with ends that terminate on the anterior and posterior walls of the esophagus and straddle the lesser curvature. Clasp fibers connect at an angle to the sling fibers (18). The sphincter is in close approximation to the diaphragmatic crura when no distortion of the phrenoesophageal membrane is present (e.g., with hiatal hernia) such that greatest pressures in the sphincter occur at the point of respiratory reversal in pressure waveform (19).

INNERVATION Extrinsic Parasympathetic Innervation Extrinsic control of esophageal motor function is provided by preganglionic parasympathetic neurons arising from a

MOTOR FUNCTION OF THE ESOPHAGUS / 915 “swallowing center” in the dorsal vagal brainstem complex (see Chapter 33) (20–23). The three functional components of this center include an afferent reception system, an efferent motor neuron system, and a complex organizing, or internuncial, neuronal system. These functions are provided predominantly by the nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMV). Peripheral afferent information enters the NTS where it can initiate the swallowing process, modify ongoing swallow activity, or affect muscle behavior independently of swallowing. Sensory information from the oropharynx and its processing are described elsewhere in this textbook (see Chapter 35). Sensory information from the esophagus and its sphincters is carried in the vagus nerve with cell bodies in the nodose ganglion. This sensory activity monitors and modulates esophageal motor events and may affect cognitive sensation through central descending pathways (24). Sensory information traversing sympathetic pathways is relevant to cognitive sensation and the appreciation of symptoms arising from the esophagus (25,26). The neural cascade ultimately affecting a swallow is initiated by esophageal sensory afferents that project to premotor neurons in the NTS. These project to other clusters of premotor neurons that ultimately reach somatic motor neurons in the nucleus ambiguous and motor neurons of the DMV for control of striated and smooth muscle esophageal regions, respectively (27) (see Chapter 33). The NTS and neighboring reticular substance serve functionally as a central pattern generator (CPG) to program the swallow (21,28,29). The CPG initiates and organizes motor events, connects to the motor neuron pools effecting the response, and integrates the swallowing process with other medullary centers that, for example, control diaphragmatic movement (30,31). Subnuclei serve both the striated muscle and smooth muscle regions of the esophagus to produce sequential activation of motor neurons (32). Sensory input from the esophagus can initiate the sequential programs at or just proximal to the location of the peripheral sensory input (33–35). Factors initiating the oropharyngeal phase of deglutition and its control mechanisms are discussed in Chapters 33 and 35. The vagus nerve receives fibers from the nucleus ambiguous and DMV to innervate the striated and smooth muscle portions of the esophagus, respectively. Two neuronal pools have been identified within the DMV for innervation of the smooth muscle esophageal body, but their roles are uncertain (36). Two motor neuron pools also have been identified in the DMV for control of the LES, one producing contraction and the other producing relaxation of the sphincter. The latter is most extensive, modulating LES resting tone and producing active sphincter relaxations (37,38). A variety of brainstem neurotransmitters have been implicated in control of esophageal body and LES motor function (39). Muscarinic cholinergic excitation and excitatory amino acids facilitate striated muscle peristalsis in rat esophagus (40–42). Both nicotinic stimulation and activation of γ-aminobutyric acid (GABA) receptors participate in

initiating peristalsis in other animal models (43,44). Nitric oxide (NO) participates in the initiation and programming of peristalsis in the smooth muscle esophagus (45). Other identified neurotransmitters that likely are relevant include excitatory amino acids activating N-methyl-D-aspartate (MMDA) and non-NMDA receptors, somatostatin, catecholamines, serotonin, thyrotropin-releasing hormone, vasopressin, and oxytocin (40,46–49). Many neurotransmitters are found in the sensory neurons of the nodose ganglion potentially modulating afferent vagal information (38). LES tone and aftercontraction appear mediated by NMDA and release of NO. Centrally acting NO also is involved in production of the transient lower esophageal sphincter relaxation (TLESR) that occurs independently of swallowing (50). Other central neurotransmitters involved in this reflex include cholecystokinin and acetylcholine. GABA (through the GABAB receptor) and opioids inhibit the TLESR, and the former has a strong effect to block inhibition of crural diaphragmatic activity (51,52).

Extrinsic Sympathetic Innervation The distribution of sympathetic fibers reaching the esophagus and its sphincters has been described earlier. These connections reach the esophagus via the vascular supply and, to a lesser extent, by intermingling with the vagus nerves (20,53). Cell bodies causing preganglionic sympathetic fibers innervating the esophagus and its sphincters originate in the anterior mediolateral columns of the spinal cord (segments T1-T10) (54). The preganglionic fibers that reach the celiac ganglion via splanchnic nerves synapse with postganglionic neurons and provide sympathetic innervation to the lower esophagus and LES (55–57).

Intramural Innervation Both a myenteric nerve plexus and a submucosal nerve plexus are found in the esophagus, although the latter is sparse with few ganglion cells (58,59). The myenteric plexus is best developed in the smooth muscle region, where it plays an important role in coordinated peristalsis. In the striated muscle region, efferent vagal fibers contact nicotinic cholinergic end plates on muscle fibers (60,61). Besides acetylcholine, these vagal fibers contain calcitonin gene–related peptide with an unknown function. The nerve plexuses in this region presumably play a sensory role and have an uncertain motor modulatory effect, but they may contain an inhibitory pathway to the LES (62,63). In contrast, efferent vagal fibers entering the smooth muscle region synapse on intramural myenteric neurons. Although numerous neurotransmitters have been identified in the plexus (38), two principle types of effector neurons innervate this region: one mediating cholinergic excitation of both muscle layers (M3 receptors), and the other mediating nonadrenergic, noncholinergic (NANC) inhibition

916 / CHAPTER 36 primarily of the circular muscle layer (NO mediated) (20,32). Cholinergic stimulation of these two neuron types from preganglionic vagal efferent fibers or interneurons occurs through both nicotinic and nonnicotinic mechanisms (64,65). Other neurotransmitters have been identified in the myenteric plexus (including purine nucleotides, serotonin, opiates, peptide hormones), and smooth muscle of the distal esophagus and LES is sensitive to a variety of substances that could influence motor behavior (histamine, prostaglandins, dopamine) (20,32,66). The esophageal interstitial cells of Cajal form intimate gap junction contact with both intramural nerves and smooth muscle cells and probably play an important role in esophageal motor function, although it is yet to be understood fully (67–70). Sympathetic nerves found in the myenteric plexus of both the striated and smooth muscle esophageal regions appear to serve as neuromodulators in the latter, a β-adrenergic effect being inhibitory and an α-adrenergic effect being excitatory on muscle contraction amplitude and LES tone (55,71). Few terminate directly on muscle cells (57,71). Sensory information arises from free vagal and spinal afferent nerve endings in the epithelium and submucosa, interganglionic laminar structures in the myenteric plexus, and intramuscular nerve arrays, because these structures are sensitive to many stimuli (32,38,72). The latter two are assumed to be mechanoreceptors responsive to both stretch and tension (72,73). Whereas pure mechanoreceptors are found only in the vagal pathway, mixed mechanonociceptors and high-threshold, rapidly adapting nociceptors are involved in the spinal afferent pathway (25). Vagal afferent information modulates contractile activity and may mediate nonnoxious sensations, such as satiety. Sympathetic spinal afferents both modulate motor activity and transmit centrally information from noxious stimulation. These afferent systems are not independent, and vagal afferents also modulate spinal afferent information, thereby impacting sensation (24,74–76). Sensory information from the esophagus is important not only in initiating and modulating motor patterns, but also in generating autonomic, emotional, and behavioral responses to esophageal symptoms. Vagal afferents with cell bodies in the nodose ganglion terminate in the NTS, their influence reaching higher brain regions predominantly via the parabrachial nucleus. Spinal afferents with cell bodies in the dorsal root ganglia predominantly travel via spinothalamic tracts and dorsal columns, although the role of the dorsal columns in esophageal sensation remains unknown. The wide distribution of afferents from each esophageal region overlaps at the spinal level with innervation from other thoracic organs, such as the heart (77,78).

COORDINATED MOTOR EVENTS Coordinated motor activity occurs both during swallowing and in the interswallow period to accomplish normal esophageal motor functions. The UES and LES relax during

swallowing, and the esophageal body appropriately sequences a peristaltic contraction to assist with aborad bolus transport into the stomach. In the interswallow period, the sphincters maintain tone to prevent orad movement of esophageal or gastric contents. The coordinated esophageal events initiated by the act of swallowing are called primary peristalsis. Progressive contraction in the esophageal body induced by distention from poorly cleared food or refluxed gastric contents is called secondary peristalsis. This process begins at or above the location of the intraluminal stimulus and closely resembles primary peristalsis. These events and their effectiveness most commonly are studied in humans using intraluminal manometry and measures of bolus transit (cinefluoroscopic monitoring of radiopaque material, radionuclide transit measurement). Surrogate markers of bolus movement (intraluminal pH, intraluminal impedance) also are used (79). Longitudinal muscle movement has been studied using cinefluoroscopic movement of radiopaque clips and, more recently, high-frequency intraluminal ultrasound (80,81). Intraluminal pressure measurements that incorporate an axial dimension to measure pressure relations across esophageal levels or a circumferential dimension to evaluate radial asymmetry at any one level have improved the understanding of coordinated esophageal motor activity. Highresolution manometry increases pressure sensor density in the axial dimension such that interpolation across sensors can be accomplished (82). Resultant data are displayed as axial pressure waveforms traversing the esophageal body in time or as three-dimensional isocontour maps of the entire pressure field. Manometric methods that increase sensor density not only in the axial direction, but also circumferentially (high-definition manometry), allow interpolation of pressure data along both dimensions. Such methods reveal axial and radial asymmetries in sphincteric regions, both during rest and relaxation. Sensor devices suitable for highdefinition methods are just appearing (83), and currently available high-definition images are restricted to composites derived from catheters with multiple radially oriented pressure measurement sites at a single level; repositioning of the catheter is required to satisfy the axial dimension (84,85). These primitive high-definition displays are referred to as vector volume maps.

Upper Esophageal Sphincter The UES is closed tonically at rest. A combination of continuous excitation and passive tone is responsible (86). The region of high pressure is 2 to 4 cm long, and there is marked radial and axial asymmetry to the region (Fig. 36-2) (7,87,88). The asymmetry partly relates to compression by adjacent laryngeal structures, because it disappears after laryngectomy (85). Because of the asymmetry, a singular normal pressure value within the sphincter is not meaningful (89). Neural discharge controlling resting pressure ceases with swallows, sleeping, and general anesthesia and increases with inspiration or coughing (90,91). Proximal esophageal

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MOTOR FUNCTION OF THE ESOPHAGUS / 917

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USEP in mm Hg

FIG. 36-2. Three-dimensional vector volume representations of the upper esophageal sphincter at rest. Axial and radial asymmetries are pronounced with pressures greatest in the anteroposterior orientation. The map is rotated 180 degrees to reveal its hidden side. UESP, upper esophageal sphincter pressure. (Reproduced from Welch and colleagues [84], by permission.)

acidification or slow distention will increase UES pressure, whereas abrupt esophageal distention reduces pressure for venting (92,93). Opening of the UES with swallowing requires both cessation of neural input (and resultant sphincter relaxation) and active opening by anterior and superior movement of laryngeal structures. Within 0.2 to 0.3 seconds after swallow initiation, neural input ceases to the UES to coordinate UES opening with pharyngeal peristalsis (Fig. 36-3). Elevation of the larynx in conjunction with sphincter relaxation produces a dramatic decline in UES resting pressure for less than 1 second to permit bolus transport into the esophageal body (94). Muscles responsible for active UES opening include the thyrohyoid, geniohyoid, and other muscles involved in elevation and anterior displacement of the larynx (38). A brief UES aftercontraction after relaxation results from neural excitation and is readily apparent on high-resolution isocontour maps of intraluminal manometry (Fig. 36-4). Laryngeal movements accompanying swallowing also protect the airway as the bolus leaves the pharynx; these events are described in further detail in Chapter 35.

Esophageal Body The peristaltic sequence traversing the esophageal body is not a seamless event. Three distinct contraction segments separated by pressure troughs are identified by high-resolution manometry (see Fig. 36-4) (13,14,95). Similar segments are found in the opossum, and pressure ensemble-averaging techniques indicate that no other major subdivision of the peristaltic sequence is present (96). The segments within the esophageal body are distinct from UES and LES aftercontraction (13,14), and relative locations of their defining

landmarks in the human are shown in Table 36-1. The two smooth muscle segments are merged in 8% of subjects, and marked interswallow variation in the appearance of these two segments occurs within subjects (13,14,95). Long segments of the esophageal body (>10 cm in midsequence) are contracting simultaneously during normal peristalsis (97). This is appreciated by viewing axial movement of the pressure wave as demonstrated by high-resolution manometry (for a video demonstration, access the Washington University School of Medicine, Department of Medicine, Division of Gastroenterology Web site at: http://gastro.wustl.edu/axial_ trans.htm). As is shown in the pressure isocontour map in Figure 36-4, contraction amplitudes within the esophageal body vary markedly depending on the location of measurement. Peak amplitudes typically are greatest in the third segment (distal smooth muscle esophagus) and normally do not exceed 200 mm Hg (98). Average duration of contraction in the distal esophagus is less than 6 seconds (98), and propagation velocity varies through the esophageal body in bimodal fashion when data are extracted using modified high-resolution techniques (Fig. 36-5) (97). Propagation velocity along the smooth muscle region averages less than 5 cm/sec, and velocity greater than 6.25 cm/sec is associated with disrupted bolus transit (99). Despite an amplitude trough subdividing the smooth muscle region into two distal segments, velocity measurements indicate smooth propagation through the smooth muscle region, unlike the interruption observed across the first two segments. Striated Muscle Region Coordination of peristalsis through the striated muscle region occurs exclusively by central coordination of sequential

918 / CHAPTER 36 t1

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FIG. 36-3. High-resolution manometry in the upper esophageal sphincter (UES) with pressure recording sites spaced at 1-cm intervals. (A) Conventional pressure tracings from the individual sensors. The interpolated axial pressure wave from time t1 (A) is shown in B. The complete pressure ensemble with interpolation across the entire recording time (from A) displays the spatiotemporal pressure relations as an isocontour map in C. Pharyngeal contraction (1) merges with UES pressure after the bolus has entered the opened sphincter. Pure white space on the map represents subatmospheric pressure (2) that occurs briefly with UES opening, which lasts ∼0.8 second during passage of the bolus. (Decrease to subatmospheric pressure may be an artifact of water-perfused manometry; it is not seen with solid-state transducers.) UES aftercontraction (3) reestablishes the high-pressure zone, but a short delay in propagation is observed at the distal margin of the UES before the peristaltic wave begins in the cervical esophagus (4). (Modified from Williams and colleagues [11], by permission.)

vagal excitation, as programmed in the swallowing center (100–102). Afferent esophageal information can influence contraction in this region; whether connections from myenteric plexus neurons to the striated muscle motor end plates also modulate this activity remains unknown (103–105). The principal pressure trough dividing the first contraction segment from the remaining two in the esophageal body occurs at about 22% esophageal length (14). This approximates the location at which equal proportions of muscle

14

i

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FIG. 36-4. An isocontour map of esophageal peristalsis using high-resolution manometry and 36 solid-state circumferential sensors spaced at 1-cm intervals. Sequential contraction begins in the pharynx (a) and is coordinated with upper esophageal sphincter (UES) contraction after opening for bolus transport into the esophagus (b). Cephalad movement of the high-pressure zone precedes UES opening, reflecting elevation of laryngeal structures. A short pressure segment in the striated muscle region (c) is separate but extends from UES activity to the first pressure trough (d) that likely represents transition from striated to smooth muscle. The second peristaltic pressure segment (e) ends in a less pronounced trough (f, second trough) that separates it from the third peristaltic segment (g). Peristalsis through the smooth muscle esophagus also is separated from the lower esophageal sphincter (LES) by the third pressure trough (h), and peristalsis culminates in LES after contraction (j). LES relaxation begins shortly after the swallow and remains relaxed through the majority of peristalsis (i).

types are found in the circular muscle layer during transition from striated to smooth muscle in the esophageal body (1). Smooth Muscle Region Three potential levels of control have been identified for producing peristalsis through the smooth muscle region. This redundancy may have teleologic importance in sustaining peristaltic activity despite disease or failure of

MOTOR FUNCTION OF THE ESOPHAGUS / 919 TABLE 36-1. Location and amplitude of pressure troughs separating peristaltic pressure segments in the esophageal body Location of pressure trough Trough First (proximal body) Second (distal body) Third (body-LES junction)

Prevalence of observation (% of subjects) 100 92 92

Esophageal length (%)a 21.7 ± 1.3 64.0 ± 2.7 96.1 ± 0.9

Distance above LES (cm) 16.3 ± 0.5 7.8 ± 0.4 0.9 ± 0.2

Trough amplitude (mm Hg) 22.4 ± 3.3 50.5 ± 6.9 41.4 ± 4.0

Values are means ± standard error. aMeasured in direction of upper-to-lower sphincters. LES, lower esophageal sphincter. Modified from Clouse RE, Staiano A. Topography of normal and high-amplitude peristalsis. Am J Physiol Gastrointest Liver Physiol 1993;265:G1098–G1107.

individual mechanisms. The first level of control is sequential firing of efferent motor fibers in the CPG (102,106). The second level of control, which is thought to be more dominant, is an intramural neural mechanism that can be demonstrated with vagal stimulation or after intraluminal balloon distention (107–110). Besides the neural mechanisms, a myogenic explanation for a propagating contraction has been proposed (111–113). Because swallow-induced peristalsis is highly atropine sensitive in the human and is augmented by cholinergic stimulation, the role of intramural excitatory cholinergic neurons in smooth muscle contraction responsible for peristalsis is not questioned. However, the essential factors for sequential contraction remain controversial. The importance of intramural mechanisms is

FIG. 36-5. Propagation velocity of the peristaltic wave front as it traverses the esophageal body from distal to the upper sphincter to 100% of esophageal length using axial reconstructions of pressure data. The bimodal wave represents propagation through the proximal striated muscle and distal smooth muscle regions with deceleration near the lower esophageal sphincter. A break in velocity is not apparent in the smooth muscle region. (Reproduced from Clouse and colleagues [97], by permission.)

emphasized in the opossum, for example, wherein peristalsis can be produced with vagal stimulation when the nerve is transected and excluded from central sequential activity (107,108,110). Under normal circumstances in the human, it is best to presume that all three control mechanisms integrate to result in normal peristaltic activity. The intramural neural control for peristalsis results from a blending of two types of progressive contraction mechanisms, one related to activation of the excitatory cholinergic neurons and the other related to activation of NANC inhibitory neurons. Both can be demonstrated in the opossum and cat by experimental vagal stimulation (107,108,110). The “on” contraction is atropine sensitive and has a propagation velocity that resembles that of swallow-induced peristalsis. The “off ” contraction is atropine resistant and has a more rapid propagation velocity. The former is attributed to cholinergic excitation, whereas the latter results from muscle depolarization and contraction after an initial hyperpolarization by NANC inhibitory neurons. Voltage-dependent Ca2+ channels are involved in contraction, K+ channels participate in maintaining membrane resting potential, and Cl−, as well as K+, channels may play a role in hyperpolarization (38). Whereas the circuitry responsible for propagation of the peristaltic contraction is unknown, regional differences in muscle and neural properties producing progressive delays of the contraction along the esophagus is the presumed mechanism—a mechanism that probably relates to the experimentally induced sequential contractions. Because the excitatory cholinergic influence is most prominent in the proximal smooth muscle region, whereas the inhibitory influence is most evident distally, the combined effect of these neural gradients directs the velocity and direction of propagation (32). Transition of neural influence occurs approximately halfway through the smooth muscle segment in the opossum at a location coinciding with the second-amplitude trough detected by high-resolution manometry in these animals (97,109). Thus, the two smooth muscle segments shown in Figure 36-4 may reflect this intramural peristaltic mechanism. As would be predicted, contraction amplitudes are more atropine sensitive proximally and decrease with

920 / CHAPTER 36

Striated muscle

NANC blockade distally, near the LES (32). Blockade of either mechanism reduces the blended effect and accelerates propagation velocity, with cholinergic blockade delaying its onset and NANC blockade leading to a rapidly occurring wave with high propagation velocity (Fig. 36-6). It is possible that high-resolution manometry can identify an unblending of these effects and separation of the waveform under some pathologic conditions (114). A set of programmed neurons in the CPG likely participates in directing the progressive delays in excitatory and inhibitory intramural neurons, although this has not been fully demonstrated (102,106). In several animal models, vagal fibers discharge with a timing corresponding to peristaltic contraction in the smooth muscle region (101,102). However, transection of the smooth muscle region markedly affects distal peristaltic progression, emphasizing importance of the integrity of the intramural mechanism (115). Regardless of its uncertain importance in sequencing peristalsis, the ability of the central nervous system to modulate peristalsis is well established. Afferent information regarding the size and nature of an intraluminal bolus affects both amplitude and velocity of contraction through central pathways (101,116,117). Thus, one important aspect of the central control mechanisms appears to be their modulation of the intramural nerves involved in local control of peristaltic activity. Because differences in intramural neural elements themselves have not provided a conspicuous explanation for the gradient in neural influence seen through the smooth muscle region, regional differences in muscle

UES

properties, such as ion channel diversity, may provide an alternative explanation for these gradients (32,118,119). In summary, esophageal peristalsis begins in the striated muscle region of the esophageal body as a progression of centrally programmed muscle activity in the pharynx and UES. Coordination of striated muscle contraction is totally under central control and is established by the swallowing center. Continuation of peristalsis to the smooth muscle esophagus represents significant transition in control to an integrated combination of central and intramural mechanisms. In the smooth muscle region, a balance exists between excitatory and NANC inhibitory intramural neurons, the latter having greater influence on the direction and velocity of the propagating wave. Central vagal input has timing features that could serve both early induction of inhibition and later excitation of the peristaltic contraction and modulates the contraction response (101,102). Although also suspected to be important, myogenic mechanisms participating together with central and intramural neural factors have an uncertain role in the regulation of peristaltic contractions. Longitudinal Muscle Sequential motor activity also occurs in esophageal longitudinal muscles that are more dependent on central control (120). Although longitudinal muscle contraction has been thought to precede circular muscle contraction at the same level (80,121–123), studies using high-frequency intraluminal

Block cholinergic Block NANC Normal (ACh) excitation (NO) inhibition peristalsis (NANC NO dominant) (ACh dominant) Transmitter influence

Smooth muscle

NANC

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FIG. 36-6. Schematic drawing of cholinergic (ACh, O) and noncholinergic (NANC, •) influences in the smooth muscle esophagus and the potential interplay of two intramural neural mechanisms in the production of peristalsis. Cholinergic influence is most prominent proximally and decreases distally; the reverse is true for the noncholinergic influence. The effects of cholinergic or NANC blockade are shown schematically. LES, lower esophageal sphincter; NANC, nonadrenergic, noncholinergic; NO, nitric oxide; UES, upper esophageal sphincter. (Reproduced from Clouse and Diamant [31], by permission.)

MOTOR FUNCTION OF THE ESOPHAGUS / 921 Proximal segment

12

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FIG. 36-7. Shortening of the esophagus in one healthy subject as measured by cinefluoroscopic monitoring of radiopaque mucosal clips placed at and 10 cm above the esophagogastric junction. A fixed proximal esophageal reference point was determined at the level of the true vocal cords for proximal segment and total esophageal measurements. Extensions represent one standard deviation from serial swallows. The proximal esophagus shortens, and then lengthens with maximal distal segment activity. (Modified from Edmundowicz and Clouse [79], by permission.)

ultrasound suggest that contraction of the two muscle layers is synchronous (123,124). Likewise, contraction strength in the two layers is correlated (123,125). The coordination of contraction between layers augments circular muscle contractile force and reduces stress on the esophageal wall (126,127). As a result of longitudinal muscle contraction, the esophagus shortens by an average of 7% or approximately 1.5 cm with each swallow (Fig. 36-7) (80). This muscle layer is more sensitive than circular muscle to acid injury, the hyperresponsiveness leading to enhanced shortening in this setting (128). In the distal esophagus, cholinergic nerves are responsible for contraction, activating muscarinic receptors on longitudinal smooth muscle.

Lower Esophageal Sphincter Like the UES, the LES is closed tonically at rest. Average intrasphincteric pressures vary with the measurement method and timing of the respiratory cycle (89). End-expiratory pressures reflecting intrinsic sphincter pressure are lowest but exceed 5 mg/Hg (89); resting pressures are greater at the level of the crural diaphragm and increase with inspiration (129,130). As with the UES, both radial and axial asymmetry can be demonstrated with high-definition manometric reconstructions (Fig. 36-8), although the degree of global asymmetry is somewhat less than that observed with the UES. LES pressure decreases within 1.5 to 2.5 seconds of

FIG. 36-8. Vector volume representations of the lower esophageal sphincter show the radial and axial asymmetry of sphincter pressure at rest. Pressures are greatest in the left posterior orientation. For the map on the left, the axis is atmospheric pressure, and greater sphincter pressures are plotted in an outward direction. The map on the right is a negative image of the conventional map on the left with greater pressures oriented inward from a cylinder set at atmospheric pressure. (Reproduced from Stein and colleagues [83], by permission.)

922 / CHAPTER 36 the swallow, remains decreased until peristalsis reaches the LES, and increases with an aftercontraction that is separated from the peristaltic complex by a pressure trough (see Fig. 36-4). Both central factors (via the vagus nerve) and intramural neural mechanisms in the smooth muscle esophageal body can influence LES motor function (62,63). Efferent vagal fibers innervating the LES enter the esophagus proximally to the sphincter, as surgical vagotomy separating the vagus nerves from up to 9 cm of distal esophagus has no effect on LES tone, relaxation, or responsiveness to cholinergic stimulation (131,132). LES resting tone results from myogenic properties of the clasp and sling fibers in conjunction with active tonic neural excitation. This process is modulated significantly by other neural and hormonal factors. Clasp fibers have dominant myogenic tone and are less responsive to cholinergic stimulation, whereas sling fibers have less resting tone but are very responsive to cholinergic stimulation (133,134). Spontaneous tone in LES circular muscle (predominantly clasp fibers) is produced by ongoing influx of extracellular calcium through plasmalemmal calcium channels, specifically the ICa,L channel (135–137). In contrast, resting tone in the sling fibers, as well as cholinergic-mediated contraction of the sling, use a non-ICa,L extracellular calcium source (135,138). Some of LES tone results from acetylcholine release from excitatory neurons, in part resulting from tonic vagal firing (139). The greater pressure in the left lateral posterior aspect of the LES (see Fig. 36-8) is most sensitive

to atropine and reflects the relevance of cholinergic stimulation to resting tone in the sling fibers (84,133,140). Blockade of NO synthase has little effect on resting tone, despite the importance of this neurotransmitter in producing LES relaxation (141–145). Endogenous myogenic NO, however, may have an inhibitory regulatory tonal role, at least in circular muscle (predominantly clasp fibers) (143). Therefore, a balance between neurogenic and myogenic factors, excitatory and inhibitory influences, and clasp and sling muscle contributions forms the resultant LES tone. Many factors affect LES pressure, including composition of a meal, intraesophageal acidification, and numerous hormones (20,38). LES pressure fluctuates with the migrating motor complex and increases with phase III motor activity in the stomach (146). Pressure increases also are seen with increases in intraabdominal pressure; this results from an excitatory cholinergic reflex (147). Phosphodiesterase type-5 inhibitors reduce degradation of NO and decrease LES pressure, presumably by affecting clasp fibers (148). Likewise, calcium-channel blocking agents reduce the myogenic component of LES tone and decrease LES pressure predominantly in clasp fibers (149). In contrast, botulinum toxin injection, a maneuver that impairs acetylcholine release from cholinergic nerve terminals, would reduce LES tone predominantly by affecting sling fibers. LES relaxation occurs with virtually all swallows, even when a peristaltic response fails to occur and allows forceful opening of the sphincter by a bolus. Both active inhibition

FIG. 36-9. Deglutitive inhibition as demonstrated by high-resolution manometry of repeated, closely spaced swallows. (Left) A second swallow 3.8 seconds after the first inhibits peristalsis as it begins to enter the smooth muscle segment. (Right) A second swallow 6.25 seconds after the first inhibits further peristaltic progression in the distal smooth muscle region. The subsequent peristalsis from the second swallow is of less contraction strength than seen in the left panel, suggesting a degree of contractile refractoriness. The lower esophageal sphincter (LES) remains relaxed through both swallows (brackets). Diaphragmatic pressure effect is apparent during LES relaxation, but crural activity and its prominent indentation on the LES pressure profile is inhibited.

MOTOR FUNCTION OF THE ESOPHAGUS / 923 by intramural NANC inhibitory neurons and cessation of tonic neural excitation are responsible (39,139). Relaxation is directed by the CPG in the swallowing center and also can occur through intramural pathways within the smooth muscle esophagus (62,63). NO (or a similar nitroso compound) is the principal neurotransmitter mediating swallow-induced inhibition (although other inhibitory neurotransmitters are present), and circular muscle (predominantly clasp fibers) is highly innervated with inhibitory motor neurons, whereas the sling is not (150). NO also is the local mediator of the TLESR. A TLESR occurs in the absence of a swallow and is an important mechanism responsible for gastroesophageal reflux events, both in healthy subjects and in patients with gastroesophageal reflux disease. A change in rate of TLESR is not as important in patients with gastroesophageal reflux disease as the increased likelihood of refluxing acid into the esophagus during the events (151,152). The TLESR is a vagally mediated reflex event that is triggered by intraesophageal or intragastric stimuli, or both (153,154). Many factors can influence the rate of TLESR; the rate is reduced after fundoplication, both at rest and with gastric distention (155). Crural diaphragmatic activity is inhibited during both swallow-induced LES relaxation and TLESR, also through central pathways (156).

Deglutitive Inhibition and Refractoriness During deglutition, the bolus forcefully enters the esophageal body through the opened UES. In upright subjects, the bolus will descend by gravity and may or may not require peristalsis for assistance in transport. This depends on the consistency of the bolus: liquids travel with sufficient rapidity in the upright position to impact on the closed LES; solid boluses progress through the esophagus in the upright position assisted by the peristaltic wave. Because completion of peristalsis may not occur for 8 to 10 seconds after the swallow, a mechanism is required to inhibit contractile activity during rapid, repeated swallowing (157,158). This deglutitive inhibition primarily is a function of the swallowing center. When the second swallow is closely spaced to the first and peristaltic activity still is occurring in the striated muscle segment, inhibition of further contraction results from cessation of excitatory discharges from the CPG. In addition to cessation of the excitatory discharges, vagal activation of NANC inhibitory neurons likely contributes to interruption of peristalsis in the smooth muscle region (Fig. 36-9), an effect similar to the NANC inhibition that participates in peristaltic sequencing and that can be demonstrated manometrically with creation of an artificial high-pressure zone using intraluminal balloon distention (39,159–161). The esophagus remains quiescent and the LES remains open with a series of closely spaced swallows; normal peristalsis resumes with the final swallow in the sequence. Longitudinal muscle also participates in deglutitive inhibition, and the degree of inhibition between both muscle layers is well correlated (162). Contractile refractoriness is

seen variably when a second peristaltic sequence is attempted shortly after a peristaltic contraction. Contraction amplitude of the second response can be reduced dramatically and propagation velocity altered, emphasizing the importance of an appropriate delay between swallows when studying the normal motor response (20–30 seconds in the human) (158,159).

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Neurophysiologic Mechanisms of Gastric Reservoir Function Jan Tack Functional Anatomy, 927 Different Phases of Gastric Motility, 927 Interdigestive Motility, 927 Gastric Reservoir Function, 928 Gastric Emptying, 928 Measurement of Gastric Reservoir Function, 928 The Barostat, 928 Volumetric Techniques to Study Proximal Gastric Function, 928 Caloric Drink Test to Estimate Gastric Accommodation, 929

Control of the Accommodation Reflex, 929 Site of Triggering of the Accommodation Reflex, 929 Role of Nitrergic Neurons, 929 Involvement of 5-Hydroxytryptamine, 930 Type of 5-Hydroxytryptamine Receptor Involved, 930 Pathophysiologic Role of Impaired Accommodation, 931 Conclusion, 931 References, 932

FUNCTIONAL ANATOMY

slow waves, which determine the timing of gastric contractions. See Chapter 20 for a more detailed discussion.

The stomach is a hollow organ in which different anatomic parts such as the cardia, the fundus, the gastric body, the antrum, and the pylorus can be distinguished. Functionally, the stomach consists of a proximal part and a distal part, with a sphincter muscle at both ends. The proximal stomach consists of the fundus and part of the gastric corpus, and its main function is to provide a reservoir to the meal. Smooth muscle cells in the proximal stomach do not display electrical oscillatory activity, and the fundus is characterized by a tonic contractile activity. The distal part of the stomach consists of the gastric antrum and the distal half of the gastric corpus, and its main function is to generate contractions that will mix and grind the food and empty the stomach. Smooth muscle cells in the distal stomach are characterized by the presence of rhythmic electrical activity, so-called

DIFFERENT PHASES OF GASTRIC MOTILITY Interdigestive Motility In the fasting state, the proximal gastrointestinal tract displays the migrating motor complex (MMC), a cyclical motor pattern of the stomach and the small intestine (1). The MMC consists of three different phases. Phase I is characterized by the absence of contractile activity, phase II displays irregular contractile activity, and phase III is a phase of intense contractions at maximal frequency, which is again followed by phase I. The average MMC cycle lasts between 90 and 120 minutes, but there is a large interindividual and intraindividual variability. The MMC originates from the stomach or from the proximal small intestine and migrates distally at a speed of 1 to 4 cm/min. In the stomach, phase III evacuates indigestible particles to the small bowel. Small intestinal phase III serves a similar goal and also effectively clears bacteria from the small bowel.

J. Tack: Department of Gastroenterology, University Hospitals Leuven, Center for Gastroenterological Research, University of Leuven, Leuven, Belgium. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

927

928 / CHAPTER 37

Fast

Accommo

Empt

FIG. 37-1. Phases of normal gastric motility. The accommodation reflex consists of a relaxation of the proximal stomach, providing the meal with a reservoir and enabling a volume increase without an increase in pressure. Subsequently, the meal is emptied from the stomach at a rate that matches the absorptive capacity of the duodenum.

Gastric Reservoir Function When food is ingested, the MMC is suppressed and upper gastrointestinal motility switches to the fed or postprandial pattern. During fasting, muscle fibers of the proximal stomach maintain a vagally mediated tonic contractile activity, which generates gastric fundus tone. During and after ingestion of a meal, a relaxation of the proximal stomach occurs, which provides the meal with a reservoir and enables a gastric volume increase without an increase in pressure (2). This also allows the stomach to retain food and to allow passage to the duodenum at a rate that matches the duodenal absorptive capacity (Fig. 37-1). Two phases can be distinguished in the gastric reservoir function, and both are vagally mediated. Immediately after deglutition, the lower esophageal sphincter and the proximal stomach relax to allow passage and storage of the food bolus. This rapid inhibitory phenomenon of gastric motility is called receptive relaxation. During gastric filling by food ingestion, a long-lasting relaxation of the proximal stomach occurs that is called adaptive relaxation or gastric accommodation. The role of tone in the gastric antrum and its ability to function as a reservoir have not been established, and many consider the antrum to act predominantly as a muscular pump that grinds the food and promotes evacuation. Observations in humans suggest that the antrum also is involved in the gastric accommodation reflex in humans (3).

Gastric Emptying In the postprandial state, circular peristaltic waves move from the midcorpus to the pylorus. In the antrum, these contractions will grind the food and mix it with gastric juices through retropulsion, to finally lead to propulsion and evacuation. Increasing tone in the proximal stomach contributes to the flow through the pylorus by contributing to the gastroduodenal pressure gradient. The flow of chyme from the stomach to the duodenum is of a pulsatile nature and is determined by the strength of

antral contraction, the degree of pyloric relaxation, and duodenal resistance. The pylorus also is involved in regulation of flow to the small intestine and helps to discriminate fluid, viscous, and solid gastric contents. When gastric content is fluid, antral contractions occlude the lumen and liquid is easily transferred to the duodenum. In the case of a more viscous or solid gastric content, contractions are not lumen obliterating, and retropulsion to the proximal stomach serves to mix and grind gastric contents. Because the storage capacity of the duodenum is limited compared with the intragastric volume of chime, duodenal contractions serve to delay further gastric emptying when the duodenum is not empty.

MEASUREMENT OF GASTRIC RESERVOIR FUNCTION The Barostat The gastric barostat consists of a computer-driven air pump connected to an oversized balloon, which can be positioned in the proximal or distal stomach. The barostat maintains a fixed pressure level within the stomach by adapting the intraballoon volume (4). Measurement of volume changes at a constant low pressure allows quantification of changes in gastric tone (Fig. 37-2), and measurement of perception at various distending pressure levels allows quantification of sensitivity to gastric distention. Measurements of volume changes at a constant distending pressure traditionally are used to quantify gastric accommodation or to study factors involved in the control of gastric accommodation.

Volumetric Techniques to Study Proximal Gastric Function Because gastric barostat studies are invasive in nature, several other approaches to assess proximal gastric motor function have been developed. Ultrasound studies of the proximal stomach, as mentioned earlier, allow noninvasive

NEUROPHYSIOLOGIC MECHANISMS OF GASTRIC RESERVOIR FUNCTION / 929 Strain gauge

Strain gauge Pressure selector

Ct P

Ct P

FIG. 37-2. Principle of the gastric barostat. The gastric barostat consists of an oversized bag and a computerized pump. The pressure within the bag is set at a fixed level. Changes in pressure during gastric relaxation or contraction are neutralized by decreasing or increasing the volume of air in the bag.

monitoring of meal-induced relaxation of the proximal stomach (5), but they appear labor intensive and are likely to be observer dependent. A systematic analysis of scintigraphic images in the early postprandial phase has been claimed to identify a group of patients with impaired accommodation who have early satiety (6), but confirmatory studies are lacking, and it appears less likely that this approach is suitable to quantify differences in accommodation within the normal range. Another approach has been the imaging of the gastric wall to measure gastric volume rather than the content of the stomach. An intravenous injection of 99mTc-pertechnetate results in uptake of the isotope by the gastric mucosal cells, and after single-photon emission computed tomography, the slices of the stomach can be reconstructed with specialized software. After the ingestion of a meal, it is possible to evaluate the accommodation of the entire stomach, as well as to evaluate the accommodation of the proximal and distal stomach separately (7). Magnetic resonance imaging also allows measurement of postprandial volumes in different regions of the stomach (8). However, there are indications that the assessment of postprandial gastric volumes by 99mTc single-photon emission computed tomography imaging is strongly influenced by the volume of the meal and might underestimate changes in tone in a nondistended stomach (9); the same limitations may apply to magnetic resonance imaging techniques.

Caloric Drink Test to Estimate Gastric Accommodation A slow caloric drinking test has been proposed as a noninvasive method to quantify meal-induced satiety (10). A peristaltic pump fills 1 of 2 beakers at a fixed rate of 15 ml/min with a liquid meal. The subjects are requested to maintain intake at the filling rate, thereby alternating the beakers as they are filled and emptied. At 5-minute intervals, they score their satiety using a graphic rating scale that combines verbal descriptors on a scale graded 0 to 5 (1 = threshold;

5 = maximum satiety). Participants are instructed to cease the meal intake when a score of 5 is reached. The end point of the test is the amount of calories ingested until the occurrence of maximum satiety (score of 5). The slow caloric drink test was compared in 25 control subjects and 37 patients with unexplained dyspeptic symptoms. All subjects completed a dyspepsia questionnaire and underwent gastric emptying and gastric barostat studies. In patients, satiety scores were greater and maximum satiety occurred at lower calories. In multivariate analysis, the amount of calories was significantly correlated to accommodation, but not to gastric emptying or sensitivity. The slow caloric drinking test was found to have good sensitivity and specificity to predict impaired accommodation. In healthy volunteers, ranging caloric density from 1.5 to 2.0 kcal/ml, we established that ingested volume rather than caloric load determined maximum satiety. These data show that a slow caloric drinking test can be used to evaluate accommodation and early satiety. It may provide a noninvasive method to predict impaired accommodation and to quantify pharmacologic influences on accommodation. Further validation of this easy noninvasive test is necessary before widespread clinical application can be advocated.

CONTROL OF THE ACCOMMODATION REFLEX Site of Triggering of the Accommodation Reflex Several studies have shown that duodenal stimulation, including balloon distention, lipid infusion, or acid infusion, is able to induce relaxation of the proximal stomach (11–15). This has been interpreted to reflect part of the accommodation reflex, and studies using duodenal lipid infusion have been used to study the control of the gastric accommodation reflex. Lipid digestion and subsequent release of CCK and activation of CCKB receptors are key factors in these events (12–14). However, during oral intake of a meal, triggering of the accommodation reflex occurs from several sites including the oropharynx and the stomach (16). Studies using an orally ingested meal could not confirm the importance of lipid digestion and CCK release (14,17). These data suggest that the main factors involved in triggering of the gastric accommodation reflex do not involve duodenal feedback, and pathways that become activated during duodenal lipid infusion are less important in the control of gastric relaxation after an orally ingested meal.

Role of Nitrergic Neurons Studies in animals have demonstrated that the gastric accommodation reflex is mediated via a vagovagal reflex pathway that activates nonadrenergic, noncholinergic neurons in the gastric wall (18,19). Several lines of evidence suggest a role for nitric oxide and vasoactive intestinal polypeptide

930 / CHAPTER 37 as inhibitory neurotransmitters mediating gastric relaxation (19–24). NG-monomethyl-L-arginine (L-NMMA) is an inhibitor of nitric oxide synthase, suitable for use in humans. Studies using this agent have been able to demonstrate the involvement of nitric oxide in the control of interdigestive motility and in mediating transient lower esophageal sphincter relaxations in humans (25,26). These properties were used to test the hypothesis that nitric oxide is involved in the control of postprandial gastric tone in humans (27). In a study in healthy volunteers, pretreatment with L-NMMA dose dependently decreased the gastric accommodation to a meal, thereby establishing that the gastric accommodation reflex in humans involves activation of nitrergic neurons.

5-HT in the ENS are enhanced shortly after pretreatment with a selective serotonin reuptake inhibitor. We used this property to investigate the role of 5-HT in the control of gastric motility in humans. In a placebo-controlled gastric barostat in healthy volunteers, short-term pretreatment with the selective serotonin reuptake inhibitor paroxetine, 20 mg/day, significantly enhanced the amplitude of the meal-induced fundus relaxation (30). This observation suggests involvement of 5-HT in the gastric accommodation reflex in humans as well. The 5-HT receptor involved and its localization cannot be determined from this study. Moreover, selective serotonin reuptake inhibitors may act on neurons that are located centrally, as well as peripherally.

Involvement of 5-Hydroxytryptamine

Type of 5-Hydroxytryptamine Receptor Involved

In the mouse and the guinea pig, involvement of 5-hydroxytryptamine (5-HT) receptors on intrinsic neurons in the vagally mediated gastric relaxation has been demonstrated (28). More recently, it was demonstrated that 5-HT– induced relaxations of the guinea pig stomach are mediated via the release of nitric oxide through activation of a 5-HT1–like receptor (23). The lack of suitable selective 5-HT ligands for in vivo use in humans hampers the investigation of this issue. However, the role of 5-HT in the control of gastric motility can be investigated in an alternative way. Similar to the central nervous system, a serotonin reuptake system is present in the enteric nervous system (ENS) (29). The serotonin reuptake system in the ENS is also inhibited by selective serotonin reuptake inhibitors (29). Therefore, 5-HT reuptake inhibitors may increase the availability of synaptically released 5-HT, not only in the central nervous system, but also at the level of the ENS. Physiologic processes involving the release of

Taken together, these studies suggest that release of 5-HT, probably at the level of the ENS, with subsequent activation of nitrergic motor neurons is involved in the control of the accommodation reflex in humans (Fig. 37-3). Animal studies have shown that intrinsic neurons in the stomach can be activated through a 5-HT1–like receptor (23). In the guinea-pig, a 5-HT1P receptor was shown to be present on nitrergic neurons in the myenteric plexus of the stomach, where it mediates a prolonged depolarization in response to the application of 5-HT (31,32). Using intracellular electrical recordings, we demonstrated that sumatriptan, a 5-HT1 receptor agonist that is used in the treatment of migraine in humans, is an agonist at 5-HT1P receptors on nitrergic myenteric neurons in the stomach (33). We therefore investigated whether sumatriptan is able to induce a relaxation of the proximal stomach in humans, and whether this relaxation is mediated through the activation of nitrergic neurons.

Vagal afferent G.I. tract CN ACh 5-HT?

S +

Vagal efferent

Interneuron

NO VIP

+

− −

Inhibitory motor neuron

−

Nicotinic receptor 5-HT receptor

FIG. 37-3. Neural control pathway of the accommodation reflex, based on animal studies. The accommodation reflex is mediated via a vagovagal reflex pathway that activates inhibitory motor neurons in the gastric wall to release nitric oxide (NO) and vasoactive intestinal polypeptide (VIP). Involvement of a 5-hydroxytryptamine (5-HT) receptor on intrinsic inhibitory neurons has been demonstrated. ACh, acetylcholine; CNS, central nervous system; GI, gastrointestinal.

NEUROPHYSIOLOGIC MECHANISMS OF GASTRIC RESERVOIR FUNCTION / 931 Using a gastric barostat, we were able to demonstrate that the administration of sumatriptan induces a relaxation of the gastric fundus in humans (34). Pretreatment with L-NMMA dose dependently decreased the gastric relaxatory response to sumatriptan, thereby establishing that the drug relaxes the proximal stomach in humans through a nitrergic pathway. Although this observation might suggest involvement of a 5-HT1P receptor, the nature of the 5-HT receptor involved remains unclear. Selective agonists or antagonists are not available for human studies. Animal studies have generated conflicting data. Studies in guinea pigs suggest that sumatriptan was acting on a 5-HT1A receptor (35), but studies in cats argue against a 5-HT1A receptor and suggest involvement of a 5-HT1F–type receptor (36,37), whereas studies in the dog suggest involvement of a 5-HT1B/D–type receptor (38). Moreover, additional studies in humans demonstrated that 5-HT1A agonists or 5-HT4 agonists are also able to enhance gastric accommodation to a meal, supporting possible involvement of one or more of these peripheral 5-HT receptors (39– 41). Although one study reported increased gastric volumes after administration of a 5-HT3 receptor agonist (42), 5-HT3 receptor antagonism did not alter gastric accommodation to a meal (43), suggesting that this type of receptor is not involved in the control of the accommodation reflex.

PATHOPHYSIOLOGIC ROLE OF IMPAIRED ACCOMMODATION Scintigraphic and ultrasonographic studies have demonstrated an abnormal intragastric distribution of food in patients with functional dyspepsia, with preferential accumulation in the distal stomach (44–46). This finding suggests defective postprandial accommodation of the proximal stomach. Consistently, gastric barostat studies have shown reduced proximal gastric relaxation in response to a meal in patients with functional dyspepsia (47,48). In view of the role of gastric accommodation in providing a reservoir during meal intake, we hypothesized that absence of normal accommodation might lead to early satiety. Using a gastric barostat in 40 consecutive dyspeptic patients, we showed that impaired gastric accommodation was present in 40% of these patients, and this impairment was independently associated with early satiety (48). The relation between impaired gastric accommodation and early satiety is also apparent from the correlation between the amplitude of the meal-induced relaxation and the amount of calories ingested at maximum satiety in patients with early satiety (48). These data suggest that impaired accommodation is the mechanism underlying the symptom of early satiety. According to this hypothesis, causing impaired accommodation in healthy subjects should induce symptoms of early satiety. Previously, we reported that gastric accommodation is inhibited by pretreatment with L-NMMA (27). Administration of the motilin agonist erythromycin was shown to cause a contraction of the proximal stomach in the postprandial

phase, thereby reducing the accommodation to a meal (49). We used these properties to investigate the hypotheses that inhibition of accommodation or administration of a funduscontracting drug might enhance meal-induced satiety in humans. In a double-blind, randomized, crossover study in healthy subjects, we demonstrated that pretreatment with erythromycin at the start of the meal decreased the amount of food ingested and significantly increased the average satiety scores for the same amount of kilocalories ingested (10). Similarly, using motilin in a double-blind, randomized, crossover study in healthy subjects, we confirmed again that inhibition of gastric accommodation, induced by motilin, was associated with increased satiety scores for the same amount of calories ingested (50). In another controlled study in healthy subjects, we confirmed that the NO synthase inhibitor L-NMMA significantly decreased the amount of food ingested to reach maximum satiety and significantly increased the average satiety scores for the same amount of kilocalories ingested (21). Similarly, pretreatment with sumatriptan and cisapride, which were shown to enhance gastric accommodation, decreased satiety scores for the same amount of calories ingested (10). These observations support the hypothesis that impaired accommodation is a mechanism underlying the symptom of early satiety. The mechanism underlying impaired postprandial relaxation of the proximal stomach is unknown. Several possible pathways can be involved. Theoretically, impaired relaxation can result from a disorder at the level of the sensory apparatus, the vagal reflex pathway, or the intrinsic inhibitory innervation. We demonstrated that patients with presumed postinfectious dyspepsia (acute-onset dyspepsia accompanied by infectious symptoms) have a high prevalence of impaired accommodation. We also observed that these patients had an impaired relaxatory response to sumatriptan, whereas the stomach relaxed adequately in response to administration of the nitric oxide donor amylnitrite (51). This finding suggests a defect at the level of gastric intrinsic nitrergic neurons.

CONCLUSION Gastric accommodation provides the meal with a reservoir and enables a gastric volume increase without an increase in pressure. Gastric accommodation in humans is controlled by a vagal reflex pathway that involves the release of serotonin and the activation of a nitrergic motor neuron. This is mimicked by administration of the 5-HT1P receptor agonist sumatriptan, which causes relaxation of the proximal stomach in humans through a nitrergic pathway. Almost half of patients with functional dyspepsia have an impaired accommodation reflex, and this is associated with early satiety and weight loss. Drug-induced inhibition of the accommodation reflex is able to induce early satiety in healthy subjects, thereby supporting the hypothesis that impaired accommodation is the mechanism underlying the symptom of early satiety. In a subset of patients, impaired

932 / CHAPTER 37 accommodation appears to occur as a prolonged sequel of an acute gastrointestinal infection. In these patients, the defect appears to occur at the level of gastric intrinsic nitrergic neurons.

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22. Boeckxstaens GE, Pelckmans PA, Bogers JJ, Bult H, De Man JG, Oosterbosch L, Herman AG, Van Maercke YM. Release of nitric oxide upon stimulation of nonadrenergic noncholinergic nerves in the rat gastric fundus. J Pharmacol Exp Ther 1991;256:441–447. 23. Meulemans AL, Helsen LF, Schuurkes JAJ. The role of nitric oxide (NO) in 5-HT induced relaxations of the guinea-pig stomach. Naunyn Schmiedebergs Arch Pharmacol 1993;384:424–430. 24. Meulemans AL, Eelen JGM, Schuurkes JAJ. NO mediates gastric relaxation after brief vagal stimulation in anesthetized dogs. Am J Physiol 1995;269:G255–G261. 25. Hirsch DP, Holloway RH, Tytgat GNJ, Boeckxstaens GEE. Involvement of nitric oxide in human transient lower esophageal sphincter relaxations and esophageal primary peristalsis. Gastroenterology 1998;115: 1374–1380. 26. Russo A, Fraser R, Adachi K, Horowitz M, Boeckxstaens G. Evidence that nitric oxide mechanisms regulate small intestinal motility in humans. Gut 1999;44:72–76. 27. Tack J, Demedts I, Meulemans A, Schuurkes J, Janssens J. Role of nitric oxide in the accommodation reflex and in meal-induced satiety in man. Gut 2002;51:219–224. 28. Bülbring E, Gershon MD. 5-Hydroxytryptamine participation in the vagal inhibitory innervation of the stomach. J Physiol 1967;192: 823–846. 29. Gershon MD, Jonakait GM. Uptake and release of 5-hydroxytryptamine by enteric 5-hydroxytryptaminergic neurons: effects of fluoxetine (Lilley 110140) and chlorimipramine. Br J Pharmacol 1979;66:7–9. 30. Tack J, Broekaert D, Coulie B, Fischler B, Janssens J. Influence of the selective serotonin reuptake inhibitor paroxetine on gastric sensorimotor function in man. Aliment Pharmacol Ther 2003;17:603–608. 31. Tack JF, Janssens J, Vantrappen G, Wood JD. Actions of 5-hydroxytryptamine on myenteric neurones in the gastric antrum of the guinea-pig. Am J Physiol 1992;263:G838–G846. 32. Michel K, Sann H, Schaaf C, Schemann M. Subpopulations of gastric myenteric neurons are differentially activated via distinct serotonin receptors: projection, neurochemical coding, and functional implications. J Neurosci 1997;17:8009–8017. 33. Tack J, Vanden Berghe P, Coulie B, Janssens J. Sumatriptan is an agonist at 5-HT1P receptors on myenteric neurones in the guinea pig gastric antrum. (Submitted). 34. Tack J, Coulie B, Wilmer A, Andrioli A, Janssens J. Effect of sumatriptan on gastric fundus tone and on the perception of gastric distension in man. Gut 2000;46:468–473. 35. Meulemans AL, Helsen LF, de Ridder WJE, Schuurkes JAJ. Effects of sumatriptan on the guinea-pig isolated stomach. Neurogastroenterol Motil 1996;8:183 (Abstract). 36. Coulie B, Tack J, Sifrim D, Andrioli A, Janssens J. Role of nitric oxide in fasting gastric fundus tone and in 5-HT1 receptor-mediated relaxation of gastric fundus. Am J Physiol 1999;276(2 pt 1):G373–G377. 37. Janssen P, Tack J, Sifrim D, Meulemans AL, Lefebvre RA. Influence of 5-HT1 receptor agonists on feline stomach relaxation. Eur J Pharmacol 2004;492(2-3):259–267. 38. De Ponti F, Crema F, Moro E, Nardelli G, Frigo G, Crema A. Role of 5-HT1B/D receptors in canine gastric accommodation: effect of sumatriptan and 5-HT1B/D receptor antagonists. Am J Physiol Gastrointest Liver Physiol 2003;285:G96–G104. 39. Tack J, Piessevaux H, Coulie B, Fischler B, De Gucht V, Janssens J. A placebo-controlled trial of buspirone, a fundus-relaxing drug in functional dyspepsia: effect on symptoms and gastric sensory and motor function. Gastroenterology 1999;116:G1423. 40. Tack J, Broeckaert D, Coulie B, Janssens J. Influence of cisapride on gastric tone and on the perception of gastric distension. Aliment Pharmacol Ther 1998;12:761–766. 41. Tack J, Vos R, Janssens J, Salter J, Jauffret S, Vandeplassche G. Influence of tegaserod on proximal gastric tone and on the perception of gastric distention. Aliment Pharmacol Ther 2003;18:1–7. 42. Coleman NS, Marciani L, Blackshaw E, Wright J, Parker M, Yano T, Yamazaki S, Chan PQ, Wilde K, Gowland PA, Perkins AC, Spiller RC. Effect of a novel 5-HT3 receptor agonist MKC-733 on upper gastrointestinal motility in humans. Aliment Pharmacol Ther 2003;18: 1039–1048. 43. Tack J, Wilmer A, Coulie B, Janssens J. Influence of ondansetron on gastric tone and on the perception of gastric distention. (Submitted). 44. Scott AM, Kellow JE, Shuter B, Cowan H, Corbett A-M, Riley JW, Lunzer MR, Eckstein RP, Holschl R, Lam S-K, Jones MP. Intragastric distribution and gastric emptying of solids and liquids in functional dyspepsia. Dig Dis Sci 1993;38:2247–2254.

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49. Bruley des Varannes S, Parys V, Ropert A, Chayvialle JA, Roze C, Galmiche P. Erythromycin enhances fasting and postprandial proximal gastric tone in humans. Gastroenterology 1995; 109:32–39. 50. Cuomo R, Tack J, Van Daele P, Coulie B, Peeters T, Depoortere I, Janssens J, Tack J. Influence of motilin on gastric fundus tone and on meal-induced satiety in man. Am J Gastroenterol 2005 (In Press). 51. Tack J, Demedts I, Dehondt G, Caenepeel P, Fischler B, Zandecki M, Janssens J. Clinical and pathophysiological characteristics of acute-onset functional dyspepsia. Gastroenterology 2002;122: 1738–1747.

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Small Intestinal Motility William L. Hasler Anatomic and Functional Considerations, 935 Small Intestinal Transit, 936 Specialized Small Intestinal Cell Types, 936 Smooth Muscle, 936 Nervous Tissues, 936 Interstitial Cells of Cajal, 938 Other Cell Types, 940 Coupling of Small Intestinal Contractions, 940 Control of Small Intestinal Peristalsis, 941 Stereotypical Small Intestinal Motor Patterns, 943 Migrating Motor Complex, 943 Fed Motor Pattern, 946 Giant Migrating Complexes, 948 Other Aborally Migrating Patterns, 948

Retrograde Peristaltic Contractions, 948 Motor Activity of the Ileocolonic Junction, 949 Extended Reflexes Involving the Small Intestine, 951 Intestinointestinal Reflex, 951 Nutrient-Evoked Small Intestinal Reflexes, 951 Reflexes Activated by Nonnutrient Small Intestinal Stimulation, 953 Reflex Intestinal Responses to Stimulation of Other Organs, 953 External Influences on Small Intestinal Motor Activity, 953 Central Nervous System Modulation, 953 Alteration by Infection or Immune Mediators, 954 References, 956

The small intestine accepts and processes luminal contents from the stomach and delivers the indigestible residues to the colon for storage and subsequent elimination from the body. Two basic local contractile activities, mixing and propulsion, as well as several programmed motor patterns subserve these functions. In the postprandial period, triturated food from the stomach is mixed with bile and pancreatic enzymes for digestion and is propelled aborally. By virtue of its ability to distribute luminal contents for variable distances, intestinal motor activity influences nutrient absorption rates (1). During fasting, mixing and propulsion clear the small intestine of undigested solids and sloughed enterocytes. The small intestine terminates at the ileocolonic junction, a sphincteric structure that prevents reflux of feces from the cecum to the ileum. However, its role in modulating delivery of ileal contents to the colon is less clear. Motility of the small intestine is controlled by myogenic characteristics, extrinsic and intrinsic nerves, interstitial cells,

and circulating hormones. Reflex responses within the gut and external influences from central nervous system activation or from immune factors modulate this contractile function (see Chapter 24).

ANATOMIC AND FUNCTIONAL CONSIDERATIONS The small intestine is approximately 600 cm long, including the duodenum, jejunum (40% of the length), and ileum (60%). Its wall has two regions of muscle tissue, the muscularis externa and muscularis mucosa. The muscularis externa consists of an outer longitudinal layer and an inner circular layer, oriented at 90-degree angles to each other. The circular layer is subdivided into inner and outer layers. The muscularis externa is the major effector of mixing and propulsion, whereas the role of the muscularis mucosa is poorly understood. Specialized circular muscle thickening is observed at the ileocolonic junction, reflecting the sphincteric function of this structure. The distinct functions of longitudinal and circular muscle in the small intestine are poorly understood. The circular layer is believed to mediate both mixing and propulsion as

W. L. Hasler: Department of Internal Medicine, Division of Gastroenterology, University of Michigan Health System, Ann Arbor, Michigan 48109. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

935

936 / CHAPTER 38 luminal occlusion and displacement of gut contents require contraction of this layer. The most basic small intestinal contractile pattern, segmentation, results from reciprocal inhibition and disinhibition of adjacent circular muscle. Longitudinal contraction shortens the gut and can increase luminal diameter to facilitate movement of large boluses. The timing of contractions in the two layers is variable, with some investigations observing concurrent motor activity and others reporting 90- to 270-degree phase lags between circular and longitudinal contractions (2,3). In guinea pig ileum, independent contractions of the two layers are noted at low intraluminal pressures, whereas simultaneous contractions occur at greater pressures (4). In a study using spatiotemporal mapping, the longitudinal layer was observed to contract immediately preceding and during the propulsive period of circular muscle contraction (5). However, when the circular contraction occluded the lumen, longitudinal lengthening occurred.

SMALL INTESTINAL TRANSIT Propulsion of small intestinal contents depends on the luminal caliber, wall tone, and amplitude of phasic contractions. Scintigraphic studies report highly variable rates of small intestinal transit ranging from 78 to 264 minutes in healthy humans (6–8). Studies suggest that intestinal transit is slower in women, especially in those who are postmenopausal or obese (8,9). The effects of age are minimal, although one investigation has reported accelerated transit in older individuals (9,10). Most flow events, measured by intraluminal impedance, propagate only short distances (<10 cm) in an antegrade direction (11). In rats, transit is more rapid across the proximal half of the intestine (30 minutes) than the distal intestine (2.5 hours) (12). In contrast with the stomach, where solids and liquids are handled differently, 131 I-containing solids and 99mTc-DTPA-water are propelled through the small intestine at similar speeds (13,14). However, the ileum may selectively retain indigestible solids such as bran, whereas allowing liquids to pass into the colon (15). Small intestinal nutrient transit depends on the caloric density and the nutrient class. Inert substances such as guar have little effect on total intestinal transit (16). In studies of different protein solutions, intestinal transit is slowed in proportion to the calories delivered, with a resultant increase in the amount of protein absorbed in the proximal intestine. This maintains a constant level of absorption across a broad range of nutrient loads. Similarly, intraluminal lipids delay intestinal transit in a manner dependent on their caloric density (17).

SPECIALIZED SMALL INTESTINAL CELL TYPES Several specialized cell types in the small intestine serve specific roles in the control of motor function in the organ.

Smooth Muscle Small intestinal smooth muscle cells are spindle shaped and uninucleate. In contrast with skeletal muscle, there are no specialized regions for neuronal interaction. Intestinal smooth muscle cells are electrically active with resting membrane potentials of −40 to −80 mV, which are maintained by Na+K+-ATPase activity, and exhibit spontaneous contractions activated by stretch. Small intestinal myoelectrical activity exhibits ubiquitous membrane potential fluctuations of 3 to 15 mV oscillating at 11 to 12 cycles/min in the duodenum and at lower frequencies in more distal regions. Extracellular recordings of this slow-wave activity show sinusoidal patterns or rapid biphasic deflections from zero potential. Intracellular recordings demonstrate rapid depolarizations followed by partial repolarizations to prolonged plateau phases of depolarization. The slow-wave cycle terminates with full repolarization to the resting membrane potential. In the small intestine, the frequencies and propagation directions of phasic contractions are controlled by the slow wave, although tonic contractions lasting 10 seconds to 8 minutes have been recorded from the circular muscle (18). In most instances, the slow wave produces insufficient membrane depolarization to initiate contraction. Neurohumoral stimuli or exogenous administration of excitatory agonists evoke motor activity by increasing the duration and amplitude of the slow-wave plateau potential or by inducing spike potentials of brief duration (10–100 milliseconds) but high amplitude (50 mV) in phase with the slow wave. Spike potentials can be generated in isolated longitudinal and circular muscle and propagate only a few millimeters. Intestinal relaxation results from removal of a contractile stimulus or application of an active relaxant agent that acts to inhibit spike potentials, reduce plateau potential amplitude or duration, or induce membrane hyperpolarization. Nervous Tissues Small intestinal smooth muscle is innervated by intrinsic nerves within the gut wall and by extrinsic nerves relaying information to and from extraintestinal ganglia, the spinal cord, and the central nervous system. The myenteric plexus provides the major intrinsic innervation to the small intestinal muscle layers, although the submucous plexus may play a minor role in some reflexes (see Chapters 21 and 24). The vagus and splanchnic nerves represent the external input to the small intestine. The number of intrinsic neurons greatly exceeds the number of vagal or splanchnic fibers. The human enteric nervous system contains 10 to 100 million neurons versus 2000 efferent fibers in the vagus nerves; thus, most motor activities are directed by intrinsic nerves, whereas extrinsic innervation provides a modulatory function. Intrinsic Innervation The density of myenteric neurons ranges from 3700 to 12,170 per square centimeter in the cat small intestine,

SMALL INTESTINAL MOTILITY / 937 approximating that of the spinal cord (19). In humans, an additional deep muscular plexus innervates the interface of the inner and outer circular muscle layers of the small intestine and receives input from the myenteric plexus. Most submucous plexus neurons project to the mucosa, where they likely mediate secretory and absorptive activities (20). However, studies have demonstrated innervation of both the circular and longitudinal muscle layers by neurons from the inner and outer submucous plexus (21). The enteric nervous system possesses the elements for complete reflex activities including afferent neurons, interneurons, and motor neurons, and thus can mediate physiologic motor patterns in the absence of extrinsic input (see Chapter 24). Myenteric ganglia resemble the central nervous system in that only neurons and glial cells are present. Blood vessels and connective tissue cells are absent; thus, neuronal nourishment diffuses from the interstitial fluid. Neurotransmitters released from axonal varicosities diffuse 20 to 100 nm to specific receptors on muscle cells or neurons. In the small intestine, motor neurons typically project 1 to 2 mm longitudinally, although some fibers extend for 30 mm. Excitatory fibers run cephalad, whereas inhibitory fibers project in a caudad direction. That some reflexes project more than 100 cm implies that extensive interneuronal connections are involved (22). Most myenteric neurons project to other myenteric neurons and the circular muscle with a lesser number supplying the submucous ganglia. The longitudinal muscle is less well supplied and receives fibers primarily from excitatory motor neurons with little inhibitory input. Eighty to 90% of myenteric neurons contain either tachykinins (40–45%) or vasoactive intestinal polypeptide (VIP) (40–45%), with no overlap between the two groups. Tachykinin neurons containing substance P, neurokinin A, and acetylcholine mediate most of the excitatory functions of intestinal smooth muscle (23). In rat intestinal longitudinal muscle, neural stimulation evokes contractions incompletely blocked by atropine (24). Atropine-resistant contractions in porcine ileum are abolished by tachykinin neurokinin-1 (NK1) receptor antagonists (25). The inhibitory supply to the small intestine and colon is provided by nitric oxide (NO)– and VIP-containing myenteric neurons (26). Inhibitors of NO synthesis block ileal relaxations evoked by electrical depolarization, serotonin, adenosine triphosphate (ATP), and γ-aminobutyric acid (GABA) (27). Furthermore, blockade of nitric oxide synthase (NOS) evokes duodenal contractions in rats, indicating tonic inhibition by NO pathways (28). Similarly, antagonists or antisera to VIP prevent neurally mediated relaxation (29,30). A related transmitter, pituitary adenylate cyclase–activating peptide (PACAP), may also act as a physiologic relaxant substance in some regions via activation of PACAP1/VIP receptors (31). NOS, the enzyme responsible for NO generation, colocalizes with VIP in some neurons, suggesting that the two transmitters perform interrelated functions (32). In some models, VIP evokes NO-dependent relaxations via an intermediate transmitter such as GABA acting on GABAA receptors (33). Some VIP neurons contain neuropeptide Y

(NPY), calcitonin gene-related peptide (CGRP), gastrinreleasing peptide (GRP), and galanin. ATP is an important inhibitory neurotransmitter for some gut activities. In rat colon, nearly all ATP-containing myenteric neurons exhibit NO synthetic capabilities (34). Finally, NO pathways interact with contractile pathways in some models. In guinea pig small intestine, NK1 receptor activation evokes both a stimulatory effect and an inhibitory effect on distention-evoked contractions, which are mediated by NO release (35). Inhibitors of NOS and guanylate cyclase increase acetylcholine release and cholinergically mediated contractions in mouse ileum, an effect not observed in neuronal NOS knockout mice (36). Intrinsic neurons distinct from tachykinin-, NO-, and VIP-containing cells likely function as interneurons to modulate gut motor activity. Opioid neurons regulate both excitatory and inhibitory transmission. Enkephalinergic neurons project orally to both muscle layers, whereas neurons containing GRP and NPY project in an aboral direction (37). Immunoreactivity for the excitatory transmitter galanin is prominent in nerve fibers and varicosities in both the longitudinal and circular muscles (38). Orphanin FQ immunoreactivity is expressed in excitatory neurons projecting both to muscle layers and to a small number of interneurons (39). Neurons containing substance P and CGRP may mediate sensory function in some regions (40). Investigations propose an inhibitory action of endogenous cannabinoid pathways on small intestinal motility via action on cannabinoid receptor subtype 1 (CB1) receptors (41). Embryonic development of neurons in the small intestinal myenteric plexus occurs as a consequence of antegrade colonization of advancing vagal neural crest cells (see Chapter 18). Migration of neural crest cells progresses in response to aborally propagating sources of glial cell line–derived neurotrophic survival factor (GDNF) in a manner dependent on the transmembrane tyrosine kinase Ret (42,43). GDNF controls the number of neurons formed during development of the enteric nervous system (44). Contained in the advancing neural crest cells are neural crest stem cells, which are multipotent and self-renewing precursors of neural progenitors, glia, and more differentiated cells in the developing enteric nervous system. In vitro, neural crest stem cells form clonal neurospheres that contain neuronal and glial cells in various stages of differentiation (45). Other Ret signaling components participate in enteric nervous system development. Neurturin helps to maintain size of mature enteric neurons and the extent of neuronal projections (44). An investigation has characterized 216 genes that are relatively enriched in mouse embryonic, hematopoietic, and neural stem cells, and that likely produce specific transcription factors and signaling molecules (46,47). Defects in neurturin, Gfra-1 (a coreceptor for Ret), endothelin-3, endothelin receptor B, endothelin-converting enzyme, Phox2b transcription factor, and the Sox10 transcription factor have been associated with disorders of failed enteric nervous system development such as Hirschsprung’s disease

938 / CHAPTER 38 (48–51). Sox10 is purported to function in maintaining stem cells, as well as neural and glial progenitors (52). Extrinsic Innervation Efferent extrinsic fibers are carried in parasympathetic and sympathetic tracts. Most efferent fibers terminate in the myenteric plexus and form connections with enteric ganglia, although some sympathetic axons terminate directly on sphincteric smooth muscle. Such connections are formed late in fetal development. Extrinsic regulation of small intestinal motor function is not demonstrable in half of preterm infants (53). The vagus nerves contain three groups of efferent fibers: preganglionic parasympathetic excitatory cholinergic nerves to the enteric plexuses, preganglionic inhibitory cholinergic nerves to the myenteric plexus, and sympathetic fibers from the cervical ganglia. Efferent vagal cholinergic neurons activate nicotinic receptors within enteric ganglia with excitation of motor activity. In general, these fibers respond with a low threshold to electrical stimulation. Cell bodies of these efferent nerves reside in the brainstem vagal dorsal motor nucleus. Those fibers exhibiting high thresholds to electrical stimulation are inhibitory to motor activity via release of VIP and NO. The efferent vagal supply is maximal in the upper gut, although anterograde tracing studies from the rat dorsal motor nucleus confirm vagal innervation to much of the colon as well (54). Sympathetic splanchnic innervation is different from vagal parasympathetic innervation in that neuronal cell bodies reside outside the gut in the prevertebral ganglia (celiac and superior mesenteric ganglia) (see Chapter 22). Preganglionic cholinergic neurons project from the spinal cord to the prevertebral ganglia. Noradrenergic postganglionic neurons then project to the enteric ganglia. Such sympathetic innervation generally inhibits excitatory cholinergic transmission in the myenteric plexus of the small intestine (55,56). The functional significance of these pathways is exemplified by the long inhibitory intestinal reflexes that decrease motility via neural arcs involving the prevertebral ganglia. A number of neurotransmitters participate in extrinsic neural control of small intestinal motor function. Peptidergic fibers containing somatostatin, substance P, cholecystokinin (CCK), NPY, enkephalins, and other transmitters are present in the vagus and splanchnic nerves. Afferent fibers outnumber efferent fibers by 10-fold in the vagus nerve and by almost 3-fold in the splanchnic nerves. Vagal afferent fibers terminate in the brainstem nucleus solitarius. Neurons in the nucleus solitarius project to the vagal dorsal motor nucleus and the nucleus ambiguus. Sensory information from the small intestine is transmitted to the dorsal horn of the spinal cord via the splanchnic nerves. From there, second-order neurons project to the brainstem and cerebral cortex. The small intestine is richly supplied with sensory fibers. Free mucosal nerve endings respond to stroking or to chemical stimuli such as hydrochloric acid. Thermoreceptor exposure

in the small intestine activates perceptual responses and reflex motor responses in the stomach (57). Mechanoreceptors are activated by passive distention or during active contractions. Excision of the mucosa, submucosa, and the inner circular muscle does not abolish responses to stretch, localizing mechanoreceptors to the outer muscle layers or the myenteric plexus (58). Mesenteric and serosal receptors respond to tension or to forceful contraction and may mediate visceral pain perception. Intrinsic afferent neurons mediating local neural reflexes project within the enteric plexuses. Afferent pathways that mediate extended reflexes involve vagal and splanchnic nerves and may be modulated by cerebral input. Afferent information involving perception of nonnoxious stimulation may be transmitted via vagal or splanchnic pathways, whereas nociceptive input is believed to be carried mainly via the splanchnic nerves. Plasticity of sensory nerve activity is shown by experiments that demonstrate reductions in ileal vagal afferent sensitivity with prior exposure of the distal ileum to short-chain fatty acids (59).

Interstitial Cells of Cajal The interstitial cells of Cajal (ICCs) are distinct from neurons, glia, fibroblasts, and smooth muscle cells, and they play important roles in the regulation of small intestinal motility (60) (see Chapter 20). ICCs are uninucleate with thin cytoplasms and numerous mitochondria indicative of high metabolic activity, as well as abundant surfacemembrane invaginations (caveolae) with prominent endoplasmic reticula inferring active ion transport. Anatomic studies characterizing the distribution of ICCs measure immunoreactivity to c-kit, a protooncogene coding for a receptor tyrosine kinase. Such immunohistochemical studies identify at least six distinct ICC populations in the gut including intramuscular ICCs, ICCs within the myenteric plexus (ICC-MYs), and ICCs in the deep muscular plexus of the small intestine (61). Evidence suggests that ICCs are important mediators of neurotransmission. In the small intestine, deep muscular ICCs are closely associated with excitatory tachykinin and inhibitory nitrergic neurons (62). In gastric tissues from mutant mice lacking intramuscular ICCs, cholinergic neural responses to electrical stimulation are profoundly reduced secondary to loss of synaptic contacts between the ICCs and motor neurons, providing a functional correlate for the histologic observations (63). A second crucial function of ICCs is generation of the electrical pacemaker activities of the small intestine. Substantial evidence points to ICC-MYs as generators of spontaneous electrical rhythmicity in the small intestine. In cat intestine, rhythmic oscillations are generated only by tissue containing ICCs (64). Intrauterine maturation of ICCs correlates with the initiation of electrical rhythmicity (65). Electrical recordings from mouse intestinal ICCs exhibit pacesetter potentials characterized by initial depolarizations followed by plateau phases with a frequency of

SMALL INTESTINAL MOTILITY / 939 14 cycles/min and mean amplitudes of 13 mV (66). Pacesetter potential activity in ICC-MYs is associated with several cyclic fluctuations in ion channel activity. Selective injury to ICCs by administration of methylene blue plus intense illumination disrupts the slow wave, providing evidence of the pacemaker capabilities of these cells (67). Cyclic fluctuations in intracellular calcium are associated with electrical rhythmicity in the small intestine (68). ICC-generated pacesetter potentials are reduced by removal of extracellular calcium or depolarization with a highpotassium solution but are insensitive to L-type calcium channel blockers (66,69). Rather, slow-wave rhythmicity is dependent on calcium release from inositol 1,4,5trisphosphate–sensitive intracellular stores and the rate of sarcoplasmic reticulum calcium refilling (Fig. 38-1) (70–72). Disruption of caveolae in ICC-MYs with agents to remove or load excess membrane cholesterol alters pacing frequencies, indicating the importance of these structures (73). In cell clusters loaded with the calcium ionophore Fluo-4, dihydropyridine-insensitive calcium oscillations are observed that also depend on calcium influx through nonselective cation channels that are mediated by the transient receptor potential-like channel 4 in caveolae of ICC-MYs (74). The plateau component of the pacesetter potential is maintained by calcium-activated chloride channels (69). ICCs also express calcium-inhibited nonselective cation channels that activate at the same frequency as pacemaker currents (75). An additional mechanosensitive sodium

channel current is present in human intestinal ICCs that may modulate slow-wave frequency (76). Finally, potassium channels in ICC-MYs regulate membrane potentials and may participate in control of intestinal pacemaking activities (77,78). Intrauterine ICC maturation requires signaling via c-kit (79). In human fetal small intestine, c-kit immunoreactivity is demonstrable at a gestational age of 13 weeks (80). A continuous layer of ICCs forms from week 17 to birth. Using staining for c-kit and smooth muscle myosin heavy chain, investigations suggest that ICCs and longitudinal muscle cells may share a common mesenchymal progenitor cell (81). c-kit is down-regulated in cells destined to become muscle cells, but it is up-regulated in cells that become ICCs (82). ICCs mature normally in GDNF knockout mice that exhibit no enteric neurons, indicating that the enteric plexuses are not required for ICC development (83). Studies measuring c-kit expression provide the most convincing support for ICCs as physiologic regulators of the slow wave. W/Wv mice with mutations in the kit gene lack ICCs and exhibit impaired peristalsis (65,84). In animals with mutations in the steel factor, the ligand for c-kit, ICCs are absent in the small intestinal myenteric plexus and slowwave cycling is not detected (79). Finally, in neonatal mice, administration of neutralizing monoclonal antibodies to c-kit abolishes small intestinal rhythmicity and reduces the number of ICCs that then instead assume the appearance of smooth muscle cells (85,86).

Control

− 64 mV + 0.5 µM Xestospongin C

− 64 mV + 1 µM Xestospongin C

− 64 mV

10 mV 5s

FIG. 38-1. Small intestinal slow-wave recordings are shown under control conditions and with different concentrations of xestospongin C, a reversible, membrane-permeable inhibitor of inositol 1,4,5trisphosphate–sensitive calcium release. With increasing concentrations of xestospongin C, there is disruption, then abolition of intestinal slow-wave activity. (Reproduced from Malysz and colleagues [72], by permission.)

940 / CHAPTER 38 Studies of mutant W/Wv mice with reduced c-kit activity and loss of ICC-MYs provide insight into genes that may be responsible for controlling ICC function. ACPL1 (acid phosphatase-like protein 1) and G28K, a protein with homology to a protein that regulates human cell division, are down-regulated in W/Wv mice (87,88). Other gene products suppressed in W/Wv mice include ARG2 (arginase II), ONZIN (encoding leukemia inhibitory factor regulated protein), COX7B (cytochrome c oxidase, subunit VIIb), and SORCIN (encoding multidrug-resistance complex, class 4), whereas proteins increased in mutant animals include ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL-8 (ribosomal protein L8), SBTB2 (spectrin, nonerythroid β subunit), p6-5 (encoding phosphoryl choline T-cell suppressor factor), and DDWMEST-1 and -91 (89,90).

Other Cell Types Other cell types are present within the smooth muscle layers and myenteric plexus that traditionally have been considered to have little modulatory role in controlling small intestinal motor function. However, calcium-activated potassium channels have been characterized in fibroblast-like cells that form gap junctions with small intestinal smooth muscle cells in mice, raising the possibility they may participate in control of intestinal motility (91).

COUPLING OF SMALL INTESTINAL CONTRACTIONS Migration of small intestinal myoelectric and motor activity is regulated by intrinsic electrical coupling properties. Intestinal slow-wave propagation requires continuity of both muscle layers. After excision of a 1-mm band of circular muscle, longitudinal slow-wave propagation is limited to distances of 3 to 6 mm (92). If more than 5 mm longitudinal muscle is removed, slow-wave propagation is abolished, whereas excision of shorter bands permits inefficient propagation. Significant coupling exists between longitudinal and circular muscle cells in the small intestine. In the circular layer, gap junctions or nexuses provide low-resistance pathways for electrical conduction and for passage of lowmolecular-weight second-messenger compounds. Close coupling also is observed in the longitudinal layer, which possesses few or no nexuses. In this layer, other structures such as peg-and-socket junctions may mediate intercellular communication (93). Interconnecting bridges between the circular and longitudinal layers may provide low-resistance coupling across the myenteric region. ICC-MYs also are mutually connected to up to 35 other ICC-MYs, most likely by gap junctions, permitting extended propagation of pacing electrical currents (94,95). Synchrony of rhythmic calcium transients in ICC-MYs and longitudinal muscle indicates coupling of these two

structures as well (96). Mixing movements within the longitudinal layer are the consequence of spontaneous calcium waves generated by pacemaker sites that spread in all directions and that terminate by collision with calcium waves from other pacing sites (97). In W/Wv mice, regular contractions are observed in the longitudinal layer but are rare and uncoordinated in the circular muscle, suggesting that ICC control of pacing differs in the two muscle layers (98). ICCs in the deep muscular plexus and within the smooth muscle layers are extensively innervated and form close electrical contacts with smooth muscle cells from both layers, supporting the postulate that these cells are responsible for coupling of neural and myogenic activities. In the circular layer, these ICCs interface with smooth muscle cells by gap junctions (99). They further interact with one to five other ICCs in the deep muscular plexus (100). In the deep muscular plexus of the circular layer, ICCs in close conjunction with smooth muscle cells exhibit NOS activity and substance P–like immunoreactive axonal varicosities (101,102). Immunohistochemistry indicates that both cholinergic and nitrergic nerves form synapses with intramuscular ICCs (103). Furthermore, ICCs express M2 and M3 muscarinic receptors, NK1 and NK3 neurokinin receptors, VIP1 receptors, and stem cell factor, indicating modulation by neuronal pathways (104,105). Electrically active regions with the greatest slow-wave frequency entrain adjacent regions in a phenomenon termed phase lock. In humans, the dominant small intestinal pacemaker extends from the pylorus to the ligament of Treitz. Slow waves propagate more rapidly in the circumferential (1.7 ± 0.8 cm/sec) than in the axial direction (1.3 ± 0.5 cm/sec) (106). In contrast, spike potentials propagate more than twice as fast longitudinally as circumferentially. Retrograde slow-wave propagation into the stomach is rare because of an electrically insulating septum at the pylorus. Uncoupling of slow-wave synchrony does develop over the long distances encountered in the small intestine, which is more pronounced in the ileum than in the duodenum (107). The human intestinal slow-wave frequency decreases from 11 to 12 cycles/min in the duodenum to 7 to 8 cycles/min in the distal ileum. In most species, the decrease in frequency from the duodenum to the ileum occurs in stepwise fashion, producing alternating frequency plateaus and regions of variable frequency. Frequency plateaus are prominent more proximally but often are undetectable in the ileum (107). Consequently, there are expansive regions of myoelectric coupling in the upper gut that promote long-distance contractile propagation, whereas uncoordinated ileal slow waves limit the migration distance. In addition to regional variations in frequency, slow-wave propagation velocities decrease by up to 50% from the duodenum to the distal ileum. These phenomena are advantageous for efficient digestion. Proximally, nutrients are propelled over a large mucosal surface area for rapid digestion and absorption, whereas, distally, retarded propulsion permits absorption of slowly digested and absorbed substances such as fats and bile.

SMALL INTESTINAL MOTILITY / 941 CONTROL OF SMALL INTESTINAL PERISTALSIS The peristaltic reflex is demonstrable in the small intestine and produces aboral propulsion of luminal contents. Stimuli that evoke the peristaltic reflex include mucosal pinching and mechanical distention with gas, liquids, or a solid bolus. The peristaltic reflex consists of two phases, an excitatory response proximal to stimulation (ascending) and a distal inhibition (descending relaxation) (108). The ascending contraction is characterized by simultaneous circular muscle shortening and longitudinal muscle relaxation, whereas descending relaxation involves simultaneous longitudinal contraction and circular relaxation. In guinea pig ileum distended to pressures of 0.5 to 1.5 cm H2O, isolated increases in longitudinal tension are observed while at 1.5 to 3.0 cm H2O; the longitudinal tension increase is followed by a propagative circular contraction indicating differential sensitivities of the two muscle layers (109). Different luminal contents modulate peristaltic reflex activity. Distention with oil or cellulose increases the length of contraction but decreases clearance from an isolated intestinal segment, and cellulose, but not oil, slows propagation velocity (110). Distention with air produces short-contraction segments with rapid propagation velocities. Small intestinal peristalsis may proceed independently of slow-wave propagation. In isolated feline duodenum, peristaltic electrical waves propagate at lower velocities than slow waves and stop conducting even when slow-wave activity persists (111). Both neural and intestinal wall factors participate in generation of the peristaltic reflex. Jejunal distention evokes initial nerve-mediated phasic pressure elevations and increases in wall tension through stretch-activated generation of circular muscle contractions (112). This is followed by pressure decreases secondary to inherent viscoelastic

A

B

properties of the intestinal wall. Tetrodotoxin and lowcalcium, high-magnesium solutions abolish peristaltic propagation indicating neural mediation, whereas the calcium channel antagonist nifedipine attenuates contractile amplitudes but does not prevent peristaltic wave propagation (Fig. 38-2) (113). Afferent neuron activation is needed to elicit the peristaltic reflex. Cell bodies of the primary sensory neurons initiating the peristaltic reflex are intrinsic to the intestinal wall (114). Radial stretch is the most potent stimulus for inducing the reflex (56). The threshold for peristalsis is reduced by more rapid stretching and by using longer intestinal preparations (115). Receptors that sense radial stretch are mucosal in location, because stripping the mucosa or applying luminal topical anesthetics abolishes the reflex. Nonmucosal receptors also are suggested by experiments in which blunted reflexes persist despite chemical destruction of the mucosa by silver nitrate or tannic acid (116). Transmitters that mediate the peristaltic reflex have been studied extensively. Ascending contractions are partly inhibited by atropine at low levels of stimulation, whereas tachykinin receptor antagonists or antisera block contractions induced by intense radial stretching, indicating dual cholinergic and tachykinin mediation (109,117). Acetylcholine, substance P, and neurokinin A are released by radial stretch (117). Muscarinic M1 receptors participate in interneuronal transmission, whereas M3 receptors mediate the smooth muscle contraction (118). In guinea pig preparations, NK1, NK2, and NK3 receptors all participate in peristalsis in concert with cholinergic pathways (119,120). The mediators of the descending relaxation likely are VIP and NO because both are released during peristalsis (121). NOS inhibitors such as Nω-nitro-L-arginine and N-nitro-L-arginine methyl ester inhibit descending relaxations elicited by colonic distention (122). Capsaicin-sensitive

C

δP/δt

Balloon pressure

0

5mN

Oral chamber

5mN

Intermediate chamber

Anal chamber

5mN 4s

FIG. 38-2. Effects of a low-calcium, high-magnesium solution on propagation of peristaltic waves in isolated guinea pig jejunal segments. Under control conditions (A), balloon distention elicited a peristaltic wave that propagated to the anal chamber. With the low-calcium, high-magnesium buffer in the intermediate chamber, propagation of the peristaltic wave is blocked. This inhibition is restored with restitution of a physiologic buffer solution. (Reproduced from Spencer and colleagues [113], by permission.)

942 / CHAPTER 38

CGRP

Ach/SP motor neurons

VIP/PACAP/NOS motor neurons

5-HT4 receptor

CGRP

5-HT

FIG. 38-3. Neural pathways underlying the peristaltic reflex. Mucosal stimulation evokes serotonin (5-hydroxytryptamine [5-HT]) release, which activates release of calcitonin gene–related peptide (CGRP), which then stimulates ascending motor pathways containing the contractile agents acetylcholine (Ach) and substance P (SP) and descending motor pathways containing the relaxant agents vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), and pituitary adenylate cyclase–activating peptide (PACAP). (Reproduced from Grider and colleagues [126], by permission.)

afferent neurons mediate both ascending and descending components of the peristaltic reflex (123). Serotonin is released with gut distention and reduces the threshold for evoking the peristaltic reflex, indicating an important role for this transmitter (124). Mucosal stimulation initiates the reflex by activating 5-hydroxytryptamine subtype 4 (5-HT4)/5-HT1P receptors on sensory neurons containing CGRP in human intestine and 5-HT4/5-HT1P and 5-HT3 (125). In isolated human, rat, and guinea pig colon tissue, 5-HT4 agonists activate CGRP pathways that evoke ascending substance P release and descending VIP release (Fig. 38-3) (126). In marmoset small intestine, it is hypothesized that 5-HT4 receptors are activated by low concentrations of serotonin, whereas greater concentrations produce 5-HT3 receptor activation (127). More recent investigations have characterized an inhibitory effect of serotonin on peristalsis that is blocked by pretreatment with a selective 5-HT7 antagonist (128). Other neurohumoral substances can modulate the peristaltic reflex. Thresholds for eliciting the peristaltic reflex are increased by clonidine, indicating inhibitory effects of α2-adrenoceptor pathways (129). Electrophysiologic testing localizes this effect to α2-adrenoceptors on ascending interneurons and orally directed intrinsic sensory neurons (130). Stretch activates aborally projecting somatostatin neurons that inhibit activity of opioid neurons with reduction in met-enkephalin release (131). The decrease in met-enkephalin

produces relaxation via VIP and NO release. Conversely, opioids acting on µ and κ receptors inhibit peristalsis in guinea pig small intestine (132). Endogenous purinergic pathways suppress peristalsis via activation of P2 receptors (133). In rats, ascending responses are augmented by activation of adenosine A1 receptors, whereas A1 receptor activation inhibits descending relaxations and A2 receptor stimulation inhibits the ascending contraction (134). Stimulation of cannabinoid CB1 receptor pathways inhibits peristalsis in guinea pig ileum (135). In rat duodenum and ileum, ascending contractions are blocked by CCK via simultaneous action on CCK1 and CCK2 receptors (136,137). Activation of endothelin ETA receptors stimulates peristalsis in guinea pig small intestine, whereas ETB receptor occupation inhibits peristalsis (138). The ability of an ETB antagonist to enhance peristalsis suggests a physiologic modulatory role for ETB receptors. Estradiol and progesterone increase the threshold pressure needed to elicit the peristaltic reflex in guinea pig small intestine (139). Different prostanoids have receptorselective effects on peristaltic activity (140). Although traditionally considered to be mediated by intrinsic nerves, some evidence suggests that extrinsic pathways may participate in the peristaltic reflex. In isolated tissues, extrinsic denervation decreases basal CGRP levels and abolishes peristaltic reflexes activated by muscle stretch but not mucosal stimulation (141). In these models, extrinsic sensory pathways are postulated to mediate responses

SMALL INTESTINAL MOTILITY / 943

D1

D2

J1

J2

J3 30 min

FIG. 38-4. Manometric recordings of the different phases of the human migrating motor complex cycle from the duodenum (D1, D2) to the jejunum (J1-3). Dashed lines show propagation of the leading edge of two phase III complexes. (Reproduced from Soffer and colleagues [154], by permission.)

to stretch whereas intrinsic pathways activate peristalsis with mucosal stimulation. That CGRP antagonists abolish peristalsis with both stimuli indicates CGRP involvement in extrinsic and intrinsic pathways.

STEREOTYPICAL SMALL INTESTINAL MOTOR PATTERNS A number of stereotypical motor patterns have been characterized that control small intestinal propulsion under physiologic and pathophysiologic conditions. Migrating Motor Complex Physiologic Characteristics The migrating motor complex (MMC) is an organized fasting pattern that propels undigested food residue and sloughed enterocytes from the proximal gut; it has been termed the intestinal housekeeper (142). Small intestinal bacterial overgrowth develops rapidly in rats given morphine to disrupt MMC cycling, which shows the importance of the complex (143). The electrical correlate of the MMC is the migrating myoelectric complex (144). The MMC consists of four phases with a total duration of 84 to 112 minutes (Fig. 38-4). Phase I is a period of motor quiescence lasting 40% to 60% of the cycle length. Phase II, occupying 20% to 30% of the cycle, exhibits irregular phasic contractions involving approximately half of slow wave cycles. The duodenal cross-sectional area is greater during phase II than phase I, possibly serving to accommodate pancreaticobiliary secretions (145). Phase III is a 5- to

10-minute period of luminally occlusive, rhythmic contractions, most of which propagate aborally. However, some duodenal phase III contractions propagate in an orad direction, indicative of a physiologic retroperistaltic pump (146). This retroperistaltic activity is associated with duodenogastric reflux of bicarbonate and immunoglobulin A, which have been proposed to reconstitute the antral mucosal barrier during fasting (147). Retrograde duodenal motor activity during late phase III directed against antral phase III pressure waves also may increase fasting duodenal pH and nocturnal antral pH, serving to protect both regions (148,149). Finally, up to 32% of phase II pressure waves are bidirectional (148,149). The maximal phase III contractile frequency is determined by the slow-wave frequency (11–12 cycles/min in the duodenum, 7–8 cycles/min in the ileum). Phase III complexes propagate more slowly (4–6 cm/min) than the slow wave because of a loss of slow-wave phase locking and shortening of frequency plateaus (150). Nevertheless, individual phase III contractions propagate over longer distances than phase II contractions (108). The length of intestine in a given phase III complex decreases from 40 to 60 cm in the duodenum to 5 to 10 cm in the ileum. Phase IV is a transition period of irregular contractions occurring between phases III and I. Combining manometry with cinefluoroscopy has permitted correlation of fasting and fed patterns with movement of intestinal contents. Intestinal transit of inert substances is four times faster in phase III than in phase I of the MMC. Fifty percent of total flow occurs during phase III, which efficiently clears the small intestine of retained material (151). Transit also occurs in phase II, with rapid propulsion occurring during the transition from phase II to III. On cineradiography, transit during phase III is characterized by

944 / CHAPTER 38 intermittent boluses of 4 to 5 cm in length separated by 1- to 2-cm ring contractions (152). Although typically present during fasting in healthy individuals, selected MMC parameters exhibit significant variability. However, serial manometric recordings performed within given patients demonstrate reproducible intraindividual MMC characteristics (153). In nearly all healthy subjects, at least one phase III develops during 6 hours of fasting (154). Seventyone percent of phase III complexes originate in the stomach, with 28% beginning in the duodenum and 1% in the proximal jejunum (155). The fraction of MMC complexes that originate in the stomach or duodenum linearly correlates with the duration of fasting (156). MMC cycle duration is nearly twice as long if the previous phase III complex originated in the stomach versus the duodenum (157). Half of MMCs propagate beyond the midjejunum, and only 10% reach the distal ileum. Infants born at term exhibit physiologic phase III activity, albeit with an increased number of nonpropagated clusters, but preterm babies often exhibit low-amplitude, disorganized clustered contractions of short duration without recognizable phase III activity (158). There are no qualitative differences in MMC characteristics between young (18–39 years old) and old (40–69 years old) individuals, although phase III propagation velocity may decrease after age 80 years (159). Phase III propagation velocities are slower and contractile amplitudes are increased during both the follicular and luteal phases of the menstrual cycle in women compared with men (160). Small intestinal motor patterns exhibit subtle differences during wakefulness and sleep. MMC complexes migrate 2.5 times faster during the day but exhibit greater contractile amplitudes at night (161). MMC periodicity and phase II duration are shorter during sleep, whereas phase I duration is prolonged (162). The summed motility index during wakefulness exceeds that during sleep. Prolonged manometric recordings show no correlation of the sleep stage with MMC cycling and no synchrony of rapid eye movement sleep with intestinal phase III (163). Neural Regulation Neural regulation of the intestinal MMC involves extrinsic and intrinsic input. In studies of W/Wv mice that lack pacemaker ICC-MYs, generation and directional propagation of MMC is preserved, indicating that the enteric nervous system possesses the programming necessary for the motor complex, and that slow waves are not requisite (164). Extrinsic innervation from the vagus and splanchnic nerves serves a modulatory function. Neither bilateral truncal vagotomy, removal of the superior and inferior mesenteric ganglia, total sympathectomy, nor complete extrinsic denervation of the small intestine prevent MMC cycling, although cycle duration and regularity may be altered (165–167). Bilateral vagotomy increases cycle duration, whereas section of the extrinsic nerves to the jejunum and ileum decreases the MMC cycle length, reduces the percentage of

phase III complexes that propagate to the ileum, and disrupts coordination of duodenal and jejunal phase III activity (165,168). Vagal cooling in dogs has no effect on intestinal phase III, but it shortens intestinal phase II activity, suggesting a vagally mediated pathway for maintenance of phase II activity and a vagally independent pathway for phase III (169). Enteric ganglia coordinate small intestinal MMC propagation. Isolated, denervated intestinal segments exhibit spontaneous phase III complexes that propagate aborally; however, cycling in the excluded segment is out of phase with the main segment, suggesting that continuity of the enteric nervous system synchronizes cycling from one intestinal region to the next (170). Likewise, intestinal transection into many segments with subsequent end-to-end reanastomosis promotes independent phase III activity in each segment (171). In this model, neuronal growth across the anastomosis is noted at 28 days and coordinated cycling resumes at 45 days, indicative of regeneration of enteric nerve connections (171,172). If a segment of colon is interposed between the small intestinal segments or if end-to-side or side-to-side intestinal anastomoses are performed, coordination does not resume, showing a requirement for continuity of small intestinal-type enteric nerves (173). In guinea pigs, longitudinal myomectomy with circumferential interruption of myenteric ganglia blocks propagation of 50% to 60% of phase III complexes, whereas complete transection with reanastomosis blocks more than 80% of complexes, indicating an important role for the myenteric plexus in MMC continuity (172). MMC cycling is disrupted, but not abolished, by serosal application of benzalkonium hydrochloride, which selectively destroys myenteric ganglia, suggesting that the deep muscular plexus and submucous plexus have some role in maintaining the MMC (174). Cholinergic and noncholinergic neural pathways participate in MMC contractions. In dogs, intravenous administration of atropine, the ganglionic blocker hexamethonium, or the neural toxin tetrodotoxin eliminate MMC cycling (175). If given via close intraarterial injection, these agents prevent MMC propagation distal to the site of infusion (176). Adrenoceptor antagonists disrupt, but do not abolish, MMC cycling. NOS inhibitors evoke premature MMC activity followed by more rapid cycling, indicating that endogenous NO may be a physiologic inhibitor of fasting motor function (177,178). Conversely, the NO donor sodium nitroprusside disrupts MMC activity in rats, inducing a postprandial-like pattern (179). Similarly, the phosphodiesterase inhibitor sildenafil, which prevents cyclic guanosine monophosphate degradation after NO stimulation, disrupts MMC cycling (180). Selective VIP receptor antagonists block the disruptive effects of NO donors, indicating that NO may mediate its actions via VIP release (181). Excitatory and inhibitory transmitters may interact in controlling MMC activity. Neurokinin A evokes phase II–like activity in rats (182,183). This response can be augmented by pretreatment with an

SMALL INTESTINAL MOTILITY / 945 NOS inhibitor (183). After small intestinal transection and reanastomosis in guinea pigs, immunoreactivities for VIP, GRP, and somatostatin are decreased distal to the anastomosis (172). Recovery of MMC cycling is associated with recovery of these peptides and nerve regrowth, suggesting that peptidergic nerves within the myenteric plexus might contribute to coordinated MMC activity. Endogenous serotonin has been promoted as a physiologic modulator of the intestinal MMC. Serotonin increases the frequency of intestinal contractions, shortens the MMC cycle length, accelerates propagation velocities, and converts mixing patterns to propulsive ones (184,185). In rats, ablation of myenteric serotonin neurons with 5, 6and 5, 7-dihydroxytryptamine prolongs MMC periodicity and decreases propagation velocities (186). Finally, 5-HT3 and 5-HT4 receptor antagonists inhibit phase III activity or prolong cycle length (187–190). The inhibitory effects of the 5-HT3 receptor antagonist alosetron are more prominent in murine intestinal preparations from female animals, suggesting sex selectivity for serotonin mediation of the MMC (190). Other serotonin receptors may also participate in control of the MMC. The 5-HT1 agonist sumatriptan prolongs MMC durations by increasing phase II length (191).

Neurohumoral Mediators of the Migrating Motor Complex Induction of phase III in the stomach and proximal small intestine results from release of the hormone motilin from the duodenal mucosa (192). Motilin receptors are expressed in human duodenal and colonic enteric neurons (193). The motilin receptor is G protein coupled and is 52% identical to the receptor for growth hormone secretagogues. Motilin also is localized to the hippocampus, thalamus, hypothalamus, amygdala, cerebellum, and vagus in animal models; however, its physiologic function in these sites is uncertain (194–197). Antral phase III complexes correlate temporally with plasma motilin increases in healthy humans, and premature antral phase III activity is inducible by motilin infusion (198). Motilin-evoked phase III is identical in duration, amplitude, and propagation velocity to spontaneous complexes (199). In dogs, gastroduodenal phase III is abolished for several hours after infusion of motilin antisera and is replaced by irregular phasic contractions (200). Likewise, duodenal excision with removal of motilin-secreting tissue alters fasting antroduodenal motility in dogs (201). In such animals, phase III activity begins ectopically in the jejunum (202). The motilin dependence of MMC cycling exhibits neural plasticity. In dogs that have undergone duodenectomy, gastroduodenal MMC-like activity recovers 1 to 4 months after surgery, which is vagally mediated and dependent on cholinergic and adrenergic efferent pathways (203). The physiologic stimulus for motilin cycling is unknown. Cyclic motilin fluctuations are blocked by atropine and

hexamethonium, suggesting regulation by cholinergic pathways (204). Motilin release is evoked by vagal stimulation, cholinergic agonists, opioid agents, and duodenal pH changes, but the roles of these stimuli in the regulation of motilin release are unclear (204–206). Similarly, NOS inhibitors evoke phase III–like antroduodenal complexes with associated increases in plasma motilin (207). There are differences in the motilin dependence of phase III complexes in the proximal and distal small intestine. Ectopic complexes originating distal to the ligament of Treitz often are not associated with plasma motilin increases (208). Furthermore, the effects of motilin antisera on distal jejunal and ileal phase III complexes are minimal (171,200). Similarly, ectopic phase III complexes form in the jejunum and ileum after resection of duodenal motilin-producing tissues (201). These findings suggest that “programming” of phase III activity in the mid and distal intestine is a motilinindependent phenomenon that is entrained by the actions of motilin on the antrum and duodenum. The only peptides other than motilin known to cycle in phase with the MMC are somatostatin, pancreatic polypeptide, and xenin (209–211). Somatostatin evokes intestinal phase III complexes every 20 to 30 minutes, but the complexes are not physiologic because gastric motor activity is suppressed (209). Furthermore, somatostatin-evoked complexes do not exhibit a phase II–like pattern of irregular contractions and delay, rather than accelerate, transit. It is conceivable that somatostatin may play a role in motilinindependent cycling in the distal intestine. Plasma levels of pancreatic polypeptide, a peptide produced by the pancreas, also peak just before phase III (212). Furthermore, pancreatic polypeptide is released by exogenous motilin administration via vagal, cholinergic pathways involving 5-HT3 receptors (213). However, pancreatic autotransplantation has no effect on intestinal phase III cycling despite its disruption of pancreatic polypeptide fluctuations (214). In addition, there is no effect on fasting intestinal motility with pancreatic polypeptide infusion to levels mimicking those during phase III (215). Plasma xenin peaks are associated with phase III of the MMC (211). Xenin infusion evokes one or more premature phase III complexes in healthy humans, which is suggestive of a possible modulatory role for the peptide in regulating physiologic interdigestive motility. Other neurohumoral factors can modify MMC activity. Morphine induces premature intestinal phase III activity, whereas the µ-opioid receptor antagonist naloxone prolongs MMC periodicity from 103 to 219 minutes (171,216). Consequently, ligands acting on µ-opioid receptors may be intermediaries in generating intestinal phase III activity and may function in recovery of phase III cycling after a meal. δ-Opioid antagonists do not inhibit MMC cycling but do decrease motility indices during the interdigestive period (217). Orexin A induces dose-dependent prolongation of MMC cycle lengths, which is inhibited by pretreatment with NOS inhibitors indicative of mediation by nitrergic

946 / CHAPTER 38

Motility index, mmHg/min

Day 6

Night

Motillty

5 4 3 2 1

Amylase output, U/min

0 1200 1000

Amylase

800 600 400 200 0 08:00

12:00

16:00

20:00

00:00

04:00 08:00

FIG. 38-5. The circadian cycling of jejunal motility (top) and amylase output (bottom). Pancreatic secretion exhibits a close correlation with motor cycling; however, differential regulation is shown by the observation that enzyme output increases at night, whereas contractile activity is unchanged. (Reproduced from Keller and colleagues [227], by permission.)

pathways (218,219). This is supported by colocalization of orexin A and neuronal NOS in duodenal myenteric neurons. Glucagon-like peptide-1 (GLP-1) prolongs MMC cycle duration with slowing of transit via activation of NOdependent pathways (220). Ileal resections reduce phase I duration, shorten the MMC cycle, and induce intestinal clusters, raising the possibility of modulatory roles for peptides specifically released by the distal small intestine (221).

Other Associated Synchronous Cyclic Phenomena The intestinal MMC cycles in phase with motor activity of the gallbladder and sphincter of Oddi, as well as with several secretory functions (222). Gastric acid and pepsin production and intestinal fluid secretion increase before duodenal phase III (223). Similarly, the release of bicarbonate, bile acids, bilirubin, pancreatic enzymes, and luminal secretory immunoglobulin A peak just before or during phase III (Fig. 38-5) (212,224,225). In healthy humans, intestinal absorptive flux is twice as high as secretory flux during phase I, but four times as high during submaximal motor activity later in the MMC cycle (226). Qualitatively, this relation persists at night; however, there is a relative reduction in motor activity with preservation of exocrine output, providing evidence for differential circadian rhythms for motility and enzyme release (227). Furthermore, duodenectomy and diseases such as chronic pancreatitis

disrupt the synchrony of MMC activity and pancreatic exocrine cycling, suggesting differential regulation of the two phenomena (201,228). Duodenectomy also disrupts cyclic motility in the sphincter of Oddi and increases total sphincteric motor activity throughout the cycle (222). Cyclic bile release into the duodenum modulates MMC periodicity. Motilin is released into the circulation after exposure of duodenal mucosa to bile (229). Furthermore, experimental bile duct occlusion or diversion of bile away from the duodenum disrupts motilin cycling with loss of duodenal phase III and induction of ectopic jejunoileal complexes (230). These effects are reversible with bile flow restoration or exogenous bile acid perfusion (231). In addition, ileal perfusion of mixed micelles shortens the time to onset of phase III activity (232). Peaks of pancreatic secretion have been observed both in phase and out of phase with phase III cycling (233). Phase III–independent secretory peaks occurred in subjects with longer MMC cycle lengths. However, other studies dispute the role of bile in MMC generation (234). Fed Motor Pattern Physiologic Characteristics After a meal, the MMC is replaced by a fed pattern of intermittent phasic contractions of varying amplitude that peak 10 to 20 minutes after eating at all levels of the small intestine (235). The number of isolated pressure waves during the fed period is greater than during a comparable period during phase II (236). The myoelectric correlate consists of random bursts of spike potentials in phase with the slow wave (237). The fed pattern serves to both mix and propel intestinal contents. After eating, the distribution of intestinal contents becomes more uniform. In dogs and humans, postprandial transit is more rapid and exhibits a smaller number of fluctuations than during fasting (151). Forty-four percent of fed contractions do not propagate. Of contractions that do, 90% migrate less than 30 cm and 66% migrate less than 9 cm. In the proximal duodenum, 40% to 50% of fed contractions are retrograde (238). This duodenogastric propulsion may modulate gastric emptying. However, most pressure waves propagate in an antegrade direction for distances of 1.5 to 4.5 cm in the distal duodenum during duodenal lipid perfusion (239). The small intestinal fed pattern is recorded in preterm infants, which is indicative of its presence throughout life (240). Other motor patterns are seen after meal ingestion. A transitional motor pattern has been observed immediately after meal ingestion characterized by highly propagative contractile clusters that expose extended regions of mucosa to nutrients (241). Intense individual migrating contractions occurring after meals have amplitudes twice those of normal fed phasic contractions and durations equal to two slowwave cycles (110). Older studies suggested that postprandial ileal contractions are weaker than in the duodenum, but more recent investigations in dogs show increased contractile

SMALL INTESTINAL MOTILITY / 947 amplitudes in the ileum (242). Intense pressure waves at frequencies of 19 to 24 cycles/min have been observed in the terminal ileum, which develop 1 to 4 hours after eating when chyme reaches the colon (243). Myoelectric recordings from dogs show that this activity is accompanied by spike bursts similar to myenteric potential oscillations in the colon. Finally, a delayed tonic relaxation of the ileum is induced by local exposure to bile acids and triglycerides not absorbed in the proximal intestine (244). The duration of the fed motor pattern is dependent on the caloric content and qualitative aspects of the meal (245). In dogs, a mixed 450 kcal meal induces a fed pattern of more than 3 hours. The threshold for inducing the fed state in humans is unknown, but a 345-kcal meal disrupts MMC cycling for longer than 90 minutes (161). Duodenal nutrient perfusion at rates as low as 0.5 kcal/min can disrupt normal MMC activity in dogs (246). Peanut oil, consisting mostly of 18 carbon triglycerides, induces fed contractions for longer periods (>8 hours) than equicaloric sucrose or milk protein meals (247). In humans, a 400-kcal meal with 9% fat disrupts MMC activity for 294 ± 21 minutes, whereas a meal with 50% fat prolongs the fed period to 410 ± 42 minutes (248). Long-chain triglycerides convert the MMC to a postprandial pattern, whereas medium-chain triglycerides in equicaloric amounts have no effect (249). Addition of guar to a glucose drink prolongs the duration of the fed pattern (250). Intravenous amino acids shorten the cycle length of the MMC but do not induce a fed motor pattern, indicating that participation of the gut lumen is requisite (251). Neural Regulation Extrinsic innervation of the small intestine plays an important role in inducing the fed pattern. In dogs, the sight or smell of food disrupts MMC cycling, suggesting a cephalic phase for initiating the fed pattern. In humans, sham feeding disrupts duodenal phase III but does not evoke fed contractions (252). Bilateral vagotomy, splanchnicectomy, mesenteric ganglionectomy, and total extrinsic denervation do not prevent induction of the fed state, but bilateral vagotomy shortens its duration and increases the latency from the time of eating to the onset of fed contractions (165,170). Complete denervation reduces the number of propagated fed contractions, the mean distance of propagation, and the rate of intestinal transit. Jejunoileal autotransplantation, which produces extrinsic denervation, increases the number of calories needed to abolish MMC cycling and initiate the fed pattern (253). Finally, vagal cooling during the early postprandial period converts the fed pattern to intermittent phase III activity (169). These findings indicate modulation of the fed state by extrinsic neural pathways. Intrinsic innervation also modulates propulsion during the fed period. Intestinal transection and reanastomosis decreases the frequency and amplitude of fed contractions in dogs with decreased propagation (254). In the absence of continuity of the enteric nervous system, the extrinsic nerves alone are sufficient to suppress fasting motility in excluded

intestinal regions. If nutrients are perfused into an isolated, but extrinsically innervated, intestinal loop, the MMC is disrupted in the unconnected main portion of the intestine (255). As with the MMC, atropine and hexamethonium abolish fed motor and spike potential activity, emphasizing the importance of cholinergic innervation (256). Neurohumoral Mediators of the Fed Pattern Much investigation has focused on CCK as a possible mediator of the fed state; however, its role has not been proved. CCK levels increase 5- to 10-fold after eating but do not persist for the duration of the fed state. CCK and its analogs increase intestinal motility, although the motor pattern exhibits nonphysiologic characteristics such as prominent retrograde contractions and a preferential stimulation of proximal intestine contractions (257). CCK inhibits MMC activity but does not prevent motilin cycling (257). In rats, duodenal perfusion of trypsin inhibitor releases endogenous CCK, which acts on vagal afferent CCK2 receptors to stimulate a central CCK1 receptor pathway to disrupt MMC cycling (258). Intravenous CCK2 receptor blockade or central administration of a CCK1 receptor antagonist prevents the disruption of MMC activity in response to nutrients in rats (259). In dogs, the CCK receptor antagonists loxiglumide and MK-329 reduce, but do not prevent, the fed response or the interruption of MMC cycling after eating (260). Thus, the role of CCK in mediating the fed response may be species dependent. Other compounds inhibit the MMC and induce complexes similar to the fed pattern including gastrin, insulin, glucagon, neurotensin, neuromedin N, enkephalins, and prostaglandin E2 (165). As with CCK, gastrin does not reproduce the fed pattern, but it does stimulate proximal intestinal motility. Gastrin release also does not persist for the duration of the fed period (257). Peanut oil, which induces a fed pattern, does not evoke gastrin or insulin release (261). Conversely, glucose increases insulin levels but does not induce a fed pattern. Indomethacin infusion induces a fed pattern in dogs, but prostaglandin E2 also disrupts MMC cycling (262). Neurotensin has been proposed as a mediator of fed motility based on its ability to convert the fasting to a fed pattern along the entire small intestine in rats and humans (263). In rats, neurotensin antagonists also reduce the duration of the fed pattern (264). Although motilin is important in initiating fasting activity, it plays no role in the fed state. After eating, motilin levels decrease drastically. If exogenous motilin is given during the fed state, no phase III activity develops (199). In addition, motilin antisera generate motor patterns similar to the fed state, suggesting that suppression of motilin release may be necessary for induction of the fed state (200).

Reversion to the Fasting Motor Pattern The mechanism for reversion to MMC cycling after completion of the fed pattern is poorly understood. The first MMC

948 / CHAPTER 38 after eating begins distal to the duodenum, implying that factors that initiate normal complexes are not yet operational (247,265). There is a strong correlation between initiation of duodenal phase III and completion of gastric emptying (266). However, in dogs, continuous intraduodenal perfusion of nutrients induces the fed motor pattern for only a finite time, after which the fasting pattern returns, indicating that the presence of intestinal nutrients does not prevent resumption of fasting motor activity. In humans, continuous duodenal feedings produce persistence of the fed pattern for at least 6 hours, whereas phase III activity resumes 4 hours after initiation of intragastric feedings, indicating differential inhibitory effects of the two regions on MMC cycling (267). The mediators of this reversion are unknown; however, the ability of infused NOS inhibitors to convert postprandial duodenal motor patterns to phase III–like activity in healthy humans suggests possible involvement of nitrergic pathways (177,268).

Giant Migrating Complexes Intense contractile waves that propagate aborally for long distances in the intestine are observed in experimental animals during hypoxia, anemia, and gangrene, and after laparotomy and death. This phenomenon has been given many names including peristaltic rush, prolonged propagated contractions, migrating action potential complexes, power contractions, and giant migrating contractions (GMCs). Small intestinal GMCs are 2 to 3 times greater in amplitude and 4 to 5 times longer in duration than individual phasic contractions and propagate at 1 cm/sec (269,270). The duration of an intestinal GMC generally is longer than that of a single slow wave, indicating dissociation of the slow-wave regulation of phasic contractile activity (269). During a GMC, myoelectric recordings document intense bursts of spike potentials lasting 4 to 16 seconds, which obscure the slow wave (270). GMCs typically begin in the jejunum or ileum during fasting and migrate to the ileocolonic junction. They are intensely propulsive of ileal contents, in contrast with phase III (269). Studies report that nearly half of GMCs are associated with propagating sequences in the cecum, indicating a coordinated evacuation mechanism in the distal gut (271). Consequently, intestinal GMCs are postulated to clear debris from the ileum and prevent coloileal reflux. Intestinal GMCs are rare (0.03 per hour) in health but are induced by noxious stimuli such as intravenous morphine, intragastric vinegar, ileal perfusion of feces or short-chain fatty acids, ionizing radiation, and infection with Vibrio cholerae, Clostridium perfringens, Clostridium difficile, noninvasive Escherichia coli, Shigella, and Trichinella spiralis (270). Induction of GMCs is associated with decreases in ileal pH, suggesting that coloileal reflux may be a physiologic stimulant of this motor pattern (272). Diarrhea, fecal urgency, and abdominal discomfort temporally correlate with GMCs during ileal inflammation, suggesting a role in symptom induction. In enteropathogenic

E. coli infection, GMCs present before the onset of diarrhea, confirming that they are not merely a response to the diarrheal state (273). Propagation of small intestinal GMC activity is controlled by enteric neural pathways, whereas extrinsic nerves regulate inhibition of motor activity orad to the complex (274). GMCs elicited by arterial infusion of CGRP are blocked by atropine, hexamethonium, and tetrodotoxin, indicating mediation by cholinergic neural pathways (Fig. 38-6) (275). Neurokinin (NK3) receptors on presynaptic neurons also may participate in GMCs in the small intestine (276).

Other Aborally Migrating Patterns Intensely propulsive contractions also occur in clusters termed migrating clustered contractions, discrete clustered contractions, or the minute rhythm (Fig. 38-7) (269,277). Discrete clustered contractions occur while fasting and after eating and consist of 3 to 10 contractions preceded and followed by 1 minute of motor quiescence. Discrete clustered contractions migrate at 5 to 10 cm/min over distances of 2 to 40 cm and are proposed, together with GMCs, to be physiologic means of emptying the ileum (269,277). Unlike GMCs, discrete clustered contractions rarely extend into the proximal colon (271). In some studies, increased discrete clustered contractions are observed in irritable bowel syndrome, intestinal pseudoobstruction, and partial smallbowel obstruction (278). An extremely propagative pattern called the rapidly migrating contraction migrates for 200 cm at more than 30 cm/sec (279). In contrast with intestinal GMCs, rapidly migrating contractions occur predominantly in the proximal small intestine and are associated with disruption or loss of slow-wave activity.

Retrograde Peristaltic Contractions After administration of emetic agents, complexes migrate orally from the mid-small intestine to the duodenum before vomiting. These retrograde peristaltic contractions (RPCs) exhibit periods of motor inhibition immediately before and after the complex, which are followed by several phasic contractions, then a second inhibitory period (280). The entire phenomenon is abolished by vagotomy, but only the RPC itself is prevented by atropine, hexamethonium, and tetrodotoxin (275,280). Although most often associated with retching or vomiting, RPCs do occur in their absence. Conversely, retching and vomiting can occur in the absence of RPCs. The purported function of RPCs is to evacuate intestinal contents into the stomach so that they may be expelled during emesis. RPCs exhibit contractile amplitudes 1.3 to 1.8 times greater than normal intestinal contractions with durations 2 to 4 times longer and migrate rapidly (8–10 cm/sec) over distances more than 100 cm (151). RPCs are preceded by slow-wave obliteration before generation of

SMALL INTESTINAL MOTILITY / 949 140g 1 min SI-382 CGRP

SI-382 Atropine

CGRP

SI-382 Hexamethonium

CGRP

SI-382 TTX

CGRP

FIG. 38-6. Calcitonin gene–related peptide (CGRP)–evoked giant migrating contractions (GMCs) in the presence of different neural inhibitors. Under control conditions, CGRP elicits a GMC followed by phasic contractions (top tracing). All motor responses to CGRP are blocked by atropine, whereas the GMCs, but not the subsequent phasic contractions, are inhibited by hexamethonium and tetrodotoxin (TTX). (Reproduced from Sarna [275], by permission.)

electrical bursts that migrate orally (281). Investigations involving autotransplantation of small intestinal segments in dogs indicate that retrograde motor activity is controlled by extrinsic neural pathways (282).

Motor Activity of the Ileocolonic Junction The ileocolonic junction exhibits characteristics distinct from the ileum and colon, having a localized high-pressure

zone in animal models not abolished by neural toxins. Manometric recordings in dogs show a basal pressure of 30 to 40 cm H2O, with phasic contractions greater than 100 cm H2O (283). In humans, a high-pressure zone is less reliably observed, although phasic contractions often are noted (284). However, studies in patients with temporary ileostomies demonstrate high-pressure regions 5 cm in length with mean pressures of 10 mm Hg (Fig. 38-8) (285). Slow waves and spike potentials migrate across the ileocolonic junction into the colon in cats and dogs (286). In dogs, more than

D1

D2

J1

J2 50 mmHg J3 30 min.

FIG. 38-7. Discrete clustered contractions (DCCs) occurring in human duodenum (D1, D2) and jejunum (J1-3). (Reproduced from Soffer and colleagues [154], by permission.)

950 / CHAPTER 38 Sidehole No. (Distance from IJC)

mm Hg 40

SH 1 (41.5 cm) 0 40 SH 2 (34 cm)

0 40

SH 3 (26.5 cm)

0 40

SH 4 (19 cm)

0 40

SH 5 (8 cm)

0 40

SH 6 (3 cm)

0 40

Sleeve (IJC)

0 40

Cecum

0

20 sec

FIG. 38-8. Pressure recordings from sideholes (SH1-6) in the ileum, a sleeve sensor in the ileocolonic junction (ICJ), and the cecum from patients with ileostomies. The ICJ exhibits increases in tone and phasic motor activity unrelated to basal ileal or cecal motility. (Reproduced from Dinning and colleagues [285], by permission.)

50% of MMCs traverse the ileocolonic junction in dogs, but in humans, the structure participates in few MMC cycles (287). In dogs, more than 80% of phase III complexes traversing the ileocolonic junction evoke colonic contractions (288). In scintigraphic studies, fasting flow across the canine ileocolonic junction is maximal before phase III, whereas in humans, changes in flow do not correlate with MMC phases (287). Eating increases motor activity and flow across the ileocolonic junction that is maximal 4 hours after a meal in dogs (289). In humans, meals increase ileocolonic junction tone and the proportion of time occupied by phasic activity (285). GMCs and discrete clustered contractions readily traverse the ileocolonic junction and decrease ileocolonic junction phasic and tonic activity (285,287).

The ileocolonic junction safeguards against fecal reflux from the colon into the ileum. Resistance is detected on attempts to propel material in retrograde fashion across the structure. In humans and animals, surgical excision of the ileocolonic junction increases ileal bacterial counts, suggesting that the structure maintains relative sterility of the ileum (290). Both intrinsic and extrinsic nerves control ileocolonic junction responses. In canine and human tissues, colonic distention evokes reflex ileocolonic junction contraction, which is not blocked by transection of vagal or pelvic nerves (284,285,291). Conversely, splanchnic nerve transection blocks the reflex, indicating the importance of extrinsic sympathetic pathways. Similarly, total extrinsic denervation increases phasic ileocolonic junction pressures (292). The anatomic conformation of the ileocolonic

SMALL INTESTINAL MOTILITY / 951 junction likely plays an important role in this response, being maintained at a constant acute angulation at its point of insertion into the cecum by fibrous connections. If this fibrous tissue is severed such that the acute angulation is lost, the ileocolonic junction becomes incompetent and coloileal reflux results (293). The role of the ileocolonic junction as a barrier to aboral flow from the ileum into the colon is less clear. Early studies showed that the structure does not impede ileal liquid transit. Some manometric studies have reported reflex ileocolonic junction relaxations with ileal distention, whereas others have noted reflex contractions (294). In one canine investigation, ileocolonic junction relaxations in response to ileal distention were abolished by extrinsic denervation (292). Colonic filling from the ileum is characterized by bolus movements separated by intervening periods of stasis, suggesting that the ileum regulates cecal delivery (295). Studies with Heidelberg capsules show lag times of 0.8 to 2.5 hours for passage of solids across the junction, but other investigations show no discrimination of solid versus liquid passage into the cecum (296). Furthermore, patients with surgical resection of the ileocolonic junction exhibit normal transit of radiolabeled beads, suggesting little importance as a regulator of forward flow (297). However, if a long small intestinal segment has been excised, removal of the ileocolonic junction accelerates transit showing that the structure can be a barrier to aboral propagation under conditions of high flow.

EXTENDED REFLEXES INVOLVING THE SMALL INTESTINE The small intestine participates in several nerve-mediated reflexes within the organ itself, as well as with more distant regions of the gastrointestinal tract.

Intestinointestinal Reflex The inhibitory intestinointestinal reflex is characterized by profound motor inhibition of up to several hundred centimeters of intestine as a consequence of abrupt stretching or dilation of a localized intestinal segment, and it represents a possible model for ileus. This is associated with slow-wave disruption on both the oral and anal side of the distention with generation of dysrhythmic myoelectric activity and slow-wave uncoupling (298). In humans, the inhibitory effects of distention are more pronounced in the proximal small intestine than in the ileum (299). The relevance of the reflex is that, when distention results from mechanical obstruction or another cause, the bowel responds with decreased motility and tone, thus serving a protective function. The intestinointestinal reflex is not mediated by mucosal receptors because excision of the mucosa and submucosa has no effect. Instead, removal of the longitudinal muscle prevents the reflex. In dogs, abrupt distention of an excluded, but not

extrinsically denervated, intestinal segment abolishes spike activity in the excluded loop and in the intact intestine via splanchnic pathways (300). The reflex is abolished by spinal cord transection below T7, indicating a need for intact thoracolumbar spinal pathways.

Nutrient-Evoked Small Intestinal Reflexes The ileal and jejunal brakes are defined as the inhibition of proximal intestinal and gastric motor activity with nutrient (usually lipid) stimulation of the ileum and jejunum, and they likely serve a protective function to prevent the distal intestine from being overwhelmed by massive caloric loads. Gastric motility is inhibited more by ileal glucose perfusion than by duodenal administration (301). Similarly, ileal shortchain fatty acid perfusion potently reduces antral contractions and reduces transpyloric flow (302,303). Ileal lipid or carbohydrate perfusion also reduces duodenal and jejunal motility and proximal intestinal transit more potently than proximal perfusions (304). Lipids are the most potent stimulators of the ileal brake, with oleate being 20-fold more potent than glucose at inhibiting gastric emptying (305). Twelve carbon chain-length fatty acids are more potent than those with 10 carbon atoms (306). Lipase inhibition with tetrahydrolipstatin attenuates the reflex effects of triglyceride on proximal motor function, indicating that products of lipid hydrolysis mediate lipid-induced intestinal braking (307). Perfusion of 150 cm intestine with glucose induces significant inhibition of gastric emptying, whereas exposure of 15 cm has no effect on gastric emptying, demonstrating the dependence of the intestinal brake on the magnitude of the stimulus (308). Duodenal peptone perfusion is more effective than equicaloric glucose perfusion (309). Several neurohumoral mediators participate in nutrient inhibition of proximal gut activity. Extrinsic innervation is necessary for inhibition of duodenal and jejunal motility by ileal triglycerides but not oleic acid (310). Furthermore, local capsaicin administration abolishes the inhibitory effects of ileal lipids on gastric emptying, indicating participation of extrinsic sensory neurons in this reflex (311). Similarly, delays in gastric emptying evoked by duodenal carbohydrate and protein perfusion are associated with increased vagal afferent activity and are inhibited by capsaicin treatment of vagal and spinal afferents (309,312). α1 and β1 adrenoceptor, opioid, and 5-HT3 antagonists blunt intestinal nutrientinduced motor inhibition (Fig. 38-9) (313–315). Naloxone only antagonizes the ileal brake when administered to the proximal intestine, indicating dependence on opioid pathways on the efferent end of the reflex (316). In some models, enterogastric reflexes are blocked by the CCK antagonist MK-329 (312,317). In dogs, the lipid-induced jejunal brake is slowed initially by the selective CCK1 receptor antagonist devazepide with later acceleration by naloxone, indicating dual mediation of this reflex by CCK and opioid pathways (318). Apolipoprotein A-IV, released by enterocytes in response to triglyceride absorption, stimulates duodenal

952 / CHAPTER 38 Vehicle

Tropisetron

75

50

Glucose 990 mM

Control

0

Glucose 660 mM

25

Glucose 330 mM

% gastric emptying

100

FIG. 38-9. The effects of the 5-hydroxytryptamine subtype 3 (5-HT3) receptor antagonist tropisetron on intestinal glucose-induced inhibition of gastric emptying. Progressive increases in glucose concentration evoked increasing delays of gastric emptying that were reversed by tropisetron. (Reproduced from Raybould and colleagues [315], by permission.)

Chylous lymph

intestinal transit mimicking the effects of ileal nutrients (322). PYY immunoneutralization accelerates intestinal transit inhibited by ileal lipids, confirming dependence of the ileal brake on endogenous PYY (324). Ileal oleic acid, but not triglyceride, elicits PYY release, indicating differential activation of the ileal brake by different nutrients (310). Inhibition of duodenal motility by ileal protein is reversed by a GLP-1 receptor antagonist, indicating a role for this peptide as well (325). Finally, nutrient-evoked intestinal

Control lymph

CCK

Vehicle

vagal afferents via CCK-dependent mechanisms (319). Similarly, chylous lymph increases discharge of CCKresponsive duodenal fibers, which are responsible for eliciting vagovagal inhibition of gastric motility (Fig. 38-10) (320). Ileal lipids and short-chain fatty acids also release GLP-1, neurotensin, and peptide YY (PYY) (321–323). GLP-1 release is selectively promoted by fatty acids with chain lengths of 12 or more carbon atoms (306). Intravenous PYY prolongs the duodenal MMC cycle length and delays

Impulses bin−1

120

80

40

0

20

40

60

40

100

Time (min)

FIG. 38-10. The firing rate of a single duodenal vagal afferent fiber. Cholecystokinin (CCK) elicited significant increases in firing rates that were mimicked by chylous lymph but not control lymph, suggesting that chylomicrons or their products release CCK, which then activates vagal afferents. (Reproduced from Glatzle and colleagues [320], by permission.)

SMALL INTESTINAL MOTILITY / 953 brakes are modulated by other nonnutrient stimuli. Slowing of intestinal transit by jejunal oleate perfusion is reversed by volume distention of the ileum by buffer perfusion (326).

Reflexes Activated by Nonnutrient Small Intestinal Stimulation Small intestinal stimuli other than nutrients also elicit reflex motor effects on other gastrointestinal regions. Intraduodenal capsaicin inhibits plasma motilin cycling with resultant abolition of gastric MMC cycling, whereas intraileal capsaicin inhibits gastrointestinal contractions via NO, 5-HT3, and opiate pathways (327,328). Duodenal distention reduces intragastric pressure, inhibits antral motility, and induces pyloric contractions (329). Capsaicin-sensitive vagal afferents mediate the inhibitory response to low volumes, whereas spinal afferents mediate the response to high volumes of duodenal inflation (330). Intrinsic pathways are involved in pyloric contractile responses because duodenal transection prevents induction of pyloric contractions by duodenal distention (330). Conversely, duodenal transection does not block inhibition of antral contractions or the delay in gastric emptying induced by duodenal distention, indicating mediation by pathways other than ascending intraduodenal nerves (330). The magnitude of enterogastric responses to nonnutrient stimulation depends on the stimulus intensity, its nature, and the length and region of intestine that is stimulated. Exposure of 15 cm of duodenum to hydrochloric acid inhibits gastric emptying, whereas lactic acid has no effect (331). Fluid distention of the duodenum increases motor activity of the sphincter of Oddi via a tetrodotoxinsensitive mechanism, a reflex that may serve to prevent duodenobiliary and duodenopancreatic reflux (332). Ileal distention evokes colonic relaxation via pathways involving the splanchnic nerves and the prevertebral ganglia (288). In dogs, effects of ileal distention on colonic tone are not solely mediated by adrenergic, nicotinic, or NO pathways.

Reflex Intestinal Responses to Stimulation of Other Organs Small intestinal motor function is reflexively modulated by stimulation of other structures. In dogs, gastric distention abolishes fasting duodenojejunal motor activity and delays intestinal transit (333). This gastroenteric reflex is unaffected by vagotomy but is reduced by celiac plexus sectioning or by small intestinal denervation, showing mediation by nonvagal pathways (334). Other investigations report mediation of this reflex by NO release in the celiac plexus (335). In humans, fundic distention evokes phasic duodenal motor activity (336). Colonic distention retards intestinal transit, decreases ileal pressures, reduces ileal phasic motility, and disrupts intestinal slow-wave cycling (337–339). Sectioning the splanchnic nerves and ablating the prevertebral ganglia abolish these reflexes, indicating mediation by extrinsic

sympathetic pathways (288). Topical anesthesia of the rectum prevents inhibition of intestinal contractions by rectal distention, indicating participation of mucosal receptors (337). Colonic perfusion of mixed nutrients inhibits duodenal fasting activity (340). Colonic distention delays the onset of MMC activity via activation of nicotinic ganglionic receptors.

EXTERNAL INFLUENCES ON SMALL INTESTINAL MOTOR ACTIVITY Central Nervous System Modulation The central nervous system has pronounced modulatory effects on small intestinal function. Intestinal phase III is reduced by depression and by acoustic and mental stress (mental arithmetic, video games, driving in rush hour traffic) (341). Phase II and discrete clustered contractions also are suppressed by stress, whereas acoustic stress prolongs the fed pattern in dogs (342). MMC disruption depends on the nature of the stressor and the time of day it is administered. Abrupt nighttime awakening does not modify the MMC, despite the subjects’ perception that this nocturnal stressor is more noxious than daytime stressors (341). Stressinduced effects on intestinal motility usually are associated with delayed transit. A complex neurohumoral response is associated with stress. Prolongation of the intestinal fed pattern with acoustic stress is associated with gastrin, pancreatic polypeptide, and somatostatin release (342). The delay in intestinal transit evoked by cold water immersion of the forearm is attenuated by β-adrenoceptor blockade (343). Similarly, in rats, the inhibitory effects of restraint stress on small intestinal motility are reversed by selective antagonism of β3-adrenoceptors (344). Several compounds administered centrally can modify intestinal motor patterns. Inhibition of NOS in the brain suppresses duodenal phase III in dogs via vagal pathways (345). Destruction of hypothalamic and locus ceruleus adrenergic pathways with 6-hydroxydopamine lengthens MMC periodicity (346). Intracerebroventricular (icv) CCK increases whereas icv somatostatin decreases MMC cycle duration (341). Intracerebroventricular calcitonin, CGRP, neurotensin, and opioids evoke intestinal phase III activity, whereas NPY delays intestinal transit (347). Intracerebroventricular neurotensin delays MMC disruption by a meal, whereas icv atropine or substance P shorten the fed state (348). Intracerebroventricular galanin restores MMC cycling during the fed period via opioid pathways (349). Injection of a GABA antagonist into the dorsomedial hypothalamic nucleus increases jejunal contractions in rats, suggesting physiologic regulation by central GABA pathways (350). Intracerebroventricular thyrotropin-releasing hormone accelerates intestinal transit in rats via cholinergic and serotonergic pathways (351). Intracerebroventricular ghrelin induces fasting duodenal motor activity in fed rats, an effect blocked by icv injection of growth hormone

954 / CHAPTER 38

Alteration by Infection or Immune Mediators Activation of the immune system elicits significant changes in motor activity of the small intestine, as evidenced by animal models of intestinal anaphylaxis. In rats antigen sensitized by intraperitoneal egg albumin injection, subsequent oral albumin evokes diarrhea associated with MMC disruption and induction of propagating, high-amplitude, clustered contractions (360). The importance of afferent pathways in mediating these responses is shown by the ability of capsaicin to prevent antigen effects in the small intestine (361). Mast-cell stabilizers and degranulation inhibitors (doxantrazole, cromoglycate, quercetin) blunt antigeninduced intestinal motor responses and diarrhea, and granulated mast cells are reduced at sites of antigen challenge, indicating an important role for mast cells (362,363). These motor responses are prevented by the serotonin antagonists methysergide and cinanserin and the prostaglandin synthesis inhibitor indomethacin (364). Effects of intestinal anaphylaxis on intestinal function in antigen-sensitized animals may involve central nervous system activation. In sensitized rats, egg albumin MMC disruption is associated with increased c-fos expression in the nucleus tractus solitarius and lateral parabrachial and paraventricular hypothalamic nuclei via vagal afferent pathways (365). In models of postoperative ileus, intestinal immune infiltrates maintain the dysmotility state. In mice that have undergone intestinal manipulation, delays in gastric emptying are associated with appearance of leukocyte infiltrates in the smooth muscle layers of the small intestine (366). Similar manipulation in rats suppresses jejunal contractions and delays gastrointestinal transit (367). These responses are associated with up-regulation of interleukin-6 (IL-6),

Control Sham CLP (1 day)

70 60

Mean±SEM N–6

50 (200X mag. field)

MPO+Neutrophils

secretagogue receptor and immunoneutralization of NPY in the brain (352). Evidence has accumulated indicating that corticotropinreleasing factor (CRF) is a physiologic mediator of stress effects on the gut. Many stressors are associated with intracerebral CRF release. In mice, cold stress effects on intestinal transit are mimicked by icv CRF (353). In rats, icv CRF inhibition of intestinal transit is prevented by vagotomy and naloxone, suggesting the importance of vagal and opioid pathways (354). MMC inhibition by icv CRF in dogs is associated with suppression of motilin cycling (355). Effects of CRF and stress on intestinal function are reversed by CRF antagonists, supporting a physiologic role for the peptide (356). Small intestinal transit also is delayed by icv injection of urocortin via induction of nonpropagating contractions, an effect blocked by truncal vagotomy (357). CRF acts through activation of CRF1 receptors, whereas urocortin acts on receptors of the CRF2 subtype (358,359). Neither urocortin antisera nor CRF antagonists affect fasting or fed small intestinal motility in the absence of stress, indicating that endogenous urocortin and CRF do not regulate basal motor activity (357).

40 30 20 10 0 Control sham CLP-1 Small intestine

Control sham CLP-1 Colon

FIG. 38-11. The number of myeloperoxidase-positive (MPO) neutrophils in the muscularis layers of the jejunum and colon for control rats, animals that underwent sham surgery, and rats with polymicrobial sepsis 1 day after cecal ligation and puncture (CLP-1). In the small intestine, neutrophils greatly increased in septic rats that had CLP-1. SEM, standard error of the mean. (Reproduced from Overhaus and colleagues [368], by permission.)

messenger RNA, tumor necrosis factor-α, cyclooxygenase-2, and NOS, and activation of signal transducer and activator of transcription proteins in the smooth muscle layers. These responses are preventable by dexamethasone pretreatment. Bacterial infections elicit significant disruptive effects on small intestinal motility. In a rat model of peritonitis and sepsis induced by cecal puncture, intestinal transit is delayed and in vivo jejunal muscle contractility is impaired (368). This is associated with muscularis mononuclear infiltrates and increases in IL-6, IL-1β, monocyte chemoattractant protein-1, and inducible NOS (Fig. 38-11). Ileal muscle strips from such animals exhibit reduced contractile responses to potassium, substance P, and cholinergic stimulation (369). These defects are reversible by pretreatment with an inhibitor of NOS. The presence of ongoing intestinal inflammation is not required for distant bacterial infection to disrupt motor function. Patients with acute cholecystitis exhibit reduced intestinal phase III activity compared with those with biliary colic without infection, whereas fed motor activity and propagating clusters are more common with cholecystitis (370). Responses to specific organisms have been characterized. In rats, Yersinia enterocolitica and E. coli alter MMC cycling via free radical generation (371). In germ-free rats, Clostridium tabificum vp 4 promotes intestinal spike-burst activity (Fig. 38-12) (372). This organism, as well as Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11, reduce the period of the MMC and accelerate small intestinal transit. Conversely, micrococcus luteus prolongs MMC periodicity. Likewise, the heat labile enterotoxin of E. coli prolongs phase II of the MMC, increases the MMC period, and accelerates phase III propagation velocities in pigs (373). In vitro, E. coli endotoxin modifies peristalsis

SMALL INTESTINAL MOTILITY / 955 40

40

40 p = 0.08

35

p <0.02

p <0.01

35

35

30

30

25

25

25

20

20

20

15

15

15

min

30

Germfree

A

Lactobacilli & difidobacteria

Germfree

B

Clostridium l.

Germfree

E. coli

C

FIG. 38-12. The intestinal migrating myoelectric complex period (in minutes) before and after introduction of specific intestinal microflora. Lactobacilli and bifidobacteria (A) and Clostridium tabificum (B) reduce the period of cycling and accelerate transit, whereas Escherichia coli (C) and micrococcus tend to prolong the periodicity of the complex and delay transit. (Reproduced from Husebye and colleagues [372], by permission.)

and blunts the inhibitory effects of adrenoceptor agents on motor function (374). Similarly, lipopolysaccharide reduces spontaneous and acetylcholine-evoked contractions in isolated rabbit small intestinal segments (375). In rats given Serratia marcescens endotoxin, ileal responses are suppressed (376). This effect is reversed by a lipoxygenase inhibitor, indicating mediation by peptidoleukotrienes. The disruptive effects of lipopolysaccharide on intestinal motility also are prevented by submandibular gland peptide T, its carboxyl-terminal fragment tripeptide FEG, and its D-isomer feG, which modulate anaphylactic reactions (377). In pigs, endotoxin infusion accelerates jejunal MMC migration and increases MMC cycling frequencies (378). Finally, in the endothelin receptor null rat model of long-segment Hirschsprung’s disease, abnormal intestinal flora with enriched Enterobacteriaceae and Bacteroidaceae colonies increase macrophage populations in the muscle layers with associated damage to the ICC-MYs and loss of electrical rhythmicity (379). Nonbacterial infections elicit similar dysmotility patterns in experimental models. In rats, fasting intestinal myoelectric activity is increased with induction of spike potential bursts by Hymenolepis diminuta infection (380). This response requires continuity of the enteric nervous system, as evidenced by their prevention by intestinal transaction (381). Gastrointestinal transit is slowed and ileal smooth muscle is thickened in mice infected with Schistosoma mansoni (382). These animals exhibit increased in vitro ileal contractility, which is mimicked by incubation with IL-1β. Nippostrongylus brasiliensis enhances jejunal motor responsiveness to carbamylcholine and neurokinin A, an effect prevented by mast-cell degranulation inhibition with BrX537A (383). Increased responses to CCK after infection

with this organism are blocked by vagotomy (384). With peak inflammation at day 14 of infection with N. brasiliensis, there is a loss of NK1 receptors in myenteric neurons and in substance P–immunoreactive nerve endings (385). By day 30 of infection, there is complete loss of ICCs in the deep muscular plexus. The greatest volume of information on the role of the immune system on gut function has been provided by study of the nematode T. spiralis, which produces trichinosis. In rats, motor disruption is prominent with generation of GMCs (386). T. spiralis reduces jejunal myenteric neuron membrane potential and increases neuronal metabolic activity (387). The organism inhibits myenteric nerve activity and reduces acetylcholine and norepinephrine release with peak effects 6 days after infection (388). T. spiralis reduces intestinal substance P by 73% and VIP levels by 59% and markedly inhibits NOS type 2 gene transcription, protein expression, and enzyme activity (389,390). Damage to ICCs with slow-wave destabilization is observed (391). In previously infected rats, subsequent exposure to T. spiralis larvae evokes muscle contraction, which is blocked by a serotonin antagonist and the mast cell stabilizer doxantrazole (392). Jejunal muscle from T. spiralis–infected rats exhibits increased responses to muscarinic agonists, CCK, and serotonin (393,394). Nerve and smooth muscle effects of T. spiralis may persist for 1 to 2 months after resolution of acute infection (395,396). Several pathways participate in the motor effects of T. spiralis infection. Macrophages and lymphocytes penetrate the intestinal longitudinal muscle, and cellular protrusions contact individual myocytes (397). Inhibition of myenteric nerve activity with T. spiralis infection is preserved in athymic rats, suggesting participation of T-cell

956 / CHAPTER 38 independent pathways. Conversely, increased contractions to muscarinic agonists and serotonin are not observed in athymic animals, indicating T-cell–dependent responses as well (388,393). Furthermore, reconstitution of CD4 T cells to mice that are athymic, CD4 cell deficient, or major histocompatibility complex II deficient restores the enhanced muscle contractility (398). IL release appears to mediate many effects of T. spiralis infection. Nerve effects of the organism are blocked by an IL-1 antagonist, whereas increases in muscle contractility are blunted in IL-5–deficient mice (399). In mice, selective in vitro cyclooxygenase-2 inhibition reduces smooth muscle responses to agonist stimulation (400). Finally, motor responses to T. spiralis infection are prevented by treatment with an antibody to nerve growth factor, although the inflammatory infiltrate is unaffected by this therapy (401).

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964 / CHAPTER 38 371. Scott RB, Gall DG, Diamant SC. Intestinal motility during acute Yersinia enterocolitica enteritis in rabbits. Can J Physiol Pharmacol 1989;67:553–560. 372. Husebye E, Hellstrom PM, Sundler F, Chen J, Midtvedt T. Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats. Am J Physiol Gastrointest Liver Physiol 2001;280:G368–G380. 373. Yao G, Wolinski J, Zabielski R. Effect of Escherichia coli heat-labile enterotoxin on the myoelectric activity of the duodenum in weaned pigs. J Vet Med A Physiol Pathol Clin Med 2004;51:106–112. 374. Fruhwald S, Herk E, Scholl G, Shahbazian A, Hammer HF, Metzler H, Holzer P. Endotoxin pretreatment modifies peristalsis and attenuates the antipropulsive action of adrenoceptor agonists in the guinea-pig small intestine. Neurogastroenterol Motil 2004;16: 213–222. 375. Rebollar E, Arruebo MP, Plaza MA, Murillo MD. Effect of lipopolysaccharide on rabbit small intestine muscle contractility in vitro: role of prostaglandins. Neurogastroenterol Motil 2002;14: 633–642. 376. Ezberci F, Tekin E, Onuk E. Protective effect of a lipoxygenase inhibitor, nordihydroguaretic acid, on the ileal motor disturbances induced by Serratia marcescens endotoxin in rats. Surg Today 2001;31:497–501. 377. Mathison R, Lo P, Tan D, Scott B, Davison JS. The tripeptide feG reduces endotoxin-provoked perturbation of intestinal motility and inflammation. Neurogastroenterol Motil 2001;13:599–603. 378. Bruins MJ, Luiking YC, Soeters PB, Akkermans LM, Deutz NE. Effect of prolonged hyperdynamic endotoxemia on jejunal motility in fasted and enterally fed pigs. Ann Surg 2003;237:44–51. 379. Suzuki T, Won KJ, Horiguchi K, Kinoshita K, Hori M, Torihashi S, Momotani E, Itoh K, Hirayama K, Ward SM, Sanders KM, Ozaki H. Muscularis inflammation and the loss of interstitial cells of Cajal in the endothelin ETB receptor null rat. Am J Physiol Gastrointest Liver Physiol 2004;287:G638–G646. 380. Dwinell MB, Bass P, Oaks JA. Intestinal myoelectric alterations in rats chronically infected with the tapeworm Hymenolepis diminuta. Am J Physiol 1994;267:G851–G858. 381. Dwinell KL, Bass P, Zou F, Oaks JA. Small intestinal transections decrease the occurrence of tapeworm-induced myoelectric patterns in the rat. Neurogastroenterol Motil 2002;14:349–356. 382. Moreels TG, De Man JG, Bogers JJ, De Winter BY, Vrolix G, Herman AG, Van Marck EA, Pelckmans PA. Effect of Schistosoma mansoniinduced granulomatous inflammation on murine gastrointestinal motility. Am J Physiol Gastrointest Liver Physiol 2001;280: G1030–G1042. 383. Gay J, Fioramonti J, Garcia-Villar R, Bueno L. Alterations of intestinal motor responses to various stimuli after Nippostrongylus brasiliensis infection in rats: role of mast cells. Neurogastroenterol Motil 2000;12:207–214. 384. Gay J, Fioramonti J, Garcia-Villar R, Bueno L. Enhanced intestinal motor response to cholecystokinin in post-Nippostrongylus brasiliensisinfected rats: modulation by CCK receptor and the vagus nerve. Neurogastroenterol Motil 2001;13:155–162. 385. Faussone-Pelligrini MS, Gay J, Vannucchi MG, Corsani L, Fioramonti J. Alterations of neurokinin receptors and interstitial cells of Cajal

386. 387. 388. 389. 390.

391.

392. 393. 394. 395.

396. 397. 398.

399.

400.

401.

during and after jejunal inflammation induced by Nippostrongylus brasilensis in the rat. Neurogastroenterol Motil 2002;14:83–95. Cowles VE, Sarna SK. Trichinella spiralis infection alters small bowel motor activity in the fed state. Gastroenterology 1991;101:664–669. Palmer JM, Wong-Riley M, Sharkey KA. Functional alterations in jejunal myenteric neurons during inflammation in nematode-infected guinea pigs. Am J Physiol 1998;275:G922–G935. Collins SM, Blennerhassett P, Vermillion DL, Davis K, Langer J, Ernst PB. Impaired acetylcholine release in the inflamed rat intestine is T-cell independent. Am J Physiol 1992;263:G198–G201. Greenwood B, Palmer JM. Neural integration of jejunal motility and ion transport in nematode-infected ferrets. Am J Physiol 1996; 271:G48–G55. Bian K, Harari Y, Zhong M, Lai M, Castro G, Weisbrodt N, Murad F. Down-regulation of inducible nitric-oxide synthase (NOS-2) during parasite-induced gut inflammation: a path to identify a selective NOS2 inhibitor. Mol Pharmacol 2001;59:939–947. Der T, Bercik P, Donnelly G, Jackson T, Berezin I, Collins SM, Huizinga JD. Interstitial cells of Cajal and inflammation-induced motor dysfunction in the mouse small intestine. Gastroenterology 2000;119:1590–1599. Vermillion DL, Ernst PB, Scicchitano R, Collins SM. Antigeninduced contraction of jejunal smooth muscle in the sensitized rat. Am J Physiol 1988;255:G701–G708. Fox-Robichaud AE, Collins SM. Altered calcium-handling properties of jejunal smooth muscle from the nematode-infected rat. Gastroenterology 1986;91:1462–1469. Torrents D, Vergara P. In vivo changes in the intestinal reflexes and the response to CCK in the inflamed small intestine of the rat. Am J Physiol Gastrointest Liver Physiol 2000;279:G543–G551. Venkova K, Palmer JM, Greenwood-Van Meerveld B. Nematodeinduced jejunal inflammation in the ferret causes long-term changes in excitatory neuromuscular responses. J Pharmacol Exp Ther 1999; 290:96–103. Barbara G, Vallance BA, Collins SM. Persistent intestinal neuromuscular dysfunction after acute nematode infection in mice. Gastroenterology 1997;113:1224–1232. Rociu E, Stoker J, Eijkemans MJ, Lameris JS. Normal anal sphincter anatomy and age- and sex-related variations at high-spatial-resolution endoanal MR imaging. Radiology 2000;217:395–401. Vallance BA, Galeazzi F, Collins SM, Snider DP. CD4 T cells and major histocompatibility complex class II expression influence worm expulsion and increased intestinal muscle contraction during Trichinella spiralis infection. Infect Immun 1999;67:6090–6097. Vallance BA, Blennerhassett PA, Deng Y, Matthaei KI, Young IG, Collins SM. IL-5 contributes to worm expulsion and muscle hypercontractility in a primary T. spiralis infection. Am J Physiol Gastrointest Liver Physiol 1999;277:G400–G408. Barbara G, De Giorgio R, Deng YK, Vallance B, Blennerhassett P, Collins SM. Role of immunologic factors and cyclooxygenase 2 in persistent postinfective enteric muscle dysfunction in mice. Gastroenterology 2001;120:1729–1736. Torrents D, Torres R, De Mora F, Vergara P. Antinerve growth factor treatment prevents intestinal dysmotility in Trichinella spiralisinfected rats. J Pharmacol Exp Ther 2002;302:659–665.

CHAPTER

39

Function and Regulation of Colonic Contractions in Health and Disease Sushil K. Sarna and Xuan-Zheng Shi Function and Spatiotemporal Characteristics of Colonic Contractions, 966 Rhythmic Phasic Contractions, 966 Giant Migrating Contractions, 967 Tonic Contraction, 969 Cellular and Molecular Regulation of Colonic Contractions, 969 Excitation–Contraction Coupling, 970 Excitation–Inhibition Coupling, 976 Enteric Neurons, 976

Colonic Motor Dysfunction, 982 Irritable Bowel Syndrome, Chronic Idiopathic Constipation, and Functional Diarrhea, 982 Inflammatory Bowel Disease and Acute Colonic Inflammation, 985 Acknowledgment, 987 References, 987

The requirements of colonic motility function are complex. Extensive stirring and turning over of luminal contents is required for uniform exposure of colonic contents to the mucosal surface for optimal absorption of water and electrolytes. The luminal contents must be propelled rather slowly in the anal direction to account for slow absorption rate of the tight colonic mucosa. The chyme emptied from the ileum must be retained temporarily in the ascending colon where the bulk of water absorption takes place. Similarly, the feces must be held temporarily in the descending and sigmoid colons so that voluntary defecation can occur under suitable conditions. In contrast with all of the above motility requirements that require sluggish propulsion, mixing, and temporary storage, the colonic contents must be moved rapidly, but infrequently, over relatively long distances (mass movements) so that they are not oversolidified by excessive absorption of water. The feces in the distal colon also must be propelled rapidly during defecation so that its expulsion is accomplished in a short time. Whereas the luminal contents emptying from

the terminal ileum to the ascending colon are mostly fluid, they get progressively harder as they are propelled distally in the colon. Therefore, the contractions that regulate colonic propulsion and turning over of colonic contents must be of adequate amplitude and duration to effectively mix and propel fluid, semisolid, and nearly solid contents. It is apparent that such diverse motility requirements could not be met by the generation of just one type of contraction by colonic smooth muscle cells. In fact, the colon smooth muscle cells generate three distinct types of contractions (1,2): (1) rhythmic phasic contractions (RPCs); (2) giant migrating contractions (GMCs); and (3) tonic contractions (TCs). The physical characteristics of these three different types of contractions and their spatiotemporal patterns, function, and regulatory mechanisms are markedly different from each other. The colon is the final major organ in the gastrointestinal (GI) tract. Its motility function has a major impact on the frequency and timing of defecation, as well as on the consistency and shape of stools. These variables differ markedly among different species. For example, humans and dogs normally defecate once or twice a day and have formed feces. In contrast, rodents and guinea pigs that are used frequently in experimental studies defecate frequently and produce pellets. Consequently, the composition of the above three types of colonic contractions differs among species.

S. K. Sarna and X-Z Shi: Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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966 / CHAPTER 39 Furthermore, the spatiotemporal characteristics of the same type of contraction may also be different in the colons of different species. For example, the GMCs occur only a few times a day in human and dog colons, but in the rat colon, these contractions occur frequently, but irregularly. It is therefore crucial to identify the type of contraction being studied in diagnostic evaluation of colonic motor function and in investigation of its mechanisms of regulation. Failure to do so may make it difficult to extrapolate the findings from animal models to human colonic function in health and disease, and it may also lead to contradictory results because the regulatory mechanisms of different types of colonic contractions differ. For example, the phasic contractions are regulated in time and space by slow waves, but the other two types of contractions, GMCs and tone, are independent of slow waves. The colon wall, like the rest of the gut, has two outer muscle layers: the circular muscle layer and the longitudinal muscle layer. In humans, the longitudinal muscle layer is bundled into three-taenia coli with a thin longitudinal muscle coat over the rest of the surface. In other species, such as dogs, the longitudinal muscle layer is a thin, uniform coat around the circumference. The contractions of the circular muscle cells partially or completely occlude the lumen; hence, they are effective in mixing, turning over, and propulsion. The contractions of the longitudinal muscle shorten the length of the colon segment, which has minimal effect on mixing and propulsive functions. For this reason, longitudinal muscle contractions are not discussed further in this chapter, although they remain of academic interest.

FUNCTION AND SPATIOTEMPORAL CHARACTERISTICS OF COLONIC CONTRACTIONS

(A) Aboral

(B) Aboral

(C) Aboral

(D) Aboral

FIG. 39-1. The role of different spatiotemporal patterns of contractions in mixing and propulsion in the gut. (A) A lumen occluding rhythmic phasic contraction (RPC) that propagates distally propels the digesta ahead of it up to the distance of its propagation. This type of contraction is seen generally in the terminal antrum and proximal small intestine. (B) A largeamplitude, long-duration, and rapidly propagating contraction, such as a giant migrating contraction (GMC), occludes a long segment of the gut and is more effective in propulsion of digesta. This type of contraction is seen after each swallow in the esophagus and infrequently in the terminal ileum and proximal colon, and it also precedes defecation. (C) An RPC that only partially occludes the lumen and propagates causes some propulsion and some mixing. This type of contraction generally is seen in the gastric corpus and most of small intestine. (D) Spatially disorganized RPCs that may or may not occlude the lumen cause much mixing with little or no propulsion. These types of contractions are seen in the terminal ileum and the entire length of the colon.

Rhythmic Phasic Contractions The spatiotemporal characteristics of contractions determine whether they propel or produce back-and-forth movements to mix, stir, and turn over the fecal inertial (3–6). The contractions that propagate and have large enough amplitude to occlude the lumen propel the luminal contents trapped ahead of them and up to the distance that they propagate (Fig. 39-1A). The propulsion is more effective with larger amplitude and longer duration of contractions (see Fig. 39-1B). The speed of propulsion is nearly the same as the velocity of propagation of the contraction. The frequency of propagated contractions determines the total volume of digesta propelled per unit time. If the amplitude of contraction is not large enough to occlude the lumen, the propulsion is less effective, because some of the digesta escapes from the partial opening of the lumen to cause mixing and turning over as the contraction propagates (see Fig. 39-1C). In contrast, if the contractions occur totally randomly, they will produce mostly mixing and turning over of luminal contents and little or no propulsion regardless of whether they occlude the lumen (see Fig. 39-1D). The spatiotemporal patterns of

RPCs in the colon are predominantly random in nature (Fig. 39-1D and 39-2). The majority of these contractions do not occlude the lumen, and either they do not propagate at all or they propagate over short distances. In species, such as humans and dogs, the RPCs are responsible for the mixing, stirring, turning over, and slow net distal propulsion of colonic contents. The colonic circular muscle cells in humans and dogs generate two subtypes of RPCs, the short-duration and longduration RPCs (7–9). These two types of phasic contractions are regulated by different mechanisms (see later). The shortduration RPCs occur most frequently, whereas the longduration RPCs occur in bursts of variable durations, generally lasting a few minutes. The purpose of longduration RPCs is to aid in the mixing and slow propulsion of feces as it solidifies. They are distinguished from the GMCs by their smaller amplitude and shorter distances of propagation. The rat colon also generates phasic contractions, but their amplitude in the intact conscious state is small compared

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 967 C-2

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FIG. 39-2. Colonic contractions recorded from human colon after a meal. The rhythmic phasic contractions are disorganized in time and space, as shown in Figure 39-1D. The numbers after C indicate the distances of the side openings in the manometric tube from the tip of the manometric tube. The proximal end of the recording tube was positioned close to the cecum.

with that of GMCs that occur frequently in this species and other rodents. The phasic contractions in the intact rat colon are often unrecordable by conventional strain transducers that concurrently record GMCs (10). The phasic contractions in the rodent colon are therefore unlikely to play a major role in their colonic motility function. The recordings of colonic motility in the intact conscious state have not been made from the mice or the guinea pig, but recordings from ex vivo segments and muscle strips in organ baths show that, in these species, the phasic contractions also are small in amplitude and inconspicuous when compared with GMCs (10–12).

Giant Migrating Contractions Giant migrating contractions in the human and canine colons are large-amplitude lumen occluding contractions that propagate rapidly (about 1 cm/sec) in the distal direction over appreciable distances to produce mass movements (13–23). In these species, the spontaneous GMCs occur randomly at about two to five times a day in the proximal, middle, or descending segments of the colon. During defecation, these contractions propagate from their point of origin to the rectum to rapidly expel the feces (Fig. 39-3). Notably, among the three types of colonic contractions, GMCs are the only ones that produce descending inhibition that results in the suppression of spontaneous contractions in the distal segment and relaxation of its tone (13,15,24). The large amplitude of GMCs provides the stimulus for descending inhibition (25). The descending inhibition accommodates the large volume of luminal contents being propelled rapidly by the GMCs. This descending inhibition also relaxes the internal

anal sphincter for easy passage of feces during defecation (24) (Fig. 39-4). By contrast, the phasic contractions move colonic contents back and forth or propel small quantities of fecal material slowly over short distances; therefore, descending relaxation is not required to accommodate them (5) (Fig. 39-5). In addition to producing the above normal physiologic functions, the GMCs may also be associated with three major symptoms of colonic motor dysfunction: diarrhea, constipation, and visceral pain (15,22,26–29). A significant increase in the frequency of GMCs and resulting mass movements rapidly propel the colonic contents and decrease their contact time with the mucosa that is required for adequate water and electrolyte absorption (15,30). In addition, if these GMCs propagate up to the rectum, they produce urgency of defecation and frequent bowel movements (19). In contrast, if the GMCs are totally absent or their frequency is reduced significantly, the prolonged contact of fecal material with colonic mucosa may harden the stools, resulting in constipation (24,26,31). Note that hardening of the feces may make it difficult for the RPCs to propel it slowly in the distal direction. The sensation of pain requires three components: (1) a mechanical or chemical stimulus that triggers the receptors at the periphery of primary afferent sensory neurons; (2) the autonomic neurons that transmit the coded signal to the central nervous system (CNS); and (3) the CNS that processes the signal for perception. GMCs provide the first component by strongly contracting the colon wall or by distending the distal segment above nociceptive threshold due to the large bolus being propelled ahead of it (15,27,32). Under normal conditions, the amplitude of

968 / CHAPTER 39 Urge

Urge

Stool expulsion

Small bowel

Cecum

Hepatic flexure

Splenic flexure

Sigmoid colon 80 mmHg 1 min

FIG. 39-3. Spontaneous giant migrating contractions (GMCs) in human colon. The second and third GMCs resulted in urge to defecate, and the fourth GMC resulted in defecation. Note descending inhibition preceding GMCs. (Reproduced from Bampton and colleagues [19], by permission.)

GMCs does not exceed the nociceptive threshold, and descending relaxation prevents the triggering of nociceptive receptors by the large bolus being propelled by the GMC (Fig. 39-6). Therefore, the infrequent occurrence of GMCs in healthy subjects is not perceived to be painful. However, there are at least three factors that may cause GMCs to be perceived as painful in motility disorders. First, their amplitude may increase beyond the sensory threshold (see Fig. 39-6). As an example, the administration of edrophonium, a cholinesterase inhibitor, enhances the amplitude and duration of GMCs in the normal esophagus in response to swallowing. Under these conditions, the subjects perceive pain of esophageal origin with the occurrence of each GMC after a swallow, whereas no pain was felt when the esophageal GMCs were of normal amplitude and duration. Second, the descending inhibitory reflex may be insufficient to inhibit the ongoing spontaneous contractions or relax the receiving segment, or both. In this case, the GMCs would attempt to propel the luminal contents against resistance produced by the spontaneous contractions, and it may overdistend the descending segment beyond the nociceptive threshold (Fig. 39-7). This situation occurs if GMCs are attempting to expel the feces during defecation and the anal sphincters fail to relax. Third, the nociceptive threshold may be decreased because of allodynia and hyperalgesia, so that GMCs of normal amplitude and duration are perceived to be painful. The above critical roles of GMCs underscore their importance in the physiology and pathophysiology of colonic motor

function. In patients with ulcerative colitis (UC) and irritable bowel syndrome (IBS), visceral pain is of intermittent nature, suggesting that it is triggered by an intermittent stimulus, such as a GMC. Note that even though patients with IBS and inflammatory bowel disease (IBD) have allodynia and hyperalgesia, they do not feel pain unless a balloon is distended in their colons. Therefore, both neuronal hypersensitivity and the occurrence of a stimulus, such as a GMC or distension in the colon, are required to produce the sensation of intermittent abdominal cramping. In contrast with the human and canine colons, the motor activity of the rat colon is composed predominantly of GMCs (10). The GMCs in the rat colon occur at a frequency of about 20 to 40 per hour (Fig. 39-8). However, unlike the human and canine colons, most GMCs in the rat colon do not propagate or propagate only over short distances, and hence are responsible for the gradual distal propulsion of the pellets (10). The human and canine colons generate GMCs in the intact conscious state. However, muscle strips or full-thickness rings prepared from the colons of these species do not generate GMCs in organ baths. Instead, they generate only tone and phasic contractions. This situation is similar to that in the stomach and small intestine where phase III activity of the migrating motor complex can be recorded in the intact conscious state but not in the anesthetized state, in in vitro muscle strips, or in ex vivo segments. By contrast, the rat and mouse colonic circular muscle continues to generate GMCs

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 969 60 s 0-80 mmHg A

T

T

D

D

D AS

of spontaneous contractions and relaxation of tone ahead of it. As noted earlier, these conditions are met by the GMCs (13,25,32). The RPCs do not produce descending inhibition/ relaxation (3,5,38). If this did not occur, the regularly occurring spontaneous phasic contractions in the sigmoid colon and rectum would relax the internal anal sphincter with each occurrence. Instead, the sphincters relax only with the occurrence of a GMC that precedes defecation (see Fig. 39-4). The original term peristalsis therefore applies only to GMCs. The phasic contractions are propagating or nonpropagating, but without descending inhibition.

Tonic Contraction The tone of the colon is increased after ingestion of a meal (39,40) (Fig. 39-9). The duration of the increase in tone depends on the volume and caloric intake of food (40). The primary effect of increase in tone is to narrow the lumen size. The decrease in lumen size by itself has little direct effect on propulsion and mixing. However, the narrowing of the lumen makes the RPCs more effective in propulsion and mixing because phasic contractions of smaller amplitude may now be able to occlude the lumen more effectively.

AS

FIG. 39-4. Descending relaxation of anal sphincter ahead of a giant migrating contraction that began in the ascending colon and propagated to the sigmoid colon. A, ascending colon; AS, anal sphincter does not relax when rhythmic phasic contractions occur in the colon; D, descending colon; S, sigmoid colon; T, transverse colon. (Reproduced from Bassotti and colleagues [24], by permission.)

in muscle strips, rings, and ex vivo segments from the rodent colon (12,33,34). These in vitro preparations also can generate phasic contractions and tone. The generation of spontaneous GMCs in ex vivo segments may be the underlying reason that the isolated segments from rodents and guinea pigs propel artificial fecal pellets (35,36), whereas the ex vivo segments of the human and the canine colons cannot do it. Thus, the rodent and guinea pig colons can serve as excellent models to investigate the cellular mechanisms of generation of GMCs. However, caution may be required to extrapolate the data on colonic propulsion from these species to humans, because in the human colon, the slow net distal propulsion occurs predominantly by the spatially uncoordinated phasic contractions. GMCs produce only infrequent mass movements. Peristalsis and Propagating Contractions Bayliss and Starling (37) initially used the term peristalsis to describe a strong contraction that begins above a local stimulus and propagates distally, whereas producing inhibition

CELLULAR AND MOLECULAR REGULATION OF COLONIC CONTRACTIONS The gut smooth muscle cells are unique in the sense that they can concurrently generate different types of contractions (see previous section). By contrast, the vascular smooth muscle and skeletal muscle cells generate mostly tone, and the cardiac muscle cells generate mostly the phasic contractions. Furthermore, the cardiac muscle cells contract all the time in a monotonous sequence, but the GI smooth muscle cells contract mostly intermittently, and their spatiotemporal patterns of contractions vary markedly from one contraction to the next. This difference between the cardiac and GI cell types requires different types of interactions between them and the neurons that innervate them. The skeletal muscle cells contract in response to a signal from the CNS by releasing excitatory neurotransmitter at the neuromuscular junction. These cells do not determine the timing, duration, or frequency of their contractions. By contrast, in gut organs, the phasic contractions have to be organized as propagating and nonpropagating contractions, which is achieved by smooth muscle cells playing an essential role in determining the timing, duration, and frequency of contractions (41–43). These comparisons indicate a more complex and multifactorial nature of regulatory mechanisms of GI smooth muscle cells when compared with those of other types of excitable cells. The following sections discuss the enteric regulatory mechanisms of regulation of colonic contractions. These mechanisms can be modulated by inputs from the spinal cord, the CNS, and endocrine hormones, but they are not included here.

970 / CHAPTER 39

Giant migrating contractions and transit

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FIG. 39-5. The first radiograph in the top left corner was taken at the beginning of the tracing and the next at the end of it. Randomly occurring rhythmic phasic contractions had little effect on the propulsion of 10 radiopaque markers (filled circles) over a period of about 2 hours. However, a GMC starting at strain gauge transducer C5 emptied all the markers in a 14-cm-long colon segment within 1 minute. Rectangles in the colon show the positions of surgically implanted strain-gauge transducers. C1 to C7 are the seven strain-gauge transducers. The numbers after these symbols indicate their distances from the ileocolonic junction. (Reproduced from Sethi and Sarna [5], by permission.) A. Normal state Threshold for visceral pain

Excitation–Contraction Coupling

B. Disease state Threshold for visceral pain

C. Disease state Threshold for visceral pain (Allodynia and Hyperalgesia)

FIG. 39-6. (A) In the normal state, the maximum amplitudes of both the rhythmic phasic contractions (RPCs) and giant migrating contractions (GMCs) is less than the sensory threshold for visceral pain. (B) The occurrence of a GMC is perceived to be painful if its amplitude increases to more than the sensory threshold because of a pharmacologic stimulus or a defect in the neuromuscular mechanisms regulating its generation. (C) Abdominal pain will be felt with the occurrence of a GMC of normal amplitude if the sensory threshold is reduced by allodynia and hyperalgesia. Note that the amplitude of RPCs is less than the sensory threshold in all three conditions.

A significant degree of our understanding of excitation– contraction coupling has developed from studies on non-GI smooth muscle cells (44–47). This section integrates the information from these studies with that from available studies on colonic smooth muscle cells to explain the generation of different types of contractions by the colon cells. The smooth muscle cells contain actin (thin filament) and myosin (thick filament). Myosin is a hexamer composed of 2 paired 230-kDa heavy chains. Each chain is composed of a head (cross-bridge) and a tail region and 2 light chains, one 20 kDa (regulatory light chain; MLC20) and the other 17 kDa (essential light chain). Smooth muscle cell contraction occurs by interaction between actin and myosin filaments by cross-bridge cycling. In the resting state, the binding of adenosine triphosphate (ATP) to myosin head dissociates it from actin (released state). The phosphorylation of MLC20 hydrolyses ATP to adenosine diphosphate (ADP) and phosphate by ATPase activity. The released energy cocks the myosin head to a new position on actin chain (cross-bridge state). The phosphate is released and the head changes its conformation, resulting in force generation and sliding of myosin and actin filaments past each other to cause cell shortening. The amount of force

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 971

FIG. 39-7. The rhythmic phasic contractions move luminal contents back and forth with slow net distal propulsion. This motion does not cause distension of the distal segment (top). Pain is perceived if the large bolus (mass movement) is being propelled by a giant migrating contraction and distends the distal segment beyond the sensory threshold. This may happen because of a defect in descending inhibition that does not allow the distal segment or the anal sphincter to inhibit its ongoing contractions and relax.

generated and the duration of cell shortening depend on the degree of cross-bridge cycling, which, in turn, depends on the degree and duration of MLC20 phosphorylation. MLC20 is phosphorylated by myosin light chain kinase (MLCK) and dephosphorylated by myosin light chain phosphatase (MLCP). The binding of cytosolic-free calcium to calmodulin activates MLCK (Fig. 39-10). The mammalian MLCP is a trimeric enzyme consisting of a catalytic 37- to 38-kDa subunit PP1c, an associated 110- to 130-kDa regulatory targeting subunit MYPT1, and a tightly bound 20-kDa subunit

C-3

C-6

C-11

C-14

5g C-17 1 min

FIG. 39-8. Spontaneous colonic motor activity recorded from an intact conscious rat by five strain-gauge transducers shows frequent spontaneous giant migrating contractions (GMCs). The arrow indicates rhythmic phasic contractions that were of much smaller amplitude than that of the GMCs. C represents colonic strain-gauge transducers; the numbers after C show their respective distances from the cecum. (Reproduced from Li and colleagues [10], by permission.)

of which the function is unknown (46–48). MYPT1, through its NH2 terminus, binds the MLCP complex to myosin II and enhances the catalytic activity of PP1c. The dephosphorylation rate of myosin II can be reduced by decreasing the binding activity of MYPT1 by phosphorylation (or thiophosphorylation) of Thr-696, by dissociating the subunits MYPT1 and PP1c of MLCP, and by reducing the catalytic activity of PP1c. MLC20 is phosphorylated constitutively in the resting state of the colon cells because of a low concentration of cytosolic-free Ca2+ (70–100 nM) that maintains the basal tone (49). In this state, there is equilibrium between the rates of basal phosphorylation of MLC20 by MLCK and dephosphorylation by MLCP. The phosphorylation of MLC20 is enhanced by increasing the activity of MLCK or by reducing the activity of MLCP. The net phosphorylation of MLC20 is determined, therefore, by the ratios of the activities of MLCK and MLCP in the stimulated state. The activities of MLCK and MLCP are regulated by electromechanical and pharmacomechanical couplings. The electromechanical coupling is caused by the increase in cytosolic-free Ca2+ by membrane depolarization and influx through membrane calcium channels. The pharmacomechanical coupling is caused by the activation of cell signaling pathways by the binding of ligands to their membrane receptors, followed by the activation of kinases that modulate the activity of MLCP or MLCK. Pharmacomechanical coupling also further increases [Ca2+]i by the opening of receptor-operated Ca2+ channels, through nonselective cation channels, and by release of Ca2+ from the intracellular stores to enhance the phosphorylation of MLC20 by MLCK. The key to the generation of different types of contractions by gut smooth muscle cells lies in different contributions of electromechanical and pharmacomechanical couplings in response to an agonist. A given increase of cytosolic-free calcium by electromechanical coupling results in a certain degree of MLC20 phosphorylation, and hence the force of cell contraction. However, if at the same time the activity of MLCP is suppressed by pharmacomechanical coupling to decrease the rate of dephosphorylation of MLC20, the net phosphorylation of MLC20 is increased, resulting in increase in amplitude of contraction. A prolonged activation of pharmacomechanical coupling enhances the duration of contraction. The contribution of pharmacomechanical coupling to the amplitude and duration of force generation is referred to as adjustment of Ca2+ sensitivity (50–53). Overall, the electromechanical coupling in colon cells caused by membrane depolarization is the initiating event for all three types of contractions, and subsequent pharmacomechanical coupling modulates the degree and duration of MLC20 phosphorylation through MLCP or other kinases to generate the different amplitudes and durations of the three types of contractions. Short-Duration Rhythmic Phasic Contractions The colonic smooth muscle cells have a resting membrane potential (RMP) of about −60 to −80 mV (54–57). Similar to

972 / CHAPTER 39 250 230

Intraballoon volume (mL)

210 190 170 150 130 110 90 70

Meal

50 −30

30 Time (min)

60

85

FIG. 39-9. Mean preprandial and postprandial colonic intraballoon volumes in healthy control subjects (filled triangles) and patients with active ulcerative colitis (filled squares). The decrease in intraballoon volume indicates an increase in colonic tone that lasted for more than 85 minutes in healthy control subjects. The meal induced a greater reduction in volume in healthy control subjects than in patients with ulcerative colitis. Also, the increase of tone in patients with ulcerative colitis was of much shorter duration than in healthy control subjects. (Reproduced from Coulie and colleagues [39], by permission.)

the smooth muscle cells in the stomach and the small intestine, the colon cells exhibit omnipresent spontaneous slow waves that periodically depolarize the membrane to the maximum range of about –40 to –50 mV. Each depolarization opens voltage-dependent Ca2+ channels in the membrane to induce calcium influx (58). As indicated earlier, the resulting increase in cytosolic-free Ca2+ binds with calmodulin to activate MLCK and phosphorylate MLC20. However, I-V curves obtained by patch clamping of colonic smooth muscle cells indicate that these channels conduct little calcium current in the membrane potential range of −40 to −50 mV (59). Therefore, there is little or no contraction associated with slow-wave depolarization alone in the intact conscious state. If this did not occur, the colonic smooth muscle cells would display continuous contractions with each slow-wave depolarization and there would be no intermittent quiescent states in colonic motor activity. By contrast, the membrane potential during each depolarization of cardiac muscle cells exceeds 0 mV, and the I-V curves for these cells indicate Imax in the voltage range of about 10 to 20 mV. Thus, each depolarization of cardiac cells generates a strong contraction. In contrast with in vivo recordings, regularly occurring RPCs of small amplitude are recorded with each colonic slow wave in in vitro muscle strips or full-thickness rings (54,58, 60,61). However, these tissues are under stretch that opens nonselective cation channels to enhance influx of Ca2+ and release Ca2+ from intracellular stores to phosphorylate MLC20 through MLCK (62–64). Even though the slow-wave depolarization by itself does not cause a colonic smooth muscle contraction of significant

amplitude, the depolarization produced by it and the accompanying Ca2+ influx are essential for colonic short-duration RPCs to occur in response to an excitatory neurotransmitter, such as acetylcholine (ACh). The Ca2+ influx induced by slowwave depolarization produces the initial phosphorylation of MLC20, which is then enhanced by ligand-initiated pharmacomechanical coupling. The established physiologic neurotransmitter at the neuroeffector junction for the stimulation of all three types of colonic contractions is ACh. Substance P is colocalized with choline acetyltransferase (ChAT) in motor neurons, and its pharmacological application can induce colonic contractions (65,66). However, it has not been established that the inhibition of its receptors in intact conscious state blocks spontaneous contractions. On the contrary, atropine, a nonselective inhibitor of muscarinic receptors, inhibits all spontaneous colonic contractions. The GI smooth muscle cells express the muscarinic M2 and M3 receptors in a ratio of about 4:1 (61,67–69). Although the expression of M2 receptors predominates, the gut smooth muscle contractions in the normal state are mediated predominantly by M3 receptors (70). The M2 receptor, however, makes more significant contribution to cell contraction in the inflamed state (70). The M3 are seven-transmembrane-spanning receptors coupled to Gαq/11 protein (71–73). This G protein is coupled to nonspecific cation channels (74), the opening of which allows additional calcium influx, further depolarization of membrane, and phosphorylation of MLC20. The activation of Gαq/11 also activates phospholipase C (PLC) to hydrolyze phosphatidylinositol biphosphate (PIP2) and produce diacylglycerol (DAG) and inositol triphosphate (IP3) (71). IP3 acting

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 973

FIG. 39-10. Potential signaling pathways for the generation of the three types of contractions in colonic circular smooth muscle cells. The electromechanical coupling initiated by slow-wave depolarization induces Ca2+ influx through voltage-gated calcium channels (VGCC; L-type or Cav1.2 channels). The binding of acetylcholine (ACh) to muscarinic M3 receptors initiates multiple signaling pathways that induce further Ca2+ influx through receptor-gated Ca2+ channels (RGCC), releases intracellular Ca2+ from inositol 1,4,5-triphosphate (IP3)–sensitive stores, inhibits myosin light chain phosphatase (MLCP), and increases the phosphorylation of 20-kDa regulatory light chain of myosin (MLC20) via protein kinase C (PKC), Rho kinase (ROK), and release of arachidonic acid (AA) to enhance the amplitude and/or duration of contraction, which is the key for the generation of three types of contractions. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CaM, calmodulin; CPI-17, PKC-potentiated inhibitor protein of 17 kDa; cPLA2, cytosolic phospholipase A2; DAG, diacylglycerol; GEFs, guanine nucleotide exchange factors; MYPT1 and PP1c, MLCP regulatory and catalytic subunits respectively; MLCK, myosin light chain kinase; NO, nitric oxide; PIP2, phosphatidyl inositol 4,5,-biphosphate; PLC, phospholipase C; RhoGDI, guanosine diphosphate (GDP) dissociation inhibitor; SR, sarcoplasmic reticulum; VIP, vasoactive intestinal peptide. (See Color Plate 22.)

on its receptors on sarcoplasmic reticulum releases Ca2+ from intracellular stores. The combined increase of Ca2+ from all three sources depolarizes the membrane beyond an excitation threshold, prolongs the slow-wave plateau potential, and induces a burst of rapidly recurring depolarizations (spikes) superimposed on it. The membrane depolarization during each spike is in the range of +10 to +30 mV, which is the range of Imax, causing maximal inward Ca2+ current through L-type calcium channels, and hence further phosphorylation of MLC20 by Ca2+-calmodulin/MLCK complex that produces a significant amplitude of contraction (59). When slow wave terminates and membrane repolarizes because of the inactivation of Ca2+ channels or the opening of Ca2+-activated K+ channels, or both, the spikes cease, cytosolic-free Ca2+ decreases, and MLCP dephosphorylates MLC20 to return the smooth muscle to its basal resting tone. This happens even if there is continued release of ACh, and that is why both enteric neurons and smooth muscle cells

together regulate the occurrence of short-duration RPCs. The duration of phasic contractions generated by this mechanism is correlated with the amplitude and duration of spike burst, and it cannot exceed the duration of the depolarization phase of the slow wave. In addition, the maximum frequency of short-duration phasic contractions cannot exceed the frequency of slow waves. The electromechanical coupling described earlier is caused by slow waves occurring within each circular muscle cell. There has been intense interest for several decades to determine whether these slow waves are generated within the circular muscle cells or are generated by other cells in the gut wall and then passively propagated through different layers of the circular muscle. This question has arisen because physical separation of the submucosal layer together with submucosal plexus from the rest of the full-thickness colonic muscle strips abolishes the slow waves in circular muscle cells (57,75–77). Later studies found interstitial cells

974 / CHAPTER 39 of Cajal at the circular muscle/submucosal plexus border (ICC-SMs), and it was hypothesized that these are the pacemaker cells for the generation of slow waves in colonic circular muscle cells (57,78–81). In support of this hypothesis, it was found that single, isolated ICCs continue to generate slow waves, whereas single, isolated circular muscle cells are quiescent (78). The idea is that the slow waves in ICC-SMs drive the slow-wave depolarizations in the innermost circular muscle cells, and from there they spread passively into the full thickness of the circular muscle layer. Notably, the total number of ICC-SMs is small when compared with the total number of driven circular muscle cells (82–85). In particular, the number of ICC-SMs is sparse in the human colon (82,85), and the ICCs in the human colon make rare gap junctions with other ICCs or to neurons (83). Therefore, the circular muscle cells would be a large sink for the current generated by the relatively few ICC-SMs. The RMP of circular muscle cells, as well as the amplitude of slow-wave depolarization recorded from in vitro muscle strips, decreases from the submucosal plexus to the myenteric plexus (54,77). Despite this, some form of regeneration of slow waves may be necessary to spread them into the entire bulk of circular muscle cells. There are also reports indicating that, even though the single dispersed circular muscle cells do not generate spontaneous slow waves, cultured cells on forming small aggregates exhibit spontaneous slow waves with superimposed spike bursts (86). These findings indicate that the smooth muscle cells are close to the threshold of generating spontaneous slow waves on their own. However, the slow waves are still absent in about 70% of the intact circular muscle strips when the submucosal layer is separated from them (57,87). Thus, even in their natural state, the circular muscle cells require some input from the submucosal border to generate slow waves. Experiments also indicate that the addition of carbachol or tetraethylammonium to the medium depolarizes the RMP that makes isolated single smooth muscle cells to generate spontaneous slow waves and spikes (87). The possibility, therefore, cannot be ruled out that the input required from the submucosal border is to depolarize the smooth muscle RMP rather than provide a trigger for the generation of slow waves. This mechanism would then generate spontaneous slow waves in smooth muscle cells without the need for them to act as a large sink for current generated by the ICC-SMs. Note that the spontaneous slow waves are absent in the circular muscle of the distal colon of lethal spotted ls/ls mice even though the ICC-SMs are normal (88). Thus, there may be factors other than the absence of ICC-SMs that can suppress spontaneous slow waves in circular muscle cells. The W/Wv mouse model that lacks specific types of ICC (ICC-MYs) in the small intestine and intramuscular ICCs (ICC-IMs) in the stomach has been used to investigate their role in the generation of slow waves in those organs (89). However, the ICC-SMs, ICC-IMs, and ICC-MYs are not absent in the colon of W/Wv mouse; therefore, this model has not proved useful in determining their in vivo role in the generation of colonic slow waves. However, spontaneous RPCs at the frequency of slow waves are present in colonic

muscle strips of the homozygous Ws/Ws rats, which lack c-kit–immunoreactive ICC-SMs and ICC-MYs (90). Despite this controversy, it is notable that the phasic contractions regulated by slow waves in the rodent colon may play only a minor role in its motility function because this function is regulated predominantly by GMCs that are not regulated by slow waves (10,34). Long-Duration Rhythmic Phasic Contractions The long-duration phasic contractions in the colon are regulated by similar electromechanical and pharmacomechanical couplings, except that the initial membrane depolarization for these contractions is not by the omnipresent slow wave. The colonic smooth muscle cells spontaneously, but intermittently, generate a burst of high-amplitude depolarizations at a frequency of about 25 to 40 cycles/min (7,8). In the presence of ACh, each depolarization in this burst is superimposed by a spike burst. Because of the high frequency of these depolarizations, the individual spike bursts nearly fuse, resulting in a long-duration contraction (7,8). In circular muscle strips devoid of the myenteric and submucosal plexuses, these high-frequency oscillations are generated by greater concentrations of BaCl2 and Bay K 8644, indicating that they may be of myogenic origin in response to excessive membrane depolarization or opening of Ca2+ channels (91). The cell signaling for the long-duration contractions has yet to be investigated. Note that the duration of these contractions is longer than that of each slow-wave depolarization; therefore, they are not regulated by them. The duration of these contractions depends on the duration of high-frequency oscillations called the contractile electrical complex (7). Giant Migrating Contractions As noted earlier, GMCs occur infrequently and randomly in human and dog colons. The duration of a GMC is severalfold longer than that of a slow-wave depolarization; therefore, its occurrence is also not initiated or regulated by the electromechanical coupling normally initiated by the slow-wave depolarization. There is also no other known spontaneous and random oscillations of membrane depolarization that could be related to GMCs. However, studies on the rat colonic circular muscle cells with intracellular electrodes show that the membrane is depolarized in the range of −40 to −50 mV that is superimposed with an intense burst of spikes that lasts throughout this depolarization during the occurrence of a GMC (92). The GMCs in the human colon are also associated with intense bursts of spikes (93,94). The duration of each spike burst is about the same as that of a GMC. The GMCs in the rat colon, as well their associated depolarizations, and the spike bursts are blocked by Cav1.2 channel inhibitor nifedipine or verapamil (10,33,92). By contrast, the slow-wave depolarizations are not blocked by Cav1.2 channel blockers, suggesting that the depolarizations associated with GMCs are caused by Ca2+ influx through Cav1.2

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 975 channels. Therefore, the membrane depolarizations associated with GMCs are entirely caused by pharmacomechanical coupling, and the increase of [Ca2+]i with them is sufficient to induce spikes on each depolarization. The separation of the longitudinal muscle-myenteric plexus layer from the rest of the colon wall abolishes the spontaneous generation of GMCs in the rat colon (92). At least in the muscle bath environment, therefore, an input from the longitudinal muscle-myenteric plexus may be required for the generation of GMCs in the rodent colon. This note of caution is necessary because several studies indicate that the cholinergic neural function in muscle strips is compromised (10,95). For example, the contractions generated in muscle strips are not blocked by muscarinic receptor antagonist atropine, but in intact animals, the spontaneous contractions are inhibited completely by atropine. The stretching of tissue in vitro may make up for the loss of normal cholinergic input to muscle cells so that they can generate the same type of contractions observed in vivo. The reductionism approach is indispensable to investigate the cellular and molecular mechanisms of regulation of motility function, but it is essential to establish the relevance and role of these findings in integrated systems before they can be used to understand the physiology and pathophysiology of gut motility function. Accumulating evidence from experiments in intact conscious animals indicates that GMCs are stimulated by the release of an excessive amount of ACh for a longer duration at the neuroeffector junction. It is well known that the normal release of ACh in short bursts stimulates RPCs, but it does not stimulate GMCs (96). In contrast, close intraarterial or intravenous administration of neostigmine stimulates GMCs in the colon and the small intestine (13,97). Neostigmine, a cholinesterase inhibitor, prevents the degradation of ACh released from neurons and causes its accumulation at the neuroeffector junction in the colon wall. Under physiologic conditions, the excessive and sustained release of ACh may occur because of a slow excitatory postsynaptic potential (sEPSP; see later). For example, in the small intestine, close intraarterial infusion of calcitonin gene–related peptide (CGRP) stimulates a GMC (98). Electrophysiologic studies indicate that CGRP also stimulates sEPSPs (99). The stimulation of GMCs by CGRP is blocked by atropine and hexamethonium, indicating that it acts on presynaptic neurons to release ACh at the neuroeffector junction. However, it must be added that in colon the sEPSPs may be stimulated by a neurotransmitter other than CGRP because its close intraarterial infusion does not stimulate GMCs in this organ (96). The evidence cited earlier suggests that GMCs are stimulated primarily by pharmacomechanical coupling triggered by the large and prolonged release of ACh at the neuroeffector junction. First, the ACh opens the receptor-operated Ca2+ channels to cause influx of calcium and long-duration membrane depolarization due to the sustained release of ACh. This influx is essential because the spontaneous GMCs are blocked by Cav1.2 calcium channel blockers (10,98,100). ACh also releases Ca2+ from IP3-sensitive intracellular stores to depolarize the membrane beyond excitation threshold,

resulting in a long train of spike bursts that causes additional influx of Ca2+ with each spike. Because of the long duration of spike bursts, the total accumulation of Ca2+ in the cytosolic compartment may be greater than that caused by shortduration spike bursts during a slow-wave depolarization. That would result in greater phosphorylation of MLC20 and a longer duration to generate greater force and duration of cell contraction, which are the defining characteristics of GMCs. The binding of ACh to its receptors on colonic smooth muscle cells triggers multiple signaling pathways to activate kinases, such as protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and Rho kinase (ROK), as well as release of arachidonic acid (AA) from membrane phospholipids and by catalysis of DAG (101–104). Each of these molecules has the ability to increase Ca2+ sensitivity and prolong the duration of MLC20 phosphorylation without any additional change in [Ca2+]i (44,47,50,51,105–108). This portion of pharmacomechanical coupling further enhances the duration and amplitude of GMCs. The potential roles of these molecules to enhance Ca2+ sensitivity and prolong the phosphorylation of MLC20 are discussed later. At least 12 different isozymes of PKC have been identified (109). The expression and function of these isozymes are cell specific. The colonic smooth muscle cells express the classical α, β, and γ, novel δ and ε, and atypical ι, λ, and ξ isozymes of PKC (102). However, only the classical α and β and the novel ε isozymes translocate to the membrane in response to ACh, indicating a possible role of these isozymes in colonic smooth muscle contraction (102). Notably, although the activation of the classical isozymes is Ca2+ dependent, that of the novel isozymes is Ca2+ independent. Therefore, PKC ε may be activated under low [Ca2+]i conditions. The PKC enzymes phosphorylate serene and threonine residues on their target proteins. Studies in other smooth muscle cells indicate that the activation of PKC isozymes by phorbol esters increases the phosphorylation of MLC20 without any concurrent increase in [Ca2+]i (106,110,111). This effect is mediated by the inhibition of dephosphorylation of MLC20 by MLCP, rather than by its increased phosphorylation by MLCK. PKC phosphorylates CPI-17, a 17-kDa peptide first isolated from the porcine aorta (112). CPI-17 is phosphorylated at Thr-38 by PKC (113,114). The degree of expression of CPI-17 is cell specific. In general, CPI-17 expression is greater in tonic smooth muscle cells than in phasic smooth muscle cells (115). However, its relative expression in colonic smooth muscle cells that can generate both tonic and phasic contractions is unknown. Evidence from studies on single smooth muscle cells and in intact dogs indicates that PKC activation plays a critical role in the generation of GMCs in colonic circular smooth muscle cells. The inhibition of PKC α, β, and ε isozymes by their respective pseudo-substrate inhibitors concentration dependently suppresses the contractile response to ACh in single dispersed cells, indicating its potential role in enhancing Ca2+ sensitivity (102). Close intraarterial infusion of substance P stimulates colonic GMCs, and this response is blocked by chelerythrine, a PKC inhibitor (116). Taken together, the in vitro and in vivo data indicate that PKC activation in

976 / CHAPTER 39 colonic circular muscle cells may be critical for the generation of GMCs by increasing Ca2+ sensitivity. The stimulation of muscarinic receptors in colonic circular smooth muscle cells also releases AA. The inhibition of AA release concentration dependently inhibits the ACh-induced cell shortening (102). The precise mechanism by which AA increases Ca2+ sensitivity in colonic circular smooth muscle cells is unknown. But, in other smooth muscle cells, AA increases Ca2+ sensitivity by the following actions: (1) dissociation of the catalytic subunit PP1c of MLCP from the regulatory targeting subunit MYPT1 (107) that reduces the affinity of PP1c for MLC20 (see Fig. 39-10); (2) phosphorylation of MYPT1 at a site other than Thr-696, which is the phosphorylation site of ROK; and (3) activation of PKC ξ that phosphorylates CPI-17, as discussed earlier (106). Currently, there is no direct evidence to indicate which types of contractions are enhanced by the release of AA in colonic smooth muscle cells. The stimulation of colonic smooth muscle cells by ACh or other agonists also activates MAPKs: extracellular signal– regulated kinase 1/2 (ERK1/ERK2), p38, and c-Jun N-terminal kinase (104,117–119). The inhibition of ERK1/2 by PD98059 or p38 by SB203580 concentration dependently inhibits ACh-induced shortening of colonic circular smooth muscle cells, indicating a potential role of these kinases in mediating pharmacomechanical coupling in these cells (118,119). The activated ERKs phosphorylate caldesmon to block its inhibitory effect on the activation of actomyosin ATPase, and hence to enhance the amplitude and increase the duration of smooth muscle contraction (120). Notably, ERKs also phosphorylate caldesmon in vascular smooth muscle cells, but that does not increase Ca2+ sensitivity (121). p38, in contrast, phosphorylates heat shock protein 27 (HSP27) through MAPK-activated protein kinase-2, a Ser/Thr kinase. The phosphorylation of HSP27 facilitates actin-myosin association, and hence smooth muscle contraction (122). Tone The stimulation of Gαq/11-coupled receptors in smooth muscle cells also activates Rho A (GTP.Rho A), a small (20-kDa) monomeric GTPase (123). Rho is retained in the cytoplasm in its inactive form, GDP.Rho A, in the resting state of smooth muscle cells (see Fig. 39-10). The activation of Rho A is catalyzed by guanine nucleotide exchange factors to exchange GTP for GDP, which translocates it to the membrane, where it binds to the regulatory binding domain of serine/threonine ROK to autophosphorylate it through a conformational change (44,105,108). The activated ROK phosphorylates MYPT1 at Thr-696, which reduces the dephosphorylation rate of MLC20, thereby enhancing Ca2+ sensitivity (see Fig. 39-10). ROK has been proposed to mediate slow TC in vascular smooth muscle cells, but its role in increasing tone in colonic smooth muscle cells is unknown. However, the sustained contraction in rabbit gastric smooth muscle cells is mediated by ROK and PKC (124–126). The TC mediated by ROK-induced phosphorylation of

MYPT1 occurs under low [Ca2+]i conditions. The low-level increase in [Ca2+]i produced by Ca2+ influx may be required to phosphorylate MLC20 because this contraction in vivo is blocked also by the inhibition of Cav1.2 channels. The sustained increase in tone is not regulated by slow waves, and it is not associated with spike bursts. The TC may, therefore, be generated primarily by pharmacomechanical coupling.

Excitation–Inhibition Coupling The excitation–inhibition coupling in smooth muscle cells is mediated by cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). These cyclic nucleotides are generated respectively by the activation of adenylate cyclase in response to vasoactive intestinal peptide (VIP) and guanylate cyclase in response to nitric oxide (NO). The inhibition of cell contraction is activated by reducing [Ca2+]i or by enhancing the activity of MLCP to dephosphorylate MLC20 (44). Therefore, basically, the excitatory neurotransmitter ACh and the inhibitory neurotransmitters NO and VIP compete for the phosphorylated state of MLC20 by using the same cell signaling pathways. Little information is available for the specific signaling pathways for excitation–inhibition coupling in colonic circular smooth muscle cells. Studies in other cells indicate, however, that there is significant cross talk between cAMP/protein kinase A (PKA) and cGMP/PKG (44,127). At low concentrations, VIP activates PKA exclusively, whereas at greater concentrations it can cross-activate PKG-I (128–130). Both kinases inhibit PLCβ1-dependent IP3 formation by phosphorylation of regulator of G protein signaling 4 and acceleration of the activation of GTP-bound Gαq/11 (125). PKG-I also inhibits IP3-induced Ca2+ release by direct phosphorylation of the sarcoplasmic IP3 receptors. These kinases can also inhibit cell contraction by Rho A–dependent and –independent signaling by targeting MYPT1, CPI-17, and Triton (44,125). As discussed in the previous section, each of these signaling pathways makes specific contributions to the generations of the three types of colonic contractions. Therefore, theoretically, it should be possible to selectively stimulate or inhibit each type of colonic contraction to normalize motility dysfunctions in colonic motor disorders (see later). Enteric Neurons Types of Enteric Neurons and Their Functional Significance Enteric neurons are classified according to function, morphology, electrophysiologic characteristics, and chemical coding (131–135; see Chapter 21). This chapter uses the functional classification of neurons to explain the generation of different types of colonic contractions discussed in previous sections: (1) motor neurons, (2) interneurons, and (3) intrinsic sensory neurons (ISNs). Morphologically, most enteric neurons are of two types, Dogiel types I and II (136). The type I neurons

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 977 are monoaxonal with lamellar dendrites. The Dogiel type II neurons are multiaxonal with or without dendrites. From a functional point of view, the type I neurons have a narrow field of influence, whereas the type II neurons have a broader field of influence because of their multiaxonal structure. Therefore, type II neurons can integrate information from a larger field and, in turn, send the effector signal over a larger field. Consequently, these neurons may be best suited to sense the state of affairs at one or more locations in the gut wall and the lumen and to coordinate the motor response over a broad field, such as that to make a contraction occur simultaneously around the circumference or over a short segment of the colon. According to electrophysiologic characteristics (see Chapter 23), the neurons are classified as S-type and AH (afterhyperpolarization)-type. Both types of neurons generate sEPSPs and fast EPSPs (fEPSPs). An fEPSP lasts only for about a few milliseconds, and it is usually associated with a single action potential. Accordingly, it would release a small quantity of ACh for a short duration, but the total release of the neurotransmitter can be enhanced by a series of fEPSPs. By contrast, the depolarization during an sEPSP lasts longer than 10 seconds. Consequently, an sEPSP may induce a long series of action potentials resulting in the continuous release of neurotransmitter for a longer period. This results in the accumulation of the neurotransmitter at the neuroeffector junction. Several studies show that cell signaling pathways activated by low and high concentrations of the same agonists may differ and they may also depend on the duration of application of the agonist to its receptors on smooth muscle cells (96,98,137). In AH-type neurons, each action potential is followed by a short duration of hyperpolarization. There is also delayed afterhyperpolarization after each sEPSP (131). The functional significance of afterhyperpolarization is to limit the maximum frequency of action potentials. Presumably, this would allow time for mobilization of neurotransmitter to be released by the next action potential. Note that this may not be required for fESPSs because they are of short duration and the number of action potentials generated by them is limited. Consequently, the S-type neurons do not exhibit afterhyperpolarization. The functional significance of delayed afterhyperpolarization is to prevent the immediate generation of another sEPSP that allows time for synthesis or mobilization of neurotransmitters for the next release. The AH neurons also exhibit an inflection on the repolarization phase of their action potentials (138). Although this may be a useful feature for the identification of these neurons, it may have no functional implication.

broaden their field of influence in this direction (138,139) (Fig. 39-11). This configuration facilitates the concurrent release of neurotransmitter around the circumference, resulting in ring-type contractions. The narrow field of influence of these neurons in the radial direction of the colon wall is desirable for close coordination of neurotransmitter release and slow waves in circular muscle cells beneath the ganglia containing the neuronal cell body. The excitatory and inhibitory inputs to the smooth muscle cells act like the accelerator and the braking system of an automobile, respectively. In the resting state, a low-level release of the inhibitory neurotransmitters keeps the smooth muscle cells in a state of low tension. Close intraarterial administration of tetrodotoxin in intact animals or its addition to muscle bath blocks conduction of enteric neural action potentials and almost immediately stimulates contractions by blocking the basal inhibitory neurotransmitter release (140,141). As discussed earlier (see Excitation–Contraction Coupling), the release of ACh from the cholinergic excitatory

Motor Neurons

FIG. 39-11. Three examples of Dogiel type I circular muscle motor neurons. Images of the cell bodies are shown at high power, and the projections are at a lower power. The axons branch to promote a circumferentially orientated plexus in the circular muscle. Some of the axons extended farther than shown here. Inset shows photomicrograph of part of the circular muscle innervation (boxed area). Scale bar = 20 µm in insets. (Reproduced from Nurgali and colleagues [139], by permission.)

Two types of motor neurons project from the myenteric plexus to the circular muscle layer: the excitatory and inhibitory motor neurons. These are primarily the Dogiel type I neurons, suggesting a narrower field of influence. However, the axons of the motor neurons branch extensively along the circumference within the circular muscle layer to

978 / CHAPTER 39 neurons overcomes the low-level inhibitory input and the cells contract. This is equivalent to stepping on the accelerator of an automobile. However, if there is also a concurrent increase in the release of the inhibitory neurotransmitters because of neural reflexes, the excitation–inhibition coupling generated by them partially or completely overcomes the excitation–contraction coupling and suppresses the contractions, as well as relaxes the tone of smooth muscle cells. This is equivalent to stepping strongly on the brake system of an automobile while it is running. Among the inhibitory neurotransmitters, both NO and VIP are physiologically important in providing inhibitory input to smooth muscle cells to compete with the excitatory input and determine the final amplitude and duration of contractions. These neurotransmitters also produce descending inhibition (142,143). Note that the descending inhibition results from the excitation–inhibition coupling exceeding the excitation– contraction coupling in the distal segment. The signal to enhance excitation–inhibition coupling in this case comes from a proximal location through the interneurons. The projection of excitatory and inhibitory motor neurons from the myenteric plexus to the circular muscle layer has been mapped by retrograde labeling with DiI (144). The tendency has been to conclude that the ChAT-containing excitatory neurons project orally from the cell body in the myenteric plexus and the NOS-containing inhibitory neurons project anally. However, the data in these studies indicate that the bulk of these projections into the muscle layers are within 2 to 4 mm oral or anal from their cell bodies in the myenteric ganglia and the center of gravity of these projections in either direction is about 2 mm (144) (Fig. 39-12).

Notably, an average colonic RPC occupies about a 10-mm length of the colon, whereas a GMC occupies a segment several-fold longer than 10 mm. It is, therefore, unlikely that the oral or anal projections of the motor neurons over a few millimeters would materially contribute to the ascending and inhibitory reflexes. Therefore, the excitatory and inhibitory motor neurons project mostly radially into the circular muscle layer to regulate contractions locally. The oral and anal communications may be conducted primarily by the interneurons that project greater than 10 mm in the 2 directions and may regenerate the signals at the oral and aboral ganglia to produce excitation or inhibition over much longer distances. Morphologic data indicate that the enteric motor neurons containing VIP, NO, and ACh have their varicosities close to both the circular muscle cells and ICCs (145–150). This has raised the question whether the colonic circular smooth muscle cells and ICC-SMs receive their neural inputs independently or ICCs are obligatory intermediates in the neural regulation of circular muscle contractility. Furthermore, if ICCs are the obligatory intermediates, how is the neural signal transmitted from the ICCs to the entire thickness of the circular muscle layer. One evidence in favor of ICCSMs acting as obligatory intermediates in the colon is that the contact distances between them and the nerve varicosities are shorter (25 nm) than those between the circular muscle cells and the nerve varicosities (25–250 nm) (80,148,150,151). However, electrical field stimulation of enteric neurons in canine colonic muscle strips generates inhibitory junction potentials (IJPs) in both the circular muscle cells and the ICC-SMs (152). Furthermore, the

70 80 70 Number of neurons labelled

Number of neurons labelled

60 50 40 30 20 10

50 40 30 20 10

0

0 −12 −10 −8 −6 −4 −2

A

60

0

2

4

6

−20 −16 −12 −8 −4

8 10 12

Oral Distance from application site (mm) Anal

B

0

4

8

12

16

Oral Distance from application site (mm) Anal

FIG. 39-12. (A) Histogram showing the distribution of choline acetyltransferase (ChAT) immunoreactivity in motor neurons in all ChAT-labeled preparations. Black bars indicate ChAT+; white bars indicate ChAT–. (B) Histogram showing the distribution of nitric oxide synthase (NOS) and vasoactive intestinal peptide (VIP) immunoreactivity in circular muscle motor neurons in all NOS/VIP-labeled preparations. Black bars indicate NOS+/VIP+; gray bars indicate NOS+/VIP–; white bars indicate NOS–/VIP–. (Reproduced from Porter and colleagues [144], by permission.)

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 979 characteristics of the IJPs in the circular muscle cells are not altered by removal of the ICC-SMs from the muscle strips by separation of the circular muscle-submucosal border. This indicates that neural stimulation generates IJPs in circular muscle cells independent of any input from the ICC-SMs. Both types of cells express VIP receptors and respond to exogenous NO by generating IJPs. The longer distances between the nerve varicosities and circular smooth muscle cells therefore are not an impediment to their direct neural regulation. In vitro experiments indicate that neurotransmitters, such as NO, can effectively diffuse over a distance of at least 225 µm, which is two-log-order magnitude greater than the maximum distance between nerve varicosities and circular smooth muscle cells (153). The varicosities in motor neurons are arranged randomly along their axons (see Fig. 39-11). There is no morphologic evidence that ICC-SMs or ICC-IMs follow these axons and make contact with the varicosities on a 1:1 basis. Direct innervation of smooth muscle cells occurs in the longitudinal layer that lacks ICC-IMs. Exogenous NO increases [Ca2+]i in freshly dissociated ICCs, but it decreases in freshly dissociated circular smooth muscle cells (153). The decrease of [Ca2+]i in smooth muscle cells is consistent with the direct inhibitory role of NO in these cells. In addition, the depolarization of dissociated ICCs by BAY K 8644 reduces [Ca2+]i in an adjacent single circular muscle cell less than 225 µm away. An NOS inhibitor blocks this effect. This effect is not observed between two adjacent circular muscle cells (same distance apart), even though immunohistochemical methods confirmed the presence of NOS in both circular muscle cells. It was postulated that ICCs amplify the NO signal by generating more NO or an NO-dependent compound to relay the inhibitory signal to smooth muscle cells. However, the limitation of this theory is that the ICCs could convey this inhibitory signal only to the smooth muscle cells with which they make gap junctions. Further transmission of this signal would stop because the process is not reproduced by smooth muscle cells. Even if this mechanism were to explain the modulatory effect of ICCs in nitrergic inhibition of smooth muscle contraction, it remains unexplained how ICCs would transmit the signals to circular smooth muscle cells in response to ACh and VIP. The ICCs are nonneural cells; there is no evidence that they can synthesize or amplify these neurotransmitters. As discussed in the previous section, pharmacomechanical coupling is essential for smooth muscle cells to generate all three types of colonic contractions. ACh and VIP must bind directly to their receptors on smooth muscle cells to activate this coupling. Any other form of signal, such as voltage or current, from the ICCs to smooth muscle cells will not activate the pharmacomechanical coupling. It appears, therefore, that the circular muscle cells and ICCs receive independent cholinergic and noncholinergic, nonadrenergic neural inputs. Interneurons The interneurons in the colon are largely Dogiel type I (154). These interneurons project greater than 10 mm in the

oral, anal, and circumferential directions to communicate signals among nearby ganglia (138). These neurons typically project more than 10 mm in these directions. The descending projections of interneurons outnumber the ascending projections by a ratio of 4:1 (138). The interneurons may synapse on excitatory and inhibitory motor neurons in the target ganglia to stimulate the release of their respective neurotransmitters, or they may synapse on other interneurons to form a relay system that can project the signal over longer distances (Fig. 39-13). About 90% of the ascending interneurons contain ChAT, and about 50% of descending interneurons contain ChAT alone or together with NOS. In both cases, the cholinergic neurons synapse on nicotinic receptors. Consequently, hexamethonium blocks oral/aboral transmission, as well as the stimulation of motor neurons in the lateral direction (97,155,156). The ascending and descending cholinergic interneurons that synapse or inhibitory motor neurons produce ascending and descending relaxation, respectively, and those that synapse or cholinergic excitatory motor neurons produce ascending or descending excitation (see Fig. 39-13). The precise role of NOS immunoreactivity in interneurons remains unknown. It has been postulated that these neurons suppress the release of ACh from the cholinergic descending interneurons (157,158). A small number of descending interneurons in the colon are also immunoreactive for VIP, tachykinins, 5-hydroxytryptamine (5-HT), neuropeptide Y, galanin, somatostatin, and met-enkephalin (159–162). Intrinsic Sensory Neurons The ISNs are also called intrinsic primary afferent neurons. However, there is some controversy about the use of this term (163; see Chapter 23). The term afferent implies projection of the peripheral neurons to the CNS, and these neurons do not do that. In addition, one cannot rule out that the enteric neurons that sense a mechanical or chemical stimulus in the lumen or in the gut wall may synapse directly on the motor neurons so that their separation into primary and secondary order neurons may not be necessary. The generic term intrinsic sensory neurons (ISNs) is used in this chapter. The word sensory indicates that these neurons detect a physiologic stimulus. The morphologic and electrophysiologic characteristics of ISNs are known for the human, mouse, and guinea pig colons (138,164–168). The cell bodies of these neurons are located in the submucosal and myenteric ganglia with axonal endings in the mucosa, muscle layers, and muscularis submucosa to detect the presence of digesta and its chemical nature, as well as wall tension due to a contraction or descending distension ahead of a GMC (132,169–171). Most of the sensory neurons are of the multiaxonal Dogiel type II/AH. In response to a chemical or mechanical stimulus from the lumen, the sensory signals are transmitted to the large field of influence of the ISNs to generate a signal for contraction simultaneously over an appreciable length of colon segment (1–2 cm). In the guinea pig small intestine,

980 / CHAPTER 39 Aboral

Oral

AII DII DEI AEI

AII DII DEI AEI

ACh

NO, VIP M3

AII DII DEI AEI

ACh

NO, VIP M3

NO, VIP

ACh M3

Excit. threshold Slow wave Contraction

Neuronal cell body Varicosity

AII = Ascending inhibitory interneuron DII = Descending inhibitory interneuron DEI = Descending excitatory interneuron AEI = Ascending excitatory interneuron

FIG. 39-13. Schematic diagram showing the regulation of colonic contractions by smooth muscle cells, motor neurons, interneurons, and intrinsic sensory neurons (ISNs). The ISNs detect digesta in the lumen or tension/distension in the gut wall and transmit the signal to the excitatory and inhibitory motor neurons. The neurotransmitter of excitatory motor neurons is acetylcholine (ACh) and of inhibitory motor neurons nitric oxide (NO) and vasoactive intestinal peptide (VIP). The binding of ACh to M3 receptor on circulai smooth muscle cells initiates excitation–contraction coupling in these cells. The smooth muscle cell contracts when there is a net increase in the phosphorylation of 20-kDa regulatory light chain of myosin (MLC20). For rhythmic phasic contractions (RPCs), the slow wave in circular smooth muscle cells regulates the timing and frequency of contractions at each location. The interneurons project orally and aborally. They synapse on both the excitatory and inhibitory motor neurons in both directions to provide ascending excitation and inhibition, as well as descending excitation and inhibition. The interneurons also synapse on other interneurons to transmit the signal to the next ganglion in the chain. The interneurons also project in the circumferential direction (not shown). Filled circles indicate neuronal cell body; open circles indicate varicosity. AEI, ascending excitatory interneuron; AII, ascending inhibitory interneuron; DEI, descending excitatory interneuron; DII, descending inhibitory interneuron.

mucosal stimuli evoke both fast and slow EPSPs on postsynaptic neurons (171). Tetrodotoxin, hexamethonium, ω-conotoxin, and low Ca2+/high Mg2+ in the medium block the fEPSPs, indicating that their generation requires neuronal conduction and at least one nicotinic synapse. The sEPSPs are blocked by CGRP antagonists but not by hexamethonium, indicating that their generation is mediated by CGRP. These data are consistent with the findings in the small intestine of intact conscious dogs that close intraarterial infusions of CGRP stimulate GMCs (98). However, CGRP infusions in the colon do not stimulate GMCs, and therefore it is likely that its sEPSPs may be mediated by another neurotransmitter, such as one of the tachykinins. Close intraarterial infusions of substance P in intact dogs stimulate GMCs, but in this case, substance P acts directly on smooth muscle cells (96).

Notably, a significant proportion of the enteric neurons (both Dogiel types I and II) exhibit spontaneous fEPSPs and sEPSPs (138). These neurons therefore release neurotransmitters at the neuroeffector junction in the absence of any sensory input. This spontaneous neuronal excitation is the source of contractions in the intact, completely empty colon. Several types of neurons generate spontaneous rhythmic or arrhythmic activity (172–174). In the stomach and the small intestine, this rhythmic activity of the enteric neurons generates the well-established pattern of migrating motor complex in the interdigestive state (156,175). Thus far, spontaneous rhythmic groups of contractions (colonic migrating motor complexes) have been reported only from the dog colon (176), and these may be generated by the rhythmic bursts of EPSPs in a subset of presynaptic colonic enteric neurons. Note that in the canine colon, the migrating motor complexes

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 981 are not disrupted by ingestion of a meal. Therefore, the activity of these spontaneously active neurons may be superimposed on that stimulated by input from sensory neurons in the postprandial state. Enteric Reflexes Enteric nervous system (ENS) in the gut is called the “little brain” (177). The term reflects the ability of the ENS to integrate sensory information from multiple locations and translate it into an appropriate signal to other parts of the same organ, to other organs, and to higher centers to produce a specific motility response. Gastrocolonic reflex is an example of interorgan reflex and it prepares the colon for ingestion of a meal. Similarly, the entry of fresh digesta in the proximal colon alters the motor activity of its entire length by generating intrinsic and extrinsic reflexes (176). In general, reflexes over shorter distances are mediated by interneurons, whereas those over longer distances are mediated by extrinsic neurons. In this regard, both excitatory and inhibitory signals can be transmitted in the oral and the anal directions. Among the various reflexes in the colon, the peristaltic reflex has received the most attention. The studies of this reflex have advanced our understanding of the oral and anal projections of interneurons, their chemical coding, orientation of ISNs, and the potential neurotransmitters of excitatory and inhibitory motor neurons. The peristaltic reflex in the gut was proposed originally by Bayliss and Starling in 1899 (37) as an ascending contraction and a descending relaxation in response to localized mucosal stimulation. Bayliss and Starling’s idea was that on sensing the presence of a bolus in the lumen the gut initiates a contraction proximal to it, which propagates distally and propels the bolus, whereas the distal segment relaxes to accommodate it. This concept has been called the “law of the intestine” because of its discovery in an era when proclamation of laws in physics was prevalent. However, physics is a precise discipline where a precise and constant relation exists between the stimulus and the response. This may not be the case for the postprandial function of the gut. Bayliss and Starling correctly identified the ascending excitation and descending inhibition in response to focal stimulation of the mucosa in an empty segment (equivalent to fasting state). However, the application of the peristaltic reflex alone to explain postprandial propulsion has been overinterpreted because of the limited understanding of the different types of contractions generated by the gut, their spatiotemporal patterns and their regulatory mechanisms at the end of the nineteenth century. In the postprandial state, the chemical and mechanical stimulation of the mucosa by the digesta is continually variable, and it is concurrently all over the length of the colon, or at least over a long segment of it. This widely dispersed and moment-to-moment variable mucosal stimulation generates the ascending excitatory and descending inhibitory signals at the same locations throughout the colon, because the descending segment of one point of stimulation is the ascending segment of another point of

stimulation distal to it. In essence, therefore, the amplitude of contractions at any single location depends on the competition between the excitation–contraction and excitation– inhibition couplings discussed earlier in this chapter. The only gut contraction that produces descending inhibition is the GMC. However, then the question arises whether the descending inhibition ahead of a GMC is produced by the strong contraction of the GMC that exceeds an excitation threshold or by a stimulus from the mucosa. It appears that the large amplitude of the GMC produces descending inhibition because this inhibition is blocked by a transaction and anastomosis in the small intestine that interrupts the interneurons, which conduct the signal for descending inhibition, but it allows propulsion of the bolus ahead of the GMC (25). According to the above, the original paradigm that Bayliss and Starling (37) proposed can be modified as follows to explain postprandial propulsion (see Fig. 39-13). First, the mucosal stimulation by the digesta stimulates ascending excitatory and descending inhibitory signals via the ISNs, interneurons, and motor neurons (see earlier). However, because of concurrent stimulations over an entire segment, the release of excitatory (ACh) and inhibitory neurotransmitters (NO and VIP/pituitary adenylyl cyclase–activating polypeptide [PACAP]) compete at each location in the segment by activating excitation–contraction and excitation–inhibition couplings in circular smooth muscle cells. However, the release of the excitatory neurotransmitter, ACh, predominates, which causes an overall increase in the frequency of contractions after the fresh digesta enters the lumen of the gut. The result is intermittent RPCs of variable amplitude caused by the continually variable mucosal stimulation by the digesta. Second, the spatial organization of slow waves and excitation–contraction coupling in smooth muscle cells determine the timing, frequency, propagation velocity, direction of propagation, and distance of propagation of RPCs. In the colon, the slow waves are highly variable in frequency and are not phase-locked at adjacent locations (7,8,178), which results in largely disorganized phasic contractions causing back and forth mixing movements and slow net distal propulsion. Third, the RPCs do not produce descending inhibition because their amplitude does not exceed the excitation threshold to stimulate the descending interneurons. The GMCs produce descending inhibition because of their large amplitude. Electrophysiologic, immunohistochemical, and contractile studies, together with the measurements of release of neurotransmitters associated with the in vitro mucosal stroking or stretch-initiated peristaltic reflex, have significantly advanced our knowledge of the characteristics of enteric neurons and their neurotransmitters (36,142,143,164,167,179–185). Mucosal stroking releases 5-HT from enterochromaffin (EC) cells to stimulate 5-HT1P/ 5-HT4 receptors on sensory nerve endings, causing the release of CGRP and ACh in myenteric ganglia (36,182,186). According to different reports, the 5-HT4 receptors are localized on nerve endings in the mucosa, where they are

982 / CHAPTER 39 stimulated together with the 5-HT1P receptors directly by 5-HT released from EC cells, or they are localized on presynaptic nerve endings in the myenteric plexus, where they modulate the release of CGRP (171). There is no evidence for or against the idea that sensory neurons synapse directly on the motor neurons or on interneurons that then transmit the signal to the motor neurons. The former situation is more likely. Studies using the three-compartment flat-sheet preparations indicate that the release of CGRP in response to mucosal stroking is mediated by ISNs, whereas that in response to muscle stretch is mediated by extrinsic primary afferent neurons, inferior mesenteric ganglia, and dorsal root ganglia (180,182). The studies of in vitro peristaltic reflex confirm that ACh and substance P are the neurotransmitters of the excitatory motor neurons in the colon. The release of ACh mediates the response to low-level mucosal stimulation, whereas both ACh and substance P mediate the response to stronger stimuli (155). This finding suggests a potential role of substance P under conditions outside the normal range. For example, in the canine colon, close intraarterial administration of substance P stimulates the large-amplitude GMCs, whereas ACh stimulates only the phasic contractions (96). VIP/ PACAP and NO are the primary neurotransmitters of descending inhibition. Nicotinic receptors are critical for synaptic transmission between the sensory neurons and interneurons/motor neurons, because hexamethonium blocks the reflex responses almost completely in response to mucosal stroking or muscle stretch in vitro and in vivo. Somatostatin acting on somatostatin receptor 1 also modulates the descending inhibition in the three-compartment flat-sheet preparations (142). Furthermore, Met-enkephalin acting on δ-opioid receptors exerts a continuous inhibitory restraint on both ascending excitatory and descending inhibitory reflexes. Neuropeptide Y acting on Y2 receptors may also exert an inhibitory influence on the release of substance P in the ascending excitatory reflex (187). Ex-vivo segments of the rodent and guinea pig colons propel an artificial pellet introduced at their oral end. This model is useful in investigations of the roles of neurotransmitters/neuromodulators and their receptors in in vitro colonic propulsion. These studies have confirmed the essential role of ACh as the neurotransmitter of excitatory motor neurons and of nicotinic receptors in synaptic transmission in colonic propulsion (35,36,188,189). Atropine and hexamethonium completely block the propulsion of fecal pellets in ex vivo segments. NK1, NK2a, and NK2b receptor antagonists also partially or completely inhibit the propulsion of fecal pellets in isolated segments of the rat colon. The above studies also indicate that the inhibition of NOS by Nω-Nitro-L-Arginine (L-NNA) or that of VIP receptors by VIP10-28 decreases the velocity of propulsion of pellets. The conclusion was that the slowing of propulsion velocity was caused by the blockade of the descending inhibition. This may not be true, because the inhibition of NO or VIP by adding their antagonists to the medium will block the inhibitory neurons simultaneously in the entire ex vivo segment, not just in the descending segment. Note that the inhibitory input to smooth muscle cells competes with the excitatory input for

the generation of contractions at all locations. Selective blockade of the inhibitory input upsets this balance and alters the spatiotemporal patterns of contractions everywhere in the ex vivo segment to delay the transit (190,191). No concurrent recordings of motor activity in the experiments on ex vivo segments are available, but these could provide valuable additional information on the types of contractions they generate and the alterations in their spatiotemporal patterns in response to test substances. The information on the role of 5-HT3 and 5-HT4 receptors obtained from this model is contradictory. The administration of 5-HT3 or 5-HT4 receptor antagonist into the lumen of an ex vivo colon segment inhibits the propulsion velocity of pellets (35). However, the addition of either one of these antagonists to the bath has no effect; but when added together, they concentration dependently inhibit the propulsion velocity (36). Thus, it appears that 5-HT release from EC cells plays a critical role in the propulsion of fecal pellets in this model, but it is not certain whether the 5-HT3 and 5-HT4 receptors are arranged in parallel or in series to regulate colonic propulsion. Currently, it has not been established that the ex vivo segments of nonrodent colons also can propel artificial pellets. The ex vivo segments and muscle strips of the rodent colons generate GMCs, whereas those of nonrodent colons do not (11,12,33,34,192). The generation of GMCs in ex vivo segments may be required for propulsion of pellets. Furthermore, the slow net distal propulsion in nonrodent colons occurs by RPCs, whereas that in the rodent colons occurs by GMCs. Consideration of these differences is important in extrapolating functional and mechanistic data from the rodent colons to the human colon.

COLONIC MOTOR DYSFUNCTION The colon is the last major organ in the GI tract. As such, its motility function plays a critical role in regulating the frequency and consistency of stools. Consequently, abnormalities in the generation and spatial organization of the three types of colonic contractions play a major role in manifesting the symptoms of irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), chronic idiopathic constipation, acute diarrhea, and chronic functional diarrhea. Furthermore, under abnormal conditions of allodynia, hyperalgesia, and impaired descending inhibition, particularly of the anal sphincters, the occurrence of GMCs in the colon may also produce the sensation of intermittent abdominal cramping. The following sections discuss the roles of the three types of colonic contractions in the above motility disorders of the colon. Irritable Bowel Syndrome, Chronic Idiopathic Constipation, and Functional Diarrhea IBS is characterized by intermittent abdominal cramping often relieved by bowel movement and accompanied by diarrhea (IBS-D), constipation (IBS-C), or alternating diarrhea and constipation (IBS-C/D). The precise cause of this

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 983 disease is not understood, but accumulating evidence suggests that the symptoms result from abnormal motility patterns and neuronal hypersensitivity. Stress and food sensitivity may also precipitate or exaggerate the symptoms of IBS (193). The symptoms of diarrhea and constipation represent the opposite extremes of motility function. Therefore, the abnormalities of the different types of colonic contractions are expected to be different among patients with IBS-D and IBS-C, and they may switch between the two states in patients with IBS-C/D. Notably, the total stool weight per 24 hours in all three subclasses of patients with IBS is within or close to that in healthy human subjects (194–196). The main difference between patients with IBS and healthy subjects is in the frequency of defecation and stool consistency. In addition, patients with IBS may also have the urgency to defecate, the feeling of incomplete evacuation during defecation, and tenesmus. The softness of stools in patients with IBS-D is because of rapid transit, whereas the hard stools in patients with IBS-C is because of slower transit in the colon when compared with that in healthy subjects (196,197) or because of outlet obstruction. Therefore, patients with IBS-D have motor-diarrhea rather than secretory-diarrhea (198). Rhythmic Phasic Contractions Of the three types of colonic contractions, the RPCs are the most difficult to record and interpret. A major difficulty arises from the highly disorganized spatial coordination of these contractions (see Fig. 39-2). Even with the availability of computer programs, it is difficult to quantitate their frequency and propagation distances, which determine propulsion rates (5,21) (see Fig. 39-1). Manometry is the most feasible and commonly used method to record these contractions from the human colon. However, this method records only the lumen occluding contractions; therefore, an unknown proportion of RPCs is missed in manometric recordings. In addition, the insertion of a manometric tube through the anus requires prior cleansing of the colon, which may alter the normal spatiotemporal patterns of contractions. Finally, the colonic motility patterns are highly variable with time, and their organization differs in different segments of the colon. Therefore, short-term recordings of a few hours and recording of motor activity only from a part of the colon may not truly represent the overall state of colonic motility. Despite these limitations, most studies have found that the overall incidence of RPCs quantified as motility index using the amplitude and duration of contractions or spike bursts is greater in patients with IBS-D than in healthy subjects (15,16,22,199,200). The enhanced contractile response of the colon to mechanical stimulation, ingestion of a meal, and pharmacologic stimulation supports the hyperactivity of RPCs in patients with IBS-D (15,26,201–203). Manometric recordings also show that patients with IBS-D have more spontaneous contractions in the jejunum during phase II activity and after a meal (204,205). The hyperactivity of RPCs in patients with IBS-D therefore may be a generalized

phenomenon in the gut. In contrast, the RPCs are suppressed in patients with constipation (16,22,206). Because of the limitations of recordings and analysis cited earlier, the alterations in the proportions of propagated and nonpropagated RPCs in patients with IBS-C or IBS-D are unknown. Therefore, the exact contribution of enhanced occurrence of RPCs in IBS-D or their suppression in IBS-C to colonic transit is unknown. If the proportions of propagated and nonpropagated contractions do not change, the enhanced occurrence of RPCs in IBS-D would contribute to more rapid transit, as well as more mixing movements; conversely, suppression of RPCs in patients with IBS-C would contribute to slower transit and less mixing movements. These effects would be different if the proportions of propagated and nonpropagated contractions also change in any of the two conditions (21). A rat model of postinfective IBS suggests that the increased RPCs in IBS-D may, in part, be caused by the hypersensitivity of excitation–contraction coupling in smooth muscle cells, rather than because of alterations in the release of excitatory or inhibitory neurotransmitters in the colon wall (207). No correlation between the occurrence of RPCs and abdominal cramping has been noted in numerous studies in patients with IBS. The RPCs are generally present somewhere in the colon at all times. If they stimulated the sensory neurons, the pain would be felt at all times. It appears, therefore, that the sensory threshold for contractions to be perceived as painful during allodynia and hyperalgesia in patients with IBS is greater than 50 mm Hg, which is about the maximum amplitude of RPCs in the human colon. The total volume of ICCs is decreased at all levels of the colon wall in patients with slow transit constipation (85), and ICC processes are blunted. However, there is no evidence that these alterations in ICC result in any abnormalities of slow waves or in neural regulation of circular muscle contractions in these patients. Giant Migrating Contractions The amplitude of GMCs in the human colon usually exceeds 100 mm Hg and can be as high as 500 mm Hg (14,15). The contractions of this amplitude generally occlude the lumen, and therefore are faithfully recorded by the manometric method. The amplitude and propagation distances of these contractions can be analyzed easily by visual inspection, which enhances their use in investigative and diagnostic studies. However, concurrent recordings of motility from as long a segment of the colon as possible, and for as a long period as possible (at least 24 hours), would enhance their diagnostic and investigative values. Several clinical studies indicate that the frequency and amplitude of spontaneous GMCs are significantly greater in patients with IBS-D than in healthy subjects (15,23,27). The increase in the frequency of GMCs is associated with an almost sixfold faster transit in the colon of patients with IBS-D compared with that in healthy subjects (15,21). The frequent mass movements produced by the increase in

984 / CHAPTER 39 frequency of GMCs are a major factor in producing diarrhea in these patients. The frequency of GMCs is also greater after awakening in the morning, after a meal, and after intravenous administration of cholecystokinin-8 (CCK-8) in patients with IBS-D than that in healthy subjects (15,16,193). These findings explain the onset of symptoms of urge to defecate and abdominal cramping after ingestion of a meal and after awakening in patients with IBS. The contractile response to CCK-8 in strips of taenia coli from patients with IBS-D is also greater than that in strips from healthy subjects, indicating hypersensitivity of smooth muscle cells to hormonal stimulation (15,207). The GMCs start more distally in the colon, and they propagate farther in patients with IBS-D than in healthy subjects, thus propelling the colonic contents closer to the rectum (23,27). The GMCs that propagate to the rectum also induce the urge to defecate, one of the major clinical symptoms of this disorder (see Fig. 39-3). Patients with IBS-D exhibit rectal hypersensitivity (208) to distension. Therefore, the accumulation of fecal material in the rectum by frequent mass movements in patients with IBS-D further enhances the stimulation of GMCs in the sigmoid colon, which relaxes the anal sphincters by descending inhibition, and at the same time provides propulsive force for rapid expulsion of feces. In contrast, if the anal sphincters fail to relax because of a defect in descending relaxation, the distension caused by the bolus ahead of the GMC produces the sensation of abdominal cramping. Complete evacuation of the distal colonic contents minimizes the luminal stimulus for the generation of GMCs, which explains the relief in abdominal cramping obtained by patients with IBS-D after defecation. Tenesmus results if GMCs occur after complete evacuation of feces from the distal colon. In contrast, the frequency of GMCs is suppressed in severely constipated patients (26,193,209–212). This means

P

P

P

less mass movements and greater contact time of feces with colonic mucosa resulting in hardening of stools. The decrease in the frequency of GMCs in patients with IBS-C may not be to the same extent as that in severely constipated patients. The inability of the internal anal sphincter to relax in patients with outlet obstruction may be because of a defect in descending inhibition, as well as the absence of GMCs in the distal colon that provide the rapid propulsive force for defecation and the stimulus for descending relaxation. The rectal sensitivity in patients with IBS-C is reduced or unaltered (213). The rectal contents are, therefore, less likely to stimulate a GMC and its accompanied descending inhibition to relax the internal anal sphincter. In the absence of the GMCs in the distal colon, the already hardened feces caused by slower transit have to be expelled by straining. The smaller number of GMCs in severely constipated or IBS-C patients may still produce the sensation of abdominal cramping because they may attempt to propel luminal contents against obstructing pellets, and hence produce distension (Fig. 39-14) (214). Tone The basal tone in the descending colon of patients with IBS does not differ from that in healthy subjects. However, the increase of postprandial tone in patients with IBS-C is less than that in healthy subjects (215). In contrast, the increase of postprandial tone is not different in patients with IBS-D compared with that in healthy subjects. Therapeutic Agents The stimulation of GMCs that rapidly and forcefully propels luminal contents and induces defecation is a potential target for the treatment of IBS-C or chronic constipation.

P

0-80 mmHg 30 s

FIG. 39-14. Each spontaneous occurrence of a GMC at the two proximal recording sites in the colon was associated with a sensation of pain (P ). (Reproduced from Bassotti and colleagues [214], by permission.)

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 985 Likewise, selective inhibition of GMCs in patients with IBS-D or functional diarrhea would reduce the frequency of mass movements and defecations, and hence provide relief of symptoms (100). In this regard, the partial 5-HT4 receptor agonists stimulate GMCs in intact animals, accelerate transit, and induce defecation (216–219). Coincidentally, the 5-HT4 agonists also reduce nociceptive threshold to colorectal distension that prevents abdominal cramping associated with their stimulation under the conditions of allodynia and hyperalgesia (220,221). A significant decrease in median time to first complete spontaneous bowel movement by 5-HT4 agonists suggests a mass movement induced by the drug. Notably, only a few GMCs are stimulated up to several hours after the oral or intravenous administration of 5-HT4 agonists (217). The long time lag and the random stimulation of only a few GMCs over several hours by these agonists may be the underlying cause of their marginal effectiveness in relieving the symptoms of constipation (222). More potent and consistent stimulation of GMCs by serotonin- or nonserotonin-based therapeutic agents is likely to provide greater relief of the symptoms of IBS-D and constipation. The role of stimulation of RPCs by the 5-HT4 agonists is largely unknown because of the difficulties of their faithful recording and analysis of their propagation characteristics cited earlier in this chapter. The 5-HT4 agonists act at a presynaptic neuronal site to stimulate GMCs in the canine colon (217), but the complete mechanisms of their stimulation, including any alterations in excitation–contraction coupling, are not understood. 5-HT4 receptors are localized on the ISNs, on other enteric neurons, and on smooth muscle cells (35,171,182,186,223). The random generation of a few GMCs after the administration of 5-HT4 agonists suggests the involvement of both genomic and nongenomic mechanisms. Studies show that mucosal 5-HT, tryptophan hydroxylase 1 messenger RNA, serotonin transporter messenger RNA, and its immunoreactivity are all reduced in the mucosal biopsies of patients with IBS-C, IBS-D, or UC (224). The anomaly is that the basal and stimulated release of 5-HT are not altered. However, the alterations in different types of contractions in the three groups of patients differ. For example, the frequency of GMCs is increased in patients with IBS-D and UC, whereas it is decreased in patients with IBS-C. The RPCs are suppressed in patients with UC and IBS-C, but they are enhanced in patients with IBS-D. Therefore, it is unlikely that the suppression of serotonin reuptake transporter (SERT) and the decrease in mucosal 5-HT alone could explain the divergent motility dysfunctions in these diseases. The alterations of colonic motor function in IBS and IBD are unlikely to be caused by changes in the expression or cell signaling of a single molecule. In the case of IBD, the alterations in neurotransmitter release from the motor neurons and changes in excitation–contraction coupling have already been established (225–227). Further work to identify the effect of suppression of SERT and mucosal 5-HT on different types of colonic contractions would help in identifying their roles in colonic motility dysfunction in these diseases.

5-HT3 receptors mediate the occurrence of spontaneous RPCs in the human colon because their antagonists concentration dependently inhibit the postprandial response (228–230). In clinical studies, 5-HT3 receptor antagonists retard colonic transit in healthy subjects, reduce the frequency of bowel movements, and improve stool consistency in patients with IBS-D (231,232). Animal studies in conscious dogs and in isolated segments of the guinea pig colon indicate that the stimulatory effect of 5-HT in the colon is mediated partially by 5-HT3 receptors located on presynaptic neurons and on smooth muscle cells (218,233). Other studies show that 5-HT3 receptor antagonists may also reduce visceral hypersensitivity (228,234,235). However, the use of 5-HT3 receptor antagonists for patients with IBS-D is restricted because of their severe negative side effects.

Inflammatory Bowel Disease and Acute Colonic Inflammation The IBD in the colon refers to Crohn’s colitis and UC. UC affects mainly the colon, whereas Crohn’s disease may affect the small intestine and the colon. The etiologies of the two diseases are different and are not understood. The classic inflammatory infiltrate in UC is confined to the mucosal and submucosal layers, whereas in Crohn’s disease, it is present in the neuromuscular layers as well. However, regardless of the potential differences in their etiologies, and the inflammatory markers that characterize them, the clinical symptoms of Crohn’s disease and UC are the same: diarrhea, abdominal cramping, and urgency of defecation. These symptoms contribute in large part to the morbidity of the disease. These symptoms also characterize acute inflammation of the colon produced experimentally by chemical exposure of the colon in animals. The frequency of defecation in patients with IBD is increased, and they have a greater incidence of nocturnal defecation during active UC than during quiescent UC (236). The stool consistency varies from unformed to formed stools, but they are not watery, like those in secretory diarrhea. Occasionally, patients with UC may pass hard stools, which may be characterized as constipation, but the frequency of bowel movements is high, and this does not fit well with the classical definition of constipation. Therefore, although the mucosal injury in inflammation blunts the epithelial transport functions, the diarrhea in UC may be predominantly caused by altered motility function (motor diarrhea) (198). The symptoms of urgency of defecation and abdominal cramping are entirely due to abnormal colonic contractions and visceral hypersensitivity. The following sections discuss the alterations in the different types of colonic contractions and their mechanisms that produce the above symptoms. Note that these alterations are more consistent in animal models of acute inflammation, where confounding factors of duration of disease; active, inactive, or partially active state of the disease; and use of medications are absent. The animal models have proved valuable in identifying the cellular and

986 / CHAPTER 39 molecular mechanisms of altered colonic motility function in inflammation. Rhythmic Phasic Contractions, Giant Migrating Contractions, and Tone The occurrence of circular muscle phasic contractions in the fasting and postprandial states is suppressed in experimental models of acute inflammation, as well as in patients with UC (29,54,237–243). In experimental models, the degree of suppression of RPCs depends on the severity of inflammation (54). The contractility of freshly dispersed single circular muscle cells taken from the inflamed colons of animals in response to ACh also is suppressed when compared with that in cells from healthy colons (102,244). Although patients with UC and IBS-D have motor diarrhea, the effects on the RPCs are the opposite of each other: in inflammation, the RPCs are suppressed, whereas in IBS-D, they are enhanced. The resting tone of the colon wall, as well as the increase of tone after a meal, also is suppressed in patients with UC when compared with that in healthy subjects (see Fig. 39-9) (39,205). In contrast with the suppression of RPCs and tone, the frequency of colonic GMCs is enhanced in the inflamed colon during the fasting and postprandial states (22,28,29). The GMCs start randomly at different locations in the inflamed colon and propagate distally. The GMCs that propagate to the rectum cause the urge to defecate or defecation. Inflammation also causes neuronal hypersensitivity in the colon (239,245). Therefore, during hyperalgesia and allodynia, the strong amplitude of the GMC contraction or distention distal to it produced by the bolus being propelled exceeds the nociceptive threshold, and they produce the sensation of abdominal cramping (see Fig. 39-6). Note that allodynia or hyperalgesia by itself does not produce the sensation of pain in the colon. In the laboratory setting, a balloon must be inflated beyond the nociceptive threshold in the colon to induce the sensation of pain. Likewise, in an ambulatory patient, there must be a GMC, with an amplitude that exceeds the nociceptive threshold, at some location in the colon to provide the stimulus for abdominal cramping. The frequent mass movements produced by the GMCs reduce the contact time of colonic contents with the mucosa. Furthermore, the suppression of RPCs reduces the mixing and turning over of luminal contents. The resulting inadequate absorption of water and ultrarapid propulsion result in diarrhea. In some studies, the rate of transit in the colon was found to be slower than that in healthy subjects. This is likely if during the study no GMCs occurred to cause rapid propulsion, whereas the RPCs and tone were suppressed, which retards normal propulsion. Cellular and Molecular Mechanisms of Altered Motility in Inflammation The observations that the contractile activities of smooth muscle strips, single dispersed cells, and neurotransmitter

release from the enteric neurons are suppressed from tissues taken from the inflamed colons indicate that these are not the pharmacologic (nongenomic) effects of the inflammatory mediators. These effects are because of the altered gene expressions of key signaling molecules that regulate different types of colonic contractions. This distinction is critical because accumulating evidence indicates that although some of these genomic effects are reversible, others are not, and they may result in more permanent conditions, such as postinfective IBS (193,207,246). Excitation–Contraction Coupling The expression of muscarinic M2 and M3 receptors is not altered by inflammation (247). However, it decreases the expression of Gαq/11 protein coupled to M3 receptor and increases that of Gαi3 coupled to M2 receptor. Consequently, the M2 receptors that contribute little to contraction in the normal state play a more prominent role in the inflamed state. In contrast, the decrease in the expression of Gαq/11 contributes to the suppression of contractility in response to ACh. Inflammation also depolarizes the RMP and reduces the amplitude and duration of slow waves, but it does not abolish them. The density of ICC is reduced in the inflamed colon, and their processes are damaged (54,248,249). The amplitude of the IJP is not altered, but its duration is decreased (54). The reduced amplitude of slow waves contributes to a smaller increase in [Ca2+]i on stimulation with ACh (244), and hence to suppression of RPCs. However, it is unclear whether the damage to ICCs contributes to the alterations in slow waves. As noted earlier, increase in [Ca2+]i plays a critical role in excitation–contraction coupling. Inflammation suppresses calcium current through L-type Ca2+ channels (Cav1.2) (59,250). The resulting decrease in [Ca2+]i reduces the Ca2+calmodulin-MLCK–dependent phosphorylation of MLC20 to reduce cell contractility (49). By contrast, inflammation does not alter the release of Ca2+ from the intracellular stores (244). The decrease of Ca2+ influx in inflammation is caused by the suppression of the pore-forming α1C subunit of Cav1.2 channels (59). Inflammation does not alter the pharmacologic and functional characteristics of the L-type Ca2+ channels, such as the amplitude of unitary currents and voltage sensitivity. By contrast, the expression of Ca2+-activated K+ channels is not affected by inflammation, but their openstate probability and voltage sensitivity are reduced in the inflamed colon cells (251). The suppression of the pore-forming α1C subunit of L-type calcium channels in intact animals, as well as in primary cultures of human colonic circular smooth muscle cells treated with tumor necrosis factor-α (TNF-α), is mediated by the activation of the nuclear factor-κB (NF-κB) (252,253). The human colonic circular smooth muscle cells express primarily the transcripts containing exon 1b of the L-type calcium channel gene. The binding of both the p50 and p65 subunits of NF-κB to two adjacent κB motifs on the promoter 5′ to exon 1b is required to suppress the transcription of α1C (253).

FUNCTION AND REGULATION OF COLONIC CONTRACTIONS IN HEALTH AND DISEASE / 987 Inflammation also alters the pharmacomechanical coupling in colonic circular smooth muscle cells. Oxidative stress induced by treatment of the colon smooth muscle cells with H2O2 reduces the expression of the classic and novel PKC α, β, and ε isozymes but enhances that of the atypical isozymes PKC ι and λ (102). The reduced expression of PKC α, β, and ε and the absence of their translocation from the cytosol to the membrane in inflammation contributes to the suppression of contractility. The release of AA in response to ACh also is suppressed in the inflamed colon cells because of the impaired activation of cytosolic phospholipase A2, which also contributes to the suppression of contractility (101). Motor Neurons Morphologic evidence indicates that the density of neurons innervating the circular smooth muscle cells is not altered by inflammation (254). However, the packaging, storage, and release of neurotransmitters from the motor neurons and sympathetic nerve endings in the myenteric plexus are impaired (225,255,256). The decrease in ACh release is mimicked by incubation of the longitudinal muscle-myenteric plexus reparations with interleukin-1β (IL-1β), suggesting the role of cytokines in producing these effects (226). Intrinsic Sensory Neurons Intracellular recordings with microelectrodes indicate that the excitability and synaptic transmission of both the AH- and S-type submucosal neurons is increased in trinitrobenzene sulfonic acid–induced inflammation in the guinea pig colon (227). Although such recordings have not been made from the neurons in the myenteric plexus, a similar increase in excitability and synaptic facilitation is expected, because of transmural inflammation in this model. The increased excitability of the neurons suggests an increase in the release of excitatory, inhibitory, or both types of neurotransmitters at the neuroeffector junction. However, this effect is contrary to the findings that neurotransmitter release is suppressed in inflammation. The potential role of increased neuronal excitability in stimulating sEPSPs, and hence GMCs, has not been investigated in this model. Myoneuroimmune Interactions The classic concept of inflammation is that it is initiated and maintained by the recruitment and activation of infiltrating and resident immunocytes. The rest of the cells in the gut wall are innocent bystanders that simply get hurt by the release of inflammatory mediators by the immune cells. Accumulating evidence over more than a decade has modified this concept (246,257). It now appears that the nonimmune cells in the gut wall, such as the enteric neurons, smooth muscle cells, epithelial cells, and glia, are not just bystanders, but rather they actively participate in the inflammatory response.

The extrinsic and enteric neurons interact bilaterally with the immune cells. The inflammatory response in colonic inflammation alters sensory perception (239,245,258,259). In contrast, the release of substance P, a neurotransmitter of the extrinsic pituitary afferent neurons, activates immune cells and modulates the inflammatory response (260,261). The bioavailability of substance P is increased in the inflamed gut because of the suppression of the tissue level of neutral endopeptidase (262,263). Inflammation also alters the neurotransmitter content of enteric neurons, as well as its release on stimulation (225,255,264,265). The smooth muscle–immune cell interactions result in alterations of excitation–contraction coupling, as discussed earlier, and in programmed release (116) of proinflammatory and anti-inflammatory mediators by the circular smooth muscle cells (266–268). In response to inflammatory mediators, TNF-α and IL-1β, the colonic circular smooth muscle cells initially secrete a panel of proinflammatory cytokines followed by the secretion of anti-inflammatory cytokine IL-11 (266). The early secretion of proinflammatory cytokines, such as intercellular adhesion molecule-1 (ICAM-1), mediates the suppression of contractility in smooth muscle cells (269), whereas the late secretion of IL-11 blunts the inflammatory response to begin the process of healing (266). The secretion of proinflammatory and anti-inflammatory mediators by smooth muscle cells is regulated by gene expression (266,269). The activation of the p65 subunit of NF-κB plays a central role in the expression of ICAM-1(269). IL-11 decreases the expression of ICAM-1 by suppressing the activation of NF-κB (266). The secretion of inflammatory mediators by colonic circular smooth muscle cells may be critical to achieving a critical mass of secretions to obtain the full inflammatory response in the neuromuscular layer. In addition, smooth muscle cells present antigen in a major histocompatibility complex II restricted to T lymphocytes, causing activation and proliferation of T cells. The expression and secretion of inflammatory mediators by colonic smooth muscle cells may also be the basis of persistence of motility dysfunction in patients with IBD in remission and in patients with postinfective IBS (207,246,270,271).

ACKNOWLEDGMENT Supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (grants DK 32346 and DK 072414 to S.K. Sarna).

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Neural Control of Pelvic Floor Muscles David B. Vodusek and Paul Enck Innervation of Pelvic Floor Muscles, 995 External Anal Sphincter Muscle Innervation Zones, 995 Somatic Motor Pathways, 996 Afferent Pathways, 998 Pelvic Floor Muscle Activity, 999 Tonic Pelvic Floor Muscle Activity, 1000 Reflex Activity of Pelvic Floor Muscles, 1001 Voluntary Activity of Pelvic Floor Muscles, 1002 Pelvic Floor Muscles and Pelvic Organ Function, 1003

Neural Control of Micturition, 1003 Neural Control of Urinary Continence, 1004 Neural Control of Anorectal Continence, 1004 Neural Control of the Sexual Response, 1005 Pelvic Floor Muscles and Neurologic Lesions, 1005 Neuromuscular Injury to the Pelvic Floor Caused by Vaginal Delivery, 1005 Pelvic Floor Activity Patterns in Nulliparous Continent and in Parous Incontinent Women, 1006 References, 1006

Neural control of pelvic floor muscles (PFMs) is affected by somatic afferent and efferent nerves of the sacral cord segments reflexly integrated at the spinal cord and brainstem level. The inputs from several higher centers influence the complex reflex control and are decisive for voluntary control and for socially adapted behavior related to excretory functions. PFMs are intimately involved in function of lower urinary tract, the anorectum, and sexual functions; therefore, their neural control transcends the primarily important somatic innervation of striated muscle, because they are directly involved in “visceral activity.” Neural control of pelvic organs is affected by a unique coordination of somatic and autonomic motor nervous systems. Sensory information from pelvic organs is supplied by visceral and somatic sensory fibers; their input also influences PFM activity through central integrative mechanisms.

INNERVATION OF PELVIC FLOOR MUSCLES External Anal Sphincter Muscle Innervation Zones Muscle fibers (motor units) are innervated at different locations. The motor unit action potentials (MUPs) propagate from the innervation zone, toward the muscle fiber endings, with a specific conduction velocity. Multichannel electromyography (EMG) signals provide important information about anatomic properties of the muscle under study (1–6). According to the concepts described elsewhere, visual analysis of the multichannel recordings allows estimation of the length of muscle fibers and the location of the innervation zones and the tendon regions. Figure 40-1 shows signals detected from the external anal sphincter (EAS), in comparison with bipolar needle EMG signals, with the visual identification of the MUP anatomic features (innervation zones and fiber length) and automated signal decomposition. The application of an automated decomposition technique (7) showed that it is possible to noninvasively identify motor units at low and high contraction levels. In many cases, it was possible to detect the same motor unit at different contraction levels, as well as the recruitment of new ones, with increasing effort. When this technique was applied to a series of healthy male and nulliparous female subjects (8) of different ages,

D. B. Vodusek: Division of Neurology, University Medical Center, Ljubljana, Slovenia. P. Enck: Department of Internal Medicine VI, University Hospitals Tuebingen, Tuebingen, Germany.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

995

996 / CHAPTER 40 significant differences between the sexes became evident. In men, a rather scattered distribution of innervation zones was found all along the anal canal, with an apparent predominance of right-left innervation, but with a large interindividual variability. In contrast, in women, this innervation pattern was observed only in the most superficial part of the EAS, whereas at a higher level, the distribution of innervation zones was more uniform around the circumference. The most proximal EAS shows innervation zones predominantly in the dorsal and ventral regions. These findings are at least in partial agreement with a more recent anatomic concept of the pelvic floor anatomy that describes the EAS as consisting of two parts: a subcutaneous part with circumferentially running muscle fibers, and a deep “loop”

A)

The motor neurons that innervate the striated muscle of the external urethral and anal sphincters and perineum originate from a localized column of cells in the sacral/spinal cord called Onuf’s nucleus (10). The Onuf’s nucleus in humans is found in the second and third sacral segments but occasionally extends into S1 (11) and is located dorsolaterally in the

SIGNALS

− DORSAL SIDE E16

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PROBE AND DETECTION SYSTEMS

that opens in the midline ventrally but demonstrates thickened muscle bundles ventrolaterally, especially in women (9). Such data are as yet unavailable for other muscles in the perineal/pelvic area.

+

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C) CONVENTIONAL BIPOLAR EMG SIGNALS E4-E12 = BIP −22.5° 200 µV

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FIG. 40-1. Examples of surface electromyography (EMG) detection from the anal sphincter muscle using different detection systems. (A) Schematic representation of the rectal probe (1), shown from cable side (see also Figure 40-2, panel 1). Multichannel EMG detection is obtained with a series of differential amplifiers (2). Conventional bipolar EMG recording is instead obtained detecting a single signal as the difference between two opposite electrodes (3), from which amplitude-based information can be extracted. (4) Effect of rotation of a bipolar probe by a one-electrode step (22.5 degrees) in counterclockwise direction. (B) Sample epoch, 100-msec long, of multichannel, single–differential, EMG signals detected from external anal sphincter muscle during maximal contraction. (C) Sample epoch of conventional bipolar EMG signals calculated from opposite electrodes on the same signal shown in B in three different probe orientations: electrodes aligned to left-right direction (BIP 0 degrees, solid line), rotated by 22.5 degrees counterclockwise (BIP −22.5 degrees), and clockwise (BIP +22.5 degrees). In this specific case, a slight (−22.5 degrees) rotation of the probe in counterclockwise direction would not produce a significant effect, whereas a rotation in the other direction would greatly affect both the amplitude (by about a twofold factor) and shape of the potentials.

Time 100 (ms)

NEURAL CONTROL OF PELVIC FLOOR MUSCLES / 997 D

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FIG. 40-1, cont’d. (D) Examples of multichannel EMG signals detected from the external anal sphincter muscle of a female subject. Signals were acquired from 1-cm depth in the anal canal in the relaxed condition and (E) during maximal voluntary contraction. Note the different vertical scales in D and E. (F) Example of the decomposition of signals recorded during 10-second-long contractions at the contraction levels 100% maximum voluntary contraction (MVC) (left) and 50% MVC (right). Superposition of the motor unit (MU) action potentials (MUAPs) belonging to each of the four MUs. Note that the same MUs (#2 and #4) are identified at the two contraction levels, and new MUs (#1 and #3) are recruited at 100% MVC. In particular, the MU 2 firing pattern is quite well reconstructed.

anterior horn. Within the Onuf’s nucleus there is some spatial separation between motor neurons concerned with the control of the urethral and the anal sphincter. Spinal motor neurons for the levator ani group of muscles often are considered also to be a part of the Onuf’s nucleus (12). It appears, however, that the areas of the sacral gray matter that innervate the anal and urethral sphincter and the levator

ani are adjacent and show some overlap. The innervation of levator ani in the female individual was demonstrated to originate from S3-S5 segment (13). Sphincter motor neurons are uniform in size and are smaller than the other α motor neurons. They are distinguished also by bundles of dendrites from multiple neurons projecting along transverse and longitudinal axes.

998 / CHAPTER 40 The neurons demonstrate high concentrations of amino acid-, neuropeptide-, norepinephrine-, serotonin-, and dopaminecontaining terminals that represent the substrate for the distinctive neuropharmacologic responses of these neurons, which differ both from those of limb muscles and from the bladder (14,15). The somatic motor fibers leave the spinal cord as the ventral radices and fuse with the dorsal radices to constitute the spinal nerve. (The sacral roots travel within the spinal canal from levels T12/L1 as the cauda equina.) After passing through to the intravertebral foramen, the spinal nerve divides into a dorsal ramus and a ventral ramus (16). Somatic fibers from the ventral rami (also called the sacral plexus) form the pudendal nerve. Traditionally, the pudendal nerve is described as being derived from the S2-S4 ventral rami, but there may be some contribution from the S1, and possibly little or no contribution from the S4 (17). The pudendal nerve continues through the greater sciatic foramen and enters in a lateral direction through the lesser sciatic foramen into the ischiorectal fossa (the Alcock’s canal). In the posterior part of the Alcock’s canal, the pudendal nerve gives off the inferior rectal nerve; then it branches into the perineal nerve and the dorsal nerve of the penis/clitoris. Some authors describe a branch at the level superior to the passage of the pudendal nerve through the greater sciatic (infrapiriformic) foramen (18), which contains fibers to the levator ani and the external urethral sphincter. Other authors have not found this branching (17). Thus, it remains controversial whether the urethral sphincter is innervated by somatic motor nerve fibers traveling with the pelvic plexus (19) or the pudendal nerve (18,20). Whether the pudendal nerve branches (also) innervate the levator ani (“from below”) is controversial; most authors agree that its main innervation is through direct branches from the sacral plexus (“from above”). Branches originating from S3-S5 sacral nerve roots and traveling on the superior surfaces of the pelvic floor (called the levator ani nerve) were proposed as the sole innervation of levator ani in female individuals (13). There appears to be significant variability of normal human neuroanatomy. This is probably why there are still controversies resulting from anatomic studies of peripheral innervation of pelvis, which as a rule are performed in a small number of cases. Little is known about asymmetries of anatomy; interestingly, anatomic studies apparently dissect specimens only unilaterally, so intersubject variability is noticed, but intrasubject variability (asymmetry) is not. Much less remains known about the finer details of central pathways to PFS, which mostly have been explored in experimental animals. Descending inputs to PFM motor neurons are manifold and mostly “indirect.” More “direct” connections to Onuf’s nucleus are from some nuclei in the brainstem (raphe, ambiguus) and from paraventricular hypothalamus. Positron emission tomography (PET) studies revealed activation of the (right) ventral pontine tegmentum (in the brainstem) during holding of urine in human subjects (21). This finding

is consistent with the location of the “L region” in cats, which has been proposed to control PFM nuclei (22). The mentioned connections serve the coordinated inclusion of PFS into “sacral” (lower urinary tract, anorectal, and sexual) functions. It needs to be mentioned that PFS not only needs to be neurally coordinated “within” a particular function (e.g., with bladder activity), but the single functions need to be neurally coordinated with each other (e.g., voiding and erection). The mentioned sacral function control system is proposed to be a part of the “emotional motor system.” This is a system derived from brain or brainstem structures belonging to the limbic system. It consists of the medial and a lateral component (23). The first represents diffuse pathways originating in the caudal brainstem and terminating on (almost all) spinal gray matter, using particularly serotonin as its neurotransmitter. This system is proposed to “set the threshold” for overall changes, such as in muscle tone. The lateral component of the emotional motor system consists of discrete areas in the hemispheres and the brainstem responsible for specific motor activities, such as micturition and mating. The pathways use spinal premotor interneurons to influence motor neurons in somatic and autonomic spinal nuclei, thus allowing for confluent interactions of various inputs to modify the motor neuron activity. PFM nuclei furthermore receive descending (corticospinal) input from the cerebral cortex (the superomedial precentral gyrus). PET studies have revealed activation of the superomedial precentral gyrus during voluntary PFM contraction, and of the right anterior cingulate gyrus during sustained PFM straining (21). PFM contraction can be obtained by (electrical or magnetic) transcranial stimulation of the motor cortex in humans (c.f. Vodusek [24] and Brostrom [25]). Studies have revealed a significantly slower conduction in the central motor pathways for sphincter muscles compared with lower limb muscles (26). The cortical surface areas that, when stimulated, produced the highest responses in PFS were the areas left and right of the central interhemispheric gap of the motor cortex (27), the area of the anogenital representation in Penfield’s homunculus. It was further shown that with transcranial magnetic stimulation, the electromechanical coupling at the level of the motor end plates, which was recorded by surface EMG electrodes from within the anal canal, reveals a specific speed of contraction of the anal sphincter muscle that reflects its fiber composition and can be distinguished from voluntary contractions and from reflex contraction (28).

Afferent Pathways Because PFM function is intimately connected to pelvic organ function, it is proposed that all sensory information from the pelvic region is relevant to discussion of PFM neural control. The afferent pathways from the anogenital and pelvic regions are commonly divided into the somatic

NEURAL CONTROL OF PELVIC FLOOR MUSCLES / 999 and visceral afferents. The visceral afferents accompany both parasympathetic and sympathetic efferent fibers; the somatic afferents accompany the pudendal nerves and direct somatic branches of the sacral plexus. Somatic afferents derive from touch, pain, and thermal receptors in skin and mucosa and from proprioceptors in muscles and tendons. Proprioceptive afferents arise particularly from muscle spindles (which are probably scarce or absent in the EAS muscles, absent in the external urethral sphincter, but present in the PFMs). Monosynaptic reflexes arising by stretch of muscle spindles are probably of little importance in PFS, but these end organs play a major role in proprioception and are relevant for as yet poorly understood perceptions from pelvic organs surrounded by PFS, particularly from rectum. All afferent neurons have their cell bodies in spinal ganglia; those accompanying somatic and parasympathetic pathways are in the sacral segments (S1/S2-S4), and those accompanying sympathetic fibers are in Th11-L2 dorsal root ganglia. The sensory neurons are bipolar and send long processes to the periphery and the central process into the dorsal column of the spinal cord or to the brainstem (16). Afferent pathways accompanying sympathetic nerves encompass sensory neurons residing in the T11-L2 dorsal root ganglia and terminating in the dorsal horn (lamina I-V) of the spinal cord. High-threshold sympathetic afferents transmit nociceptive (pain) information (29). Afferent pathways accompanying somatic motor pathways include several groups of sensory fibers. Somatic afferents from the external urethral sphincter mechanism, the distal vaginal mucosa, and the anogenital region travel in the pudendal nerves, have cell bodies in S1/S2-S3/S4 dorsal root ganglia, and terminate in the sacral segments of the spinal cord in regions that overlap with those of afferents accompanying parasympathetic fibers in the pelvic nerve from the bladder (29). Our own studies with selective direct intraoperative recordings made from the dorsal sacral roots (on electrical stimulation of the dorsal clitoral and penile nerves) have demonstrated that, in most patients, pudendal afferent activity is present in the S2 and S3 roots bilaterally; but in some patients, a small contribution also can be demonstrated in the S1 roots. Variability and asymmetry of afferents was demonstrated (30,31). Similar findings also were obtained by mapping anal mucosal afferents, and it also was noted that dominant afferents from the genitals and the anal mucosa may not necessarily be located on the same root or side (32). Visceral afferent fibers of the pelvic and pudendal nerves enter the cord and travel rostrocaudally within Lissauer’s tract. Axon collaterals from Lissauer’s tract distribute transversely around the lateral and medial edges of the dorsal horn. These distributions (termed the lateral and medial collateral pathways of Lissauer’s tract, respectively), carry axons to deeper layers of the spinal cord. The terminals of pudendal nerve afferents are found ipsilaterally, but also bilaterally, with ipsilateral predominance (32). Muscle and cutaneous afferents in the pudendal nerve terminate in

different regions of the cord. Projections of pudendal nerve afferents from the external urethral sphincter muscle have been shown to overlap with those of visceral afferents (from the pelvic nerve). Proprioceptive afferent fibers from the levator ani muscle are distributed most densely in medial lamina VI. This region is known to contain second-order interneurons that mediate reflexes initiated by Golgi tendon organs and muscle spindles. The proprioceptive afferents not only form synaptic contacts in the spinal cord, but also have collaterals (“primary afferent collaterals”) that run ipsilaterally in the dorsal spinal columns to synapse in the gracilis (dorsal column) nuclei in the brainstem. This pathway transmits information about the sensation of innocuous sensations from the PFMs. The lateral columns of the spinal cord transmit information concerning pain sensations from perineal skin, as well as sexual sensations. In humans, this pathway is situated superficially just ventral to the equator of the cord and is probably the spinothalamic tract (29). The spinal pathways that transmit sensory information from the visceral afferent terminations in the spinal cord to more rostral structures can be found in the dorsal, lateral, and ventral spinal cord columns. Electrophysiologic recordings of somatosensory-evoked potentials (SEPs) have demonstrated differences of afferent innervation of the distal and proximal urethra suggesting that the sensory innervation of the proximal urethra is provided by pelvic nerve afferents and the distal urethra by pudendal nerve afferents (33). The distribution of cerebral SEPs on pudendal nerve stimulation shows largest amplitude potentials over the central (Cz) area corresponding to the Penfield’s sensory homunculus (i.e., representing the anogenital region on the medial surface of the postcentral gyrus in the primary somatosensory cortex) (33). When during magnetoencephalography evoked magnetic fields were used to identify cortical areas processing sensations from within the anal canal, a twofold representation of the sphincter muscle became evident: the above-mentioned anogenital region at the interhemispheric rim, as well as the insular cortex (34). The latter appears to process all afferent input from the viscera (35), hence transmitted via sympathetic/parasympathetic fibers, and current magnetoencephalography technology does not allow topographical differentiation (36). Primary processing of anal afferents were also found in the cerebellum in close proximity to the lip representation (37).

PELVIC FLOOR MUSCLE ACTIVITY The histochemical characteristics of PFMs are different in humans than in quadrupled animals; this is related to the upright body posture in humans which has produced switching contractile and metabolic properties of the PFMs. However, the evolutionary aspects of PFMs remain a controversial issue (38). Morphometry and histochemistry of EAS, levator

1000 / CHAPTER 40

200 µV

1s

FIG. 40-2. Synchronous recruitment of motor units on voluntary contraction in the levator ani muscle (the pubococcygeus part) in a healthy adult nulliparous continent woman. On the right (top beam), continuous firing of motor units is present (tonic activity). (Reproduced from Deindl and colleagues [40], by permission.)

ani, and puborectalis muscles of normal human subjects showed that the three muscles present a marked predominance of type 1 fibers, a feature of tonic slow-twitch muscles (39). Comparisons with other nonpelvic tonic muscles show that the mean diameter of type 1 fibers is smaller in PFMs. The function of pelvic floor/sphincter lower motor neurons is organized quite differently from other groups of motor neurons. The neurons innervating each side have to work in harmony and synchronicity. This is distinctly different from the reciprocal innervation that is common in limb muscles. Thus, the pelvic floor is primarily seen as a functional unit (in the closure system of the excretory tracts, the support system for pelvic viscera, and a “participating unit” in the sexual response). In fact, it has been demonstrated in normal continent women that both pubococcygei act in unison (40) (Fig. 40-2), and so do both halves of the sphincter muscles. It would appear that it is not possible to activate the right or left half of PFMs separately or to strictly separate activation of individual “pairs of muscles,” for instance, to activate voluntarily either both levator ani muscles or only the urethral sphincter. However, dissociation has been described in defecatory pathologies (see later). But each muscle in the pelvis has its own (bilateral) peripheral innervation, and flexible activation patterns could, in principle, be possible! Differences in activation patterns have, in fact, been reported, even between the intraurethral and periurethral sphincters in healthy female individuals (41), and between levator ani and the urethral sphincter (42). Surface EMG recording by multielectrode arrays (MEAs) that allow identification of the generation of MUPs and their characterization demonstrated—when applied to sphincter muscles (43) and the puborectalis (44) (see later)—that asymmetric distribution of innervations zones is the rule rather than the exception. This may not be of functional relevance for appropriate closure of the anal and urethral orifice, as long as muscle fibers cross the body’s midline and encircle the entire sphincter; this was shown by unilateral lesion studies (45) in animals and by MEA technology in humans (7). The differences in evolutionary origin of the sphincter muscles and levator ani furthermore imply that unilateral activation may be less of an impossibility for the PFMs than for sphincters, which evolved as a part of cloaca, whereas

the PFMs have a different, although as yet controversial, origin. It thus can be postulated that the neural mechanisms controlling the different muscles involved in sphincter mechanisms and pelvic organ support may not be as uniform as has been assumed. How much variability there is in normal activation patterns of PFS is simply not yet clarified. In disease, the coordination between individual PFMs can suffer definitively. It had been noted during clinical diagnostic work-up of patients with fecal incontinence by means of measuring the pudendal nerve terminal motor latency (46) that occasionally the responses received from the anal sphincter after right- or left-side pudendal stimulation were significantly different, and some authors have attributed this to “unilateral pudendal neuropathy” (47,48). However, the same asymmetry of response can be found in a subgroup of healthy continent subjects (49), and this has fostered the hypothesis that peripheral sensory and motor innervations of PFMs may not be symmetric in nature, but that dominance of one side may exist in some cases (50). It still needs to be shown whether these subjects are more prone to experience incontinence symptoms in cases of PFM trauma (51).

Tonic Pelvic Floor Muscle Activity The normal striated sphincter muscles (as revealed by kinesiologic EMG) demonstrate some continuous motor unit activity at rest (which may be increased voluntarily or reflexly); such activity has been recorded continuously for up to 2 hours (52) and even after subjects have fallen asleep during the examination (53). Whether this activity is continuous during all sleep stages remains controversial. This physiologic spontaneous activity may be called tonic, and it depends on prolonged activation of certain motor units (tonic motor units) and not interchanging activation and inactivation of various motor units (Fig. 40-3) (54). The number of motor unit activities actually recorded depends on physiologic and technical factors. It usually, but not always, increases with bladder filling (also depending on the rate of filling). The amount of recorded activity also depends on technical factors (the selectivity of the electrode). With the concentric needle electrode, activity from one to five motor units usually is recorded per detection site. Typically, small-amplitude, “low-threshold” MUPs that

NEURAL CONTROL OF PELVIC FLOOR MUSCLES / 1001 (n) 250

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FIG. 40-3. Regular discharges of two (tonic) motor units from the external urethral sphincter of a healthy adult woman. The second motor unit is discharging at a slow rate. (Only a short segment of a prolonged recording made with a single-fiber electromyographic electrode is shown.)

fire rather regularly at lower frequencies constitute the “tonic activity” in the PFMs. In a study of 39 such motor units from the anal sphincter in 17 subjects (inclusion criterion: rhythmic spontaneous firing for 2 minutes before onset of measurement), the range of discharge rates was found to be 2.5 to 9.4 Hz (3.5 ± 1.8 Hz, mean ± standard deviation) (54) (Fig. 40-4). Any reflex of voluntary activation is mirrored first in an increase of the firing frequency of these motor units. Inhibition of firing is apparent on initiation of (also simulation of) voiding. With any stronger activation or increase in abdominal pressure (and only for a limited length of time), new motor units are recruited and may be called “phasic” motor units. As a rule, they have potentials of higher amplitudes, and their discharge rates are higher and irregular. A small percentage of motor units with an “intermediate” activation pattern can also be encountered (54) Apart from differences in amplitude, the different types of MUPs may also differ in duration, as evidenced by EMG frequency analysis (55), and multi-MUP analysis (56). Others have also found low firing rate of EAS motor

neurons (3–5 Hz) (57). This rate is far lower than in antigravity muscles such as the soleus (discharging rates typically are 8–10 Hz in the standing posture). This fact may be connected to the important connections of the sphincter muscle with skin afferents. It has been determined that the threshold to elicit a response with cutaneous electrical stimulation is significantly lower in the EAS than in the levator ani muscle (58). For tonic activity, sphincters differ from some other perineal muscles; tonic activity is encountered in many, but not all, detection sites for the levator ani muscle (40,54) and is practically never seen in the bulbocavernosus muscle (54). In the pubococcygeus in the normal female individual there is some (but not invariable) increase of activity during bladder filling and reflex increases in activity during any activation maneuver performed by the subject (talking, deep breathing, coughing). On voiding, the external urethral sphincter muscle—and also PFMs—relaxes (the tonic activity is inhibited). This can be detected as a disappearance of all EMG activity (this precedes detrusor contraction). Similarly, the striated anal sphincter relaxes with defecation (and also micturition) (59).

Reflex Activity of Pelvic Floor Muscles Typical muscle spindles have been reported not only in PFMs, but also in the EAS muscle—in several, but not all, quadruped animal species (60). In humans, they are present in some PFMs, but are rare or absent in sphincter muscles (61). As known from animal experiments, monosynaptic reflexes are not a feature of either of these muscle groups. Mackel (62) found that monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in sphincteric motor neurons by dorsal root stimulation typically had amplitudes of less than 1 mV; those evoked by pudendal nerve stimulation rarely exceeded 0.5 mV. Indeed, less than half of motor neurons received monosynaptic EPSPs. Regarding central integrative mechanisms, apart from the related specificities of scarce or absent muscle spindle input, motor neurons from the striated urethral sphincter do not receive Renshaw’s cell inhibitory inputs or disynaptic inhibitory inputs; they also exhibit shorter afterhyperpolarizations than might be

R.: 50 µV./div.

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A Voluntary contraction

FIG. 40-4. Frequency histogram of the discharge rate of a continuously firing (tonic) motor unit from the anal sphincter of a healthy adult woman. The values on the y-axis denote number of discharges. The regularity of the motor unit discharge can be seen from the Gaussian distribution of its frequency histogram. (Reproduced from Vodusek [57], by permission.)

1002 / CHAPTER 40 expected from motor neurons innervating slow muscle (62). The resting discharge of these motor units (the “tonic activity”) may be related to these properties. In fact, the separate discussion of “tonic” and “reflex” PFM activity is arbitrary, because the continuous firing of motor units in PFMs is related to an appropriate excitatory setup of their motor neurons, which is derived from suprasegmental and segmental (“reflex”) inputs. The sacral motor neurons for the PFMs receive crossed primary afferents. In the interneuronal system of the PFMs (as well as in the micturition reflex) there appears to be a gating mechanism that allows or disallows stimuli from a number of sources to excite (or inhibit) these motor neurons. Their reflex activity is generated by (light) cutaneous stimuli, by pelvic organ distension, and by increasing intraabdominal pressure; excitatory reflex activity is regulated at the spinal level. Although sphincter inhibition during bladder contractions or distensions are mediated by a spinal reflex, a looped pathway through the brainstem cannot be excluded, particularly if the stimuli are noxious (63). Inhibitory descending controls are obtained from brainstem (raphe, spinal, reticulospinal) pathways and from the corticospinal tract. PFMs are involved in involuntary (reflex) activity presumably particularly within “patterns” of activity that correspond to their functional role as pelvic organ supporters, as sphincters for the lower urinary tract and anorectum, and as an effector in the sexual arousal response, orgasm, and ejaculation. For the timed relaxation pattern of sphincter activity during voiding, the descending pathway from pontine tegmentum is crucial. Inhibitory input to sphincter neurons is received from the pontine micturition center (PMC) via the commissural nucleus in the sacral/spinal cord segments (64). In fact, the “sphincter relaxation pattern” is just one part of a complex motor (behavioral) sequence including not only coordinated bladder and sphincter activity, but also body posture and so forth. It is interesting that inhibition of urethral sphincter activity can be achieved voluntarily in the absence of actual voiding (57,65). The reflex activity of PFMs is evaluated clinically by eliciting the bulbocavernosus and anal reflex either mechanically or electrophysiologically (66). The sacral reflex evoked on nonpainful squeeze of glans or electrical dorsal penile or clitoral nerve stimulation is the “bulbocavernosus reflex”; the sacral reflex evoked on painful (pinprick or strong electrical) perianal stimulation is called the anal reflex. Physiology of these reflex responses in PFMs have been studied in humans with the aid of single-fiber EMG, which allows for selective observation of single motor neuron reflex discharges (by detecting single muscle fiber potentials, which are marking individual motor unit behavior) (67). Reflex latency variability can be used to characterize the complexity of its central integration. Using this technique, Trontelj and colleagues (68) reported levator ani responses obtained by intramuscular electrical stimulation. By relating their latencies and their variability, they defined direct, recurrent, and reflex responses. The latency variability

values of reflex responses (and therefore the complexities of the respective reflex arcs) were comparable with the H-reflex and the first and the second components of the blink reflex (i.e., monosynaptic, oligosynaptic, and polysynaptic central integration), respectively. The bulbocavernosus reflex is elicited by mucocutaneous afferents and was shown to be a complex response, often forming two components; the first (with a typical latency of 30 msec) is stable and does not habituate (69). Both absence of habituation and relatively low latency variability of the early latency reflex responses within the bulbocavernosus reflex response are suggestive of an oligosynaptic reflex pathway (69) (Fig. 40-5). The latency variability for the longer latency component of bulbocavernosus reflex is typical for polysynaptic reflexes (69). The existence of two unilateral bulbocavernosus reflex arcs has been demonstrated using unilateral dorsal penile nerve blocks (70,71).

Voluntary Activity of Pelvic Floor Muscles PFMs are, in principle, under voluntary control; that is, it is possible to voluntarily activate or inhibit the firing of their motor units. By intracortical microstimulation, Dubrovsky (72) was able to demonstrate, in the motor cortex of the cat, the presence of neuronal groups as they selectively activate both the EAS and levator ani muscles. Nakagawa (73) has provided histologic evidence for a direct cortical input to Onuf’s nucleus; the projection is bilateral, that is, the motor neurons also receive uncrossed corticospinal tracts (74). There have been some claims that, in humans, the sphincter muscles can be contracted voluntarily but not relaxed at will. EMG studies have shown, however, that the activity of motor units in the urethral sphincter can be extinguished at both low and high bladder volumes even without initiating micturition (57,65). Nevertheless, the voluntary control of both sphincter and other perineal muscles and PFMs is not as straightforward and “reliable” as for limb muscles. It is well known that many otherwise neurologically healthy women cannot contract their PFMs on command, and this is also an experience of those performing kinesiologic EMG recordings. Men appear to be able to contract their PFMs on command. It may be that the observed differences in the ability to contract PFMs between men and woman is because men appear to regularly “squeeze” the last drops of urine from the urethra, whereas the way women perform their sacral functions does not call for regular voluntary activation of PFMs. Proprioceptive information is crucial for striated muscle motor control both in the “learning” phase of a certain movement and for later execution of overlearned motor behaviors. Proprioceptive information is passed to the spinal cord by fast-conducting, large-diameter, myelinated afferent fibers and is influenced not only by the current state of the muscle, but also by the efferent discharge the muscle spindles receive from the nervous system via γ efferents.

NEURAL CONTROL OF PELVIC FLOOR MUSCLES / 1003

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FIG. 40-5. Bulbocavernosus reflex responses (top and middle) as recorded by single-fiber electromyography. (Suprathreshold single electrical pulse stimulation of the dorsal penile nerve with surface electrodes recorded from the bulbocavernosus muscle in a healthy adult man.) (Bottom) Direct muscle responses from the same muscle in the same subject are recorded (on single electrical pulse stimulation of the ipsilateral pudendal nerve). Superimposition of 10 consecutive responses is shown in each channel (note the difference in time scales!). The single muscle fiber action potentials indicate single motor unit reflex responses, that is, single motor neuron reflex responses. The latency variability of the responses can be seen as the “width” of the superimposed responses indicating the “jitter” of responses. In the study, the latency variability of at least 50 consecutive responses was measured and expressed as standard deviation of the mean latency. (The latency was short for the low-variability reflex responses (typically 33 msec) and longer for the large-variability reflex responses (typically 42 msec). The typical latency variability was 26 µsec for the direct responses, 500 µsec for the bulbocavernosus responses with shorter latencies, and 2000 µsec for the longer latency bulbocavernosus reflex responses above (see also Vodusek and Janko [69]).

To work out the state of the muscle, the brain must take into account these efferent discharges and make comparisons between the signals it sends out to the muscle spindles along the γ efferents and the afferent signals it receives from the primary afferents. Essentially, the signal from the muscle spindles is thought to be compared with the copy of the motor command (the “corollary discharge” or “efferents copy”) sent to the muscle spindle intrafusal fibers by the central nervous system via γ efferents, and the differences are used in deciding the state of the muscle. Experiments were done in limb muscles (75), but it has been suggested

(12) that similar principles rule in bladder neurocontrol: the brain would know which efferent discharges were caused by distension and which were caused by contractions because the latter would be initiated by the central nervous system. We propose that this is generally true, but that the mechanism, although present, is quite “weakly developed” for PFMs. Relevant to discussing the differences in neural control of PFMs compared with other muscles is that, in addition to the mentioned control mechanisms (proprioceptive afferent information and “corollary discharges” of efferent commands), there is more than just one sensory channel information for limb movement, eye movement, and, to make a closer comparison, to some extent even for bladder fullness and micturition. From these more “direct” experiences of limb and eye movement the brain can build a complex “awareness” of such activity. Because of lacking concomitant information, such awareness cannot be reliably built up for PFMs, although they may contain the same somatic motor/proprioceptive afferent mechanisms as other muscles.

PELVIC FLOOR MUSCLES AND PELVIC ORGAN FUNCTION Neural Control of Micturition The lower urinary tract is innervated by three sets of peripheral nerves: parasympathetic nerves, which arise at the sacral level (S2-S4) of the spinal cord, excite the bladder, and relax the urethra (pelvic nerves); sympathetic nerves (Th11-L2), which inhibit the bladder body and excite the bladder base and urethra in animals (hypogastric and pelvic nerves); and somatic nerves (S1-4), which excite the external urethral sphincter and PFMs (pudendal and levator ani nerves). These nerves contain afferent (sensory) axons and motor (efferent) nerve fibers. Central nervous system integrative (reflex) centers responsible for micturition and urinary continence are located in the rostral brainstem in all species studied including humans, their activity being modulated by higher centers in the hypothalamus and other brain areas including the frontal cortex. Centers in the pons (brainstem) coordinate micturition as such, but centers rostral to the pons are responsible for the timing of the start of micturition. The PMC coordinates the activity of motor neurons of the urinary bladder and the urethral sphincter (both nuclei located in the sacral spinal cord), receiving afferent input via the periaqueductal gray. The central control of lower urinary tract function is organized as an “on”/“off ” switching circuit (or rather a set of circuits) that maintains a reciprocal relation between the urinary bladder and urethral outlet. PMC has been well studied in experimental animals, and it also has been demonstrated by PET in the right dorsomedial pontine tegmentum in human subjects (apparently it is more active on the right) (21). PMC sends direct excitatory (glutaminergic) projections to the parasympathetic detrusor

1004 / CHAPTER 40 nucleus and (possibly through the same pathway) to the GABAergic commissural nucleus at the S2-S3 spinal level (23). The premotor interneurons from the commissural nucleus inhibit the urethral sphincter (64). PMC receives descending input from brain areas and afferent input from lower urinary tract, the latter being received indirectly via the periaqueductal gray (in the brainstem) (76). Without PMC and its spinal connections, coordinated bladder/ sphincter activity is not possible (detrusor-sphincter dyssynergia develops in patients with spinal cord injury). Patients with lesions above the pons do not show detrusor-sphincter dyssynergia. However, these patients experience urge incontinence, that is, detrusor overactivity and an inability to delay voiding to an appropriate place and time.

Neural Control of Urinary Continence Coordinated bladder/PFM activation patterns are important for voiding and for continence. Continence is assured by a competent sphincter mechanism and an adequate bladder storage function. The kinesiologic sphincter EMG recordings in health show continuous activity of MUPs at rest, increasing with increasing bladder fullness. Reflexes mediating excitatory outflow to the sphincters are organized at the spinal level. It has been suggested that the L region (which is well described in the cat) (22) is present in humans as well; it also has been called the “storage center” (21). This area was active (laterally in the pons on the sight) in PET studies of those volunteers, who could not void but contracted their PFMs. The L region is thought to exert a continuous exciting effect on the Onuf’s nucleus during the storage phase. The urethral and anal sphincters, as well as the other PFMs (e.g., pubococcygei), are reflexly activated during any stress. This activation is, as a rule, preset in particular “motor patterns,” such as coughing and so forth, and is not just a reflex response to increased intraabdominal pressure. The latter is, of course, an additional factor in sustaining reflex activation of PFMs. In addition, PFMs can be activated voluntarily, but typically for less than 1 minute (40,77). Bladder neck support is considered important for urinary continence and is effected particularly by PFMs. Timely activation of PFMs has been demonstrated to be an important aspect of stable bladder neck support; its activation precedes activity of other muscles in the cough reflex (78).

Neural Control of Anorectal Continence The gastrointestinal tract has its own intrinsic innervation—the enteric nervous system—regulating both enteric smooth muscle activity and secretory and absorptive functions of the mucosa (see Chapters 23 and 24). It has its “own” afferents. The colon is innervated by the parasympathetic system, the vagus and pelvic nerves. The latter originate from S2-S4 cord segments and innervate the descending and sigmoid

colon and anorectum including the smooth muscle part of the anal sphincter apparatus, the internal anal sphincter. Excitation promotes peristalsis, local blood flow, and intestinal secretion, but it relaxes the resting tone of the internal anal sphincter. Its basal tone plays a major role in the rectoanal continence. Conversely, internal anal sphincter relaxation is vital for the successful rectoanal inhibitory reflex or the defecation reflex. Nitric oxide plays a major role in internal anal sphincter relaxation, but it involves multiple other inhibitory neurotransmitters, such as vasoactive intestinal peptide, and carbon monoxide (79). The basic intracellular mechanism(s) responsible for the basal tone in the internal anal sphincter smooth muscle is unknown. Feces stored in colon are transported past the rectosigmoid “physiologic sphincter” into the normally empty rectum, which can store up to 300 ml of contents. Rectal distension causes regular contractions of the rectal wall (effected by the intrinsic myenteric plexus) and prompts the desire to defecate (80). Rectal distension causes reflex relaxation of the smooth internal anal sphincter (the rectoanal inhibitory reflex), allowing the rectal contents to come into contact with the sensitive anal canal. Simultaneously the EAS contracts to ensure continence. This physiologic process is called “anal sampling reflex,” and it is thought to allow for identification of the rectal contents (gas, fluid, solid). Indeed, it has been demonstrated to occur regularly (up to seven times per hour), even during sleep (81). Stool entering the rectum is detected by stretch receptors in the rectal wall and PFMs; their discharge leads to the urge to defecate. It starts as an intermittent sensation that becomes more and more constant. Contraction of PFS may interrupt the process, probably by concomitant inhibitory influences to the defecatory neural “pattern generator,” but also by “mechanical” insistence on sphincter contraction and the propelling of feces back to the sigmoid colon (80). PFMs are intimately involved in anorectal function. Apart from the “sensory” role of PFMs and the EAS function, the puborectalis muscle is thought to maintain the “anorectal” angle that facilitates continence and has to be relaxed to allow defecation. Current concepts suggest that defecation requires increased rectal pressure coordinated with relaxation of the anal sphincters and PFMs. Pelvic floor relaxation allows opening of the anorectal angle and perineal descent, facilitating fecal expulsion. Puborectalis and EAS activity during evacuation (measured by EMG) generally is inhibited. However, observations by EMG and defecography suggest that the puborectalis may not always relax during defecation in healthy subjects. Puborectalis activity measured by EMG was unchanged in 9% and increased in 25% of healthy subjects (82). Thus, although “paradoxical” puborectalis contraction during defecation is used to diagnose pelvic floor dyssynergia in patients with typical symptoms, this finding might be related to variations from normal function; as an alternative explanation, it might be that paradoxical contraction of PFMs during attempted defecation in the laboratory

NEURAL CONTROL OF PELVIC FLOOR MUSCLES / 1005 situation produce false-positive results because of psychologic inhibition to perform such a maneuver in the “public.” Dissociations between EAS and PFM activity, especially the puborectalis and levator ani, also have been reported in patients with defecation disorders: failure to relax one muscle may coexist with normal function of the other (83).

Neural Control of the Sexual Response In principle, genitals have similar peripheral innervation from the same spinal cord segments by three sets of nerves as the lower urinary tract. Erection, seminal emission, ejaculation, and orgasm are part of the complex mating behavior integrated by the forebrain (medial amygdala, septal region, etc.) and hypothalamus (medial preoptic area, paraventricular nucleus). Nucleus paragigantocellularis has been implicated in inhibition of climax. This nucleus, as well as the periaqueductal gray, receives afferent input from genitals. Sexual arousal in men has been associated with activation of the insula and inferior frontal cortex in the right brain hemisphere (84). PFMs are actively involved in the sexual response; their activation has been mostly explored in male individuals during ejaculation, when they are responsible for the expulsion of semen from the urethra (particularly the bulbospongiosus and the bulbocavernosus muscles). Little is known about PFM activity patterns during other parts of the human sexual response cycle. It is assumed that apart from general changes in muscle tone set by the emotional motor system, it is the sacral reflex circuitry that governs much of the PFM activity during the sexual response cycle. The bulbocavernosus reflex behavior, as known from other studies (66), would allow for reflex activation of PFMs during genital stimulation. Tonic stimulation of the reflex is postulated to hinder venous outflow from the penis/clitoris, thus helping erection. PFM reflex contraction should conceivably contribute to the achievement of the “orgasmic platform” (contraction of the levator ani and, in the female individual, the circumvaginal muscles). Climax in humans (in both sexes, and in experimental animals) elicits rhythmic contractions of the PFS/perineal muscles, which in the male individual drives the ejaculate from the urethra (assisted by a coordinated reflex bladder neck closure).

PELVIC FLOOR MUSCLES AND NEUROLOGIC LESIONS Coordinated detrusor-sphincter activity is retained after suprapontine lesions (such as stroke, Parkinson’s disease, etc.); however, normal behavioral patterns of sacral function may be altered and voluntary control of PFMs attenuated or lost. Coordinated detrusor-sphincter activity is lost with lesions between the lower sacral segments and the upper pons, and sphincter inhibition is no longer seen preceding

detrusor contractions. In contrast, detrusor contractions are associated with increased sphincter EMG activity. This pattern of activity is called detrusor-sphincter dyssynergia (85). Sphincter contraction (or failure of relaxation) during involuntary detrusor contractions also has been reported in patients with Parkinson’s disease (86). This neurogenic, uncoordinated, sphincter behavior has to be differentiated from “voluntary” contractions that may occur in poorly complying patients; the sphincter contractions of the so called nonneuropathic voiding dyssynergia may be a learned abnormal behavior (see Rudy and Woodside [87]). Paradoxical EAS activation (“anismus”) during defecation has been described in Parkinson’s disease (88), multiple sclerosis (89), and other “higher” neurologic lesions such as spinal cord injury (90). Nonrelaxation of the puborectalis muscle or the EAS, or both, in the absence of a neurologic disorder has been claimed as a major cause of obstipation, but this is controversial. PFM spasm has been described in women as a nonneurologic functional problem in painful intercourse; the entity “vaginismus” is, however, controversial. No EMG changes have been found in one study (91), and increased basal activity has been reported by another (92). Peripheral lesions—either of the motor nuclei in spinal conus, the nerve roots within cauda equina, or the peripheral nerves (the levator ani nerve, the pudendal nerve)—lead to denervation injury of PFM. This can be partial or total, and it results in atrophy. Collateral reinnervation after partial lesions is the rule. Axonal reinnervation after total denervation may not occur for several physiologic or anatomic reasons. Denervation and reinnervation may be diagnosed by concentric needle EMG (93). Nuclear lesions are encountered in patients with neurodegenerative diseases (multisystem atrophy), whereas other lower motor nuclei are relatively preserved (10). The reverse situation occurs in amyotrophic lateral sclerosis, where the Onuf’s nucleus is relatively preserved (94).

Neuromuscular Injury to the Pelvic Floor Caused by Vaginal Delivery The pelvic diaphragm has evolved in Homo sapiens to a weight-supporting function and an anal, vesical, and vaginal sphincter-controlling function. In the quadrupedal posture, because the pressure produced by distended organs is transmitted to the abdominal wall and not to the pelvic diaphragm, there is no problem in retaining the contents of the bladder or rectum, or both. In the erect posture, these viscera, even when only moderately distended, exert a direct pressure on the anal or vesical aperture. Switching from quadrupedalism to bipedalism therefore requires a more complex control of the visceral apparatus/apertures, and furthermore a change in the neural control of PFMs. It would appear that, in humans, because of the intraabdominal pressure exerted from above in the erect posture, all the muscles of the pelvic floor contract and have a tendency to narrow as much as possible the surrounding ring into which

1006 / CHAPTER 40 R

L

FIG. 40-6. Bilateral recording from the levator ani (pubococcygeus part) in a parous woman with stress incontinence. During coughing a paradoxical unilateral inhibition of motor unit recruitment on the right has been demonstrated as a reproducible abnormal asymmetrical reflex pattern of pelvic floor muscle activation. (Reproduced from Deindl and colleagues [99], by permission.)

they are inserted. The smaller the ring, the better the support, because the total pressure exerted on the pelvic floor is directly proportional to its area. The important evolutionary result is a medial migration of the ischial spine in comparison with quadruped mammals (95). This narrowing of the bispinal diameter of the mid-pelvis is, in contrast, recognized as the most important factor responsible for painful, prolonged, and difficult vaginal delivery. The evolutionary compromise appears to have settled for a state that allows both (difficult) vaginal delivery and an easily compromised continence mechanism (96). In addition, the latter is further jeopardized by vaginal deliveries, which may cause pelvic floor neuromuscular injury. Many studies using different techniques have demonstrated both neurogenic and structural damage to pelvic floor sphincter muscles (c.f. Vodusek [97]). (There remains a role for other lesion mechanisms, such as muscle ischemia.) The implication of all these studies was that PFMs become weak; this, indeed, can be demonstrated (98). Thus, the sphincter mechanisms and pelvic organ support become functionally impaired, with stress incontinence and prolapse being a logical consequence. Although muscle weakness may well be a consequence of childbirth injury, there appear to be further possibilities for deficient PFM function, because it is not only the strength of muscle contraction that counts. Normal neural control of muscle activity leads to coordinated and timely responses to ensure appropriate muscle function as required. Thus, although weakness caused by denervation may be a factor in a selected group of more severely affected women, studies comparing kinesiologic EMG in nulliparous women who are continent and parous women with stress incontinence showed abnormalities of “muscle behavior” in the latter group that would conceivably be functionally relevant (40,99).

Pelvic Floor Activity Patterns in Nulliparous Continent and in Parous Incontinent Women Kinesiologic EMG measurements were performed in a group of healthy nulliparous women (by painless intramuscular recording with wire electrodes) to obtain the activity patterns of the pubococcygeus (40). Good recruitment of motor units on voluntary and reflex activation could be

detected at all registration sites. The overall activation or inhibition of the motor unit firing was always found to be concordant in both pubococcygeal muscles. No isolated unilateral activation or inhibition could be demonstrated. The recruitment patterns could be divided into two types: a gradual crescendo-decrescendo (spindle-like) pattern, and a brisk (sudden onset, sudden offset) activity pattern. Two different patterns of motor activity could be distinguished during relaxation of the muscles. In most recording sites, continuous rather than regular firing of the motor units was observed. In few recording sites, a complete absence of any activity was registered. We named these different behavior patterns the “tonic” and the “phasic” pattern, respectively. The phasic and tonic patterns are probably related to the grouping of motor units with fast- and slow-twitch fibers or to the differences in the gain of excitability of different pools of motor neurons, or to both. Individual muscle activation patterns in parous women with urinary stress incontinence were, in principle, similar to those obtained in continent nulliparous women. However, significant quantitative and qualitative differences were noted. The duration of recruitment of motor units on voluntary maximal activation was significantly shorter in the urinary stress incontinent parous group than in the nulliparous group, with the bladder both empty and full (99). In some of the parous women with urinary stress incontinence, pubococcygeus activation disturbances were demonstrated, such as only unilateral reflex recruitment of motor unit, or, in fact, concomitant activation and inactivation of the right and left muscles (Fig. 40-6). All of the described dysfunctional patterns were well reproducible. We termed them either reflex or voluntary coordination disturbances of pubococcygeus muscle activity, respectively. Our initial findings were corroborated by further studies in women with stress incontinence (100). A delay in PFM activation on coughing also has been demonstrated in stress-incontinent patients versus continent women (78).

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79. Rattan S. The internal anal sphincter: regulation of smooth muscle tone and relaxation. Neurogastroenterol Motil 2005;17(suppl 1): 50–59. 80. Bartolo DCC, Macdonald ADH. Fecal continence and defecation. In: Pemberton JH, Swash M, Henry MM, eds. The pelvic floor. Its function and disorders. London: WB Saunders, 2002;77–83. 81. Enck P, Eggers E, Koletzko S, Erckenbrecht JF. Spontaneous variation of anal “resting” pressure in healthy humans. Am J Physiol 1991; 261:G823–G826. 82. Fucini C, Ronchi O, Elbetti C. Electromyography of the pelvic floor musculature in the assessment of obstructed defecation symptoms. Dis Colon Rectum 2001;44:1168–1175. 83. Fernandez-Fraga X, Azpiroz F, Malagelada J-R. Significance of pelvic floor muscles in anal incontinence. Gastroenterology 2002;123:1441–1450. 84. Stoleru S, Gregoire MC, Gerard D, Decety J, Lafarge E, Cinotti L, et al. Neuroanatomical correlates of visually evoked sexual arousal in human males. Arch Sex Behav 1999;28:1–21. 85. Blaivas JG, Sinha HP, Zayed AA, Labib KB. Detrusor-external sphincter dyssynergia: a detailed electromyographic study. J Urol 1981;125:545–548. 86. Pavlakis AJ, Siroky MB, Goldstein I, Krane RJ. Neurourologic findings in Parkinson’s disease. J Urol 1983;129:80–83. 87. Rudy DC, Woodside JR. Non-neurogenic neurogenic bladder: the relationship between intravesical pressure and the external sphincter electromyogram. Neurourol Urodyn 1991;10:169. 88. Mathers SE, Kempster PA, Law PJ, Frankel JP, Bartram CI, Lees AJ, et al. Anal sphincter dysfunction in Parkinson’s disease. Arch Neurol 1989;46:1061–1064. 89. Chia YW, Gill KP, Jameson JS, Forti AD, Henry MM, Swash M, et al. Paradoxical puborectalis contraction is a feature of constipation in patients with multiple sclerosis. J Neurol Neurosurg Psychiatry 1996;60:31–35. 90. Pannek J, Greving I, Tegenthoff M, Nedjat S, Boetel U, May B, et al. Urodynamic and rectomanometric findings in patients with spinal cord injury. Neurourol Urodyn 2001;20:95–103. 91. van der Velde J, Laan E, Everaerd W. Vaginismus, a component of a general defensive reaction. An investigation of pelvic floor muscle activity during exposure to emotion-inducing film excerpts in women with and without vaginismus. Int Urogynecol J Pelvic Floor Dysfunct 2001;12:328–331. 92. Graziottin A, Bottanelli M, Bertolasi L. Vaginismus: a clinical and neurophysiological study. Urodinamica 2004;14:117–121. 93. Vodusek DB, Fowler CJ. Pelvic floor clinical neurophysiology. In: Binnie C, Cooper R, Mauguiere F, Osselton J, Prior P, Tedman B, eds. Clinical neurophysiology, volume 1. Revised and enlarged edition. EMG, nerve conduction and evoked potentials. Amsterdam: Elsevier, 2004;281–307. 94. Iwata M, Hirano A. Current problems in the pathology of amyotrophic lateral sclerosis. In: Zimmerman HM, ed. Progress in neuropathology. New York: Raven Press, 1979;277–298. 95. Abitbol MM. Evolution of the ischial spine and of the pelvic floor in the Hominoidea. Am J Phys Anthropol 1988;75:53–67. 96. Deindl FM, Vodusek DB, Schüssler B, Hofmann R, Hartung U. Pelvic floor activity patterns in urinary stress incontinent women: evolutionary, neurophysiological and diagnostic considerations. Int Urogynecol J Pelvic Floor Dysfunct 1995;6:175–179. 97. Vodusek DB. The role of electrophysiology in the evaluation of incontinence and prolapse. Curr Opin Obstet Gynecol 2002;14:509–514. 98. Verelst M, Leivseth G. Are fatigue and disturbances in preprogrammed activity of pelvic floor muscles associated with female stress urinary incontinence? Neurourol Urodyn 2004;23:143–147. 99. Deindl FM, Vodusek DB, Hesse U, Schussler B. Pelvic floor activity patterns: comparison of nulliparous continent and parous urinary stress incontinent women. A kinesiological EMG study. Br J Urol 1994;73:413–417. 100. Peschers UM, Vodusek DB, Fanger G, Schaer GN, DeLancey JO, Schuessler B. Pelvic muscle activity in nulliparous volunteers. Neurourol Urodyn 2001;20:269–275.

CHAPTER

41

Pathophysiology Underlying the Irritable Bowel Syndrome Jackie D. Wood Neuropathy in the Brain-in-the-Gut, 1009 Enteric Motor Neurons, 1010 Inhibitory Motor Neurons, 1010 Disinhibitory Motor Disease, 1011 Neurogenic Secretion: Diarrhea and Constipation, 1013 Secretomotor Neurons, 1013 Abdominal Pain and Discomfort, 1015 Peripheral Neuropathologic Factors, 1015 Postinfectious Irritable Bowel Syndrome, 1015 Serotonin Receptors on Sensory Afferents, 1016 Receptors for Inflammatory Mediators on Sensory Afferents, 1017 Central Neuropathologic Factors, 1017 Intestinal Motility and Abdominal Pain, 1021

Psychological Stress, 1022 Neuroimmunophysiologic Paradigm for Irritable Bowel Syndrome–Like Symptoms, 1022 Immunoneural Communication, 1022 Sources of Immunoneural Signals, 1022 Enteric Mast Cells, 1023 Mast Cells and the Brain–Gut Connection, 1024 Mast-Cell Signal Substances, 1025 Brain–Mast Cell Connection: Implications for Irritable Bowel Syndrome, 1026 References, 1027

The irritable bowel syndrome (IBS) is a functional disorder that affects 10% to 20% of the population worldwide (1–3). A functional disorder is one for which no abnormal physical or metabolic processes can be found that explain the symptoms. Predominant symptoms of IBS are abnormal defecation and abdominal pain, both of which may be exacerbated by emotional stress (4). Abnormal defecation can be diarrhea or constipation, and a subgroup of patients with IBS might alternate from one to the other over time. Urgency to stool and liquid incontinence often accompany the diarrheal state. Patients with the constipation-predominant form of IBS might report bloating, straining, and sensations of incomplete evacuation. A significant subpopulation of patients with IBS is hypersensitive to distension of the rectosigmoid region of

the large bowel, and the hypersensitivity can extend to other regions of the digestive tract (e.g., esophagus) (4). The basic gastrointestinal physiology in this chapter deals with the normal physiology of the systems from which IBS symptoms emerge. Knowledge of integrated systems physiology is necessary for understanding the pathophysiology that underlies the symptoms of IBS and for rational therapeutic strategies. The systems involved in the defecation-related symptoms are the intestinal secretory glands, the musculature, and the nervous system that controls their activity. Abdominal pain and discomfort, which arise from these systems, falls into the sphere of sensory neurophysiology (see Chapter 25). This chapter aims to explore the pathophysiology of IBS in terms of current concepts of the physiology of intestinal secretion, motility, nervous control, and sensing functions.

J. D. Wood: Departments of Physiology and Biology and Internal Medicine, The Ohio State University, College of Medicine, Columbus, Ohio 43210.

NEUROPATHY IN THE BRAIN-IN-THE-GUT

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Intestinal behavior reflects the integrated function of the musculature, mucosal epithelium, and vasculature. The enteric nervous system (ENS) organizes and coordinates the activity

1009

1010 / CHAPTER 41 of the three effector systems to generate functionally effective patterns of behavior. The ENS is a local minibrain with a library of programs for different patterns of intestinal behavior. Mixing in the digestive state, the migrating motor complex in the interdigestive state, and emetic patterns of smallintestinal motor behavior are examples of outputs from three of the neural programs in the ENS library. During emesis, the program includes reversal of peristaltic propulsion in the upper jejunum and duodenum to rapidly propel the contents toward the open pylorus and relaxed antrum and corpus. Structure, function, and neurochemistry of the ganglia of the enteric nervous system are unlike other autonomic ganglia, most of which function mainly as relay-distribution centers for signals from the central nervous system (CNS). Neurons in the ganglia of the ENS are interconnected to form an independent nervous system with mechanisms for integration and processing of information similar to those in the brain and spinal cord. Instead of localizing neural control entirely within the CNS, a minibrain has evolved within the walls of the digestive tract distributed in close proximity to the effector systems that must be controlled and regulated along several meters of bowel. The ENS contains as many neurons as the spinal cord. The large number of neurons, required for program control of digestive functions, would greatly expand the CNS if placed there. Rather than packing the neural control circuits exclusively within the skull or vertebral column and transmitting control signals over long transmission lines to the gut, vertebrate animals have evolved with most of the neural networks required for automatic feedback control spatially distributed along the digestive tract in close relationship to the effector systems that must be controlled and integrated for wholeorgan function. Like the CNS, the ENS has interneurons and motor neurons that are interconnected by chemical synapses into functional neural networks. Also like the CNS, the ENS networks receive input from sensory neurons. The sensory neurons, most of which have their cell bodies in spinal dorsal root ganglia and terminals in the intestinal wall and spinal cord, have receptor regions specialized for detecting changes in thermal, chemical, or mechanical stimulus energy. The receptor regions transform changes in stimulus energy into signals coded by action potentials that subsequently are transmitted along sensory nerve fibers to be received and processed in both the ENS and CNS. Interneurons are connected by synapses into networks that process sensory information and control the behavior of motor neurons. Multiple connections among many interneurons use the same mechanisms of chemical neurotransmission as the CNS to form “logic” circuits that decipher inputs from sensory neurons. Some of the circuits are reflex circuits that organize reflex responses to sensory inputs, and others are integrative circuits that contain the programs for motor behavioral patterns, such as the migrating motor complex, haustral formation in the colon, and postprandial mixing movements in the small intestine. Motor neurons are the final common output pathways for transmission of control signals from the interneuronal networks to the effector systems.

Enteric Motor Neurons The ENS pool of motor neurons includes both excitatory and inhibitory motor neurons. Enteric excitatory motor neurons release neurotransmitters that evoke contraction of the musculature and secretion from mucosal glands. Acetylcholine and substance P are the primary neurotransmitters released from excitatory motor neurons to stimulate muscle contraction. Acetylcholine, vasoactive intestinal polypeptide, and adenosine triphosphate (ATP) are excitatory neurotransmitters responsible for evoking secretion from the intestinal crypts of Lieberkühn. Enteric inhibitory motor neurons release neurotransmitters that act to suppress contractile activity of the musculature. Vasoactive intestinal polypeptide, nitric oxide, and ATP are among the neurotransmitters implicated as inhibitory neurotransmitters at neuromuscular junctions in the digestive tract.

Inhibitory Motor Neurons Enteric inhibitory motor neurons are a central focus in consideration of ENS neuropathy because their loss is manifest as profound changes in contractile behavior of the intestinal musculature. The changes in motor behavior associated with degeneration of inhibitory motor neurons reflect the specialized physiology of the musculature. The musculature of the digestive tract is described as a self-excitable electrical syncytium consisting of interstitial cells of Cajal (ICCs) that function as pacemakers for the circular muscle coat, which generates contractile forces for propulsion (see Chapter 20). The term electrical syncytium implies that action potentials and pacemaker potentials spread by way of gap junctions from smooth muscle fiber to muscle fiber in three dimensions in much the same way as in cardiac muscle. The action potentials trigger contractions as they spread through the musculature. The ICCs are a nonneural pacemaker system of electrical slow waves that are electrically coupled to the musculature and account for the selfexcitable characteristics of the muscle (5–8). Within this organization, the electrical slow waves are an extrinsic factor to which the circular muscle responds. Consideration of these physiologic characteristics of the musculature evokes the questions of why the circular muscle fails to respond with action potentials and contractions to each and every pacemaker cycle and why action potentials and contractions do not spread in the syncytium throughout the entire length of intestine whenever they occur at any point along the bowel. The answer is that the circular muscle in a segment of intestine can respond only to invading electrical slow waves from ICCs when the inhibitory motor neurons in the ENS of that segment are inactivated by input from the control circuits formed by interneurons. Likewise, action potentials and associated contractions can propagate only into regions of musculature where the inhibitory motor neurons are inactivated. Therefore, activity of inhibitory motor neurons determines when the omnipresent slow waves

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1011 initiate a contraction, as well as the distance and direction of propagation once the contraction has begun. Because some of the inhibitory motor neurons to the circular muscle fire continuously, action potentials and contractions of the muscle are permitted only when the inhibitory neurons are inactivated by input from interneurons in the control circuits (9,10). Firing behavior of inhibitory motor neurons to smooth muscle sphincters (e.g., lower esophageal and internal anal sphincters) is opposite to that of the intestinal circular muscle coat. Inhibitory motor neurons to the sphincters are normally quiescent and are switched to firing mode with timing appropriate for coordinated opening of the sphincter with physiologic events in adjacent regions. When inhibitory motor neurons fire, they release inhibitory neurotransmitters that relax ongoing muscle contraction in the sphincteric muscle and prevent excitation-contraction in the musculature on either side of the sphincter from spreading into and closing the sphincter. In nonsphincteric circular muscle, the activity state of the inhibitory innervation determines the length of a contracting segment by controlling the distance of spread of action potentials within the three-dimensional electrical geometry of the smooth muscle syncytium. Contraction can occur in segments in which ongoing inhibition is inactivated, whereas adjacent segments with continuing inhibitory input cannot contract. The boundaries of the contracted segment reflect the transition zone from inactive to active inhibitory motor neurons. The directional sequence in which the inhibitory motor neurons are inactivated establishes the direction of propagation of the contraction. Normally, inhibition is progressively inactivated in the aboral direction, resulting in contractile activity that propagates in the aboral direction. During emesis, the inhibitory motor neurons must be inactivated in a reverse sequence to account for small-intestinal propulsion that travels toward the stomach. In general, any treatment or condition that ablates the intrinsic inhibitory neurons results in spastic contractile behavior of the intestinal circular muscle coat. Several conditions associated with ablation of enteric inhibitory neurons are associated with conversion from a hypoactive contractile condition of the circular muscle to a hyperactive contractile state. Observations of this nature, usually made by manometric recording methods in vivo, reinforce the evidence that a subset of the pool of inhibitory motor neurons is tonically active, and that blockade or ablation of these neurons releases the circular muscle from the inhibitory influence (9–11). The behavior of the muscle in these cases is tonic contracture and recurring, disorganized, phasic contractile activity.

Disinhibitory Motor Disease The physiology of inhibitory neuromuscular relations in the intestine predicts that spasticity and “achalasia” (i.e., failure to relax) will accompany any condition where inhibitory motor neurons are destroyed. Without ENS inhibitory neural control, the self-excitable smooth muscle contracts continuously and

behaves as an obstruction. This happens because the muscle is freed to respond to the pacemaker ICCs (i.e., electrical slow waves) with contractions that propagate without amplitude, distance, or directional control. Contractions spreading in the uncontrolled syncytium collide randomly, resulting in chaotic-ineffective behavior in the affected intestinal segment, which is reminiscent of fibrillation in the myocardium. Loss or malfunction of inhibitory motor neurons is the pathophysiologic starting point for disinhibitory motor disease, which includes several forms of chronic intestinal pseudo-obstruction and sphincteric achalasia. Neuropathic degeneration of the ENS includes loss of inhibitory motor neurons and is a progressive disease that, in its early stages, may be manifest as symptoms that could be interpreted as a functional gastrointestinal disorder (i.e., IBS). Intestinal pseudo-obstruction is a pathophysiologic condition in which the symptoms resemble those of a mechanical obstruction to forward propulsion, but without the presence of a mechanical obstruction (e.g., intussusception and strictures). Chronic intestinal pseudo-obstruction can be myopathic or neuropathic. The neuropathic form of chronic intestinal pseudo-obstruction is a form of disinhibitory motor disease linked with neuropathic degeneration in the ENS. Failure of propulsive motility in the affected length of bowel reflects loss of the neural microcircuits that program and control the repertoire of motility patterns required for the necessary functions of that region of bowel. Pseudo-obstruction occurs partly because contractile behavior of the circular muscle is hyperactive but disorganized in the denervated regions (12,13). Hyperactivity determined manometrically in vivo is a diagnostic sign of the neuropathic form of chronic small-bowel pseudo-obstruction. The hyperactive and disorganized contractile behavior reflects the absence of inhibitory nervous control of the muscles that are autogenic when released from the braking action imposed by the inhibitory motor neurons of the ENS. Chronic pseudoobstruction is therefore symptomatic of the advanced stage of a progressive enteric neuropathy. Retrospective review of patient records suggests that some IBS-like symptoms can be an expression of early stages of the neuropathy (14–16). Degenerative noninflammatory and inflammatory ENS neuropathies are two kinds of disinhibitory motor disorders that culminate in pseudo-obstruction. Noninflammatory neuropathies can be either familial or sporadic (17). The mode of inheritance can be autosomal recessive or dominant. In the former, the neuropathological findings include a marked reduction in the number of neurons in both myenteric and submucosal plexuses, and the presence of round, eosinophilic intranuclear inclusions in about 30% of the residual neurons. Histochemical and ultrastructural evaluations show the inclusions not to be viral particles, but rather proteinaceous material forming filaments that, in some ways, are reminiscent of Alzheimer’s disease in the brain (18,19). Members of two families have been described with intestinal pseudo-obstruction associated with the autosomal dominant form of ENS neuropathy (20,21). The numbers of enteric neurons were decreased in these patients, with no

1012 / CHAPTER 41 alterations found in the CNS or parts of the autonomic nervous system outside the gut. Degenerative inflammatory ENS neuropathies are characterized by a dense inflammatory infiltrate confined to enteric ganglia. Paraneoplastic syndrome, Chagas’ disease, and idiopathic degenerative disease are recognizable forms of pseudo-obstruction related to inflammatory neuropathies. Paraneoplastic syndrome is a form of pseudo-obstruction where commonality of antigens between small-cell carcinoma of the lungs and enteric neurons leads to autoimmune attack, which results in loss of neurons (22). Most patients with symptoms of pseudo-obstruction in combination with small-cell lung carcinoma have IgG autoantibodies that react with their enteric neurons (23). These antibodies react with a variety of molecules expressed on the nuclear membrane of neurons, including Hu and Ri proteins, and with cytoplasmic antigens (Yo protein) of Purkinje cells in the cerebellum (24–27). Immunostaining with sera from paraneoplastic patients shows a characteristic pattern of staining in enteric neurons (24). The detection of anti-enteric neuronal antibodies is a means to a specific diagnosis. Induction of apoptosis is involved in the mechanisms by which the antibodies damage the neurons (28). The association of enteric neuronal loss and symptoms of pseudo-obstruction in Chagas’ disease also reflects autoimmune attack on the neurons with accompanying symptoms that mimic the situation in sphincteric achalasia and paraneoplastic syndrome. Trypanosoma cruzi, the bloodborne parasite that causes Chagas’ disease, has antigenic epitopes similar to enteric neuronal antigens (29). This antigenic commonality

activates the immune system to assault the ENS coincident with its attack on the parasite. Idiopathic inflammatory degenerative ENS neuropathy occurs unrelated to neoplasms, infectious conditions, or other known diseases (30–34). De Giorgio and colleagues (30) and Smith and coworkers (32) described two small groups of patients with early reports of symptoms similar to IBS that progressively worsened and were later diagnosed as idiopathic degenerative inflammatory neuropathy based on fullthickness biopsies taken during exploratory laparotomy, which revealed chronic intestinal pseudo-obstruction. Each patient had inflammatory infiltrates localized to the myenteric plexus. Sera from the two cases reported by Smith and colleagues (32) contained antibodies against enteric neurons similar to those found in secondary inflammatory neuropathies (i.e., anti-Hu), but with different immunolabeling patterns characterized by prominent cytoplasmic rather than nuclear staining. Recognition of the brainlike functions of the ENS leads to a conclusion that early neuropathic changes are expected to be manifest as symptoms that worsen with progressive neuronal loss. In diagnostic motility studies (e.g., manometry), degenerative loss of enteric neurons is reflected by hypermotility and spasticity (12) because inhibitory motor neurons are included in the missing neuronal population. The observations in patients with autoimmune attack on the ENS suggest that early stages of an enteric neuropathy might be expressed as IBS-like symptoms. The disease in these individuals appears to progress from IBS-like symptoms to symptoms of chronic intestinal pseudo-obstruction in parallel with progressive loss of neurons from their ENS (Fig. 41-1).

Enteric neuropathy Autoimmune attack Normal enteric Nervous system

Normal bowel

A

Progressive neuronal degeneration

Progressive neuronal degeneration Functional bowel symptoms

End-Stage neuropathy

Chronic intestinal pseudoobstruction

B FIG. 41-1. Enteric neuropathy. (A) Idiopathic myenteric ganglionitis. Histologic section of the myenteric plexus obtained from a full-thickness small-intestinal biopsy acquired during exploratory laparotomy for intestinal obstruction. A diagnosis of neuropathic small-intestinal pseudo-obstruction was made from neuronal degeneration associated with an inflammatory infiltrate localized to the patient’s myenteric plexus. The patient had a multiyear history of symptoms suggestive of irritable bowel syndrome (IBS). (Histologic section and patient history provided by Dr. Claudio Fiocchi, University Hospitals, Cleveland, OH.) (B) Parallel progression of enteric neuronal degeneration and functional gastrointestinal disorders (e.g., IBS). Most individuals start postnatal life with a normal enteric nervous system (ENS) and normally functioning bowel. Neuronal degeneration mediated by autoimmune attack directed to the enteric nervous system (e.g., paraneoplastic syndrome, Chagas’ disease, idiopathic forms) can progress to a stage where ENS function is lost. Functional bowel symptoms appear as neurons are lost from the ENS microcircuits required for integrated function of the whole organ. Chronic intestinal pseudo-obstruction of the neuropathic form occurs when the loss of neurons progresses to a stage where the neuronal circuits for propulsive motility are no longer functional.

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1013 NEUROGENIC SECRETION: DIARRHEA AND CONSTIPATION Disordered defecation in IBS is related directly to the physiology of enteric secretomotor neurons. Secretomotor neurons are excitatory motor neurons in the submucosal plexus of the ENS, which innervate and stimulate secretion from the intestinal crypts of Lieberkühn (Fig. 41-2).

Secretomotor Neurons Secretomotor neurons are uniaxonal with characteristic shapes described as Dogiel type I and electrophysiologic behavior similar to S-type enteric neurons (39,40) (see Chapter 23). When they fire, they release acetylcholine and vasoactive intestinal polypeptide at the neuroepithelial junctions. Collateral projections from secretomotor axons innervate submucosal arterioles (see Fig. 41-2). Collateral innervation of the blood vessels links secretomotor activity in the crypts to submucosal blood flow (35). Active firing of secretomotor neurons releases acetylcholine simultaneously at

neuroepithelial and neurovascular junctions. Acetylcholine acts at the blood vessels to release nitric oxide from the endothelium, which, in turn, dilates the vessels and increases blood flow in support of stimulated secretion (35). Secretomotor neurons have receptors that receive excitatory and inhibitory synaptic input from other neurons in the integrative circuitry of the ENS and from sympathetic postganglionic neurons (36–38). Their excitability can be greatly influenced also by paracrine messages from nonneural cell types in the mucosa and submucosa (e.g., enterochromaffin cells and immune/inflammatory cells). Activation of the excitatory receptors on secretomotor neurons stimulates the neurons to fire and release their transmitters at the junctions with the crypts and regional blood vessels. The overall result of secretomotor neuronal firing is stimulation of the secretion of H2O, NaCl, bicarbonate, and mucus from the intestinal glands into the intestinal lumen.

Inhibitory Input to Secretomotor Neurons Inhibitory inputs from other neurons hyperpolarize the membrane potential of secretomotor neurons, which decreases

FIG. 41-2. Intestinal crypts are innervated by secretomotor neurons. Neurotransmitters (e.g., acetylcholine [ACh] and vasoactive intestinal peptide [VIP]), which evoke secretion, are released at the neuro-crypt junctions when secretomotor neuron fire. Axon collaterals to blood vessels simultaneously dilate submucosal vessels to increase blood flow in support of stimulated secretion. Noradrenergic input from the sympathetic nervous system and somatostatinergic input from intrinsic enteric nervous system neurons suppress firing of secretomotor neurons, and thereby inhibit secretion. Inflammatory mediators act to increase excitability of secretomotor neurons and at presynaptic inhibitory receptors to suppress release of norepinephrine from sympathetic nerves and somatostatin from intrinsic neurons. Stimulation of secretomotor neurons by paracrine signals is expected to evoke secretion and can account for neurogenic secretory diarrhea. Presynaptic inhibition of norepinephrine and somatostatin release facilitates secretion by removing the braking action of sympathetic and intrinsic nerves, which might contribute to diarrheal symptoms. IPSP, inhibitory postsynaptic potential.

1014 / CHAPTER 41 overstimulated by circulating VIP released from VIPomas, excessive serotonin release from mucosal enterochromaffin cells, or histamine release from inflammatory/immune cells in the mucosa/submucosa (41). Release of histamine or other inflammatory mediators associated with diarrhea not only stimulates the firing of secretomotor neurons, but also acts simultaneously at presynaptic inhibitory receptors to suppress the release of norepinephrine from the postganglionic sympathetic axons that provide inhibitory input to secretomotor neurons (37). Thus, two pathologic factors are involved in the production of neurogenic secretory diarrhea. One is overstimulation of secretomotor neuronal firing, and the other is presynaptic suppression of norepinephrine release from sympathetic postganglionic neurons that results in removal of the sympathetic braking action on the neurons (see Fig. 41-2).

the probability of firing (see Fig. 41-2). The physiologic effect of inhibiting secretomotor firing is suppression of mucosal secretion. Postganglionic neurons of the sympathetic nervous system are an important source of inhibitory input to the secretomotor neurons. Norepinephrine released from sympathetic nerve terminals in the submucosal plexus acts at α2 noradrenergic receptors to inhibit firing of the secretomotor neurons there. Inhibition of secretomotor firing reduces the release of excitatory neurotransmitters at the junctions with epithelial cells in the crypts. The end result is reduced secretion of water and electrolytes. Suppression of secretion in this manner is part of the mechanism involved in sympathetic nervous shutdown of gut function in homeostatic states where blood is shunted from the splanchnic to systemic circulation. Neurogenic Secretory Diarrhea and Neurogenic Constipation

Excitatory Input to Secretomotor Neurons

Knowledge of the neurobiology of submucous secretomotor neurons is central to understanding the pathophysiology of secretory diarrhea, as well as constipation. In general, secretomotor hyperactivity is associated with neurogenic secretory diarrhea; hypoactivity is associated with decreased secretion and a constipated state. Suppression of secretomotor firing by antidiarrheal agents (e.g., opiates, clonidine, and somatostatin analogs) is manifest as harder, drier stools. Stimulation by chemical mediators, such as vasoactive intestinal peptide (VIP), serotonin, and histamine, is manifest as more liquid stools. Watery diarrhea may be caused by several different pathophysiologic events. For example, secretomotor neurons may be

Secretomotor neurons have excitatory receptors for several neurotransmitters, which include acetylcholine, VIP, substance P, and serotonin (see Chapter 27). One of the serotonergic receptors belongs to the 5-hydroxytryptamine subtype 3 (5-HT3) receptor (42). Efficacy of blockade of 5-HT3 receptors by a 5-HT3 antagonist in the treatment of diarrhea in the diarrhea-predominant population of women with IBS (43,44) suggests that overstimulation of secretomotor neurons by serotonin is a significant factor in this form of IBS (Fig. 41-3). Observations that IBS symptoms of cramping abdominal pain, diarrhea, and fecal urgency are commonly exacerbated in the postprandial state, (4,45) and evidence for increased postprandial release of serotonin (46,47) raises

A

B FIG. 41-3. Efficacy of a serotonergic 5-hydroxytryptamine subtype 3 (5-HT3) receptor blocking drug, alosetron, in treatment of the diarrhea-predominant form of irritable bowel syndrome (IBS) in women. (A) Treatment with the 5-HT3 receptor antagonist resulted in firmer stools relative to placebo after the first week, which persisted throughout the treatment period. (B) Assessment of adequate relief of abdominal pain and discomfort. Improvement relative to placebo reached significance after 4 weeks and persisted throughout the treatment period. Beneficial effects for pain and discomfort and stool consistency returned to baseline within 1 week of discontinuation of the drug. Stool consistency was scored as follows: 1 = very hard; 2 = hard; 3 = formed; 4 = loose; 5 = watery. *P < 0.05 (Data courtesy of GlaxoSmithKline, Inc.)

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1015 suspicion that overactive release of serotonin from the enterochromaffin cell population underlies the diarrheal symptoms of IBS. This is reinforced by suggestions of increased numbers of serotonin-containing enterochromaffin cells and also mast cells in the mucosa of patients with IBS (48–50).

ABDOMINAL PAIN AND DISCOMFORT

Percent reporting pain

Primary causes of abdominal pain of digestive tract origin are distension and exaggerated muscular contraction. Stimuli such as pinching and burning of the mucosa applied via fistulae do not evoke pain. Hypersensitivity of the mechanosensors for stretch (distension) and contractile tension are implicated as pain factors in IBS. Hypersensitivity to distension is present in a substantial subset of patients with IBS (51–54). Filling of a balloon placed in the rectosigmoid region evokes sensations of discomfort and pain at lower distending volumes for patients with IBS than for healthy subjects (Fig. 41-4). Hypersensitivity to distension is not restricted to the distal bowel. Patients who are diagnosed with functional dyspepsia also experience discomfort and pain at lower distending volumes in the stomach than healthy subjects (55,56), and hypersensitivity to distension is present in the esophagus of patients with noncardiac chest pain (57). Patients with IBS experience the same kind of hypersensitivity to electrical stimulation applied to the lining of the rectosigmoid region as they do during balloon distension in the rectosigmoid (58). The hypersensitivity to direct electrical stimulation of the sensory innervation implicates abnormal sensory neurophysiology rather than mechanical factors (e.g., wall tension and compliance) as underlying the decreased sensory thresholds present in patients with IBS. Identification of the sensory defect is an area of current active investigation and is yet to be determined satisfactorily. The lower sensory threshold in IBS might reflect the following: (1) sensitization of intramural mechanosensors; (2) sensitization of neurotransmission in the spinal cord; (3) abnormal processing of arriving sensory information in the brain; and (4) a combination of all three possibilities.

60

Irritable bowel patients

50

Normal subjects

40 30 20 10

Peripheral Neuropathologic Factors Mechanosensitive primary afferents in the walls of the specialized organs of the digestive tract detect and signal contraction and distension of the musculature. The mechanosensing structures at the afferent terminals behave as if they are attached “in series” with the long axes of the smooth muscle fibers (Fig. 41-5). The in-series arrangement accounts for activation of the sensors by either muscle contraction or distension. The cell bodies of the intestinal afferents are located in nodose ganglia and dorsal root spinal ganglia (Fig. 41-6). Mechanosensitive information is transmitted along spinal afferents to the spinal cord by way of dorsal root ganglia and along vagal sensory afferents to the brainstem by way of the nodose ganglia. Vagal and spinal afferent fibers are predominantly unmyelinated C-fibers or thinly myelinated A-delta fibers, which transmit different modalities of sensory information at low-conduction velocity. In general, vagal afferents transmit physiologic information on the nature and composition of the luminal contents, the presence or absence of motility, and contractile tension in the musculature, whereas spinal afferents transmit pathophysiologic information related to potentially noxious mechanical or chemical stimuli arising through tissue injury, ischemia, or inflammation. Sensations of pain and discomfort of digestive tract origin reflect transmission in spinal afferents and information processing in the spinal cord and brain. Sensory information transmitted by the vagus nerves appears not to reach the level of conscious sensation because patients with high spinal cord transections and intact vagal pathways experience little or no sensations of digestive tract origin. In contrast, the persistence of conscious perception of sensations from the digestive tract after a surgical vagotomy (e.g., as outdated treatment for peptic ulcer disease) reflects transmission over spinal afferents. The streams of mechanosensory information that are transmitted from the small and large intestines to the CNS are generated by low-threshold, high-threshold, and silent afferents (see Chapter 25). Low-threshold afferents respond to weak changes in wall tension and are thought to transmit the minute-to-minute information required for functional autonomic negative-feedback control. High-threshold afferents require stronger changes in wall tension, which might be produced by distension of the lumen or strong contraction of the musculature, for activation. The high-threshold afferents are postulated to be responsible for the range of sensations from mild to severe pain that are associated with excessive distension or exceptionally strong muscle contraction. Silent afferents are of pathophysiologic interest because they become active and highly sensitive to stimuli during inflammatory states (59,60).

0 20

60 80 100 120 Distension of rectosigmoid region (ml)

FIG. 41-4. Patients with irritable bowel syndrome have increased sensitivity to balloon distension in the large intestine. (Modified from Whitehead and colleagues [52], by permission.)

Postinfectious Irritable Bowel Syndrome A significant proportion of patients experience IBS-like symptoms after an acute bout of infectious enteritis (61–64).

1016 / CHAPTER 41

Mechanoreceptor (e.g., Filling)

Muscle contraction

Distension (e.g., Balloon)

FIG. 41-5. Mechanoreceptors are connected “in series” with the long axes of the smooth muscle fibers that form the intestinal circular muscle layer. Either contraction of the muscle or distension of the intestinal wall distorts the receptors, leading to increased firing in the afferent fibers connected to the receptor and transmission of sensory input to the spinal cord or dorsal vagal motor complex (see Fig. 41-6).

Hypochondriasis and adverse life events are reported to double the risk for development of postinfective IBS (62). Nevertheless, whether the association between acute infectious enteritis and IBS reflects low-level inflammation (e.g., microscopic enteritis) and chronic exposure of the neural and glial elements of the ENS to increased levels of serotonin, histamine, or other inflammatory mediators is yet to be fully resolved.

Serotonin Receptors on Sensory Afferents Application of 5-HT evokes increased firing in extrinsic afferents via 5-HT3 receptors that can be blocked by selective

antagonists (e.g., alosetron) (65,66). Intramural terminals of both spinal and vagal sensory nerves express 5-HT3 receptors (see Fig. 41-6). Reported efficacy of alosetron in the treatment of abdominal pain and discomfort in the diarrheapredominant form of IBS in women (see Fig. 41-3) suggests that these symptoms might reflect overactive endogenous release and increased levels of 5-HT (44,67). Persistence of 5-HT at its receptors, because of a defective serotonin transporter, is a second possibility for hyperstimulation of intramural serotoninergic receptors. Active uptake mediated by the serotonin transporter, which is expressed by enteric neurons and the mucosal epithelium, restricts accumulation and action of 5-HT at its receptors after its release (68). Down-regulation of both transporter

Brain

Sensory afferent

Dorsal root ganglion Ascending spinal tracts

Adenosine

5-HT3

Histamine

Receptors for paracrine mediators (Examples)

Sensory receptor

Spinal cord

FIG. 41-6. The terminals of sensory afferents in the intestinal wall express receptors for paracrine mediators. Stimulation of the receptors by paracrine mediators changes the responsiveness of the sensory receptor (e.g., mechanoreceptors), thereby altering the flow of information into the enteric and central nervous systems. 5-HT3, 5-hydroxytryptamine subtype 3 receptor.

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1017 messenger RNA expression and expression of immunoreactivity for the serotonin transporter were reported to be present in mucosal biopsies taken from the large intestine of patients with IBS (69). Watery stools and enhanced propulsive motility occurred in mice with a deletion in the serotonin transporter gene, and the mice sometimes alternated between diarrhea and constipation in a way reminiscent of the subset of patients with IBS who are classified as “alternators” (70).

Receptors for Inflammatory Mediators on Sensory Afferents In addition to 5-HT3 receptors, receptors for bradykinin, ATP, adenosine, prostaglandins, leukotrienes, and mast-cell proteases are expressed on spinal sensory nerve terminals (71) (see Fig. 41-6). Any one of these mast-cell mediators has potential for increasing the sensitivity of intestinal sensory nerves, especially in the disordered conditions of inflammation or ischemia. This is reinforced by findings that a reduced threshold for painful responses to balloon distension in the large bowel is associated with degranulation of mast cells in animal models. Treatment with mast-cell stabilizing drugs prevents the reduction of the pain threshold during mucosal inflammation in the animal models (72) and suggests that mast-cell stabilization could be an efficacious treatment in human IBS (73).

Central Neuropathologic Factors Much of the evidence points to altered sensory transduction as the underlying cause of the exaggerated sensitivity to distension found in patients with IBS. Mechanosensitive primary afferents in the gut wall become hypersensitive to mechanical stimuli and, as a result, transmit nociceptive information to the CNS. In contrast, there might be cases where normally functioning mechanosensors transmit accurate information, which is then misinterpreted in processing circuits in the spinal cord, brain, or both to evoke conscious perceptions of abnormal sensations from the gut. Ascending Sensory Pathways in the Spinal Cord Ascending spinal pathways involved in the transmission of nociceptive signals from the digestive tract are the spinothalamic, spinohypothalamic, spinosolitary, spinoreticular, and spinoparabrachial tracts (Fig. 41-7). A recently realized pathway in the dorsal columns also transmits visceral pain information to higher processing centers in the brain (74–81). Spinal afferents, which transmit nociceptive information, synapse directly with second-order neurons in the dorsal column nuclei (i.e., nucleus gracilis and nucleus cuneatus). Second-order neurons in the spinal dorsal horn also provide input to the dorsal column nuclei (see Fig. 41-7). From the dorsal column nuclei, the pain signals are transmitted via

Brainstem Reticular Formation

FIG. 41-7. Multiple ascending spinal pathways transmit nociceptive information to processing centers in the brain.

1018 / CHAPTER 41 the ipsilateral dorsal column nuclei to the contralateral ventroposterolateral nucleus of the thalamus (75,82–84). This pathway is now considered to be more important in visceral nociceptive transmission than the spinothalamic and spinoreticular tracts. A midline myelotomy that severs axons in the human dorsal columns attenuates otherwise intractable visceral pain (76,81). Stimulation of the dorsal columns in patients with severe IBS evokes an immediate increase in the intensity of their abdominal pain (85). These observations in humans are consistent with experimental results in animals. Severing the axons of the dorsal columns in rats or monkeys reduces the neuronal responses to colorectal distension in the ventral posterolateral nucleus of the thalamus and of neurons in the dorsal column nuclei, particularly in the nucleus gracilis (77,82). Sensory Processing in the Cerebral Cortex Modern methods of brain imaging are applied to investigate questions related to abnormal processing of sensory information in the cerebral cortex of patients with IBS (86–89). Functional magnetic resonance imaging (fMRI) and positron emission tomography (PET) are the methods used to study information processing in the higher brain centers that underlie an individual’s experience of conscious sensations (see Chapter 26). Results from imaging studies suggest that unlike somatic sensation, which has a main homuncular representation in the primary somatosensory cortex, visceral sensation is mainly represented in the secondary somatosensory cortex (88,90). Differences in processing in these specialized regions might account for the vagueness of an individual’s ability to localize visceral sensation in relation to somatic sensation. Beyond the sensory cortices, fMRI and positron emission tomography images show representation of both somatic and visceral sensation to be similar in the limbic and paralimbic regions of the cortex (e.g., anterior insular, anterior and posterior cingulate, prefrontal and orbitofrontal cortices) (86,87). These areas are known to be involved in the individual’s motivational and emotional mood states and in the cognitive components of visceral sensations. Sex differences are present in the cortical representation of balloon distension in the rectosigmoid region in healthy subjects (91). Whereas activation in the sensorimotor and parietooccipital areas does not differ between sexes, more extensive activation appears in the anterior cingulate and prefrontal cortices in women. The volume of evoked cortical activity in women is greater than in men for perception levels in the range from the urge to defecate to sensations of fullness, mild discomfort, and pain. The functional significance of these sex differences is unclear; nevertheless, they are reminiscent of the greater perceptual responses reported in female patients with functional gastrointestinal disorders (e.g., IBS) (92). In healthy subjects, either painful distension by a balloon in the rectosigmoid region or even anticipation of a potentially painful distension is associated with increased blood flow in the anterior cingulate cortex. In patients with IBS, activation

of the anterior cingulate cortex was reported not to occur in response to painful distension or the anticipation of painful distension of the balloon in response to a warning from the investigator (86,89). In contrast, an fMRI study found that patients with IBS showed enhanced activation of the midcingulate cortex in response to rectal distension (93). Selective activation of the prefrontal cortex appeared to take place coincident with decreased activation of the anterior cingulate cortex in the study of Silverman and colleagues (86). The cingulate cortex attracts attention because it is generally thought to be an integrative center for both emotional experience and specific representation for pain, which might account for linkage of pain and emotional state. This cortical region is formed around the rostrum of the corpus callosum and has projections into the motor regions of the cortex (Fig. 41-8). The “affective” areas of the cingulate cortex have extensive connections with the amygdala and periaqueductal gray matter and with autonomic nuclei in the brainstem (94).

FIG. 41-8. Medial view of the brain showing organization of the cingulate cortex. Perigenual, midcingulate, posterior, and retrosplenial regions are important structural and functional regions of the cingulate cortex: (1) Perigenual anterior cingulate cortex surrounds the rostral portion of the cingulate cortex and is primarily involved in affect with a subsector devoted to visceromotor control via projections to the cranial parasympathetic and spinal sympathetic divisions of the autonomic nervous system. (2) Midcingulate cortex is involved in response selection and motivation and has a skeletomotor control subsector with neurons that project directly to the spinal cord. (3) Retrosplenial cingulate cortex is associated with memory recall. (4) Posterior cingulate cortex overlays the entire surface of the cingulate gyrus and has a caudomedial subsector that overlaps with the retrosplenial cingulate cortex. The retrosplenial cingulate cortex and posterior cingulate cortex are both involved in memory and activation of the caudomedial sector of the posterior cingulate cortex during memory of emotional events (e.g., emotional events associated with affect and gut function) provide an important imaging framework for assessment of visceral function and disorder.

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1019 The cingulate cortex integrates autonomic and endocrine functions and is involved in recall of emotional experiences. Cognition appears to reside in the caudal portion of the anterior cingulate cortex together with circuits delegated to premotor functions and processing of nociceptive information. Knowledge of the functional neuroanatomy of the cingulate cortex stimulates consideration of the well-documented association of psychosocial disturbances (e.g., negative life events) with more severe cases of IBS (92). Drossman and colleagues (95) reported that rectal distension in individuals with histories of severe sexual or physical abuse selectively activates the perigenual region of the anterior cingulate cortex (see Fig. 41-8). A case report was presented for a middleaged woman whose low pain threshold for rectal distension and diarrhea-predominant IBS improved after she was extricated from an abusive psychosocial situation. Brain imaging with fMRI showed activation of the midcingulate cortex during rectal distension before treatment and resolution of activation associated with improvement in the patient’s psychosocial situation. Sensory Processing in the Spinal Cord Nociceptive afferents, not projecting to dorsal column nuclei, terminate in the dorsal horn of the spinal cord. They synapse with second order nociceptive neurons that are located mainly in laminae I and II. Most of the neurons in laminae I and II receive direct synaptic input from A-delta and nonmyelinated C-fibers. Large numbers of neurons in lamina I respond exclusively to noxious stimulation and project the information to higher brain centers (see Fig. 41-7).

nociceptive neurons. A related phenomenon occurs in the rat spinal cord where excitability of second-order nociceptive neurons becomes sensitized to distension-evoked input after inflammation of the colon produced by application of acid to the mucosa (Fig. 41-9). The same phenomenon is evident in dorsal column nuclei where firing of second-order neurons to colorectal distension becomes sensitized after inflammation of the colon produced by application of mustard oil to the mucosa (97). These effects of inflammation are characterized by a decrease in threshold and an increase in firing rate of the neurons in the dorsal horn and dorsal column nuclei in response to distension of the large intestine. This is believed to reflect central sensitization secondary to peripheral sensitization and increased firing of sensory nerve terminals in the inflamed intestinal wall. In a study that is reminiscent of the connection between sexual and physical abuse in early childhood and IBS in adult humans, Al-Chaer and colleagues (98) found that central sensitization may also be induced in animal models in the absence of inflammation. Neonatal rats in this study were subjected daily to noxious colorectal distension or intracolonic injection of mustard oil beginning 8 days after birth and lasting for 21 days. When tested in adulthood, the rats that were “abused” as neonates were hypersensitive to colorectal distension as reflected by a lower threshold and increased intensity of reflex responses indicative of abdominal pain. Single-unit electrophysiologic recording from dorsal horn neurons in the lumbar and sacral regions of the spinal cord of the adult animals found averaged background firing rates

80

Increased excitability in nociceptive dorsal horn neurons underlies spinally mediated hyperalgesia, which is termed central sensitization to distinguish it from sensitization that occurs at nociceptive terminals in the periphery. In conditions of severe tissue injury and persistent injury, nociceptive C-fibers fire repetitively and the excitability of the secondorder neurons in the dorsal horn increases progressively in response to the increased synaptic input. This effect is sometimes called wind-up and reflects the synaptic release of glutamate from the incoming C-fibers and activation of N-methyl-D-aspartate–type glutamate receptors expressed by the second-order neurons. These long-lasting changes in the excitability of dorsal horn neurons are like a memory imprint of the nociceptive input. Accumulating evidence suggests that the spinal wind-up phenomenon may be operational and be an underlying factor in the intestinal hypersensitivity associated with IBS. One piece of suggestive evidence for wind-up hypersensitivity is the finding that second-order neurons in the dorsal horn show induction of the early gene c-fos in response to noxious balloon distension of the colon in rats (96). Changes in gene expression in this case are expected to underlie postsynaptic excitability changes in the second-order

Dorsal horn neurons firing frequency (spikes S−1)

Central Sensitization (“Wind-Up”) 60

40 Inflammation 20 Non-inflamed

0 0

25 50 75 Colonic distension (mmHg)

100

FIG. 41-9. Firing frequency in dorsal root neurons to distension of the colon is increased during experimentally induced inflammation of the colon in rats. Increased sensitivity is reflected by a decreased firing threshold and increased gain (increased slope) of the relation of firing frequency to degree of distension. (Data extracted from Cervero F, Laird JMA. Irritable bowel disease: possible role of spinal mechanisms. In: Krammer HJ, Singer MV, eds. Neurogastroenterology from the basics to the clinics. Falk Symposium 112 ed. Boston: Kluwer Academic Publishers, 2000;664–673.)

1020 / CHAPTER 41 that were significantly greater in the animals that were “abused” as neonates and enhanced frequency responses to colorectal distension when compared with nonabused control animals. Histologic examination found no evidence of an inflammatory state in the large intestine in either the adult “abused” animals or their control animals. Limited evidence for central sensitization to distension of the large bowel has been obtained for humans who meet diagnostic criteria for IBS (53). Repetitive inflation of a balloon in the sigmoid colon to noxious levels of stimulation was reported to alter the processing of afferent information entering the spinal cord from the rectum. What was interpreted as altered central processing in the human studies occurred in the patients with IBS and not in healthy subjects. Altered processing was reflected by development of hyperalgesia in response to rectal distension and spontaneously developing hyperalgesia over an extended period in the rectosigmoid region in the absence of applied distension. Descending Spinal Modulatory Pathways Nociceptive processing in the spinal cord is subject to descending modulatory influences from supraspinal structures (e.g., periaqueductal gray, nucleus raphe magnus, nuclei reticularis gigantocellularis, and the ventrobasal complex of the thalamus; Fig. 41-10). The descending modulation can be either inhibitory, facilitatory, or both, depending on the context of the visceral stimulus or the intensity of the descending stimulation. The descending pathways that originate in the brainstem and higher centers influence the modulation and integration of visceroceptive signals in the dorsal horn.

Brain Midline Cerebral cortex

Hypothalamus

Periaquaductal gray matter

Reticular formation

Midbrain raphe nulcei

Dorsal horn

Spinal cord FIG. 41-10. Multiple descending spinal pathways from the brain modulate the transmission of nociceptive signals after they enter the spinal cord.

Serotonergic, noradrenergic, and to a lesser extent, dopaminergic networks make up the descending pathways. Descending modulation of visceral nociceptive processing at the spinal cord level is continuous and includes both inhibitive and facilitative influences on synaptic transmission in the dorsal horn microcircuits. Results that correlate behavioral responses and neuronal electrical and synaptic behavior find that activity in descending modulatory pathways influences both neuronal activity at the spinal cord level and the individual’s behavior in situations involving acute and persistent pain (99). The descending modulation includes facilitative and inhibitive influences, both of which can alter the sensing of pain of visceral origin. Electrical stimulation or chemical activation (i.e., intracerebral injection of neurotransmitters or blocking drugs) of several different supraspinal centers modulates the neuronal and behavioral responses to visceral stimuli. For example, responses to intraperitoneal injection of hypertonic saline in rats are attenuated by electrical stimulation in the periaqueductal gray matter (100). Chemically induced activation of neuronal cell bodies in the rostroventral medulla attenuates responses to visceral stimulation (101,102). Electrical stimulation or glutamate microinjection into the periaqueductal gray matter, nucleus raphe magnus, or the nuclei reticularis gigantocellularis suppresses spinal dorsal horn neuronal responses in spinoreticular and spinothalamic tract neurons to visceral afferent fiber stimulation or noxious colorectal distention (103–105). Descending inhibition from the rostroventral medulla is mediated mainly by pathways traveling in the dorsolateral spinal cord (106). Electrical stimulation of the ventrobasal complex of the thalamus suppresses the responses of dorsal horn neurons to colorectal distension in normal rats (107). In chronically sensitized rats (e.g., by irritation of the colon in the neonate), the descending pathway from the thalamus has pathways that are facilitative, whereas others are inhibitory. In adult rats with colorectal hypersensitivity produced by irritation of their colon when they were neonates, thalamic stimulation facilitates neuronal responses to colorectal distension in more than 60% of the neurons in which action potential discharge can be recorded with microelectrodes. This form of facilitation occurs regardless of the intensity of electrical stimulation and is facilitated by electrical stimulation of the dorsal columns (107). Inhibition in response to thalamic stimulation is found in less than 40% of the neurons in chronically sensitized rats. This emphasizes the conclusion that descending modulatory pathways do not exclusively exert inhibitory or facilitative actions in the dorsal horn. The release of individual transmitters and their actions in the dorsal horn differ depending on the type of neuron targeted and the kind of neuronal receptor activated (107). Nevertheless, there is evidence for tonic descending inhibition under normal circumstances, which when suppressed, results in increases in spontaneous activity or responses of spinal neurons to visceral stimuli, or both. Reversible cold block at the level of the cervical spinal cord increases the excitability of spinal viscerosomatic neurons, which is evidence that the viscerosomatic

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1021 Reversible spinal blockade by injection of lidocaine or irreversible transection of spinal funiculi indicated that descending facilitatory influences from the rostroventral medulla were transmitted in the ventrolateral/ventral funiculus, and that descending inhibitory influences were carried by the dorsolateral funiculi. Descending modulation, whether inhibitive or facilitative, was linked to a reflex response to noxious stimulation; neither electrical stimulation nor glutamate injection changed the resting activity in the abdominal musculature in the absence of colorectal distension.

neurons are under tonic descending inhibitory influences from supraspinal structures (108–110). Sensory visceral input to the dorsal horn is also subject to descending facilitative modulation. Descending facilitation might enhance conscious perception of the bowel in the absence of any noxious stimulation and explain the hypersensitivity sometimes reported to be present in IBS. Several lines of evidence support the presence of tonic descending facilitative influences in animals. In cats, reversible blockade of descending pathways by cervical cooling suppresses the responses of a subset of visceral neurons to stimulation of the splanchnic nerves (110). Zhuo and Gebhart (106) investigated the effects of electrical or chemical stimulation in the rostroventral medulla on the electromyogram of the abdominal musculature (i.e., external oblique muscles) of the rat in response to noxious high-pressure colorectal distension. At 22 places in the rostroventral medulla, electrical stimulation facilitated the reflex responses in the abdominal musculature at low-stimulus intensities of 5, 10, and 25 µA and suppressed the reflex at stimulus intensities greater than 50 µA. Electrical stimulation at all intensities tested (5–200 µA) in other sites in the rostroventral medulla only inhibited or only facilitated reflex response to noxious colorectal distention. Microinjection of glutamate into the rostroventral medulla mimicked the effects of electrical stimulation.

Intestinal Motility and Abdominal Pain Powerful contractions of the intestinal circular muscle coat during intestinal power propulsion (Fig. 41-11) underlie the sensation of cramping abdominal pain (16,111,112). Power propulsion, which is one of the patterns of motility stored in the program library of the ENS, occurs more frequently in patients with IBS than in healthy subjects, and the circular muscle contractions are significantly stronger than normal in patients with IBS (113). Power propulsion is more prevalent in the colon of patients with IBS than in healthy individuals after a meal, and power propulsion in the colon is often associated with their diarrhea (41).

Force sensors

0

0.5 1.0 1.5 Time (minutes)

2.0

FIG. 41-11. Power propulsion is a protective response to the presence of food allergens or other threatening agents in the intestinal lumen. It is an enteric nervous system programed motility pattern that can be recorded with sensors (e.g., manometric catheters or strain gages) as strong, long-lasting contractions of the circular muscle that propagate rapidly for extended distances along the intestine. Power propulsion quickly strips the lumen clean as it travels along extended lengths of intestine. Abdominal cramping sensations, urgency, diarrhea, and threat of incontinence are associated with this motor program. Application of irritants to the mucosa, introduction of luminal parasites, enterotoxins from pathogenic bacteria, allergic reactions, and exposure to ionizing radiation all trigger the propulsive motor program.

1022 / CHAPTER 41 Power propulsion generally starts in the proximal colon and strips the lumen clean as it travels rapidly toward the rectosigmoid (see Fig. 41-11). In diarrheal states, large volumes of watery stool may be propelled quickly into the distal large bowel. Rapid distension of the rectosigmoid by the advancing luminal contents triggers the rectoanal reflex. Relaxation of the internal anal sphincter and conscious need for contraction of the external sphincter and puborectalis muscle occurs at this time coincident with the sensation of urgency and concern about incontinence. The sensation of urgency is derived from mechanosensory information transmitted to the CNS along primary sensory afferents from the rectosigmoid region and pelvic floor musculature. The pain and discomfort of a patient with IBS during the powerful contractions of power propulsion may be explained in three ways, either separately or in combination. One explanation is for the exceptionally powerful circular muscle contractions to activate high-threshold mechanoreceptors that transmit the information centrally where it is processed and projected to consciousness as the perception of pain and discomfort (see Figs. 41-5 and 41-6). A second is for the mechanoreceptors to become sensitized in the patients with IBS (e.g., by inflammatory mediators or other paracrine signals) and send erroneously coded information to processing centers in the spinal cord and brain. A third explanation is for accurately coded sensory information in primary sensory afferents to be misinterpreted as it is decoded in the spinal cord and central processing centers of the brain.

PSYCHOLOGICAL STRESS Psychological stress and negative life experiences are recognized as psychosocial factors in IBS (2,89,92,95). Stress often exacerbates symptoms of cramping abdominal pain, diarrhea, and urgency in patients with IBS. These stressexacerbated symptoms in IBS are similar, if not identical, to the abdominal pain, diarrhea, and urgency associated with enteric allergic responses, infectious enteritis, radiationinduced enteritis, and noxious mucosal irritation (e.g., senna laxatives). Advances in the basic science of brain-to-gut and immune cells-to-ENS signaling have introduced fresh insight into mechanisms underlying the effects of psychological stress on the intestinal tract.

Neuroimmunophysiologic Paradigm for Irritable Bowel Syndrome–Like Symptoms The human enteric immune system is developed at birth and is colonized by populations of immune/inflammatory cells that will change continuously in response to luminal conditions and pathophysiologic states throughout life. The enteric immune system is positioned to provide security at one of the most contaminated borders between the interior of the body and the outside world. It deals continuously with dietary antigens, parasites, bacteria, viruses, and toxins as they

appear in the warm, dark, damp, anaerobic environment of the intestinal lumen. The system is challenged continuously because physical and chemical barriers at the epithelial interface never exclude the large antigenic load in its entirety. Furthermore, stress in the form of threatening environmental conditions in animal models or handling of the gut during laparotomy in humans opens the barrier (114–116).

Immunoneural Communication Insight into how chemical communication between the mucosal immune system and the ENS takes place is derived from electrophysiologic recording in enteric neurons in intestinal preparations from antigen-sensitized animal models (117–119). Signals from enteric immune/inflammatory cells activate a neural program for defensive intestinal behavior in response to circumstances within the lumen that are threatening to the functional integrity of the whole animal (41). The signaling mechanism is chemical in nature (i.e., paracrine) and incorporates specialized sensing functions of intestinal mast cells for antigens together with the capacity of the ENS for intelligent interpretation of antigen-evoked mast-cell signals (41,112,119). Immunoneural integration progresses sequentially beginning with immune detection, followed by signal transfer to the ENS, followed by neural interpretation, and then by selection of a specific neural program of coordinated mucosal secretion and powerful motor propulsion (i.e., power propulsion) that effectively clears the threat from the intestinal lumen. IBS-like symptoms of cramping lower abdominal pain, fecal urgency, and diarrhea accompany operation of the immunoneural defense program.

Sources of Immunoneural Signals Lymphoid and myeloid cells colonize the gastrointestinal tract in numbers that continuously fluctuate with changing luminal conditions and pathophysiologic states (120). Several cell types including polymorphonuclear leukocytes, lymphocytes, macrophages, dendrocytes, and mast cells are present in continuously varying numbers in the intestinal mucosa, lamina propria, and smooth muscle and are potential sources of immunoneural signals. Each of these cell types may be situated in close histoanatomic association with the neuronal elements of the ENS, vagal nerve fibers, and spinal sensory nerves (48,50,114,121–123). Signaling from the cells of the enteric immune/inflammatory system to the ENS establishes a first line of defense against foreign invasion at the vulnerable interface between the body and the outside environment. In inflammatory states, close histoanatomic proximity of increased numbers of lymphocytes and polymorphonuclear leukocytes to enteric nerve elements suggests that inflammatory mediators released by these cells might access and influence the ENS. Electrophysiologic studies in enteric neurons confirm that inflammatory mediators released in paracrine fashion alter

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1023 electrical and synaptic behavior of enteric neurons (124–126).

Enteric Mast Cells All kinds of immune/inflammatory cells are putative sources of paracrine signals to the ENS. Nevertheless, most of what is known is about signaling is between enteric mast cells and the neural elements of the ENS. Enteric mast cells are filled with granules that are sites of storage for a broad mix of preformed chemical mediators. Antigens stimulate the mast cells to release the mediators, which then diffuse into the extracellular space inside the ENS. Enteric mast cells express high-affinity receptors for IgE antibodies or other immunoglobulins on their surfaces. A deluge of multiple mediators is released from the mast cells when antibodies to a sensitizing antigen occupy the receptors and cross-linking occurs by interaction of the sensitizing antigen with the bound antibody (Fig. 41-12). Infections with nematode parasites stimulate proliferation of intestinal mast cells in animal models (121,122). These nematode-infected models and food allergy models have proved to be valuable for studies of mast-cell involvement in enteric immunoneural communication. In the sensitization

models, a second exposure to antigen isolated from the infectious agent (e.g., intestinal nematode) or to a food antigen results in predictable integrated intestinal motor and secretory behavior (117,118,124–127). Recognition of the antigen by antibodies bound to the sensitized mast cells triggers degranulation and release of the mediators of the mast cells. After release, the mediators become paracrine signals to the ENS, which responds by suspending operation of other programs in its library and running a defense program designed to eliminate the antigen from the lumen. Copious secretion and increased blood flow followed by orthograde power propulsion of the luminal contents are the behavioral components of the program. Abundant evidence supports a hypothesis that mast cells are equipped and strategically placed to recognize foreign agents that threaten whole-body integrity and to signal the ENS to program a defensive response that eliminates the threat. The immunoneural defensive program in the lower half of the intestinal tract is reminiscent of the emetic program, which provides a similar defense for the upper gastrointestinal tract. Mast-cell function in immunoneural communication is an immune counterpart of sensory detection and information coding in sensory neurophysiology (see Chapter 25). In sensory physiology, sensory neurons are genetically programed to express detection mechanisms for specific

FIG. 41-12. Heuristic model for brain–gut interaction in stress. The enteric nervous system (ENS) is a minibrain located in close apposition to the gastrointestinal effectors it controls. Enteric mast cells are positioned to detect foreign antigens and signal their presence to the ENS. Stimulated mast cells release several paracrine mediators simultaneously. Some of the mediators signal the ENS, whereas others act as attractant factors for polymorphonuclear leukocytes responsible for acute inflammatory responses. The ENS responds to the mast-cell signal by initiating a program of coordinated secretion and propulsive motility that expels the source of antigenic stimulation from the bowel. Symptoms of abdominal pain and diarrhea result from operation of the neural program. Neural inputs to mast cells from the brain stimulate simultaneous release of chemoattractant factors for inflammatory cells, which establishes inflammatory surveillance at a dangerous interface and chemical signals to the ENS with effects that mimic the symptoms of antigenic stimulation. Stress activates the brain–mast cell connection.

1024 / CHAPTER 41 stimuli (e.g., touch, temperature, or light) that remain fixed throughout the life of the individual. Mast cells, in contrast, acquire specific detection capabilities through the flexibility of recognition functions inherent in synthesis of specific antibodies by the immune system. Detection specificity for foreign antigens is acquired and reinforced throughout life because of formation of new antibodies that bind to and remain on immunoglobulin receptors on mast cells. The output signals from mast cells, which are triggered by cross-linking of antigens with the attached antibodies, are chemical in nature and analogous to chemical output signals (i.e., neurotransmitters) from sensory neurons to second-order neurons in the brain and spinal cord. Both mast cells and sensory neurons ultimately code information on the sensed parameter by releasing a chemical message that is decoded by information-processing circuits in the nervous system.

Mast Cells and the Brain–Gut Connection In addition to their sensing function, enteric mast cells couple the CNS and ENS. This is a brain–gut interaction in which central psychological status might be linked to irritable states of the digestive tract by way of mast-cell degranulation and release of mediators. Mast-cell degranulation evoked by psychological stress activates the ENS “alarm program” to produce the same symptoms of diarrhea and abdominal distress as antigen-evoked degranulation. Evidence for a brain–mast cell connection appears in reports of Pavlovian conditioning of enteric mast-cell degranulation (128). Release of mast-cell proteases into the systemic circulation and intestinal lumen is a marker for degranulation of enteric mucosal mast cells. Release of proteases into the circulation occurs as a conditioned response in laboratory animals to either light or auditory stimuli after pairing with antigenic sensitization (128). In humans, release of mastcell proteases into the jejunal lumen occurs as a conditioned response to stress (129), which, similar to the animal studies, reflects a brain–enteric mast cell connection. CNS influence on mast cells in the upper gastrointestinal tract is suggested by a close histologic association between vagal afferents and enteric mast cells (130) and by increased expression of histamine in intestinal mast cells in response to vagal nerve stimulation (131). Stimulation of neurons in the brainstem by intracerebroventricular injection of thyrotropinreleasing hormone (TRH) evokes degranulation of mast cells in the rat small intestine and adds support for the brain–mast cell connection (132). Intracerebroventricular injection of TRH in the rat brain evokes the same kinds of inflammation and erosions in the stomach as cold restraint stress. In the large intestine, restraint stress exacerbates nociceptive responses to distension that are associated with increased release of histamine from mast cells (133). Similar to the effects of central TRH on gastric mucosal pathology, intracerebroventricular injection of corticotropin-releasing factor mimics large intestinal responses to stress (see Chapters 28–30). Injection of a

corticotropin-releasing factor receptor antagonist or pretreatment with mast-cell stabilizing drugs suppresses stress-evoked responses in the lower gastrointestinal tract. The brain–mast cell connection is significant because the gastrointestinal symptoms associated with mast-cell degranulation are expected to be the same whether the mast cells are degranulated by antigen–antibody cross-linking in allergies or by input from the brain during stress. Mast-cell degranulation releases mediators that sensitize “silent” nociceptors (see Chapter 25) in the large intestine. In animals, degranulation of enteric mast cells results in a reduced threshold for pain responses to intestinal distension; this is prevented by treatment with mast-cell stabilizing drugs (133). Receptors for mast-cell mediators are expressed on the terminals of vagal and spinal sensory afferent neurons (see Chapter 25) (134–137). Actions of mast-cell mediators to sensitize sensory nerves calls to mind the characteristic hypersensitivity to intestinal distension found in a subset of patients with IBS (52,53,56). Colonic mucosal biopsies from patients with IBS contain increased numbers of mast cells, which raises the question whether the hypersensitivity to distension reflects sensitization of intramural endings of spinal mechanosensitive nerves by mast-cell mediators (49,50). Degranulating mast cells release mediators to signal the ENS that degranulation has taken place, and at the same time, to attract immune/inflammatory cells into the intestinal wall from the mesenteric circulation. Placement of purified Clostridium difficile toxin A into intestinal loops stimulates influx of acute inflammatory cells coincident with activation of the ENS “alarm program.” Activation of the alarm program is evident as copious mucosal secretion and power propulsion. Blockade of enteric nerves by tetrodotoxin, treatment with tachykinin neurokinin-1 receptor antagonists, or treatment with mast-cell stabilizing drugs prevents the acute inflammatory response to the toxin (138–141). Responses to C. difficile toxin A do not occur in mast cell– deficient mice (142). Substance P is a recognized mediator in the chain of events leading to toxin A–induced mast-cell degranulation and release of chemoattractant factors for inflammatory cells (143,144). The neuropeptide is expressed in enteric and spinal sensory afferent neurons. It is a putative neurotransmitter in the ENS and for axonal reflexes mediated by spinal sensory afferents. Substance P is a secretagogue for histamine and cytokine release from mast cells (144–145). The excitatory action of toxin A on enteric neurons is expected to release neuronal substance P, which, in turn, acts to degranulate mast cells in the neighborhood where it is released. Exposure of enteric myenteric and submucosal neurons to toxin A depolarizes the membrane potential and increases excitability. This occurs coincident with presynaptic suppression of nicotinic fast excitatory synaptic transmission in both plexuses and with suppression of inhibitory noradrenergic neurotransmission to secretomotor neurons in the submucosal plexus (146). Suppression of noradrenergic neurotransmission removes sympathetic braking action from secretomotor neurons, which facilitates stimulation of

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1025 secretion (see Fig. 41-2). Together with toxin A–evoked excitation of secretomotor neurons, this is undoubtedly an underlying factor in the diarrhea associated with C. difficile overgrowth in the large intestine.

Mast-Cell Signal Substances Several mast cell–derived mediators have neuropharmacologic actions on electrical and synaptic behavior of neurons in the ENS. Some key mediators known to act at their receptors on neural elements in the ENS are: 1. Histamine 2. Interleukin-6 3. Leukotrienes 4. 5-HT 5. Platelet-activating factor 6. Mast-cell proteases 7. Adenosine 8. Interleukin-1β 9. Prostaglandins 10. Nitric oxide Histamine Histamine is not synthesized by enteric neurons and is not considered to be a neurotransmitter in the ENS (147). Mast cells and neutrophils are sources of histamine in the intestine. Knowledge of histaminergic actions on ENS neurons comes from results obtained in electrophysiologic and immunohistochemical studies on single enteric neurons in animals. Application of histamine, to mimic release from mast cells and neutrophils, excites neurons in the small and large intestinal myenteric and submucosal plexuses of the guinea pig (148,149). Unlike in the intestine, enteric neurons in the guinea pig stomach do not express histamine receptors and do not respond to experimental applications of histamine (119). Histamine has three significant actions on neural elements in the guinea pig intestine. One action, which occurs at the level of neuronal cell bodies, is long-lasting excitation mediated by histamine H2 receptors (148,149). The second is at fast excitatory nicotinic synapses, where it acts at presynaptic inhibitory histamine H3 receptors to suppress cholinergic synaptic transmission (37,150). The third action is to prevent inhibition by the sympathetic innervation of the intestine of secretomotor-evoked mucosal secretion (see Fig. 41-2). Histamine acts at presynaptic histamine H3 inhibitory receptors on sympathetic noradrenergic fibers to suppress sympathetic inhibitory input to submucosal secretomotor neurons, and at H3 presynaptic terminals of enteric somatostatinergic neurons that also provide inhibitory input to secretomotor neurons (37). Mast cells in colonic mucosal biopsies from patients with diarrhea-predominant IBS release more histamine than healthy subjects (50). Increased release of histamine onto the neural networks that control the secretomotor innervation of the intestinal crypts in this set of patients with IBS might

enhance intestinal secretion, leading to secretory diarrheal symptoms such as those associated with infectious agents and food allergies. Histaminergic receptor antagonists have been used effectively in the past to treat watery diarrhea symptoms associated with mastocytosis and microscopic colitis (151). The H2 receptor antagonist cimetidine was used effectively for treatment of diarrhea associated with short-bowel syndrome in patients with Crohn’s disease (152). Serotonin 5-HT (serotonin) is another preformed mediator that is known to be released during degranulation of enteric mast cells and mucosal enterochromaffin cells to form a neuromodulatory overlay on the ENS in animal models and humans. Two receptors mediate the excitatory actions of serotonin at the cell bodies of guinea pig enteric neurons. One of the receptors, identified as a 5-HT1P receptor, is a metabotropic receptor linked to intraneuronal signal transduction cascades by a G protein (153–155). The response to stimulation of 5-HT1P receptors, similar to stimulation of histamine H2 receptors, evokes long-lasting excitatory responses in the time range of minutes in ENS neurons. The second receptor, identified as a 5-HT3 receptor, is an ionotropic receptor directly coupled to nonselective cation channels (156). Binding of 5-HT to the 5-HT3 receptor evokes “fast” depolarizing responses that quickly desensitize and occur in the millisecond time range, similar to those evoked by nicotinic receptor stimulation. Expression of two kinds of serotonergic receptors with different mechanisms of postreceptor signal transduction differs from the situation for histamine where only a single metabotropic receptor subtype is expressed by the neuronal cell body. Histamine and 5-HT both act at presynaptic inhibitory receptors on cholinergic axons to suppress fast neurotransmission at nicotinic synapses in the enteric neural networks (150,157). Presynaptic inhibition by both neuromodulators is mediated by a different receptor subtype than the one that evokes excitatory responses in the neuronal cell bodies. Identification of the presynaptic inhibitory 5-HT receptor is equivocal; the evidence suggests that it might be a 5-HT1 receptor (158,159). In addition to presynaptic inhibitory action at enteric neuronal synapses, histamine and 5-HT each acts presynaptically to suppress the release of norepinephrine from sympathetic postganglionic fibers in the ENS (37,160). Serotonin and Irritable Bowel Syndrome Infusion of 5-HT either intravenously or into the intestinal lumen evokes copious secretion of H2O, electrolytes, and mucus from the intestinal secretory glands in dogs and rodents (161–163). The stimulatory action of 5-HT underlies its action as a diarrheogenic agent and its involvement in diarrheogenic syndromes in humans (164). Efficacy of blockade of the 5-HT3 serotonergic receptor subtype by the selective 5-HT3 receptor antagonist alosetron in the treatment of diarrhea and abdominal pain in the diarrhea-predominant

1026 / CHAPTER 41 population of women with IBS (44,67) suggests that enhanced stimulation of neural elements in the ENS by 5-HT might be a pathophysiologic factor in this form of IBS (see Fig. 41-3A). The IBS symptoms of cramping abdominal pain, diarrhea, and fecal urgency are exacerbated in the postprandial state (4,45,165). Increased appearance of 5-HT in the hepatic portal circulation in the postprandial state reflects stimulated release from mucosal enterochromaffin cells (46). The normal postprandial release of 5-HT is reported to be augmented in patients with IBS (47), and this contributes to the suspicion that overactive release of serotonin might be an underlying factor in the symptoms of patients with diarrhea-predominant IBS. This suspicion is reinforced by findings of increased numbers of mast cells and enterochromaffin cells, both of which contain 5-HT, in colonic mucosal biopsies from patients with IBS (49,50). Reports that a significant proportion of patients with IBS progress to the development of IBS-like symptoms after an acute bout of infectious enteritis are reminiscent of the basic research findings for the actions of inflammatory mediators, including histamine and serotonin, in the ENS. Gwee and colleagues (61) reported that 23% of patients who experienced an acute bout of gastroenteritis progressed to IBS-like symptoms within 3 months. Hypochondriasis and adverse life events are reported to double the risk for development of postinfective IBS and may account for the increased proportion of women who acquire the syndrome (62). Nevertheless, whether the association between acute infectious enteritis and IBS reflects low-level inflammation (e.g., microscopic enteritis) and chronic exposure of the neural and glial elements of the ENS to increased levels of serotonin, histamine, or other inflammatory mediators remains to be fully resolved. Exposure to 5-HT evokes increased firing in sensory nerves that leave the intestine and project to the spinal cord (65). The excitatory action of 5-HT on intestinal sensory nerves is mediated by the 5-HT3 receptor subtype and is blocked by selective 5-HT3 receptor antagonists (e.g., alosetron) (66). Intramural terminals of both spinal and vagal sensory nerves express 5-HT3 receptors. Reported efficacy of the 5-HT3 receptor blocking drug, alosetron, in the treatment of abdominal pain and discomfort in the diarrheapredominant form of IBS in women (44,67) suggests that the pain and discomfort, like the diarrhea and fecal urgency, reflect disordered endogenous release of 5-HT and its action at the 5-HT3 receptors present on sensory terminals in the intestinal wall (see Fig. 41-3B). Serotonergic 5-HT3 receptors are expressed in the brain; nevertheless, there is little available evidence suggesting a central site of action for alosetron in the alleviation of IBS-associated pain and discomfort. Receptors on Sensory Nerve Terminals Aside from 5-HT3 receptors, the sensory nerve terminals in the intestine express receptors for several other putative signal substances, including inflammatory mediators. Functional receptors for bradykinin, ATP, adenosine,

prostaglandins, leukotrienes, and mast-cell proteases are expressed on spinal sensory nerve terminals (71). Intramural pooling of any one of these mediators has potential for increasing the sensitivity of intestinal sensory nerves, especially in the disordered conditions of inflammation or ischemia. Mediators, released when mast cells degranulate, are known to sensitize nociceptors in the large intestine (59). Mast-cell degranulation in animal models results in a reduced threshold for pain responses to balloon distension in the large bowel. Treatment with mast-cell stabilizing drugs prevents decline of the pain threshold during mucosal inflammation in the animal models (72). The results obtained from animals lead to the unresolved question of whether the association between an acute bout of infectious enteritis and the follow-up IBS symptoms of abdominal pain and increased sensitivity to distension reflects chronic sensitization of intestinal sensory nerves as they function in an environment with low-level inflammation (see Fig. 41-6).

Brain–Mast Cell Connection: Implications for Irritable Bowel Syndrome The brain–mast cell connection holds clinical relevance for the well-known relation between stress and IBS-like bowel symptoms. Existence of a brain–mast cell connection implies a mechanism linking central psycho-emotional status to irritable states of the digestive tract. The irritable state of the bowel (i.e., abdominal discomfort, diarrhea, and urgency), known to result from degranulation of intestinal mast cells and release of signals to the ENS, is expected to occur regardless of the mode of stimulation of the mast cells (see Fig. 41-12). Degranulation and release of mediators evoked by neural input to the mast cells will have the same effect of triggering a program of secretion and power propulsion as degranulation triggered by antigen detection. This might explain the similarity of bowel symptoms between those associated with noxious insults in the lumen and those associated with psychogenic stress in susceptible individuals. The immunoneurophysiologic evidence reinforces the hypotheses that moment-to-moment behavior of the gut, whether normal or pathologic, is determined primarily by integrative functions of the ENS. The enteric minibrain processes input signals derived from immune/inflammatory cells (e.g., mast cells), sensory receptors, and the CNS. Enteric mast cells use the capacity of the immune system for detection of new antigens and long-term memory that permits recognition of the antigen if it ever reappears in the gut lumen. Should the antigen reappear, the mast cells signal its presence to the enteric minibrain. The minibrain interprets the mast cell signal as a threat and calls up from its program library secretory and propulsive motor behavior organized for quick and effective eradication of the threat. Operation of the program protects the integrity of the bowel and the individual, but at the expense of the side effects of abdominal distress and diarrhea. The same symptomatology is expected to result from activation of neural pathways that

PATHOPHYSIOLOGY UNDERLYING THE IRRITABLE BOWEL SYNDROME / 1027 link psychological states in the brain to degranulation of mast cells in the gut. The immunoneurophysiology in this respect is suggestive of mechanisms with susceptibility to malfunctions that could result in symptoms resembling IBS. A lingering issue for the enteric neuroimmunophysiologic paradigm is the question of the functional significance of the brain–mast cell connection. What were the selective pressures that led to evolution of central signaling to mast cells in the gut? A hypothesis emerges from recognition that the barrier between an extremely “dirty” luminal environment and the interior of the body is a single epithelial cell layer. Probability of a break in the barrier is greater during physical stress and the potential for trauma (e.g., “predacious attack, fright and flight”). Stress factors are known to increase the permeability of the barrier in animals (166). The potential for threats to breach the mucosal barrier suggests a need for immune surveillance at a dangerous interface in the body. Increased immune surveillance would include an influx of acute inflammatory cells into the intestinal lamina propria and mucosa. The brain has evolved to program homeostatic adjustments to environmental stressors and the emotional stress associated with negative life events. These adjustments include cardiovascular, hormonal, and metabolic changes and probably the targeting of inflammatory cells into the gut from the systemic circulation. The only apparent mechanism available to the brain for selective targeting of inflammatory surveillance to the gut is to transmit nervous signals that degranulate enteric mast cells and release chemoattractant factors to “call-in” inflammatory cells from the circulating blood (see Fig. 41-12). Studies of colitis in a nonhuman primate (cotton-top tamarin) implicate two coexisting factors as being necessary for the initiation and progression of inflammatory bowel disease (ulcerative colitis) in this model. One of the factors is environmental stress, and the other is the large intestinal microflora (120,167–169). Colitis does not exist in cottontop tamarins living in a natural “stress-free” environment and with the normal microflora present in the large intestine. Movement of colitis-free tamarins out of their natural environment and into a stressful environment leads to an acute inflammatory response in the large intestine only if the feces are present. Studies in which the fecal stream was diverted from loops of large intestine found that colonoscopic and histologic features of colitis disappeared from the loops and progressed in the colon in the same animal. These changes occurred while the tamarins in the study remained in a colitisinducing environment. Results of a preliminary study in which putative neural input to colonic mast cells was suppressed by pharmacologic blockade of neurokinin receptors showed suppression of the inflammatory response in cotton-top tamarins held in a colitis-inducing (i.e., stressful) environment (170).

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144. Cocchiara R, Albeggiani G, Lampiasi N, Bongiovanni A, Azzolina A, Geraci D. Histamine and tumor necrosis factor-alpha production from purified rat brain mast cells mediated by substance P. Neuroreport 1999;10:575–578. 145. Cocchiara R, Bongiovanni A, Albeggiani G, Azzolina A, Lampiasi N, Di Blasi F, Geraci D. Inhibitory effect of neuraminidase on SP-induced histamine release and TNF-alpha mRNA in rat mast cells: evidence of a receptor-independent mechanism. J Neuroimmunol 1997;75:9–18. 146. Xia Y, Hu HZ, Liu S, Pothoulakis C, Wood JD. Clostridium difficile toxin A excites enteric neurones and suppresses sympathetic neurotransmission in the guinea pig. Gut 2000;46:481–486. 147. Panula P, Kaartinen M, Macklin M, Costa E. Histamine-containing peripheral neuronal and endocrine systems. J Histochem Cytochem 1985;33:933–941. 148. Nemeth PR, Ort CA, Wood JD. Intracellular study of effects of histamine on electrical behaviour of myenteric neurones in guinea-pig small intestine. J Physiol (Lond) 1984;355:411–425. 149. Frieling T, Cooke HJ, Wood JD. Histamine receptors on submucous neurons in guinea pig colon. Am J Physiol 1993;264:G74–G80. 150. Tamura K, Palmer JM, Wood JD. Presynaptic inhibition produced by histamine at nicotinic synapses in enteric ganglia. Neuroscience 1988; 25:171–179. 151. Baum CA, Bhatia P, Miner PB Jr. Increased colonic mucosal mast cells associated with severe watery diarrhea and microscopic colitis. Dig Dis Sci 1989;34:1462–1465. 152. Aly A, Barany F, Kollberg B, Monsen U, Wisen O, Johansson C. Effect of an H2-receptor blocking agent on diarrhoeas after extensive small bowel resection in Crohn’s disease. Acta Med Scand 1980; 207:119–122. 153. Wood JD, Mayer CJ. Slow synaptic excitation mediated by serotonin in Auerbach’s plexus. Nature (Lond) 1979;276:836–837. 154. Wood JD, Mayer CJ. Serotonergic activation of tonic-type enteric neurons in guinea pig small bowel. J Neurophysiol 1979;42:582–593. 155. Mawe GM, Branchek TA, Gershon MD. Peripheral neural serotonin receptors: identification and characterization with specific antagonists and agonists. Proc Natl Acad Sci U S A 1986;83:9799–9803. 156. Derkach V, Surprenant A, North RA. 5-HT3 receptors are membrane ion channels. Nature (Lond) 1989;339:706–709. 157. North RA, Henderson G, Katayama Y, Johnson SM. Electrophysiological evidence for presynaptic inhibition of acetylcholine release by 5-hydroxytryptamine in the enteric nervous system. Neuroscience 1980;5:581–586. 158. Tack JF, Janssens J, Vantrappen G, Wood JD. Actions of 5-hydroxytryptamine on myenteric neurons in guinea pig gastric antrum. Am J Physiol 1992;263:G838–G846. 159. Galligan JJ, Surprenant A, Tonini M, North RA. Differential localization of 5-HT1 receptors on myenteric and submucosal neurons. Am J Physiol 1988;255:G603–G611. 160. Surprenant A, Crist J. Electrophysiological characterization of functionally distinct 5-hydroxytryptamine receptors on guinea-pig submucous plexus. Neuroscience 1988;24:283–295. 161. Hansen MB, Jaffe BM. 5-HT receptor subtypes involved in luminal serotonin-induced secretion in rat intestine in vivo. J Surg Res 1994; 56:277–287. 162. McFadden D, Jaffe BM, Zinner MJ. Secretory effects of intravenous serotonin on the proximal jejunum of the awake dog. Am J Surg 1985; 149:60–64. 163. McFadden D, Jaffe BM, Zinner MJ. Effect of luminally administered serotonin and substance P on jejunal handling of water and electrolytes. Am J Surg 1986;151:81–86. 164. Jaffe BM. Prostaglandins and serotonin in diarrheogenic syndromes. Adv Exp Med Biol 1978;106:285–295. 165. McKee DP, Quigley EM. Intestinal motility in irritable bowel syndrome: is IBS a motility disorder? Part 1. Definition of IBS and colonic motility. Dig Dis Sci 1993;38:1761–1772. 166. Soderholm JD, Perdue MH. Stress and the gastrointestinal tract II. Stress and intestinal barrier function. Am J Physiol 2001;280: G7–G13. 167. Stonerook MJ, Weiss HS, Rodriguez-M MA, Rodriguez-M JV, Hernandez-C JI, Peck OC, Wood JD. Temperature-metabolism relations in the cotton-top tamarin (Saguineus oedipus) model for ulcerative colitis. J Med Primatol 1994;23:16–22. 168. Wood JD, Peck OC. Colitis and colon cancer in the cotton-top tamarin. In: Guglietta A, Lirussi F, eds. Infectious pathogens in gastrointestinal

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170. Wood JD, Peck OC, Sharma HS, Hernandez JI, Rodriquez JVRMA, Stonerook MJ, Tefend KS, Gardeazabal J, Nassar F. A non-peptide neurokinin 1 (NK-1) receptor antagonist suppresses initiation of acute inflammation in the colon of the cotton-top tamarin model for spontaneous colitis and colon cancer. Gastroenterology 1996; 110:A1047.

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Index A ABCA1, gallbladder cholesterol metabolism, 1547–1548 ABCG5/G8 gallbladder cholesterol metabolism, 1548 intestinal cholesterol transport, 1725–1727 sitosterolemia role, 1726 Abdominal pain, see Irritable bowel syndrome; Visceral hypersensitivity Abetalipoproteinemia, see Microsomal triglyceride transfer protein ACAT2, see Acyl CoA cholesterol acyltransferase 2 Ace, secretory function effects, 1168–1169 Acetylcholine bile formation regulation, 1519 gallbladder emptying regulation, 844 gastric acid secretion regulation, 1243–1244 pancreas electrolyte regulation, 1387, 1390 presynaptic inhibition mediation in enteric nervous system, 657 stress effect mediation in mucosa, 770 Acetylcholine receptor gastric expression, 1232–1233 knockout mouse mutants, 1303 ligand binding, 1232 signaling, 1233 structure, 1232 N-Acetyltransferase, phase II metabolism, 1489 Acid secretion, see Gastric acid Acinar cell endocytosis, 1362 protein synthesis and trafficking endoplasmic reticulum to Golgi transport, 1323–1324 folding, 1322–1323 Golgi to trans-Golgi network transport, 1324–1326 misfolding and degradation, 1323 overview, 1319, 1321 signal peptide cleavage, 1322 trans-Golgi network to zymogen granule transport, 1326 receptors apical membrane expansion and retrieval, 1332–1333 calcium signaling agonist studies, 1341, 1343 cell–cell communication, 1345 clearance, 1349–1350 cytosolic, 1327–1329 diacylglycerol, 1350 inositol phosphate signaling, 1339–1341, 1345–1347 nicotinic acid dinucleotide phosphate-induced release, 1349 nuclear, 1329–1330 ryanodine receptor-induced release, 1347–1349 spatial properties, 1343 temporal properties, 1341, 1343 cyclic AMP regulation, 1351–1352 cyclic GMP regulation, 1352–1353 exocytosis, 1331–1332, 1358–1362 G protein-coupled receptors, 1326, 1338 mechanisms, 1330–1331 membrane effectors, 1339, 1354–1358 protein extrusion, solubilization, and delivery into duct system, 1333 protein phosphorylation, 1357–1358 second messenger interactions, 1353 secretory function, 1355–1356 Actin acinar cell exocytosis role, 1361 transport meditation in polarized epithelial cells, 1614 vesicular trafficking in gastric acid secretion, 1210 Acyl CoA cholesterol acyltransferase 2, intestinal cholesterol absorption regulation, 1728

Adaptive immunity definition, 1033 Helicobacter pylori, 1098 innate immunity comparison, 1033–1034 Adenosine presynaptic inhibition mediation in enteric nervous system, 657 vasodilation induction in intestine, 1639 Adenosine monophosphate, secretory function, 1173 Adenosine receptors, see Purinergic receptors Adherens junctions, pathogen receptors, 1181–1182 Adhering zonule, pancreas, 1318–1319 Adhesion molecules cadherins, 1142 cellular adhesion molecules, 1140 endothelial–blood cell interaction mediation, 1143–1145 integrins, 1141–1142 leukocyte movement through interstitium, 1151–1152 platelet glycoproteins, 1142–1143 selectins, 1138–1140 table, 1138 Aging adaptation bowel resection, 423–424 fasting and sepsis, 423 angiodysplasia, 425 definition, 405 demographics, 405 esophagus Barrett’s esophagus, 407 prospects for study, 407 structural and functional properties, 406–407 gastrointestinal cancer studies carcinogen susceptibility, 417–418 epidemiology, 414–415 mutational events, 418–419 premalignant events apoptosis inhibition, 416–417 epithelial growth and renewal, 415–416 prospects for study, 419–420 hypopharyngeal intrabolus pressure effects, 898 intestine contractility and motility, 412–413 fecal incontinence, 413–414 gut peptides, 412 immunologic effects, 412 prospects for study, 414 structural and functional properties, 411–412 mucosal growth regulation gene expression patterns, 423 growth factors and protein kinases, 421–423 intracellular mechanisms, 420 nutritional and hormonal factors, 420–421 prospects for study, 423 stomach mucosal injury and repair, 408–410 prospects for study, 411 secretion and digestion, 410–411 structural and functional properties, 407–408 surgical diseases of gut, 424–425 AH-type neuron action potentials, 645 cellular physiology, 644 metabotropic signal transduction, 650–652 mucosal projections, 642 neuronal gating function, 642–644 resting membrane potential, 644–645 slow excitatory postsynaptic potentials, 648–652 terminology, 644 Akt cell growth signaling, 443–444 target genes, 444 Alcohol dehydrogenase, phase I metabolism, 1487–1488 Aldehyde dehydrogenase, phase I metabolism, 1488

I-1

Aldosterone, chloride–bicarbonate exchange regulation, 1886 Amidation, protein processing, 47–48 Amino acids, see also Protein digestion and absorption basolateral membrane transport systems A, 1677–1678 Asc, 1680 glycine transporter 1, 1678 L, 1679–1680 y+, 1679 y+L, 1680 cell growth regulation, 450 cholangiocyte transport, 1512 enterocyte transport systems ASC, 1674–1675 β, 1674 B0, 1671–1673 b0,+, 1673 B0,+, 1673 IMINO, 1673–1674 overview, 1671–1672 PAT, 1675 − , 1674 XAG gallbladder transport, 1549 genetic disorders of transport, 1681–1683 ontogenic regulation of transport, 383 pancreatic secretion regulation, 1402, 1404 regulation of transport developmental regulation, 1684–1685 dietary regulation, 685 diurnal rhythm, 1686 hormonal regulation, 1685–1686 sodium–proton exchanger regulation, 1686 Aminopeptidases, protein processing, 46–47 AMP, see Adenosine monophosphate Amphiregulin discovery, 198 expression, 198 function, 198 structure, 198 Amylin, food intake regulation, 884 Anal canal defecation integrated control by enteric nervous system, 681 rectoanal reflex, 680 sensory innervation, 679 spinal motor control, 680–681 supraspinal motor control, 681 Anaphylaxis, afferent sensitivity effects, 711 Angiodysplasia, aging, 425 Angiogenins, innate immunity, 1055 Angiotensin II pancreas electrolyte regulation, 1388 vasoconstriction induction in small intestine, 1631–1632 Anoikis, characteristics, 363–364 Antral pump cycles, 667 neural control, 667–668 APC β-catenin binding, 248–249 destruction complex regulation, 256–258 mutation in familial adenomatous polyposis, 248 Apolipoprotein, gallbladder cholesterol metabolism, 1546 Apolipoprotein A-I, intestinal lipoprotein biogenesis role, 1723–1724 Apolipoprotein A-IV, intestinal lipoprotein biogenesis role, 1723–1724 Apolipoprotein B domains, 1713–1714 familial hypobetalipoproteinemia mouse models, 1719–1720 mutations, 1717–1718 phenotypic mechanisms, 1717, 1719

I-2 / INDEX Apolipoprotein B (Continued) lipoprotein assembly first step, 1714–1715 overview, 1714–1715 presecretory degradation, 1715 primordial lipoprotein particle formation, 1715–1716 second step, 1716 messenger RNA editing functional importance, 1723 molecular machinery and tissue-specific regulation, 1722 secretion rates, 1715 structure, 1712–1714 Apoptosis accessory molecules, 346–347 characteristics versus necrosis, 345–346 effectors, 346 gastric mucosa ethanol induction, 366 Helicobacter pylori induction, 364–365 nonsteroidal anti-inflammatory drug induction, 365–366 hepatocytes, 1493–1494 insulin-like growth factor-I inhibition, 209 intestinal epithelium anoikis, 363–364 cell studies, 358–363 induced apoptosis inducers, 351–352, 440 ischemia–reperfusion injury, 354 mechanisms, 352–353 small bowel resection, 353–354 pathways and regulation, 358–362 polyamine depletion effects, 359–360 prevention cytokines, 357–358 growth factors, 355–356 lysophosphatidic acid, 357 polyamines, 356–357 prostaglandins, 356 spontaneous apoptosis crypt, 350–351 villus, 351 pathways extrinsic pathway, 347 intrinsic pathway, 348–349 overview, 347, 437–438 regulators, 347 Appetite food intake and energy balance, 877 hypothalamic systems in feeding control, 878–881 interactions between gut peptide and hypothalamic signaling, 887–888 leptin and food intake regulation, 878 peptide YY effects, 147 within-meal feedback signaling across-meal peptide signaling, 885–887 gut neural signaling, 882–883 gut peptide signaling, 883–884 overview, 881–882 AP proteins, vesicle budding in polarized epithelial cells, 1607–1609 Aquaporins, see also Water transport angiogenesis and cell migration role, 1834–1835 AQP3 function in skin, 1834 AQP7 function in fat cells, 1834 brain swelling and neural signal transduction role, 1833 cholangiocytes, 1512–1513 corneal edema and transparency role, 1834 fluid transport in organs colon, 1840–1841 gallbladder, 1838 liver, 1837–1838 salivary gland, 1835–1836 small intestine duodenum, 1839 ileum, 1839–1840 jejunum, 1839–1840 stomach, 1836–1837 gallbladder, 1540 gastrointestinal tract expression, 1835 kidney tubule function near-isomolar fluid absorption and secretion, 1833 urine concentrating, 1833

Aquaporins (Continued) pancreas electrolyte secretion, 1381, 1384 prospects for study, 1841 selectivity, 1832 Arfs Arf1 and vesicle budding in polarized epithelial cells, 1607 Arf6 and vesicular trafficking in gastric acid secretion, 1216 G protein-coupled receptor signaling, 72 Arginine vasopressin, pancreas electrolyte regulation, 1390–1391 Argosome, Wnt transport, 249 Arrestins, G protein-coupled receptor desensitization, 77–81 Ascorbic acid, see Vitamin C ATP bile formation regulation, 1521–1522 organic anion transporter regulation, 1467 pancreas electrolyte regulation, 1387–1388, 1391 sensory signaling role, 748 sympathetic neuron neurotransmission in blood vessels, 821–822 ATP-binding cassette transporters, liver detoxification, 1490–1492 Axin, destruction complex regulation, 254–255 B BabA, Helicobacter pylori virulence factor, 1102–1103 Bacteroides fragilis, barrier function effects, 1176 Barrett’s esophagus, aging, 407 Base excision repair, genetic instability in cancer, 489–490 Bcl-2, apoptosis regulation, 347 Belching, see Pharyngeal motor function BETA2, Notch regulation, 298 Betacellulin, features, 199 Bicarbonate chloride exchange, see Chloride–bicarbonate exchanger corticotrophin-releasing hormone stimulation small intestine, 802–803 stomach, 798 gastroduodenal epithelium protection cystic fibrosis paradox, 1268–1269 duodenal secretion, 1270–1273 gastric secretion, 1269–1270 principles, 1268 Bile acids chloride–bicarbonate exchange regulation, 1885 concentration in gallbladder, 1450–1451 enterohepatic circulation anatomy, 1444–1445 bacterial biotransformation during recycling, 1441–1443 efficiency, 1446 transport, 1444, 1446 functions colon, 1455 overview, 1437–1438 small intestine, 1454–1455 intestinal absorption and transport ileal basolateral transport, 1452 ileal sodium-dependent uptake, 1451–1452 overview, 1451 regulation bile acids, 1453–1454 development, 1452–1453 gene expression, 1452 inflammation effects, 1454 ontogenic development of absorption, 391 pancreatic secretion regulation, 1403 physical chemistry, 1443–1444 secretion canalicular bile flow, 1447 overview, 1446–1447 structure–physiochemical relations, 1444 synthesis, 1438–1441 therapeutic applications agonists, 1455 antagonists, 1456 transport canalicular transport overview, 1450, 1471 regulation, 1472–1473 trafficking of transporters, 1473–1474 transporters, 1471–1472

Bile acids (Continued) cholehepatic shunt pathway, 1450 ductal modifications, 1450 hepatocyte sinusoidal membrane transport sodium-dependent uptake and regulation, 1448–1449, 1469–1471 sodium-independent uptake, 1449 transporters, 1449 overview, 1447–1448 transporters, 1444, 1446 Bile duct architecture, 1506 cholangiocyte, see Cholangiocyte microscopic anatomy, 1506–1507 Bile formation, see Bile acids; Cholangiocyte Bile salts export pump and detoxification, 1491 gallbladder transport, 1549 mucin secretion regulation in gallbladder, 1550–1551 Bilirubin gallbladder transport, 1548–1549 organic anion transporter regulation, 1468 Biotin functions, 1802 intestinal transport mechanism, 1802–1804 molecular components, 1804–1805 regulation, 1805–1807 Blood flow, see Gastrointestinal circulation BMPs, see Bone morphogenetic proteins Bombesin bile formation regulation, 1518–1519 cell growth regulation, 452–453 pancreas electrolyte regulation, 1389 satiety signaling, 884 BON cell, see Enterochromaffin cell Bone morphogenetic proteins mutation and enteric nervous system lesions, 509–510 receptors, 326 stem cell regulation, 326–327 Bowel resection adaptation in aging, 423–424 apoptosis induction, 353–354 small-bowel resection and gastric acid secretion, 1246 Brain, see Sensory signals, brain processing Brainstem, gastric function control acid secretion, 856 efferent autonomic overlay, 852–854 history of study, 851–852 motility, 855–856 vago-vagal control reflex components and characteristics circulating factors, 864 corticotrophin-releasing hormone activation of vagal nonadrenergic, noncholinergic path, 862 descending central nervous system pathways and circulating agents, 860–861 dorsal vagal complex chemosensitivity, 864–866 dorsal vagal complex inputs from Raphe nuclei, 861 gastric accommodation reflex, 859–860 nucleus of the solitary tract, 857–858 oxytocin inputs to dorsal vagal complex from paraventricular nucleus, 861–862 receptive relaxation reflex, 858–859 short-term receptor trafficking and plasticity, 863–864 tumor necrosis factor-α regulation, 866–869 vagal efferent neurons in dorsal motor nucleus of vagus nerve, 856–857 visceral afferent inputs to brainstem, 854 C Cadherins, types and functions, 1142 cag pathogenicity island CagA and barrier function effects, 1178–1179 Helicobacter pylori virulence factor, 1099–1101 Calcitonin gene-related peptide, primary afferent neuron neurotransmission in blood vessels, 827–828 Calcium dietary requirements, 1953–1954 intestinal absorption diffusion, 1962 extrusion across basolateral membrane, 1962–1963

INDEX / I-3 Calcium (Continued) overview, 1954–1957 paracellular pathway, 1957–1958 regulation calcium, 1964 calmodulin, 1966 estrogens, 1964 growth hormone, 1966–1967 parathyroid hormone, 1965 protein–protein interactions, 1965–1966 S100A10, 1966 stanniocalcin, 1964 thyroid hormone, 1964 vitamin D, 1963–1964 transcellular pathway enterocyte uptake, 1958–1959 transient receptor potential family proteins, 1959–1962 intestinal transport diffusion, 1762 extrusion at basolateral surface, 1762 knockout mouse studies, 1762–1763 overview, 1761 TRPV ion channels, 1761–1762 vitamin D regulation of transporter expression, 1763–1764 ontogenic development of absorption, 386 pancreas electrolyte regulation, 1388 pancreatic secretion regulation, 1403–1404 Calcium-activated chloride channels, see Chloride channels Calcium/calmodulin-activated kinases acinar cell secretory function, 1355–1356 calcium-activated chloride channel activation, 1920 Calcium channels extrinsic sensory afferent nerve voltage-gated calcium channels, 695–696 smooth muscle responses to slow waves and neural inputs L-type calcium channels, 551, 555–556 T-type calcium channels, 556 Calcium flux acinar cell signaling agonist studies, 1341, 1343 cell–cell communication, 1345 clearance, 1349–1350 cytosolic, 1327–1329 diacylglycerol, 1350 inositol phosphate signaling, 1339–1341, 1345–1347 nicotinic acid dinucleotide phosphate-induced release, 1349 nuclear, 1329–1330 ryanodine receptor-induced release, 1347–1349 spatial properties, 1343 temporal properties, 1341, 1343 cholangiocyte signaling, 1514–1515 mucin secretion regulation in gallbladder, 1550 Calcium-sensing receptors gastric expression, 1234 ligand binding, 1233 signaling, 1234 structure, 1233–1234 Calmodulin, calcium absorption regulation, 1966 cAMP, see Cyclic AMP Cancer, gastrointestinal, see also specific cancers aging studies carcinogen susceptibility, 417–418 epidemiology, 414–415 mutational events, 418–419 premalignant events apoptosis inhibition, 416–417 epithelial growth and renewal, 415–416 prospects for study, 419–420 cardinal features growth inhibitory signaling defects, 482–484 overview, 481 proliferation signal self-sufficiency, 481–482 unlimited replication, 484–485 carcinogen exposure, 479 gene mutations, 477–478 genetic instability base excision repair, 489–490 chromosomal instability, 486–487 epigenetic mechanisms, 9, 490–492 microsatellite instability, 487–489 overview, 485–486

Cancer (Continued) Hedgehog signaling defects, 282–283 heredity, 478–479 inflammation role, 479–480 multistep carcinogenesis, 480–481 sporadic carcinogenesis, 480 transforming growth factor-β role, 191–192 Wnt/β-catenin signaling defects esophagus, 263 familial adenomatous polyposis, 262–263 liver, 264 pancreas, 264 sporadic colorectal cancer, 261–262 stomach adenocarcinoma, 263–264 adenoma, 263 fundic gland polyps, 263 Carbohydrate forms in diet, 1653 sugar absorption, see Glucose transporters; Sodium/glucose cotransporters Carbonic anhydrase, pancreas electrolyte secretion, 1384 Carboxypeptidases, protein processing, 45–46 Carotenoids dietary sources, 1737 intestinal absorption carotenoid interactions, 1744 differential transport, 1743–1744 kinetics of transport, 1742–1743 overview, 1741–1742 proteins, 1748 stereoselectivity, 1743 retinoid conversion cleavage enzymes, 1739–1740 nutritional factor regulation, 1740 solubilization, 1738–1739 synthesis, 1736 types and structures, 1735–1736 Caspases, apoptosis regulation, 346 Catalase, antioxidant protection, 1494 β-Catenin, see Wnt/β-catenin signaling Cathelicidins, innate immunity, 1052–1053 Caveolae, protein sorting in polarized epithelial cells, 1611–1612 CCK, see Cholecystokinin CD36, fatty acid uptake, 1698–1699 CD44, stem cell function regulation, 321 Cdx genes developmental regulation of gastrointestinal function, 393 stem cell function regulation, 319–320 Celiac disease, intestinal epithelial tight junction barrier and permeability index, 1583–1584 Cell cycle growth inhibitory signaling defects in cancer, 482–484 regulation, 437 Cell migration, see Wound healing CFTR, see Cystic fibrosis transmembrane conductance regulator Chagas’ disease, disinhibitory motor disorders, 641 ChIP, see Chromatin immunoprecipitation Chloride absorption chloride–bicarbonate exchange, see Chloride–bicarbonate exchanger electroneutral sodium chloride absorption, 1882–1883, 1936–1938 intestinal secretion regulation, 1932–1935 Chloride–bicarbonate exchanger basolateral membrane transport, 1203–1204 cholangiocytes, 1510 gallbladder, 1538 intestinal distribution and function, 1883–1884 knockout mice, 1306 membrane topology, 1888–1889 pancreas electrolyte secretion, 1380 regulation aldosterone, 1886 bile acids, 1885 infection, 1886 inflammation, 1885 nitric oxide, 1885–1886 protein kinase C, 1886–1887 reactive oxygen species, 1887 serotonin, 1886

Chloride–bicarbonate exchanger (Continued) short-chain fatty acids, 1884–1885 structure, 1893 SLC4 family features ammonium activation, 1892 expression regulation, 1891–1892 gene family, 1887–1888 pH effects, 1892 phosphorylative regulation, 1892 SLC4A1, 1889–1890 SLC4A2, 1890 SLC4A3, 1890–1891 SLC4A4, 1891 SLC26 family features gene family, 1892–1893 SLC26A2, 1898–1899 SLC26A3 expression, 1896 mutations in congenital chloride diarrhea, 1893–1896 protein–protein interactions, 1896–1897 regulation, 1900 SLC26A6 functions, 1897–1898 regulation, 1900 tissue distribution and expression, 1898 SLC26A7, 1899–1900 Chloride channels, see also Cystic fibrosis transmembrane regulator calcium-activated chloride channels ClC-2, 1919–1920 ClC-3, 1920 ClC-4, 1920 pancreas electrolyte secretion, 1382–1383 cholangiocytes, 1509–1510 smooth muscle responses to slow waves and neural inputs calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 Cholangiocyte bile formation regulation acetylcholine, 1519 adrenergic agonists, 1519–1520 amino acids, 1523 ATP, 1521–1522 bile acids, 1522 bile osmolarity and flow, 1523 bombesin, 1518–1519 corticosteroids, 1519 dopaminergic agonists, 1520 endothelin, 1520–1521 gastrin, 1520 glucose, 1522–1523 integrated model, 1524–1525 overview, 1515–1517 secretin, 1516, 1518–1521 somatostatin, 1520 vasoactive intestinal peptide, 1519 biochemical heterogeneity, 1507–1508 distribution, 1505 functional heterogeneity, 1524 morphology, 1506–1507 signaling calcium, 1514–1515 cyclic AMP, 1514 cyclic GMP, 1515 G protein-coupled receptors, 1514 purine receptors, 1515 transport systems amino acids, 1512 aquaporins, 1512–1513 bile acids, 1511 chloride–bicarbonate exchanger, 1510 chloride channels, 1509–1510 glucose transporters, 1511–1512 heterogeneity, 1513 organic ions, 1513 overview, 1508–1509 potassium channels, 1510–1511 proteins, 1513 proton-ATPase, 1511 sodium–bicarbonate cotransporter, 1511 sodium/potassium ATPase, 1511 sodium–potassium–chloride cotransporter, 1511 sodium–proton exchangers, 1510–1511 vesicular trafficking of proteins, 1523–1524

I-4 / INDEX Cholecystokinin aging effects in intestine, 412 assays, 102–103 excess and deficiency in disease, 106 functions gall bladder contraction, 105 gastric emptying, 105 gastric secretory effects, 105–106 overview, 100–101, 104 pancreas, 104–105 gallbladder emptying regulation, 843–844 gastrin-cholecystokinin double-knockout mice, 1301 gene structure and expression, 101 metabolism, 103 pancreas electrolyte regulation, 1389 pancreas secretion regulation, 1412–1415, 1418 postprandial hyperemia role, 1642–1643 posttranslational processing, 101 release, 103 satiety signaling, 883–884 structure, 101 tissue distribution, 101–102 vago-vagal control reflex regulation, 864 Cholecystokinin receptors gastric cell regulation, 1235 gastric expression, 1234–1235 ligand binding, 1234 overview, 96–97, 103–104 structure, 1234 vagal afferents, 636 Cholecystokinin-releasing factor, pancreas secretion regulation, 1418–1419 Cholehepatic shunt pathway, 1450 Cholera toxin secretion induction, 758 secretory function ontogeny studies, 377 Cholestasis bile canalicular transport effects, 1473 hepatocyte bile acid transport effects, 1471 Cholesterol gallbladder absorption and efflux cholesterolosis, 1545–1546 crystallization and inflammation, 1552 history of study, 1544–1545 mechanisms ABCA1, 1547–1548 ABCG5, 1548 ABCG8, 1548 apolipoproteins, 1546 cubulin, 1548 megalin, 1548 scavenger receptor type B class I, 1546–1547 oxysterol formation, 1553 intestinal absorption, 1711–1712, 1727–1728 intestinal transport, 1724–1728 Chromatin, see Epigenetics Chromatin immunoprecipitation, principles, 18 Chromium, absorption and transporters, 1998 Chromogranin A, enterochromaffin-like cell secretion, 1230 Chromosomal instability, cancer, 486–487 Chronic intestinal pseudo-obstruction, disinhibitory motor disorders, 640–641 Chymotrypsin, protein digestion, 1669 Ciliary neurotrophic factor, receptor mutation and enteric nervous system lesions, 509 Cingulate cortex, gastrointestinal sensory signal processing, 730, 734 Circulation, see Gastrointestinal circulation Clathrin G protein-coupled receptor endocytosis regulation, 81–82 secretory vesicle formation, 42 vesicle budding in polarized epithelial cells, 1607 vesicular trafficking in gastric acid secretion, 1214–1215 Claudins, tight junctions, 1567–1568 Clostridium botulinum, barrier function effects C2 toxin, 1178 C3 toxin, 1178 overview, 1166 Clostridium difficile barrier function effects overview, 1166 toxins, 1176–1177 secretion pathobiology, 759–760

Clostridium difficile (Continued) secretory function effects inflammation and neuropeptide mediation, 1172 overview, 1164 Clostridium perfringens, barrier function effects enterotoxin, 1177–1178 overview, 1166 CNTF, see Ciliary neurotrophic factor Cobalamin forms, 1813 functions, 1813 gastrointestinal absorption cubulin expression regulation, 1818–1819 structure and function, 1817–1818 malabsorption acquired causes, 1815 inherited causes, 1815–1816 molecular components, 1816–1817 mechanism, 1813–1815 regulation of component expression, 1818–1819 Colitis, see Intestinal inflammation Colon corticotropin-releasing hormone modulation of function hypersensitivity, 808–809 inflammation, 807 motility, 804–807 permeability, 807 development, 295–296 integrated control by enteric nervous system defecation, 681 musculature, 670 peristaltic propulsion, 670–673 peristaltic retropropulsion, 673 physiologic ileus, 673 power propulsion, 674 motility, see Intestinal motility sodium–proton exchange, 1871–1873 water transport, 1840–1841 Colon cancer, vitamin D prevention, 1765–1766 Constipation colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 ion transport alterations, 1944 COPII, vesicle budding in polarized epithelial cells, 1606–1607 Copper absorption and transporters, 1995–1996 sequestration, 1498 Corticosteroids, bile formation regulation, 1519 Corticotropin-releasing factor binding protein, 795 central distribution hormone and functional significance, 796 receptors, 796–797 colonic function modulation hypersensitivity, 808–809 inflammation, 807 motility, 804–807 permeability, 807 gastric function modulation acid secretion inhibition, 797–798 bicarbonate secretion stimulation, 798 gastric emptying inhibition, 798–800 motility, 800–802 gastrointestinal motility modulation overview, 781–782 peripheral receptor and colonic motor alterations, 785 type 1 receptor activation and stimulation, 784 type 2 receptor activation and inhibition, 784 visceral hyperalgesia mediation by receptors, 786 receptors antagonists, 794–795 pharmacology, 794 structures and variants type 1 receptor, 793–794 type 2 receptor, 794 related peptides, 793–794 sequence homology between species, 792–793 small intestine function modulation

Corticotropin-releasing factor (Continued) anti-inflammatory action, 804 bicarbonate secretion stimulation, 802–803 jejunoileal absorption, 803 motility, 803–804 stress effect mediation in mucosa, 771 COX, see Cyclooxygenase CRF, see Corticotropin-releasing factor Crohn’s disease, see Inflammatory bowel disease Cryptdins, secretory function, 1172–1173 Cubulin expression regulation, 1818–1819 gallbladder cholesterol metabolism, 1548 structure and function, 1817–1818 Cyclic AMP acinar cell secretory function, 1351–1352 cholangiocyte signaling, 1514 gallbladder transport control, 1540–1541 hepatocyte bile acid transport regulation, 1470 mucin secretion regulation in gallbladder, 1550 pancreas electrolyte regulation, 1386–1387 secretion signaling, 744–745, 756–757 smooth muscle contraction regulation, 528–529 Cyclic GMP acinar cell secretory function, 1352–1353 cholangiocyte signaling, 1515 pancreas electrolyte regulation, 1387 Cyclooxygenase bicarbonate secretion regulation in duodenum, 1272–1273 chloride secretion mediation, 1172 Helicobacter pylori gastritis role, 1098–1099 intestinal inflammation role, 1120 Cystic fibrosis, bicarbonate paradox, 1268–1269 Cystic fibrosis transmembrane conductance regulator chloride–bicarbonate exchanger regulation, 1900–1901 experimental models, 1922 gallbladder, 1538–1539, 1551 intestinal channel diseases, 1918 structure and function, 1918–1919 pancreas electrolyte secretion, 1381–1382 pancreatic channel, 1919 secretory function ontogeny, 376–377 Cystinuria, pathophysiology, 1682 Cytochalasins, intestinal epithelial tight junction barrier regulation, 1569–1570 Cytochrome P450, phase I metabolism, 1484–1487 D D cell, see Somatostatin Defecation, integrated control by enteric nervous system, 681 Defensins α-defensins, 1049–1051 β-defensins, 1051–1052 Delta, Notch ligand, 289 Deltex, Notch regulation, 294 Dendritic cell antigen sampling, 765–766 intestinal inflammation role, 1116 Desert Hedgehog, see Hedgehog Desmosome, pancreas, 1318–1319 Detoxification, see Liver Diabetes mellitus dipeptidyl peptidase-IV inhibitors in treatment, 170 glucagon-like peptide-1 receptor antagonists for diabetes type 2 treatment, 169–170 Diarrhea congenital chloride diarrhea, 1893–1896 functional diarrhea and colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 ion transport alterations in infectious diarrhea, 1942–1943 Digestive function, ontogeny, 377–379 Dipeptidyl peptidase-IV, inhibitors in diabetes treatment, 170 Dishevelled, destruction complex regulation, 255–256 Disulfide bond, formation, 38–39 Divalent metal ion transporter 1, iron transport, 1984–1985

INDEX / I-5 Dkk proteins Dkk1 and stem cell function regulation, 321–322 Wnt/β-catenin signaling inhibition, 252 DMT1, see Divalent metal ion transporter 1 DNA, see Epigenetics; Gene DNA-binding proteins domains, 12–13 motifs, 13–14 phosphorylative regulation, 15 transactivation, 14–15 DNA microarray, principles, 19 Dopamine, pancreas secretion regulation, 1409 Dorsal vagal complex, see Brainstem Drug-induced colitis, clinical manifestations, 1123 Duodenal ulcer, gastric acid secretion, 1246 Dynamin G protein-coupled receptor endocytosis regulation, 81–82 vesicle scission in polarized epithelial cells, 1609–1610 vesicular trafficking in gastric acid secretion, 1216 Dynorphin, protein processing, 45 E ECL, see Enterochromaffin-like cell EGF, see Epidermal growth factor Elastase, protein digestion, 1669 Emesis, vagal afferents, 713 Endoplasmic reticulum posttranslational modifications of proteins disulfide bond formation, 38–39 folding, 39 glycosylation, 39 myristoylation, 39 protein trafficking, 39–41 protein transport overview, 35–36 signal peptide removal, 38 structure and function, 36 signal recognition particle, 36–37 signal recognition particle receptor, 37 translocon, 37–38 Endothelial cell activation products, 1145–1146 blood cell interactions adhesion molecule mediation, 1143–1145 auxiliary cells, 1146–1148 Endothelin B receptor, Hirschsprung’s disease mutations, 505–506 Endothelin bile formation regulation, 1520–1521 intestinal pathology endotoxic shock, 1633 gut strangulation, 1632–1633 Hirschsprung’s disease, 1633 inflammatory bowel disease, 1633 neonatal necrotizing enterocolitis, 1633 isoforms, 1632 postprandial hyperemia, see Postprandial hyperemia processing, 1632 receptors, 1632 vasoconstriction induction in small intestine, 1632–1633 Enteric nervous system AH-type neurons action potentials, 645 cellular physiology, 644 mucosal projections, 642 neuronal gating function, 642–644 resting membrane potential, 644–645 terminology, 644 blood vessel innervation, see Gastrointestinal circulation central nervous system interactions, 499–500 colonic contraction regulation enteric reflexes, 981–982 interneurons, 979 intrinsic sensory neurons, 979–981 motor neurons, 977–979 overview, 976–977 conceptual model, 630–631, 666 development crest-derived stem cells in bowel, 502–503 functions laminin, 511–512 netrins, 512–513 β-neurexin, 513–514 neuroglin, 513–514

Enteric nervous system (Continued) Notch signaling, 302 overview, 501–502 disinhibitory motor disorders Chagas’ disease, 641 chronic intestinal pseudo-obstruction, 640–641 paraneoplastic syndrome, 641 sphincteric achalasia, 641–642 extrinsic innervation of gut wall parasympathetic innervation, 591–592 sympathetic innervation, 592 extrinsic sensory nerve endings, see Extrinsic sensory afferent nerves fast excitatory postsynaptic potentials nicotinic receptors, 646 purinergic receptors, 646–647 rundown, 647 serotonin receptors, 647 significance, 648 functional overview, 577, 629 histoanatomy, 631–632 inhibitory postsynaptic potentials ionic mechanisms, 654 significance, 655 slow potential mediators, 654–655 integrated control, see Anal canal; Colon; Pelvic floor; Small intestine; Stomach interneurons ascending interneurons, 586–587 descending interneurons, 587–588 interplexus interneurons, 590–591 overview, 586, 636–637 intestinal inflammation effects, 1128–1129 intrinsic sensory neurons of gut wall functional implications, 583 myenteric intrinsic primary afferent neurons, 582–583 submucosal intrinsic primary afferent neurons, 582 late-acting factor defects and disease bone morphogenetic proteins, 509–510 ciliary neurotrophic factor receptor, 509 Mash1, 508–509 neurotrophin-3, 508 overview, 507 pathology, 511 serotonin receptors, 510–511 motor neurons circular muscle motor neurons, 584–585 excitatory musculomotor neurons, 638–639 inhibitory musculomotor neurons, 639–640 longitudinal muscle motor neurons, 585–586 overview, 583–584 secretomotor neurons excitatory receptors, 638 inhibitory input, 638 intestinal content liquidity control, 638 overview, 589–590, 637–638 specialized motor neurons, 586 neurochemical hierarchy, 594–595 neuron abundance, 629 neuron classification biophysical characteristics, 580–581 histochemistry, 580 morphology, 579–580, 632–633 plasticity gastric reflexes, 674 Hirschsprung’s disease, 674–675 histaminergic neuromodulation, 675–676 pattern generator, 677–678 presynaptic inhibitory modulation, 677 secretomotor control, 674 serotonergic neuromodulation, 676–677 presynaptic facilitation, 655–656 presynaptic inhibition mediators, 656–657 prevertebral ganglia, see Prevertebral sympathetic ganglia prospects for study, 595 secretory reflexes, see Secretory reflexes sensory function, see also Extrinsic sensory afferent nerves central processing, see Sensory signals, brain processing chemoreception cholecystokinin receptors on vagal afferents, 636 serotonin receptors in irritable bowel syndrome, 636

Enteric nervous system (Continued) mechanoreception high-threshold, 635 low-threshold, 635 visceral hypersensitivity in irritable bowel syndrome, 635–636 visceral pain, 635 overview, 633–635 slow excitatory postsynaptic potentials AH-type neuron, 648–652 mediators, 649–650 metabotropic signal transduction adenosine receptors, 652 AH-type neurons, 650–652 calcium influx suppression, 652 S-type neurons, 652–654 S-type neuron, 649 significance, 654 structural organization ganglia, 578 overview, 577–578 plexuses, 578 structural basis of intestinal motility, 579 structure–function relations with other anatomic ganglia, 632 S-type neurons action potentials, 646 general features, 646 viscerofugal neurons, 588–589 Enterochromaffin-like cell activation and growth regulation epinephrine, 1229 galanin, 1228 gastrin, 1227–1228 interleukin-1, 1229 norepinephrine, 1229 peptide YY, 1228 pituitary adenylate cyclase-activating peptide, 1228 prostaglandins, 1229 transforming growth factor-α, 1228 afferent sensitivity effects, 711–712 distribution, 1226 gastrin regulation, 98–99 Helicobacter pylori effects on function, 1231 morphology, 1226–1227 secretory products chromogranin A, 1230 fibroblast growth factor-2, 1231 gastrocalcin, 1231 histamine, 1229–1230 pancreastatin, 1230 regenerating protein 1α, 1231 transforming growth factor-α, 1231 WE-14, 1230–1231 transgenic mouse studies, 1307–1308 Enterocyte amino acid transport, see Amino acids fatty acid transport, see Fatty acids Ephrin system, stem cell function regulation, 317–318 Epidermal growth factor cell growth regulation, 447 developmental regulation of gastrointestinal function, 393 expression, 194–195 family definitive criteria, 193–194 functions, 195 gastric acid secretion signaling, 1208–1209 gene, 194 mucosal growth regulation in aging, 421–423 nonmitogenic activity, 197–198 peptide shedding, 200 receptors ErbB1, 200–201 ErbB2, 204 ErbB3, 204 ErbB4, 204 expression, 203–204 knockout mouse studies, 204–205 overview, 193, 200 signaling, 201, 203 therapeutic targeting, 205 transactivation, 205 wound healing regulation, 463 Epigen, features, 199–200

I-6 / INDEX Epigenetics cancer role, 9, 490–492 chromatin-binding proteins, 8–9 definition, 5 developmental gene regulation, 9 DNA methylation, 8 histone acetylation, 6–7 methylation, 7 phosphorylation, 7–8 structure, 5–6 Epinephrine, enterochromaffin-like cell regulation, 1229 Epiregulin, features, 199 Epithelial cell, see Polarized epithelial cell; Tight junctions ER, see Endoplasmic reticulum ErbB receptors, see Epidermal growth factor Erythromycin, motilin receptor binding, 140 Escherichia coli absorption and enteropathogenic Escherichia coli, 1171 barrier function effects cytotoxic necrotizing factors, 1174–1175 enterohemorrhagic Escherichia coli, 1180 enteropathogenic Escherichia coli, 1179–1180, 1182 overview, 1166 serine protease autotransporters of Enterobacteriaceae, 1175–1176 secretory function effects heat-stable enterotoxins, 1170 LTI, 1170 LTII, 1170 overview, 1164 Esophageal body, coordinated motor activity longitudinal muscle, 920–921 pressure isocontour map, 917–918 smooth muscle region, 918–920 striated muscle region, 917–918 Esophagus aging studies Barrett’s esophagus, 407 prospects for study, 407 structural and functional properties, 406–407 coordinated motor activity deglutition inhibition and refractoriness, 923 esophageal body longitudinal muscle, 920–921 pressure isocontour map, 917–918 smooth muscle region, 918–920 striated muscle region, 917–918 lower esophageal sphincter, 921–923 techniques for study, 916 upper esophageal sphincter, 916–917 Hedgehog signaling in development, 275–276 innervation extrinsic parasympathetic innervation, 914–915 extrinsic sympathetic innervation, 915 intramural innervation, 915–916 neuromuscular behavior esophageal body, 914 gross anatomy, 913–914 lower esophageal sphincter, 914 upper esophageal sphincter, 914 Estrogens calcium absorption regulation, 1964 organic anion transporter regulation, 1467 Ethanol, apoptosis induction in gastric mucosa, 366 Exenatide, diabetes type 2 treatment, 169–170 Exocrine pancreas, see Acinar cell; Pancreas Exocyst complex, exocytosis regulation in polarized epithelial cells, 1618 External anal sphincter defecation integrated control by enteric nervous system, 681 rectoanal reflex, 680 spinal motor control, 680–681 supraspinal motor control, 681 Extrinsic primary afferent nerves, intestinal inflammation effects, 1129 Extrinsic sensory afferent nerves development axonal guidance and survival molecules, 686–687 dorsal root ganglion, 686 jugular/nodose ganglion complex, 686

Extrinsic sensory afferent nerves (Continued) distribution and morphology of peripheral afferent terminals mucosa, 689 muscle, 690 serosa/mesentery, 690 electrophysiology afferent recording, 692–693 calcium-activated potassium channels, 695 dissociated cell studies, 693 hyperpolarization-activated currents, 695 voltage-gated calcium channels, 695–696 voltage-gated potassium channels, 694–695 voltage-gated sodium channels, 693–694 integrative physiology food intake regulation, 706–710 pathophysiology, 710–716 sensory function, see also Enteric nervous system chemical sensitivity G protein-coupled receptor signaling, 704–706 ligand-gated ion channels, 702–704 general properties, 696 mechanosensitivity measurement, 696–698 mechanisms, 699–701 mucosal afferents, 698 muscle afferents, 698–699 neurotransmitter release, 701–702 serosal afferents, 699 transient receptor potential channels, 700–701 modulators anaphylaxis, 711 enterochromaffin cells, 711–712 mast cells, 711 visceral pain and hypersensitivity, 713–716 spinal afferents central projections, 691–692 overview, 593–594 peripheral projections, 688–689 vagal afferents central projections, 690–691 food intake regulation, 706–710 overview, 592–593 pathophysiology, 710–716 peripheral projections, 687–688 F Familial adenomatous polyposis, see APC Familial hypobetalipoproteinemia, see Apolipoprotein B Fanconi–Bickel syndrome, GLUT2 mutations, 1662 Fatty acids, see also Short-chain fatty acids amides in food intake regulation, 708–709 delivery systems, 1694 enterocyte uptake CD36, 1698–1699 fatty acid transport proteins, 1696–1698 fatty-acid binding protein expression regulation, 1701–1702 isoforms, 1700 ligand binding, 1700–1701 plasma membrane protein, 1696 proximal intestine functions, 1702–1704 structure, 1700 intracellular transport, 1699–1700 overview, 1695–1696 prospects for study, 1704 facilitated membrane transfer, 1694–1695 uptake, 1693–1694 Fed motor pattern neural regulation, 947 neurohumoral mediators, 947 physiologic characteristics, 946–947 Ferritin, metal detoxification, 1498 FGFs, see Fibroblast growth factors Fiber, cell growth regulation, 450 Fibroblast growth factors classification, 216–217 enterochromaffin-like cell secretion of FGF-2, 1231 expression patterns, 217 functions angiogenesis, 220 cell growth regulation, 447–448 development, 219–220 knockout mouse studies, 219 tissue repair, 220 tumorigenesis, 221

Fibroblast growth factors (Continued) genes, 216 receptors ligands, 216 signaling, 218–219 structure, 217 types, 217 stem cell regulation, 327–330 Flavin monooxygenase, phase I metabolism, 1487 FldA, Helicobacter pylori virulence factor, 1103 fMRI, see Functional magnetic resonance imaging Folate functions, 1792 intestinal transport mechanism, 1792–1795 molecular components, 1795–1796 reduced folate carrier, 1795–1797 regulation, 1796–1797 Food allergy immunoglobulin E role, 1082 stress in pathogenesis, 775–776 Food intake, see Appetite Formins, exocytosis regulation in polarized epithelial cells, 1617–1618 Fox11 knockout mouse, 1306 stem cell function regulation, 322 Frizzled destruction complex regulation, 255–256 Wnt interactions, 249–252, 441 Fructose absorption, 1654–1656 malabsorption and GLUT5 mutations, 1662–1663 Functional diarrhea, colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 Functional magnetic resonance imaging, gastrointestinal sensory signal processing studies, 730–734 Furin, protein processing, 43 G Galactose, absorption, 1654–1656 Galanin enterochromaffin-like cell regulation, 1228 presynaptic inhibition mediation in enteric nervous system, 657 Galanin-1 receptor, secretory function, 1173 Gallbladder amino acid transport, 1549 bile acid concentrating, 1450–1451 bile salt transport, 1549 bilirubin transport, 1548–1549 cholecystokinin effects on contraction, 105 cholesterol absorption and efflux cholesterolosis, 1545–1546 crystallization and inflammation, 1552 history of study, 1544–1545 mechanisms ABCA1, 1547–1548 ABCG5, 1548 ABCG8, 1548 apolipoproteins, 1546 cubulin, 1548 megalin, 1548 scavenger receptor type B class I, 1546–1547 oxysterol formation, 1553 electrolyte and water transport aquaporins, 1540 chloride–bicarbonate exchanger, 1538 cystic fibrosis transmembrane conductance regulator, 1538–1539 gallstone formation roles bile acidification, 1543–1544 calcium, 1542–1543 potassium channels, 1539–1540 regulation cell volume control, 1542 cyclic AMP, 1540–1541 neural control, 1541–1542 sodium/potassium-ATPase, 1539 sodium–proton exchanger, 1537–1538 functional overview, 1535–1536 infection and inflammation, 1551–1553

INDEX / I-7 Gallbladder (Continued) morphology, 1536 mucin secretion regulation bile salts, 1550–1551 calcium flux, 1550 cyclic AMP, 1550 cystic fibrosis transmembrane conductance regulator, 1551 gallstone formation, 1551 overview, 1549–1550 transcriptional regulation, 1551 neural control emptying mechanisms acetylcholine, 844 cholecystokinin, 843–844 neurokinins, 844 filling, 844–845 ganglia and neurons, 841–843 interprandial motility, 845 protein absorption and secretion, 1551 secretory function, 1544 techniques for study, 1536–1537 water transport, 1838 GALT, see Gut-associated lymphoid tissue Gap junction, pancreas, 1319 Gastric accommodation caloric drink test, 929 definition, 928 pathophysiology, 931–932 reflex, 859–860 reflex control nitrergic neurons, 929–930 serotonin system, 930–931 triggering site, 929 Gastric acid aging effects, 410–411 apical channel proteins, 1202–1203 basolateral membrane transport chloride–bicarbonate exchanger, 1203–1204 conductance, 1205 homeostasis, 1203 sodium–potassium–2chloride cotransporter, 1205 sodium–proton exchanger, 1204–1205 brainstem control of secretion, 856 cell biology of secretion enterochromaffin-like cell, 1225–1231 parietal cell, 1225 concentration, 1189 corticotropin-releasing hormone and secretion inhibition, 797–798 disorders duodenal ulcer, 1246 gastritis, 1246 histamine overproduction, 1246 small-bowel resection, 1246 Zollinger–Ellison syndrome, 1246 gastric gland and cell isolation, 1205–1206 gastrin regulation, 98 parietal cell membrane recycling hypothesis, 1193–1194 morphology and ultrastructure, 1191–1193 phases of secretion basal secretion, 1238 cephalic phase, 1238–1240 gastric phase, 1240–1241 intestinal phase, 1241–1243 receptors and signal transduction acetylcholine receptors, 1232–1233 calcium-sensing receptors, 1233–1234 cholecystokinin receptors, 1234–1235 cholinergic pathways of activation, 1207–1208 epidermal growth factor, 1208–1209 gastrin-releasing peptide receptors, 1235–1236 histamine receptors, 1236–1237 pituitary adenylate cyclase-activating polypeptide receptors, 1237 protein kinase A, 1206–1207 somatostatin receptors, 1237–1238 transforming growth factor-α, 1208–1209 regulation of secretion acetylcholine, 1243–1244 gastrin, 1244–1245 histamine, 1243, 1246 somatostatin, 1245–1246 regulation histamine, 1301–1302

Gastric acid (Continued) inhibition, 1297–1298 stimulation, 1296–1297 transgenic and knockout mouse studies, 1298–1306 secretin regulation, 123–124 sodium/potassium-ATPase crystal structure, 1199 functional transport, 1194–1195 identification as proton pump, 1194 inhibitors, 1199–1200, 1202 kinetics, 1196 membrane changes with acid secretion, 1195–1196 subunit structure, 1196–1199 vesicular trafficking machinery actin, 1210 actin-binding proteins, 1210 Arf6, 1216 clathrin and adaptors, 1214–1215 dynamin, 1216 myosin II, 1210–1211 Rab11, 1211–1213 Rab13, 1211–1213 SNAREs, 1213–1214 Gastric emptying cholecystokinin effects, 105 corticotropin-releasing hormone inhibition, 798–800 neural control, 669 peptide YY effects, 143–144 secretin regulation, 124 Gastric motility, see also Gastric accommodation; Gastric emptying; Gastric reservoir function; Gastrointestinal motility brainstem control, 855–856 phases gastric emptying, 928 gastric reservoir function, 928 interdigestive motility, 927 Gastric mucosa, see also Gastroduodenal mucosal defense apoptosis ethanol induction, 366 Helicobacter pylori induction, 364–365 nonsteroidal anti-inflammatory drug induction, 365–366 Helicobacter pylori colonization, see Helicobacter pylori injury and repair in aging, 408–410 transgene expression in mice enterochromaffin-like cells, 1307–1308 gastrin promoter and G cell transgenics, 1308 parietal cells, 1307 promoters, 1306–1308 transgenic and knockout mouse studies, 1306–1308 Gastric reservoir function, see also Gastric accommodation measurement barostat, 928 caloric drink test, 929 volumetric techniques, 928–929 overview, 928 Gastrin bile formation regulation, 1520 cell growth regulation, 451 enterochromaffin-like cell regulation, 1227–1228 excess and disease, 100 functions acid secretion control, 98 enterochromaffin-like cell regulation, 98–99 gastric epithelial cell regulation, 99 Gly-gastrin, 99–100 overview, 92, 97–98 progastrin, 100 target genes, 99 gastric acid secretion regulation, 1244–1245 gene regulation, 4 gene structure and expression, 93 knockout mice gastrin-cholecystokinin double-knockout mice, 1301 pathway mutants, 1299, 1301 metabolism, 96 posttranslational processing, 93–95 preprogastrin posttranslational processing, 48–51 radioimmunoassay, 95 receptors, 96–97 release, 95–96

Gastrin (Continued) secretin interactions, 124 structure, 92–93 tissue distribution, 95 transgenic mice gastrin promoter and G cell transgenics, 1308 overexpression, 1301 Gastrin-releasing peptide, see also Bombesin pancreas secretion regulation, 1407–1408 satiety signaling, 884 Gastrin-releasing peptide receptors gastric expression, 1236 ligand binding, 1235 signaling, 1236 structure, 1236 Gastritis, see Helicobacter pylori Gastrocalcin, enterochromaffin-like cell secretion, 1231 Gastroduodenal mucosal defense epithelium bicarbonate protection cystic fibrosis paradox, 1268–1269 duodenal secretion, 1270–1273 gastric secretion, 1269–1270 principles, 1268 intracellular pH acidification mechanism, 1267–1268 measurement and regulation, 1265–1267 permeability regulation apical membranes, 1264 intercelullar junctions, 1264–1265 injury and restitution animal models of injury, 1281–1282 cell exfoliation and mucoid cap, 1280 epithelial integrity challenges, 1279 epithelial restitution, 1280–1281 imposed mucosal damage, 1279–1280 mucous layer composition, 1260–1261 pH, 1261–1263 overview, 1259–1260 prospects for study, 1282 subepithelial defense duodenal acid sensing and blood flow, 1277–1278 overview, 1273–1274 prostaglandins, 1274–1276, 1278–1279 Gastroesophageal reflux disease, esophagoglottal closure reflex, 904–905 Gastrointestinal cancer, see Cancer, gastrointestinal Gastrointestinal circulation blood vessel innervation enteric neurons neurotransmitters, 819–820, 825 stimulation effects, 823–825 multiplicity of neurons, 819 parasympathetic neurons neurotransmitters, 819, 823 stimulation effects, 822–823 primary afferent neurons neurotransmitters, 820, 827–829 overview, 820 pathophysiology, 829–830 stimulation effects, 825–827 sympathetic neurons neurotransmitters, 819, 821–822 stimulation effects, 820–821 interactive control enteric and sympathetic vasomotor neurons, 830–831 local and paracrine mediators, 831–832 primary afferent and enteric vasodilator neurons, 831 primary afferent and sympathetic vasomotor neurons, 831 organization, 817–818 physiologic relevance, 817 prospects for study, 832 Gastrointestinal motility, see also Gastric emptying; Intestinal motility coordination of secretion and motility, 755–756 corticotropin-releasing hormone effects colon, 804–807 small intestine, 803–804 stomach, 800–802 stress effects animal studies, 783 autonomic dysfunction, 784–785 corticotropin-releasing hormone modulation

I-8 / INDEX Gastrointestinal motility (Continued) overview, 781–782 peripheral receptor and colonic motor alterations, 785 type 1 receptor activation and stimulation, 784 type 2 receptor activation and inhibition, 784 visceral hyperalgesia mediation by receptors, 786 healthy subjects, 782 irritable bowel syndrome patients, 782–783 prospects for study, 786–787 sex differences in visceral hypersensitivity, 786 visceral perception studies, 785–786 Gastrointestinal tube formation, 295 Hedgehog signaling in development, 275 G cell, see Gastrin GDNF, see Glial-derived neurotrophic factor Gene classification, 2 coregulatory proteins, 15–16 DNA-binding proteins, 12–15 DNA composition and structure, 1–2 5’-flanking sequences, 4 number in human genome, 1 organization, 2–4 promoter, see Promoter transcription, see Transcription GERD, see Gastroesophageal reflux disease Ghrelin assays, 110 excess and deficiency in disease, 111 food intake regulation, 886 functions, 109–111 gene structure and expression, 109 metabolism, 110 posttranslational processing, 109 receptors, 110 release, 110 structure, 109 tissue distribution, 109–110 Giant migrating contractions excitation–contraction coupling, 974–976 excitation–inhibition coupling, 976 features, 948 features, 967–969 inflammatory bowel disease dysfunction, 986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction, 983–984 GIP, see Glucose-dependent insulinotropic polypeptide GIPR, see Glucose-dependent insulinotropic polypeptide receptor Glial cells, intestinal inflammation effects, 1129 Glial-derived neurotrophic factor, Hirschsprung’s disease mutations, 505–507 GLP-1, see Glucagon-like peptide-1 GLP-2, see Glucagon-like peptide-2 Glucagon derived peptides, see Glucagon-like peptide-1; Glucagon-like peptide-2; Glycentin; Oxyntomodulin functions, 166–167 gene regulation of expression, 162–163 structure, 161–162 metabolism and clearance, 164 pancreas secretion regulation, 1420–1421 receptor, 165 release regulation, 164 secretion regulation, 164 therapeutic application, 167 vasodilation induction in intestine, 1640 Glucagon-like peptide-1 cell growth regulation, 451–452 food intake regulation, 885 functions, 167–169 metabolism and clearance, 164–165 pancreas secretion regulation, 1421 processing, 162 receptor, 165–166 receptor antagonists for diabetes type 2 treatment, 169–170 release regulation, 164 Glucagon-like peptide-2 cell growth regulation, 452 functions, 170–171

Glucagon-like peptide-2 (Continued) metabolism and clearance, 164–165 processing, 162 receptor, 166 release regulation, 164 stem cell regulation, 331 therapeutic applications, 171 Glucocorticoids, stress effect mediation in mucosa, 771 Glucose absorption, 1654–1656 dorsal vagal complex chemosensitivity, 865 Glucose-dependent insulinotropic polypeptide functions, 171–172 receptor features, 166 secretion, 171 synthesis, 171 therapeutic applications, 172 Glucose-galactose malabsorption, SGLT1 mutations, 1662 Glucose transporters cholangiocytes, 1511–1512 gene family, 1654 glucose, galactose, and fructose absorption, 1654–1656 GLUT2 function, 1656 mutation in disease, 1662 ontogenic regulation of intestinal monosaccharide absorption, 383 sugar selectivity, 1657 GLUT5 function, 1656 mutation in disease, 1662–1663 ontogenic regulation of intestinal monosaccharide absorption, 383 sugar selectivity, 1657 kinetics, 1661 regulation of expression, 1663 Glutaminyl cyclase, protein processing, 47 Glutathione antioxidant protection, 1494–1495 metal detoxification, 1497 transport in small intestine, 1681 Glutathione peroxidase, antioxidant protection, 1494 Glutathione S-transferase, phase II metabolism, 1489–1490 GLUTs, see Glucose transporters Glycentin, functions, 167 Glycine transporter 1, amino acid transport, 1678 GMC, see Giant migrating complex Golgi acinar protein trafficking endoplasmic reticulum to Golgi transport, 1323–1324 Golgi to trans-Golgi network transport, 1324–1326 trans-Golgi network to zymogen granule transport, 1326 posttranslational modifications glycosylation, 41 phosphorylation, 41 sulfation, 41–42 protein trafficking, 39–41 GPCRs, see G protein-coupled receptors G protein-coupled receptors abundance, 63 accessory proteins, 69 acinar cells, 1326, 1338 cholangiocytes, 1514 classification class A, 66–67 class B, 67 class C, 68 desensitization by receptor kinases and arrestins, 77–81 dimerization, 68 domains, 63–64 endocytosis, 81–82 extrinsic sensory afferent nerve signaling, 704–706 G proteins GTPases Arf, 72 Rab, 72–73 Ras, 72 Rho, 72 signaling mediation, 71–72 states, 70–71 types, 70

G protein-coupled receptors (Continued) internalized receptor signaling, 83 ligands classification, 71 interactions, 65, 67 lysosomal trafficking arrest of signal transduction, 83–85 microdomains in signaling, 73–74 mitogen-activated protein kinase signaling, 71, 73–74 prospects for study, 85 receptor tyrosine kinase signaling dimerization, 74 G protein signaling, 76–77 recruitment, 74–76 transactivation, 76 recycling and resensitization, 82–83 rhodopsin features, 64–65 smooth muscle contraction signaling desensitizaton, 526–527 initial phase of contraction, 524–525 sustained phase of contraction, 525–526 relaxation signaling PACAP receptor, 528 vasoactive intestinal peptide receptors, 527–528 structure, 64 GRKs, G protein-coupled receptor desensitization, 77–79 Growth factors, see also specific growth factors apoptosis inhibition, 355–356 compartmentalization of function, 185 definition, 184 intestinal transport in neonatal period, 380–381 redundancy in families, 185–186 signaling paradigms, 184–185 Growth hormone, calcium absorption regulation, 1966–1967 GRP, see Gastrin releasing peptide Gut-associated lymphoid tissue intestinal immunity, 1148–1149 leukocyte movement through interstitium adhesion molecules, 1151–1152 chemoattractant regulation, 1152–1153 lymphocyte homing and activation, 1149–1151 H Hand2, Hirschsprung’s disease mutations, 507 Hartnup disease, pathophysiology, 1681–1682 HB-EGF, see Heparin-binding epidermal growth factor-like growth factor Heat-stable enterotoxin, secretory function effects, 1169 Hedgehog cancer defects, 282–283 development signaling esophagus, 275–276 gastrointestinal tube, 275 hindgut, 278–279 midgut, 278 pancreas, 277–278 stomach, 277 thyroid, 276–277 trachea, 275–276 VACTERL signaling, 279 genes, 272–273 homeostasis role Indian Hedgehog, 280–282 morphostasis, 279–280 Sonic Hedgehog, 280–281 processing, 273–274 prospects for study, 283 receptor signaling, 274 Sonic Hedgehog in gastric gland maintenance, 445–446 stem cell function regulation, 324–326 target genes, 274–275 Helicobacter pylori aging studies esophagus, 406 stomach, 408, 410–411 apoptosis induction in gastric mucosa, 364–365 barrier function effects CagA, 1178–1179 overview, 1166 cell-mediated immunity, 1098 enterochromaffin-like cell function effects, 1231 gastric mucosa colonization, 1092–1094 gastrin induction, 93

INDEX / I-9 Helicobacter pylori (Continued) gastritis cyclooxygenase role, 1098–1099 cytokine release, 1095–1096 interleukin-8 expression regulation, 1096–1097 nitric oxide role, 1099 gene polymorphisms and disease susceptibility, 1104–1106 humoral response, 1097–1098 iceA strains and disease, 1103 immune evasion, 1094–1095 inflammatory response, 1091–1092 secretory function effects overview, 1164 VacA, 1170–1171 virulence factors BabA, 1102–1103 cag pathogenicity island, 1099–1101 FldA, 1103 novel factors, 1103–1104 VacA, 1101–1102 Heparin-binding epidermal growth factor-like growth factor discovery, 198 functions, 199 receptors, 199 synthesis, 198 tissue distribution, 198–199 Hepatocyte bile acid transport physiology, 1468 sodium-dependent uptake and regulation, 1448–1449, 1469–1471 sodium-independent uptake, 1449 transcriptional regulation of transporter expression, 1474–1475 transporters biochemical studies, 1468 molecular biology studies, 1469 overview, 1449 detoxification, see Liver Kupffer cell clearance, 1499 nonbile acid organic anion uptake albumin binding effects, 1463–1464 driving forces, 1464 physiologic modulation, 1464 transporters biochemical studies, 1464–1465 biochemistry, 1465, 1467 function, 1467 molecular biology studies, 1465 regulation, 1467–1468 sequence alignment, 1466 substrate specificity, 1468 regeneration mitogen and cytokine regulation, 1500 oval cells, 1500–1501 overview, 1500 Hepatocyte growth factor discovery, 213 functions cell growth regulation, 448 development, 214–215 tissue repair, 215 tumorigenesis, 216 gene, 213 processing, 213 protein–protein interactions, 213–214 receptors, 214 signaling, 214 structure, 213 Hepcidins, innate immunity, 1052 Hereditary hemochromatosis, pathophysiology, 1498–1499 Hes1, Notch regulation, 299, 322–323 HGF, see Hepatocyte growth factor Hirschsprung’s disease aganglionosis, 499–500 endothelin role, 1633 enteric nervous system plasticity, 674–675 epidemiology, 503 gene mutations, 503–507 pathogenesis, 499 Histamine enterochromaffin-like cell secretion, 1229–1230 gastric acid secretion regulation, 1243, 1246, 1301–1302

Histamine (Continued) knockout mouse pathway mutants, 1302–1303 mast cell signaling in irritable bowel syndrome, 1025 pancreas electrolyte regulation, 1388 pancreas secretion regulation, 1416 vasodilation induction in intestine, 1640 Histamine receptors gastric expression, 1236–1237 ligand binding, 1236 signaling, 1237 structure, 1236 Histone acetylation, 6–7 methylation, 7 phosphorylation, 7–8 structure, 5–6 Histone-derived peptides, innate immunity, 1055 Hormone, definition, 91–92 Hydrochloric acid secretion, see Gastric acid I ICAMs, see Intercellular adhesion molecules IGF-I, see Insulin-like growth factor-I IGF-II, see Insulin-like growth factor-II IGFBPs, see Insulin-like growth factor binding proteins IL-1, see Interleukin-1 IL-8, see Interleukin-8 Ileum bile acid transport ileal basolateral transport, 1452 sodium-dependent uptake, 1451–1452 water transport, 1839–1840 Immune system, see Adaptive immunity; Immunoglobulins; Innate immunity Immunoglobulins Helicobacter pylori humoral response, 1097–1098 immunoglobulin E FcεRII low-affinity receptor, 1083–1084 transcytosis of immunoglobulin E, 1084 food allergy, 1082 intestinal anaphylaxis, animal models, 1082–1083 intestinal functions, 1082, 1084–1085 immunoglobulin G FcRn antigen uptake pathway mediation, 1081–1082 binding model, 1078–1079 discovery, 1077 structure, 1077–1078 transport, 1079–1081 mucosal physiology and function, 1081 transport, 1079–1081 inflammatory bowel disease levels, 1082 polymeric receptor expression, 1073–1074 structure, 1074–1075 trafficking, 1075–1076 secretory immunoglobulin A functions, 1068–1070 mucosal plasma cell origins, 1070–1072 polymeric immunoglobulin structure, 1072–1073 Indian Hedgehog, see Hedgehog Infection, chloride–bicarbonate exchange regulation, 1886 Infectious colitis, clinical manifestations, 1123 Inflammation, see also Intestinal inflammation; Leukocyte recruitment bile acid transport effects, 1454 cancer role, 479–480 chloride–bicarbonate exchange regulation, 1885 gallbladder, 1551–1553 leukocyte trafficking acute inflammation bacterial toxins, 1154–1155 oxidative stress-dependent models, 1154 chronic inflammation, 1155–1157 presynaptic inhibition mediation in enteric nervous system, 656–657 stem cell effects, 332–335 systemic inflammation and behavior, 710 trefoil factor family role, 212–213 Inflammatory bowel disease clinical manifestations, 1122 colonic contraction dysfunction excitation–contraction coupling, 986–987 intrinsic sensory neurons, 987 motor neurons, 987

Inflammatory bowel disease (Continued) myoneuroimmune interactions, 987 overview, 985–986 Crohn’s disease and intestinal epithelial tight junction barrier function effects, 1584–1585 endothelin role, 1633 immunoglobulin G levels, 1082 ion transport alterations, 1943–1944 stress in pathogenesis, 775 Innate immunity adaptive immunity comparison, 1033–1034 angiogenins, 1055 cathelicidins, 1052–1053 defensins α-defensins, 1049–1051 β-defensins, 1051–1052 definition, 1033 hepcidins, 1052 histone-derived peptides, 1055 lysozyme, 1053–1054 molecular pattern recognition, 1035 nitric oxide, 1055–1057 nucleotide-binding oligomerization domain proteins NOD1, 1045–1047 NOD2, 1047–1049 structure, 1045 phospholipase A2, 1054 Toll-like receptors structure, 1035 TLR1, 1039–1040 TLR2, 1039–1040 TLR3, 1043–1044 TLR4, 1035–1039 TLR5, 1042–1043 TLR6, 1039–1040 TLR7, 1044 TLR8, 1044 TLR9, 1040–1042 TLR10, 1044 Inositol phosphates, see Phosphoinositides Insula, gastrointestinal sensory signal processing, 730, 733–734 Insulin-like growth factor-I cancer role, 209 functions apoptosis inhibition, 209 cellular proliferation, 208–209, 447 profibrogenic actions, 209 gastrointestinal distribution, 207–208 gene, 206 knockout mouse studies, 209 receptors, 206, 209 stem cell regulation, 330 Insulin-like growth factor-II cancer role, 209 gastrointestinal distribution, 207–208 gene, 206 receptors, 206 stem cell regulation, 330 Insulin-like growth factor binding proteins functions, 207 structures, 207 types, 206 Integrins, types and functions, 1141–1142 Intercellular adhesion molecules, types and functions, 1140–1141 Interleukin-1 enterochromaffin-like cell regulation, 1229 polymorphisms in Helicobacter pylori disease, 1104–1105 Interleukin-8, expression regulation in Helicobacter pylori gastritis, 1096–1097 Intermediate filaments, transport mediation, 1614–1615 Interneuron ascending interneurons, 586–587 colonic contraction regulation, 979 descending interneurons, 587–588 interplexus interneurons, 590–591 intestinal motility role, 979 overview, 586, 636–637 secretory reflexes, 740 Interstitial cell of Cajal animal model for study, 537, 549–550 defects in human disease, 539–540, 549 intestinal inflammation effects, 1128 intramuscular cells, 534–535

I-10 / INDEX Interstitial cell of Cajal (Continued) morphology, 535 neurotransmission innervation by enteric motor neurons, 546–547 motor neurotransmission acetylcholine, 547 nitric oxide, 547 neural control of slow wave activity, 547 neurotransmitter mechanism of action, 547–548 pacemaker activity action propagation of slow waves, 542–544 calcium flux, 538, 540–542 cultured cells, 538 Kit knockout studies, 537, 549 mechanism, 538, 540–542 spontaneous electrical rhythmicity, 535 unitary potentials, 544–546 slow wave origins, 533–534 small intestine motility regulation, 938–940 smooth muscle responses to slow waves and neural inputs chloride conductances calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 integration of electrical activity, 569 ion channels, 552–554 nonselective cation channels currents activated by ATP, 568 currents activated by muscarinic and peptide agonists, 567–568 currents activated by stretch, 568–569 overview, 550–551 voltage-dependent inward currents inward rectifier currents, 556 L-type calcium channels, 551, 555–556 sodium channels, 556 T-type calcium channels, 556 voltage-dependent outward currents A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 ether-a-go-go-related currents, 560–561 inward rectifier potassium channels, 559–560 overview, 556–557 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 stretch receptor function, 548–549 Intestinal epithelium apoptosis anoikis, 363–364 cell studies, 358–363 induced apoptosis inducers, 351–352 ischemia–reperfusion injury, 354 mechanisms, 352–353 small bowel resection, 353–354 pathways and regulation, 358–362 polyamine depletion effects, 359–360 prevention cytokines, 357–358 growth factors, 355–356 lysophosphatidic acid, 357 polyamines, 356–357 prostaglandins, 356 spontaneous apoptosis crypt, 350–351 villus, 351 electrolyte transport, see specific electrolytes growth and differentiation, 349–350 Intestinal inflammation, see also Inflammation; Inflammatory bowel disease chronic inflammation, 1121–1122 clinical manifestations drug-induced colitis, 1123 infectious colitis, 1123 inflammatory bowel disease, 1122 microscopic colitis, 1122–1123 radiation enteritis, 1122 environmental factors, 1123 functional effects digestive dysfunction, 1127 epithelial cell apoptosis, 1125–1126

Intestinal inflammation (Continued) motor disturbances enteric nervous system, 1128–1129 extrinsic primary afferent nerves, 1129 glial cells, 1129 interstitial cells of Cajal, 1128 nerve/mast cell interactions, 1124–1125 nutrient absorption, 1128 secretagogue hyporesponsiveness, 1126–1127 secretomotor neurons, 1124 sensory nerves, 1124 smooth muscle phenotype changes, 1129–1130 tight junctions, 1125 genetic factors, 1123 luminal triggers dendritic cells, 1116 effectors arachidonic acid metabolites, 1120 cytokines and chemokines, 1118–1119 neutrophil products, 1120 nitric oxide, 1120–1121 proteases and activated receptors, 1121 Toll-like receptors, 1116 transcription factors nuclear factor-κB, 1116–1117 STATs, 1117–1118 overview, 1115 prospects for study, 1130 resolution phase, 1121 Intestinal motility, see also Gastrointestinal motility abdominal pain in irritable bowel syndrome, 1021–1022 colonic contractions enteric neuron regulation enteric reflexes, 981–982 interneurons, 979 intrinsic sensory neurons, 979–981 motor neurons, 977–979 overview, 976–977 excitation–contraction coupling giant migrating contractions, 974–976 overview, 970–971 rhythmic phase contractions, 971–974 tonic contractions, 976 excitation–inhibition coupling, 976 giant migrating contractions, 967–969 inflammatory bowel disease dysfunction excitation–contraction coupling, 986–987 intrinsic sensory neurons, 987 motor neurons, 987 myoneuroimmune interactions, 987 overview, 985–986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 overview, 965–966 rhythmic phase contractions, 966–967 tonic contractions, 969 motilin regulation, 138–139 small intestine central nervous system modulation, 953–954 contraction coupling, 940 extended reflexes intestino-intestino reflex, 951 non-nutrient stimulation, 953 nutrient-evoked reflexes, 951–953 reflex stimulation by other organs, 953 fasting motor pattern reversion, 947–948 fed motor pattern neural regulation, 947 neurohumoral mediators, 947 physiologic characteristics, 946–947 functional anatomy, 935–936 giant migrating complex, 948 ileocolonic junction motor activity, 949–951 infection effects and immune mediation, 954–956 interstitial cell of Cajal regulation, 938–940 migrating motor complex associated synchronous cyclic phenomena, 946 neural regulation, 944–945 neurohumoral mediators, 945–946 physiologic characteristics, 943–944

Intestinal motility (Continued) nervous tissue extrinsic innervation, 938 intrinsic innervation, 936–938 peristalsis control, 941–943 retrograde peristaltic contractions, 948–949 smooth muscle, 936 transit, 936 structural basis, 579 Intestinal mucosa barrier function antigen-sampling cells dendritic cells, 765–766 follicle-associated epithelium, 765 M cells, 765 ion and fluid transport, 766 mucus secretion, 766 overview, 763–764 tight junctions, 764 transcellular uptake of protein antigens, 765 inflammation effects nerve/mast cell interactions, 1124–1125 secretomotor neurons, 1124 sensory nerves, 1124 stress effects animal studies acute stress mediators, 770–772 barrier function, 769–770 chronic stress studies, 772–773 early life stress, 774–775 flora changes, 773 ion secretion, 769 models, 768–769 mucus secretion, 769 food allergy pathogenesis, 775–776 inflammatory bowel disease pathogenesis, 775 irritable bowel syndrome pathogenesis, 776 physical stress, 767–768 prospects for study, 776 psychological stress, 768 Tcf-4 regulation cell cycle progression, 442 intestinal cell position along crypt-villus axis, 442–443 Intestinal transport, see also specific nutrients hormones and growth factors in neonatal period, 380–381 macromolecules during neonatal and suckling periods, 379–380 ontogeny along vertical and horizontal gut axes, 391–392 postnatal development amino acid transport, 383 bile acid absorption, 391 calcium absorption, 386 lipid absorption, 383–384 lipid-soluble vitamin transport, 391 monosaccharide absorption, 381–383 phosphate absorption, 386–388 potassium absorption, 386 sodium chloride absorption, 384–386 trace mineral absorption, 388–389 water transport, 384 water-soluble vitamin transport, 389–391 Intestine, see Colon; Small intestine Ion channels, see also specific channels acinar cell exocytosis role, 1361–1362 alterations in disease constipation, 1944 infectious diarrhea, 1942–1943 inflammatory bowel disease, 1943–1944 therapeutic implications, 1944 analysis effectors, 1923–1924 experimental models, 1922 permeabilization, 1922 permeable supports, 1925 structure–function studies, 1925 transfection studies, 1924–1925 Ussing chamber studies, 1923 inhibitors, 1167 interactions between transport mechanisms, 1938–1939 intestinal ion transport regulation at tissue level acute regulation, 1939–1941 chronic regulation, 1941–1942

INDEX / I-11 Ion channels (Continued) secretory function ontogeny, 376–377 smooth muscle responses to slow waves and neural inputs chloride conductances calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 integration of electrical activity, 569 ion channels, 552–554 nonselective cation channels currents activated by ATP, 568 currents activated by muscarinic and peptide agonists, 567–568 currents activated by stretch, 568–569 overview, 550–551 voltage-dependent inward currents inward rectifier currents, 556 L-type calcium channels, 551, 555–556 sodium channels, 556 T-type calcium channels, 556 voltage-dependent outward currents A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 ether-a-go-go-related currents, 560–561 inward rectifier potassium channels, 559–560 overview, 556–557 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 Iron deficiency, 1986–1987 intestinal absorption, 1983–1985 overload ferroportin disease, 1989 hemochromatosis, 1987–1989 hyperabsorption disorders, 1990 iatrogenic overload, 1990 plasma protein defects, 1989–1990 transfusional overload, 1990 plasma trafficking, 1985 systemic homeostasis, 1985–1986 Irritable bowel syndrome abdominal pain and discomfort central neuropathologic factors cerebral cortex sensory processing, 1018–1019 spinal ascending sensory pathways, 1017–1018 spinal cord sensory processing, 1019–1020 spinal descending modulatory pathways, 1020–1021 inflammatory mediator receptors on sensory afferents, 1017 intestinal motility, 1021–1022 peripheral neuropathologic factors, 1015 postinfectious irritable bowel syndrome, 1015–1016 presentation, 1015 serotonin receptors on sensory afferents, 1016–1017 colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 enteric nervous system neuropathy disinhibitory motor disease, 1011–1012 inhibitory motor neurons, 1010–1011 motor neurons, 1010 overview, 1009–1010 secretomotor neurons excitatory input, 1014–1015 inhibitory input, 1013–1014 neurogenic secretory diarrhea and neurogenic constipation, 1014 projections, 1013 epidemiology, 1009 psychological stress immunoneural signal sources, 1022–1023 mast cells brain–gut connection, 1024–1027 histamine signaling, 1025 immunoneural communication, 1023–1024 serotonin signaling, 1025–1026 neuroimmunophysiologic paradigm, 1022

Irritable bowel syndrome (Continued) serotonin receptors, 636 sex differences, 786 stress in pathogenesis, 776 visceral hypersensitivity, 635–636 Ischemia, afferent sensitivity effects, 712–713 Ischemia–reperfusion injury, apoptosis induction, 354 J Jagged, Notch ligand, 289–290 Jejunum, water transport, 1839–1840 K Keratinocyte growth factor, cell growth regulation, 448–449 KGF, see Keratinocyte growth factor KLFs, see Krüppel-like transcription factors Knockout mouse acetylcholine receptor mutants, 1303 basolateral membrane transporters, 1306 epidermal growth factor receptor studies, 204–205 fibroblast growth factor studies, 219 Fox11 knockout, 1306 gastrin-cholecystokinin double-knockout mice, 1301 gastrin pathway mutants, 1299, 1301 generation, 1295–1296 gene regulation studies, 17 histamine pathway mutants, 1302–1303 insulin-like growth factor-I studies, 209 Kit studies, 537, 549 somatostatin receptor knockout, 1303–1304 transforming growth factor-β studies, 192–193 trefoil factor knockout, 1306 TRPV calcium transporter studies, 1762–1763, 1967 Krüppel-like transcription factors, epithelial cell growth regulation, 445 Kupffer cell, hepatocyte clearance, 1499 L Laminin, enteric nervous system development role, 511–512 Large intestine, see Colon Laryngeal motor function nasopharyngeal motor function during swallowing and belching, 901–902 overview of deglutitive function, 900–901 Leptin, food intake regulation, 878 LES, see Lower esophageal sphincter Leukocyte recruitment gut-associated lymphoid tissue lymphocyte homing and activation, 1149–1151 inflammation acute inflammation bacterial toxins, 1154–1155 oxidative stress-dependent models, 1154 chronic inflammation, 1155–1157 movement through interstitium adhesion molecules, 1151–1152 chemoattractant regulation, 1152–1153 overview, 1137 Lipid, see also Fatty acids; Lipoproteins cell growth regulation polyunsaturated fatty acids, 450–451 short-chain fatty acids, 450 ontogenic regulation of absorption, 383–384 pancreatic secretion regulation, 1402–1404 wound healing promotion, 464 Lipid raft G protein-coupled receptor signaling, 74 protein sorting in polarized epithelial cells, 1610–1611 Lipoproteins, intestinal assembly apolipoprotein A-I role, 1723–1724 apolipoprotein A-IV role, 1723–1724 apolipoprotein B, see Apolipoprotein B first step, 1714–1715 microsomal triglyceride transfer protein, see Microsomal triglyceride transfer protein novel pathways and defects, 1728–1729 overview, 1712 prechylomicron transport, 1717 second step, 1716 Liraglutide, diabetes type 2 treatment, 170 Liver anatomy and function, 1483–1484 detoxification

Liver (Continued) ATP-binding cassette transporters, 1490–1492 liver health effects on metabolism and elimination, 1492 metabolism principles, 1484 phase I metabolism alcohol dehydrogenase, 1487–1488 aldehyde dehydrogenase, 1488 cytochrome P450, 1484–1487 flavin monooxygenase, 1487 phase II metabolism glutathione S-transferase, 1489–1490 N-acetyltransferase, 1489 sulfotransferase, 1489 thiopurine methyltransferase, 1490 UDP glucuronosyltransferase, 1488–1489 metal detoxification copper sequestration, 1498 ferritin, 1498 glutathione binding and elimination, 1497 metallothionein, 1497–1498 pathology hereditary hemochromatosis, 1498–1499 Wilson’s disease, 1499 toxicity mechanisms, 1497 oxidative damage antioxidant protection catalase, 1494 glutathione, 1494–1495 glutathione peroxidase, 1494 superoxide dismutase, 1494 thioredoxin, 1495 vitamin C, 1495 vitamin E, 1495 hepatocyte necrosis versus apoptosis, 1493–1494 pathology biochemical pathway stimulation, 1496 cancer, 1496–1497 chronic liver disease, 1496 drug toxicity, 1495–1496 reactive oxygen species sources, 1492–1493 redox cycling, 1493 toxicity, 1492 sodium–proton exchange, 1867–1868 water transport, 1837–1838 Low-density lipoprotein receptor-related proteins destruction complex regulation, 254–255 Wnt interactions, 249, 251–252, 441 Lower esophageal sphincter anatomy, 914 coordinated motor activity, 921–923 LRP, see Low-density lipoprotein receptor-related protein Lysinuric protein intolerance, pathophysiology, 1682–1683 Lysophosphatidic acid, apoptosis inhibition, 357 Lysozyme, innate immunity, 1053–1054 M Macula adherens, see Desmosome Magnesium dietary requirements, 1953–1954 intestinal absorption physiology, 1972–1973 regulation, 1975 sites, 1971–1972 transient receptor potential family proteins, 1973, 1975 pancreatic secretion regulation, 1403 Malignancy, see Cancer, gastrointestinal Manganese, absorption and transporters, 1998 MAPKs, see Mitogen-activated protein kinases Mash1 mutation and enteric nervous system lesions, 508–509 Notch regulation, 302 Mast cell afferent sensitivity effects, 711 irritable bowel syndrome and psychological stress brain–gut connection, 1024–1027 histamine signaling, 1025 immunoneural communication, 1023–1024 serotonin signaling, 1025–1026 nerve interactions in intestinal inflammation, 1124–1125 stress effect mediation in mucosa, 770–771

I-12 / INDEX Mastermind, Notch regulation, 294 Math1, Notch regulation, 300, 322 M cell, antigen sampling, 765 MCH, see Melanin-concentrating hormone MDR3, see Multidrug resistance gene 3 Megalin, gallbladder cholesterol metabolism, 1548 Melanin-concentrating hormone, feeding control, 881 Melanocortins, feeding control, 879–880 α-Melanocyte stimulating hormone, feeding control, 879 Messenger RNA nuclear transport, 21–22 processing polyadenylation, 19–20 splicing alternative splicing, 20 regulation, 20–21 spliceosome, 20 ribonucleoprotein complex, 31 stability regulation, 35 structure, 3 transcription, see Transcription translation, see Translation Metallothionein, metal detoxification, 1497–1498 N-Methyl-D-aspartate receptor, extrinsic sensory afferent nerves, 703 Microsatellite instability, cancer, 487–489 Microscopic colitis, clinical manifestations, 1122–1123 Microsomal triglyceride transfer protein abetalipoproteinemia gene mutations, 1720–1721 mouse models, 1721 lipoprotein assembly role, 1716 structure, 1714 Microtubule, transport in polarized epithelial cells, 1612–1614 Micturition, pelvic floor neural control, 1003 Migrating motor complex associated synchronous cyclic phenomena, 946 neural regulation, 944–945 neurohumoral mediators, 945–946 physiologic characteristics, 943–944 Mitogen-activated protein kinases cell growth regulation, 445 epidermal growth factor receptor signaling, 201 G protein-coupled receptor signaling, 71, 73–74 MMC, see Migrating motor complex Monocarboxylate transporter 1, short-chain fatty acid transport, 1904, 1906 Monosaccharides, postnatal development of absorption, 381–383 Motilin antibiotic binding to receptor, 140 functions, 137–139 mechanism of action, 139 pathophysiology, 140 receptor, 137 release, 139–140 structure, 137–138 tissue distribution, 137 Motor neuron, see Enteric nervous system Mouse genetic engineering, see Knockout mouse; Transgenic mouse mRNA, see Messenger RNA MRPs, see Multidrug resistance proteins α-MSH, see α-Melanocyte stimulating hormone mTOR cell growth regulation, 444 phosphatidylinositol 3-kinase interactions, 444–445 rapamycin inhibition, 444 MTTP, see Microsomal triglyceride transfer protein Mucins, gallbladder secretion regulation bile salts, 1550–1551 calcium flux, 1550 cyclic AMP, 1550 cystic fibrosis transmembrane conductance regulator, 1551 gallstone formation, 1551 overview, 1549–1550 transcriptional regulation, 1551 Mucosa, see also Gastric mucosa; Gastroduodenal mucosal defense; Intestinal mucosa apoptosis, see Apoptosis cell cycle regulation, 437 cell growth regulation bombesin, 452–453 epidermal growth factor receptor, 447

Mucosa (Continued) fibroblast growth factors, 447–448 gastrin, 451 glucagon-like peptide-1, 451–452 glucagon-like peptide-2, 452 hepatocyte growth factor, 448 insulin-like growth factor-I, 447 keratinocyte growth factor, 448–449 Krüppel-like transcription factors, 445 luminal nutrients and secretions, 449–451 mitogen-activated protein kinases, 445 neurotensin, 452 peptide YY, 452 phosphatidylinositol 3-kinase/Akt pathway, 443–445 prospects for study, 453 somatostatin, 453 transforming growth factor-β, 446–447 trefoil factors, 448 Wnt signaling, 440–443 growth regulation in aging gene expression patterns, 423 growth factors and protein kinases, 421–423 intracellular mechanisms, 420 nutritional and hormonal factors, 420–421 prospects for study, 423 organization, 536–437 repair, see Wound healing Multidrug resistance gene 3, detoxification, 1491 Multidrug resistance proteins, detoxification, 1491–1492 Musashi1, Notch regulation, 300 c-Myc, stem cell function regulation, 318–319 Myosin II, vesicular trafficking in gastric acid secretion, 1210–1211 N NEC, see Neonatal necrotizing enterocolitis Necrosis, hepatocytes, 1493–1494 Neonatal necrotizing enterocolitis, endothelin role, 1633 Netrins, enteric nervous system development role, 512–513 β-Neurexin, enteric nervous system development role, 513–514 Neuroglin, enteric nervous system development role, 513–514 Neurokinins gallbladder emptying regulation, 844 primary afferent neuron neurotransmission in blood vessels, 828–829 Neuromedin B, satiety signaling, 884 Neuropeptide Y feeding control, 879–881, 887 functions, 134–135, 137 presynaptic inhibition mediation in enteric nervous system, 657 receptors, 134, 142–143 release, 137 structure, 134, 136 sympathetic neuron neurotransmission in blood vessels, 82 tissue distribution, 134, 136–137 Neurotensin cell growth regulation, 452 functions, 130, 132–133 pancreas electrolyte regulation, 1389 pancreas secretion regulation, 1415 receptors, 131–132 release, 133–134 structure, 130–131 tissue distribution, 131 Neurotrophin-3, mutation and enteric nervous system lesions, 508 NHEs, see Sodium–proton exchangers Niacin functions, 1812 intestinal absorption, 1812 sources, 1812 Nicotinic receptors extrinsic sensory afferent nerves, 703–704 inhibitory postsynaptic potentials in enteric nervous system, 646 Nitric oxide bicarbonate secretion regulation in duodenum, 1271–1272 chloride–bicarbonate exchange regulation, 1885–1886 chloride secretion mediation, 1172

Nitric oxide (Continued) endothelial nitric oxide synthase regulation activity regulation, 1635 posttranslational modification, 1635 transcription, 1634–1635 gallbladder function effects, 1552 gastric accommodation reflex control, 929–930 Helicobacter pylori gastritis role, 1099 innate immunity, 1055–1057 intestinal circulation localization, 1635 vasodilation induction, 1635–1636 synthesis, 1634 NO, see Nitric oxide NODs, see Nucleotide-binding oligomerization domain proteins Nonsteroidal anti-inflammatory drugs apoptosis induction in gastric mucosa, 365–366 intestinal epithelial tight junction barrier function effects, 1586 Norepinephrine enterochromaffin-like cell regulation, 1229 presynaptic inhibition mediation in enteric nervous system, 656 sympathetic neuron neurotransmission in blood vessels, 821 Notch developmental signaling enteric nervous system, 302 enteroendocrine system, 298–300 mucosal immune system, 301 overview, 295–297 time course, 297–298 ligands Delta, 289 functions, 292–293 Jagged, 289–290 Numb regulation, 294 posttranslational modifications glycosylation, 293 phosphorylation, 293 ubiquitination, 293–294 prospects for study, 302 receptors functions, 292–293 processing, 288 structures, 288–289 signaling crosstalk, 294–295 overview, 287–288 regulation, 290 target genes, 291–292 stem cell function regulation, 322–324 structure, 288 NPC1L1, intestinal cholesterol transport, 1724, 1727–1728 NPY, see Neuropeptide Y NSAIDs, see Nonsteroidal anti-inflammatory drugs Nuclear factor-κB, intestinal inflammation role, 1116–1117 Nucleosome, structure, 5 Nucleotide-binding oligomerization domain proteins NOD1, 1045–1047 NOD2, 1047–1049 structure, 1045 Nucleotides, cell growth regulation, 450–451 Nucleus of the solitary tract, pars centralis portion, 858–859 vago-vagal control reflex, 857–858 Numb, Notch regulation, 294 O Obesity, peptide YY deficiency, 145 Opioids, stress effect mediation in mucosa, 771–772 Orexins, functions, 172 Organic anion transporters, see Hepatocyte Oval cell, hepatocyte regeneration, 1500–1501 Oxidative stress, see Reactive oxygen species Oxygen metabolic vascular regulation and postprandial hyperemia, 1642 parenchymal demand versus vascular supply metabolic feedback, 1639 metabolic theory, 1637 microvascular studies, 1638–1639 whole-organ studies, 1637–1638 vascular smooth muscle effects, 1636–1637

INDEX / I-13 Oxyntomodulin metabolism and clearance, 164–165 functions, 167 P PACAP, see Pituitary adenylate cyclase-activating peptide PAF, see Platelet-activating factor Pancreas acinar cell, see Acinar cell cholecystokinin effects, 104–105 development, 1314–1316 electrolyte secretion apical transport anion exchangers, 1381 aquaporins, 1384 calcium-activated chloride channels, 1382–1383 carbonic anhydrase, 1384 cystic fibrosis transmembrane conductance regulator, 1381–1382 paracellular cation transport, 1384 potassium channels, 1383 sodium–bicarbonate cotransporter, 1383–1384 sodium–proton exchanger, 1383 basolateral transport aquaporins, 1381 chloride–bicarbonate exchanger, 1380 potassium channels, 1380 proton–ATPase, 1379 sodium–bicarbonate cotransporter, 1379–1380 sodium/potassium-ATPase, 1380–1381 sodium–potassium–2chloride cotransporter, 1380 sodium–proton exchanger, 1379 duct cell preparations and techniques for study, 1375–1377 model, 1384–1385 prospects for study, 1391 regulation acetylcholine, 1387, 1390 angiotensin II, 1388 arginine vasopressin, 1390–1391 ATP, 1387–1388, 1391 bombesin, 1389 calcium, 1388 cholecystokinin, 1389 cyclic AMP, 1386–1387 cyclic GMP, 1387 histamine, 1388 neurotensin, 1389 proteinase-activated receptor-2, 1388 secretin, 1390 serotonin, 1390 substance P, 1389–1390 species-dependent patterns, 1371–1372 structural basis, 1372–1374 exocrine pancreas organization, 1313–1314 extracellular matrix, 1316–1317 function testing blood enzymes, 1424 metabolic activity, 1423 pancreatic polypeptide, 1424 stool enzymes, 1424 Hedgehog signaling in development, 277–278 hormonal regulation of secretion cholecystokinin, 1412–1415, 1418 cholecystokinin-releasing factor, 1418–1419 feedback regulation, 1417–1420 glucagon, 1420–1421 glucagon-like peptide-1, 1421 histamine, 1416 inhibitors, 1420–1423 neurotensin, 1415 pancreastatin, 1422 pancreatic polypeptide, 1422 peptide YY, 1422 proteinase-activated receptor-2, 1416–1417 secretin, 1410–1412, 1420 somatostatin, 1421–1422 xenin, 1415–1416 intercellular junctions adhering zonules, 1318–1319 desmosomes, 1318–1319 gap junctions, 1319 tight junctions, 1317–1318 neural regulation of secretion adrenergic nerves, 1408–1409

Pancreas (Continued) dopaminergic nerves, 1409 gastrin-releasing peptide, 1407–1408 inhibitory neuropeptides, 1408 serotonergic nerves, 1409–1410 vagal innervation, 1404–1407 vasoactive intestinal peptide, 1407 peptide YY effects, 143 secretion patterns absorbed nutrient phase, 1403–1404 basal secretion, 1397–1398 cephalic phase, 1400 gastric phase, 1400–1401 integrated response to meals, 1398–1400 intestinal phase bile acid regulation, 1403 fat contributions, 1402 gastric acid role, 1401–1402 ions, 1403 overview, 1401 protein, peptide, and amino acid contributions, 1402 sodium–proton exchange, 1868–1869 Wnt/β-catenin signaling defects in cancer, 264 Pancreastatin enterochromaffin-like cell secretion, 1230 pancreas secretion regulation, 1422 Pancreatic polypeptide dorsal vagal complex chemosensitivity, 865 food intake regulation, 885 functions, 172 pancreas function regulation, 1424 pancreas secretion regulation, 1422 presynaptic inhibition mediation in enteric nervous system, 657 Pantothenic acid, functions and absorption, 1812–1813 PAR-2, see Proteinase-activated receptor-2 Paraneoplastic syndrome, disinhibitory motor disorders, 641 Parathyroid hormone, calcium absorption regulation, 1965 Parietal cell, see also Gastric acid membrane recycling hypothesis, 1193–1194 morphology and ultrastructure, 1191–1193 sodium/potassium-ATPase crystal structure, 1199 functional transport, 1194–1195 identification as proton pump, 1194 inhibitors, 1199–1200, 1202 kinetics, 1196 membrane changes with acid secretion, 1195–1196 subunit structure, 1196–1199 transgenic mouse studies, 1307 Patterning, development, 271–272 Pax4, digestive function ontogeny, 378–379 Pax6 digestive function ontogeny, 378–379 glucagon gene expression regulation, 163 PCR, see Polymerase chain reaction Pdx1, digestive function ontogeny, 379 Pelvic floor innervation afferent pathways, 998–999 external anal sphincter muscle innervation zones, 995–996 somatic motor pathways, 996–998 muscle activity overview, 999–1000 reflex activity, 1001–1002 tonic activity, 1000–1001 voluntary activity, 1002–1003 musculature, 678–679 neural control anorectal continence, 1004–1005 micturition, 1003 sexual response, 1005 urinary continence, 1004 neuromuscular injury in vaginal delivery, 1005–1006 rectoanal reflex, 680 sensory innervation, 679 spinal motor control, 680–681 supraspinal motor control, 681 PEPT1 mutations, 1683 peptide transport, 1676–1677

Peptide histidine isoleucine, functions, 130 Peptide histidine methionine, functions, 130 Peptide transport, see Protein digestion and absorption Peptide YY aging effects in intestine, 412 cell growth regulation, 452 dorsal vagal complex chemosensitivity, 865–866 enterochromaffin-like cell regulation, 1228 food intake regulation, 885–886 functions, 140, 143–145 pancreas secretion regulation, 1422 pathophysiology, 147–148 presynaptic inhibition mediation in enteric nervous system, 657 receptors, 142–143 release, 145–146 structure, 140 tissue distribution, 140–142 trophic actions, 146–147 PET, see Positron emission tomography Pharyngeal motor function airway protection during belching, 909–910 cerebral cortical representation of pharyngeal/reflexive and volitional swallow, 906–907 esophagoglottal closure reflex, 904–905 inhibitory reflexes, 908–900 laryngeal motor function nasopharyngeal motor function during swallowing and belching, 901–902 overview of deglutitive function, 900–901 musculature, 897 pharyngoglottal adduction reflex, 908 pressure phenomenon in relation to swallowed material, 898–900 pressure profile, 896–898 secondary swallow, 905–906 swallowing phases, 895–896 upper esophageal sphincter laryngo-upper esophageal sphincter contractile reflex, 908 opening, 903–904 pharyngolaryngeal and esophageal reflexes, 904 pharyngo-upper esophageal sphincter contractile reflex, 907–908 pressure phenomena, 902–903 PHI, see Peptide histidine isoleucine PHM, see Peptide histidine methionine Phosphate dietary requirements, 1953–1954 intestinal absorption mechanisms, 1967 regulation, 1971 sites, 1967 SLC34A2, 1970 transporters, 1968–1970 ontogenic development of absorption, 386–388 Phosphatidylinositol 3-kinase cell growth signaling, 443–444 epidermal growth factor receptor signaling, 203 mTOR interactions, 444–445 target genes, 444 Phosphoinositides acinar cell secretory function, 1339–1341, 1345–1347 protein sorting role in polarized epithelial cells, 1612 Phospholipase A2, innate immunity, 1054 Phospholipase C enterochromaffin cell signaling, 743–744 epidermal growth factor receptor signaling, 203 Phox2b, Hirschsprung’s disease mutations, 505 Pituitary adenylate cyclase-activating peptide enterochromaffin-like cell regulation, 1228 functions, 130 receptors gastric expression, 1237 ligand binding, 1237 signaling, 1237 structure, 1237 PKC, see Protein kinase C Platelet-activating factor, vasoconstriction induction in small intestine, 1634 Platelet glycoproteins, types and functions, 1142–1143 Polarized epithelial cell cytoarchitecture cell junctions, 1596–1597

I-14 / INDEX Polarized epithelial cell (Continued) cytoskeleton, 1597 membrane compartments, 1597 overview, 1595–1596, 1599–1600 protein sorting actin-mediated transport, 1614 caveolae, 1611–1612 intermediate filaments, 1614–1615 lipid rafts, 1610–1611 microtubule-mediated transport, 1612–1614 phosphoinositides, 1612 pre-trans Golgi network, 1597 prospects for study, 1618–1619 proteolipids, 1610–1611 Rab GTPase regulation, 1615–1616 selective retention, 1599 SNAREs in vesicle fusion, 1616–1617 sorting signals, 1600–1601, 1604–1605 spatial regulation of exocytosis exocyst complex, 1618 formins, 1617–1618 septins, 1618 techniques for study, 1600, 1602–1604 trans Golgi network, 1597–1599 vesicle budding AP protein adaptors, 1607–1609 clathrin-mediated budding, 1607 coat protein II-mediated budding, 1606–1607 coat-based budding model, 1606 overview, 1605 scission, 1609–1610 Polyamines apoptosis inhibition, 356–357 depletion and apoptosis induction, 359–360 Polymerase chain reaction, principles, 3–4 Positron emission tomography, gastrointestinal sensory signal processing studies, 730–734 Postprandial hyperemia chyme constituents, 1641 discovery, 1640–1641 mechanisms cholecystokinin, 1642–1643 metabolic vascular regulation, 1642 overview, 1646–1647 prostaglandins, 1643–1644 small mesenteric artery dilation, 1644, 1646 sodium-induced hyperosmolarity, 1644 sites, 1641 Potassium, ontogenic regulation of absorption, 386 Potassium channels apical membrane channels, 1921–1922 basolateral membrane channels, 1920–1921 calcium-activated potassium channels, 695 cholangiocytes, 1510 cholangiocytes, 1510–1511 extrinsic sensory afferent nerve voltage-gated potassium channels, 694–695 gallbladder, 1539–1540 gastric acid secretion role, 1202–1203 pancreas electrolyte secretion, 1380, 1383 smooth muscle responses to slow waves and neural inputs A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 inward rectifier potassium channels, 559–560 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 transgenic mouse mutants, 1305 PP, see Pancreatic polypeptide Prevertebral sympathetic ganglia chemical coding nerve fibers, 611 neurons, 610 neurotransmitters and neuromodulators, 605 distribution, 603 electrophysiology of neurons, 620–622 functional overview, 603–604 gut targets of neurons, 614 imaging, 603 innervation, 604, 607–610 morphology, 605–607 pacemaker neurons, 622–623

Prevertebral sympathetic ganglia (Continued) prospects for study, 623 symnpathetic motor innervation, organization in gut cat, 613 dog, 613 guinea pig, 612 overview, 611–612 pig, 613 rat, 612–613 visceral afferent neurons, 614–616 viscerofugal neuron chemical coding dog, 620 guinea pig, 619 pig, 620 rat, 619–620 mechanosensory neurons, 620 morphology, 616–617, 619 Prohormone convertases glucagon processing, 162 preprogastrin processing, 50 Promoter DNA elements, 9–12 identification, 3–4 RNA polymerase initiation complex, 10–11 structure, 9–10 Proopiomelanocortin, peptides in feeding control, 878–879 Prostaglandins apoptosis inhibition, 356 bicarbonate secretion regulation in duodenum, 1271–1272 duodenal protection, 1278–1279 enterochromaffin-like cell regulation, 1229 gastric protection, 1274–1276 postprandial hyperemia role, 1643–1644 prostaglandin E and chloride secretion mediation, 1172 Protein digestion and absorption, see also Amino acids absorbed amino acid and peptide fates, 1677 enterocyte active transport energetics, 1670–1671 peptidases, 1669 genetic disorders of transport, 1681–1683 overview, 1667–1668 peptide transport basolateral membrane transport, 1680 chain length effects, 1676 developmental regulation, 1684–1685 dietary regulation, 685 diurnal rhythm, 1686 energetics, 1675 hormonal regulation, 1685–1686 independence from amino acid transport, 1675 nutritional, clinical, and pharmacologic relevance, 1683–1684 PEPT1, 1676–1677, 1683 sodium–proton exchanger regulation, 1686 prospects for study, 1686–1687 proteases, 1668–1669 sites of absorption, 1669–1670 Protein kinase A acinar cell secretory function, 1354–1355 gastric acid secretion signaling, 1206–1207 Protein kinase B, see Akt Protein kinase C acinar cell secretory function, 1355 chloride–bicarbonate exchange regulation, 1886–1887 G protein-coupled receptor signaling, 79, 81 Protein phosphatases, acinar cell secretory function, 1356–1357 Protein trafficking, see Acinar cell; Cholangiocyte; Endoplasmic reticulum; Gastric acid; Golgi; G protein-coupled receptors; Immunoglobulins; Polarized epithelial cell Proteinase-activated receptor-2 pancreas electrolyte regulation, 1388 pancreas secretion regulation, 1416–1417 Proton-ATPase cholangiocytes, 1511 pancreas electrolyte secretion, 1379 Psychological stress, see Stress Purinergic receptors cholangiocytes, 1515 enterochromaffin cell excitatory adenosine receptors, 747–748

Purinergic receptors (Continued) extrinsic sensory afferent nerves, 702–703 inhibitory postsynaptic potentials in enteric nervous system, 646–647 secretion disorders, 76 PYY, see Peptide YY R Rab GTPases acinar cell exocytosis role, 1359–1360 exocytosis regulation in polarized epithelial cells, 1615–1616 G protein-coupled receptor signaling, 72–73 vesicular trafficking in gastric acid secretion, 1211–1213 Radiation enteritis, clinical manifestations, 1122 Ras, G protein-coupled receptor signaling, 72 Reactive oxygen species chloride–bicarbonate exchange regulation, 1887 endothelial cell activation products, 1145–1146 liver damage antioxidant protection catalase, 1494 glutathione, 1494–1495 glutathione peroxidase, 1494 superoxide dismutase, 1494 thioredoxin, 1495 vitamin C, 1495 vitamin E, 1495 hepatocyte necrosis versus apoptosis, 1493–1494 pathology biochemical pathway stimulation, 1496 cancer, 1496–1497 chronic liver disease, 1496 drug toxicity, 1495–1496 reactive oxygen species sources, 1492–1493 redox cycling, 1493 toxicity, 1492 Receptor tyrosine kinase acinar cell secretory function, 1356 dimerization, 74 G protein signaling, 76–77 recruitment, 74–76 transactivation, 76 Reduced folate carrier, folate transport, 1795–1797 Regenerating protein 1α, enterochromaffin-like cell secretion, 1231 RET, Hirschsprung’s disease mutations, 503–505 Retinoids, see Carotenoids; Vitamin A Rho, G protein-coupled receptor signaling, 72 Rhythmic phase contractions excitation–contraction coupling long-duration contractions, 974 short-duration contractions, 971–974 excitation–inhibition coupling, 976 features, 966–967 inflammatory bowel disease dysfunction, 986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction, 983 Riboflavin functions, 1811 intestinal transport mechanism, 1811 regulation, 1811–1812 RNA polymerase initiation complex, 10–11 types and functions, 2 RNA silencing, mechanism, 34–35 ROS, see Reactive oxygen species Rotavirus barrier function effects overview, 1166 nonstructural protein 4, 1171, 1181 secretory function effects, 1164 RPCs, see Rhythmic phase contractions RTK, see Receptor tyrosine kinase RTX, barrier function effects, 1173 Runx3, transforming growth factor-β activation, 447 S S100A10, calcium absorption regulation, 1966 Salivary gland sodium–proton exchange, 1866–1867 water transport, 1835–1836 Salmonella enterica, barrier function effects, 1166, 1181 Satiety, see Appetite

INDEX / I-15 Scavenger receptor type B class I, gallbladder cholesterol metabolism, 1546–1547 SCFAs, see Short-chain fatty acids Secreted frizzled-related proteins, Wnt/β-catenin signaling inhibition, 252 Secretin bile formation regulation, 1516, 1518–1521 discovery, 121–122 functions, 123–125 gastrointestinal localization, 122 pancreas electrolyte regulation, 1390 pancreas secretion regulation, 1410–1412, 1420 pathobiology, 126 receptor, 122 release, 125–126 structure, 122–123 Secretomotor neuron excitatory receptors, 638 inhibitory input, 638 intestinal content liquidity control, 638 intestinal inflammation effects, 1124 irritable bowel syndrome excitatory input, 1014–1015 inhibitory input, 1013–1014 neurogenic secretory diarrhea and neurogenic constipation, 1014 projections, 1013 overview, 589–590, 637–638 Secretory function, ontogeny, 376–377 Secretory reflexes ATP role in sensory signaling, 748 coordination of secretion and motility, 755–756 cyclic AMP signaling and secretion, 756–757 distension, 751–752 humans, 752–754 modulation of endogenous secretory tone, 738 mucosal stroking reflexes in rodent intestine, 749–751 neuroanatomy AH neurons, 739 extrinsic primary afferent neurons, 741 interneurons, 740 overview, 738–739 secretomotor neurons, 740–741 pathobiology cholera toxin-induced secretion, 758 Clostridium difficile enteritis, 759–760 purines, 760 sensory enterochromaffin cells BON cell model, 741–742 chemosensitive stimulation of serotonin release, 741 excitatory adenosine receptors, 747–748 mechanosensitivity, 741, 743 signal transduction adenosine as physiologic brake, 745–747 cyclic AMP, 744–745 phospholipase C, 743–744 synaptic transmission mediators, 754–755 Secretory vesicle formation, 42 processing reactions acetylation, 42 amidation, 47–48 aminopeptidases, 46–47 carboxypeptidases, 45–46 dibasic cleavage, 42–44 glutaminyl cyclase, 47 monobasic cleavage, 44–45 Selectins, types and functions, 1138–1140 Selenium, absorption and transporters, 1998 Sensory signals, brain processing functional imaging studies, 730–734 integrated view, 734–735 neuroanatomy cingulate cortex, 730, 734 insula, 730, 733–734 overview, 728, 730 somatosensory cortex, 729–730, 733–734 thalamus, 728–729, 732–733 Septins, exocytosis regulation in polarized epithelial cells, 1618 Serine protease autotransporters of Enterobacteriaceae, barrier function effects, 1175–1176 Serotonin chemosensitive stimulation of release in enterochromaffin cells, 741 chloride–bicarbonate exchange regulation, 1886

Serotonin (Continued) gastric accommodation reflex control, 930 irritable bowel syndrome role, 1025–1026 mast cell signaling in irritable bowel syndrome, 1025–1026 pancreas electrolyte regulation, 1390 pancreas secretion regulation, 1409–1410 Serotonin receptors extrinsic sensory afferent nerves, 702 fast excitatory postsynaptic potentials in enteric nervous system, 647 inhibitory postsynaptic potentials in enteric nervous system, 647 irritable bowel syndrome sensory afferents, 1016–1017 mutation and enteric nervous system lesions, 510–511 presynaptic inhibition mediation in enteric nervous system, 657 Sexual response, pelvic floor neural control, 1005 SFRPs, see Secreted frizzled-related proteins SGLUTs, see Sodium/glucose cotransporters Shigella flexneri, barrier function effects overview, 1166 serine protease autotransporters of Enterobacteriaceae, 1176 tight junction effects, 1180–1181 Short-chain fatty acids anion exchange bicarbonate exchange apical exchange, 1902 basolateral exchange, 1903 diffusion comparison, 1901–1902 chloride exchange, 1902–1903 chloride–bicarbonate exchange regulation, 1884–1885 intestinal absorption regulation, 1906–1907 nonionic diffusion, 1903–1904 transporters monocarboxylate transporter 1, 1904, 1906 SL16 gene family, 1904–1905 Signal peptide removal, 38 structure and function, 36 Signal recognition particle function, 36–37 receptor, 37 Sitosterolemia, ABCG5/G8 role, 1726 Smad, transforming growth factor-β signaling, 188–189 Small intestine absorption, see specific nutrients barrier function, see also Tight junctions apical junction complex, 1561–1562 electrical resistance, 1563–1564 elements, 1560 permselectivity charge discrimination, 1564–1565 size discrimination, 1565 transport pathways, 1560 corticotropin-releasing hormone modulation of function anti-inflammatory action, 804 bicarbonate secretion stimulation, 802–803 jejunoileal absorption, 803 motility, 803–804 integrated control by enteric nervous system musculature, 670 peristaltic propulsion, 670–673 peristaltic retropropulsion, 673 physiologic ileus, 673 power propulsion, 674 microvascular anatomy artery–arteriolar transition, 1627–1628 mucosal and villous microcirculation, 1629 muscularis, 1628–1629 physiologic considerations, 1629–1630 motility, see Intestinal motility sodium–proton exchange, 1870–1871 vasoconstriction induction angiotensin, 1631–1632 endothelin, 1632–1633 myogenic response, 1631 platelet-activating factor, 1634 somatostatin, 1633–1634 vasodilators adenosine, 1639 glucagon, 1640 histamine, 1640

Small intestine (Continued) nitric oxide, 1634–1636 oxygen, 1636–1639 water transport duodenum, 1839 ileum, 1839–1840 jejunum, 1839–1840 Smooth muscle contraction signaling pathways G protein-coupled receptors desensitizaton, 526–527 initial phase of contraction, 524–525 sustained phase of contraction, 525–526 ligands and receptors, 524–525 electrical activity, 534–535 pacemaker cells, see Interstitial cells of Cajal relaxation signaling pathways cyclic nucleotide regulation, 528–529 G protein-coupled receptors PACAP receptor, 528 vasoactive intestinal peptide receptors, 527–528 initial phase of contraction, 529 ligands and receptors, 527–528 sustained phase of contraction, 529–530 responses to slow waves and neural inputs chloride conductances calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 integration of electrical activity, 569 ion channels, 552–554 nonselective cation channels currents activated by ATP, 568 currents activated by muscarinic and peptide agonists, 567–568 currents activated by stretch, 568–569 overview, 550–551 voltage-dependent inward currents inward rectifier currents, 556 L-type calcium channels, 551, 555–556 sodium channels, 556 T-type calcium channels, 556 voltage-dependent outward currents A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 ether-a-go-go-related currents, 560–561 inward rectifier potassium channels, 559–560 overview, 556–557 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 signal transduction calcium flux, 534–535 cross-talk, 530–531 overview, 523–524 SNAREs acinar cell exocytosis role, 1358–1361 vesicle fusion role in polarized epithelial cells, 1616–1617 vesicular trafficking in gastric acid secretion, 1213–1214 Sodium electrogenic absorption, 1935–1936 electroneutral absorption, 1882–1883, 1936–1938 Sodium–bicarbonate cotransporter cholangiocytes, 1511 pancreas electrolyte secretion, 1379–1380, 1383–1384 Sodium channels experimental models, 1922 extrinsic sensory afferent nerve voltage-gated sodium channels, 693–694 intestinal function, 1920 smooth muscle responses to slow waves and neural inputs, 556 Sodium chloride electroneutral sodium chloride absorption, 1882–1883, 1936–1938 ontogenic development of absorption, 384–386 Sodium/glucose cotransporters gene family, 1654 glucose, galactose, and fructose absorption, 1654–1656

I-16 / INDEX Sodium/glucose cotransporters (Continued) SGLT1 cation selectivity, 1657–1658 function, 1656 kinetics modeling, 1659–1660 presteady-state kinetics, 1660–1661 reversibility and asymmetry, 1658–1659 stoichiometry, 1658 mutation in disease, 1662 ontogenic regulation of intestinal monosaccharide absorption, 381–383 regulation of expression, 1663 sugar selectivity, 1656–1657 water transport studies, 1830–1831 Sodium–phosphate cotransporter, ontogenic development of phosphate absorption, 387–388 Sodium/potassium-ATPase cholangiocytes, 1511 crystal structure, 1199 functional transport, 1194–1195 gallbladder, 1539 glucocorticoid induction, 383 identification as proton pump in gastric acid secretion, 1194 inhibitors, 1199–1200, 1202 kinetics, 1196 membrane changes with acid secretion, 1195–1196 ontogenic development of potassium absorption, 386 pancreas electrolyte secretion, 1380–1381 subunit structure, 1196–1199 transgenic mouse mutants, 1305 Sodium–potassium–2chloride cotransporter basolateral membrane transport, 1205 cholangiocytes, 1511 pancreas electrolyte secretion, 1380 Sodium–proton exchangers amino acid and peptide transport regulation, 1686 ATP dependence, 1853 basolateral membrane transport, 1204–1205 chloride dependence, 1853–1854 cholangiocytes, 1510–1511 colon physiology, 1871–1873 domains, 1852–1853 gallbladder, 1537–1538 history of study, 1847 inhibitors, 1854 knockout mice, 1306 liver physiology, 1867–1868 mammalian genes, 1848–1849 membrane homology, 1851–1852 NHE1 functions, 1855 pathophysiology, 1856 posttranscriptional regulation, 1856 structure, 1855 tissue distribution and cellular localization, 1855 transcriptional regulation, 1855 NHE2 functions, 1857 pathophysiology, 1858 posttranscriptional regulation, 1858 structure, 1856 tissue distribution and cellular localization, 1856–1857 transcriptional regulation, 1857–1858 NHE3 functions, 1859 pathophysiology, 1862 posttranscriptional regulation, 1860–1862 structure, 1858 tissue distribution and cellular localization, 1859 transcriptional regulation, 1859–1860 NHE4 functions, 1863 structure, 1863 tissue distribution and cellular localization, 1863 NHE5 features, 1863 NHE6 functions, 1864 structure, 1863 tissue distribution and cellular localization, 1863–1864 NHE7 features, 1864 NHE8 functions, 1865

Sodium–proton exchangers (Continued) structure, 1864–1865 tissue distribution and cellular localization, 1865 NHE9 functions, 1865–1866 pathophysiology, 1866 structure, 1865 tissue distribution and cellular localization, 1865 ontogenic development of sodium chloride absorption, 384–386 pancreas electrolyte secretion, 1379, 1383 pancreas physiology, 1868–1869 salivary gland physiology, 1866–1867 sequence homology between human types, 1850 small intestine physiology, 1870–1871 species differences, 1848 stomach physiology, 1869–1870 substrate specificity, 1853 Somatosensory cortex, gastrointestinal sensory signal processing, 729–730, 733–734 Somatostatin assays, 107 bile formation regulation, 1520 cell growth regulation, 453 cholecystokinin effects on D cells, 105–106 excess and deficiency in disease, 108–109 functions, 106, 108, 126 gastric acid secretion regulation, 1245–1246 gene structure and expression, 106–107 intestinal hormone, 126 metabolism, 108 pancreas secretion regulation, 1421–1422 posttranslational processing, 107 receptors, 108, 126 release, 107–108, 126 structure, 106 tissue distribution, 107, 126 vasoconstriction induction in small intestine, 1633–1634 Somatostatin receptors gastric expression, 1238 knockout mouse, 1303–1304 ligand binding, 1237–1238 signaling, 1238 structure, 1238 Sonic Hedgehog, see Hedgehog Sox10, Hirschsprung’s disease mutations, 504 SOX9, stem cell function regulation, 320–321 SPATEs, see Serine protease autotransporters of Enterobacteriaceae Sphincter of Oddi, neural control ganglia and neurons, 845–846 interprandial motility, 846–847 resistance decrease mechanisms, 846 Sphincteric achalasia, disinhibitory motor disorders, 641–642 Spinal cord ascending sensory pathways, 1017–1018 descending modulatory pathways, 1020–1021 sensory processing and central sensitizaton, 1019–1020 SRP, see Signal recognition particle Stanniocalcin, calcium absorption regulation, 1964 STATs epidermal growth factor receptor signaling, 203 intestinal inflammation role, 1117–1118 Notch signaling crosstalk, 294–295 STAT3 and polyamine depletion-induced apoptosis, 362 Stem cells bone morphogenetic protein signaling in regulation, 326–327 crest-derived stem cells in bowel, 502–503 definition, 308 discovery, 307 fibroblast growth factor regulation, 327–330 gastrointestinal structural/proliferative units clonality studies chimeric organisms, 310–312 stem cell mutagenesis assays, 312–313 X-chromosome inactivation, 312 colon, 308 gastric epithelium, 308–309 morphogenesis, 309–310 small intestine, 308 glucagon-like peptide-2 regulation, 331

Stem cells (Continued) Hedgehog signaling in regulation, 324–326 hierarchy, 314 inflammation mediator effects, 332–335 insulin-like growth factor regulation, 330 markers, 315 niche, 315–316 Notch signaling in regulation, 322–324 number assay, 313 regulation, 314–315 plasticity, 335–336 prospects for study, 336 therapeutic prospects, 307–308 transforming growth factor-β regulation, 331–332 Wnt/β-catenin/Tcf-4 signaling in regulation overview, 316–317 signal-modifying genes Dkk1, 321–322 Fox11, 322 target genes CD44, 321 Cdx genes, 319–320 Ephrin system, 317–318 c-Myc, 318–319 SOX9, 320–321 Stomach acid secretion, see Gastric acid aging studies mucosal injury and repair, 408–410 prospects for study, 411 secretion and digestion, 410–411 structural and functional properties, 407–408 brainstem control, see Brainstem emptying, see Gastric emptying epithelial organization, 1189–1191, 1298 functional anatomy innervation, 1225 structure, 1223 vasculature, 1225 Hedgehog signaling in development, 277 integrated control by enteric nervous system antral pump cycles, 667 neural control, 667–668 brainstem/enteric nervous system cooperative integration, 666–667 gastric emptying, 669 gastric reservoir neural control, 668–669 vasovagal reflexes, 667 mucosa, see Gastric mucosa sodium–proton exchange, 1869–1870 water transport, 1836–1837 Stress gastrointestinal motility effects animal studies, 783 autonomic dysfunction, 784–785 corticotropin-releasing hormone modulation overview, 781–782 peripheral receptor and colonic motor alterations, 785 type 1 receptor activation and stimulation, 784 type 2 receptor activation and inhibition, 784 visceral hyperalgesia mediation by receptors, 786 healthy subjects, 782 irritable bowel syndrome patients, 782–783 prospects for study, 786–787 sex differences in visceral hypersensitivity, 786 visceral perception studies, 785–786 history of study, 791–792 irritable bowel syndrome and psychological stress immunoneural communication, 1022–1023 immunoneural signal sources, 1022–1023 mast cells brain–gut connection, 1024–1027 histamine signaling, 1025 immunoneural communication, 1023–1024 serotonin signaling, 1025–1026 neuroimmunophysiologic paradigm, 1022 mucosal function effects animal studies acute stress mediators, 770–772 barrier function, 769–770 chronic stress studies, 772–773 early life stress, 774–775

INDEX / I-17 Stress (Continued) flora changes, 773 ion secretion, 769 models, 768–769 mucus secretion, 769 food allergy pathogenesis, 775–776 inflammatory bowel disease pathogenesis, 775 irritable bowel syndrome pathogenesis, 776 physical stress, 767–768 prospects for study, 776 psychological stress, 768 neuroendocrinology, 766–767, 791–792 S-type neurons action potentials, 646 general features, 646 metabotropic signal transduction, 652–654 slow excitatory postsynaptic potentials, 649 Substance P pancreas electrolyte regulation, 1389–1390 primary afferent neuron neurotransmission in blood vessels, 828–829 Sugar absorption, see Glucose transporters; Sodium/glucose cotransporters Sulfate, intestinal absorption, 1881–1882 Sulfotransferase, phase II metabolism, 1489 Superoxide dismutase, antioxidant protection, 1494 Swallowing, see also Laryngeal motor function; Pharyngeal motor function phases, 895–896 Sympathetic nervous system, see Enteric nervous system; Prevertebral sympathetic ganglia System A, amino acid transport, 1677–1678 System ASC, amino acid transport, 1674–1675 System Asc, amino acid transport, 1680 System β, amino acid transport, 1674 System B0, amino acid transport, 1671–1673 System b0,+, amino acid transport, 167 System B0,+, amino acid transport, 1673 System IMINO, amino acid transport, 1673–1674 System L, amino acid transport, 1679–1680 System PAT, amino acid transport, 1675 − , amino acid transport, 1674 System XAG System y+, amino acid transport, 1679 System y+L, amino acid transport, 1680 T T cell, Notch in mucosal immunity development, 301 Tcf-4, see Wnt/β-catenin signaling TCs, see Tonic contractions TFF, see Trefoil factor family TGF-α, see Transforming growth factor-α TGF-β, see Transforming growth factor-β Thalamus, gastrointestinal sensory signal processing, 728–729, 732–733 Thiamin functions, 1797–1798 intestinal absorption mechanism, 1798 molecular components, 1798–1800 regulation, 1800–1801 thiamin transporter protein molecular biology, 1801–1802 Thiopurine methyltransferase, phase II metabolism, 1490 Thioredoxin, antioxidant protection, 1495 Thyroid, Hedgehog signaling in development, 276–277 Thyroid hormone, calcium absorption regulation, 1964 Tight junctions apical junction complex, 1561–1562 electrical resistance, 1563–1564 intestinal inflammation effects, 1125 intestinal pathology celiac disease and permeability index, 1583–1584 clinical assessment, 1583 Crohn’s disease, 1584–1585 nonsteroidal anti-inflammatory drugs, 1586 pancreas, 1317–1318 pathogen receptors, 1181–1182 permeability regulation in gallbladder, 1542 permselectivity charge discrimination, 1564–1565 size discrimination, 1565 protein components claudins, 1567–1568 PDZ domain scaffolding proteins, 1566 signaling proteins, 1566–1567 transmembrane proteins, 1565–1566

Tight junctions (Continued) regulation in intestinal epithelium cytochalasins, 1569–1570 cytokines, 1576–1579 infection, 1579–1582 luminal osmolarity and solvent drag effect, 1571–1573 overview, 1568–1569 sodium–nutrient cotransport, 1573–1576 Shigella effects on barrier function, 1180–1181 TLRs, see Toll-like receptors Tocopherols, see Vitamin E Toll-like receptors intestinal inflammation role, 1116 structure, 1035 TLR1, 1039–1040 TLR2, 1039–1040 TLR3, 1043–1044 TLR4, 1035–1039 TLR5, 1042–1043 TLR6, 1039–1040 TLR7, 1044 TLR8, 1044 TLR9, 1040–1042 TLR10, 1044 Tonic contractions excitation–contraction coupling, 976 excitation–inhibition coupling, 976 features, 969 inflammatory bowel disease dysfunction, 986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction, 984 Trace elements, see also specific elements absorption chromium, 1998 copper, 1995–1996 general properties, 1993 genetic variation, 1998 manganese, 1998 mechanisms, 1994 selenium, 1998 zinc, 1996–1998 diet composition, 1993–1994 ontogenic development of absorption, 388–389 Trachea, Hedgehog signaling in development, 275–276 Transcription analysis animal models, 17 cell culture models, 16–17 chromatin immunoprecipitation, 18 constituted transcription systems, 16 DNA microarray, 19 knockouts, 17 proteomics, 19 transgenic animals, 4–5, 17 coregulatory proteins, 15–16 DNA-binding proteins, 12–15 gastrointestinal peptides, 19 mRNA, see Messenger RNA overview, 2–3 promoter, see Promoter RNA polymerase initiation complex, 10–11 transcription factor types and functions, 10–11 Transforming growth factor-α discovery, 195 enterochromaffin-like cell regulation, 1228 secretion, 1231 expression, 195–197 gastric acid secretion signaling, 1208–1209 mucosal growth regulation in aging, 421–423 nonmitogenic activity, 197–198 processing, 195 Transforming growth factor-β activation, 186–187 cancer role, 191–192 discovery, 186 expression, 191 functions cell growth regulation, 190, 446 cell migration and angiogenesis, 191 extracellular matrix formation induction, 190 immune function, 190–191 genes, 186 isoforms, 186 receptors

Transforming growth factor-β (Continued) interacting proteins, 187–188 signaling, 187 types, 187 Runx3 activation, 447 Smad signaling, 188–189, 446 stem cell regulation, 331–332 transgenic and knockout mice studies, 192–193 wound healing regulation, 463–464 Transgenic mouse gastric mucosa transgene expression enterochromaffin-like cells, 1307–1308 gastrin promoter and G cell transgenics, 1308 parietal cells, 1307 promoters, 1306–1308 gastrin overexpression, 1301 generation, 1294–1295 gene regulation studies, 4–5, 17 potassium channel mutants, 1305 sodium/potassium ATPase mutants, 1305 transforming growth factor-β studies, 192–193 Transient receptor potential vanilloids intestinal calcium entry mediation, 1761–1762, 1959–1960 knockout mouse studies, 1762–1763, 1967 TRPV6 biophysical properties, 1960–1961 calcium regulation, 1961 pharmacology, 1961–1962 S100A10 regulation, 1966 structure, 1962 vitamin D regulation, 1961 vitamin D regulation of expression, 1763–1764, 1961 Translation elongation, 34 endoplasmic reticulum transport, see Endoplasmic reticulum transport initiation and regulation, 32, 34 localized regulation, 34 posttranslational processing, 35, 38–39, 41–42 RNA silencing, 34–35 termination, 34 Trefoil factor family functions brain, 213 cell growth regulation, 448 development, 212 inflammation, 212–213 mucosal defense, 211 tissue repair, 211–212 tumor suppression, 213 gastroduodenal mucosal defense, 1260–1261 genes, 210 knockout mice, 1306 receptors and binding proteins, 211 signaling, 211 structure, 209–210 types, 210–211 wound healing regulation, 464 TRPVs, see Transient receptor potential vanilloids Trypsin, protein digestion, 1669 Tumor necrosis factor-α intestinal epithelial tight junction barrier regulation, 1576–1579 polymorphisms in Helicobacter pylori disease, 1105 vago-vagal control reflex regulation, 866–869 wound healing regulation, 464 U UDP glucuronosyltransferase, phase II metabolism, 1488–1489 UES, see Upper esophageal sphincter Ulcerative colitis, see Inflammatory bowel disease Unfolded protein response, acinar cell proteins, 1323 Upper esophageal sphincter anatomy, 914 coordinated motor activity, 916–917 laryngo-upper esophageal sphincter contractile reflex, 908 opening, 903–904 pharyngolaryngeal and esophageal reflexes, 904 pharyngo-upper esophageal sphincter contractile reflex, 907–908 pressure phenomena, 902–903 Urination, see Micturition

I-18 / INDEX Urocortin central distribution, 796 colonic motility effects, 804–807 Ussing chamber, ion channel studies, 1923 V VacA Helicobacter pylori virulence factor, 1101–1102 secretory function effects, 1170–1171 Vagus nerve afferents central projections, 690–691 food intake regulation, 706–710 overview, 592–593 pathophysiology, 710–716 peripheral projections, 687–688 anti-inflammatory pathway, 710–711 enteric nervous system–central nervous system communication, 500 pancreas innervation, 1404–1407 systemic inflammation and behavior, 710 vago-vagal gastric control reflexes, see Brainstem Vasoactive intestinal polypeptide bile formation regulation, 1519 functions, 126, 129 pancreas secretion regulation, 1407 receptors, 128–129 release, 129–130 structure, 127–128 tissue distribution, 127–128 VCC, secretory function effects, 1169 Ventromedial nucleus, gastrointestinal sensory signal processing, 729 Ventroposterolateral nucleus, gastrointestinal sensory signal processing, 728 Vibrio cholerae, see also Cholera toxin barrier function effects hemagglutinin/protease, 1173–1174 overview, 1166 RTX, 1173 Zot, 1174 secretory function effects Ace effects, 1168–1169 cholera toxin mechanisms, 1165, 1167–1168 heat-stable enterotoxin effects, 1169 overview, 1164 VCC effects, 1169 Vibrio parahemolyticus, secretory function effects overview, 1164 thermostable direct hemolysin, 1169–1170 thermostable direct hemolysin-related hemolysin, 1169–1170 VIP, see Vasoactive intestinal polypeptide Visceral hypersensitivity corticotropin-releasing hormone signaling in irritable bowel syndrome, 808–809 extrinsic sensory afferent nerves, 713–716 hyperalgesia mediation by corticotropin-releasing hormone receptors, 786 irritable bowel syndrome, 635–636 irritable bowel syndrome, see Irritable bowel syndrome Viscerofugal neuron chemical coding dog, 620 guinea pig, 619 pig, 620 rat, 619–620 function, 588–589 morphology, 616–617, 619 Vitamin A dietary sources, 1737 intestinal absorption cellular retinol binding proteins, 1745–1746 portal secretion, 1744–1745 proteins, 1748 reesterification and lymphatic secretion, 1746–1747 retinoid conversion from carotenoids cleavage enzymes, 1739–1740 nutritional factor regulation, 1740 retinyl ester digestion intestinal enzymes, 1741 pancreatic enzymes, 1740–1741 solubilization, 1738–1739 storage, 1736–1737 synthesis, 1736 Vitamin B1, see Thiamin

Vitamin B2, see Riboflavin Vitamin B3, see Niacin Vitamin B6 forms, 1809–1810 functions, 1810 intestinal transport mechanism, 1810–1811 regulation, 1810 Vitamin B12, see Cobalamin Vitamin C antioxidant protection, 1495 forms, 1807 functions, 1807 intestinal transport cell biology, 1808–1809 mechanism, 1807 molecular components, 1807–1808 regulation, 1808 Vitamin D calcium absorption regulation, 1963–1964 calcium and phosphorous homeostasis regulation, 1755–1756 calcium transporter expression regulation, 1763–1764 colon cancer prevention, 1765–1766 discovery, 1754 functions, 1753–1754, 1766 receptor cofactor recruitment, 1759–1760 discovery and cloning, 1756 experimental proof of function, 1760–1761 retinoid X receptor heterodimerization, 1757–1760 steroid receptor gene family, 1756–1757 target gene interactions, 1758 transactivation, 1760 transcriptional modulation, 1758–1759 synthesis, activation, and degradation, 1754–1755 therapeutic applications, 1765 Vitamin E antioxidant protection, 1495 deficiency status assessment, 1779 treatment, 1779–1780 functions, 1777–1778 intestinal absorption overview, 1774–1775 α-tocopherol transfer protein and regulation of serum levels, 1775–1776 malabsorption and deficiency causes and symptoms, 1778–1779 oxidation and metabolism, 1776–1777 structure and biochemistry, 1773–1774 toxicity, 1780–1781 Vitamin H, see Biotin Vitamin K deficiency status assessment, 1784–1785 treatment, 1785 dietary sources and requirements, 1781–1782 functions, 1782–1784 intestinal absorption, 1782 malabsorption and deficiency causes, 1784 metabolism, 1782 structure and biochemistry, 1781 toxicity, 1785 W Water transport, see also Aquaporins epithelial fluid-transporting mechanisms countercurrent multiplication, 1831–1832 luminal hypertonicity driven by acidification, 1832 overview, 1828, 1832 solute recirculation, 1830 standing gradient model, 1828, 1830 transcellular versus paracellular fluid transport, 1830 water pumps, 1830–1831 postnatal development, 384 volumes in gastrointestinal tract, 1827–1828 WE-14, enterochromaffin-like cell secretion, 1230–1231 WIF1, Wnt/β-catenin signaling inhibition, 252 Wilson’s disease, pathophysiology, 1499 Wnt/β-catenin signaling cancer defects esophagus, 263 familial adenomatous polyposis, 262–263 liver, 264 pancreas, 264

Wnt/β-catenin signaling (Continued) sporadic colorectal cancer, 261–262 stomach adenocarcinoma, 263–264 adenoma, 263 fundic gland polyps, 263 β-catenin APC binding, 248–249 nuclear action, 258–259 subcellular distribution, 258 destruction complex overview, 252–254 regulators APC, 256–258 Axin, 254–255 Dishevelled, 255–256 Frizzled, 255–256, 441 low-density lipoprotein receptor-related proteins, 254–255, 441 discovery, 248 gastrointestinal roles development, 260 homeostasis and architecture, 260–261 ligands, receptors and inhibitors, 249, 441–442 noncanonical Wnt signaling, 260, 440 overview, 249 stem cell function regulation via Tcf-4 overview, 316–317 signal-modifying genes Dkk1, 321–322 Fox11, 322 target genes CD44, 321 Cdx genes, 319–320 Ephrin system, 317–318 c-Myc, 318–319 SOX9, 320–321 target genes, 259–260, 441–442 Tcf-4 regulation intestinal cell cycle progression, 442 intestinal cell position along crypt-villus axis, 442–443 Wound healing assays chemical injury, 468–469 fence assay, 467–468 non-gastrointestinal model systems, 469 organ culture, 469 polarized cultures and chemotaxis assays, 468 scrape wounding, 467 subconfluent cultures, 468 two-photon microscopy, 469 cell migration altered migration and signaling, 469–470 chemotaxis versus restitutional migration, 461 gene transcriptional regulation, 466 inside-out signaling, 467 mechanisms, 460–461 phases, 462 signal transduction pathways, 465–466 gastroduodenal injury and restitution animal models of injury, 1281–1282 cell exfoliation and mucoid cap, 1280 epithelial integrity challenges, 1279 epithelial restitution, 1280–1281 imposed mucosal damage, 1279–1280 prospects for study, 470 regulation cytokines, 464 extracellular matrix, 464–465 growth factors, 463–464 promotion by lipids, 464 trefoil factors, 464 X Xenin, pancreas secretion regulation, 1415–1416 Y Yersina pseudotuberculosis, barrier function effects, 1182 Z Zinc, absorption and transporters, 1996–1998 Zollinger–Ellison syndrome, gastric acid secretion, 1246 Zonula adherens, see Adherens junctions Zonula occludens, see Tight junction Zot, barrier function effects, 1174

Color Plate 1. Flask-shaped peptide YY cell of the open type in the rat colonic mucosa (original magnification × 360). (See FIG. 5-25.) (Modified from Jeng and colleagues [495], by permission.)

Villi

Crypts

Color Plate 2. The intestinal stem cell niche. Illustration of the stem cell hierarchy of the crypt. Chimeric mice can be generated by injecting pluripotent embryonic stem cells from one genetic background into blastocysts representing another genetic background. The small intestine of the resulting mouse contains cells representing both genetic backgrounds. The image is a section prepared from the proximal small intestine of an adult chimeric mouse. The finger-shaped villus is supplied with epithelial cells from two adjacent crypts. One crypt is populated entirely by blue epithelial cells, representing one genetic background. The other crypt is populated entirely by nonblue cells, representing the other genetic background. Crypts do not contain a mixture of cells from both genetic backgrounds. Thus, they are monoclonal, that is, composed of epithelial cells ultimately derived from a single progenitor. This progenitor occupies the highest position in the stem cell hierarchy. The precise number of progenitorderived multipotent stem cells that are active in each crypt is uncertain. Note that cells from each crypt migrate up the villus in an orderly fashion, forming distinct columns with discrete borders. Migration is completed in 3 to 5 days. The highly organized migration is illustrated further in the inset that shows a whole-mount preparation of intestine from a chimeric mouse. Striped villi contain differentiating epithelial cells derived from monoclonal blue crypts and nonblue (white) crypts. (See FIG. 11-3A.)

Color Plate 3. Notch signaling pathway. (A) Low-power 4-mm section of adult murine small intestine. Precursor cells (brown) are stained for cyclin proliferating cell nuclear antigen (PCNA); enterocytes (red) express intestinal alkaline phosphatase (IAP); goblet cells (blue) secrete mucins. Inset shows highpower image of small intestine enterocytes and goblet cells. (B) Math1, a component of the Notch signaling pathway, influences intestinal epithelial cell fate decisions. In crypt progenitor stem cells that express high levels of Notch, the Hes1 transcription factor is switched on, and the expression of Math1 and other “prosecretory” genes is blocked. The result is that the precursor cells become enterocytes. In cells expressing low amounts of Notch, levels of Delta are high, production of Hes1 is blocked, and Math1 expression is induced. Production of the Math1 helix-loop-helix transcription factor allows precursor cells to make a choice whether to become goblet cells, Paneth cells, or enteroendocrine cells. Cr, crypt; Vi, villus. (See FIG. 11-7.) (Reproduced from van den Brink GR, de Santa Barbara P, Roberts DJ. Development. Epithelial cell differentiation—a Mather of choice. Science 2001;294: 2115–2116, by permission.)

A

B

C

D

BG

Color Plate 4. Fibroblast growth factor receptor-3 (FGFR-3) expression in the suckling mouse intestine. Sections were incubated with rabbit anti–FGFR-3. (A) Immunoreactive FGFR-3 in the proximal jejunal crypts from a 14-day-old mouse (original magnification ×100). Open arrowheads point to Paneth cells, while the black arrowheads indicate FGFR-3–positive ganglion cells. The inset shows a higher magnification view of a crypt from the section in A (original magnification ×400). Staining is localized to the basolateral membranes of undifferentiated epithelial cells in the lower half of the crypt. Black arrow indicates the crypt-villus junction. Arrowheads denote Paneth cells showing barely detectable staining. (B) The specificity of the anti–FGFR-3 immunohistochemistry is demonstrated in a section from the proximal jejunum incubated with normal rabbit IgG in place of the primary anti–FGFR-3 antibody. (C) In the duodenum (original magnification ×100), staining is confined to the crypt epithelium with no staining in the adjacent Brunner’s glands (BG). (D) FGFR-3 immunoreactivity also is detectable in the lower third portion of colonic crypt epithelium (original magnification ×200). (See FIG. 11-10.) (Reproduced from Vidrich and colleagues [115], by permission.)

A

B

C

Color Plate 5. Cell-specific patterns of fibroblast growth factor receptor-3 (FGFR-3) expression in mouse proximal jejunum. Proximal jejunum from 21-day-old FVB/n mice (A–C) received BrdUrd 2 hours before tissue harvest. (A) S-phase cells incorporating BrdUrd (red fluorescence) are found primarily in the lower two-thirds crypt. Solid white arrows indicate the location of the crypt-villus junction. (B) FGFR-3 is expressed along the basolateral surfaces of epithelial cells in the lower crypt (green fluorescence). Open arrows indicate the basilar surface of a crypt. (C) Colocalization of BrdUrd and FGFR-3. The S-phase cells in the upper portions of the crypt epithelium do not stain with anti–FGFR-3 (e.g., white arrowhead ). (See FIG. 11-11.) (Modified from Vidrich and colleagues [115], by permission.)

GLP-2

A

KGF

Control

A

B

C

D

Mucous progenitor clones

Columnar progenitor clones

Stem cell clones

C 2.0 1.5 1.0 0.5 0.0 Columnar Mucos Stem

Normalized mucous cell density

Normalized clone size

B

3 2

GLP-2 KGF Control

1 0 Stem Clones Epithelium

Color Plate 6. Progenitor behavior detected by specific progenitor assay by somatic mutation (SPASM) after glucagon-like peptide-2 (GLP-2) or keratinocyte growth factor (KGF) administration. (A) Photomicrographs of typical NEUinduced Dlb-1+ clones derived from mucous progenitors, columnar progenitors, or stem cells. Dlb-1+ cells, labeled with horseradish peroxidase–conjugated lectin are stained brown. The tissue is counterstained with Alcian blue to aid identification of mucous cells (which are blue [arrow], unless Dlb-1+ [arrowhead] ). Columnar cells are pale blue (arrow), unless Dlb-1+ (arrowhead). Dlb-1+ mucous (arrowhead) and columnar (arrow) cells are both seen in stem cell clones. Original magnification ×320. (B) After GLP-2 treatment, columnar cell and stem cell clones are larger in comparison with those in control samples. Mucous cell clones were larger after KGF treatment. Control clones had 129.2 ± 28.3, 3.4 ± 0.3, 12.0 ± 3.5 (mean ± standard error of the mean) cells for stem cell, mucous cell, and columnar cell clones, respectively. (C) GLP-2 treatment decreases, but KGF treatment increases mucous cell density in the epithelium and stem cell clones. Control mucous cell density was 0.039 ± 0.001 in the epithelium and 0.032 ± 0.003 in stem cell clones. (See FIG. 11-12.) (Reproduced from Bjerknes and Cheng [88], by permission.)

Color Plate 7. Altered lineage allocation and crypt morphology accompany ileitis in SAMP1/YitFc mice. Ileitis in SAMP1/YitFc mice is characterized by the presence of a marked transmural acute and chronic inflammatory infiltrate and crypt abscesses. Loss of villi, marked crypt elongation, and increased numbers of Paneth cells and intermediate cells containing eosinophilic granules (A, open arrows ) are evident throughout the crypt of SAMP1/YitFc mice at 20 weeks of age. Note the eosinophilic granules in numerous cells located from the base of the crypt to the crypt-villus junction. In control AKR or C57BL/6 mice, Paneth cells are normally restricted to cell positions 1 to 4 at the crypt base. Periodic acid-Schiff-Alcian blue staining demonstrates increased numbers of goblet cells in areas of inflammation (B). Electron microscopic analysis of crypt epithelium of 10-week-old SAMP1/YitFc mice shows cells in the mid and upper crypt containing immature Paneth cell granules characteristic of intermediate cells (C). Electron micrograph of Paneth cells with mature granules from 10-week-old AKR mice is shown for comparison (D). (See FIG. 11-13.) (Reproduced from Vidrich and colleagues [260], by permission.)

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Color Plate 8. Regeneration of crypts in SAMP1/YitFc mice after radiation injury. The appearance of regenerating crypts is shown in hematoxylin and eosin–stained sections of ileum from 40-week-old SAMP1/YitFc mice (A) and age-matched AKR (B) mice 3.5 days after 14 Gy γ-irradiation. In adjacent sections, S-phase cells were detected by immunohistochemistry with anti-BrdUrd (C, D). A regenerative crypt (microcolony) is observed if one or more clonogenic stem cells within the crypt survive radiation injury and are able to subsequently replicate. Note that the number of regenerating crypts is markedly increased in the SAMP1/YitFc (C) mice compared with the age-matched AKR control mice (D). Crypt stem cell survival was globally increased in both inflamed (ileum, E) and noninflamed (proximal jejunum, F) regions of the small intestine in SAMP1/YitFc mice compared with AKR or C57BL/6 control mice (*, p < 0.01). Crypt stem cell survival after 14 Gy γ-irradiation (G) increases in parallel to the severity of the inflammatory response (H). (See FIG. 11-14.) (Reproduced from Vidrich and colleagues [260], by permission.)

50 µm

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1 µm

20 µm

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Color Plate 9. Features of interstitial cells of Cajal (ICC) in gastrointestinal (GI) muscles. (A) The ICC between the circular and longitudinal muscle layers (ICC-MY) network in the canine gastric antrum labeled with antibodies against the receptor tyrosine kinase, Kit (19). (B) Electron micrograph of ICC-MY in the canine antrum. Cells contain few contractile filaments and an abundance of smooth endoplasmic reticulum, mitochondria, and caveolae. ICC-MY form gap junctions with each other and with neighboring circular smooth muscle cells. (C) Higher power electron micrograph of the area outlined in B. A small gap junction between ICC-MY and a circular smooth muscle cell is shown (arrow). (D) Close apposition between varicose nerve terminals and intramuscular ICC (ICC-IM) in the canine antrum. (E) Higher power image of the area outlined in D reveals synaptic-like contacts with presynaptic and postsynaptic membrane densifications in the nerve varicosity and in the ICC-IM. (F, G) Close apposition between vesicular acetylcholine transporter (vAChT) and nitric oxide synthase–like immunoreactive nerves and ICC-IM in the circular muscle layer of the murine antrum. Nerve fibers preferentially target ICC-IM (51,52). (See FIG. 20-1.)

Color Plate 10. Spatial arrangement of neurons in guinea pig inferior mesenteric ganglion (IMG). Sixteen neurons in the same IMG were microinjected with biocytin, visualized with streptavidinCy5, and volume rendered. The visualized neurons (false-colored) are viewed from 0-degree rotation in A. A planar arrangement of the neurons was observed when the entire data set was rotated 90 degrees along the long axis (x-axis) of the ganglion (B). (C, D) Images were made at higher magnification of three neurons located in the top left of A. (B, D) Note the mingling of dendritic arbors of neurons located side by side in the same plane, whereas dendritic arbors of neurons located in different layers did not intermingle. The volume-reconstructed images were made combining 32 sets of images (375-µm/side). Each set contained 78 sequential confocal images recorded at 1.2-µm-depth increments. The total number of sequential consecutive confocal sections used to render and volume reconstruct the neurons was 2496. Each neuron was reconstructed separately, tiled, and placed in the same space it occupied in the intact ganglion relative to the other neurons. Scale bars = 1 mm (A, B); 200 µm (C, D). (See FIG. 22-1.) (Reproduced from Ermilov and colleagues [5], by permission.)

A

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Color Plate 11. Spatial (three-dimensional) distribution of presynaptic vasoactive intestinal peptide (VIP)–like-immunoreactive (IR), vesicular acetylcholine transporter (VAChT)–like-IR appositions (yellow, A and B, respectively), and monoclonal antibody 35 (MAb35)–IR (putative nicotinic acetylcholine receptors [nAChRs], red) on the surface (C) and inside the same neuron (D) in guinea pig inferior mesenteric ganglion (IMG). Note that the majority of VIP–like-IR presynaptic endings (55.9 ± 5.7% of the total neuron-associated, VIP–like-IR–containing structures) are on secondary and tertiary dendritic branches of the neuron, and that the majority of VAChT–like-IR (marker of the presynaptic acetylcholine-containing terminals) and MAb35 sites (marker of nAChRs) also are on secondary and tertiary dendrites. The presence of MAb35–like-IR within the IMG neuron (D) most likely represents nAChR subunits associated with intracellular organelles during biosynthesis and trafficking of nAChRs. IMG neurons were intracellularly injected with Lucifer Yellow (LY), immunostained with appropriate antibodies, visualized with either Cy3 or Cy5, and imaged using a confocal microscope. Images of the LY-filled neurons (gray) and associated immunoreactive sites (red) were volume reconstructed and superimposed using ANALYZE software. Scale bars = 50 µm. (See FIG. 22-4.) (From Ermilov and colleagues [18], by permission.)

A

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F

Color Plate 12. Spatial distribution of monoclonal antibody 35 (MAb35)-LI immunoreactivity (IR) (a marker for nicotinic acetylcholine receptors, red) in two viscerofugal neurons in guinea pig distal colon retrogradely labeled with DiI and filled by microinjection with Lucifer Yellow. (A–C) The viscerofugal neuron has Dogiel type I morphology; (D–F) the viscerofugal neuron has Dogiel type II morphology. In both neurons, putative nicotinic acetylcholine receptors are located on the surface membrane (B, E) and clusters of putative receptors covered the cell bodies and processes (A, D). Cytoplasmic staining also can be observed in both types of neurons (C, F). The three-dimensional volume-reconstructed images were made of neurons in whole-mount preparations of guinea pig distal colon. Viscerofugal neurons were intracellularly injected with Lucifer Yellow after retrograde labeling with DiI, immunostained labeled with MAb35, and imaged using a confocal microscope. Images were superimposed using ANALYZE software. Scale bars = 25 µm. (See FIG. 22-8.) (Reproduced from Ermilov and colleagues [156], by permission.) Longitudinal muscle

lar

rcu

Ci

Myenteric plexus

cle

s mu

Submucosal plexus

Mucosa

A A

Enteric nervous system

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Color Plate 13. Spatial (three-dimensional) distribution of presynaptic pituitary adenylate cyclase–activating peptide (PACAP)-LI immunoreactivity (IR) (A) and neuronal type I PACAP receptor (PAC1)-LI-IR (B) on guinea pig inferior mesenteric ganglion (IMG) neurons. The IMG neurons were intracellularly injected with Lucifer Yellow, and the preparations were subsequently immunostained with appropriate antibodies visualized with either Cy3 or Cy5 fluorescent dyes. Preparations were imaged using a confocal microscope. The images were volume reconstructed and superimposed using ANALYZE software. Voxel counting revealed that the majority (54.0 ± 9.4%, n = 6 neurons) of PACAP–like-IR appositions (red, A) and the majority (66.7 ± 4.9%, n = 7 neurons) of PAC1-receptor–like IR (red, B) were distributed along secondary and tertiary dendrites of IMG neurons (gray). Scale bars = 50 µm. (See FIG. 22-11.) (Reproduced from Ermilov and colleagues [18], by permission.)

B

Longitudinal axis

Longitudinal axis

Submucosal plexus

Myenteric plexus

C

Color Plate 14. Histoanatomic organization of the enteric nervous system (ENS). (A) Ganglia and interganglionic fiber tracts of the myenteric and submucosal plexuses are prominent features of the ENS. The myenteric plexus is distributed between the circular and longitudinal muscle coats; the submucous plexus is between the mucosa and circular muscle coat. (B) Tyrosine hydroxylase stain of the submucosal plexus as it appears in a preparation obtained from guinea pig small intestine. (C) Tyrosine hydroxylase stain of the myenteric plexus as it appears on the underside of the longitudinal muscle coat after microdissection from guinea pig small intestine. The long dimension of myenteric ganglia in the small intestine is in the circumferential direction; no such orientation occurs for the submucosal plexus. Asterisks identify ganglia; diamonds identify interganglionic fiber tracts. (See FIG. 23-2.)

∆Afferent discharge (spikes/s)

80

Color Plate 15. Transient receptor potential vanilloid receptor 1 (TRPV1) plays a role in the mechanosensitivity of gastrointestinal afferents. (A) The pressure–response curve of multiunit activity from mesenteric nerve during ramp distensions in wild-type and TRPV1 knockout mice. (B) TRPV1 immunoreactivity in vagal afferent endings (A–D), nodose (E), and dorsal root ganglion (F) neurons supplying the rat gastrointestinal tract. IGLE, intraganglionic laminar ending; IMA, intramuscular array. (See FIG. 25-12.) (A: Reproduced from Rong and colleagues [194], by permission; B: Reproduced from Ward and colleagues [268], by permission.)

Wild type TRPV1 knockout

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Color Plate 17. Pseudocolor images of a touch-evoked Fluo-4/AM Ca2+ response in a BON cell. (See FIG. 27-6B.)

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Color Plate 16. The Orexin receptor OX-R1 in human nodose ganglia colocalizes with Ob-R, cholecystokinin (A) (CCK-A), cocaine and amphetamine regulatory transcript (CART), or calcitonin gene–related peptide (CGRP). (A, D, G, J) Individual neurons expressing OX-R1. Middle panels show the corresponding neurons exhibiting (B) Ob-R, (E) CCK-A, (H) CART, or (K) CGRP immunoreactivity. (C, F, I, L) Overlay of the relevant left and middle panels. Cells colocalizing both labels appear in yellow. Bars = 25 µm. (See FIG. 25-16.) (Modified from Burdyga and colleagues [325], by permission.)

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Color Plate 18. Purinergic laser scanning confocal microscopy (LSM) Fluo-4/AM Ca2+ imaging in intact submucous plexus of rat distal colon. (A) Basal Ca2+ response. (B) Response to high K+ depolarization (75 mM). (C) The P2Y1R agonist 2MeSADP increased [Ca2+]i in three neurons (arrows). (D) Substance P (SP) increased intracellular free Ca2+ levels in six neurons and three of them also had Ca2+ responses to 2MeSADP; arrowheads designate cells with only SP responses. (See FIG. 27-13A–D.)

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Color Plate 19. The P2Y1 antagonist MRS 2179 or tetrodotoxin (TTX) abolish the touch-induced Ca2+ fluorescence response; representative Ca2+ images from a single ganglion. (See FIG. 27-14D–G.)

Color Plate 20. Endoscopic views of the pharynx, descending from the skull base (A) to the upper esophageal sphincter (D). B, Pharyngeal cavity proximal to uvula. C, Pharyngeal cavity at uvula. (See FIG. 35-2.) Color Plate 21. A representative example of cerebral cortical functional magnetic resonance imaging (fMRI) activity during volitional swallowing (A) and reflexive swallowing (B). There were substantial differences in cortical activity regions between volitional and reflexive swallow. Although the former was represented in the primary sensorimotor area, the prefrontal, cingulated, insula, and parietooccipital regions, the later was represented in the primary sensorimotor cortex. (See FIG. 35-9.) (Modified from Kern and colleagues [143].)

Cortical regions of activity Volitional swallowing – Young subject left

Reflexive swallowing – Young subject left

right

right

A Color Plate 22. Potential signaling pathways for the generation of the three types of contractions in colonic circular smooth muscle cells. The electromechanical coupling initiated by slow-wave depolarization induces Ca2+ influx through voltage-gated calcium channels (VGCC; L-type or Cav1.2 channels). The binding of acetylcholine (ACh) to muscarinic M3 receptors initiates multiple signaling pathways that induce further Ca2+ influx through receptor-gated Ca2+ channels (RGCC), releases intracellular Ca2+ from inositol 1,4,5-triphosphate (IP3)– sensitive stores, inhibits myosin light chain phosphatase (MLCP), and increases the phosphorylation of 20-kDa regulatory light chain of myosin (MLC20) via protein kinase C (PKC), Rho kinase (ROK), and release of arachidonic acid (AA) to enhance the amplitude and/or duration of contraction, which is the key for the generation of three types of contractions. ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CaM, calmodulin; CPI-17, PKC-potentiated inhibitor protein of 17 kDa; cPLA2, cytosolic phospholipase A2; DAG, diacylglycerol; GEFs, guanine nucleotide exchange factors; MYPT1 and PP1c, MLCP regulatory and catalytic subunits respectively; MLCK, myosin light chain kinase; NO, nitric oxide; PIP2, phosphatidyl inositol 4,5,-biphosphate; PLC, phospholipase C; RhoGDI, guanosine diphosphate (GDP) dissociation inhibitor; SR, sarcoplasmic reticulum; VIP, vasoactive intestinal peptide. (See FIG. 39-10.)

B

PHYSIOLOGY OF THE GASTROINTESTINAL TRACT FOURTH EDITION

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Physiology of the

Gastrointestinal Tract FOURTH EDITION

Volume 2 Editor-in-Chief Leonard R. Johnson Department of Physiology University of Tennessee College of Medicine Memphis, Tennessee

Associate Editors

Kim E. Barrett Department of Medicine University of California, San Diego, School of Medicine University of California San Diego Medical Center San Diego, California

Fayez K. Ghishan Department of Pediatrics Steele Children’s Research Center University of Arizona Health Sciences Center Tucson, Arizona

Juanita L. Merchant Departments of Internal Medicine and Molecular and Integrative Physiology University of Michigan Ann Arbor, Michigan

Hamid M. Said Departments of Medicine and Physiology/Biophysics University of California Irvine, California, and VA Medical Center-151 Long Beach, California

Jackie D. Wood Departments of Physiology and Biology and Internal Medicine The Ohio State University College of Medicine Columbus, Ohio

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Library of Congress Cataloging-in-Publication Data Physiology of the gastrointestinal tract / editor-in-chief, Leonard R. Johnson ; associate editors, Kim Barrett ... [et al.].-- 4th ed. p. ; cm. Includes bibliographical references and index. ISBN 0-12-088394-5 (set : alk. paper) -- ISBN 0-12-088395-3 (v.1 : alk. paper) ISBN 0-12-088396-1 (v.2 : alk. paper) 1. Gastrointestinal system--Physiology. I. Johnson, Leonard R., 1942[DNLM: 1. Gastrointestinal Tract--physiology. WI 102 P578 2006] QP145.P492 2006 612.3′2--dc22

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Volume 1: ISBN 13: 978-0-120883950 • ISBN 10: 0-12-088395-3 Volume 2: ISBN 13: 978-0-120883967 • ISBN 10: 0-12-088396-1 Two Volume Set: ISBN 13: 978-0-120883943 • ISBN 10: 0-12-088394-5

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Contents Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Preface to the First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii VOLUME 1

Section I. Basic Cell Physiology and Growth of the GI Tract 1. Transcriptional and Epigenetic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Juanita L. Merchant and Longchuan Bai

1

2. Translation and Posttranslational Processing of Gastrointestinal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cheryl E. Gariepy and Chris J. Dickinson

31

3. Transmembrane Signaling by G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claire Jacob and Nigel W. Bunnett

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4. Gastrointestinal Hormones: Gastrin, Cholecystokinin, Somatostatin, and Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . Graham J. Dockray

91

5. Postpyloric Gastrointestinal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Ella W. Englander and George H. Greeley Jr. 6. Gastrointestinal Peptide Hormones Regulating Energy and Glucose Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Daniel J. Drucker 7. Growth Factors in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 John A. Barnard and Kirk M. McHugh 8. Developmental Signaling Networks Wnt/β-Catenin Signaling in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . 247 Guido T. Bommer and Eric R. Fearon 9. Hedgehog Signaling in Gastrointestinal Morphogenesis and Morphostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Gijs R. van den Brink, Maikel P. Peppelenbosch, and Drucilla J. Roberts 10. Developmental Signaling Networks: The Notch Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Guy R. Sander, Hanna Krysinska, and Barry C. Powell 11. Physiology of Gastrointestinal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Alda Vidrich, Jenny M. Buzan, Sarah A. De La Rue, and Steven M. Cohn 12. Apoptosis in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Leonard R. Johnson 13. Molecular Aspects and Regulation of Gastrointestinal Function during Postnatal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 James F. Collins, Liqun Bai, Hua Xu, and Fayez K. Ghishan 14. Effect of Aging on the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Adhip P. N. Majumdar and Marc D. Basson 15. Regulation of Gastrointestinal Normal Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Mark R. Hellmich and B. Mark Evers

v

vi / CONTENTS 16. Mucosal Repair and Restitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Mark R. Frey and D. Brent Polk 17. Mechanisms of Gastrointestinal Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 John Lynch and Anil K. Rustgi

Section II. Neural Gastroenterology and Motility 18. Development of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Michael D. Gershon and Elyanne M. Ratcliffe 19. Cellular Physiology of Gastrointestinal Smooth Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523 Gabriel M. Makhlouf and Karnam S. Murthy 20. Organization and Electrophysiology of Interstitial Cells of Cajal and Smooth Muscle Cells in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 Kenton M. Sanders, Sang Don Koh, and Sean M. Ward 21. Functional Histoanatomy of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Simon J. H. Brookes and Marcello Costa 22. Physiology of Prevertebral Sympathetic Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Joseph H. Szurszewski and Steven M. Miller 23. Cellular Neurophysiology of Enteric Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629 Jackie D. Wood 24. Integrative Functions of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 Jackie D. Wood 25. Extrinsic Sensory Afferent Nerves Innervating the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 Michael J. Beyak, David C. E. Bulmer, Wen Jiang, C. Keating, Weifang Rong, and David Grundy 26. Processing of Gastrointestinal Sensory Signals in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727 Anthony Hobson and Qasim Aziz 27. Enteric Neural Regulation of Mucosal Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Helen Joan Cooke and Fedias Leontiou Christofi 28. Effect of Stress on Intestinal Mucosal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763 Johan D. Söderholm and Mary H. Perdue 29. Effect of Stress on Gastrointestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Michèle Gué 30. Central Corticotropin-Releasing Factor and the Hypothalamic-Pituitary-Adrenal Axis in Gastrointestinal Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791 Yvette Taché and Mulugeta Million 31. Neural Regulation of Gastrointestinal Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817 Peter Holzer 32. Neural Control of the Gallbladder and Sphincter of Oddi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841 Gary M. Mawe, Gino T. P. Saccone, and María J. Pozo 33. Brainstem Control of Gastric Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851 Richard C. Rogers, Gerlinda E. Hermann, and R. Alberto Travagli 34. Neural and Hormonal Controls of Food Intake and Satiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877 Timothy H. Moran

CONTENTS / vii 35. Pharyngeal Motor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 Reza Shaker 36. Motor Function of the Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913 Ray E. Clouse and Nicholas E. Diamant 37. Neurophysiologic Mechanisms of Gastric Reservoir Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jan Tack

927

38. Small Intestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William L. Hasler

935

39. Function and Regulation of Colonic Contractions in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sushil K. Sarna and Xuan-Zheng Shi

965

40. Neural Control of Pelvic Floor Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David B. Vodusek and Paul Enck

995

41. Pathophysiology Underlying the Irritable Bowel Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009 Jackie D. Wood VOLUME 2

Section III. Gastrointestinal Immunology and Inflammation 42. Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033 Lars Eckmann 43. Biology of Gut Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067 Finn-Eirik Johansen, Elizabeth H. Yen, Bonny Dickinson, Masaru Yoshida, Steve Claypool, Richard S. Blumberg, and Wayne I. Lencer 44. Mechanisms of Helicobacter pylori–Induced Gastric Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091 Dawn. A. Israel and Richard M. Peek Jr. 45. Mechanisms and Consequences of Intestinal Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1115 Wallace K. MacNaughton 46. Recruitment of Inflammatory and Immune Cells in the Gut: Physiology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137 D. Neil Granger, Christopher G. Kevil, and Matthew B. Grisham 47. Physiology of Host–Pathogen Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163 Kim Hodges, V. K. Viswanathan, and Gail Hecht

Section IV. Physiology of Secretion 48. The Cell Biology of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189 Curtis Okamoto, Serhan Karvar, and John G. Forte 49. Regulation of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223 Arthur Shulkes, Graham S. Baldwin, and Andrew S. Giraud 50. Gastroduodenal Mucosal Defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259 Marshall H. Montrose, Yasutada Akiba, Koji Takeuchi, and Jonathan D. Kaunitz 51. Genetically Engineered Mouse Models of Gastric Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293 Linda C. Samuelson 52. Structure–Function Relations in the Pancreatic Acinar Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313 Fred S. Gorelick and James D. Jamieson

viii / CONTENTS 53. Stimulus-Secretion Coupling in Pancreatic Acinar Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1337 John A. Williams and David I. Yule 54. Cell Physiology of Pancreatic Ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1371 Barry E. Argent, Michael A. Gray, Martin C. Steward, and R. Maynard Case 55. Regulation of Pancreatic Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1397 Rodger A. Liddle 56. Bile Formation and the Enterohepatic Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1437 Paul A. Dawson, Benjamin L. Shneider, and Alan F. Hofmann 57. Mechanisms of Hepatocyte Organic Anion Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463 Allan W. Wolkoff 58. Mechanisms of Hepatocyte Detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483 Karen F. Murray, Donald J. Messner, and Kris V. Kowdley 59. Physiology of Cholangiocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505 Anatoliy I. Masyuk, Tatyana V. Masyuk, and Nicholas F. LaRusso 60. Gallbladder Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1535 Sum P. Lee and Rahul Kuver

Section V. Digestion and Absorption 61. Tight Junctions and the Intestinal Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559 Thomas Y. Ma and James M. Anderson 62. Protein Sorting in the Exocytic and Endocytic Pathways in Polarized Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . 1595 V. Stephen Hunt and W. James Nelson 63. Physiology of the Circulation of the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627 Philip T. Nowicki 64. Sugar Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 Ernest M. Wright, Donald D. F. Loo, Bruce A. Hirayama, and Eric Turk 65. Protein Digestion and Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667 Vadivel Ganapathy, Naren Gupta, and Robert G. Martindale 66. Role of Membrane and Cytosolic Fatty Acid Binding Proteins in Lipid Processing by the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693 Nada Abumrad and Judith Storch 67. Genetic Regulation of Intestinal Lipid Transport and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711 Zhouji Chen and Nicholas O. Davidson 68. Digestion and Intestinal Absorption of Dietary Carotenoids and Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1735 Alexandrine During and Earl H. Harrison 69. Vitamin D3: Synthesis, Actions, and Mechanisms in the Intestine and Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1753 J. Wesley Pike, Makoto Watanuki, and Nirupama K. Shevde 70. Vitamin E and Vitamin K Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1773 Ronald J. Sokol, Richard S. Bruno, and Maret G. Traber 71. Intestinal Absorption of Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1791 Hamid M. Said and Bellur Seetharam 72. Water Transport in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827 Jay R. Thiagarajah and A. S. Verkman

CONTENTS / ix 73. Na+-H+ Exchange in Mammalian Digestive Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1847 Pawel R. Kiela and Fayez K. Ghishan 74. Intestinal Anion Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1881 Pradeep K. Dudeja and K. Ramaswamy 75. Ion Channels of the Epithelia of the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917 John Cuppoletti and Danuta H. Malinowska 76. Integrative Physiology and Pathophysiology of Intestinal Electrolyte Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . 1931 Kim E. Barrett and Stephen J. Keely 77. Molecular Mechanisms of Intestinal Transport of Calcium, Phosphate, and Magnesium . . . . . . . . . . . . . . . . . . . . 1953 James F. Collins and Fayez K. Ghishan 78. Iron Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1983 Nancy C. Andrews 79. Trace Element Absorption and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1993 Robert J. Cousins

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Contributors Nada Abumrad, Departments of Medicine and Nutritional Sciences, Washington University, Campus Box 8031, 660 South Euclid Avenue, Saint Louis, Missouri 63110

Marc D. Basson, John D. Dingell Veterans Affairs Medical Center, Department of Surgical Service, Wayne State University, Detroit, Michigan 48201 Michael J. Beyak, Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario, Canada, and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

Yasutada Akiba, Brentwood Biomedical Research Institute, Building 114, Suite 217, West Los Angeles VAMC, Los Angeles, California 90073 James M. Anderson, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 6312 MBRB, 103 Mason Farm Road, Chapel Hill, North Carolina 27599-7545

Richard S. Blumberg, Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Hepatology, and Endoscopy, Harvard Digestive Diseases Center, Laboratory of Mucosal Immunology, Thorn 1419, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115

Nancy C. Andrews, Department of Basic Sciences and Graduate Studies, Harvard Medical School, and Department of Pediatrics, Children’s Hospital, Howard Hughes Medical Institute, Gordon Hall, 25 Shattuck Street, Boston, Massachusetts 02115

Guido T. Bommer, Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan School of Medicine, LSI 5-183A, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216

Barry E. Argent, Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom

Simon J. H. Brookes, Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042

Qasim Aziz, University of Manchester, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom

Richard S. Bruno, Department of Nutritional Sciences, University of Connecticut, 3624 Horsebarn Road Ext, Unit 4017, Storrs, Connecticut 06269-4017

Liqun Bai, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073

David C. E. Bulmer, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

Longchuan Bai, Department of Internal Medicine, University of Michigan, 1150 West Medical Center Drive, 3510 MSRB I, Ann Arbor, Michigan 48109

Nigel W. Bunnett, Departments of Surgery and Physiology, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, California 94143-0660

Graham S. Baldwin, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia

Jenny M. Buzan, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708

Kim E. Barrett, Department of Medicine, University of California, San Diego, School of Medicine, University of California San Diego Medical Center 8414, 200 West Arbor Drive, San Diego, California 92103-8414

R. Maynard Case, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Zhouji Chen, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, Campus Box 8046, 660 South Euclid Avenue, Saint Louis, Missouri 63110

John A. Barnard, Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, 700 Children’s Drive WA2011, Columbus, Ohio 43205

xi

xii / CONTRIBUTORS Fedias Leontiou Christofi, Departments of Anesthesiology and Physiology and Cell Biology, The Ohio State University, 226 Tzagounis Medical Research Facility, Columbus, Ohio 43210

Bonny Dickinson, Department of Pediatrics, Research Institute for Children, Children’s Hospital, Research and Education Building Room 2231, 200 Henry Clay Avenue, New Orleans, Louisiana 70118

Steve Claypool, Laboratory of Mucosal Immunology, Thorn 1419, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115

Chris J. Dickinson, Division of Pediatric Gastroenterology, University of Michigan, D3252 MPB, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718

Ray E. Clouse, Departments of Medicine and Psychiatry, Washington University, 660 South Euclid Avenue, Campus Box 8124, Saint Louis, Missouri 63110

Graham J. Dockray, Department of Physiology, School of Biomedical Sciences, Crown Street, University of Liverpool, PO Box 147, Liverpool, L69 3BX United Kingdom

Steven M. Cohn, Digestive Health Center of Excellence, University of Virginia, 2 Jefferson Park Ave, Room 2091, Charlottesville, Virginia 22908-0708 James F. Collins, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Helen Joan Cooke, Department of Neuroscience, The Ohio State University, 4066D Graves Hall, 333 West Tenth Avenue, Columbus, Ohio 43210 Marcello Costa, Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042 Robert J. Cousins, Boston Family Professor of Nutrition, Center for Nutritional Sciences, University of Florida, PO Box 110370, Gainesville, Florida 32611-0370 John Cuppoletti, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way ML 0576, Cincinnati, Ohio 45267-0576 Nicholas O. Davidson, Division of Gastroenterology, Washington University School of Medicine, Campus Box 8124, 660 South Euclid Avenue, Saint Louis, Missouri 63110 Paul A. Dawson, Department of Internal Medicine, Section of Gastroenterology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157 Sarah A. De La Rue, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708 Nicholas E. Diamant, Departments of Medicine and Physiology, University of Toronto, 6B Fell 6-176, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8

Daniel J. Drucker, Banting and Best Diabetes Centre, Toronto General Hospital, University of Toronto, 200 Elizabeth Street MBRW 4R-902, Toronto, Ontario, Canada M5G 2C4 Pradeep K. Dudeja, Department of Medicine, University of Illinois at Chicago College of Medicine, Research and Development, Jesse Brown VAMC, MP151, 820 South Damen Avenue, Chicago, Illinois 60612 Alexandrine During, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 Lars Eckmann, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0665 Paul Enck, Department of Internal Medicine VI, University Hospitals Tuebingen, Osianderstr. 5, 72076 Tuebingen, Germany Ella W. Englander, Department of Surgery, The University of Texas Medical Branch, 815 Market Street, Galveston, Texas 77550 B. Mark Evers, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536 Eric R. Fearon, Division of Molecular Medicine and Genetics, Departments of Internal Medicine, Human Genetics, and Pathology, University of Michigan School of Medicine, LSI 5-183A, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216 John G. Forte, Department of Molecular and Cell Biology, University of California, 241 LSA, MC 3200, Berkeley, California 94720 Mark R. Frey, Department of Pediatric Gastroenterology, Hepatology, and Nutrition, S4322 MCN Vanderbilt University, Nashville, Tennessee 37232-2576

CONTRIBUTORS / xiii Vadivel Ganapathy, Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1459 Laney-Walker Boulevard, Augusta, Georgia 30912-2100 Cheryl E. Gariepy, Division of Pediatric Gastroenterology, University of Michigan, D3252 MPB, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718 Michael D. Gershon, Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, 630 West 168 Street, New York, New York 10032 Fayez K. Ghishan, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Andrew S. Giraud, Department of Medicine, University of Melbourne, Western Hospital, Footscray 3011, Australia Fred S. Gorelick, Department of Medicine, VA Healthcare CT, and Yale University School of Medicine, 950 Campbell Avenue, West Haven, Connecticut 06516 D. Neil Granger, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 Michael A. Gray, Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom George H. Greeley Jr., Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0725 Matthew B. Grisham, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 David Grundy, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Michèle Gué, Department of Physiology, Université Paul Sabatier, IFR31, Institut Louis Bugnard, BP 84225, INSERM U388, Laboratoire de Pharmacologie Moléculaire et Physiopathologie Rénale, 31432 Toulouse, Cedex 4, France Naren Gupta, Department of Surgery, University of Virginia Health System, Charlottesville, Virginia 22908

Earl H. Harrison, Phytonutrients Laboratory, United States Department of Agriculture Human Nutrition Research Center, Building 307 C, Room 118 BARC-East, Beltsville, Maryland 20705 William L. Hasler, Department of Internal Medicine, Division of Gastroenterology, University of Michigan Health System, 3912, Taubman Center, Box 0362, Ann Arbor, Michigan 48109 Gail Hecht, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 Mark R. Hellmich, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536 Gerlinda E. Hermann, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808 Bruce A. Hirayama, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 Anthony Hobson, University of Manchester, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom Kim Hodges, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 Alan F. Hofmann, Department of Medicine MC 0813, Division of Gastroenterology, University of California, San Diego, La Jolla, California 92093-0813 Peter Holzer, Department of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, A-8010 Graz, Austria V. Stephen Hunt, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5435 Dawn A. Israel, Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Room 1012A MRB IV, 2215 Garland Avenue, Nashville, Tennessee 37232 Claire Jacob, Departments of Surgery and Physiology, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, California 94143-0660

xiv / CONTRIBUTORS James D. Jamieson, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Wen Jiang, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Finn-Eirik Johansen, Institute of Pathology, University of Oslo, Department of Pathology, Rikshospitalet University Hospital, Sognsvannsveien 20, N-0027 Oslo, Norway Leonard R. Johnson, Department of Physiology, University of Tennessee College of Medicine, 894 Union Avenue, Memphis, Tennessee 38163 Serhan Karvar, Department of Molecular and Cell Biology, University of California, 245 LSA, MC 3200, Berkeley, California 94720 Jonathan D. Kaunitz, Department of Medicine, Division of Digestive Diseases, West Los Angeles VAMC, and UCLA School of Medicine, Building 114, Suite 217, Los Angeles, California 90073 C. Keating, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Stephen J. Keely, Department of Medicine, University of California, San Diego, School of Medicine, University of California San Diego Medical Center 8414, 200 West Arbor Drive, San Diego, California 92103-8414 Christopher G. Kevil, Department of Pathology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 Pawel R. Kiela, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Sang Don Koh, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Kris V. Kowdley, University of Washington, Box 356174, 1959 NE Pacific Street, Seattle, Washington 98195 Hanna Krysinska, Child Health Research Institute, North Adelaide, 72 King William Road, South Australia 5006, Australia Rahul Kuver, Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Box 356424, 1959 NE Pacific Street, Seattle, Washington 98195

Nicholas F. LaRusso, Departments of Medicine, Biochemistry, and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Sum P. Lee, Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Box 356424, 1959 NE Pacific Street, Seattle, Washington 98195 Wayne I. Lencer, Department of Pediatrics, Harvard Medical School, and Division of Pediatric Gastroenterology and Nutrition, Harvard Digestive Diseases Center, Gastrointestinal Cell Biology Laboratories, Enders 720, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115 Rodger A. Liddle, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 Donald D. F. Loo, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 John Lynch, Division of Gastroenterology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 600 Clinical Research Building, Philadelphia, Pennsylvania 19104 Thomas Y. Ma, Division of Gastroenterology and Hepatology, Departments of Medicine, Cell Biology and Physiology, Inflammatory Bowel Disease Program, University of New Mexico, MSC10 5550, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001 Wallace K. MacNaughton, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada Adhip P. N. Majumdar, John D. Dingell Veterans Affairs Medical Center, Department of Medicine, Karmanos Cancer Center, Wayne State University, Research Service, Room-B-4238, 4646 John R, Detroit, Michigan 48201 Gabriel M. Makhlouf, Department of Medicine, Virginia Commonwealth University Medical Center, Sanger Hall, Room 12-003, 1101 East Marshall Street, Richmond, Virginia 23298 Danuta H. Malinowska, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576

CONTRIBUTORS / xv Robert G. Martindale, Department of Surgery, Oregon Health and Science University, Portland, Oregon 97239 Anatoliy I. Masyuk, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Tatyana V. Masyuk, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Gary M. Mawe, Department of Anatomy and Neurobiology, The University of Vermont, D403A Given Building, 89 Beaumont Avenue, Burlington, Vermont 05405

Karnam S. Murthy, Departments of Medicine and Physiology, Virginia Commonwealth University Medical Center, Sanger Hall, Room 12-003, 1101 East Marshall Street, Richmond, Virginia 23298 W. James Nelson, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5435 Philip T. Nowicki, Departments of Pediatrics and Physiology, Center for Cell and Vascular Biology, Columbus Children’s Research Institute, College of Medicine and Public Health, The Ohio State University, Children’s Hospital, 700 Children’s Drive, Columbus, Ohio 43205

Kirk M. McHugh, Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, 700 Children’s Drive WA2011, Columbus, Ohio 43205

Curtis Okamoto, Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033

Juanita L. Merchant, Departments of Internal Medicine and Molecular and Integrative Physiology, University of Michigan, 1150 West Medical Center Drive, 3510 MSRB I, Ann Arbor, Michigan 48109

Richard M. Peek Jr., Division of Gastroenterology, Departments of Medicine and Cancer Biology, Vanderbilt University Medical Center, Room 1012A MRB IV, 2215 Garland Avenue, Nashville, Tennessee 37232

Donald J. Messner, Bastyr University, 14500 Juanita Drive, NE, Kenmore, Washington 98028

Maikel P. Peppelenbosch, Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Steven M. Miller, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905

Mary H. Perdue, Intestinal Disease Research Program, HSC-3N5C, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N3Z5, Canada

Mulugeta Million, CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, CURE Building 115, Room 117, 11301 Wilshire Boulevard, Los Angeles, California 90073

J. Wesley Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706

Marshall H. Montrose, Department of Molecular and Cellular Physiology, University of Cincinnati, Medical Science Building, Room 4253, 231 Albert Sabin Way, Cincinnati, Ohio 45267

Barry C. Powell, Child Health Research Institute, North Adelaide, and School of Pharmacy and Medical Sciences, University of South Australia; Department of Pediatrics, University of Adelaide, 72 King William Road, South Australia 5006, Australia

Timothy H. Moran, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Avenue, Baltimore, Maryland 21205 Karen F. Murray, Children’s Hospital and Regional Medical Center, University of Washington School of Medicine, 4800 Sand Point Way, NE, PO Box 5371/A5950, Seattle, Washington 98105

D. Brent Polk, Departments of Cell and Developmental Biology and Pediatric Gastroenterology, Hepatology, and Nutrition, Digestive Disease Research Center, S4322 MCN Vanderbilt University, Nashville, Tennessee 37232-2576

María J. Pozo, Department of Physiology, Nursing School, University of Extremadura, Avda Universidad s/n, 10071 Cáceres, Spain K. Ramaswamy, Department of Medicine, University of Illinois at Chicago College of Medicine, 840 South Wood Street (m/c 716), Chicago, Illinois 60612

xvi / CONTRIBUTORS Elyanne M. Ratcliffe, Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Columbia University, 3959 Broadway, CHN 702, New York, New York 10032

Reza Shaker, Division of Gastroenterology and Hepatology, Digestive Disease Center, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, Wisconsin 53226

Drucilla J. Roberts, Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114

Nirupama K. Shevde, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706

Richard C. Rogers, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808

Xuan-Zheng Shi, Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, 9.138E Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1064

Weifang Rong, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Anil K. Rustgi, Division of Gastroenterology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 600 Clinical Research Building, Philadelphia, Pennsylvania 19104 Gino T. P. Saccone, Department of General and Digestive Surgery, Flinders Medical Centre, Flinders Drive, Bedford Park, South Australia 5042, Australia Hamid M. Said, Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California, and VA Medical Center-151, Long Beach, California 90822 Linda C. Samuelson, Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109-0622 Guy R. Sander, Child Health Research Institute, North Adelaide, and Department of Pediatrics, University of Adelaide, 72 King William Road, South Australia 5006, Australia Kenton M. Sanders, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Sushil K. Sarna, Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, 9.138C Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1064 Bellur Seetharam, Departments of Medicine and Biochemistry, Medical College of Wisconsin and Clement Zablocki VA Medical Center, Research 151, 5000 National Avenue, Milwaukee, Wisconsin 53295

Benjamin L. Shneider, Department of Pediatrics, Box 1656, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029 Arthur Shulkes, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia Johan D. Söderholm, Department of Biomedicine and Surgery, Linköping University Hospital, Linköping SE-581 85, Sweden Ronald J. Sokol, Department of Pediatrics, Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Pediatric General Clinical Research Center, University of Colorado School of Medicine, The Children’s Hospital, 1056 East 19th Avenue, Box B290, Denver, Colorado 80218 Martin C. Steward, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Judith Storch, Department of Nutritional Sciences, Rutgers University, 96 Lipman Drive, New Brunswick, New Jersey 08901-8525 Joseph H. Szurszewski, Department of Physiology and Biomedical Engineering, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905 Yvette Taché, CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, CURE Building 115, Room 117, 11301 Wilshire Boulevard, Los Angeles, California 90073

CONTRIBUTORS / xvii Jan Tack, Department of Gastroenterology, University Hospitals Leuven, Center for Gastroenterological Research, University of Leuven, Herestraat 49, 3000 Leuven, Belgium Koji Takeuchi, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan Jay R. Thiagarajah, Departments of Medicine and Physiology, University of California San Francisco, 1246 Health Sciences East Tower, San Francisco, California 94143-0521 Maret G. Traber, Department of Nutrition and Exercise Sciences, Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, Oregon 97331-6512 R. Alberto Travagli, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808 Eric Turk, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 Gijs R. van den Brink, Laboratory for Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands A. S. Verkman, Departments of Medicine and Physiology, University of California San Francisco, 1246 Health Sciences East Tower, San Francisco, California 94143-0521 Alda Vidrich, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708 V. K. Viswanathan, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 David B. Vodusek, Division of Neurology, University Medical Center, Ljubljana 1525, Slovenia Sean M. Ward, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

Makoto Watanuki, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706 John A. Williams, Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0522 Allan W. Wolkoff, Marion Bessin Liver Research Center and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 Jackie D. Wood, Departments of Physiology and Biology and Internal Medicine, The Ohio State University College of Medicine, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210-1218 Ernest M. Wright, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, 10833 Le Conte Avenue, 53-263 Center for Health Sciences, Los Angeles, California 90095-1751 Hua Xu, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Elizabeth H. Yen, Harvard Medical School Fellowship in Pediatric Gastroenterology and Nutrition, Gastrointestinal Cell Biology, Enders 720, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115 Masaru Yoshida, Frontier Medical Science in Gastroenterology, International Center for Medical Research and Treatment, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan David I. Yule, Departments of Pharmacology and Physiology, University of Rochester Medical School, Rochester, New York 14642

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Preface to the First Edition As with any publishing venture and especially one of this magnitude, one must first ask, “Why?” The Associate Editors and I were motivated primarily to collect in one set of volumes the most up-to-date and comprehensive knowledge in our field. Nothing comparable has been attempted in the area of gastrointestinal physiology during the past fourteen years. During this time, there has been a rapid expansion of knowledge and many new areas of investigation have been initiated. More than fifty leading scientists—physiologists, clinical specialists, morphologists, pharmacologists, immunologists, and biochemists—have contributed chapters on their various areas of expertise for these volumes. Our original goal was to review the entire field of gastrointestinal physiology in one work. After examining all of the chapters, however, it was apparent that the final product encompassed more than physiology. The chapters reflect the backgrounds of the authors and the approaches of their different disciplines. As such, these volumes contain information for not only the investigator working in these fields but for the clinician or graduate student interested in the function of the gastrointestinal tract. Anyone involved in teaching gastrointestinal physiology of pathophysiology can readily find the latest and most pertinent information on any area in the discipline. This work is divided into five sections. The first consists of topics such as growth, the enteric nervous system, and gastrointestinal peptides, each of which relates to all areas of the gastrointestinal tract. The second section contains material describing smooth muscle physiology and gastrointestinal motility. The third section presents treatment of the functions of the stomach and pancreas. The fourth series of chapters treats the entire field of digestion and absorption. These chapters vary from basic electrophysiology and membrane transport to reviews of mechanisms leading to clinical conditions of malabsorption. The final section contains chapters on areas peripheral to physiology (such as immunology, parasitology, and prostaglandins) yet necessary for a comprehensive understanding of the subject. No one person can presume to organize and edit a scientific work of this scope. I was fortunate to enlist the aid of four preeminent scientists whose expertises cover the entire field. James Christensen was primarily responsible for the chapters on smooth muscle and motility. Eugene D. Jacobson solicited and edited most of the chapters dealing with secretory mechanisms as well as those covering many of the general topics. Chapters relating to regulation were primarily handled by Morton I. Grossman, and those covering aspects of digestion and absorption were organized and reviewed by Stanley G. Schultz. I am exceedingly grateful to these four men without whom this work would not have been possible. L.R.J.

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Preface This fourth edition of Physiology of the Gastrointestinal Tract follows 12 years after the third edition. The delay was mainly due to buyouts and mergers of the involved publishing houses, certainly not to a lack of new information. On the contrary, the explosion of information at the cellular level, made possible, in part, by the continued emergence of powerful molecular and cellular techniques, has resulted in a greater degree of revision than that of any other edition. Section I, now titled “Basic Cell Physiology and Growth of the Gastrointestinal Tract,” contains numerous new chapters on topics such as transcriptional regulation, signaling networks in development, apoptosis, and mechanisms in malignancies. Most of the chapters in the first section have been edited by Juanita L. Merchant. Section II has been renamed “Neural Gastroenterology and Motility” and has been expanded from 7 chapters with rather classic titles to more than 20 chapters encompassing not only the movement of the various parts of the digestive tract but also cell physiology, neural regulation, stress, and the regulation of food intake. Almost all of the chapters in the second section have been recruited and edited by Jackie D. Wood. Section III is entirely new and contains chapters on “Gastrointestinal Immunology and Inflammation,” which were edited by Kim E. Barrett. Section IV, “Physiology of Secretion,” consists of chapters with familiar titles but with completely updated information to reflect the advances in our understanding of the cellular processes involved in secretion. Section V, “Digestion and Absorption,” contains new chapters on the intestinal barrier, protein sorting, and ion channels, together with those focusing on the uptake of specific nutrients. These chapters have been recruited and edited by Hamid M. Said and Fayez K. Ghishan. The original purpose of the first edition of this textbook—to collect in one set of volumes the most current and comprehensive knowledge in our field—was also the driving force for this edition. As mentioned earlier, this edition includes completely new chapters that cover many new areas. Although the number of chapters has increased by 15, some chapters from the previous edition have been eliminated, some with identical titles have been written by different authors, and a few have been updated by the original authors. The final product again encompasses more than physiology. The information provided is relevant not only to the researcher in the various specialized areas but also to the clinical gastroenterologist, the teacher, and the student. The authors have done an excellent job of presenting their knowledge in a style that is readable and understandable. Much of the effort in organizing and editing these volumes has come from five preeminent scientists whose interest and expertise cover the entire field. Drs. Barrett, Ghishan, Merchant, Said, and Wood met with me to decide on chapter topics, authors, and the overall organization of the material. They were responsible for recruiting authors and for the scientific editing of most chapters. The enthusiasm and abilities of these individuals simplified my task as editor, and without them this work would not have been possible. I also am especially grateful to Philip Carpenter of Elsevier, who contacted authors, tracked submissions, and assisted me in many ways. My Associate Editors and I are all grateful to the contributing authors who were generous enough to make their expert knowledge available. Their efforts have made this work more than a mere review of past contributions to a field. The various chapters synthesize and criticize this accumulated knowledge and identify voids in it, pointing out future directions for research; many of them are superb presentations of information in fields that have been reviewed nowhere else. L.R.J.

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Acknowledgments From the organization stage to actual production the following people provided invaluable assistance, helpful suggestions, and a great deal of support. Their role and efforts have been much more than what is normally provided by a publisher, and I express my thanks to them. Jasna Markovac (Senior Vice President, Global Academic & Customer Relations) Julie Eddy (Publishing Services Manager) Lisa Royse (Production Editor) Mara Conner (Publishing Editor) Judy Meyer (Publishing Editor) Tari Broderick (Senior Publishing Editor) Cate Rickard Barr (Design Manager) Andrea Lutes (Book Designer) Patricia Howard (Senior Marketing Manager) Trevor Daul (Senior Marketing Manager) Philip Carpenter (Developmental Editor)

xxii

CHAPTER

42

Innate Immunity Lars Eckmann Sensor Molecules, 1034 Toll-Like Receptors, 1035 Nucleotide-Binding Oligomerization Domain Proteins, 1044 Summary: Sensor Molecules of Innate Immunity, 1049 Effector Molecules, 1049 Defensins, 1049 Hepcidins, 1052 Cathelicidins, 1052

Lysozyme, 1053 Secreted Phospholipase A2, 1054 Angiogenins, 1055 Other Proteins with Antimicrobial Activity, 1055 Nitric Oxide and Reactive Oxygen Species, 1055 Summary: Effector Molecules of Innate Immunity, 1057 References, 1057

The emergence of multicellular organisms more than 1,000 million years ago brought with it a critical need to defend against invasion and destruction by the abundant and diverse unicellular organisms in the environment, particularly for those multicellular organisms that have a body cavity free of microbes. In addition, many animals acquire nutrients in the form of bacteria, or at least in unavoidable conjunction with them, leading to close contact with bacteria, and hence a requirement for increased vigilance against bacterial invasion. Not surprisingly, mechanisms of host defense are found in the most ancient plants and animals. In general, such mechanisms involve detection of microbes and their subsequent removal or destruction by gene products encoded in the genome. Based on molecular and functional characteristics, the gene products and processes that constitute such host defenses are commonly divided into those comprising the “innate immune system” and those of the “adaptive immune system.” The innate immune system is characterized by an inborn capacity to recognize microbes and mount antimicrobial defenses before encounters with any specific microbe. The host molecules involved in these processes are coded for in the

germ line of the organism and are produced in functional form by many host cells, which facilitates rapid (within hours) innate immune responses to microbial infection. Furthermore, the involved molecules, and the genes coding for them, are not fundamentally altered on specific microbial encounters. Immunologic memory does not develop in the innate immune system. Historically, innate immunity also has been referred to as nonspecific immunity, but this term is imprecise and no longer appropriate given the discovery that recognition of microbial molecules by many of the sensors of the innate immune system is highly specific. Most, if not all, multicellular organisms have an innate immune system, some elements of which appear to have been present in ancient ancestors reaching back as far as the common unicellular ancestor of plants and animals more than 1,500 million years ago (1) (Fig. 42-1). The hallmark of adaptive immunity (also termed specific or acquired immunity) is the capacity to develop exquisite specificity against a wide range of antigens and to establish immunologic memory. This is achieved by generating new sensor and effector molecules that can detect and defend against microbial threats, but are not encoded in the genome of the germ line. Through a process of somatic DNA recombination, segments of specific groups of immune genes are reshuffled and edited in the host, giving rise to new genes encoding unique new proteins in the form of specific B- (immunoglobulins) and T-cell receptors in individual immune cells. Cells expressing receptors directed against a specific antigen are selected by clonal expansion and give

L. Eckmann: Department of Medicine, University of California, San Diego, La Jolla, California 92093. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1033

1034 / CHAPTER 42 Million years ago 2000+ |

1500 |

1000 |

500 |

0 | Eubacteria Archaea Plants Fungi Athropods, mollusks, nematodes Protochordates (lancelets, sea squirts) Jawless vertebrates (hagfish, lamprey)

Innate immunity

Cartilaginous fish (sharks, rays) Bony fish (zebrafish, salmon) Adaptive immunity

Reptiles (snakes, turtles, crocodiles) Mammals (humans, mice)

FIG. 42-1. Phylogeny of innate and adaptive immunity. The depicted phylogenetic tree shows major branch points in the phylogeny of different kingdoms and selected phyla, together with estimates of the time when these events occurred. Examples of species in the different phyla are given in parentheses. Innate immune mechanisms, as defined by genome-encoded receptors and effectors present in the germ line and directed against microbes, and inducible antimicrobial host defense mechanisms, are found in most, if not all, multicellular organisms (dotted rectangle), of which more than a million species are estimated to exist. Adaptive immunity, characterized by the presence of rearranging receptor and effector genes not encoded in the germ line and major histocompatibility complex genes for antigen presentation, has only been identified in jawed vertebrates (dashed rectangle), which comprise an estimated 100,000 species.

rise to immunologic memory. Only few cells with the requisite receptors exist before antigen encounter, so that the development of primary adaptive immune responses entails significant delay (several days). Subsequent encounters with the same antigen induce a more rapid and effective immunologic response. In contrast to innate immunity, adaptive immune defenses are more restricted phylogenetically, occurring only in jawed vertebrates and their descendants, which include cartilaginous and bony fish, reptiles, birds, and mammals (2,3) (see Fig. 42-1). Although innate and adaptive immunity differ in important ways, the two systems show functional overlap and interdependence. In particular, adaptive immunity is grafted onto the phylogenetically older innate immune system and is controlled and assisted by it. Innate immunity is the sole immune system of most multicellular organisms (with the exception of jawed vertebrates, which make up only a small fraction of all animals) and is fully operational in the absence of adaptive immunity. In contrast, adaptive immunity offers only weak protection without a functional innate immune system. Neither system is required for normal development in the absence of microbial challenges. In principle, any system capable of defending the host against microbial attack and overgrowth requires specific recognition of microbes as distinct from the host and subsequent measures to control and defeat them. These sensor and effector functions are mediated by groups of gene products characteristic for the immune system involved.

Innate immunity uses multiple, but at most a few hundred, germ line–encoded gene products that function as sensors for microbial signature structures and as effectors with antimicrobial activity. This chapter discusses these gene products in detail. By contrast, adaptive immunity uses products coded for by millions of somatically rearranged immunoglobulin and T-cell receptor genes as sensors and effectors, as well as class I and II molecules of the major histocompatibility complex (MHC), to present antigens to B and T cells.

SENSOR MOLECULES The gastrointestinal tract, more than any other organ in the human body, is constantly in contact with an abundant commensal bacterial microbiota and is frequently exposed to foodborne and waterborne microbes, both harmless and potentially pathogenic. This poses a great challenge for the intestinal immune system to contain and control enteric bacteria, and prevent their access to the remainder of the body, which is essentially free of microbes under normal conditions. Effective control of intestinal microbes can occur without specific recognition (e.g., killing by gastric acid), but ultimately requires recognition by the host when excessive nonspecific control mechanisms would compromise host functions (e.g., acid hypersecretion). Recognition of enteric bacteria is mediated by several mechanisms, the most important of which rely on host receptors specific for

INNATE IMMUNITY / 1035 conserved bacterial structures not found in the host. Such structures are termed molecular patterns, and when associated with pathogens are known as pathogen-associated molecular patterns (PAMPs), although commensal microbes also possess PAMPs. Examples of PAMPs include cell wall components such as lipopolysaccharide (LPS), peptidoglycans, and lipoteichoic acid, as well as bacterial flagellin and unmethylated bacterial DNA. The receptors that recognize PAMPs are referred to as pattern-recognition receptors (PRR), which on engagement activate cellular signal transduction pathways that trigger innate defense mechanisms. The two major groups of human PRRs currently recognized are the Toll-like receptors (TLRs) and the nucleotide-binding oligomerization domain (NOD)–containing proteins.

Toll-Like Receptors The fruit fly, Drosophila melanogaster, possesses a protein, Toll (named after the German word toll meaning “awesome,” based on the phenotype of the flies lacking this protein), which was originally discovered as a key regulator in embryonal development (4), but was later found also to be required for mounting fly host defense against fungal pathogens (5). Homologues of the Drosophila Toll protein with host defenserelated functions have been observed in almost all multicellular organisms, attesting to the evolutionary importance of these proteins (1,6,7). TLRs are single-spanning (type I), integral membrane glycoproteins and members of a receptor superfamily that also includes the receptors for interleukin-1 (IL-1) and IL-18, as well as several cytoplasmic adaptor molecules important for TLR and IL-1 receptor (IL-1R) signaling (8). The group members share a structurally conserved “Toll/IL-1R” (TIR) motif in the cytoplasmic domain, but they differ markedly in their extracellular domain, because TLRs contain numerous leucine-rich repeat (LRR) motifs, whereas IL-1Rs contain three immunoglobulin-like domains (Fig. 42-2). Humans have 10 TLRs (TLR1-10), whereas mice appear to have 11 Tlr genes (Tlr1-9, Tlr11, and Tlr12) (7). Human TLRs fall into several groups based on their degree of sequence homology, including the TLR2 subfamily (TLR1, TLR2, TLR6, and TLR10) and the TLR9 subfamily (TLR7-9) (Fig. 42-3). The different TLRs are variably expressed by different cell types in the gastrointestinal tract and most other organs (9), and they can recognize distinct microbial signature structures and activate innate host defense pathways. Individual TLRs are discussed in the order of their historical discovery and the available body of knowledge. TLR4 The prototypic and best-studied mammalian TLR is TLR4, the functional importance of which was first recognized through its identification as the gene responsible for genetic hyporesponsiveness to LPS in certain strains of inbred mice (10). TLR4 is predominantly expressed by hematopoietic

Leucine-rich repeats Ig-like domains

Cytoplasm

TIR domain

TLR

IL-1R

FIG. 42-2. Structure of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). TLRs and IL-1Rs are singlespanning type I transmembrane glycoproteins that share a common intracellular domain, the TLR/IL-1 receptor domain (TIR), but have unique extracellular domains. TLRs possess multiple leucine-rich repeats in their extracellular region, whereas IL-1Rs have immunoglobulin (Ig)-like domains.

cells, particularly macrophages, dendritic cells, and B cells, throughout the body, although intestinal macrophages have only low TLR4 levels under normal conditions (11,12). Stromal cells in different organs can also express the receptor. For example, endothelial cells isolated from the small intestine, colon, and liver produce functional TLR4 on their surface (13,14), as do intestinal myofibroblasts (15). Intestinal and gastric epithelial cells have been studied extensively for their TLR4 phenotype. Most commonly used human colon epithelial cell lines produce little TLR4 messenger RNA (mRNA) or protein, whereas a few other lines display significant constitutive expression (16,17). In vivo, human intestinal epithelium has low levels of TLR4 normally (11,12,18), but expression is increased under inflammatory conditions (12). Consistent with this, interferon-γ (IFN-γ) (Table 42-1), whose expression is increased in regions of inflammation, can induce TLR4 expression in certain colon epithelial cell lines (16,18). In the gastric epithelium, TLR4 appears to be expressed constitutively in antrum and corpus, and levels are increased during acute gastritis associated with Helicobacter pylori infection (19). TLR4 is required for recognition of LPS, the major constituent of the outer leaflet of the outer membrane of gram-negative bacteria (Fig. 42-4). LPS is a complex phospholipid composed of lipid A, core oligosaccharides, and sets of repeating oligosaccharides that make up the O-antigen characteristic of different groups of bacteria. Lipid A, the biologically active component of LPS also termed endotoxin, is a phosphorylated disaccharide of N-acetylglucosamine

1036 / CHAPTER 42 TLR subfamily

TLR2

Known activators

TLR

TLR1

Tri-acyl lipopeptides

TLR6

Di-acyl lipopeptides

TLR10

?

TLR2

Lipopeptides, peptidoglycans

TLR4

LPS, anthrolysin O

TLR7

ssRNA

TLR8

ssRNA

TLR9

CpG DNA

TLR3

dsRNA

TLR5

Flagellin

TLR4

TLR9

TLR3

TLR5

FIG. 42-3. Human Toll-like receptor (TLR) family. Humans have 10 TLRs, which can be grouped into 5 subfamilies based on their structural similarities (dashed boxes). Examples for major known activators of the respective TLRs are shown in the right column. CpG DNA, DNA containing unmethylated CpG motifs; dsRNA, double-stranded RNA; LPS, lipopolysaccharide; ssRNA, single-stranded RNA.

acylated with four to seven hydroxy fatty acids and serves as the hydrophobic membrane anchor of LPS (Fig. 42-5). TLR4 alone is insufficient for the recognition of lipid A and requires a complex with the secreted protein, MD-2 (20). Cells that lack either TLR4 or MD-2, or both, are unresponsive to LPS. This point is illustrated in human colon epithelial cell lines, which produce little TLR4 or MD-2. These cells cannot be stimulated with LPS, but forced transgenic expression of TLR4 and MD-2 in combination, but not individually, renders them LPS-responsive (17). MD-2 is not expressed in normal human colonic epithelium in vivo, which is consistent with the reported hyposensitivity of the cells to LPS (18). MD-2 binds LPS directly (21), but lacks a transmembrane or signaling domain. In contrast, TLR4 possesses transmembrane and intracellular domains that can transmit signals into the cell, but it is unclear whether it can bind LPS directly (22,23). In addition to LPS, TLR4 was also shown to recognize anthrolysin O, a cholesterol-dependent cytolysin from Bacillus anthracis, and other members of this family of cytolysins in gram-positive bacteria, indicating that TLR4 can recognize not only products from gram-negative bacteria, but also from gram-positive bacteria (24). TLR4 may also recognize a variety of host-derived, endogenous ligands, but this contention is controversial because unrecognized contamination with microbial products can be a serious complication in such studies (25). Although TLR4 and MD-2 are necessary and sufficient for specific LPS recognition, two other proteins, LPS-binding protein (LBP) and CD14, are involved in highly sensitive LPS detection. LBP is a liver-derived serum protein that binds LPS with high specificity and serves to concentrate it and convert it from an aggregate to a monomeric form (23). Only the latter is recognized by the TLR4/MD-2 receptor. LPS bound to LBP is delivered to CD14, which is produced by most

TABLE 42-1. Important cytokines in immunity and inflammation Cytokine

Alternative name

IL-1α IL-1β IL-6 IL-8 IL-10 IL-12 IL-18 IFN-α IFN-β IFN-γ TNF-α

CXCL-8

Type I interferon Type I interferon Type II interferon

Major functions and characteristics Induction of many genes with products that have proinflammatory functions, strong activator of IKK/NF-κB Induction of many genes with products that have proinflammatory functions, strong activator of IKK/NF-κB, cytokine requires processing by caspase-1 for biological activity Induction of acute-phase response, macrophage activation Chemoattractant for neutrophils and T cells Anti-inflammatory functions, deactivation of macrophages Induction of IFN-γ and Th1-type T-cell differentiation Induction of IFN-γ Induction of antiviral responses, suppression of inflammation Induction of antiviral responses, suppression of inflammation Induction of Th1-type immune responses, activation of macrophages and neutrophils Induction of many genes with products that have proinflammatory functions, strong activator of IKK/NF-κB

Cytokines are secreted proteins involved in communication between different cells. A selection of major cytokines important in regulating immune and inflammatory responses is listed. Only a subset of critical functions is given for each cytokine. CXCL, C-X-C chemokine ligand; IKK, inhibitor of κB kinase; IL, interleukin; IFN, interferon; NF-κB, nuclear factor-κB; TNF, tumor necrosis factor.

INNATE IMMUNITY / 1037 n

O antigen

Core

LPS

Lipid A

Outer membrane

Peptidoglycan

Periplasm

Membrane-derived oligosaccharides Inner membrane

Cytoplasm

Phospholipid

Protein

FIG. 42-4. Structure of the gram-negative bacterial cell wall. The cell wall of gram-negative bacteria is composed of two membranes, outer and inner, separated by a periplasmic space containing polymers of peptidoglycans and membrane-derived oligosaccharides. The outer leaflet of the outer membrane consists mostly of lipopolysaccharide (LPS), which has three major domains, designated lipid A, core domain, and O antigen. Lipid A is the innermost, membrane-anchoring domain and is composed of a phosphorylated and acylated disaccharide. The core domain is attached to lipid A and consists of a short chain of sugars, at least one of which, 2-keto-3-deoxyoctonic acid, is characteristic for LPS. The O antigen is the outermost and most hydrophilic region, which is linked to the core polysaccharide domain and is composed of up to 40 repeating oligosaccharide subunits with 3 to 5 sugars.

N-acetylglucosamines OH O O HO P O O O HO O NH O O Phosphate O O O group

Fatty acid

O HO O HO

O O

O NH O P OH O OH HO

3-Hydroxy fatty acids

FIG. 42-5. Structure of lipid A. Lipid A, the lipid and membrane-anchoring domain of lipopolysaccharide in gramnegative bacteria, consists of a disaccharide of phosphorylated N-acetylglucosamine acylated with four to seven saturated fatty acids of different chain lengths, of which some are attached directly to the sugars and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of lipid A is highly conserved among gram-negative bacteria and virtually constant among Enterobacteriaceae such as Salmonella and Escherichia coli.

macrophages and neutrophils, either in the form of a secreted protein or tethered to the cell membrane through a glycosylphosphatidylinositol anchor. It exhibits high affinity for LPS and, like LBP, acts as a sentinel to concentrate LPS and enhance its recognition by TLR4/MD-2. CD14 is not present in intestinal epithelial cells and is in only a small fraction (<10%) of macrophages in the normal intestinal lamina propria, which is paralleled by a general lack of LPS responsiveness in these cell populations (17,26). However, acute colonic inflammation is accompanied by an increased number and proportion of CD14-expressing macrophages in the mucosa, which is probably caused by the influx of CD14+ cells from the circulation (27–29) and is accompanied by enhanced LPS responses of those cells (30). These results indicate that intestinal epithelial cells, as well as intestinal macrophages, are hyporesponsive to LPS in the normal state, which is an attractive concept because it can explain why luminal exposure of the epithelium to LPS from the commensal microbiota is not associated with frank mucosal inflammation. Under inflammatory conditions, in contrast, expression of the critical components of the LPS receptor complex and resulting LPS responsiveness can be induced, indicating that mucosal

1038 / CHAPTER 42 sensitivity to LPS might be controlled, at least in part, by the mucosal microenvironment. The LPS receptor, TLR4/MD-2, is membrane-bound and expressed at the cell surface of macrophages and other myeloid cells, where it binds LPS. Although intestinal epithelial cells express low levels of TLR4 under normal conditions, those cells positive for TLR4 express significant quantities of the protein intracellularly in the Golgi apparatus (31). Accordingly, binding of LPS and activation of cell signaling occurs at the cell surface in macrophages, but appears to require internalization in intestinal epithelial cells (32). Engagement of TLR4/MD-2 leads to activation of two major signaling pathways; one pathway is dependent on the central signal adapter molecule, myeloid differentiation factor 88 (MyD88), whereas the other is not (Fig. 42-6). TLR4-mediated activation of MyD88 requires another adapter molecule, TIR homology domain–containing adapter protein (TIRAP; also termed MyD88-adaptor–like, Mal), and proceeds through interleukin-1 receptor–associated kinases (IRAK) 1 and 4 and tumor necrosis factor receptor–associated factor (TRAF) 6, culminating in activation of inhibitor of κB kinase (IKK) and nuclear factor (NF)κB, as well as mitogen-activated protein kinase (MAPK) and activator protein 1 (AP-1) (7). In a second pathway, TLR4 links to TIR homology domain–containing adapter inducing IFN-β (TRIF; also termed TIR homology domain–containing adapter molecule-1 [TICAM-1]) via the adapter molecule, TRIF-related adapter molecule (TRAM), which leads to activation of interferon regulatory factor 3 (IRF-3), and ultimately production of IFN-β, as well as TRAF6 leading to activation of IKK/NF-κB and MAPK/AP-1 (33). Accordingly, LPS stimulation of TLR4/MD-2–expressing cells up-regulates expression of a wide range of NF-κB and AP-1 target genes, including multiple proinflammatory and chemotactic cytokines, such as IL-8, tumor necrosis factor-α (TNF-α), and IL-6, as well as the type I IFN, IFN-β (see Table 42-1). These LPS responses are strongly enhanced by the presence of LBP and CD14. In addition to sensitizing cells to LPS activation, CD14 is required for sensing certain LPS chemotypes by TLR4/MD-2, because activation of TRIF and type I IFN production by the minimal LPS structure, lipid A, is CD14 dependent, whereas that of MyD88 and NF-κB is not (34). LPS released by gram-negative bacteria during the course of an infection, or administered directly, has profound effects on the mammalian host. Most importantly, when given at high doses, it causes endotoxic shock and death due to the release of inflammatory mediators such as TNF-α. Recognition of LPS by TLR4/MD-2 is critical for this response, because mice deficient for TLR4 or MD-2 are resistant to the lethal effects of LPS (35,36). Similarly, mice lacking LBP or CD14 survive challenge with LPS doses lethal for normal mice (37,38). Furthermore, individuals with a polymorphism of the TLR4 gene (Asp299Gly), which codes for a receptor with impaired LPS recognition, are more resistant to the airway effects of inhaled endotoxin (39). It would appear from these studies that the LPS receptor mediates mostly detrimental effects in the infected host,

MyD88-dependent TLR5/7/8/9

TLR1/2/6 TIRAP

MyD88-independent TLR4

TLR3 TRAM

MyD88 TRIF IRAK4/1 IRF-3 TRAF6 IRF-7 MAPK/AP-1

IKK/NF-κB

Pro-inflammatory cytokines

IFN-α/β

FIG. 42-6. Toll-like receptor (TLR) signaling pathways. Stimulation of different TLRs leads to recruitment and activation of multiple adapter proteins and signaling pathways, as depicted in the scheme. AP-1, activator protein-1; IFN, interferon; IKK, inhibitor κB kinase; IRAK, IL-1 receptor–associated kinase; IRF, interferon regulatory factor; MAPK, mitogenactivated protein kinase; MyD88, myeloid differentiation factor 88; TIR, Toll-like receptor/IL-1 receptor; NF-κB, nuclear factorκB; TIRAP, TIR homology domain–containing adapter protein (Mal, MyD88-adaptor-like); TRAF, tumor necrosis factor receptor–associated factor; TRAM, TRIF-related adapter molecule; TRIF, TIR homology domain–containing adapter inducing IFN-β (TICAM-1, TIR homology domain–containing adapter molecule-1).

but this is not true, because recognition of LPS serves a critical function in defense against gram-negative bacteria. Mice deficient for TLR4, MD-2, or LBP are highly susceptible to infection with the enteric bacteria Salmonella typhimurium and Escherichia coli (36,37,40–43), and Klebsiella pneumoniae, a common cause of hospital-acquired urinary tract infections, pneumonia, and burn wound infections (44). Conversely, transgenic overexpression of TLR4 promotes enhanced survival of mice upon S. typhimurium challenge (45). Individuals carrying the TLR4 Asp299Gly allele are more susceptible to severe bacterial infections (46) and septic shock caused by gram-negative bacterial infections (47). Although TLR4 contributes to immune defense against gram-negative bacteria, it is not beneficial, and may even be detrimental, in defense against other microbial pathogens. For example, successful eradication of gram-positive bacterial pathogens such as Listeria monocytogenes or Streptococcus pneumoniae in mice is not affected by the loss of TLR4 or LBP, observations consistent with the absence of LPS in the cell wall of these bacteria (48,49). Lack of TLR4 promotes immune-dependent expulsion of the intestinal nematode, Trichuris muris, from experimentally infected mice, indicating that TLR4 actually inhibits host defense against this pathogen (50). This inhibition is related to the propensity of LPS, and hence TLR4/MD-2, to enhance T helper cell 1 (Th1)type and suppress Th2-type immune defenses, the latter of which are required for expulsion of T. muris and other

INNATE IMMUNITY / 1039 intestinal nematodes. In contrast, this propensity to promote Th1-type, and inhibit Th2-type, immune responses also assigns a role to TLR4 in limiting the occurrence of Th2-type– mediated food allergies, as suggested by the observation that TLR4-deficient mice develop a more severe allergic response against a foodborne peanut allergen than normal mice (51). In addition to host defense against specific gram-negative bacterial pathogens, TLR4 may play a role in the regulation of inflammatory responses promoted by poorly defined, and possibly multiple, commensal bacteria. Several studies have demonstrated that TLR4 deficiency exacerbates experimentally induced colitis in murine models, in which the intestinal microbiota contributes to the development of disease (52,53). Consistent with a protective function of TLR4, a synthetic agonist of TLR4 was reported to attenuate inflammation in murine colitis (54). Other reports, in contrast, have found little effect of TLR4 loss on colitis severity, or even improvement (55–58). The reasons for the apparent discrepancies currently are unclear, but they may relate to the varying importance of the microbiota in modulating mucosal inflammation under different, often incompletely understood experimental conditions. This situation is not unlike that in patients with inflammatory bowel disease (IBD), where some benefit from antibiotic therapy and others do not (59). Furthermore, individuals with the Asp299Gly allele of TLR4 exhibit a significantly increased risk for development of Crohn’s disease (CD) or ulcerative colitis (UC), yet disease does not develop in most individuals with the mutation (60,61). Together, these data suggest that LPS recognition by TLR4/MD-2 can have a spectrum of functions in intestinal inflammatory disease, ranging from protection to exacerbation, the relative importance of which is probably determined by the specific pathogenetic mechanisms and the involvement of the microbiota in these processes. Any protective function of TLR4 might appear counterintuitive given the strong induction of proinflammatory mediators by LPS in cultured cells, yet is made plausible by the important role of the LPS receptor in host defense against live gram-negative bacteria, which can contribute to the development of intestinal inflammatory disease in a susceptible host (62). TLR2, TLR1, and TLR6 TLR2 functions in cooperation with other TLRs, namely, TLR1 and TLR6, which makes it relatively unique among TLRs. All three of these TLRs are constitutively expressed in monocytes/macrophages, dendritic cells, and B cells in most organs (63,64), whereas TLR2 levels are low in macrophages and dendritic cells in the normal human colon, but inducible in inflamed tissue (11,12,65). Human intestinal myofibroblasts also express TLR2 and TLR6 (15), and activated and memory, but not quiescent, murine CD4 T lymphocytes display TLR2 (9,66). TLR2 is not expressed, or is expressed at levels too low for functional relevance, in the intestinal epithelium under normal or inflamed conditions (11,12,67); also, functionally relevant TLR2 levels cannot be detected, with few exceptions, in most of the commonly

used human colon epithelial cell lines (67–69). Similarly, TLR6 expression is undetectable in normal human small intestine and only at low levels in the whole colon (9), although isolated colon epithelial cells appear to have readily detectable TLR6 mRNA expression (67). Among all of the TLRs, TLR2 can perhaps sense the broadest range of bacterial products. In particular, it is involved in recognition of diverse lipopeptides and peptidoglycans (7,70), which are complex, high-molecular-weight molecules of varying composition found in the cell wall of gram-positive and -negative bacteria (see Fig. 42-4). TLR2 also recognizes, among other microbial structures, the plasma membrane constituent of gram-positive bacteria, lipoteichoic acid (71), the mycobacterial glycolipid, lipoarabinomannan (72), and zymosan, an insoluble polysaccharide fraction of fungal cell walls (73). Most bacterial lipopeptides, which are characterized by a triacylated N terminus, bind directly to TLR2 at the cell surface in the presence of CD14 and activate cell signaling, whereas recognition of some diacylated forms of lipopeptides, such as mycoplasmal macrophage-activating lipopeptide-2, requires a combination of TLR2 and TLR6 (64,74–77). Similarly, TLR2 sensing of bacterial peptidoglycans appears to depend on intact TLR6 based on in vitro transfection studies (64,67,76), although this function is only modestly attenuated in TLR6-deficient murine macrophages (77). TLR1 cooperates with TLR2 in recognizing a subset of lipopeptides different from those sensed by TLR2/TLR6 (75,78). Taken together, these results indicate that TLR1 and TLR6 control the fine specificity of TLR2 for different lipopeptides and peptidoglycans, which forms the basis for the concept that heterodimerization of different TLRs diversifies the recognition specificity of these microbial sensors (76). Activation of TLR2 in conjunction with TLR6 or TLR1 initiates the recruitment of the adapter molecules, TIRAP, MyD88, IRAKs, and TRAF6, subsequent activation of MAPK and IKK (see Fig. 42-6), and increased expression of their respective target genes, which include TNF-α, IL-6, IL-8, IL-12, and inducible nitric oxide synthase (iNOS), depending on the cell type under study (7,79,80). TLR2 engagement can also induce apoptosis in macrophages and concomitant release of mature, biologically active IL-1β (81). Agonists of TLR2 are poor stimulators of type I IFN expression, which is in contrast with the findings with activators of TLR4 (82). Given the recognition of many diverse microbial products by TLR2, it might be expected that this receptor has critical functions in host defense against a wide range of pathogens, but this prediction is only partly borne out by the experimental evidence. In general, TLR2 is important for immune defense against gram-positive bacteria and fungal pathogens, but it has limited importance, with some exceptions, in immunity against gram-negative bacteria, which comprise most gastrointestinal bacterial pathogens. To illustrate this point, in one study, TLR2-deficient mice exhibited decreased clearance and increased mortality upon systemic infection with the enteric pathogen L. monocytogenes (83), although another study, using a different Listeria strain, could not find a unique role for this receptor in host

1040 / CHAPTER 42 defense against these bacteria (84). Both reports show that TLR2-dependent recognition of Listeria in macrophages is needed for maximal production of TNF-α and IL-12 (see Table 42-1), which are cytokines known to be important for induction of effective immunity against this pathogen (83,84). Similarly, effective host defense against the grampositive pathogens, S. pneumoniae and Staphylococcus aureus, which are major causes of meningitis in adults and of nosocomial infections in children, respectively, requires intact TLR2 functions (85,86). In contrast, eradication of the gram-negative enteric pathogen, Salmonella, is independent of TLR2 in mice (42). The apparent greater importance of TLR2 in host defense against gram-positive compared with gram-negative bacteria might be related to overlap and redundancy in TLR signaling pathways, because LPS in the cell wall of the latter, but not the former, can activate TLR4, and thus many of the same signaling pathways used by TLR2 (see Fig. 42-6). Furthermore, cell wall peptidoglycans, which are potent TLR2 agonists, are more abundant and accessible in gram-positive compared with gram-negative bacteria. In addition to antibacterial defense, TLR2 is indispensable for effective immune defense against certain fungal pathogens, such as Cryptococcus neoformans (87,88), which is consistent with the observation that the fungal cell wall fraction, zymosan, is a strong TLR2 activator (73). In contrast, TLR2 may not be universally protective against all fungal pathogens, because TLR2 deficiency appears to be associated with decreased susceptibility of mice to infection with Candida albicans in some, but not all, studies (88,89). TLR2 also has been investigated for its role in regulating inflammation in the colon and other organs in experimental models. In one report, mice lacking TLR2 were more susceptible to colon injury induced by feeding dextran sulfate sodium (52), whereas another study did not observe a significant protective function of TLR2 in colitis (55). Such differences in TLR2 involvement, as also discussed for TLR4, may be related to the relative importance of specific bacterial species of the intestinal microbiota for induction and maintenance of colitis under different experimental conditions (90). Furthermore, TLR2 can play a differential role in induction of specific types of Th responses, because TLR2 agonists can promote the development of asthma, a chronic inflammatory condition of the lungs characterized by a predominance of Th2-type immune responses (91). Such considerations may also apply to Th-dependent forms of intestinal inflammation (92). TLR9 Among human myeloid cells, TLR9 is expressed constitutively in B cells and plasmacytoid dendritic cells, but only at low levels or not at all in monocytes/macrophages, T cells, natural killer cells, or myeloid dendritic cells (63,93). Plasmacytoid and myeloid dendritic cells represent two major subsets of dendritic cells, which differ in their TLR repertoire and ability to induce different types of innate and adaptive immune responses. In particular, plasmacytoid

dendritic cells produce large amounts of type I IFNs (i.e., IFN-α and -β) in response to viruses, and thus play an important role in antiviral immunity. In comparison with humans, mice express TLR9 on a wider range of myeloid cells, which in addition to B cells and plasmacytoid dendritic cells, include monocytes/macrophages, natural killer cells, and myeloid dendritic cells (7). TLR9 also is found in epithelial cells in stomach and colon and in follicle-associated epithelial cells including M cells in Peyer’s patches of the small intestine (19,94,95). Expression of TLR9 is upregulated in B cells on cross-linking of surface immunoglobulins and in monocytes by IFN-γ stimulation or infection with Yersinia pestis (96), whereas epithelial TLR9 levels appear to be little affected by immune and inflammatory activation in colon or stomach (19,97). Systemic infection of mice with S. typhimurium caused a marked increase in TLR9 mRNA expression in the liver, suggesting that changes in receptor expression may affect TLR9 responses during the course of infection with this enteric pathogen (98). TLR9 recognizes an unmethylated cytidine-phosphateguanosine (CpG) motif (Fig. 42-7) within the context of particular hexamer sequences in bacterial DNA (99). In contrast, vertebrate DNA lacks TLR9-activating potential because of the absence of such CpG-containing motifs (100,101). Two major mechanisms account for this observation. First, the relative abundance of CG dinucleotides is significantly (fourfold to fivefold) less in vertebrate DNA than expected by chance alone (a phenomenon referred to as “CG underrepresentation” or “CG suppression”), whereas bacterial DNA, with few exceptions (e.g., Borrelia burgdorferi, the causative agent of Lyme disease), has the expected, random frequency of such dinucleotides (1/16) (102). Secondly, 70% to 80% of CG dinucleotides in vertebrates are modified by methylation of the cytosine (see Fig. 42-7), which suppresses recognition by TLR9 (100,103). Methylation of cytosine is the only known physiologic modification of DNA in human cells and is an important mechanism of epigenetic regulation, that is, stable changes in gene expression that are not related to alterations in the primary structure of DNA (104). A notable exception to the widespread DNA methylation in vertebrate DNA are the CpG islands, which are relatively small (0.5–2 kb) regions rich in unmethylated CG dinucleotides present in the upstream region of about half of all human and other vertebrate genes (105). CpG-containing DNA sequences are taken up by receptormediated endocytosis into TLR9-expressing immune cells and bind directly to TLR9 within an endosomal compartment (106–108). Binding is optimal under acidic conditions (pH ≤ 6.5), which explains the requirement for endosomal acidification (also termed endosomal maturation) for TLR9 activation (106). Single-stranded DNA stimulates TLR9 more effectively than its double-stranded form, suggesting that double-stranded DNA released from bacteria requires degradation and denaturing for optimal TLR9 activation (106). Single-stranded oligodeoxynucleotides (ODNs) containing a CpG motif are as potent as bacterial DNA in stimulating TLR9. In particular, synthetic ODNs in

INNATE IMMUNITY / 1041 NH2

R1: H, Cytosine (C) CH3, 5-Methyl cytosine (mC)

R1 N

O

N 5

CH2

O

4

O

1 N 3

O

N

2

O P

N O

CH2

O

Guanine (G)

N

NH2

R2 R2: O−, Phosphodiester S−, Phosphorothioate

O

FIG. 42-7. Dinucleotide motif recognized by Toll-like receptor 9 (TLR9). TLR9 recognizes the unmethylated dinucleotide, 5′-CG-3′, in which R1 is H and R2 is O-. Methylation of cytosine at the R1 position abolishes recognition. Phosphorothioated oligodeoxynucleotides, in which O- at R2 is replaced with S-, are more stable in aqueous solution and can also be recognized by TLR9.

which an oxygen is replaced with a sulfur in the phosphodiester backbone to yield a phosphorothioate linkage (see Fig. 42-7) are used commonly for TLR9 studies because they are resistant to hydrolytic degradation by ubiquitous nucleases. Such phosphorothioated (S)-ODNs exhibit species specificity, because TLR9-positive human cells are optimally stimulated by S-ODNs containing the hexamer motif, 5′-GTCGTT-3′, whereas murine cells are best stimulated by S-ODNs with the motif purine-purine-C-G-pyrimidinepyrimidine (e.g., 5′-GACGTT-3′ or 5′-AACGTT-3′) (109,110). Furthermore, sequences adjacent to the hexamer motif in ODNs can influence the uptake efficiency and possibly the type of cellular responses that are activated. For example, class A ODNs have centrally located palindromic CpG phosphodiester sequences and phosphorothioated G-rich ends. Although such sequences do not occur naturally, they are strong inducers of IFN-α by plasmacytoid dendritic cells and potent activators of natural killer cells, but not B cells (111). Class B ODNs are phosphorothioated sequences without palindromes that are strong B-cell activators but relatively weak inducers of IFN-α secretion (111). These chemical and biological characteristics are important in the design of stable and potent ODNs for future clinical applications (111,112). Stimulation of TLR9 leads to recruitment of the adapter molecules MyD88 and TRAF6, as well as members of the IRAK family, resulting in activation of the downstream signaling kinases, IKK and MAPK, and the corresponding transcription factors, NF-κB and AP-1, as well as IRFs (113–115) (see Fig. 42-6). Accordingly, bacterial DNA and

stimulatory ODNs induce expression of a wide range of target genes of these signaling pathways, particularly those coding for proinflammatory cytokines and other mediators, as well as type I IFNs. For example, bacterial DNA and stimulatory ODNs increase IL-8 expression in cultured human colon epithelial cell lines, although the induction is modest (twofold to threefold) compared with more potent stimuli such as TNF-α (97,116,117). In TLR9-expressing dendritic cells, CpG-containing DNA stimulates production of NF-κB target genes such as IL-6, and IL-12, and the IRF target genes IFN-α and IFN-β (see Table 42-1) (115,118). Release of IFN-α can activate expression of additional immune-related genes, such as the T cell chemokine IFN-inducible protein-10 (IP-10, also termed CXCL10), in an autocrine or paracrine manner (119). TLR9 activation promotes the survival and maturation of dendritic cells with a bias toward providing signals that commit Th cells to the Th1-type lineage, which is characterized by strong IFN-γ responses and activation of cell-mediated immune defenses (120–122). Bacterial DNA stimulates IFN-γ production in natural killer cells and enhances their cytotoxic activity (123,124). In B cells, unmethylated, CpG-containing DNA stimulates proliferation and activation, sensitizes the cells to antigen-dependent activation through surface immunoglobulins, and protects them from apoptosis (125–127). In addition to changes in gene expression, activation of TLR9 by CpG-containing ODNs triggers the degranulation of Paneth cells in the small intestine, a process associated with the release of defensins and other antimicrobial molecules into the lumen (94).

1042 / CHAPTER 42 Given the multitude of immune-related activities on cultured cells in vitro, it is not surprising that unmethylated CpGcontaining DNA has profound adjuvant effects on the immune system in vivo. Systemic administration enhances T- and B-cell responses to coadministered protein antigens (128,129) and protects against systemic infection with several pathogens, including bacteria such as L. monocytogenes (130,131) and Francisella tularensis (131), viruses such as cytomegalovirus (132,133), and fungal pathogens such as Cryptococcus neoformans (134). Common to these events is that CpG DNA promotes the development of Th1-type immune responses characterized by enhanced IL-12 and IFN-γ production, as well as the release of type I IFNs important for antiviral defenses. Moreover, CpG DNA acts as a mucosal adjuvant, because intranasal immunization of mice with a protein antigen and CpG-containing ODN, either as a mixture or as a covalently bonded conjugate, induces antigen-specific IgA production, as well as the generation of specific CD4 and CD8 T cells (135,136). Immunomodulation by CpGcontaining DNA is also observed in the context of intestinal inflammation, because administration of such DNA protects against experimentally induced murine colitis when administered before, or in the early phase of, induction (137,138), whereas treatment during severe acute colitis may exacerbate inflammation (138,139). The protective effects of CpGcontaining DNA can be mediated by TLR9 through the induction of type I IFNs acting as anti-inflammatory agents (55,140), or the development of regulatory CD4 T cells that can suppress antigen-specific immune responses (141). Together, these studies indicate that unmethylated CpGcontaining DNAs hold great promise as immunoregulatory and adjuvant agents for vaccinations and treating immunemediated diseases in humans, although it must be noted that daily long-term administration was reported to cause immunosuppression and lymphoid follicle destruction in mice, which cautions against potential side effects of drugs based on selective activation of TLR9 (142). In contrast, long-term inactivation of TLR9 appears to have no major physiologic consequences under normal conditions, because mice deficient for TLR9 are viable, healthy, and fertile (99,140). In addition to mediating the effects of exogenously administered CpG-DNA, TLR9 has critical functions in immune defense against certain viruses. Mice lacking functional TLR9 are highly susceptible to infection with cytomegalovirus, a double-stranded DNA virus that causes acute systemic infection often followed by prolonged persistence in the host, and show impaired infection-induced secretion of IFN-α/β and natural killer cell activation (132,133). Similarly, dendritic cells from TLR9-deficient mice fail to mount a normal type I IFN response on infection with herpes simplex virus (143,144), a common double-stranded DNA virus that targets stratified squamous epithelia lining the oral mucosa, conjunctiva, cornea, and skin in humans. These data suggest that TLR9 can play an important role in viral sensing, possibly through recognition of viral DNA intermediates within infected cells.

TLR5 TLR5 is expressed by epithelial cells in stomach and colon, as well as the lungs and skin (12,19,145). In addition, microvascular endothelial cells in the colon were shown to produce TLR5 (146), and the receptor is expressed by dendritic cells and macrophages, but not B cells, throughout the body and presumably in the intestinal tract, although resident intestinal macrophages may have low TLR5 levels under resting conditions (11). Epithelial expression of the receptor is constitutive, showing little regulation under conditions of colonic inflammation in vivo (12), whereas intestinal macrophages were found to express increased TLR5 levels when isolated from the inflamed intestinal mucosa of patients with CD (11). TLR5 recognizes a bacterial protein, flagellin, the major component of bacterial flagella (147). These complex appendages of the outer bacterial membrane confer motility, which is an important virulence factor in many bacterial pathogens, although some nonpathogenic bacteria are also motile. As a membrane protein at the cell surface, TLR5 binds directly to a conserved motif present in soluble flagellin monomers but not accessible in polymerized flagella (148). Ligand binding leads to activation of signaling without a requirement for intracellular uptake of the receptor (149). Signaling through this receptor is dependent on the adapter molecule, MyD88, but not TRIF (see Fig. 42-6), and leads to activation of IKK/NF-κB and MAPK/AP-1 in epithelial and other TLR5-expressing cells (147,150,151). Given the constitutive TLR5 expression by intestinal epithelial cells, particular attention has been paid to the role of these cells in the host response to flagellated gastrointestinal bacterial pathogens, such as Salmonella and H. pylori. Flagellin stimulation of cultured human colon epithelial cells induces the expression of several proinflammatory gene products, including the chemokines IL-8 and macrophage inflammatory protein (MIP)-3α, which are chemotactic for neutrophils, and immature dendritic cells or T cells, respectively (150–153). One report showed that epithelial cytokine production occurred only after basolateral, but not apical, exposure of polarized T84 human colon epithelial cells to flagellin, which was paralleled by basolateral, but not apical, expression of TLR5 (152). In contrast, other studies have found that polarized intestinal epithelial monolayers can be stimulated with flagellin from both the basolateral and apical sides, with no apparent difference in the kinetics or sensitivity of the responses (145,153). Similarly, instillation of flagellin into ligated small-intestinal loops in mice stimulated cytokine expression (145), also suggesting that the intestinal epithelium is responsive to apical flagellin exposure. The reasons for these different results are unclear, but might relate to the use of different cell culture models or culture conditions. The notion of selective apical hyporesponsiveness of intestinal epithelia to flagellin, in principle, is attractive, because the luminal presence of flagellin from commensal bacteria would not be expected to elicit constitutive

INNATE IMMUNITY / 1043 epithelial inflammatory responses under normal conditions. Only after disruption of the epithelial barrier, or on bacteriamediated transcytosis of the protein through the epithelium (154), would flagellin be expected to have access to basolateral TLR5, and thus be able to activate epithelial cells. In contrast, flagellin is not likely to be as ubiquitous as LPS in the normal microbiota; thus, it is possible that apical epithelial hyporesponsiveness may not be required to explain the apparent lack of an ongoing epithelial inflammatory response to flagellin under normal conditions. This is in contrast to the situation with the LPS receptor. Alternatively, a lack of responsiveness may also be accounted for by the possibility that prolonged exposure to apical flagellin could desensitize the epithelium to this bacterial product (155). Flagellin is produced by a wide range of gram-positive and -negative motile bacteria and is important for their motility, yet not all flagellins are equally potent in activating TLR5. Flagellin isolated from two important human gastrointestinal pathogens, H. pylori and Campylobacter jejuni, has minimal capacity to stimulate TLR5-dependent cytokine production and NF-κB activation in cultured epithelial cells (156,157). Evasion from TLR5 recognition is caused by mutations in an 8-amino-acid motif within the conserved TLR5 recognition site of flagellin, because replacement of this motif in TLR5-activating Salmonella flagellin with the motif from H. pylori flagellin abolished TLR5 recognition (157). Interestingly, this replacement also destroyed bacterial motility, indicating that compensating mutations in other flagellin regions exist to maintain motility in H. pylori (157). Thus, H. pylori and C. jejuni have evolved to evade TLR5 detection by the human host, yet remain highly motile, properties that are likely to contribute to their ability to colonize humans for prolonged periods and cause chronic inflammatory disease. Despite the extensive insights generated from cell culture studies, the physiologic importance of TLR5 is only beginning to be understood. Flagellin is a potent immunologic adjuvant on systemic administration (158), suggesting that recognition through TLR5 is probably important for mediating this function. Consistent with this notion, flagellin induces maturation and enhanced chemokine production in human dendritic cells, which are key sentinels for capturing antigens and activating T lymphocytes (159). A role of flagellin, and hence presumably TLR5, in human disease pathogenesis is also suggested by studies in patients with CD, because IgG and IgA in patient sera were shown to detect bacterial flagellin significantly more frequently than in healthy control subjects or patients with UC (160). Similarly, antibodies in sera from mice with experimentally induced colitis were more reactive with flagellins compared with those from normal mice (161). Concomitantly, flagellin-specific CD4 T lymphocytes, but not a polyclonal population of broadly specific CD4 T lymphocytes, induced colitis on adoptive transfer to immunodeficient recipient mice (161). Together, these reports suggest that flagellins can act as immunodominant antigens that stimulate pathogenetic intestinal immune reactions.

A single-nucleotide polymorphism has been found in the human TLR5 gene, C1174T, which codes for a truncated, nonfunctional protein, TLR5392STOP (162). This inactivating mutation, which functions in a dominant-negative fashion, occurs in 5% to 10% of healthy people, indicating that TLR5 is not required for normal development and health, and it may even be detrimental under certain conditions. However, TLR5 contributes to host defense against at least some flagellated bacteria, because individuals carrying inactive TLR5 exhibited increased susceptibility to pneumonia caused by the flagellated, gram-negative bacteria, Legionella pneumophilia (162). In contrast, TLR5 deficiency did not increase the risk for acquiring typhoid fever due to enteric infection with the flagellated, gram-negative bacteria, Salmonella typhi (163), suggesting that TLR5 has no unique role in host defense against these particular bacteria. A more complete understanding of the physiologic functions of TLR5 will come from additional clinical studies, as well as work in suitable animal models, although mice genetically deficient for Tlr5 have not yet been reported. TLR3 TLR3 is expressed in human dendritic cells, particularly in the immature myeloid subset (164,165), but expression is low or absent in monocytes, neutrophils, T and B lymphocytes, and natural killer cells (166). It is also produced in epithelial cells in the normal human small intestine and the surface and upper crypts in the colon (12,167), making TLR3 one of the few TLRs, together with TLR5 and TLR9, with constitutive intestinal epithelial expression under normal conditions. Mucosal inflammation associated with CD, but not UC, was reported to down-regulate epithelial TLR3 levels (12). TLR3 recognizes double-stranded RNA, either in the form of viral RNA or as synthetic RNA such as polyriboinosinicpolyribocytidylic acid [poly(I:C)], but not DNA or singlestranded RNA (168). Double-stranded RNA is considered a molecular signature of infections with many viruses, not only double-stranded RNA viruses such as rotavirus, an important cause of diarrheal disease in children, but also singlestranded RNA and DNA viruses, because many of the latter generate double-stranded RNA at some point during their life cycle. In addition to viral RNA, it has been reported that messenger RNA, released from dying host cells, can also activate TLR3 by virtue of its ability to form secondary structures containing double-stranded sequences (169), supporting the still controversial concept that TLRs can recognize host-derived ligands (25). Stimulation of TLR3 leads to strong induction of type I IFNs in dendritic and other cells, as well as multiple other cytokines, including IL-6, IL-8, IL-12, and TNF-α (see Table 42-1) (168–170). In contrast to other TLRs, with the partial exception of TLR4, induction of gene expression by TLR3 occurs through a pathway that depends on TRIF, but not MyD88 (33,171) (see Fig. 42-6). Activated TRIF recruits and activates IRF-3, leading to type I IFN production,

1044 / CHAPTER 42 and IKK, which controls NF-κB–dependent expression of multiple genes encoding proinflammatory mediators such as IL-8 and IL-12 (see Table 42-1). In bronchial epithelial cells, which also express TLR3 constitutively, activation of NF-κB by influenza A virus, a single-stranded RNA virus, or by poly(I:C) required intact TLR3 and TRIF, but not MyD88, indicating that the TLR3/TRIF pathway is critical for viral sensing in these cells (172). In contrast, recognition of poly(I:C) by cultured intestinal epithelial cells is independent of TLR3 and mediated instead by the double-stranded RNA-dependent protein kinase, protein kinase R (PKR), another sensor of viral RNA (170). A third sensor of doublestranded RNA is the RNA helicase, retinoic acid–inducible gene I (RIG-I), the activation of which induces IRF-3 and IKK in a manner independent of TLR3 or TRIF (173). These data demonstrate that several independent mechanisms exist to sense double-stranded RNA as a viral signature molecule, suggesting that the ability to recognize viral infection is of great evolutionary importance in host defense. This point is underlined by the observation that certain pathogenic viruses have evolved strategies to circumvent viral recognition pathways in the host. For example, hepatitis C virus encodes a serine protease, NS3/4A, that cleaves and thereby inactivates TRIF, with the consequence that infected cells exhibit attenuated production of cytokines with antiviral activities (174). The physiologic functions of TLR3 have been explored in murine models. Mice deficient for TLR3 were shown in one report to exhibit increased susceptibility to infection with cytomegalovirus, which was associated with reduced induction of protective cytokines, such as type I IFNs, IL-12, and IFN-γ (132). In contrast, another study could not detect a difference in susceptibility to the same virus in TLR3deficient and wild-type mice, nor did the mutant mice show increased susceptibility to three other viruses that make double-stranded RNA intermediates (175). Thus, the physiologic importance of TLR3 in antiviral host defense may vary depending on the exact experimental conditions, such as inoculum size and time after infection, and probably is also affected by functional redundancy with other sensing mechanisms for viral RNA (176). TLR3-dependent immune protection, when it occurs, can be mediated by activation of dendritic cells and induction of immunoregulatory cytokines such as IL-12 (168). In addition, TLR3 is involved in cross-presentation of antigens in dendritic cells (177,178). Presentation of antigen classically depends on the localization of the antigen relative to the antigen-presenting cells. Thus, antigen not made (“exogenous”) but taken up by an antigen-presenting cell is shuttled into a pathway that leads to presentation by MHC class II molecules on the cell surface, and hence antigen-specific activation of CD4 T cells. In contrast, antigens produced by, and present in the cytoplasm of, an antigen-presenting cell (e.g., after viral infection of that cell) are presented by MHC class I molecules at the cell surface, which stimulates antigenspecific, frequently cytotoxic CD8 T cells. In the process of cross-presentation, exogenous antigen is taken up by an antigen-presenting cell such as a dendritic cell and presented

on MHC class I (rather than MHC class II) for priming of antigen-specific CD8 T cells, the cytotoxic variety of which are important for virus eradication by lysing infected target cells. This process is stimulated by TLR3 agonists, indicating that TLR3 activation in dendritic cells broadens their capacity to process and present antigens of viruses that do not infect these cells (178). Other Toll-like Receptors: TLR7, TLR8, and TLR10 TLR7 is produced constitutively in the normal human small intestine and colon, whereas TLR8 is expressed only in the colon and not in the small intestine (9). Dendritic cells and macrophages are the main producers of TLR7 and TLR8 (179,180), although human airway epithelial cells and primary cardiac cells can be induced to express these receptors on infection with RNA viruses (181,182). Murine TLR7 and human TLR8 mediate recognition of single-stranded RNA, either of synthetic origin or in the context of an RNAcontaining virus, which leads to activation of NF-κB and its target genes, such as IL-6 and IL-12, and production of type I IFNs (see Table 42-1) (179,180,183). Both of these processes are dependent on MyD88 (180,183). Infection of human airway epithelial cells with parechovirus or of primary cardiac cells with Coxsackie B virus, both of which are singlestranded RNA viruses, induces the production of type I IFNs and IL-6 in a manner dependent on either TLR7 or TLR8, indicating that these TLRs act as viral sensors in these cells (181,182). Given their constitutive expression in the intestinal tract, it appears likely that TLR7 and TLR8 play a role in host defense against intestinal infection with RNAcontaining enteroviruses such as group A and B coxsackieviruses, or perhaps poliovirus or echoviruses, but this possibility is speculative at this time. Little is known about the functions of human TLR10. It is highly expressed in purified B cells and weakly expressed in plasmacytoid dendritic cells (184) and in the whole human colon and small intestine under normal conditions (9). The receptor appears to be functional because forced receptor dimerization in transfected cells activates NF-κB in a MyD88dependent fashion (184). However, no natural or synthetic activators of TLR10 have been described to date. Its homology with TLR1, TLR2, and TLR6 (see Fig. 42-3) and its ability to form heterodimers with TLR1 and TLR2 (184) suggest that such activators might be lipid derivatives, but this remains to be demonstrated. Notably, such studies cannot be conducted in the murine system, because mice do not possess a functional Tlr10 gene because of inactivating gaps and insertions (184).

Nucleotide-Binding Oligomerization Domain Proteins The human NOD-containing protein family consists of some 25 members related to the Ced-4/Apaf-1 family of apoptosis regulators. Ced-4 is a protein in the nematode Caenorhabditis elegans required for normal developmental

INNATE IMMUNITY / 1045 cell death. Its mammalian homologue, Apaf-1, plays a key role in mediating apoptotic cell death in response to diverse environmental and host-derived signals. Searches for other genes with structural homologies to Ced-4/Apaf-1 led to the identification of NOD1 and NOD2, two of the best characterized members of the NOD gene family (185,186). A large number of NOD proteins contain LRRs, leading to the alternative term NOD-LRR proteins (185). A further designation used for this family of proteins is CATERPILLER, based on the different structural and functional features of the family members, which can contain caspase recruitment domains, regions that act as transcription enhancers, bind purines, have similarities to pyrin domains, and contain lots of leucine repeats (186). NOD proteins have a characteristic tripartite domain architecture, consisting of an N-terminal effector domain involved in protein–protein interactions, a centrally located nucleotide-binding domain, and a C-terminal series of LRRs implicated in ligand recognition and autoregulation (Fig. 42-8). The NOD genes have structural homologies to a large family of disease resistance genes in plants, termed R genes. The proteins encoded by R genes are highly polymorphic, but structurally conserved, and mediate the recognition of plant pathogen-derived molecules (187). They constitute the major immune defense against bacteria, viruses, and fungi in plants, which lack the adaptive immune responses of higher animals. Although mammals have far less NOD genes (~25) than plants have R genes (>100), the paradigm developed for the latter appears to apply to the former, because several NOD proteins are involved in recognition of microbial signature structures. In this regard they resemble the TLRs, but in contrast to the membrane-bound TLRs, NOD proteins are localized in the cytoplasm, thus constituting a cytoplasmic surveillance system of innate immunity in the mammalian host (188). The NOD proteins are expressed in many organs including the gastrointestinal tract, although their physiologic functions in different sites are only beginning to

CARD

Human NOD1 (also termed CARD4) is a 953-amino-acid protein composed of an N-terminal caspase recruitment domain (CARD), a central NOD, and 10 C-terminal LRRs (see Fig. 42-8). NOD1 is expressed in multiple tissues, most prominently in the heart, skeletal muscle, pancreas, spleen, and ovary, as well as the placenta, lung, kidney, testis, and intestinal tract (189). In the intestine, NOD1 is produced constitutively in epithelial cells of the stomach and colon (190–192), and probably the small intestine, although that has not been shown directly (189). Its epithelial expression has been reported to be inducible by IFN-γ (190), but that observation was not confirmed in another study (191). Currently, little is known about intestinal NOD1 expression during the course of infection or inflammation. NOD1 is located in the cytoplasm, where it functions as a microbial sensor molecule. It mediates recognition of a specific diaminopimelic acid–containing dipeptide or tripeptide motif present in the cell wall of gram-negative bacteria and a few gram-positive bacteria, which is absent in eukaryotes (193–195) (Fig. 42-9). Recognition of these peptidoglycan motifs is thought to occur through the LRR domain of NOD1, although direct binding has not been demonstrated and may require other cofactors in the cell. NOD1 associates through its N-terminal CARD domain with the protein kinase, RIP2/RICK, leading to activation of IKK and NF-κB and subsequent induction of proinflammatory cytokines and other mediators (189) (Fig. 42-10). NOD1-dependent NF-κB activation can occur in the `absence of external stimulation, but is strongly enhanced by the presence of diaminopimelic acid–containing dipeptides or tripeptides (193,194). In addition, NOD1 associates with and activates caspase-9, a proteolytic enzyme involved in mediating apoptotic responses

Ligand recognition

NOD

LRRs

1

953 15

CARD1 NOD2

NOD1

Nucleotide binding and oligomerization

Protein-protein interaction

NOD1

be understood. Two of the best characterized NOD proteins relevant to intestinal physiology are NOD1 and NOD2.

106

171

CARD2

490

657

NOD

931 LRRs

1

1040 28

127

273

577

744 R702W

1020 G908R

1007fs

FIG. 42-8. Structure of human nucleotide-binding oligomerization domain 1 (NOD1) and NOD2. NOD1 and NOD2 have a tripartite structure, with one or two caspase recruitment domains (CARD) at the N-terminal end, a centrally located NOD, and a C-terminal set of 10 leucine-rich repeats (LRRs). The three major NOD2 mutations associated with CD affect the C-terminal portion at sites depicted by arrows. Numbers designate amino acid positions in the mature human proteins.

1046 / CHAPTER 42 MDP (NOD2)

GlcNAc

MurNAc

GlcNAc

L-Ala D-Glu mDAP

GlcNAc

MurNAc

D-Glu-mDAP (NOD1)

D-Ala

MurNAc

L-Ala

L-Ala

D-Glu

D-Glu

mDAP

D-Ala

mDAP

D-Ala

mDAP

D-Ala

D-Glu L-Ala

GlcNAc

MurNAc

GlcNAc

Lysozyme

MurNAc

Lysozyme

GlcNAc

MurNAc

Lysozyme

FIG. 42-9. Bacterial cell wall structures recognized by nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and lysozyme. The bacterial cell wall contains peptidoglycan polymers composed of an alternating sequence of N-acetylglucosamine (GlcNAc) and N-acetyl-muraminic acid (MurNAc), cross-linked with other peptidoglycan polymers by a bridge made of amino acids and amino acid derivatives, including L-alanine (L-Ala), D-glutamic acid (D-Glu), meso-diaminopimelic acid (mDAP), and D-alanine (D-Ala). The minimal motifs recognized by NOD1 and NOD2 are outlined in boxes (D-Glu-mDAP and muramyl dipeptide [MDP], respectively), although the tripeptide, L-Ala/D-Glu/mDAP, is recognized with threefold greater avidity by NOD1 than the minimal motif, D-Glu/mDAP. D-Glu and mDAP are absent from eukaryotes, making the dipeptide composed of these amino acids a simple signature structure of bacterial origin. Recognition and cleavage sites for the antibacterial enzyme, lysozyme, are indicated by arrows.

to diverse stimuli. Consequently, overexpression of NOD1 in epithelial cells promotes caspase-9–dependent apoptosis (189), although concurrent activation of NF-κB–dependent survival genes may limit this response under physiologic conditions. Through its CARD domain, NOD1 also forms a complex with caspase 1 (also known as IL-1β–converting enzyme), the critical protease required for cleavage of pro– IL-1β into the mature, biologically active form of IL-1β (see Table 42-1). Association of NOD1 with caspase-1 promotes processing and activation of the caspase and subsequent release of mature IL-1β without affecting apoptosis (196). The importance of NOD1 in detecting bacterial pathogens has been studied in cell line models. Infection of cultured epithelial cells with Shigella flexneri, invasive gram-negative bacteria that enter the host cell cytoplasm, causes oligomerization of NOD1 and its association with RIP2, leading to activation of IKK and c-Jun N-terminal kinase (JNK) and expression of proinflammatory cytokines (197). Mutants of NOD1 lacking CARD, which act in a dominant-negative fashion, prevent IKK and JNK activation on S. flexneri infection, indicating that NOD1 mediates important aspects of the cellular response to these bacteria (197). Similarly, in vitro infection of intestinal epithelial cells with enteroinvasive E. coli induces NF-κB activation and secretion of the neutrophil chemokine, IL-8 (see Table 42-1) (191). These responses are

abolished by overexpression of a CARD-deficient, dominantnegative mutant of NOD1, whereas the same mutant has no effect on NF-κB activation induced by stimulation with IL-1 or flagellin (191). Therefore, important epithelial responses to at least some invasive gram-negative bacteria, or their cell wall components, are dependent on NOD1, suggesting that this molecule acts as a sensor or “trip wire” of intestinal epithelial cells for a clinically important class of enteric bacterial pathogens. A similar concept may also apply for noninvasive bacterial pathogens that can attach to the gastrointestinal epithelium and deliver bacterial virulence factors into the host cell cytoplasm. The gastric bacterial pathogen H. pylori adheres to gastric epithelial cells and has a bacterial type IV secretion apparatus that mediates translocation of protein effector molecules from the bacteria into the cytoplasm of host cells. Attachment of H. pylori to epithelial cells induces NF-κB activation and secretion of NF-κB target genes such as IL-8 in humans and MIP-2 in mice (198,199). This response is partly dependent on an intact bacterial type IV secretion system and expression of NOD1 in the host cells and correlates with the presence of bacterial peptidoglycan inside the cells (192), suggesting that bacterial cell wall components are transported into the host cell cytoplasm where they are recognized by NOD1. This recognition, and the subsequent

INNATE IMMUNITY / 1047 D-Glu-mDAP Invasive bacteria (e.g., Shigella) D-Glu-mDAP

NOD1 Pro-IL-1β Caspase 1

Caspase 9 JNK

RIP2

IKK/NF-κB

IL-1β

Caspase 3

IL -1β Anti-apoptotic gene products (e.g., Bcl-XL, cIAP)

Proteolysis Proteolysis

NF-κB target genes AP1 target genes

Pro-inflammatory gene products (e.g., IL-8)

FIG. 42-10. Signaling pathways linked to nucleotide-binding oligomerization domain 1 (NOD1). Cytoplasmic NOD1 mediates recognition of the dipeptide, D-glutamic acid/meso-diaminopimelic acid (D-Glu/mDAP), derived from peptidoglycan in the bacterial cell wall. The dipeptide enters the cytoplasm after bacterial invasion into the cytoplasm and degradation of the cell wall, or possibly by direct uptake from the extracellular space. Activated NOD1 associates with and activates several components of downstream signaling pathways. Caspase-9 is a protease that activates caspase-3, a central executioner caspase, which hydrolyzes cytoplasmic and nuclear protein targets and causes cell apoptosis. c-Jun N-terminal kinase (JNK) leads to transcriptional activation of multiple target genes of the transcription factor complex, activator protein-1 (AP1). RIP2 (also termed RICK or CARDIAK) is a serine-threonine kinase that can activate IkB kinase (IKK), leading to translocation of the transcription factor, nuclear factor-kB (NF-kB), in its active form into the nucleus and subsequent transcription of its target genes with products that have antiapoptotic and proinflammatory functions. Caspase-1 converts the proform of interleukin-1b (Pro-IL-1b) into mature, biologically active IL-1b, which is released into the extracellular space.

triggering of signaling pathways, appears to contribute to effective host defense against certain strains of H. pylori, because mice deficient for Nod1, which are normally healthy and fertile with no gross abnormalities (194), exhibited greater gastric colonization on oral inoculation (192). NOD2 Human NOD2 (also termed CARD15) is composed of two N-terminal CARDs, a central NOD, and 10 C-terminal LRRs (see Fig. 42-8). Thus, NOD2 resembles NOD1 structurally with the exception that it contains two CARD domains rather than one at its N-terminal end. It is expressed predominantly in myeloid cells, particularly macrophages, neutrophils, and dendritic cells (200), as well as in Paneth cells in the small intestine (201,202), which makes its distribution

more restricted than that of NOD1. Expression of NOD2 can be induced by the proinflammatory cytokines, TNF-α and IFN-γ, in cultured intestinal epithelial cells (203). Accordingly, its expression in enterocytes is low under normal conditions, but increased under inflamed conditions (201,203). NOD2, like NOD1, is localized in the host cell cytoplasm and serves as a microbial sensor. It differs from NOD1 in its recognition specificity, because NOD2 is involved in recognition of muramyl dipeptide (MDP), a ubiquitous peptidoglycan motif present in the cell wall of gram-positive and -negative bacteria (195,204,205) (see Fig. 42-9). Thus, NOD2 senses a structural motif found in a wider range of bacteria than NOD1. Although NOD2 is commonly referred to as the MDP receptor, direct and specific binding of MDP to NOD2 has not been demonstrated. NOD2 can associate through its CARD domains with the RIP2 protein kinase (206),

1048 / CHAPTER 42 leading to activation of IKK and NF-κB and subsequent induction of genes encoding proinflammatory cytokines. NOD2 can activate NF-κB by itself in a dose-dependent manner, a function further enhanced on incubation of NOD2-expressing cells with MDP (200,207). In intestinal epithelial cells, NOD2 also interacts with GRIM-19 (208), a subunit of the NADH:ubiquinone oxidoreductase complex (209) and a suppressor of signal transducer and activation of transcription 3 (STAT3) activity (210). This interaction contributes to NOD2-dependent NF-κB activation in these cells (208). Furthermore, NOD2 has been reported to interact with transforming growth factor-β–activated kinase 1, TAK1 (211), which plays a role in IL-1– and TNF-α–induced IKK and NF-κB activation (212). Normal TAK1 function is required for NOD2-dependent activation of NF-κB, yet NOD2 appears to inhibit the ability of TAK1 to activate NF-κB in the absence of RIP2 (211). Although the ability of NOD2 to act as a microbial sensor is well recognized, and several potential signaling pathways involved in inflammation and innate immunity linked to NOD2 activation were identified in cultured cells, the physiologic functions of NOD2 are less well understood. Genetargeted mice deficient for NOD2 were reported to be healthy and fertile, indicating that the protein has no critical developmental functions (213,214). Young Nod2 knockout mice were more resistant to systemic challenge with a high dose of bacterial endotoxin (LPS) (213), whereas adult knockout mice showed a smaller difference in LPS susceptibility (213,214). However, these mice survived systemic challenge with a combination of LPS and MDP significantly better than wild-type mice (214). Furthermore, adult Nod2 knockout mice were more susceptible to oral, but not systemic, infection with the enteric bacterial pathogen L. monocytogenes (214). This was paralleled by decreased expression of the Paneth cell α-defensins, Defcr4 and Defcr-rs10, which are antimicrobial peptides that can kill Listeria and other enteric bacteria (214). Together, these data suggest that normal NOD2 mediates systemic responses to the bacterial cell wall component, MDP, and can play a role in innate intestinal defense against at least one enteric bacterial pathogen, Listeria. In support of the latter, transfection of normal NOD2 into cultured intestinal epithelial cells was shown to limit uptake or growth of another invasive enteric bacteria, Salmonella, suggesting that NOD2 can exert, directly or indirectly, antimicrobial functions in epithelial cells (215), although it should be noted that this protein is normally expressed only in Paneth cells and not other intestinal epithelial cells. NOD2 has gained prominence through its association with increased susceptibility to several clinically important human inflammatory diseases (216–219). In particular, mutations in the NOD2 gene are strongly associated with an increased risk for development of CD in North American and European populations (220). CD is one of two major forms of IBD, which is characterized by diarrhea, abdominal pain, fever, and weight loss caused by chronic, relapsing intestinal inflammation. Of the more than 60 reported NOD2 sequence

variants, three main variants, R702W, G908R, and 1007fs, exhibit the strongest CD association (220). All three variants alter the C-terminal third of the gene product and are located within or close to a region of LRRs thought to be involved in ligand recognition (see Fig. 42-8). CD-associated mutations have not been found in the 5′ end of the gene, a region that codes for two protein–protein interaction domains. Importantly, no mutations that prevent production of a nearly full-length NOD2 protein have been identified. Individuals with one of the three major disease-associated alleles have a 2- to 4-fold increased risk for development of CD, whereas homozygous or compound heterozygous carriers have a 15- to 40-fold increase in risk (220). These associations show a strong gene dosage effect and have been interpreted as being consistent with a recessive disease phenotype, although the modest but significant increase in disease risk in heterozygotes would suggest a more complex interpretation, perhaps involving dominant and recessive aspects in the functions of the variant NOD2 proteins. Despite their strong association with CD, mutated NOD2 alleles are neither sufficient nor necessary for the development of CD, because they occur in healthy individuals, with estimates most commonly around 0.5% to 2% in the general population, and 60% to 70% of patients with CD show no NOD2 mutations (221). Therefore, mutant NOD2 is a predisposing risk factor for CD, which is likely to act in synergy with other factors, both genetic and environmental, in causing disease. In contrast with CD, NOD2 mutations are not found in patients with UC, the other major form of IBD (220). Different NOD2 mutations were reported to show an association with increased susceptibility to psoriatic arthritis, a severe form of psoriasis that occurs in up to 30% of patients with psoriasis (219), although other studies did not observe such an association (222). In addition, NOD2 mutations were found in patients with Blau syndrome, a rare autosomaldominant disorder characterized by early-onset granulomatous arthritis, uveitis, and skin rash (218). Interestingly, the Blau syndrome–associated mutations affect the central nucleotide-binding domain of NOD2, rather than the Cterminal portion, suggesting an important difference in the molecular pathogenesis of Blau syndrome and CD. Furthermore, some studies have suggested a possible association between NOD2 mutations and the development of colon and breast cancer (223,224). Such association was not found in another study (225); thus, the ultimate significance of these observations remains to be established. The pathogenetic mechanisms underlying the activities of the CD-associated NOD2 variants are unclear, but the mutations are confined to the LRR domain and were reported to abolish the ability to sense MDP and activate NF-κB in transient transfection experiment (207). This suggests that the CD-associated NOD2 mutations result in a loss-of-function phenotype. Consistently, most of these mutations appear to act in a recessive manner (220). Furthermore, monocytes isolated from patients with CD carrying the 1007fs mutation were reported to exhibit defects in production of proinflammatory cytokines TNF-α, IL-6, and IL-8, as well as the

INNATE IMMUNITY / 1049 anti-inflammatory cytokine IL-10 (226,227). However, CD is associated with the presence of epithelial cells and lamina propria macrophages with activated NF-κB in their nuclei, and these cells overexpress products of NF-κB target genes, including the proinflammatory cytokines TNF-α, IL-1β, and IL-6 (see Table 42-1), under inflamed conditions (228,229). These findings appear to be inconsistent with the presumed loss of NF-κB inducibility by the NOD2 variants. Several not mutually exclusive hypotheses have been advanced to reconcile these observations and provide a mechanistic explanation for the apparent inflammation-promoting functions of the CD-associated NOD2 variants (230). They can be divided conceptually into those advocating that NOD2 variants are defective in performing critical functions required for limiting inflammation (“loss of function”), and those proposing that the mutant protein directly activates proinflammatory signaling pathways (“gain of function”). For example, lack of normal NOD2 appears to promote the production of IL-12 in macrophages stimulated with bacterial peptidoglycan (231). This cytokine is an important regulator of T-cell proliferation and promotes intestinal inflammation (232). In contrast, a CD-associated NOD2 variant was shown in a murine model to stimulate release of mature IL-1β in macrophages, which can contribute to mucosal inflammation (233). The pathophysiologic importance of these different hypotheses is an area of active investigation.

Summary: Sensor Molecules of Innate Immunity The foregoing discussion highlights the physiologic importance of multiple, genome-encoded sensor molecules in innate immune defense against a wide spectrum of gastrointestinal and other microbial pathogens. Through sensitive and specific detection of a range of molecules characteristic for whole classes of microbes, such as conserved components of the bacterial cell wall, innate immune sensors are indispensable for rapid activation of defenses against microbes that threaten the health and survival of the host.

EFFECTOR MOLECULES Host defense against commensal and pathogenic microbes requires not only the ability to detect their presence, but also mechanisms of controlling and killing them. In fact, few, if any, enteric bacteria reach systemic sites under normal conditions. This remarkably effective barrier is achieved through multiple layers of physical separation and chemical host defense. Mammals possess a diverse set of antimicrobial molecules, including inorganic “disinfectants” (e.g., hydrogen peroxide, nitric oxide), and specialized peptides (e.g., defensins) and proteins (e.g., lysozyme). Prominent among these are evolutionarily conserved, small antimicrobial peptides, which fall into two major groups based on structural features, peptides with a β-sheet structure, and peptides with a linear α-helical conformation (234). The former is by far the

larger group in mammals and consists of defensins and hepcidins, whereas the latter has fewer mammalian members, composed mostly of the cathelicidins. They all have broadspectrum antimicrobial activities, and many are expressed in epithelial cells within the intestinal tract.

Defensins Defensins are small (2–6 kDa) cationic peptides with antimicrobial activity. They have three characteristic pairs of intramolecular disulfide bonds and a β-sheet structure (Fig. 42-11). Although the specific mechanisms of action vary somewhat between different defensins, they all cause microbial “death by lysis” through disruption of the integrity of bacterial membranes, which differ from host cell membranes in their lack of cholesterol and high content of negatively charged phospholipids. Based on the arrangement and spacing of disulfide bonds, defensins are divided into two major groups, α- and β-defensins (see Fig. 42-11). a-Defensins and Cryptdin-Related Sequences Comparative genome analysis has shown that humans carry 11 α-defensin genes, 6 of which (DEFA1-6) are expressed, whereas the remaining 5 (DEFA7P-11P) are definitely or most likely pseudo-genes (235). All 11 genes are located in a continuous cluster spanning 132 kb on chromosome 8 (235). Mice carry a larger repertoire of α-defensin genes (termed cryptdins), consisting of 26 genes, Defcr1-26, and at least one pseudo-gene, Defcr-ps1 (235). In addition, mice, but not humans, have a family of cryptdin-related sequence (CRS) peptide genes that comprise some 20 members with similarities to α-defensin genes (235,236). CRS peptides can form covalently linked homodimers and heterodimers after synthesis, which further increases the structural diversity of this group of antimicrobial peptides (236). Expression of α-defensins is restricted to only a few cell types, which in humans are predominantly neutrophils and Paneth cells located in the crypts of the small intestine. Of the six expressed human α-defensin genes, four (DEFA1-4, coding for human neutrophil peptides, HNP 1-4) are found in neutrophils, whereas DEF5 (coding for the human defensin, HD5) and DEFA6 (coding for HD6) are expressed predominantly in Paneth cells (237). HD5 also can be found in smallintestinal intermediate cells, which are villous epithelial cells with a resemblance to goblet cells (238). In addition, HNP 1-3 have been demonstrated in intestinal epithelial cells in patients with active IBD (239), although it is unclear whether these results indicate that enterocytes up-regulate α-defensin expression in response to inflammatory signals or take up peptides from the microenvironment after release by neutrophils. In mice, Paneth cells produce multiple cryptdins, as well as CRS peptides, whereas their neutrophils do not synthesize appreciable amounts of α-defensins (240). α-Defensins are synthesized as precursor proteins that need to be processed to become biologically active. For example,

1050 / CHAPTER 42 α-defensins - - - Cys1 - - - Cys2 - - - - - - Cys3 - - - - - - - - - - - - - - Cys4 - - - - - - - - - Cys5 - Cys6 -

β-defensins - - - Cys1 - - - Cys2 - - - - - - Cys3 - - - - - - - - - - - - - - Cys4 - - - - - - - - - Cys5 - Cys6 -

Hepcidins/LEAP

- - - - - - Cys1 - - Cys2 Cys3

- Cys4 Cys5

- - - - Cys6 - - Cys7 Cys8

--

FIG. 42-11. Secondary structure of mammalian β-sheet–containing antimicrobial peptides. Antimicrobial peptides with a β-sheet conformation are characterized by pairs of cysteines that form intramolecular disulfide bonds. Defensins have three disulfide bonds, whereas hepcidins (also termed LEAP [liver-expressed antimicrobial peptides]) have four. The depicted localization of the disulfide bonds in the defensins determines the subgroup to which each defensin belongs.

HD5 is stored as an inactive propeptide in human Paneth cells and is processed on secretion from the cells. The processing enzyme is trypsin, because it colocalizes with HD5 in the small intestine and cleaves the propeptide to mature forms identical to those found in vivo (241). In contrast, in mouse Paneth cells, matrix metalloproteinase-7 (also termed matrilysin) mediates constitutive processing and activation of α-defensins (242). Mice deficient for matrilysin lack mature, bioactive cryptdin peptides in the small intestine (243). In addition to posttranslational processing, defensin production also can be regulated at the expression level. For example, oral infection of mice with virulent S. typhimurium caused down-regulation of cryptdin 1 expression in the small intestine within less than 24 hours (244), indicating that enteric pathogens may have developed strategies to attenuate the chemical barrier formed by α-defensins in the small intestine. Functional studies of α-defensins to date have focused primarily on in vitro approaches. These peptides exhibit antimicrobial activity against a wide range of gram-negative and -positive bacteria, as well as certain fungi, protozoan parasites, and enveloped viruses. For example, the human Paneth cell α-defensin, HD5, is microbicidal for the gramnegative bacteria E. coli and S. typhimurium, the gram-positive bacteria L. monocytogenes, and the fungus C. albicans (245). One or several of the human neutrophil α-defensins, HNP1-3, kill the protozoan small-intestinal parasite Giardia lamblia (246) and inactivate enveloped viruses, such as Herpes simplex virus, but not nonenveloped viruses (247). Murine cryptdins are bactericidal for E. coli, L. monocytogenes, and attenuated strains, but less so for virulent strains, of S. typhimurium (248). The antimicrobial activity of α-defensins is little affected by changes in pH or the presence

of proteases, such as trypsin, that are active in the smallintestinal lumen into which Paneth cell defensins are secreted (245). Furthermore, many α-defensins are bactericidal at low micromolar concentrations (10–100 µg/ml), which is well below the levels predicted to be present inside phagolysosomes of neutrophils or in the lumen of small-intestinal crypts upon Paneth cell activation (249). Surprisingly, physiologic salt concentrations (e.g., 100 mM NaCl) markedly inhibit the bactericidal activity of purified α-defensins (245,246), which raises the question how these evolutionarily conserved peptides can be active under physiologic conditions. A definitive answer to this question currently is unavailable, but it is possible that the local salt concentrations are low in those sites in which α-defensins might be active, such as the lumen of small intestinal crypts, or that other factors present in such sites modify the salt sensitivity of α-defensins. α-defensins have activities other than their established antimicrobial actions. For example, a mixture of HNP1-3 inhibited endothelial cell adhesion, migration, and proliferation in culture and capillary-like tube formation in a threedimensional fibrin gel (250). These data indicate that neutrophil-derived α-defensins might control neovascularization, and hence provide a link between inflammation and angiogenesis. Murine cryptdin 3 was shown to induce secretion of the neutrophil chemokine IL-8 in cultured human intestinal epithelial cells, suggesting that Paneth cell cryptdins can act as paracrine regulators of innate epithelial inflammatory responses (251). The physiologic relevance of these findings remains to be established. Such studies are complicated by the unavoidable complexities of using in vivo models for defining the physiologic importance of a large group of closely related peptides that are likely to exhibit varying

INNATE IMMUNITY / 1051 degrees of functional overlap. Disruption of the α-defensin– processing enzyme, matrix metalloproteinase-7 in mice, leads to increased acute susceptibility to oral Salmonella infection because of the lack of bioactive cryptdin peptides in the small intestine (243). Conversely, transgenic expression of the human α-defensin, HD5, in mouse Paneth cells was shown to increase resistance of mice to oral challenge with virulent Salmonella (252). Together, these data demonstrate a physiologic role of α-defensins, as a group, in innate immune defense against an important enteric pathogen. Currently, the functions of individual α-defensins and of CRS peptides, particularly as they pertain to intestinal physiology, are poorly understood.

b-Defensins A definitive human and murine genome analysis of β-defensin genes has not yet been reported, but humans have at least 10, and possibly as many as 31, β-defensin genes (DEFB) located in five separate gene clusters (253). Several of these are most likely pseudo-genes. Mice have at least 14 validated and expressed β-defensin genes (253). Interestingly, β-defensin genes are subject to strong positive selection pressures, as indicated by high rates of synonymous versus non-synonymous substitutions among family members (253). Consequently, β-defensins are among the most divergent groups of genes between humans and mice, suggesting that the evolutionary pressures in the form of microbial challenges facing these species varied considerably. β-Defensins are prominently expressed in organs with epithelial surfaces, particularly the intestine, lungs, skin, kidneys, and testis, suggesting that they protect the host from exposure to environmental microbes. In the intestinal tract, DEFB1 (coding for human β-defensin, hBD-1) is constitutively expressed in small-intestinal and colonic epithelial cells, but not up-regulated by proinflammatory stimuli or infection with invasive bacteria (254). Infection of intestinal epithelial cells with the protozoan parasite, Cryptosporidium parvum, in vitro and in vivo caused down-regulation of hBD-1 or its murine counterpart mBD-1 (255). Combined with the demonstration that hBD-1 can kill C. parvum sporozoites in vitro (255), these data suggest that suppression of epithelial β-defensin expression might be a virulence strategy used by this clinically important pathogen of the small intestine. hBD-2 differs in its regulation from hBD-1, because it is not expressed constitutively in the normal intestinal mucosa, but it is strongly inducible by infection with enteric pathogens or stimulation with proinflammatory mediators such as IL-1 and under conditions of intestinal inflammation, particularly in patients with UC (254,256). Consistent with its inducibility by inflammatory mediators, hBD-2 functions as a target gene for NF-κB in epithelial cells, one of the key transcription factors responsible for controlling expression of a wide range of inflammation-associated gene products (254). Discrepant data have been reported on the intestinal expression of hBD-3. Two studies used polymerase chain reaction analysis of RNA from whole intestinal biopsies to

show that it is rarely expressed in normal human colon (256,257), but is strongly induced in inflamed areas of patients with CD and UC (256). In contrast, a different study applied the same methodology to isolated epithelial cells, and also used in situ hybridization techniques, to demonstrate that hBD-3, as well as hBD-4, is expressed constitutively in normal small-intestinal and colonic epithelium, with greater expression in crypt compared with surface cells (258). Expression of both defensins was markedly increased in epithelial cells from patients with UC, but there was significantly less or no expression in patients with CD (258). Differences in the starting materials and assay sensitivity may account for the apparent discrepancy in the results from normal tissues (256,258). In addition to hBD-1 to -4, hBD9 and hBD-31 appear to be expressed in the small intestine but not colon under normal conditions (259), whereas little is known about the expression of other β-defensins in the human intestinal tract under normal or inflamed conditions. Taken together, these data indicate that expression of the β-defensins analyzed in detail so far appear to fall into two categories in regard to their regulation within the intestinal tract: hBD-1 is a highly expressed constitutive epithelial β-defensin with little capacity for further up-regulation, whereas hBD-2, hBD-3, and hBD-4 exhibit varying degrees of constitutive epithelial expression and strong inducibility by microbial and inflammatory stimuli. β-defensins, like α-defensins, can kill a wide spectrum of microbes in vitro, although this activity of some of these peptides is diminished in the presence of physiologic salt concentrations (257,260). The apparent salt sensitivity of some β-defensins raises questions about the physiologic functions of these peptides, which currently have not been resolved. Mice deficient for mBD-1 (mDefb1) are healthy and fertile, but have increased numbers of bacteria in the urine at baseline, particularly Staphylococcus species (261,262), and greater numbers of Haemophilus influenzae in the lungs 24 hours after intranasal challenge (262). The mice showed no impairment in the clearance of Staphylococcus aureus from the airways (261). These data suggest that at least one of the β-defensins, mBD-1, appears to play a nonredundant role in innate immune defense against specific bacteria, although no studies have been reported to date on the intestinal phenotype of these mice. Given the large number of β-defensin genes and their similar antimicrobial activities in vitro, it appears likely that more detailed studies will reveal varying degrees of functional overlap between the different β-defensins depending on the microbes, route of challenge, and inflammatory status of the tissue. In addition to their antimicrobial activity, some β-defensins have immunoregulatory actions. For example, mBD-2 was reported to act directly on immature dendritic cells as an endogenous ligand for TLR4, inducing expression of costimulatory molecules and cell maturation (263). hBD-2 is chemotactic for immature dendritic cells and memory T lymphocytes, and this effect is mediated by the CC chemokine receptor 6 (264). Interestingly, several chemokines, particularly CXCL9-11, have antimicrobial activity in vitro (265),

1052 / CHAPTER 42 suggesting that a functional overlap exists between defensins and chemokines (266).

Hepcidins Hepcidins, also termed liver-expressed antimicrobial peptides (LEAPs), are 20- to 25-amino-acid-long peptides characterized by four pairs of cysteines that form intramolecular disulfide bridges (see Fig. 42-11). Humans produce two peptides, hepcidin/LEAP-1 (267,268) and LEAP-2 (269), whereas mice have at least three of these peptides, hepcidin 1, hepcidin 2, and LEAP-2 (269,270). All three hepcidins are expressed at high constitutive levels by hepatocytes in the liver (267,269,270) and are found at varying levels in serum and urine (267,271). Although the liver is the major hepcidin producer, hepcidin/LEAP-1 also is expressed in kidney epithelial cells in humans and mice (272), hepcidin 2 in the murine pancreas (270), and LEAP-2 in the human kidney and small intestine (269). Furthermore, expression of hepcidin/LAP-1 is up-regulated by iron overload (273) and inflammatory signals, particularly those associated with the induction of an IL-6–dependent acute-phase response (274–276), whereas hypoxia suppresses liver hepcidin expression in mice (277). Hepcidin/LEAP-1 and LEAP-2 have fairly broad in vitro antimicrobial activity against gram-positive bacteria, such as Streptococci and Staphylococci, and fungi including Candida albicans, Aspergillus, and Saccharomyces cerevisiae, whereas their activity against gram-negative bacteria is limited to certain species such as E. coli, but not others such as Pseudomonas aeruginosa (267,268). Little is known about the physiologic significance of these antimicrobial activities of hepcidins. However, hepcidin/LEAP-1 does have a physiologic function that is unrelated to its direct antimicrobial activity. Thus, mice lacking this protein because of a defect of a critical transcription factor required for its constitutive expression exhibit an iron overload syndrome (278). Similarly, inactivating mutations in human hepcidin/LEAP-1 are associated with severe juvenile hemochromatosis, an inheritable form of an iron overload syndrome (279). Conversely, mice engineered to overexpress hepcidin in the liver show anemia caused by iron deficiency (280). These results indicate that hepcidin/LEAP-1 is a negative regulator of iron hemostasis in vivo. This activity can be explained by the ability of hepcidin to down-regulate iron uptake in intestinal epithelial and other cells (281). Thus, induction of hepcidin by inflammatory signals constitutes a mechanistic link between the host inflammatory response and hypoferremia and anemia that are commonly associated with microbial infection and inflammation (275,282). The iron-depleting functions of hepcidin/LEAP-1 may also be important in antimicrobial host defense, because many microbial pathogens depend on iron scavenging from the host for growth and virulence (283). Limiting the availability of free iron in blood and tissue reduces the opportunity for microbes to establish infection.

Cathelicidins Cathelicidins are cationic antimicrobial peptides in which a highly conserved N-terminal structural domain, cathelin, is linked to a C-terminal peptide with antimicrobial activity (Fig. 42-12) (284,285). Some 30 different cathelicidins have been identified in mammals, but only one in humans, human cationic protein 18 kDa (hCAP18)/LL-37, and one in mice, cathelin-related antimicrobial peptide (CRAMP). LL-37, the C-terminal part of the human hCAP18/LL-37 molecule that is cleaved and has antimicrobial activity, has a linear amphipathic α-helical structure (234). hCAP18/LL-37 is expressed in neutrophils, mast cells, and epithelial cells. In the gastrointestinal tract, the protein is produced constitutively in differentiated surface and upper crypt epithelial cells in the colon and in Brunner’s glands in the duodenum, but not in the deeper regions of colon crypts or epithelial cells of the small intestine (286). Constitutive LL-37 production in differentiated intestinal epithelial cells does not require a commensal microbiota, because it occurs in human colon xenografts free of bacteria (286). Furthermore, its epithelial expression is not upregulated in response to a range of inflammatory stimuli in vitro or under inflamed conditions in vivo (286), which is in contrast with the findings in keratinocytes (287). LL-37 is also produced in the stomach by surface epithelial cells, as well as chief and parietal cells, and is found in gastric juice (288), suggesting that it might be active on passage into the proximal small intestine. In addition to epithelial cells, LL-37 is found in neutrophils when they are present in inflamed areas of colon mucosa.

Conserved preproregion Signal peptide Pre

Variable antimicrobial domain

Cathelin domain Pro

29 -30 aa Signal peptidase

Peptide

94 -114 aa

12 -100 aa Processing enzyme

Stored in granules

FIG. 42-12. Structure of mammalian cathelicidins. Mammalian cathelicidins are composed of three domains, signal peptide, cathelin domain, and antimicrobial domain; their lengths are given as approximate ranges. The signal peptide (Pre) is required for intracellular targeting into granules and is cleaved off by a signal peptidase. The conserved cathelin domain (Pro), of which the function is poorly understood, remains attached to the antimicrobial domain during granule storage. Appropriate cell stimulation induces cleavage between the cathelin and antimicrobial domains by a specific processing enzyme and release of the biologically active antimicrobial peptide. Several processing enzymes have been identified in different species and tissues, including proteinase 3 in human leukocytes and gastricsin in human vaginal fluid. aa, amino acids.

INNATE IMMUNITY / 1053 Proteolytic cleavage of the preproprotein hCAP-18/LL-37 is required for formation of bioactive antimicrobial peptides (see Fig. 42-12). In human neutrophils, LL-37 is derived by proteolytic processing through proteinase 3 (289), whereas different or additional proteases appear to be important in other sites. For example, serine proteases present in human sweat generate additional, shorter peptides from LL-37 with increased antimicrobial activity (290). In the vagina, the serum protease gastricsin can process hCAP-18 into ALL-38, a slightly longer C-terminal peptide with antimicrobial activity (291). Although little currently is known about LL-37 processing in the intestinal tract, the findings in skin and the vagina suggest that postsecretory processing of hCAP-18/ LL-37 can enhance antimicrobial potency and perhaps alter the spectrum of microbial targets. LL-37 kills a wide range of gram-positive and -negative bacteria, fungi, and protozoa through disruption of microbial membranes (292–294). Besides their antimicrobial activity in vitro, the physiologic functions of human and mouse cathelicidin have been studied more extensively than those of most other antimicrobial peptides. This has been done for several reasons, foremost of which is that humans and mice possess only single cathelicidin genes, hCAP-18/ LL-37 and CRAMP, respectively. For mice, this has allowed the generation of a knockout mutant mouse with little complications because of functional compensation through other, closely related gene products. Furthermore, neutrophils in mice produce CRAMP but not defensins, further simplifying the functional analysis of this protein. Mice lacking CRAMP are more susceptible to cutaneous infection with group A Streptococci, most likely because of the inability of neutrophils to kill the bacteria (295). In addition to neutrophils, murine macrophages also express CRAMP, and production is increased on infection with the invasive enteric bacteria S. typhimurium (296). Macrophages lacking CRAMP have an impaired ability to restrict Salmonella division, and hence survival inside the cells, indicating that this peptide is an important component of the antimicrobial arsenal of macrophages (296). Furthermore, CRAMP is expressed constitutively in colon epithelial cells and contributes to acute innate host defense against bacteria that can adhere closely to the epithelium and form attaching/ effacing lesions (297). LL-37 has several host-autonomous activities not related to its effects on microbes. It has been shown to bind bacterial LPS; act as a chemoattractant for monocytes, neutrophils, mast cells, and certain T-lymphocyte subsets in vitro (284), and promote angiogenesis through the formyl peptide receptor-like 1 expressed on endothelial cells (298). The latter data suggest that LL-37 is a multifunctional antimicrobial peptide that links host defense and inflammation with angiogenesis (285). In addition, dendritic cells cultured in the presence of LL-37 display increased endocytic capacity and costimulatory molecule expression, modified phagocytic receptor expression and function, and enhanced Th1-inducing cytokine secretion (299). These data indicate that LL-37 found at inflammatory sites may be a potent modifier of

dendritic cell differentiation, and thus a link between innate and adaptive immunity. Besides its interaction with formyl peptide receptor-like 1, LL-37 can activate additional host cell receptors on monocytes and epithelial cells. Incubation of human airway epithelial cells with LL-37 activates MAPK/ extracellular signal–regulated kinase (ERK) and stimulates IL-8 secretion through transactivation of the epidermal growth factor receptor (300). Similarly, LL-37 induces expression of IL-8, as well as several other chemokines, in isolated human keratinocytes (301). Furthermore, LL-37 stimulation of LPSprimed human monocytes induced maturation and release of IL-1β via the purinergic receptor, P2X7 (302). Together, these results suggest that LL-37 can promote acute inflammatory events through direct or indirect activation of chemokine expression, a potentially attractive concept given that LL-37 is expressed by colon epithelial cells and may be released from the cells on microbial contact. However, it is unlikely that every in vitro activity revealed to date for LL-37 has a relevant physiologic counterpart in vivo; thus, mechanistic studies in suitable animal models will be required to place in vitro findings into proper perspective. This is particularly true for the physiologic functions of LL-37 or CRAMP in the intestinal tract.

Lysozyme Lysozyme (also termed N-acetyl-muramylhydrolase or muramidase) is a small (14-kDa) cationic protein that exerts antibacterial activity through its ability to hydrolyze peptidoglycans in the bacterial cell wall. Humans possess a single lysozyme gene, whereas mice have two closely related genes, lysozyme M and P (303–305). The former is the orthologue of the human gene, whereas the latter is a closely related gene with 92% sequence identity to lysozyme M and an adjacent chromosomal localization, suggesting that it arose through a relatively recent gene duplication event (305). Lysozyme is produced in human neutrophils and macrophages, as well as at mucosal surfaces, particularly in alveolar type II epithelial cells in the lungs and Paneth cells in the small intestine. In patients with UC, CD, or celiac disease, lysozyme expression is induced in goblet cells in the colon, small intestine or both (306,307), indicating that production is inducible in intestinal epithelial cells other than Paneth cells. In mice, lysozyme M is expressed constitutively in neutrophils and macrophages, and at epithelial surfaces with the exception of the small intestine, where lysozyme P predominates. Expression can be regulated under certain conditions, because oral infection of mice with a virulent strain of S. typhimurium down-regulated small intestinal lysozyme expression within 24 hours (244). These findings show that enteric pathogens may have strategies to attenuate lysozyme-dependent host defense. Lysozyme hydrolyzes the glycosidic linkage between the two major building blocks, N-acetylglucosamine and N-acetylmuramic acid, in the peptidoglycan of the bacterial cell wall (see Fig. 42-9). This structure is exposed and readily

1054 / CHAPTER 42 accessible to the enzyme in gram-positive bacteria, but not in gram-negative bacteria, which are enveloped by a lipid membrane layer that prevents access to the underlying cell wall peptidoglycans (see Fig. 42-4). In addition, peptidoglycans make up a much greater portion of the cell wall of gram-positive compared with gram-negative bacteria (90% vs 10–20% by weight). Consequently, lysozyme generally is inhibitory to gram-positive but not gram-negative bacteria, although exceptions exist for both groups of bacteria (308). The lysozyme-mediated loss of a rigid cell wall, which functions as a bacterial exoskeleton, renders the bacteria highly susceptible to osmotic and mechanical stresses commonly encountered in the intestinal lumen and upon uptake into professional phagocytes. In addition to its hydrolytic activity, lysozyme can inhibit or kill bacteria by nonenzymatic means. Its highly cationic nature promotes interactions with bacterial cell wall components that lead to activation of bacterial enzymes, called autolysins, which can degrade the cell wall (309). Furthermore, partial proteolytic degradation of lysozyme can liberate small antimicrobial peptides (310), suggesting that lysozyme, on release by Paneth cells into the intestinal lumen, can continue to exert some inhibitory activity on enteric bacteria despite proteolytic attack by digestive enzymes. Lysozyme can also synergize with other antimicrobial molecules, such as lactoferrin, in killing a range of enteric bacteria, particularly gram-negative ones such as Vibrio cholerae, Salmonella, and E. coli (311). Genetic defects in lysozyme have not been reported in humans, but genetically engineered mice lacking lysozyme M are more prone to prolonged subcutaneous infection with the gram-positive bacteria, Micrococcus luteus (312). These bacteria are normally harmless, indicating that lysozyme contributes to constitutive host defense against nonpathogenic bacteria in the skin. Likewise, transgenic overexpression of lysozyme M in the respiratory epithelium protected mice against intratracheal infection with the gram-positive pathogenic bacteria, group B streptococci, as well as the gramnegative bacteria, Pseudomonas aeruginosa (313). Given that lysozyme is a major secretory product of small intestinal Paneth cells, it is likely that it exerts protective functions against colonization with enteric bacteria, although direct evidence for this notion currently is lacking. Beyond its physiologic importance, lysozyme, particularly hen egg white lysozyme, is used as a food preservative because of its broad antimicrobial range, high physical and chemical stability, and proven safety (308).

Secreted Phospholipase A2 Phospholipase A2 (PLA2) is an enzyme that catalyzes the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids. Humans possess some 20 PLA2 isoforms, many of which show only limited sequence identity and vary widely in their expression pattern and functions in lipid metabolism and cell signaling (314–316). Based on structural characteristics, calcium

requirements, and cellular localization, PLA2 is classified into three major groups: (1) low-molecular-weight (13- to 17-kDa), calcium-dependent, secreted enzymes (sPLA2); (2) high-molecular-weight (50- to 100-kDa), calcium-dependent, cytosolic enzymes (cPLA2); and (3) calcium-independent, intracellular enzymes (iPLA2). Of particular interest for intestinal physiology is the group of sPLA2 genes, which has at least 11 members in humans (and 1 pseudo-gene) and 12 members in mice (315). One of these, group Ib sPLA2, is produced by pancreatic acinar cells and secreted in the pancreatic juice into the intestinal lumen where it aids in fat digestion, particularly under conditions of a high-fat diet (317). Another PLA2, group IIa sPLA2, is expressed predominantly in Paneth cells in the normal small intestine and released into the lumen on stimulation with cholinergic agonists or bacterial LPS (318). The gene also is expressed in enterocytes in the small intestine and colon under conditions of intestinal inflammation (319,320), as well as in macrophages and platelets in different organs, in lacrimal gland cells, and in hepatocytes as an acute-phase protein (316). Group IIa sPLA2 is a small (14-kDa) cationic protein that exerts antibacterial functions by virtue of its ability to degrade phospholipids in the bacterial membrane (321). It has a marked preference for anionic phospholipids, such as phosphatidylglycerol, characteristically present in bacterial, but not eukaryotic, membranes. Consequently, the enzyme has minimal PLA2 activity against membrane lipids in eukaryotic cells. Like lysozyme, group IIa sPLA2 is most active on gram-positive bacteria such as L. monocytogenes and S. aureus, where it can gain direct access to the bacterial membrane, but it is less active in gram-negative bacteria where the outer lipid layer prevents contact with the inner membrane (321–323). However, in synergy with other host-derived molecules, such as bactericidal/permeabilityincreasing protein or complement components, which permeabilize the outer membrane of the gram-negative bacterial cell wall, group IIa sPLA2 can also exert antibacterial activity on gram-negative bacteria (324). Furthermore, sublethal doses of cell wall–weakening antibiotics, or lysozyme, enhance the bactericidal effects of the enzyme on gram-positive bacteria. These observations underline the fact that innate host defenses often act synergistically in killing bacteria, a concept that also has great therapeutic relevance for the optimal use of antibiotics, particularly as more drug-resistant bacterial strains emerge (321). The physiologic functions of group II sPLA2 in vivo are less well understood than their in vitro activities. The C57BL/6 strain of mice is naturally deficient in group II sPLA2, but has no apparent immunodeficiency. Transgenic overexpression of human group IIa sPLA2 in these mice renders them more resistant to systemic challenges with the gram-positive bacteria S. aureus (325), as well as the gram-negative E. coli (326), although it must be noted that serum enzyme levels in uninfected transgenic mice were 100-fold greater than those found in healthy humans (321). Nonetheless, these results indicate that human group IIa sPLA2 can contribute to innate host defense against different bacteria.

INNATE IMMUNITY / 1055 Angiogenins Angiogenins are a family of host defense–related ribonucleases, which were originally discovered as angiogenesisstimulating proteins (327). The family has four members in mice, Ang1-4, but only one immediate member in humans, ANG, although additional human genes exist with products that exhibit related activities (328). Through comparison of intestinal mRNA expression profiles in germ-free and bacterially colonized mice, Ang-4 mRNA expression was found to be induced in the small intestine by microbial colonization (329). The gene product was localized to the secretory granules of Paneth cells and secreted from the cells on LPS stimulation. Synthetic Ang-4 killed a selective spectrum of bacteria in vitro (329). In addition to Paneth cells, angiogenin can also be expressed constitutively in colon cancer cells, with further induction on stimulation with the proinflammatory cytokines TNF-α and IL-1 (330). Greater levels of tumor angiogenin expression in patients with colorectal cancer were correlated with a greater density of microvessels and a worse prognosis, suggesting that this protein contributes to disease progression by promoting tumor vascularization (330). Furthermore, consistent with angiogenin inducibility by inflammatory signals, patients with active IBD show increased serum levels of angiogenin (331). These data suggest that angiogenins not only belong to the repertoire of microbicidal proteins produced by Paneth cells, but also function as regulators of vascularization under conditions of neoplasia and perhaps inflammation.

Other Proteins with Antimicrobial Activity In addition to specialized antimicrobial peptides such as defensins and lysozyme, a number of other proteins that can inhibit or kill bacteria have been identified in the intestine. Through the combined use of chromatographic analysis and radial diffusion assays, it was shown that the normal human colon mucosa contains several proteins with antimicrobial activities, including ubiquicidin; ribosomal proteins L30, L39, and S19; histones H1.5 and H2B; and eosinophil cationic protein (332,333). Although the physiologic significance of these proteins in intestinal host defense is unknown, several studies suggest a potential role in innate immunity. For example, ubiquicidin is a widely expressed macrophage product with marked antimicrobial activity against the enteric pathogens, L. monocytogenes and S. typhimurium (334). It is produced by posttranslational processing of the Fau protein, a 133-amino-acid fusion protein consisting of the ribosomal protein S30 linked to an unusual peptide with significant homology to ubiquitin (334). Different histone-derived peptides from evolutionarily diverse species ranging from fish to mammals have antimicrobial properties against enteric bacteria and can neutralize endotoxin (335). In humans, villous epithelial cells in the small intestine contain the histone, H1, not only in the nucleus, but also in the cytoplasm, from where it can be released on

induction of apoptosis and kill bacteria, suggesting a link between epithelial cell death and antimicrobial defense (336). Histones differ from typical antimicrobial proteins in that they have an important, if not critical, role in basic cellular physiology, rather than being specialized host defense molecules. For practical matters, the indispensable nature of such proteins for basic cell function complicates the analysis of the physiologic importance of their antimicrobial activities because ablation may interfere with cell viability.

Nitric Oxide and Reactive Oxygen Species Mammals synthesize two major classes of inorganic “disinfectant” molecules, those derived from oxygen and those from nitrogen (337). The former are called “reactive oxygen species” (ROS), whereas the latter are referred to as “reactive nitrogen species” (RNS). The rate-limiting step of ROS production is the generation of superoxide (O2−) from molecular oxygen (O2) by phagocyte oxidase, phox, whereas that for RNS production is the generation of nitric oxide (NO) from arginine by NOS (Fig. 42-13). Superoxide is converted to hydrogen peroxide by superoxide dismutase and ultimately water. NO is spontaneously oxidized to nitrite (NO2−) and nitrate (NO3−). Superoxide and NO, when combined, react to form the highly toxic peroxynitrite (ONOO−). Whereas ROS production is largely limited to neutrophils and macrophages, RNS are generated in phagocytes and many other cell types including intestinal epithelial cells. NOS exists in three isoforms, neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), each encoded by a separate gene in humans and mice (338,339). nNOS and eNOS require calcium for their activity, whereas iNOS is calcium-independent. nNOS is expressed in nerves throughout the length of the gastrointestinal tract and in other organs, but also has been detected in nonneuronal cells, including myocytes, epithelial cells, mast cells, and neutrophils (339). Several splice variants of nNOS have been identified in the rat small intestine, but their differential distribution and functional significance are poorly understood (340). iNOS can be produced by many cell types, most prominently epithelial cells and macrophages in the intestine. Its expression in these cells can either be constitutive, as in mouse ileum (341) and isolated normal human duodenocytes (342), or inducible in vivo during colonic inflammation (343) or in vitro by cytokines or in response to infection with invasive bacteria (344–347). eNOS is most prominently expressed in endothelial cells in the intestinal tract and other organs, but can also be found in smooth muscle cells, platelets, and T lymphocytes (339). Of the three NOS isoforms, iNOS is responsible for the majority of intestinal NO production, particularly under inflamed conditions (348). Mice lacking either one of the three NOS isoforms are viable, fertile, and generally healthy (349–351), although eNOS deficiency leads to decreased vascular relaxation and increased arterial blood pressure (351), and nNOS-deficient mice exhibit decreased esophageal and pyloric

1056 / CHAPTER 42 Reactive nitrogen species (RNS) COOH

H2N CH

CH2

Reactive oxygen species (ROS)

L-arginine CH2 CH2

Oxygen

O2

NH C

Phagocyte oxidase Superoxide

HN

O2−

NO synthase Superoxide dismutase

Hydrogen peroxide

Citrulline

NO

Nitric oxide

H2O2 ONOO− Peroxynitrite

Water

NH2

H2O

NO2− Nitrite

NO3− Nitrate

FIG. 42-13. Reactive oxygen (ROS) and reactive nitrogen species (RNS) in mammalian cells. ROS, superoxide, and hydrogen peroxide are generated enzymatically from molecular oxygen by phagocyte oxidase (phox). Hydrogen peroxide decomposes into water. RNS are produced by nitric oxide (NO) synthase from one of the guanidino nitrogens of L-arginine (boxed). NO is degraded by oxidation into nitrite and nitrate. Reaction of superoxide and NO yields peroxynitrite (ONOO−), which is a potent and versatile oxidizing and nitrating agent for lipids and proteins.

sphincter relaxation, and consequently gastric dilation and pyloric hypertrophy (350). The latter phenotype resembles the clinical symptoms found in patients with defective gastrointestinal nNOS synthesis or activity due to genetic abnormalities, such as infantile hypertrophic pyloric stenosis (352). NO production in most cells depends on extracellular arginine despite the presence of sufficient arginine inside the cells, a phenomenon referred to as the “arginine paradox” (353,354). Efficient transport of extracellular arginine into cells is facilitated by several transporters, including those of the cationic amino acid transporter (CAT) family of transporters, which are found in close association with some NOS isoforms and are thought to form a functional complex close to the membrane (353). In intestinal epithelial cells, iNOS is largely confined to the apical side of the cell underneath the cell membrane (341,355), suggesting that functional CAT/iNOS complexes are also limited to that side of the cell, although such complexes remain to be demonstrated in epithelial cells. In addition to enzymatic generation by the host, NO can be generated nonenzymatically from nitrate and nitrite in food and saliva under acidic conditions, and from nitrate by nitrate reductase made by commensal bacteria in the oral cavity and colon. Analysis of in vivo luminal NO in rats has shown that

the greatest levels were found in the stomach followed by the cecum, whereas the small intestine and colon had low levels (356). In contrast, germ-free rats lacking any microbial colonization of the gastrointestinal tract had low NO levels throughout the gastrointestinal tract. Feeding of nitratecontaining food increased gastric NO levels, but had no effect on colonic levels, whereas treatment with an iNOS inhibitor reduced NO levels in the colon, but not the stomach (356). These data show that the intestinal microbiota is important for constitutive intestinal NO production and that dietary nitrate is a substrate for gastric NO generation. NO is a simple and small (molecular weight, 30 Da) molecule that is chemically unstable because it is a highly reactive radical with an unpaired electron. Its modes of action are manifold and depend on the particular RNS involved and the chemical microenvironment at the site of action. RNS can interact with multiple targets in microbial cells including thiols, metal centers, protein tyrosines, nucleotides, and lipids (337,357). For example, RNS inhibit bacterial DNA replication through a mechanism that involves zinc mobilization from DNA-binding metalloproteins (358). RNS are antimicrobial for a wide range of bacterial and parasitic commensals and pathogens. In the intestinal tract, RNS have two major sites of action, in phagocytic cells and in

INNATE IMMUNITY / 1057 epithelial cells. In neutrophils and macrophages, RNS are active in phagocytic vesicles that harbor bacteria on uptake. Phagocytes lacking iNOS, and hence the ability to produce NO, are impaired in their ability to kill enteric bacteria such as Salmonella (344). Consequently, iNOS-deficient mice exhibit increased susceptibility to infection with this pathogen (359). In addition, NO released by macrophages or other cells may affect the viability of extracellular bacteria such as Citrobacter rodentium, a murine model of human enteropathogenic E. coli, and can contribute to bacterial eradication (360). Polarized intestinal epithelial cells up-regulate iNOS expression and NO synthesis after cytokine stimulation or bacterial infection (346,347,361). The stable NO end products nitrite and nitrate are preferentially detected on the apical side of the cells (347,362), suggesting that relevant targets for epithelial cell–derived NO and its metabolites may be located close to the apical domain. For example, growth and differentiation of the protozoan, small-intestinal parasite Giardia lamblia, which resides luminally or in close apposition to the epithelium, is inhibited by NO in culture (362), although the physiologic importance of this potential host defense mechanism is unknown. In addition to its direct antimicrobial functions, NO has activities on host cells in the intestinal tract that can affect host/microbe interactions. It can cause dilation of tight junctions between epithelial cells in polarized intestinal monolayers and contribute to loss of epithelial barrier function, and induction of apoptosis, on infection with enteroinvasive bacteria (361). These NO-mediated events might be expected to facilitate bacterial penetration into the mucosa. In contrast, NO can also promote chloride, and hence fluid secretion, in polarized epithelial cells (361,363), which may reduce bacterial numbers at the level of the epithelium, and thus protect the host. In addition, NO may play an indirect role in regulating the host response to bacterial infection by regulating mucosal inflammatory events, although the direction of this effect depends on the underlying pathogenetic mechanisms (348,364).

Summary: Effector Molecules of Innate Immunity The innate immune system possesses numerous genomeencoded effector molecules that exhibit antimicrobial activity either directly or indirectly through the enzymatic generation of molecules with such activity. These molecules are active against many microbial targets, making them highly effective for controlling infections with not only commonly encountered pathogens, but also those not previously faced by the host. Therefore, the cooperation between sensor and effector molecules of the innate immune system provides an initial line of host defense, which is critical for rapidly controlling microbial attacks until the adaptive immune system can develop potent immune defenses directed against unique microbial components.

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339. Shah V, Lyford G, Gores G, Farrugia G. Nitric oxide in gastrointestinal health and disease. Gastroenterology 2004;126:903–913. 340. Huber A, Saur D, Kurjak M, Schusdziarra V, Allescher HD. Characterization and splice variants of neuronal nitric oxide synthase in rat small intestine. Am J Physiol 1998;275:G1146–G1156. 341. Hoffman RA, Zhang G, Nussler NC, Gleixner SL, Ford HR, Simmons RL, Watkins SC. Constitutive expression of inducible nitric oxide synthase in the mouse ileal mucosa. Am J Physiol 1997;272: G383–G392. 342. Murray IA, Daniels I, Coupland K, Smith JA, Long RG. Increased activity and expression of iNOS in human duodenal enterocytes from patients with celiac disease. Am J Physiol Gastrointest Liver Physiol 2002;283:G319–G326. 343. Singer, II, Kawka DW, Scott S, Weidner JR, Mumford RA, Riehl TE, Stenson WF. Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease. Gastroenterology 1996;111:871–885. 344. Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischiropoulos H, Fang FC. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J Exp Med 2000;192:227–236. 345. Fang FC, Vazquez-Torres A. Nitric oxide production by human macrophages: there’s NO doubt about it. Am J Physiol Lung Cell Mol Physiol 2002;282:L941–L943. 346. Salzman AL, Eaves-Pyles T, Linn SC, Denenberg AG, Szabo C. Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells. Gastroenterology 1998;114:93–102. 347. Witthoft T, Eckmann L, Kim JM, Kagnoff MF. Enteroinvasive bacteria directly activate expression of iNOS and NO production in human colon epithelial cells. Am J Physiol 1998;275:G564–G571. 348. Vallance BA, Dijkstra G, Qiu B, van der Waaij LA, van Goor H, Jansen PL, Mashimo H, Collins SM. Relative contributions of NOS isoforms during experimental colitis: endothelial-derived NOS maintains mucosal integrity. Am J Physiol Gastrointest Liver Physiol 2004; 287:G865–G874. 349. MacMicking JD, Nathan C, Hom G, Chartrain N, Fletcher DS, Trumbauer M, Stevens K, Xie QW, Sokol K, Hutchinson N, Chen H, Mudgett JS. Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 1985;81:641–650. 350. Huang PL, Dawson TM, Bredt DS, Snyder SH, Fishman MC. Targeted disruption of the neuronal nitric oxide synthase gene. Cell 1993;75:1273–1286. 351. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 1995;377:239–242. 352. Takahashi T. Pathophysiological significance of neuronal nitric oxide synthase in the gastrointestinal tract. J Gastroenterol 2003;38:421–430. 353. McDonald KK, Zharikov S, Block ER, Kilberg MS. A caveolar complex between the cationic amino acid transporter 1 and endothelial nitric-oxide synthase may explain the “arginine paradox.” J Biol Chem 1997;272:31213–31216. 354. Lee J, Ryu H, Ferrante RJ, Morris SM Jr, Ratan RR. Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox. Proc Natl Acad Sci U S A 2003;100: 4843–4848. 355. Islam D, Veress B, Bardhan PK, Lindberg AA, Christensson B. In situ characterization of inflammatory responses in the rectal mucosae of patients with shigellosis. Infect Immun 1997;65:739–749. 356. Sobko T, Reinders C, Norin E, Midtvedt T, Gustafsson LE, Lundberg JO. Gastrointestinal nitric oxide generation in germ-free and conventional rats. Am J Physiol Gastrointest Liver Physiol 2004;287:G993–G997. 357. Nathan C, Shiloh MU. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 2000;97:8841–8848. 358. Schapiro JM, Libby SJ, Fang FC. Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress. Proc Natl Acad Sci U S A 2003;100:8496–8501. 359. Mastroeni P, Vazquez-Torres A, Fang FC, Xu Y, Khan S, Hormaeche CE, Dougan G. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. II. Effects on microbial proliferation and host survival in vivo. J Exp Med 2000;192:237–248. 360. Vallance BA, Deng W, De Grado M, Chan C, Jacobson K, Finlay BB. Modulation of inducible nitric oxide synthase expression by the

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CHAPTER

43

Biology of Gut Immunoglobulins Finn-Eirik Johansen, Elizabeth H. Yen, Bonny Dickinson, Masaru Yoshida, Steve Claypool, Richard S. Blumberg, and Wayne I. Lencer Secretory Immunoglobulin A and the Polymeric Immunoglobulin Receptor, 1068 Secretory Immunity, 1068 Function of Secretory Immunoglobulin A in Luminal Secretions, 1069 Origin of Mucosal Plasma Cells, 1070 Structure of Polymeric Immunoglobulins, 1072 The Polymeric Immunoglobulin Receptor: Expression, 1073 Structure of Polymeric Immunoglobulin Receptor, 1074 Cell Biology and Trafficking of Polymeric Immunoglobulin Receptor, 1075 Immunoglobulin G and the Neonatal Fc Receptor FcRn, 1077 The Immunoglobulin G Receptor FcRn, 1077 Cell Biology of Immunoglobulin G Transport, 1079 Intracellular Itinerary, 1079 Physiology and Function of Immunoglobulin G at Mucosal Surfaces, 1081

Pathway for Antigen Uptake from Lumen Mediated by FcRn, 1081 Immunoglobulin G and Inflammatory Bowel Diseases, 1082 Immunoglobulin E, 1082 Function of Immunoglobulin E in the Mammalian Intestine, 1082 Food Allergy, 1082 Animal Models of Intestinal Anaphylaxis, 1082 CD23 (FcεRII): Low-Affinity Receptor for Immunoglobulin E, 1083 CD23-Dependent Transcytosis of Immunoglobulin E: In Vitro Studies, 1084 Immunoglobulin E Function in Intestinal Immunity and Disease, 1084 Concluding Remarks, 1085 References, 1085

F-E. Johansen: Institute of Pathology, University of Oslo, and Department of Pathology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E. H. Yen: Harvard Medical School Fellowship in Pediatric Gastroenterology and Nutrition, Gastrointestinal Cell Biology, Children’s Hospital Boston, Boston, Massachusetts 02115. B. Dickinson: Department of Pediatrics, Research Institute for Children, Children’s Hospital, New Orleans, Louisiana 70118. M. Yoshida: Frontier Medical Science in Gastroenterology, International Center for Medical Research and Treatment, Kobe University School of Medicine, Kobe 650-0017, Japan. S. Claypool: Laboratory of Mucosal Immunology, Brigham and Women’s Hospital, Boston, Massachusetts 02115. R. S. Blumberg: Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Hepatology, and Endoscopy, Harvard Digestive Diseases Center, Laboratory of Mucosal Immunology, Brigham and Women’s Hospital, Boston, Massachusetts 02115.

W. I. Lencer: Department of Pediatrics, Harvard Medical School, and Division of Pediatric Gastroenterology and Nutrition, Harvard Digestive Diseases Center, Gastrointestinal Cell Biology Laboratories, Children’s Hospital Boston, Boston, Massachusetts 02115.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Host defense at mucosal surfaces depends on many factors. One of these factors is humoral immunity (1), and all of the immunoglobulins contribute (2,3). This chapter reviews the biology, transport, and function of each class of immunoglobulins in the intestine. As in all other transporting epithelia such as those found in the lung and kidney, a single layer of columnar epithelial cells forms the protective barrier between host and environment in the intestine (4). This single-cell-thick epithelial layer functions to separate two physically distinct compartments. Such a clear distinction between the outside and inside worlds

1067

1068 / CHAPTER 43 is essential for the intestine to absorb and secrete nutrient solutes and water efficiently against high electrochemical potentials. Because of this barrier, not all nutrient solutes, much less large and stably folded proteins such as the immunoglobulins, can be absorbed or secreted. For many years, we have known that secretory IgA (SIgA) crosses the intestinal barrier to function in luminal secretions by binding to the polymeric immunoglobulin receptor (pIgR). pIgR binds polymeric IgA and mediates vesicular transport of this immunoglobulin across the intestinal epithelial cell for secretion into the gut lumen. This process of vesicular transport that carries select cargos across polarized cells is termed transcytosis (2). SIgA is the predominant antibody in intestinal secretions. Some mucosal secretions in humans also contain IgG (Fig. 43-1), where it likely functions together with SIgA in host defense (3,5). In humans, IgG concentrations predominate over SIgA in the lumen of the lower respiratory and female genital tracts (6,7), and rectal secretions contain IgG in concentrations that may exceed 700 µg/ml (8). Systemically administered IgG protects against mucosal infection by respiratory syncytial virus in the human lung (9) and against HIV in the monkey intestine and vagina (10,11). Humans deficient in IgG exhibit an increased incidence and severity of mucosal and systemic infections, particularly infections caused by microbes that invade or colonize the respiratory tract. The mechanism by which IgG may cross epithelial

100% 80% IgG

60%

IgM

40%

IgA

20% 0% Duodenum/ jejunum

A

Ileum

Colon

Ig concentration (mg/ml)

1.0 0.8 IgG

0.6

IgM

0.4

IgA

0.2 ND

ND

Jejunal fluid

Colonic fluid

0

B

Duodenal fluid

FIG. 43-1. Isotype distribution of gut immunoglobulins. (A) Percentage of immunoglobulin-producing cells of the various isotypes in the lamina propria at different anatomic locations of the small and large intestines in healthy human subjects. Immunoglobulin D (IgD)- and IgE-producing plasma cells make up less than 1% in the normal situation. (B) Concentrations of immunoglobulins sampled at different anatomic locations. ND, not determined. (Data are based on articles reviewed by Kaetzel and colleagues [30]).

barriers to function in mucosal secretions has only recently been described, and its potentially diverse functions in the intestinal lumen are still emerging. Even more recent studies have shown that some antigens may cause hypersensitivity reactions in the intestine via transport across the epithelial barrier by IgE. Like the other immunoglobulins, IgE also appears to bind a cell-surface receptor that carries it across the cell by transcytosis.

SECRETORY IMMUNOGLOBULIN A AND THE POLYMERIC IMMUNOGLOBULIN RECEPTOR Secretory Immunity The adult human gastrointestinal tract is covered by a monolayered epithelium with a surface area of approximately 300 m2, compared with about 1.5 m2 of skin. To protect this thin and sensitive surface, an elaborate set of mechanisms prevents attachment, colonization, and possible damage by pathogenic agents. Numerous innate physicochemical mechanisms cooperate with a first-line adaptive immune defense that depends on active export of SIgA and, to some extent SIgM, and other antibodies (12) (Fig. 43-2). A high antigenic load in the intestine, composed of innocuous antigens, such as food products and components of normal flora, as well as potential pathogens warrants the existence of a mucosal immune system that operates largely independently of systemic immunity. The major immunoglobulin in intestinal secretions is SIgA. SIgA is generated by the concerted action of two distinct cell types: polymeric IgA (pIgA)–producing plasma cells and pIgR-expressing mucosal epithelial cells. SIgA production relies on a predominant class switch to IgA and a high expression level of the joining (J) chain in gut lamina propria plasma cells (13). The J chain is an inherent part of dimers and larger polymers of IgA (collectively called pIgA), as well as pentamers of IgM, but it becomes degraded intracellularly when coexpressed with other immunoglobulin classes (14). Furthermore, active transport of secretory antibodies to the luminal surface is mediated by the pIgR. Therefore, the pIgR expression level determines the epithelial capacity for pIgA and pentameric IgM export. The crucial importance of the J chain and the pIgR for epithelial transport of dimeric IgA is highlighted by the absence of SIgA in knockout mice deficient for either of these genes (15–17). SIgA is a major antibody in all exocrine secretions, primarily because IgA-producing plasma cells dominate at all mucosal sites, whereas IgM and IgG plasma cells make up a variable and much smaller portion. However, IgG antibodies may also provide mucosal protection, particularly at sites with low proteolytic activity such as the respiratory and reproductive tracts. More than 80% of human and mouse plasma cells are located in the mucosal lamina propria of the gastrointestinal and respiratory tracts, and most of these produce IgA with incorporated J chain, thus being committed to pIgA production (1). The capacity for external IgA transport in the

BIOLOGY OF GUT IMMUNOGLOBULINS / 1069 Lamina propria

Secretory epithelium

Lumen

1

IgA+J 2

Plasma cell 3 IgA+J

Key: IgA J chain plgR/SC Ag Virus LPS

Activation of proinflammatory gene repertoire

4

FIG. 43-2. Four suggested noninflammatory modes of polymeric immunoglobulin A/polymeric immunoglobulin receptor (pIgA/pIgR)–mediated host defense at mucosal surfaces. (1) Secretory IgA (SIgA) generated from pIgR-driven export of locally produced dimeric IgA with J chain (IgA+J) functions as blocking antibodies in immune exclusion at mucosal surfaces. (2) Intracellular neutralization of an infecting virus by specific dimeric IgA, perhaps followed by transport of viral products to the lumen (this kind of neutralization requires occupation of the pIgR transport vesicles by virus). (3) Intracellular neutralization of endotoxin (lipopolysaccharide [LPS]) from gram-negative bacteria, which inhibits potentially harmful activation of the proinflammatory nuclear factor-κB pathway in the epithelial cell. (4) Stromal clearance of antigens that have somehow breached the mucosal barrier. Ag, antigen.

gut is calculated to be approximately 3 g/day in an adult human (18). Thus, the production of SIgA exceeds that of all other antibody classes combined.

Function of Secretory Immunoglobulin A in Luminal Secretions Clinical and experimental studies provide ample evidence for a protective role of both SIgA and SIgM (19). Both classes of secretory antibodies may exert their protective action at different levels (20) and cooperate with mechanical and chemical innate defense mechanisms such as mucin, peristalsis, gastric acid, lysozyme, lactoferrin, and defensins. Immune exclusion refers primarily to the ability of antibodies to hamper microbial colonization and to dampen penetration of soluble macromolecules through mucosal surface

epithelia (21). It has been shown that SIgA can block the interaction between microbial adhesins and their receptors on the apical surface of epithelial cells and reduce absorption of soluble antigens, including chemical carcinogens. Immune exclusion can also be performed by IgG antibodies (22), which reach external secretions either by passive paracellular bulk leakage of interstitial proteins through surface epithelia, particularly after breakdown of the intestinal mucosal barrier (23), or by receptor-mediated transport by the neonatal IgG Fc receptor, FcRn (see later) (24,25). IgG antibodies appear to be especially important for immune exclusion in the respiratory and genital tracts, where they are more stable than in gastrointestinal fluids. In addition to performing immune exclusion, pIgA and pentameric IgM may also interact with and neutralize viruses and their products within pIgR-expressing epithelial cells (20,26). A prerequisite for intracellular neutralization would be the physical interaction between specific IgA antibodies and the antigen within the epithelial cell. Colocalization of IgA and Sendai virus has been documented at the electron microscopic level (27). There is also evidence that this unique reinforced surface protection helps to clear rotavirus infection in vivo (28). A similar mechanism may also operate to down-regulate proinflammatory signals elicited by lipopolysaccharide (LPS) of gram-negative luminal bacteria (29). LPS taken up by pinocytosis can interact with Toll-like receptor 4 normally expressed intracellularly in epithelial cells and can activate nuclear translocation of the nuclear factor-κB (NF-κB) transcription factor, which, in turn, activates a proinflammatory gene repertoire. Specific anti-LPS pIgA was shown to interfere with LPS-mediated NF-κB activation in a pIgR-dependent manner. Cell culture studies moreover have suggested that pIgA and pentameric IgM can interact with soluble antigens in the mucosal lamina propria and shuttle the immune complexes across the secretory epithelium to the exterior of the body by pIgR-mediated transport (30). Immune complexes containing antigen, pIgA, and IgG could be transported in this way by human pIgR-transfected Madin–Darby canine kidney (MDCK) cells. Furthermore, mice that were immunized mucosally against horseradish peroxidase to obtain high pIgA titers against this antigen were shown to be capable of transporting it from the lamina propria to the lumen after intravenous challenge (31). Thus, pIgA and pentameric IgM antibodies may operate at three levels in immune protection at mucosal surfaces: in external secretions, within pIgRexpressing epithelial cells, and in the lamina propria. The immune protection mediated by SIgA and SIgM is principally noninflammatory at all of these levels, which is important for the maintenance of local immunological homeostasis in the mucosa. Secretory component (SC) was identified by Tomasi and coworkers (32) in the mid-1960s as a unique component of IgA polymers isolated from external secretions; it was initially called “transport piece” or “secretory piece.” This glycoprotein also was shown to occur in a free form (free SC of 80 kDa; derived from transcytosis of unoccupied pIgR) and to

1070 / CHAPTER 43 be expressed by secretory epithelia. Its cellular nature as a transmembrane epithelial receptor for J chain–containing pIgA and pentameric IgM was proposed by Brandtzaeg in the early 1970s (33,34); currently, it is most commonly referred to by its functional name, pIgR (identical to membrane SC [35]). Although the primary function of the pIgR is to transport pIgs across mucosal epithelia, it was recognized early on that SC might have additional functions (36). The covalent attachment of SC protects the IgA heavy chain from proteolytic degradation in the harsh environment of the gut lumen (37,38). Similarly, an excess of free SC in secretions stabilizes noncovalently bound pentameric IgM. Furthermore, glycosylation of SC makes it mucophilic, anchoring SIgA to the mucus, where it can trap potential pathogens (39). SC has also been shown to interact directly with several bacteria or their products, suggesting that it may have an innate immune function as well (40). A specific interaction with a Streptococcus pneumoniae surface protein (SpsA/ CbpA) is particularly intriguing because the interaction appears to be protective when free SC binds SpsA/CbpA (41). However, the pneumococcus can also bind the pIgR apically on the epithelium, and it may co-opt the transcytotic pathway to penetrate and disseminate (42). Thus, the balance of free SC to apical surface-expressed pIgR might influence the outcome of nasopharyngeal pneumococcal colonization. Because J-chain incorporation into pIgA and pentameric IgM is essential for their binding to the pIgR (see later), knockout mice deficient for J chain or deficient for pIgR have similar phenotypes (15–17,43). Somewhat surprisingly, both knockouts are fully viable, even though their ability to form pIgA with affinity for pIgR (in the former case) or ability to transport pIgA to exocrine secretions (in the latter case) is completely abrogated. In rodents, a significant amount of intestinal SIgA is derived from hepatobiliary secretions via transport by pIgR, and in the absence of pIgR, serum IgA levels are increased 10- to 40-fold. Even more surprisingly, serum IgG levels were also increased in the pIgR knockout mice. The fact that increased levels of serum albumin were found in saliva and feces suggested to some investigators that pIgR−/− mice may have reduced epithelial barrier function, but the evidence for this is inconclusive. Increased serum IgG antibodies against Escherichia coli, but not against a dietary protein (gluten), suggested increased penetration of bacterial antigens across the bowel, presumably causing a systemic immune response (16). Disturbed mucosal homeostasis was also evidenced by an increased level of IgA-producing plasma cells and increased numbers of intraepithelial lymphocytes in pIgR knockout mice (44,45). These findings suggest a hyperactive protective immune response in pIgR knockout mice that may be accompanied by an increased propensity for tolerance induction (our unpublished observations). If so, this might explain why pIgR knockout mice have unaltered levels of anti–gluten IgG in the face of an apparent increase in passive intestinal solute transport. In light of the apparent redundancy of mucosal immunity, animal models of enteric or other mucosal infections in which

secretory immunity is solely responsible for protection are probably few. However, there is one example where this idea is tested experimentally. Studies show that protection against the watery diarrhea caused by cholera toxin (CT) may depend strongly on secretory immunity. Mice orally immunized with a low dose of CT produce high amounts of CT-specific SIgA antibodies and are protected against intestinal fluid accumulation when later challenged with a high dose of CT. However, both J-chain– and pIgR-deficient mice failed to show any protection in this enterotoxin model (43) (our unpublished observations). Furthermore, although pIgR knockout mice acquired immunity to influenza virus when immunized, the absence of SIgA antibodies abrogated cross protection against strains of influenza virus that were related to, but distinct from, the original vaccine immunogen. These results suggest that systemic immunity can protect against a previously encountered pathogen, but that secretory immunity extends the repertoire of protection to cover related pathogens, probably by immune exclusion (46). A similar immune exclusion effect was seen when nasopharyngeal pneumococcus carriage was examined in pIgR knockout mice (47). Whereas systemic immunity controlled pneumococcus proliferation in multiple organs in immunized animals, SIgA antibodies were required to effectively eliminate luminal colonization by the bacteria.

Origin of Mucosal Plasma Cells The mucosal immune system can be divided into two functionally distinct arms (1) (Fig. 43-3): organized mucosaassociated lymphoid tissue, which constitutes the inductive sites where antigens sampled from mucosal surfaces stimulate cognate naive T and B lymphocytes, and effector sites, where effector cells perform their action, such as pIgA production by lamina propria plasma cells (1,12). The gut is the largest antibody-producing organ in humans, and most of its plasma cells are primed initially in the gut-associated lymphoid tissue (GALT). GALT includes Peyer’s patches, the appendix, and scattered solitary or isolated lymphoid follicles (ILFs). A unique feature of these structures is the absence of afferent lymphatics. Antigen sampling in the Peyer’s patch relies instead on specialized M cells situated within the follicle-associated epithelium above the germinal centers where initial B-cell priming occurs. Postgerminal center B cells exit through draining lymphatics and “home” to secretory effector sites such as the intestinal lamina propria (48). Peyer’s patches occur mainly in the ileum and less frequently in the jejunum. By definition, they consist of at least 5 aggregated lymphoid follicles, but can contain up to 200 such organized structures. Human Peyer’s patch anlagen can be seen at 11 weeks gestation, but no germinal centers appear until shortly after birth, reflecting dependency on stimulation from the environment. Macroscopically visible Peyer’s patches in humans increase from approximately 100 at birth to 250 in the mid-teens, then diminish to about 100 between 70 and 95 years of age (49). Human intestinal mucosa also

BIOLOGY OF GUT IMMUNOGLOBULINS / 1071 MALT structure

APC

Ag Ag

GC reaction

Lymph drainage Regional lymph node

Ag

APC

Homing via peripheral blood

Effector site

APC

SIgA

FIG. 43-3. Schematic steps in secretory immunoglobulin A (IgA) generation. (1) Naive mucosal B cells are stimulated in mucosa-associated lymphoid tissue (MALT) such as ileal Peyer’s patches and isolated lymphoid follicles in the large bowel. Germinal center (GC) B cells undergo somatic hypermutation and class switch recombination before exit via lymph to regional lymph nodes and further homing via the thoracic duct and peripheral blood to mucosal effector sites such as the intestinal lamina propria. (2) Mucosal plasma blasts receive local signals (broken arrow) for terminal differentiation and express high levels of the J chain; most of them are therefore committed to production of dimeric IgA (A +J), and some to production of pentameric IgM (M+J). Most of the rare mucosal IgG-producing cells also express J chain, although in a nonassociated form (IgG ±J). (3) Secretory epithelial cells express the polymeric immunoglobulin receptor (pIgR) basolaterally, thereby facilitating the export of dimeric IgA (and pentameric IgM) to provide secretory IgA (SIgA; and SIgM) antibodies for defense functions, as illustrated in Figure 43-1. Ag, antigen; APC, antigen-presenting cell.

harbors at least 30,000 ILFs, increasing in density distally (50). Peyer’s patches and ILFs are functionally similar structures, but evidence from mice suggests that ILFs may play a special role in the induction of SIgA responses that control the commensal microbiota (51). The homing of activated lymphoid cells to the intestinal lamina propria depends on high surface expression of α4β7 integrin and absence of L-selectin (CD62L). This allows binding of the circulating cells to unmodified mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) expressed

apically on endothelial cells of the lamina propria microvasculature (48,52). Many plasma blasts or plasma cells, or both, continue to express high levels of α4β7 after migration into the human intestinal lamina propria, and it is possible that this integrin also contributes to local retention of effector cells. In addition, migration (and/or retention) of both T and B cells into the small intestinal lamina propria appears to depend on the CC chemokine receptor 9 (CCR9) and its ligand CCL25 (i.e., thymus-expressed chemokine). CCL25 is selectively produced by small intestinal crypt epithelium in both humans and mice (53). Although α4β7 is also required for extravasation of lymphocytes into the large intestinal lamina propria, the chemokine receptor-chemokine pair of key importance here appears to be CCR10-CCL28 (i.e., mucosal epithelial chemokine) (54). The peritoneal cavity is recognized as a source of mucosal B cells independent of GALT. This self-renewing pool of surface IgM+ B1 cells (CD5+) may provide up to 40% to 50% of the intestinal IgA-producing plasma cells in mice (55). Macpherson and coworkers (56) first showed that these B1 cells caused poly-reactive (“natural”) pIgA antibodies, particularly directed against polysaccharide antigens from commensal bacteria in a T cell–independent fashion. However, the relation of B1 cells to the conventional bone marrow–derived B2 cells remains elusive, and their contribution to gut SIgA production in humans remains controversial (57–60). Although considerable levels of poly-reactive SIgA antibodies directed against self, as well as microbial antigens, do occur in human external secretions, this could be caused by microbial polyclonal activation of conventional B cells. Local production of immunoglobulins is dominated by IgA in the gut lamina propria (see Fig. 43-1A). However, the situation in intestinal fluids is somewhat different showing similar levels of IgA and IgG (see Fig. 43-1B). This might be, in part, due to contribution from serum immunoglobulins. At least three possible explanations for the domination of pIgA-producing plasma cells in gut lamina propria exist: (1) B cells stimulated in GALT activate J-chain expression and class switching to IgA before they exit via local lymph and home to the gut effector sites (61); (2) IgA-producing plasma blasts are selectively recruited to the intestinal lamina propria (53); or (3) the lamina propria microenvironment favors in situ class switch to IgA of recruited B cells (51). Some reports suggest that all three mechanisms may operate under certain conditions (59,62). However, class switch to IgA in the diffuse lamina propria is most likely restricted to the T cell–independent B1-cell population derived from the peritoneal cavity (58). This unusual switch process may circumvent the normal requirement of CD40 ligation on B cells by CD154 (CD40L) on cognate T helper (Th) cells. Instead, proliferation-inducing cytokines such as BLyS/ BAFF (B-lymphocyte stimulator/B-cell–activating factor of the tumor necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) secreted by activated dendritic cells (DCs) may provide necessary B-cell activation (59,63). However, currently, there is no evidence for this mechanism in the human gut (58), which does not appear to

1072 / CHAPTER 43 receive a significant number of B1 cells from the peritoneal cavity (57).

Structure of Polymeric Immunoglobulins pIgA and pentameric IgM are unique antibody isotypes because in addition to their heavy and light chains, they contain the short polypeptide J chain (see earlier) (Fig. 43-4). The J chain was identified independently by Mestecky and coworkers (65) and by Halpern and Koshland (64) as an additional peptide common to SIgA and IgM. The J chain is not mandatory for polymer formation but regulates the structure and function of the polymers formed. The ability of IgA and IgM to polymerize is determined by features of their Fc region, as well as enzymes that catalyze disulfide exchange reactions (65a,65b), and provides secretory antibodies with high avidity, a property particularly useful for agglutinating microbes in external secretions. IgA forms mostly dimers when J-chain expression is high, although J-chain–containing trimeric and tetrameric

Fab Fc

IgG

IgE

J

Pentameric IgM

Dimeric IgA (plgA)

J

Secretory IgA (SIgA)

FIG. 43-4. Schematic diagram of the different immunoglobulin isotypes. Class switching at the heavy chain locus exchanges the constant region domains (C). The light chain variable domain (V) and C domain (which can be κ or λ), as well as the VH domain, can be expressed with different isotypes. The Fab region and the Fc region are indicated for immunoglobulin G (IgG) only, but are present in all the isotypes. Cysteines involved in intermonomeric unit disulfide bonds in pentameric IgM and dimeric IgA are indicated. Cysteines in the J chain (J) and in secretory component (SC; the cleaved extracellular portion of the pIgR) that form covalent bonds to IgM or IgA are indicated. Secretory IgA (SIgA) contains a covalent bond between IgA and SC, whereas no such bonding occurs with SIgM. IgM operating in secretions is therefore more likely to dissociate from the SC molecule.

IgA may be generated as well (66). However, the identification of naturally occurring dimers and larger polymers of IgA without J chain suggested that J chain might not be obligatory in IgA polymer formation (66). J-chain knockout mice exhibited an increased ratio of monomeric/dimeric IgA, but some dimers were nevertheless present in their serum (15). However, there was no evidence of active pIgR-mediated transport in these mice nor transport of partially purified pIgA from their serum by polarized pIgR-expressing MDCK cells in vitro (15,67). It has been suggested that J chain is covalently incorporated into pentameric IgM late in the polymerization process and closes the ring structure by bridging the first and fifth monomeric unit, thereby excluding addition of a sixth IgM monomer (68). This model is supported by the fact that when J chain is limited, a greater proportion of hexameric IgM is formed than when J chain is readily available (69–72). Nevertheless, J-chain–negative IgM plasma blasts or plasma cells have been reported to secrete both pentamers and hexamers (72–74). All in all, cumulative evidence suggests that increasing levels of J-chain expression in IgM-producing B-cell lymphomas correlate with secretion of an increasing proportion of IgM pentamers. The J chain is associated with pIgA and pentameric IgM via disulfide bonds involving the penultimate cysteine residue in the secretory tail piece (stp) of the α or the µ heavy chain, respectively (14). To delineate the motifs in the α and µ chains that regulate class-specific polymerization and J-chain incorporation, several research groups have constructed chimeras between IgM/IgG, IgA/IgG, and IgM/IgA, as well as point mutations of specific amino acids in the heavy chains (14). The α stp substituted effectively for the µ stp on an IgM backbone (75), whereas introducing the µ stp to IgA led to the formation of larger than wild-type IgA polymers, including pentamers and hexamers (75,76). Domain-swap mutants have demonstrated the importance of the most C-terminal domain for isotype-specific polymerization of IgM and IgA, but additional structures in the penultimate domain contribute as well (76–79). Three cysteine residues in IgM, Cys337, Cys414, and Cys575, are available for intermolecular disulfide bonds. Cys337 in Cµ2 forms a disulfide bridge between two heavy chains in the IgM monomeric units, whereas Cys414 and Cys575 (the latter located in the stp) are involved in intermonomeric bridges: Cys414 between µ heavy chains and Cys575 to the J chain (80). However, the precise intermolecular pattern of disulfide bonds in pentameric IgM is still unclear. Cys495 in the α stp also forms a disulfide bond with the J chain, whereas Cys309 (sometimes denoted Cys311 and equivalent to Cys414 in IgM) forms a covalent bond with Cys467 in the pIgR during transcytosis (81). This covalent linkage remains intact in secretions and gives unique properties to SIgA that makes it particularly well suited to operate in the gut lumen (see earlier). The number of J-chain molecules per polymer remains a disputed issue. It has often been assumed that pIgA and pentameric IgM contain only one J chain. However,

BIOLOGY OF GUT IMMUNOGLOBULINS / 1073 immunochemical studies have indicated that dimeric IgA contains two and pentameric IgM three to four J chains (14). Characterization of J chain released from dimeric, trimeric, and tetrameric IgA purified from the same myeloma serum showed that the molar J chain/α chain ratio increased with the size of the polymer (66). The mature human J chain is 137 amino acids long and contains 8 cysteine residues: 2 (Cys15 and Cys69) are involved in disulfide bridges with the α or µ chains, and 6 are involved in intrachain disulfide bridges (14). The J chain is well conserved among vertebrates, being present also in cartilaginous fish, which apparently lack a secretory immune system (82). The eight cysteines are conserved among all mammalian species characterized, suggesting that they are important for proper J-chain function (14). Site-directed mutagenesis of each cysteine individually and coexpression with IgA showed that intra–J-chain disulfide bonds were dispensable for promoting IgA polymerization, but two (of three) of these bonds were essential for binding to the pIgR (83). Surprisingly, mutations of either Cys15 or Cys69 did not abrogate polymer formation under native conditions, suggesting that noncovalent interactions between the J chain and the heavy chain promote polymer formation, and that these polymers are subsequently stabilized by covalent bonding. J-chain–containing pIgA and pentameric IgM, but not J-chain–deficient polymers or monomers of IgA, IgM, or other Ig isotypes, bind stably to human pIgR (14). The dissociation constant for human pIgR/pentameric IgM complex is approximately 8 nM, whereas that for the pIgR/pIgA complex is about 40 nM (84). An apparent correlation between the amount of J chain in polymeric forms of Ig and their ability to bind free SC or pIgR raised the possibility that the J chain contributes directly to the pIgR/SC binding site (85). Furthermore, it was demonstrated that antiserum to J chain inhibited the pIgR-mediated translocation of pIgA both in vitro and in vivo, because transcytosis by human pIgRexpressing MDCK cells, as well as hepatobiliary transport of injected human pIgA in rats, was blocked (66). Although the J chain is clearly required for SIgA formation, its exact role in polymer formation, and in creating a docking site for the pIgR, still needs to be determined. It has been shown that the Cα3, and perhaps the Cα2, domain of human pIgA contributes to its noncovalent interaction with free SC in vitro (86). A characterization of domainswap mutants and mutated human pIgA suggested that a loop in the Cα3 domain is particularly important for the ability of pIgA to bind pIgR/SC (77,87). Furthermore, a peptide sequence with homology to this loop in Cα3 was isolated from a phage-display library, based on its interaction with MDCK cells expressing rabbit pIgR (87). An interaction between J-chain–negative murine pIgA and murine SC, but not between monomeric IgA and SC, was also observed with filter-immobilized IgA (88). Furthermore, purified CHOproduced pIgA without J chain showed marginal but significant affinity for free SC (83). These data support a direct interaction between pIgR and elements in the α chain in the absence of J chain, but the high expression level of J chain

in lamina propria plasma cells, as well as the much greater affinity of J-chain–containing pIgA for the pIgR, imply that in vivo transport of J-chain–deficient pIgA is negligible. Similar studies are not available for pentameric IgM, but this ligand has higher affinity for the pIgR than pIgA. Mucosal defense mediated by pIgA and pentameric IgM is principally noninflammatory. Intraepithelial neutralization occurs without cytolytic activity, and inhibition of NF-κB nuclear translocation will in itself reduce proinflammatory signals. Another important feature of pIgs subjected to active export is their intrinsic low capacity for complement activation. J-chain–positive pentameric IgM is at least 15- to 20-fold less efficient than J-chain–negative hexameric IgM in activating complement (74,89). Furthermore, polymers (and monomers) of IgA are unable to activate complement through the classical pathway, although they can mediate some complement activation through the lectin pathway (90,91). Thus, it appears that primarily immunoglobulins with low or no complement-activating potential are actively transported to the gut lumen, where the antigenic load is high.

The Polymeric Immunoglobulin Receptor: Expression The pIgR is a sacrificial receptor in that it is specifically cleaved to release secretory immunoglobulins with bound SC in the gut lumen (see Fig. 43-2). For this reason, it is constitutively expressed by mucosal epithelial cells at quite high levels in the small and large bowels. Expression is also seen in lacrimal, salivary, and lactating mammary glands, as well as in the lung, kidney, pancreas, and endometrium (40). One notable difference between rodents and the rabbit versus humans is that only the former species show significant pIgR expression in hepatocytes (40). The consequence of this difference is that rabbits and rodents obtain most of their proximal intestinal SIgA content from hepatobiliary transfer of circulating dimeric IgA, whereas for humans, nearly all SIgA (~98%) in the gut lumen is derived from pIgA produced by lamina propria plasma cells (18). These biological differences may lead to stronger compartmentalization of secretory antibody responses in humans than in rabbits and rodents. Moreover, in humans, distinct expression of pIgR is seen at 20 weeks gestation, but in rodents, it is not seen until the time of weaning, which suggests dependency on exogenous stimuli (40). The mechanistic explanation for this species difference remains unknown. In addition to its constitutive expression, pIgR is transcriptionally up-regulated by a number of mediators, such as cytokines, hormones, and bacterial products, that activate innate immunity. The expression of human pIgR was upregulated in the colonic adenocarcinoma cell line HT-29 by stimulation with interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-4, and TNF-α [40]. Similar regulation by IFN-γ and IL-4 has been demonstrated in a lung adenocarcinoma cell line, Calu-3. Enhanced pIgR expression in polarized Calu-3 cells grown on permeable filters was followed by an increased capacity of these cells to transport pIgA from the basolateral

1074 / CHAPTER 43 to apical medium (92). A similar functional link between receptor expression and transport capacity may operate in vivo in situations with increased epithelial pIgR expression, such as in celiac disease and chronic gastritis. Cytokine-mediated increased pIgR expression appears with relatively slow kinetics, reaching a plateau after 24 hours of stimulation. Increased expression has been demonstrated to occur primarily as a result of an increased transcription rate of the gene, and thus can be seen at both the messenger RNA (mRNA) and protein levels (40). The rate of pIgR gene transcription was measured by nuclear run-on assays at different time intervals after IFN-γ, IL-4, and TNF-α stimulation of HT-29 cells, and the maximal rate of transcription was achieved between 8 and 20 hours after cytokine stimulation (93). However, a small increase in the transcription rate was detected as early as 2 hours after cytokine stimulation, the early onset being most pronounced for TNF-α treatment. In accordance with the slow kinetics, up-regulation of pIgR mRNA levels depended on de novo protein synthesis demonstrated in experiments with the translational inhibitor cycloheximide (CHX). Consistent with the in vitro–induced pIgR up-regulation obtained in cell cultures, increased expression of pIgR is seen in situ in various mucosal and glandular diseases (94). This is presumably caused by local production of IFN-γ and other cytokines secreted from activated local T cells and macrophages, and thus represents a biological link between local immune responses (including increased production of pIgA and pentameric IgM) and enhanced external transport of secretory antibodies. Transient transfection assays have identified several DNA elements capable of binding nuclear factors present in pIgRpositive cell lines, which modulate the expression of the pIgR gene. Constitutive pIgR transcription depends primarily on an E-box located about 75 bp upstream of the transcription start site (13). The E-box is a target for members of the basic helix-loop-helix-leucine zipper transcription factor family, and the family members upstream stimulatory factor (USF)-1 and USF-2 have been shown specifically to bind the pIgR E-box in vitro (95,96). Furthermore, overexpression of USF-1 or USF-2 activated expression of luciferase reporter genes linked to the pIgR promoter (95). Regulatory DNA elements that mediate cytokine-modulated expression have been extensively studied in the proximal promoter region, as well as in the first intron of the human pIgR gene. An interferon-stimulated response element (ISRE) in the first exon and two in the proximal promoter have been implicated in transcriptional activation in response to IFN-γ and TNF (97,98). The exon 1 ISRE has been shown to bind both interferon regulatory factor 1 (IRF-1) and IRF-2. The expression of these factors is induced by IFN-γ and TNF, which supports the role of these two proinflammatory cytokines in up-regulated pIgR expression. An enhancer region in the extensive (5.7 kb) first intron of the human pIgR gene that is sufficient for induction by IL-4, and also contributes significantly to TNF responsiveness, has been characterized in some detail (13). The minimal enhancer for IL-4–induced transcription is a 250-bp region

that contains at least 7 target elements for different DNAbinding factors (99,100). A binding site for signal transducer and activator of transcription 6 (STAT-6) is a crucial element in this enhancer, and STAT6 has been shown to have a dual role in IL-4–activated pIgR transcription: (1) STAT6 binding to the intronic enhancer was required for IL-4–enhanced expression of pIgR-derived reporter genes; and (2) STAT6 activation induced the synthesis of additional factor(s) required for enhanced pIgR transcription. Other functional DNA elements in the intronic enhancer include an NF-κB site required for TNF-α activation and a binding site for hepatocyte nuclear factor-1, a tissue-specific transcription factor with an expression pattern that largely overlaps with that of pIgR (99,101). The mechanism of TNF-α stimulation of pIgR transcription appears to differ from the classical NF-κB activation involved in expression of proinflammatory gene repertoire as exemplified by IL-8. First, the kinetics of induction of pIgR and IL-8 differ substantially. Second, sustained pIgR transcription depends on de novo synthesized RelB, explaining why mRNA for this protein appears more slowly and is sensitive to CHX inhibition. The human intronic enhancer was highly conserved in the murine pIgR gene, suggesting that similar modes of regulation occur in this species. Notably, the conservation of the intronic enhancer extends 3′ to that required for the IL-4– induced transcriptional response, suggesting that this enhancer may also mediate pIgR transcription induced by other signals. It has been demonstrated that the murine pIgR gene is activated by the intestinal commensal flora (102). It is tempting to speculate that the proximity of regulatory elements for TNF-α and IL-4 within this enhancer is important for synergistic expression seen by coincubation with both cytokines.

Structure of Polymeric Immunoglobulin Receptor Although SC had been proposed to be derived from a transmembrane receptor several years earlier, this was not proved until after the molecular cloning of rabbit pIgR by Mostov and colleagues in 1984 (103). Its functional expression in MDCK cells as a transcytotic receptor for pIgA was shown by Mostov and Deitcher only 2 years later (104). The pIgR has an N-terminal extracellular ligand-binding domain, a single membrane-spanning α-helix, and a 103-amino-acid cytoplasmic C-terminal tail that contains all the information for proper routing (Fig. 43-5). The extracellular region is composed of five domains (D1-5) that are homologous in structure to the variable immunoglobulin domains (Ig domains). pIgR exists in all characterized mammalian species, which include the rabbit, human, rat, bovine, mouse, and possum (40). In addition, rabbit pIgR also occurs as a smaller variant in which D2 and D3 are deleted by alternative splicing. This shorter, three-domain rabbit pIgR is fully functional, in contrast with a similar version of the human pIgR constructed by recombinant DNA technology (40). The degree of conservation within the extracellular domains is in

BIOLOGY OF GUT IMMUNOGLOBULINS / 1075

Cleavage site for n release of SC

D1

D3

D4

Basolateral targeting (H647, R648, V651) Endocytosis (Y668, Y725)

D5 Plasma membrane

Non-covalent pIgA and pentameric IgM binding

Covalent plgA binding

Transcytosis P-S655

FIG. 43-5. Schematic diagram of the human polymeric immunoglobulin receptor (pIgR), indicating motifs important for various functions. Numbering corresponds to human pIgR with numbering of the equivalent amino acid in rabbit pIgR in parentheses

the order D1 > D3 ≈ D4 > D5 > D2, and the overall homology is 73%. The high degree of homology of the first domain among species is because of the important function of D1 in ligand binding (see later). The transmembrane segment and the cytoplasmic tail are the most highly conserved domains of the receptor, reflecting their importance for proper function. Cleavage of the pIgR occurs in a flexible region of the molecule between domain 5 and the plasma membrane (sometimes referred to as domain 6); this results in release of the extracellular region of the molecule (SC) to the intestinal lumen. Secondary structure analysis of free human SC suggests that the D2 and D3 domains of pIgR form a rigid structure, and that the other interdomain regions are more flexible in nature (105). Human pIgR contains 20 cysteines confined to the 5 extracellular domains, each being stabilized with 1 (D2) or 2 (D1, D3, D4, and D5) disulfide bridges. An additional disulfide bond in D5 is highly reactive and forms a covalent link to Cys309 of one α-chain in human SIgA. The pIgR is heavily glycosylated and may contain up to 25% carbohydrate (106,107). The pattern of glycosylation, however, is not particularly well conserved among species, and the ligand-binding function of pIgR does not depend on glycosylation. Several complementary approaches have shown that the initial interaction between pIgR and the pIgs occur within D1 of the receptor. Early immunochemical characterization identified the so-called I (inaccessible) determinant exposed only on free SC, which later was found to be located within D1 of pIgR (40). Mapping of the initial binding site with synthetic receptor peptides and monoclonal antibodies demonstrated that a highly conserved sequence within D1 (comprising residues 15–37 of human SC) was particularly important for the initial ligand interaction (108). However, a synthetic peptide covering these amino acids showed relatively low affinity for pIgA and pentameric IgM, as well as promiscuous binding to monomeric IgA and IgG. Thus, specific binding to pIgs is likely to depend on other elements in D1. Point mutation of the first complementarity determining region (CDR)-like loop of D1 (present within the earlier

mentioned peptide) identified Arg37, Arg40, and Lys41 as crucial for the binding of human pIgA to rabbit pIgR (109). However, the same study suggested that other loops in D1 were also involved in pIgA binding. The ability of pentameric IgM to bind the pIgR is differently preserved among species. Human and bovine pIgR have high affinity for pentameric IgM, mouse and rat pIgR bind pentameric IgM poorly, and rabbit pIgR shows virtually no such binding capacity (40). The affinity of the pIgR for pentameric IgM appears to be inversely correlated with the degree of hepatobiliary transfer of pIgA occurring in that species, suggesting that the affinity for pentameric IgM has been lost to avoid “draining” serum of this antibody class, which is important for primary immune responses. The species disparity among pentameric IgM binding has been used to map the binding site for human pentameric IgM in human pIgR (84). A chimeric pIgR with human D1 and rabbit D2to-D5 bound pentameric IgM with the same affinity as human pIgR, whereas the converse chimera (rabbit D1, human D2-to-D5) did not bind pentameric IgM. Thus, the ability of pIgR from some species to bind this polymer is also mediated by D1. Smaller D1 chimeras showed a particular importance of the CDR2-like region. Although IgA is much more abundant than IgM in healthy adults, IgM is believed to be of particular importance in the neonatal period and in patients with selective IgA deficiency. The three-dimensional structure of D1 of the human pIgR was solved by X-ray crystallography (110). Although the general topology of D1 concurred with the typical immunoglobulin fold, several unusual structural features were noticed. A helical region was identified within the first CDR-like loop, postulated to be directly involved in pIgA binding. This structure showed that Arg31 (corresponding to Arg37 in rabbit pIgR; see earlier) was solvent exposed and could interact with pIgA. Arg34 and Lys35 (corresponding to positions 40 and 41 in the rabbit) were both found in the C strand of the immunoglobulin fold in positions unlikely to interact directly with pIgA. However, these amino acids were probably important for pIgA binding because their side chains formed hydrogen bonds with amino acids in the CDR1-like loop and the CDR3-like loop, respectively. The CDR2-like loop of pIgR D1 was unusually short comprising only two amino acids, whereas the CDR3-like loop was tilted away from the other CDRs, unlike the structure seen in antigen-binding sites of immunoglobulins where the three CDRs usually form a continuous binding surface.

Cell Biology and Trafficking of Polymeric Immunoglobulin Receptor The pIgR carries dimeric IgA across the epithelial barrier by a vesicular transport process termed transcytosis. Such intracellular trafficking of the pIgR has been studied in some detail both in rat hepatocytes and liver-derived cell lines and in rabbit pIgR-transfected polarized MDCK cells (111,112) (Fig. 43-6). The latter system has allowed for a

1076 / CHAPTER 43

TJ

ARE

AEE

TGN CRE

Nucleus BEE

FIG. 43-6. Transcytosis of polymeric immunoglobulin receptor/polymeric immunoglobulin A (pIgR/pIgA) in polarized epithelial cells. Synthesis and the transcytotic pathway of the pIgR are shown. (1) The pIgR is translated in the endoplasmic reticulum and targeted via the trans-Golgi network (TGN) to the basolateral membrane where it can complex with ligand. (2) Bound (and unoccupied) pIgR moves to clathrincoated pits and (3) is endocytosed to basolateral early endosomes (BEE). This compartment contains EEA1 and Rab5. (4) The pIgR next enters the common recycling endosome (CRE), but a small portion can recycle to the basolateral surface (4b). (5) Further trafficking leads to the apical recycling endosome (ARE) positive for Rab11, Rab17, and Rab25. (6) The pIgR is exocytosed by fusion of the ARE with the plasma membrane and cleaved to release free or bound secretory component (SC) (7). A substantial portion of the receptor is reendocytosed from the apical surface and subsequently delivered back to the same surface (apical recycling; steps 7b and 7c). Ligand-stimulated transcytosis is mediated via a reduced interaction between occupied pIgR and Rab3b in the CRE and/or the ARE. Tight junctions (TJs) are essential for the epithelial cell to be able to polarize and for the integrity of the epithelial barrier.

detailed mutational analysis of the rabbit pIgR and determination of the role of specific motifs in the receptor for proper trafficking. At this time, the cell biology of pIgR represents the paradigm for transcytosis across a polarized epithelium. The pIgR is synthesized in the endoplasmic reticulum and delivered via the trans-Golgi network directly to the basolateral membrane. A basolateral targeting signal is located in the cytoplasmic tail close to the transmembrane region. The residues His656, Arg657, and Val660 are particularly important for basolateral targeting in the biosynthetic pathway (113).

After delivery to the basolateral membrane, the pIgR is available for binding to its ligands, pIgA and pentameric IgM, that are produced by lamina propria plasma cells in the subepithelial space (see detailed discussion earlier in this chapter). Receptor-mediated endocytosis of the pIg/pIgR complex, and also the unoccupied receptor, occurs by clathrinmediated endocytosis. The tyrosine-based endocytosis motifs located at Tyr668 and Tyr734, as well as phosphorylation of Ser726, are important for this process (114,115). The pIgR can then be found in the early endosomal compartment containing the proteins EEA-1 and Rab5. Some recycling back to the basolateral membrane occurs from this compartment, but the bulk of receptor is shuttled in an actinand microtubulin-dependent manner into the perinuclear endosomal compartment of MDCK cells. This compartment is variably known as the common endosome, the recycling endosome, or the common recycling endosome. Segregation of cargo destined for transcytosis from cargo destined for basolateral recycling (such as transferrin [Tfn] receptor) occurs in the common recycling endosome. In MDCK cells, the pIgR is next delivered to the apical recycling endosome (ARE). This compartment contains the proteins Rab11 and Rab25 (116,117). Release of the SC/pIg complex to the intestinal lumen occurs after fusion of the ARE with the apical plasma membrane and cleavage of the pIgR by a leupeptinsensitive endoprotease. Cleavage of unoccupied pIgR causes free SC, which exerts a number of functions in external secretions (see earlier). Cleavage of the pIgR is relatively slow, and a considerable fraction is recycled to apical early endosomes. It is subsequently transported back to the apical plasma membrane via the ARE. Paired immunofluorescence staining of human large bowel for IgA and pIgR in the epithelium showed intracellular vesicles positive for both, as well as perinuclear staining positive only for the pIgR. Whether the double-positive vesicles correspond to the common recycling endosome or the ARE, or both, currently is unknown. The pIgR-positive, IgA-negative perinuclear region presumably reflects newly synthesized pIgR in the endoplasmic reticulum and the Golgi zone. Transcytosis of the pIgR is regulated or modulated by a number of mediators and second messengers that affect the cytoskeleton and intracellular trafficking (2). Interestingly, pIgA binding to the pIgR stimulates the rate of pIgR transcytosis; this is seen both in vitro in rabbit pIgR-transfected MDCK cells (118), and in vivo during hepatobiliary transfer in rodents (119). Binding of pIgA to its receptor on the basolateral surface initiates a signaling cascade involving the Src family tyrosine kinase, p62yes, and phospholipase Cγ, the latter being phosphorylated after pIgA binding (120,121). Phospholipase Cγ cleaves phosphatidylinositol 4,5-bisphosphate to generate the second messengers inositol 1,4,5triphosphate (IP3) and diacylglycerol (DAG). IP3, in turn, causes release of intracellular Ca++. Evidence from MDCK cells suggests that these second messengers then activate effectors that act on pIgR/pIgA-containing endosomes late in the transcytotic pathway. Increased intracellular Ca++ concentration and DAG promote activation of protein kinase C

BIOLOGY OF GUT IMMUNOGLOBULINS / 1077 epsilon (PKCε) and its association with pIgR/pIgAcontaining endosomes (122). The pIgR must itself be properly sensitized to respond to the signal elicited by pIgA binding. Sensitization involves Arg657 in the pIgR tail and apparently effects a direct interaction between the small GTPase Rab3b and this region of the tail (123,124). Rab3b binds preferentially to unoccupied pIgR and acts as a negative regulator of transcytosis, inhibiting apical exocytosis of pIgRcontaining endosomes (124). Thus, in the presence of a positive signal, these endosomes are released more rapidly if the pIgR is complexed with its ligand. Experiments with human pIgR-transfected MDCK cells, however, have failed to demonstrate ligand-mediated enhanced transcytosis for pIgR (perhaps a finding specific to human transgene expression in this species) (125,126). Second messengers in this cell type were produced on pIgA binding, suggesting that initiation of signaling was conserved, but the transcytotic rate was not increased in response to these signals (125). Whether the human pIgR is not subjected to ligand stimulation in vivo, or whether the failure of such stimulation in MDCK cells is caused by a species incompatibility between human pIgR and signaling molecules or effectors present in canine cells, has not been determined. In either case, salivary SC levels in individuals with selective IgA deficiency are of a similar magnitude as the total SC levels (free and bound) in healthy control subjects, suggesting that the bulk of SC released apically is derived from constitutive transcytosis of the pIgR (40). Ligand binding in vivo may thus have little or no effect on the rate or efficiency of transcytosis in humans.

IMMUNOGLOBULIN G AND THE NEONATAL Fc RECEPTOR FcRn In addition to SIgA, intestinal secretions contain IgG that may function in immune surveillance and host defense. This immunoglobulin is transported bidirectionally across the epithelial barrier by binding to the Fc receptor FcRn. FcRn is not a typical Fc receptor. First, it has a structure like major histocompatibility class (MHC) class I molecules rather than immunoglobulin molecules. Second, it binds IgG at acidic but not neutral pH. Third, it acts as a trafficking receptor for IgG that diverts the molecule away from the lysosome and back to the cell surface. This explains why IgG exhibits a long half-life (longer than all other serum proteins) and why it can move across epithelial barriers to function at mucosal surfaces. The identification of FcRn stems from two discoveries that were made in the 1960s and 1970s. First, Brambell (127), in his experiments on IgG metabolism, found that infusion of excess IgG in vivo caused the half-life of IgG to decrease. This suggested the existence of a saturable receptor responsible for salvaging IgG from catabolism. Second, the ability of suckling neonatal rodents to absorb maternal IgG from breast milk was discovered by Rodewald (128–133) to be explained by a process of pH-dependent, receptor-mediated endocytosis and transcellular transport. Only later was the molecule that carries IgG across the neonatal rat intestine

identified at the molecular level by Simister and Mostov (134) and Simister and Rees (135). This property of IgG transport by the rodent was noted to be restricted functionally to neonatal life and decreased significantly after weaning. Quantitative methods of RNA analysis indeed showed that FcRn decreased 1000-fold in the intestinal epithelium of rodents at weaning (136). However, Blumberg and colleagues (137) showed that FcRn expression continues beyond neonatal life into adulthood by functional and biochemical detection of FcRn in rat hepatocytes. This led to the modern notion that FcRn is functionally important in adult life. Based on available evidence, we now believe that FcRn accounts for the transfer of maternal IgG from mother to fetus across the placenta (138), and, in both children and adults, FcRn explains the long IgG serum half-life (139,140) and how IgG can function at mucosal surfaces (24,141–145). The Immunoglobulin G Receptor FcRn FcRn Is a Major Histocompatibility Class I–like Molecule In 1989, Simister and Mostov (134) cloned the rat complementary DNA for FcRn from a library prepared from the duodenum and jejunum of 11-day-old rats. Analysis of the predicted amino acid sequence indicated that FcRn is a type I membrane glycoprotein containing three extracellular domains highly related to MHC class I molecules (α3: homology of 35–37%; α2: 22–29%; α1: 27–30%), but appended to unique transmembrane and cytoplasmic tail domains. Like MHC class I heavy chain, the FcRn heavy chain is also noncovalently assembled with β2 microglobulin as evidenced by both coimmunoprecipitation and cross-linking studies (134,135,146). The mature extracellular domain extends to amino acid 274, and the transmembrane region spans amino acids 275 to 298. The 44-amino-acid cytoplasmic tail of FcRn contains two well-described endocytosis motifs and several sites for phosphorylation (147–150). Crystallization of the extracellular domain of both rat and human FcRn, in association with their respective β2 microglobulin chains, confirmed that FcRn is structurally related to MHC class I molecules (151,152). As in Class I MHC molecules, the heavy chain of FcRn is composed of a α1-α2 platform linked to the immunoglobulin-like α3 domain. The α1-α2 platform consists of two antiparallel α-helices overlying an eight-stranded β-sheet. The interaction of the β2 microglobulin light chain with the heavy chain of FcRn resembles other class I MHC molecules, contacting the underside of the α1-α2 platform and the side of the α3 domain. In another situation analogous to MHC class I molecules, stable folding of FcRn in the endoplasmic reticulum requires the presence of β2 microglobulin (153). In the absence of β2 microglobulin, FcRn heavy chains form disulfide-bonded homodimers and are trapped in the endoplasmic reticulum (153,154). An important structural difference between FcRn and MHC class I molecules is the lack of the peptide binding groove that typifies MHC class I. Stable folding and

1078 / CHAPTER 43 cell-surface expression of MHC class I molecules are dependent on binding of endogenous peptide between the α1 and α2 helices (155). The two pockets critical for anchoring the peptide termini into the groove of classical MHC class I molecules are physically occluded in rat and human FcRn. On one side of the groove, the pocket is blocked by an arginine side chain (Arg162 in human, Arg164 in rat); on the other side, the second pocket is occluded by the close alignment of the two α-helices that are stabilized in this configuration by a salt bridge (Lys71 and Glu151 in human, Lys71 and Glu153 in rat) and hydrophobic interactions (Leu74 and Trp141 in human, Leu74 and Trp143 in rat). Leucine 74 in both species blocks a third binding pocket that accepts a side chain in class I MHC molecules (151,152,156). Although FcRn exhibits significant structural homology to MHC class I molecules, it is encoded outside of the MHC gene locus, on chromosome 7 in mice (157) and on chromosome 19 in humans (158,159). The FCGRT gene that encodes the heavy chain of FcRn has similar exon/intron organization in mouse and human, consistent with the evolution of FcRn before mammalian speciation (157,160,161). The coding region of human FCGRT shares 63% sequence homology with the mouse gene (159). Structural Basis for pH-Dependent Binding of FcRn to Fc One of the most important functional characteristics of FcRn that distinguishes the molecule from the other Fcbinding receptors is that FcRn binds the Fc domain at acidic but not neutral pH (143). At pH less than 6.5, FcRn binds the Fc portion of IgG with nanomolar affinity. It then releases IgG at neutral pH (156,162). The pH dependence of IgG binding by FcRn suggested that histidine residues, which contain imidazole side chains that deprotonate between pH 6.0 and 7.0, might be involved in this interaction (134). Initial investigations of the FcRn binding site on IgG revealed this to be the same locus at which Staphylococcal Protein A binds Fc, namely, at the Cγ2/Cγ3 domain interface (163,164). Two histidine residues at positions 310 and 435 are located in the Cγ 2/Cγ3 region of IgG, and these, together with isoleucine 253, have been confirmed by site-directed mutagenesis studies to be crucial for binding FcRn (162,165). Resolution of the crystal structure (Fig. 43-7) of FcRn with bound Fc provides further details of the interaction between FcRn and IgG. The interaction site on FcRn is located on the C-terminal portion of the α2 domain α-helix and the first residues of β2 microglobulin (163,166). Binding is stabilized by salt bridges between Fc His-310/FcRn Glu-117 and Fc His-435/FcRn Glu-132 (156,166,167). A strong hydrophobic interaction between Trp 133 on FcRn and Ile 253 on Fc further stabilizes the FcRn/Fc interface (167). Elucidation of the Binding Model Early work on the intestinal Fc receptor in neonatal rats by Simister and Rees (135) provided the first evidence that

FcRn/hdFc structure (2.8 Å)

A

Glu117

Glu132

Transmembrane 275-295 Cytoplasmic tail

B Trp133

Trp 311 Leu322, 323

FIG. 43-7. (A) Crystal structure of FcRn bound to the Fc Fragment of immunoglobulin G (IgG). (B) Schematic of FcRn heavy chain structure. (A: Courtesy P. J. Bjorkman, W. L. Martin, and A. P. West.)

FcRn may function as a heterodimer in vivo. Since then, several lines of evidence have confirmed the 2:1 FcRn-to-Fc stoichiometry. In the current binding model, two FcRn molecules form a dimer via their α3 and β2 microglobulin chains, with their respective α1-α2 domains pointing away from each other, and presumably, their cytoplasmic tails lying in close proximity. The Fc portion of IgG interacts with the binding site on only one of the FcRn molecules of the dimer (the “lying-down” model) (152,163,166,168–171). Before the solution of the rat FcRn crystal structure by Burmeister and colleagues (151) in 1994, using a screen of site-directed FcRn mutants, Raghavan and colleagues (163) identified a histidine pair present in the α3 domain of FcRn that drastically reduced the affinity of FcRn for binding IgG when exchanged for alanines. The binding reaction of this mutant FcRn to IgG was, however, still pH dependent, suggesting that these histidine residues in FcRn could not be the only residues responsible for binding IgG. In fact, the α3 histidine residues were eventually found not to even contact IgG (166). Evidence for this comes from solution of the FcRn structure at pH 6.5 versus 8.0. These studies showed slightly improved stability of the FcRn dimer at pH 6.5 relative to pH 8.0, which was caused by a side chain movement permitting more favorable interactions between a protonated histidine on one FcRn α3 domain and an anionic pocket on the associated α3 domain of the second FcRn molecule making up the dimer (156). Thus, the role of the α3

BIOLOGY OF GUT IMMUNOGLOBULINS / 1079 domain–associated histidine pair in high-affinity binding of FcRn to IgG is to induce stabilization of the FcRn dimer. Further support for the FcRn dimer-binding model was provided by the observation that high-affinity binding to IgG (Kd = 22 nM) occurred only under conditions where FcRn was allowed to dimerize (170). The affinity between FcRn and IgG as determined by surface plasmon resonance (BIAcore International AB, Sweden) analysis was higher when FcRn was immobilized (169). The binding affinities ranged from 17 to 93 nM and were consistent with previous affinity determinations using intact cells (163,169). The affinities determined using immobilized IgG to capture soluble FcRn were lower (Kd = 74–740 nM), similar to previous determinations using soluble FcRn (172). Thus, it appears to be important for FcRn to be constrained in two dimensions to bind IgG with high affinity, as would be predicted to be the case for membrane-associated FcRn. When BIAcore analysis was performed under conditions allowing FcRn dimerization, the binding affinity for IgG was 100-fold greater than when FcRn was allowed to bind as a monomer. These results support a model of FcRn dimerization at the plasma or endocytic membrane when binding IgG. Whether the dimer is formed before binding IgG or develops as a result of IgG binding has not been determined. Dimer formation upon IgG binding could have important implications for intracellular signaling and trafficking.

Cell Biology of Immunoglobulin G Transport The mechanism of FcRn-mediated IgG transport across epithelial cells at mucosal surfaces differs from that defined for the pIgR. First, transcytosis by FcRn is bidirectional (24,141). Second, unlike the pIgR, FcRn does not undergo proteolysis after delivering its ligand to the apical cell surface. Rather, FcRn may reenter the cell for return to the opposite membrane. Thus, unlike the pIgR, which is committed to only one round of pIgA transport, the same FcRn molecule may transport IgG for multiple rounds in both luminal and abluminal directions. Third, unlike the pIgR, FcRn may initially bind its ligand within acidic endosomes, rather than at the cell surface. This concept is not new. In the rat and mouse neonatal intestines, where the luminal gastrointestinal secretions are somewhat acidic, it was initially presumed that maternal IgG binds FcRn at the apical cell surface (133). However, FcRn also transports IgG across the rat yolk sac that divides two compartments, both at neutral pH. Based on these results, it was proposed that IgG can also bind FcRn within acidic endosomes after fluid-phase uptake of IgG from the cell surface (173). Also, Benlounes and colleagues (174) have observed transepithelial IgG transport across isolated neonatal rat intestine in which both the serosal and mucosal solutions were clamped at neutral pH, and thus were inhibitory for IgG binding to FcRn at the cell surface (174). Many other in vitro cell models have confirmed this idea (24,141).

Intracellular Itinerary The exact intracellular itinerary for FcRn transport across polarized epithelial cells is unknown. The bulk of FcRn resides intracellularly. Available evidence indicates that FcRn traffics similarly to the recycling Tfn receptor in the recycling pathway, but nothing yet is known regarding the determinants of transcytosis. The nascent FcRn heavy chain requires association with β2 microglobulin for proper folding, sorting, and function (153,154,175). hFcRn uses calnexin, calreticulin, and ERp57 in the endoplasmic reticulum, similar to MHC class I, but not tapasin or the transporter associated with antigen processing, consistent with the absence of peptides in association with FcRn (X. Zhu and R. S. Blumberg, unpublished studies). Once the FcRn heterodimer is assembled, it traffics through the Golgi apparatus, where it acquires a mature glycosylation pattern. Only hFcRn with mature glycosylation patterns has been detected on the cell surface, but both forms retain the ability to bind IgG (154). It is unclear whether the immature form of FcRn is physiologic or if it plays a significant role in intracellular IgG transport. Intracellular sorting signals typically are contained in the cytoplasmic domain of transmembrane proteins, and in the case of FcRn, two endocytosis motifs have been identified. The first endocytosis signal identified experimentally was the dileucine motif encompassing Leu-322 and Leu-323 (176) and requiring aspartic acid at positions 317 and 318 (150). This sequence conforms to the D/EXXXLL/I sequence, a well-described endocytosis and endosome sorting motif (177). Wu and Simister (150) also described a novel tryptophan-based endocytosis motif in experiments where the mutation W311A reduced the rate of endocytosis to a similar degree as mutations of the dileucine motif, and they hypothesized that the Trp 311–containing sequence WLSL was a type of YXXΦΦ endocytosis signal. YXXΦΦ is a motif that binds to the µ2 subunit of the adaptor protein-2 (AP-2) clathrin adaptor, and thus directs membrane receptors into clathrin-coated pits for endocytosis. Recent in vitro studies show that, as predicted, the WLSL sequence bound the µ2 subunit of the AP-2 clathrin adaptor complex through the YXXΦΦ binding site on µ2 (178). Mutations in either the WLSL or dileucine endocytosis motifs alone moderately reduce the rate of FcRn endocytosis. However, mutation of both endocytosis motifs dramatically reduces the rate of endocytosis. Surprisingly, this effect was greater than that caused by a complete deletion of the cytoplasmic tail of FcRn. In this double mutant, the steady-state cell-surface polarity of FcRn also appears to be affected in that the distribution of FcRn is 95% apical, in contrast with the wild-type condition in which most cell-surface FcRn is basolateral (150). The ability of the W311A/L322A/L323A mutant to transcytose IgG from the apical to basolateral surfaces is diminished, but surprisingly, basolateral to apical transcytosis did not appear to be affected (150). The interpretation is that FcRn may require clathrin-mediated endocytosis from the apical surface, but not necessarily from the basolateral surface. Because the FcRn triple mutant cannot

1080 / CHAPTER 43 bind the AP-2 complex, it can no longer associate with clathrin to undergo clathrin-mediated endocytosis. This would also explain why the polarity of the triple mutant is primarily apical. In the absence of clathrin-mediated endocytosis from the apical membrane, the continuous and nonclathrin-mediated endocytosis from the basolateral membrane, followed by transcytosis, is suggested to result in accumulation of the triple-mutant FcRn on the apical cell surface. McCarthy and colleagues (148) investigated the role of phosphorylation of the cytoplasmic tail in transcytosis of the receptor and found a requirement for Ser313. Although accounting for only 27% of FcRn cytoplasmic tail phosphorylation, Ser313 appears to be necessary for apical-tobasolateral transcytosis. When this residue was mutated to an alanine (S313A), which cannot be phosphorylated, FcRn moved through the transcytotic pathway less than half as efficiently as wild-type FcRn (148). A deficiency in endocytosis, however, does not explain why the S313A FcRn mutant exhibits decreased transcytosis in the apical-to-basolateral direction (148). Interestingly, Ser313 lies within the WLSL endocytosis signal of FcRn. Apparently, however, this substitution does not disrupt the ability of the WLSL moiety to bind µ2. Thus, phosphorylation of S313 is not a requirement for endocytosis, but it may be a requirement for the apical-tobasolateral transcytotic route. The presence of FcRn in apical endocytic vesicles and tubules was first specifically shown with immunoelectron microscopy of rat jejunal epithelial cells (146,179). Immunoelectron microscopy also showed FcRn in endocytic vesicles near the apical and basolateral surfaces of rat yolk sac endodermal cells (173). Although most FcRn is located in intracellular compartments near the apical surface (Fig. 43-8) (24,146,180), FcRn does traffic through the apical and basolateral plasma membrane, and a small fraction of FcRn can be detected on the plasma membrane at any given time. Rodewald (129,131,181) was the first to demonstrate the presence of the pH-dependent IgG receptor on the apical surface of neonatal rat intestinal epithelium, and then on the basolateral surface of isolated neonatal rat intestinal cells. More recently, studies of FcRn function in cell-culture models have been highly informative. A variety of cell lines have been used in culture to express FcRn of various species, and cell-surface expression of FcRn has varied among published models. As one might expect, there are some differences in biology that may be explained by the different cell models used or by the species of FcRn used, or by both. In MDCK cells (a well-described polarized epithelial cell model), for example, expression of human FcRn results in a predominantly basolateral cell-surface distribution (182). Similar observations were made in human cell lines, suggesting that this distribution may be physiologic. In contrast, in a rat renal intramedullary collecting duct cell line, FcRn may be expressed on both cell surfaces in varying degrees (148,150). Because FcRn is located primarily in intracellular compartments and binds IgG at acidic pH, it is believed that FcRn may bind IgG after fluid-phase uptake into the acidic endosome, and then release it at neutral pH after moving back

A

C

D B FIG. 43-8. Immunocytochemistry showing expression of FcRn in the adult human intestinal villus (A), crypt (B), and bronchial epithelium (C). (D) Same section stained with nonspecific antibody. (See Color Plate 23.) (Reprinted by permission of Spiekerman and colleagues [142].)

to the cell surface (Fig. 43-9) (144). This model of FcRndependent IgG trafficking is typical of recycling membrane proteins, but is almost the complete opposite of the classical pathway for the pH-dependent uptake of nutrient solutes, as typified by the low-density lipoprotein (LDL) and Tfn receptors (for review, see Maxfield and McGraw [183]). Unlike LDL and Tfn receptors, however, little FcRn is detected at the cell surface (146). Studies using fluorescence microscopy to localize FcRn in nonpolarized endothelial cells show that the molecule is present within EEA-1–positive early endosomes, and that it does not colocalize with lysosomal markers (184). FcRn is also found in larger, presumably sorting, endosomes, together with Tfn. Remarkably, FcRn rapidly sorts away from this compartment and away from the late endosome/ lysosome in smaller vesicular and tubular structures that also contain Tfn (184,185). FcRn appears to colocalize with Rab4 and Rab11 vesicles in both fusion and fission events with the sorting endosome of a nonpolarized endothelial cell (186). Exocytosis events were associated with Rab11, but not Rab4, which is consistent with the known function of Rab11 in recycling cargo back to the plasma membrane from both the recycling endosome and trans-Golgi network (186) (reviewed in Maxfield and McGraw [183]). Ward and colleagues (186) noted that the majority of Rab11/FcRn exocytosis events were associated with the release of Tfn, suggesting that the association between Rab11 with FcRn occurs in the recycling, and not the biogenesis pathway. Using total internal reflection microscopy, Ober and colleagues (185) visualized exocytosis of FcRn with bound

BIOLOGY OF GUT IMMUNOGLOBULINS / 1081 cell-surface FcRn was also visualized diffusing into the pit of the “prolonged-release” vesicle, possibly providing a method of rapid recovery of FcRn from the extracellular space.

TJ

ARE

AEE

TGN CRE

Nucleus BEE

FIG. 43-9. Synthesis and the transcytotic pathway of FcRn are shown. (1) FcRn is translated in the endoplasmic reticulum and delivered via the trans-Golgi network (TGN) to an intracellular compartment, presumably the common endosome, and to both the basolateral and apical membranes. The sequence of delivery to these compartments is not completely defined. (2) FcRn undergoes endocytosis from both apical and basolateral membranes by clathrin-mediated mechanisms to enter the early endosome (AEE and BEE). The site where FcRn may initially bind immunoglobulin G (IgG) is likely in the early acidic endosome after fluid-phase uptake of IgG. FcRn may also bind IgG at the apical plasma membrane where the microenvironment in the small intestine is acidic because of Na-H exchange. (3) FcRn is transferred from the EE to the common or sorting endosome that contains the transferrin receptor. (4) Sorting out of the common/recycling endosome to the cell surface occurs in Rab4- and Rab11-containing vesicles. Trafficking through the apical recycling endosome is not yet clear for FcRn. (5) FcRn moves all the way to the cell surface to release IgG. Exocytosis and then recycling may occur by a unique kiss-and-run–like mechanism presumably for return of FcRn to the recycling/ common endosome. (6) Such trafficking causes FcRn to traffic away from the late endosome and lysosome, thus explaining why IgG has a long half-life in the circulation.

IgG occurring in two patterns—the classical exocytosis pathway and a slower, “prolonged release.” In classical exocytosis, the exocytotic vesicle fuses completely with the cell membrane and its contents are dispersed. In the “prolongedrelease” pathway, the vesicle only partially fuses with the cell membrane, and IgG is slowly released into the extracellular space. During this “prolonged-release” mode, FcRn can also be seen diffusing into the plasma membrane. Interestingly,

Physiology and Function of Immunoglobulin G at Mucosal Surfaces Studies from our group have shown that FcRn transports IgG in both directions across mucosal surfaces in vivo (25,142). FcRn can therefore direct the secretion of IgG into the intestinal lumen (25). In turn, IgG/antigen complexes are also absorbed via this pathway (25), as suggested previously by studies in neonatal rodents (130). Importantly, we have shown that these complexes are directed into mucosal DCs that can traffic to regional lymphoid structures such as mesenteric lymph node (MLN) and liver (25). Thus, FcRn expression may integrate luminal antigenic events with the function of the immune system. Finally, absorption of Fc-fusion proteins across the respiratory epithelium has now been demonstrated in both adult humans and nonhuman primates (187,188). However, the biological functions of IgG, once present in luminal secretions at mucosal surfaces, are still emerging.

Pathway for Antigen Uptake from Lumen Mediated by FcRn There are several pathways for antigen uptake from the lumen into the lamina propria. Most food antigens are taken up by intracellular pathways and possibly paracellular pathways. However, these pathways appear to be inefficient for the transport of luminal antigen into the lamina propria because most antigens are degraded into nonimmunogenic determinants. Another pathway involves M cells, which are the specialized epithelial cell type present in the follicle-associated epithelium covering the Peyer’s patch. M cells have been considered the major “professional” antigen sampling cell type that is capable of delivering luminal antigens into local DCs in the lamina propria (189). In yet another pathway, intestinal DCs underneath intestinal epithelial cells have been demonstrated to capture luminal antigens present in the intestinal lumen by extending dendrites through interepithelial tight junctions. These emerge into the intestinal lumen for antigen capture and retrieval (190). In addition to these pathways, in the case of the FcRn, the intestinal antigen is delivered to the subepithelial space as a complex with IgG, as described previously (25). These studies used mouse/human chimeric mice in which hFcRn, driven by its endogenous promoter, and hβ2m were expressed in mice lacking mouse FcRn (191). We found that hFcRn can transport not only IgG (rabbit anti–OVA IgG) into the lumen, but also intestinal luminal antigens (OVA administered per oral route) as a complex with IgG across epithelial barriers into the lamina propria. Here the antigen was processed by CD11c+ cells, presumably DCs. Lamina propria cells efficiently capture these IgG/antigen complexes, which is consistent with studies

1082 / CHAPTER 43 that have demonstrated that immune complexes can be captured by DC (192). After capturing antigens in the intestinal lamina propria, DCs have been demonstrated to rapidly migrate into other tissues such as MLN (193), where antigen presentation likely occurs. Compatible with these observations, OVA/anti–OVA IgG complexes that are transported from the lumen by hFcRn can induce an OVA-specific CD4+ T-cell response within the regional lymphoid structures such as MLN and liver. The physiologic meaning of this pathway remains to be established. However, mouse FcRn overexpressed in intestinal epithelial cells under the control of an intestinal fatty acid binding protein promoter can transport IgG into the lumen for the delivery of luminal bacterial antigens into antigen-presenting cells within lamina propria (M. Yoshida and R. S. Blumberg, unpublished data). It is known that luminal bacteria play a central role in the induction and progression of various animal models of colitis and likely human inflammatory bowel disease (IBD), and that luminal bacteria stimulate increased levels of specific IgG (194,195). Moreover, the bacterial antigens that drive the production of specific IgG often are identical to the antigens that drive the pathogenic T-cell response, as shown for bacterial flagellins (196). These studies suggest that FcRn and IgG might have broad functions in immune surveillance, host defense, mucosal tolerance, and inflammation within mucosal tissue.

Immunoglobulin G and Inflammatory Bowel Diseases Increased levels of IgG against self-antigens or luminal bacterial antigens, or both, can be detected in human IBD, especially in patients with ulcerative colitis (UC) (197). Interestingly, the increased IgG levels observed in IBD assumes the phenotype of a Th1 response in Crohn’s disease with increased IgG2 levels (198,199) and a Th2 response in UC with increased IgG1 levels (198–202). Moreover, the excessive IgG antibodies observed in UC are likely to be transported to the apical cell surface and possibly the lumen in human UC. This is suggested by studies that show evidence for IgG and complement deposition on the apical cell surface of epithelial cells in these diseases (203). Increased IgG production can also be detected in animal colitis models such as TCR-α–deficient mice (204), SAMP1/YitFc mice (205), and C3H/HejBir mice (194). Moreover, the locally produced IgG has been reported to show specificity for intestinal microorganisms (194,204,206), colonic epithelial cells (202), and other self-antigen(s) (195,201,204). However, although these increases in IgG have been appreciated for a long time, the functional role of the increased IgG levels in the lumen and tissues has yet to be explained. Previous studies show that intravenously administered IgG (195,207) may affect experimental colitis in animals and possibly IBD in humans (208). Thus, IgG may play a role in the regulation of colitis of patients with IBD. Whether this is caused by effects of excess nonspecific IgG on the catabolism of pathogenic antibodies, by interfering with FcRn function (191),

by blocking ingress of pathogenic bacterial antigens from the lumen (195), or by binding inhibitory Fc receptors (209) remains to be defined. Based on these observations, it can be suggested that IgG and its regulation by FcRn in epithelial cells, macrophages, or DCs (210) may be an important factor in the pathogenesis of IBD.

IMMUNOGLOBULIN E Function of Immunoglobulin E in the Mammalian Intestine Compared with the well-described functions of IgA, IgM, and IgG in host defense at mucosal surfaces, little is known about how IgE acts at these sites. In humans, IgE is normally found in low concentrations in the respiratory and gastrointestinal secretions at equilibrium. However, in people with chronic allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, and food allergy, IgE concentrations in luminal secretions are significantly increased (211–213). In such allergic diseases, IgE-producing plasma cells are located with greater frequency in mucosal lymphoid tissues compared with the major lymphoid organs (spleen and peripheral lymph nodes), and presumably they contribute the bulk of IgE delivered to mucosal surfaces. In the mammalian intestine, IgE is thought to play a role in food allergy, immunity against intestinal parasites, and IBD. The following section addresses each of these functions with a particular focus on the role of IgE in food allergy.

Food Allergy Emerging evidence suggests that food allergy, often manifest as nonspecific gastrointestinal symptoms, is one outcome of IgE-dependent transport of allergens across the intestinal mucosa (214,215). Intestinal anaphylactic symptoms develop rapidly (i.e., peanut allergy) and are mediated by mast-cell activation induced by antigen cross-linking of IgE bound to the high-affinity IgE receptor, FcεRI, on the mast-cell surface. Receptor cross-linking activates the mast cell and results in the release of inflammatory mediators such as histamine, serotonin, and prostaglandins that affect net ion secretion from intestinal epithelial cells and recruit other inflammatory cells (neutrophils) from the periphery (216,217).

Animal Models of Intestinal Anaphylaxis Although numerous studies have documented an increase in the flux of specific and bystander antigens, and other lowmolecular-weight molecules, across the intestine during intestinal anaphylaxis (218), the mechanisms of such transport are only now being elucidated. As modeled in vivo, transepithelial transport of antigen in sensitized animals is rapid and inducible (219–221). In these model systems of food allergy,

BIOLOGY OF GUT IMMUNOGLOBULINS / 1083 mice or rats are sensitized to a protein antigen by subcutaneous or intraperitoneal injection of antigen in the presence of adjuvant (pertussis toxin, CT, or alum), a process known to induce the production of antigen-specific IgE antibodies. Ten to twelve days after systemic sensitization, animals are killed, the jejunum is resected and mounted in an Ussing chamber, and the luminal surface is challenged with the sensitizing antigen or an irrelevant control antigen. The results of these studies show a rapid flux of the antigen across the jejunum that occurs in two distinct phases (219,220,222,223). Phase I of transport occurs within 2 minutes of antigen exposure, and electron micrographs of challenged tissue show the specific antigen within endosomal vesicles throughout the enterocyte and extending into the basal region of the cell. The antigen is also visualized in the lamina propria, where it may interact with and activate resident mast cells located in the subepithelial space. Within 3 minutes of antigen challenge, an increase in short-circuit current (Isc) and tissue conductivity (∆G) can be measured, a result of mast-cell activation and release of mediators. Mast-cell activation also initiates phase II of antigen transport, which occurs as early as 30 minutes after antigenic challenge. This phase is characterized by opening of tight junctions with a measurable increase in both paracellular and transcellular transport of proteins. Importantly, the rapid transport of antigen observed in phase I of challenge has the following characteristics. First, transport is antigen-specific in that irrelevant antigens are not transported, and it is dependent on sensitization to the antigen, because naive animals or animals primed with an irrelevant antigen do not transport significant amounts of the challenge antigen. Second, antigen transport occurs via a transcellular and not a paracellular transport pathway across the intestinal epithelial barrier. It is dependent on the presence of antigen-specific IgE, because the transport responses can be reproduced if antigen-specific IgE is passively transferred from sensitized animals. Finally, antigen transport is mast cell-independent. Phase II of antigen transport, in contrast, occurs by a combination of transcellular and paracellular mechanisms, which results in the nonspecific transport of both irrelevant bystander antigens and the sensitizing antigen across the intestinal barrier (219). Mast cells are required for the second phase of antigen transport in sensitized animals, because mast cell–deficient animals do not respond to luminal antigen with an increase in paracellular conductance. Several studies suggest a role for the low-affinity IgE receptor, CD23, in IgE-mediated transepithelial transport of cognate antigens (220,224,225). In sensitized rodents, CD23 presumably binds IgE/antigen complexes in the intestinal lumen, and then carries the antigen/antibody complex across the epithelial barrier by transcytosis. Somehow, the IgE/antigen complex is released from CD23 and binds high-affinity FcεRI present on the surface of mast cells and basophils. This results in degranulation and release of mediators that act on enterocytes to relax the tight junctions, thereby allowing nonspecific paracellular antigen transport (phase II). Similar to FcRn, CD23 appears to protect the IgE/antigen

complex from degradation by trafficking the complex away from the late endosome and lysosome (225). Studies in CD23 knockout mice provide the strongest evidence to date for this model (223,226). Unlike wild-type mice, CD23-deficient mice do not exhibit enhanced transepithelial transport (phase I) and transmucosal antigen flux (phase II) when sensitized to antigen, and tissues are subsequently challenged in vitro. Subsequent studies showed that anti-CD23 antibodies decreased antigen flux across intestinal segments of sensitized mice (225), and a soluble form of CD23 also inhibited intestinal anaphylaxis (227). Thus, CD23 appears to be the receptor that carries IgE across the intestinal barrier by transcytosis.

CD23 (FcεRII): Low-Affinity Receptor for Immunoglobulin E CD23 is a type II integral membrane protein with an extracellular C-terminal domain that is homologous to C-type lectins. It is expressed at high levels on enterocytes of the rodent and human (228,229). CD23 is constitutively cleaved from the cell surface to generate a soluble form of the receptor. The released form corresponds almost completely with the extracellular domain of the molecule (230). CD23 exhibits biphasic binding to IgE with moderately high (2–7 × 10−7 M) and low (2–7 × 10−6 M) affinities (229). There are two forms of CD23 in humans and rodents, CD23a and b, which share the same C-terminal extracellular domain, but differ in their N-terminal intracytoplasmic regions (228). The two forms result from alternative transcription initiation sites and alternate RNA splicing (231,232). CD23a is constitutively expressed by B cells, where it may play a role in the internalization of IgE-bound antigens, resulting in antigen processing and MHC class II presentation by B cells (233,234). CD23b is expressed in a variety of cell types including monocytes, macrophages, and eosinophils. CD23b expression in B cells and monocytes is not constitutive; rather, it is induced by the cytokine IL-4, an IgE switch factor. CD23b, but not CD23a, is also expressed by enterocytes, and this expression is regulated by antigen sensitization (see later) (223). CD23a and b are functionally distinct. CD23a contains a classical tyrosine-based endocytosis signal in the tail domain that mediates receptor uptake into clathrin-coated pits, and this regulates B-cell functions such as proliferation and adhesion. In contrast, CD23b lacks aromatic residues in the tail domain required for endocytosis, but contains critical asparagine and proline residues that regulate IgE-dependent phagocytosis, an event required for IgEmediated immune function (232). Thus, the functions of CD23 in endocytosis and phagocytosis of IgE/antigen complexes are dissociated in the two isoforms of human CD23a and b. Two additional forms of CD23b have been identified that lack either exon 5 (CD23b∆5) or exon 6 (CD23b∆6) (224,226). Each of these exons encodes a region in the stalk domain of the receptor that resides between the transmembrane and the IgE-binding lectin head domains. High-affinity

1084 / CHAPTER 43 ligand binding is a function of the stalk region of the receptor and, specifically, the region encoded by exon 6. Mutant receptors with a deletion of exon 6 exhibit only low-affinity binding to IgE. In contrast, mutants lacking exon 5 behave like classical CD23b, with a biphasic affinity for IgE. Both classical CD23b and CD23b∆5 are localized to the apical plasma membrane in transfected epithelial cell lines and in enterocytes of antigen-sensitized mice. Antigen sensitization up-regulates the expression of both classical CD23b and CD23b∆5. These data suggest a role for CD23b and CD23b∆5 in enhanced antigen transport across the intestine.

CD23-Dependent Transcytosis of Immunoglobulin E: In Vitro Studies Classical CD23b and splice variant CD23b∆5 have now been shown to function differently in the transcytosis of free IgE and IgE/allergen complexes across polarized epithelial cell monolayers in vitro (224). When expressed in MDCK cells, classic CD23b preferentially transports IgE/allergen complexes in the apical-to-basolateral direction, but it transports only small amounts of free IgE. In contrast, CD23b∆5 efficiently transports free IgE, but not IgE/allergen complexes. CD23b∆5 binds IgE with a similar biphasic affinity as the classical form, demonstrating that the absence of the stalk region encoded by exon 5 does not influence ligand binding, and therefore cannot explain the functional differences between the two receptors. Furthermore, both forms of the receptor localize to the apical plasma membrane of transfected MDCK cells, indicating that receptor polarity for the splice variant is like that of CD23b. Unlike classical CD23b, however, CD23b∆5 is constitutively internalized from the cell membrane via clathrin-coated pits, and endocytosis was stimulated by both anti-CD23 antibodies and free IgE (226). Small interfering RNA knockdown of the clathrin AP-2 complex significantly impaired internalization of an antiCD23 monoclonal antibody in HeLa cells expressing CD23b∆5 but not classical CD23b. These results suggest that exon 5 may negatively regulate clathrin-mediated endocytosis of CD23b, but how this region of the receptor may function in this way is unknown. CD23-mediated IgE/antigen transcytosis is a regulated event. First, expression of both classic CD23b and splice variant b∆5 is induced by antigen sensitization in vivo and by the cytokine IL-4 in vitro (224,226). Consistent with these findings, the CD23 gene contains an IL-4 enhancer element, and IL-4 is produced in excess by intestinal mucosal T cells after antigen sensitization. IL-4 has multiple roles in the pathogenesis of allergic inflammation in the human intestine. In humans, IL-4 is predominantly secreted by CD4+ Th2 cells and stimulates B-cell proliferation and synthesis of IgE. It also stimulates histamine and leukotriene release from mast cells and basophils and primes T cells to the Th2 phenotype. Thus, IL-4 may be a potent target for therapeutic intervention in allergic diseases.

Interestingly, in humans, CD23 is present on both the apical and basolateral membranes of enterocytes at steady state, whereas the rodent receptor is restricted to the apical plasma membrane after antigen sensitization (235). This is consistent with the observation that IgE binding is not detectable at this surface and basolateral-to-apical transcytosis IgE has not been documented for rodent CD23 (224). Thus, human and rodent CD23 likely have different functions. In humans, the receptor may function to transport IgE and IgE/allergen complexes back and forth across the mucosal barrier, similar to how FcRn may work for IgG transport. In the rodent, however, it is unclear how IgE may be transported into the intestinal lumen after production by plasma cells in the lamina propria. Because of these species differences, studies on CD23 in the rodent may not necessarily be generalized to the human.

Immunoglobulin E Function in Intestinal Immunity and Disease A physiologically relevant role for IgE in intestinal secretions is well documented. Thus, antigen-specific IgE has been shown to be protective against a number of intestinal parasites. In mice infected with the intestinal parasite Trichinella spiralis, passively transferred IgE antibodies directed against the nematode result in rapid clearance of the parasite from the intestine (236). These data suggest that IgE is transported from the systemic circulation across the intestinal epithelium to interact with the parasite in the lumen. In a similar study, rats infected with the same organism specifically transported intravenously administered radiolabeled IgE, but not IgG1, to the intestinal lumen, whereas uninfected rats transported neither antibody (237). The proportion of IgE that entered the gut of infected rats was 50% greater than in uninfected control animals, and the small intestine accounted for 86% of this difference. In addition, IgE was found intact in intestinal wash fluids collected from infected rats, indicating that fully functional IgE is transported across the intestine. The absence of significant IgG transport argues that parasite-induced damage or inflammation causing breakdown of the intestinal barrier, and thus “leakage” of plasma proteins, cannot explain how IgE is transferred into the gut. The receptor responsible for IgE transport was not identified in this study, but this is likely CD23, as discussed earlier. Consistent with this idea, the study just described also showed that the cytokine IL-4, likely produced by CD4+ Th2 T cells induced by parasite infection, affected the selective transport of IgE. Rats infused with recombinant IL-4 transported more radiolabeled IgE across the intestine than mock-treated animals. Thus, IgE, like IgA and IgG, is also a secreted immunoglobulin that acts in host defense at mucosal surfaces. IgE transport is up-regulated by IL-4, the cytokine principally involved in stimulating IgE production in B cells. Studies in humans, however, suggest that normal human intestinal

BIOLOGY OF GUT IMMUNOGLOBULINS / 1085 secretions lack IgE (238), and in rodents, IgE-containing plasma cells are largely absent from the lamina propria of nematode-infected rats (239). These observations must somehow be reconciled with the strong evidence that IgE acts mucosally. Perhaps the system is operative only in allergic disease. There is also some evidence that IgE may play a role in chronic intestinal inflammatory diseases such as Crohn’s disease, UC, celiac disease, and possibly even irritable bowel syndrome (217,235,240–243). A series of studies have shown that IgE-mediated activation of mast cells affect barrier function and ion and water transport in the human intestine and also in people with IBD (216,221,244,245). Finally, although strong evidence now exists to show that IgE and IgE/antigen complexes, like IgA and IgG, affect mucosal immunity in the intestine, it is not known whether IgE can be transferred from mother to fetus in utero or is acquired after birth from breast milk. IgE is present in amniotic fluid and colostrum, and CD23-bearing cells are present in the fetal intestine (246). It would be interesting to know if maternal IgE may affect the developing intestine of children born in both developing and developed areas of the world.

10.

11.

12. 13. 14. 15.

16.

17.

CONCLUDING REMARKS Available evidence indicates that the immunoglobulins have important roles in immune surveillance and host defense at mucosal surfaces including the intestine. These large, multisubunit proteins are transported, across polarized epithelial cells, intact and stably folded, by a process termed transcytosis. Such transport has no effect on epithelial barrier function and operates in parallel with other mechanisms of transepithelial solute transport that depend on intercellular tight junctions and a highly restricted paracellular transport pathway.

18. 19. 20. 21. 22.

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44

Mechanisms of Helicobacter pylori–Induced Gastric Inflammation Dawn A. Israel and Richard M. Peek Jr. Colonization of the Gastric Mucosa, 1092 Evasion of the Host Immune Response by Helicobacter pylori, 1094 Development of Gastritis, 1095 Helicobacter pylori Contact-Mediated Cytokine Release and the Development of Acute Inflammation, 1095 Humoral Responses to Helicobacter pylori, 1097 Cell-Mediated Adaptive Immune Responses to Helicobacter pylori, 1098 Additional Host Effectors That Regulate Helicobacter pylori–Induced Inflammation and Progression to Disease, 1098 Helicobacter pylori Strain Variation, Gastric Inflammation, and Disease, 1099

The cag Pathogenicity Island, 1099 Helicobacter pylori Vacuolating Cytotoxin, 1101 Helicobacter pylori BabA and Additional Outer Membrane Proteins, 1102 Helicobacter pylori iceA and Disease, 1103 FldA and Risk for Gastric Mucosa-Associated Lymphoid Tissue Lymphoma, 1103 Novel Helicobacter pylori Virulence Components, 1103 Human Genetic Polymorphisms That Influence the Propensity toward Development of Disease, 1104 Conclusion, 1106 References, 1106

Helicobacter pylori induces a persistent inflammatory response within the human stomach, and chronic gastritis is the strongest known risk factor for peptic ulceration and distal gastric cancer. However, only a minority of people harboring this organism ever acquire clinically apparent disease. H. pylori isolates possess substantial genotypic diversity, which engenders differential host inflammatory responses that influence clinical outcome. H. pylori strains that contain the cag pathogenicity island and secrete a functional cytotoxin induce more severe gastric injury and further augment the risk for development of ulcer disease and cancer of the stomach. However, pathologic sequelae are not completely

dependent on bacterial virulence factors, because clinical disease also is influenced by host genetic diversity, particularly within immune response genes. It is important to gain insight into the mechanisms that regulate H. pylori–induced gastritis, not only to develop more effective treatments for diseases associated with this organism, but also because such knowledge may serve as a paradigm for the role of chronic inflammation in the genesis of other pathologic lesions that arise within the gastrointestinal tract. H. pylori is a gram-negative bacterial species that selectively colonizes human gastric mucosa, and coexisting gastritis develops in virtually all individuals colonized by this organism. A signature feature of the inflammatory response to H. pylori is its capacity to persist for decades, which is in marked contrast with inflammatory reactions induced by other gram-negative enteric pathogens, such as Salmonella, that either resolve within days or progress to eliminate the host. H. pylori has evolved numerous strategies to facilitate its persistence including limiting the bactericidal effects of proinflammatory molecules (1), varying the antigenic repertoire of surface-exposed proteins (2), and/or actively suppressing the host adaptive immune response (3–5).

D. A. Israel: Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232. R. M. Peek Jr.: Division of Gastroenterology, Departments of Medicine and Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1091

1092 / CHAPTER 44 Presence of cag island

H. pylori

Non-colonized mucosa

High-expression IL-1β allele

Atrophic gastritis

Superficial gastritis weeks

Intestinal metaplasia

Dysplasia

Gastric adenocarcinoma

years

FIG. 44-1. Progression to intestinal-type gastric adenocarcinoma. Helicobacter pylori colonization usually occurs during childhood and over a period of weeks leads to superficial gastritis. The presence of the cag pathogenicity island within H. pylori strains and host polymorphisms that promote high expression levels of the cytokine interleukin-1β augment the risk for gastric adenocarcinoma over the course of many years.

However, microbial persistence implies linkage in which signals of the colonizing organism affect signals of the host, and during its coevolution with humans, H. pylori has developed the ability to send and receive signals to and from gastric epithelium, allowing host and bacteria to participate in a dynamic equilibrium. There are biological costs incurred by long-term relationships between H. pylori and humans in that chronic inflammation confers a significantly increased risk for serious disease, including peptic ulceration (gastric, duodenal, or both), gastric adenocarcinoma, and non–Hodgkin’s lymphoma of the stomach (6–23). On the basis of epidemiologic studies in humans and experimental studies in rodents, the World Health Organization has designated H. pylori as a class I carcinogen for gastric cancer, and because virtually all infected individuals have superficial gastritis, it is likely that the organism plays a causative role early in this progression (Fig. 44-1). Eradication of H. pylori significantly decreases the risk for development of gastric adenocarcinoma in colonized individuals without premalignant lesions, providing additional evidence that this organism influences early stages in gastric carcinogenesis (24). Although persistent H. pylori infection is the strongest identified risk factor for peptic ulcer disease or malignancies that arise within the stomach, only a small percentage of colonized individuals ever experience development of clinical sequelae. This raises the hypothesis that

disease risk involves specific and well-choreographed interactions between pathogen and host, which, in turn, are dependent on strain-specific bacterial factors or inflammatory responses governed by host genetic diversity, or both. Evidence also has indicated that H. pylori–induced chronic gastritis, particularly when localized to the acid-secreting corpus, is associated with a reduced risk for development of gastroesophageal reflux disease, Barrett’s esophagus, and Barrett’s-associated esophageal adenocarcinoma (25–30). Thus, a comprehensive understanding of how H. pylori initiates ulcer disease or gastric cancer necessitates an understanding of how H. pylori induces gastritis; therefore, this chapter focuses on specific mechanisms through which H. pylori colonization leads to gastric inflammation and injury via manipulation of the host immune response.

COLONIZATION OF THE GASTRIC MUCOSA Gastric acidity and peristalsis normally inhibit bacterial colonization of the human stomach. However, natural selection has provided H. pylori with several mechanisms to elude these primary defenses and establish persistent infection within the gastric niche (Table 44-1). Acute ingestion of H. pylori in humans leads to transient hypochlorhydria (31,32), but in untreated hosts, microbial-dependent inhibition

TABLE 44-1. H. pylori components that permit gastric colonization or evasion of the host response Bacterial product

Functions

Targeted host defense

Urease Flagellae Adhesins Lewis antigens

Neutralize acidity/regulate inflammation Motility/evade TLR5 Adherence to gastric epithelium Adherence to gastric epithelium/ molecular mimicry Decreases nitric oxide Antioxidants Evade TLR4 Suppresses T-cell activation Antigenic variation

Gastric acid Gastric peristalsis/immune response Gastric peristalsis Host immune response

Arginase AhpC, NapA LPS VacA CagY

LPS, lipopolysaccharide; TLR, Toll-like receptor.

Inflammation Oxidative stress Immune response Immune response Immune recognition

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1093 of acid secretion resolves within several months and intraluminal pH decreases to within the normal range (32). In some individuals, acid production continues to increase, which is likely to result from a compensatory increase in serum gastrin and decrease in mucosal somatostatin levels induced by gastric inflammation (33–36). Another pH-altering mechanism within the H. pylori armamentarium is production of urease, a nickel-containing hexadimer composed of two different subunits (60 and 27 kDa) (see Table 44-1) (37–39). Urease enzymatic activity is conserved among all known Helicobacter species (40), and the primary structure of urease shows little divergence among H. pylori strains; these characteristics are consistent with the hypothesis that urease is a necessary factor for the establishment of chronic infection. There are seven genes in the H. pylori urease gene cluster: ureA and ureB encode the structural subunits of urease, whereas ureE, ureF, ureG, and ureH encode accessory proteins necessary for assembly and Ni2+ insertion required to form active urease (41). Urease activity increases 10- to 20-fold as the pH declines from 6.0 to 5.0, and thereafter remains constant down to a pH of 2.5 (42,43). Acid activation of cytoplasmic urease is mediated by expression of the third gene in the urease gene cluster, ureI, which encodes an H+-gated urea channel. UreI increases the permeability of the bacterial membrane to urea by at least 300-fold as the pH of the surrounding medium becomes acidic (44), and the presence of this acid-activated urea channel within the H. pylori inner membrane is necessary for efficient use of urea present in gastric juice. UreA/B forms a membrane complex with UreI, and assembly of the urease apoenzyme at the membrane surface facilitates access of urea to urease within the cytoplasm, which permits rapid neutralization of the periplasmic space (45). These data explain the absolute requirement for both urease and UreI for survival of H. pylori at a medium pH of less than 4.0, as well as for successful colonization of animal models (46,47). A potential novel function for urease has been identified by Gobert and colleagues (48) using H. pylori isogenic deletion mutants and recombinant proteins in H. pylori/macrophage coculture systems. These investigators determined that up-regulation of inducible nitric oxide synthase (iNOS) and release of nitric oxide (NO), a proinflammatory molecule, were dependent on ureA expression, raising the hypothesis that urease may not only be required for ammonia production, but may also regulate inflammation (48). To facilitate locomotion within gastric mucus and to counteract peristalsis, H. pylori possesses five or six polar flagella that are composed of two major structural subunits: a 53-kDa FlaA and a 54-kDa FlaB (49,50). The genes encoding these two flagellins are located at distant sites on the H. pylori chromosome and are regulated transcriptionally by different promoters (49,51). Expression of flaB and a gene encoding a component of the flagellar basal body export apparatus (flhA), peaks early during the bacterial growth phase and precedes expression of flaA (52). Flagellin-dependent motility also is regulated at a posttranscriptional level by an H. pylori bicistronic operon composed of neuA, encoding a cytidylyl

monophosphate-N-acetylneuraminic acid synthetase, and flmD, which encodes a putative glycosyl transferase gene (53). Inactivation of flmD results in a motility-defective H. pylori mutant that is incapable of glycosylating FlaA (53). The essential role for both flaA and flaB in establishment of persistent colonization has been demonstrated previously by Eaton and colleagues (51), who showed that a flagellate H. pylori strains only transiently colonized gnotobiotic piglets. Ultrastructural studies have demonstrated that the H. pylori flagellum is cloaked by a sheath that is an extension of the bacterial outer membrane, which likely protects the acidlabile flagellar structure from gastric contents (54). McGee and colleagues (55) have now reported that FlbA, a component of the flagellar secretion apparatus that regulates flagellar biosynthesis, also mediates urease activity; thus, FlbA may couple urease production and motility, bacterial phenotypes that are necessary for establishment and maintenance of gastric colonization. Most H. pylori bacteria in colonized hosts are free living, but approximately 20% bind to gastric epithelial cells (56). Adherence properties are likely to be important for acquisition of nutrients from the host or for resistance to shedding of the mucous gel layer, or both. Adhesion by H. pylori to gastric epithelium is highly specific in vivo, and when H. pylori is found in the duodenum, it only overlays islands of gastric metaplasia and not intestinal-type cells (57). Several H. pylori and host ligands involved in adherence have been described. These include a 20-kDa H. pylori hemagglutinin encoded by hpa that binds to sialic acid–containing components of erythrocyte membranes (58) and a 63-kDa exoenzyme S-like protein that binds phosphatidylethanolamine and gangliotetraosylceramide in vitro (59–61). H. pylori can also bind to nonsialylated ligands, which include laminin, fibronectin, various collagens, heparin sulfate, sulfatide, and trefoil factor 1 (TFF1) (62–67). Another adherence-related H. pylori product is HopZ, and inactivation of the gene encoding this protein dramatically decreases the ability of H. pylori to bind gastric epithelial cells in vitro (68). Similarly, inactivation of genes encoding AlpA and AlpB attenuates binding of H. pylori to epithelial cells, indicating that these molecules function as adhesins (69). The genes encoding HopZ, AlpA, and AlpB are present in most, if not all, H. pylori isolates (68,69), suggesting that they may have important roles in colonization. The O antigen of H. pylori lipopolysaccharide (LPS) contains different human Lewis antigens, including Lewisx, Lewisy, Lewisa, and Lewisb (70–73), and inactivation of the bacterial genes encoding Lewisx and Lewisy results in an inability of H. pylori to colonize mice (74), suggesting that H. pylori Lewis antigens may also mediate adhesion (see Table 44-1). In vitro, preincubation of H. pylori with antiLewisx monoclonal antibodies inhibits bacterial binding to gastric epithelial cells (75,76). H. pylori strains that strongly express Lewisx/y are associated with enhanced neutrophilic infiltration and higher colonization densities than strains that only weakly express Lewisx/y (77). Because H. pylori Lewis antigens undergo phase variation (i.e., random and reversible

1094 / CHAPTER 44 high-frequency alteration of phenotype) (78–82), a functional role for such changes in Lewis phenotype may be detachment of H. pylori from host epithelial cells to subsequently facilitate transfer to a new host (83). Studies that have focused on adherence of H. pylori to gastric epithelium have also provided mechanistic insights through which this organism colonizes different parts of the stomach. For example, Syder and colleagues (84) studied bacterial tropism in transgenic mice deficient in acid-secreting parietal cells. Mice were colonized for 8 weeks with an H. pylori strain that expresses adhesins recognized by epithelial NeuAcα2,3Galβ1,4 glycan receptors. In control mice, H. pylori exhibited tropism for gastric mucosa that did not contain parietal cells (e.g., the boundary between the squamous epithelium and the proximal glandular epithelium), and lymphocytic infiltration was found preferentially in this area. In mice with a genetic ablation of parietal cells, epithelial progenitor cells synthesized NeuAcα2,3Galβ1,4 glycans, which was accompanied by an expansion of bacterial colonization and lymphoid aggregates within the glandular epithelium (84). Histopathologic studies in infected human subjects have consistently noted that H. pylori binds to particular areas within the gastric epithelium, and that bacterial density is greatest in the upper portion of the gastric glands. This pattern of colonization mirrors the distribution of TFF1, and Clyne and coworkers (67) have now shown that H. pylori binds avidly to TFF1 dimers, suggesting that TFF1 may act as a receptor for this organism in vivo. A specific component of gastric mucin (α1,4-linked N-acetylglucosamine O-glycan) that is confined to the deeper glandular regions rarely colonized by H. pylori, has now been shown to exert antimicrobial effects against H. pylori in vitro, by inhibiting the biosynthesis of cholesteryl-D-glucopyranoside, a major H. pylori cell wall component (85). These findings indicate that the host has evolved mechanisms to restrict H. pylori to certain regions of the gastric mucosa.

EVASION OF THE HOST IMMUNE RESPONSE BY HELICOBACTER PYLORI If a bacterial species is to colonize a mammalian host persistently, its most formidable challenge is to evade immune clearance. One mechanism through which H. pylori may persist is by limiting the bactericidal effects of proinflammatory molecules, such as NO (1). H. pylori possesses a gene, rocF, that encodes a functional arginase (86) that effectively siphons L-arginine away from a competing host enzyme, iNOS. This, in turn, limits the production of iNOSgenerated NO by limiting the availability of the L-arginine substrate (1). Activated neutrophils generate reactive oxygen or nitrogen species that can induce oxidative DNA damage via formation of DNA adducts, and these reactive species not only injure host tissue, but also have the potential to damage infecting organisms. Therefore, studies have focused on the capacity of H. pylori to withstand environmental stress. Olczak and colleagues (87,88) identified several genes

involved in H. pylori oxidative stress resistance. ahpC encodes a protein that catalyzes the reduction of organic peroxides to alcohols, whereas napA encodes an iron-binding neutrophilactivating protein. Inactivation of each of these genes resulted in enhanced sensitivity to either paraquat and cumene hydroperoxide (ahpC−) or oxygen (napA−), respectively, whereas a double-knockout mutant (ahpC−/napA−) exhibited the greatest sensitivity to organic peroxides and oxygen (87). Interestingly, a novel H. pylori protein (HP0630) was up-regulated in the ahpC−/napA− mutant, suggesting that it may function as a compensatory oxidative stress resistance constituent (87). This same group investigated whether ahpC or another gene involved in antioxidant defense, tpx (encoding thiolperoxidase), was required for in vivo colonization (88). Inactivation of either gene led to a severe impairment of the ability of H. pylori to infect mice, suggesting that oxidative resistance is a critical factor for successful colonization (88). Another level of host defense that may be circumvented by H. pylori is innate immunity. Toll-like receptors (TLRs) are an evolutionarily conserved family of eukaryotic receptors that function in innate immunity via recognition of invariant regions in bacterial molecules termed pathogen- or microbe-associated molecular patterns (Fig. 44-2) (89–91). Eleven different TLRs have been identified in mammals, and although the bacterial ligands for TLRs are distinct, signaling pathways used by these receptors all appear to eventuate in nuclear factor-κB (NF-κB) activation and proinflammatory gene expression (see Fig. 44-2). It is becoming increasingly clear, however, that H. pylori has evolved strategies to avoid global activation of this system. For example, TLR4 recognizes bacterial LPS, yet H. pylori LPS is relatively anergic compared with that of other enteric bacteria (e.g., Escherichia coli) (92–94). This is primarily attributable to lipid A core modifications since H. pylori LPS contains only four fatty acids compared with the more typical six-component fatty acid structure present in other bioactive LPS molecules (95–97). Moreover, in contrast to flagellins expressed and secreted by gram-negative mucosal pathogens such as Salmonella or E. coli, which robustly activate TLR5-mediated proinflammatory responses, H. pylori flagellin is not secreted and is noninflammatory (98,99). H. pylori has been reported to interfere with interleukin-4 (IL-4)–dependent STAT6 signaling and to blunt interferon-γ (IFN-γ)–mediated activation of gastric epithelial cells in vitro (100,101), and H. pylori infection is associated with increased mucosal levels of IL-10 (102–105), an anti-inflammatory peptide that inhibits secretion of proinflammatory chemokines from macrophages and neutrophils. Experimental infection of IL-10−/− mice with either H. pylori or a related Helicobacter species, Helicobacter felis, leads to a significant decrease in colonization in conjunction with a more severe inflammatory response compared with infected IL-10+/− heterozygotes or wild-type control mice (106,107), suggesting that microbially induced IL-10 may attenuate an appropriately targeted gastric inflammatory response that has the capacity to eradicate H. pylori from the stomach.

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1095

TLR4

TLR5

TLR9

TLR3

FIG. 44-2. Innate immune receptors that recognize microbe-associated molecular patterns. IκB, inhibitory κB protein; IL-8, interleukin-8; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; PGN, peptidoglycan; TLR, Toll-like receptor.

H. pylori can also facilitate its own persistence by varying the antigenic repertoire of surface-exposed proteins such as CagY (2) and by actively suppressing the host adaptive immune response (3–5). Because Lewis antigens are expressed on gastric epithelial cell surfaces, a potential biological role for these molecules on the H. pylori cell surface is molecular mimicry (see Table 44-1) (83). This may allow H. pylori to escape host immunologic detection by preventing the formation of antibodies directed against shared bacterial and host epitopes, thus facilitating persistent infection. Initially, human and animal studies supported this paradigm by demonstrating a significant concordance between the Lewis phenotype of the host and the infecting Helicobacter strain (108–111). However, two studies have challenged these earlier observations by failing to show a significant relation between the human host Lewis phenotype and that of the corresponding colonizing H. pylori isolates (77,112). Moreover, strains that express both Lewisx and Lewisy can be isolated from a single human host (113). Thus, the role for H. pylori Lewis antigens in evasion of host responses has not been established conclusively. Finally, H. pylori can survive and replicate within epithelial cells and macrophages (114–116), thereby evading host clearance. The ability of H. pylori to invade epithelial cells has been investigated using differential interference contrast video and immunofluorescence microscopy (114). Using these techniques, investigators found H. pylori to reside within large cytoplasmic vacuoles, and after vacuole evolution, the reappearance of H. pylori in the extracellular environment was observed in parallel with the disappearance of intravacuolar bacteria, suggesting release from intravacuolar sites (114).

At the ultrastructural level, the H. pylori entry process was reported to occur via a zipper-like mechanism, as internalized bacteria were bound within phagolysosomes in close association with condensed filamentous actin (117). Zheng and Jones (115) reported that virulent H. pylori strains can inhibit phagosome development after bacterial ingestion by macrophages. However, despite these multiple strategies for evading host clearance, substantial immune activation still occurs as manifested by epithelial proinflammatory cytokine release, infiltration of the gastric mucosa by inflammatory cells, and cellular and humoral recognition of H. pylori antigens (94).

DEVELOPMENT OF GASTRITIS Helicobacter pylori Contact-Mediated Cytokine Release and the Development of Acute Inflammation The gastric inflammatory response induced by H. pylori consists of neutrophils, lymphocytes (T and B cells), plasma cells, and macrophages, together with varying degrees of epithelial cell degeneration and injury (118). One potential mechanism for induction of inflammation is that H. pylori secretes substances that stimulate mucosal inflammation from afar. For example, urease has been detected within the gastric lamina propria, and the urease complex of H. pylori stimulates chemotaxis by both monocytes and neutrophils and activates mononuclear cells as well (119). Similarly, H. pylori porins and low-molecular-weight molecules, such as Paf-acether, possess chemotactic properties (120,121). Finally, H. pylori water extracts promote neutrophil/endothelial cell

1096 / CHAPTER 44 interactions in vitro and increase leukocyte adherence via CD11a/CD18 and CD11b/CD18 interactions with intercellular adhesion molecule-1 (ICAM-1) (122,123). The presence of acute inflammatory components within H. pylori–infected mucosa suggests that soluble mediators that are capable of attracting polymorphonuclear cells (PMNs) are key regulators in the development of gastritis. Therefore, another mechanism through which H. pylori may induce inflammation is via direct contact with gastric epithelial cells and stimulation of cytokine release. Gastric epithelium from infected individuals contains increased levels of IL-1β, IL-2, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) compared with gastric mucosa from uninfected individuals (102,103, 124–126), and within this group of proinflammatory mediators, IL-8 has been studied most intensively as a mediator of H. pylori pathogenesis. IL-8 is a potent neutrophil-activating chemotactic cytokine (chemokine) that is secreted by gastrointestinal epithelial cells in response to infection with pathogenic bacteria (127). IL-8 produced by activated enterocytes binds to the extracellular matrix and establishes a haptotactic gradient that directs inflammatory cell migration toward the epithelial cell surface (128–131). Consistent with these observations for infected intestinal epithelial cells, expression of IL-8 within H. pylori–colonized gastric mucosa localizes to gastric epithelial cells (132), and levels of IL-8 are directly related to the severity of gastritis (102). In vitro, H. pylori stimulates IL-8 expression and release from gastric epithelial cells, and these events are dependent on an active interplay between viable bacteria and epithelial cells (133–135). Thus, a paradigm for the acute component of H. pylori– induced gastric inflammation is that contact between bacteria and epithelial cells stimulates IL-8 secretion, which then regulates neutrophil infiltration into the gastric mucosa. Molecular Regulation of Helicobacter pylori–Induced Interleukin-8 Gene Expression Proinflammatory cytokine expression often is regulated at the mRNA level by soluble transcription factors, and the human IL-8 gene contains several motifs within its promoter region including binding sites for NF-κB, NF-IL6, activator protein-1 (AP-1) (which is composed of the binding elements c-fos and c-jun), as well as a recently identified novel element

that is homologous to an interferon-stimulated response element (ISRE) (Fig. 44-3) (136–139). NF-κB constitutes a family of transcription factors sequestered in the cytoplasm, the activation of which is tightly controlled by a class of inhibitory proteins termed IκBs (140). Multiple signals, including microbial contact, stimulate phosphorylation of IκB by IκB kinase β (IKKβ), which leads to proteasomemediated degradation of phospho-IκB, thereby liberating NF-κB to enter the nucleus, where it regulates transcription of a variety of genes, including immune response genes (see Fig. 44-3) (141). Stimulation and activation of NF-κB does not require protein synthesis, thereby allowing efficient activation of target genes, such as IL-8. This system is used particularly in immune and inflammatory responses where rapid activation of defense genes after exposure to pathogens such as bacteria is critical for survival of an organism. Several studies have demonstrated that contact between H. pylori and gastric epithelial cells results in brisk activation of NF-κB, which is followed by increased IL-8 expression (see Fig. 44-3) (136,139,142–144). Smith and colleagues (145) have delineated additional upstream mediators of H. pylori–induced NF-κB activation by transfecting HEK293 cells with TLR2, as well as an NF-κB reporter gene. Cotransfection of TLR2 conferred the ability to activate NF-κB and to induce IL-8 production in response to different H. pylori strains (145). The ability of H. pylori to activate NF-κB in vitro has been corroborated in vivo, as activated NF-κB is present within gastric epithelial cells of infected, but not uninfected, patients (142), which mirrors the distribution pattern of increased IL-8 protein within colonized mucosa. Mitogen-activated protein kinases (MAPKs) also mediate H. pylori–dependent IL-8 expression. MAPKs participate in are signal transduction networks that target transcription factors such as AP-1 and regulate a diverse array of cellular functions, including cytokine expression (146–148). MAPK cascades are organized in three-kinase tiers consisting of an MAPK, an MAPK kinase (MAPKK), and an MAPK kinase kinase (MAPKKK), and transmission of signals occurs by sequential phosphorylation and activation of components specific to a respective cascade (Fig. 44-4). In mammalian systems, at least five MAPK modules have been identified and characterized to date; these include extracellular signal– regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK) (146–148). H. pylori activates p38, ERK1/2, and

AP-1

ISRE-like −145

−132

c-fos c-jun −126 −120

IL-8 NF-IL6 −94

−80

NF-κB −70

FIG. 44-3. Binding motifs for transcription factors within the human interleukin-8 (IL-8) gene promoter. AP-1, activator protein-1; ISRE, interferon-stimulated response element; NF-κB, nuclear factor-κB.

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1097 Epithelial cell cytoplasm

cag+ HB-EGF

IKKβ pro-HB-EGF IκB/NFκB

MKK4

EGFR MEK1/2 Ras

JNK

ERK1/2

c-fos

c-jun

Nucleus

IL-8

c-fos

FIG. 44-4. Helicobacter pylori strain-specific components activate multiple pathways in gastric epithelial cells. AP-1, activator protein-1; EGFR, epidermal growth factor receptor; ERK, extracellular signal-related kinase; HB-EGF, heparin-binding epidermal growth factor; IKKβ, inhibitory κB kinase β; IL-8, interleukin-8; JNK, c-Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase/ERK kinase; MKK, mitogen-activated protein kinase kinase; NF-κB, nuclear factor-κB.

JNK in gastric epithelial cells in vitro (149–151) (see Fig. 44-4), and activation of ERK1/2 is dependent on transactivation of the EGF receptor, a receptor tyrosine kinase (152). Cell culture studies have demonstrated that H. pylori– induced IL-8 expression is dependent on both NF-κB and MAPK activation of AP-1 (136), and cross talk between NF-κB and MAPK pathways has been demonstrated previously. For example, MEKK1, a hierarchical MAPK kinase, can directly activate the IκB kinase signalsome (141,153). However, inhibition of ERK1/2 attenuates H. pylori– induced IL-8 secretion without affecting NF-κB (149), raising the possibility that synergistic interactions between AP-1 and NF-κB occur within the IL-8 promoter. Consistent with these data, H. pylori activation of ERK1/2 results in enhanced c-fos transcription (150,151), indicating that ERK1/2 may exert regulatory effects on IL-8 production that are primarily dependent on AP-1 (see Fig. 44-4). Another layer of complexity is added when one considers that maximal H. pylori–induced IL-8 gene transcription requires activation of NF-κB, AP-1, and ISRE elements within the IL-8 promoter (see Fig. 44-3) (139). Collectively, these data indicate that the mucosal inflammatory reaction that develops in response to H. pylori involves multiple different pathways converging on the IL-8 promoter, and that H. pylori–mediated host signaling is of central importance for understanding the inflammatory response to this pathogen, which, if left

untreated over decades, may progress to peptic ulceration or gastric cancer.

Humoral Responses to Helicobacter pylori Although H. pylori colonization induces an exuberant systemic and mucosal humoral response directed at multiple antigens (154–159), antibody production does not result in eradication, even though this organism is susceptible in vitro to antibody-dependent, complement-mediated phagocytosis and killing (160,161). The ineffective humoral response generated against H. pylori and its components have led some investigators to speculate that this may actually contribute to pathogenesis. For example, a potential consequence of H. pylori Lewis antigen expression is induction of a humoral response that enhances gastric inflammation and injury (83). H. pylori infection in mice induces anti-Lewisx antibodies with an affinity for parietal cells, and immunization of mice leads to the development of anti-Lewisx/y antibodies that cross-react with host Lewisx/y antigens present on the β-chain of the gastric H+,K+-ATPase located in parietal cell canaliculi (162,163). In humans, H. pylori infection similarly induces autoantibodies directed against the canaliculi of gastric parietal cells (164,165). IgM antibodies generated by immortalized B cells obtained from H. pylori–colonized

1098 / CHAPTER 44 gastric mucosa also recognize gastric epithelium (166). These findings indicate that the humoral response to H. pylori may contribute to the development of distinct histologic lesions. For example, an autoimmune reaction against parietal cells may lead to gastric atrophy with a concomitant reduction in gastric acidity; conversely, immunoglobulin mediated destruction of epithelial cells may initiate or maintain mucosal inflammation and epithelial cell injury.

Cell-Mediated Adaptive Immune Responses to Helicobacter pylori The ability of the gastrointestinal tract to discern pathogenic bacteria from commensals is regulated through T-cell–dependent responses. CD4+ T cells can be broadly divided into two functional subsets, type 1 (Th1) and type 2 (Th2) T helper cells, each of which are defined by distinct patterns of cytokine secretion. Th1 cells produce IL-2 and IFN-γ and promote cell-mediated immune responses, whereas Th2 cells secrete IL-4, IL-5, IL-6, and IL-10 and induce B-cell activation and differentiation (167). The type of immune response to a particular microbial agent is governed by preferential expansion of one T helper cell subset accompanied by a corresponding relative down-regulation of the other (167). In general, most intracellular bacteria induce Th1 responses, whereas extracellular pathogens stimulate Th2-type responses. The importance of CD4+ T cells in H. pylori–induced inflammation is underscored by studies using genetic models of immunodeficiency. Compared with wild-type mice, severe combined immunodeficiency mice, T-cell–deficient mice, or recombinase activating gene– deficient mice infected with Helicobacter experience less severe mucosal injury despite similar levels of bacterial density, whereas infected B-cell–deficient mice are no different from wild-type littermates in the progression to gastric inflammation and injury (168–170). Although the acquired immune response to H. pylori involves both Th1- and Th2-type cells, cytokine profiles indicate a Th1 predominance, because the majority of H. pylori antigen-specific T-cell clones isolated from infected gastric mucosa produce greater levels of IFN-γ than IL-4 (171). Consistent with these observations, H. pylori stimulates production of IL-12 in vitro, a cytokine that promotes Th1 differentiation (105). This is somewhat counterintuitive based on the premise that H. pylori is noninvasive and that infection is accompanied by an exuberant humoral response, but additional studies now suggest that this Th1-biased response may actually play an important role in pathogenesis. H. pylori infection of IFN-γ–deficient mice (that fail to mount an appropriate Th1 response) leads to decreased levels of gastric inflammation and atrophy compared with wild-type mice (172–174). In vivo neutralization of IFN-γ in mice infected with H. felis similarly reduces the severity of gastritis (175), whereas gastric inflammation and atrophy can correspondingly be induced by delivering infusions of IFN-γ, even in the absence of Helicobacter infection (176).

Certain strains of mice (C57/BL6) infected with H. felis that mount a polarized Th1-type response experience extensive gastric inflammation, whereas other genetically distinct murine strains (Balb/c) that respond to infection with a Th2-like response experience only minimal gastritis (177). Adoptive transfer of Th1-type cells from Helicobacterinfected donor mice into infected recipients increases the severity of gastritis, whereas transfer of Th2-type lymphocytes reduces colonization density (178). Consistent with these results, antecedent challenge with a helminth (Heligmosomoides polygyrus) that induces a Th2-type mucosal reaction significantly attenuates the development of Th1-mediated gastritis and atrophy in response to H. felis (179), whereas induction of a Th1 response in a Th2responding host (Balb/c mice) enhances gastric injury after infection with H. felis (180). Regulatory T cells (Treg) also have been implicated in modulating the immune response directed against H. pylori, as two studies have demonstrated that CD4+ CD25+ Treg cells may limit the host inflammatory response by decreasing activation of CD4+ T cells within inflamed mucosa (181,182). Surface receptors on dendritic cells such as C-type lectins may also be involved in the host response to H. pylori. C-type lectins recognize carbohydrate structures and, on binding, internalize pathogens for Ag processing and presentation to T cells. The dendritic cell–specific C-type lectin ICAM-3–grabbing nonintegrin (DC-SIGN, CD209) is involved in binding to the HIV-1 envelope glycoprotein to enhance infection of T cells. Common features of pathogens that interact with DC-SIGN include chronicity and the ability to manipulate the Th1/Th2 response, and binding of mannosylated and fucosylated surface glycans of H. pylori to DC-SIGN has now been reported by Appelmelk and colleagues (183). H. pylori also induces the release of Th1-type cytokines (e.g., IL-12) from dendritic cells in vitro (184), raising the hypothesis that interactions between H. pylori and dendritic cells may influence Ag presentation, as well as cytokine secretion, thereby contributing to the persistent inflammatory response induced by this pathogen. Collectively, these data are consistent with a model in which an inappropriate host T-cell response toward H. pylori facilitates the development of gastric inflammation and injury.

Additional Host Effectors That Regulate Helicobacter pylori–Induced Inflammation and Progression to Disease Cyclooxygenases (COXs) catalyze key steps in the conversion of arachidonic acid to endoperoxide (PGH2), a substrate for a variety of prostaglandin synthases that, in turn, catalyze the formation of prostaglandins and other eicosanoids (Fig. 44-5) (185). Prostaglandins regulate a diverse array of physiologic processes including immunity, maintenance of vascular tone and integrity, nerve development, and bone metabolism (185). Three isoforms of COX have now been identified, each possessing similar activities, but differing in

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1099 Arachidonic acid

injury (201), which may contribute to long-term sequelae such as gastric cancer.

HELICOBACTER PYLORI STRAIN VARIATION, GASTRIC INFLAMMATION, AND DISEASE

FIG. 44-5. Cyclooxygenases (COX) catalyze key steps in the prostaglandin pathway. PG, prostaglandin; NSAIDs, nonsteroidal anti-inflammatory drugs; TXA2, thromboxane A2.

expression characteristics and inhibition profiles by nonsteroidal anti-inflammatory drugs. COX-1 and COX-3 (a splice variant of COX-1) are expressed constitutively in many cells and tissues. In contrast, COX-2 expression can be induced by a variety of growth factors and proinflammatory cytokines such as TNF-α, IFN-γ, and IL-1 in a number of pathophysiologic conditions (186). COX-2 expression is increased in epithelial cells cocultured with H. pylori (187,188) and within gastric mucosa of H. pylori–infected individuals (189,190). COX-2 expression is further increased within H. pylori–induced premalignant (atrophic gastritis and intestinal metaplasia) and malignant (adenocarcinoma) lesions (191,192), and COX inhibitors such as aspirin and other nonsteroidal anti-inflammatory drugs have been shown to decrease the risk for distal gastric cancer (193,194). H. pylori also activates phospholipase A2, an enzyme that catalyzes the formation of the prostaglandin precursor, arachidonic acid, in vitro and in vivo (195,196). The inflammatory response induced by H. pylori leads to the production of mutagenic substances, such as products of iNOS, which promote oncogenesis (190,197). iNOSgenerated NO can be converted to reactive nitrogen species, which nitrosylate a variety of cellular targets including DNA and proteins. Superoxide anion radicals generated by PMNs also induce DNA damage through the formation of DNA adducts (198). Serum levels of vitamin C, a scavenger of reactive oxygen species and nitrates, are inversely proportional to the prevalence of gastric cancer (199). Eradication of H. pylori has been reported to increase gastric intraluminal ascorbic acid levels (200), thus the presence of H. pylori also affects gastric mucosal antioxidant defenses. Long-term infection by H. pylori can also engender a gastric environment conducive to overgrowth by non-Helicobacter pH-sensitive bacteria. For example, a histologic consequence of persistent H. pylori colonization is the development of gastric atrophy, which leads to a reduction in gastric acidity, a fundamental barrier against infection of the stomach. In the setting of hypochlorhydria, other bacterial species, such as Acinetobacter, can induce gastric inflammation and

Colonization of humans by H. pylori is relatively common, but the development of ulcer disease or cancer follows in only a fraction of infected individuals. H. pylori strains from different individuals are extremely diverse because of point mutations, gene insertions, or deletions (202–205), and genetically unique derivatives of a single strain are present simultaneously within an individual human host (206). Although this extraordinary diversity has hindered characterization of bacterial factors associated with various disease outcomes, different genetic loci have been identified for which individuals harboring particular alleles have different risks for disease. These markers are not completely independent of each other and, importantly, are not absolutes, but instead reflect degrees of risk.

The cag Pathogenicity Island The most well-characterized H. pylori virulence determinant is the cag pathogenicity island, a horizontally acquired 27-gene locus that is present in approximately 60% of U.S. strains (202,203,207,208). Although all H. pylori strains induce gastritis, cag+ strains significantly augment the risk for severe gastritis, peptic ulcer disease, atrophic gastritis, and distal gastric cancer compared with that incurred by cag− strains (102,157,158,209–218). Several cag genes encode products that bear homology to components of a type IV bacterial secretion system that functions as a molecular syringe to export proteins, and transmission electron microscopy has now permitted detailed visualization of the secretion apparatus (219). The product of the terminal gene in the island (CagA) is translocated into host epithelial cells by the cag secretion system after bacterial attachment, where it undergoes tyrosine phosphorylation by members of the Src family of kinases such as c-Src (Fig. 44-6) (220–230). Phospho-CagA subsequently activates a eukaryotic phosphatase (SHP-2), as well as ERK, a member of the MAPK family, leading to morphologic changes (e.g., cell scattering) that are reminiscent of unrestrained stimulation by growth factors (see Fig. 44-6). These in vitro observations mirror events occurring within inflamed mucosa as CagA is translocated into and phosphorylated within epithelial cells and binds SHP-2 in vivo (231). It is likely no coincidence that manipulation of the IL-6 family coreceptor gp130, leading to altered SHP-2 signaling, similarly culminates in the development of intestinal-type gastric adenocarcinoma in genetically engineered mice (232,233). CagA also has been shown to bind to the c-Met receptor, which stimulates a motogenic response in gastric epithelial cells that is dependent on ERK signaling (234).

1100 / CHAPTER 44

Dephosphorylation of host cell proteins

FIG. 44-6. Molecular signaling alterations induced by intracellular delivery of Helicobacter pylori CagA.

Because of the ability of CagA to commandeer multiple host pathways, investigators have sought to define differences in the biological activity of CagA proteins that are present in different H. pylori strains. Higashi and colleagues (228) demonstrated that the number of tyrosine phosphorylation motifs (TPMs) within CagA proteins isolated from people residing in Western countries with low rates of gastric cancer can vary substantially, and that SHP-2 binding affinity and induction of cell scattering are potentiated by an increasing number of such motifs (228). In contrast, TPMs within CagA proteins from H. pylori strains isolated from high-risk cancer regions such as East Asia are unique, and the sequences flanking East Asian–type TPMs perfectly match the consensus binding site for SHP-2 (228). As expected, binding of SHP-2 and cell scattering are induced more potently by CagA proteins containing East Asian–type TPMs compared with Western-type TPMs. Consistent with these in vitro findings, clinical studies have demonstrated that H. pylori strains with a functional cag island and an increased number of CagA TPMs are more closely associated with gastric atrophy and gastric cancer (235–237), which may explain, in part, the strikingly different rates of gastric cancer in these regions. In addition to the effects of phospho-CagA on signaling pathways that alter cellular morphology, CagA phosphorylation also results in activation of C-terminal Src kinase, which inhibits the activity of Src, leading to a reciprocal decrease in the level of phosphorylated CagA (229,238). Inactivation of c-Src also leads to dephosphorylation of cortactin, an actin-binding protein, which is subsequently redistributed to cellular protrusions (238). Although this negative feedback

loop likely contributes to the long-term equilibrium between H. pylori and its host, emerging data indicate that unphosphorylated CagA can also exert effects within the cell that contribute to pathogenesis. For example, unmodified CagA binds to growth factor receptor bound 2 (Grb2), which results in activation of the Ras/MEK/ERK MAPK pathway (239). Translocation, but not phosphorylation, of CagA leads to disruption of apical-junctional complexes in polarized epithelial cells and a loss of cellular polarity, alterations that have been shown previously to play a role in carcinogenesis (240). Finally, CagA can activate the serum response element, leading to increased epithelial gene transcription, in a phosphorylation-independent manner (241). A CagA-independent consequence of cag-mediated epithelial contact is secretion of IL-8. Clinical observations that the cag island represented a disease-associated locus led to subsequent molecular investigations and, not surprisingly, the first H. pylori strain-specific constituent identified as being required for IL-8 production was a component of the cag secretion apparatus, cagE (242). Inactivation of cagE not only attenuates IL-8 expression, but also decreases activation of NF-κB and MAPK in vitro (242). Numerous cag island genes (cagG, cagH, cagI, cagL, and cagM), but not cagA, have now been demonstrated to be required for NF-κB– and MAPK-mediated IL-8 production (Fig. 44-7) (149–151, 243–245), and clinical cag+ strains are more potent in stimulating IL-8 production than cag− strains (133–135). Consistent with these findings, H. pylori cag− strains induce an enhanced IL-8 and inflammatory response in human tissue (102,103), and inactivation of cagE or the entire cag locus, or both, attenuates the development of gastritis and atrophy in a rodent model of H. pylori–induced gastric adenocarcinoma, Mongolian gerbils (246,247). The effects of cag island components on additional host responses also have been explored. Using eukaryotic microarrays, Guillemin and colleagues (248) transcriptionally profiled AGS gastric epithelial cells that were cocultured with H. pylori wild-type or isogenic cag mutant strains. An isogenic cagA− mutant induced expression of less cytoskeletal-related genes compared with the parental strain, and as expected, inactivation of cagE resulted in a loss of the ability to up-regulate cagA-dependent genes, as well as a number of immune response genes (248). However, the isogenic cagE mutant retained the ability to induce expression of a subset of host genes that function in cellular recognition events including IFN-γ, plasminogen, endothelin-1, and TFF1 (248). These genes were not induced by an H. pylori cag island deletion mutant, indicating that host cells respond differently to strains lacking the entire cag island and those with a defective secretory system (248). Until recently, the substrate translocated into the host cell by the cag island that induced NF-κB–mediated gene expression remained undefined. However, exciting data reported by Viala and colleagues (249) demonstrate that H. pylori peptidoglycan delivered by the cag secretion system is recognized by intracellular Nod1, a member of a new family of intracytoplasmic pathogen-recognition molecules

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1101

FIG. 44-7. Induction of interleukin-8 (IL-8) is dependent on numerous components of the Helicobacter pylori cag pathogenicity island. Induction of IL-8 for individual isogenic cag island mutant strains of H. pylori is shown compared with the wild-type (WT) strain (100%). cagA and cagE are highlighted

(see Fig. 44-2). Nod1 sensing of H. pylori peptidoglycan activates NF-κB, and Nod1-deficient mice are more susceptible to infection by H. pylori cag+ strains compared with wild-type mice (249). Collectively, these data indicate that cag+ strains are disproportionately represented among hosts who acquire serious sequelae of H. pylori infection, and that genes within the cag island are necessary for induction of pathogenic epithelial responses that may heighten the risk for disease, particularly over prolonged periods of colonization.

Helicobacter pylori Vacuolating Cytotoxin The aforementioned studies have contributed to the genesis of a molecular portrait through which CagA and the cag island manipulate host signaling pathways that regulate cellular responses and cytokine production. However, most individuals colonized by cag+ strains remain completely asymptomatic. This paradox has fostered the need for studies to identify other microbial factors that may influence disease. An independent H. pylori locus linked with pathologic outcomes such as peptic ulcer disease and gastric cancer is vacA, which encodes a secreted bacterial toxin (VacA) (250–255). When added to epithelial cells in vitro, VacA induces multiple structural and functional alterations in cells, the most prominent of which is the formation of large intracellular vacuoles (256). vacA encodes a precursor protein of approximately 140 kDa, which is processed to yield a mature 87-kDa protein (VacA) (250–254). Mature VacA monomers then assemble into oligomeric structures that are composed of 12 to 14 subunits (257,258). When subjected to acid or alkaline conditions, the VacA oligomer disassembles, but retains the capacity to reanneal after pH neutralization (258).

Acid activation also enhances the interaction of VacA with cellular membranes in vitro by increasing its insertion into artificial lipid bilayers, where it forms hexameric anion channels (259–262). Cytoplasmic internalization of VacA is mediated by active cellular processes and is required for cytotoxicity and vacuole formation (263). Vacuoles have been suggested to originate from an intermediate intracellular compartment juxtaposed between the lysosomal and late endosomal stage because vacuoles express the late endosomal marker Rab7 and their formation depends on the presence of subvacuolating concentrations of weak bases such as ammonia (264–268). There is a strong correlation between vacuolating cytotoxin activity and the presence of cagA. However, vacA and cagA map to two distinct loci on the H. pylori chromosome, and mutation of cagA does not affect toxin production, indicating that expression of the two proteins is independent (269). Unlike the cag island, vacA is present in virtually all H. pylori strains examined (251,270); however, strains vary considerably in production of cytotoxin activity, and this diversity is primarily caused by variations in vacA gene structure (Fig. 44-8). The regions of greatest diversity are localized near the 5′ end of vacA (allele types s1a, s1b, s1c, or s2) and in the midregion of vacA (allele types m1 or m2) (see Fig. 44-8) (270–272). H. pylori strains that possess a type s1/m1 vacA allele are associated with an increased risk for peptic ulcer disease and gastric cancer (273–276) and enhanced gastric epithelial cell injury (277,278) compared with vacA s2/m2 strains. The relation between s1/m1 alleles and gastric cancer is consistent with the distribution of vacA genotypes throughout the world. In regions where the background prevalence rate of distal gastric cancer is high, such as Colombia and Japan, most H. pylori strains contain type

1102 / CHAPTER 44 vacA

Signal sequence allelic types

Mid-region allelic types

s1a

m1

s1b

m2a

s1c

m2b

s2

FIG. 44-8. Schematic representation of vacA, the gene encoding the Helicobacter pylori vacuolating cytotoxin. vacA is distinguished by mosaicism within the signal sequence (types s1a, s1b, s1c, and s2) and midregion (types m1 and m2).

s1/m1 alleles (272), whereas the converse is true in regions of the world with low rates of distal gastric adenocarcinoma, underscoring the importance of vacA as a bacterial locus related to high-grade host responses within the gastric niche. Most type s1 VacA toxins possess detectable cytotoxic activity in vitro, whereas type s2 VacA proteins possess little, if any, cytotoxic activity (270). This is attributable to the presence of a 12-amino-acid hydrophilic segment at the N-terminus of type s2 toxins, which abolishes cytotoxic activity (279,280). The midregion of VacA contains a cell-binding site; m1-type toxins exhibit higher binding affinities to host cells than do m2-type toxins, and VacA has been demonstrated to bind to a unique receptor-type protein tyrosine phosphatase, PTPξ, a member of a family of receptor-like enzymes that regulate cellular proliferation, differentiation, and adhesion (281). Oral delivery of purified VacA induces gastric inflammation, hemorrhage, and ulcers, but only in PTPξ+/+ mice; and in vitro, VacA treatment of PTPξ+/+, but not PTPξ−/−, cells induces cellular detachment, which may contribute to H. pylori–induced ulcerogenesis in humans (281). In addition to vacuolation and cellular detachment, VacA induces gastric epithelial cell apoptosis, a form of programmed cell death that likely contributes to the development of ulcer disease and gastric cancer (282–286). Cover and colleagues (285) demonstrated that VacA-induced apoptosis is dependent on motifs present within the NH2 terminus of VacA, suggesting that differences in levels of apoptosis among H. pylori–infected individuals may result from straindependent variations in VacA structure. The eukaryotic mediators of toxin-induced apoptosis also have been identified, as treatment of cells with purified VacA leads to the release of mitochondrial cytochrome c, implicating the mitochondrial apoptotic pathway in VacA-induced programmed cell death (286). VacA also functions as transmembrane pore, permeabilizing host epithelial cells to urea, which, in turn, may allow H. pylori to manipulate the pH of its environment by generating ammonia. When added to polarized epithelial cell monolayers, acid-activated VacA increases

paracellular permeability to organic molecules, iron, and nickel. Cellular extracts from toxigenic H. pylori strains increase vascular permeability, leukocyte adhesion, platelet aggregation, and vasoconstriction within the gastric mucosal microcirculation of rats (287). VacA also has been shown to actively suppress T-cell proliferation and activation in vitro (3–5), which may contribute to the longevity of H. pylori colonization (see Table 44-1). Collectively, these data indicate that VacA can induce multiple physiologic consequences that may contribute to pathogenesis.

Helicobacter pylori BabA and Additional Outer Membrane Proteins Sequence analysis of the genomes from H. pylori strains 26695 and J99 has shown that an unusually high proportion (1%) of identified open reading frames are predicted to encode outer membrane proteins (OMPs), which may represent novel H. pylori adhesins; consequently, attention has been directed toward a possible role of these OMPs in H. pylori pathogenesis (202,203,288,289). BabA is a membrane-bound adhesin encoded by the strain-selective gene babA2 that binds the blood-group antigen Lewisb present on gastric epithelial cell membranes, and H. pylori strains possessing babA2 are associated with increased risk for gastric adenocarcinoma (273). The presence of babA2 is associated with cagA and vacA s1; strains that possess all three of these genes convey the greatest risk for gastric cancer (273). H. pylori babA2+ strains are associated with an increased risk for glandular atrophy, intestinal metaplasia, and enhanced epithelial cell proliferation compared with strains that lack babA2, which may partly explain the augmentation in gastric cancer risk associated with these strains (290). Another adherence-related H. pylori OMP is HopQ, which is encoded by a gene that has been shown to co-vary with the cag pathogenicity island. Cao and Cover (291) identified two distinct families of hopQ alleles and demonstrated that type I alleles are found significantly more commonly in H. pylori cag+ vacA s1 strains isolated from ulcer patients than in cag+ vacA s2 strains harvested from patients without ulceration, suggesting that this locus may have an important role in virulence. Mahdavi and colleagues (292) identified sialyldimeric-Lewisx glycosphingolipid as a receptor for H. pylori and demonstrated that bacterial colonization induced expression of sialyl-Lewisx antigens in human and monkey gastric mucosa. The H. pylori adhesin required for binding was identified as SabA, and because sialyl-Lewisx antigens are established tumor antigens and markers of gastric dysplasia that are up-regulated by chronic gastric inflammation, these results further emphasize the pivotal role of H. pylori adherence in the induction of gastric inflammation and injury. Another H. pylori OMP that may mediate disease is a 34-kDa proinflammatory protein encoded by oipA (293). Yamaoka and colleagues (293) were the first to demonstrate that most cag+ strains isolated from patients in East Asia have an in-frame copy of oipA, and when cocultured with

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1103 gastric epithelial cells in vitro, these strains were found to induce high levels of IL-8. Inactivation of oipA decreased IL-8 levels by approximately 40% (293), whereas inactivation of both oipA and cagE in the same strain completely abolished IL-8 production. An analysis of IL-8 promoter activation in gastric epithelial cells showed that maximal H. pylori–induced IL-8 transcription requires the presence of NF-κB, AP-1, and the ISRE-like element, and that OipA selectively induced STAT1 phosphorylation, which functions as an upstream mediator of ISRE activation (see Fig. 44-3) (139). H. pylori strains that contain an in-frame or functional copy of oipA are linked with more severe gastric inflammation, higher bacterial colonization density, enhanced mucosal levels of IL-8, and duodenal ulcer disease (294). In a murine model of H. pylori–induced gastritis, the ability of H. pylori to successfully colonize C57Bl/6 mice was shown to be dependent on the in-frame status of not only OipA, but also several other OMPs (HopZ, HopO, and HopP), and strains that possessed more than two out-of-frame OMPs were completely unable to establish infection (295). Infection with an isogenic oipA mutant led to a significant decrease in the severity of gastritis, as well as a reduction in chemokine production, compared with levels induced by the parental wild-type H. pylori strain (295), indicating a potential role of OipA in H. pylori pathogenesis.

Helicobacter pylori iceA and Disease An independent, strain-specific H. pylori locus associated with disease is iceA (296). iceA was originally identified as a gene with transcription that was up-regulated after adherence to gastric epithelial cells (296). iceA exists in two major allelic sequence variants, iceA1 and iceA2, yet only iceA1 is induced after contact with epithelial cells. The deduced H. pylori iceA1 product demonstrates strong homology to a restriction endonuclease, NlaIII, in Neisseria lactamica (297); however, mutations including insertions and deletions found in the majority of iceA1 sequences preclude translation of a full-length homolog. In contrast to iceA1, iceA2 has no homology to known proteins and its structure indicates patterns of repeated peptide cassettes. In its most common form, iceA2 can encode a protein of 59 amino acids with 2 conserved outer domains of 14 and 10 amino acids that flank 3 internal peptide domains of 13, 16, and 6 amino acids, respectively (298). Sequence analysis of iceA2 from several H. pylori strains has demonstrated that the internal 35-amino-acid cassette (composed of the 13-, 16-, and 6-amino-acid domains) may be absent or repeated up to 3 times, resulting in deduced proteins of 24, 59, 94, or 129 amino acids (298). That iceA genes constitute two distinct families of alleles significantly different from each other suggests there may be a biologically relevant function for both types of iceA products. H. pylori iceA1 strains are significantly associated with the presence of peptic ulceration and distal gastric adenocarcinoma in certain populations (296,299,300), and levels of iceA1

expression within colonized human gastric mucosa are directly related to the severity of acute inflammation and IL-8 expression (301,302). iceA1 is associated with the presence of cagA and the vacA s1 allele, but is found in only 25% of U.S. H. pylori isolates, which approximates the percentage of infected individuals who progress to peptic ulceration or gastric cancer, or both (296). The association of iceA1 with increased tissue damage and disease, its allelic distribution in a minority of clinical strains, its linkage disequilibrium with cagA and vacA s1 alleles, and its induction by physiologic events that induce pathologic responses (adherence), collectively suggest that iceA1 may be a marker for strains that induce more severe gastric inflammation and injury.

FldA and Risk for Gastric Mucosa-Associated Lymphoid Tissue Lymphoma Another H. pylori antigen that is significantly associated with specific clinical sequelae of long-term colonization is FldA, a putative flavodoxin protein. Chang and colleagues (303) reported that this 19-kDa protein is recognized by sera from patients infected with H. pylori who have mucosaassociated lymphoid tissue (MALT) lymphoma significantly more frequently than by sera from patients colonized with H. pylori who do not have lymphoma. These investigators also found that an 11-amino-acid truncation of the FldA protein was more common among H. pylori strains isolated from patients with MALT (9/9) than in control strains (6/17) (303). However, FldA antibodies are not universally present among patients with MALT lymphomas and not universally absent among control subjects, indicating that they are neither necessary nor sufficient for disease to occur. Furthermore, it is unclear why a truncation of FldA would affect host production of serum antibodies. Therefore, it remains to be determined whether FldA plays an important causal role in MALT development or, alternatively, simply may be a marker for strains associated with this neoplasm.

Novel Helicobacter pylori Virulence Components Identification of additional strain-specific genes related to pathogenesis has been facilitated by the use of H. pylori whole-genome microarrays (205,304) that contain representations of each known H. pylori gene (202,203). Microarraybased comparisons of genetic content between strains allow each gene within the genome to be sampled simultaneously, and because evolutionary pressures tend to select for the coinheritance of genes involved in common pathways, identification of genes that segregate with known H. pylori virulence-related loci may reveal functional relations that are not evident from sequence data alone. Indeed, a subset of genes has now been identified that co-vary with the presence of the cag island, including two that encode predicted OMPs: omp27 and babA (176,205). Thompson and colleagues (305) examined H. pylori genes that were altered transcriptionally

1104 / CHAPTER 44 during transition from log-to-stationary phase. Expression of napA, flaA, and pfr (encoding an iron storage protein) were significantly increased during the late log phase, suggesting that the virulence capacity of H. pylori is greatest at this point in the growth phase. Using a similar microarray approach, Forsyth and colleagues (306) identified seven genes that were regulated by HP0164, which encodes an H. pylori sensory histidine kinase. H. pylori arrays also have been used to identify putative virulence components that may regulate inflammation and injury in vivo. In one study, 25 gastric cancer strains and 71 control H. pylori strains were phenotyped based on their ability to bind 2 different classes of epithelial cell receptors (307). From this group, 13 binding and 2 nonbinding strains were selected for microarray analysis that identified a panel of variable genes (n = 370), and then each strain was used to challenge germ-free transgenic mice that had been shown previously to develop enhanced pathology in response to H. pylori infection. Eighteen variable genes were directly related to the severity of the host response, and the proportion of genes encoding HsdS homologs was significantly greater in this pool compared with the pool of variable genes that were not related to pathology, suggesting that HsdS homologs may regulate expression of H. pylori virulence constituents within the gastric niche (307). Graham and colleagues (308) used an RNA capture technique in conjunction with microarrays to identify H. pylori genes that were selectively expressed within gastric tissue. A small subset of H. pylori open reading frame (ORF)s was consistently present within infected mucosa that was not detected in bacteria grown in vitro, and the majority of these transcripts encoded factors unique to H. pylori (308). Although many of in vivo–selective RNAs encode hypothetical proteins, one (HP0228) is predicted to encode a membrane protein involved in the transport of sulfate anion (308), which is likely to be important for the in vivo survival of H. pylori. Collectively, these results have identified a group of candidate genes that may be expressed by H. pylori in response to environmental signals encountered in the stomach, many of which can be examined further using appropriate in vitro and rodent models of H. pylori/host interactions. The ability of additional pathogenesis-related determinants to influence disease also has been examined in rodent models of infection. Andermann and colleagues (309) investigated the role of two predicted chemoreceptors, tlpA and tlpC, in establishing infection in mice. When challenged into mice alone, isogenic tlpA− and tlpC− mutants colonized to the same extent as the parental strain. However, coinfection of either mutant with the wild-type strain resulted in an increased recovery fraction of wild-type strains, suggesting that tlpA− or tlpC −-encoded products may contribute partially to in vivo colonization (309). Similarly, inactivation of H. pylori ruvC, which encodes a Holliday junction endonuclease that functions in recombination, increased the threshold dose required for successful infection of mice by nearly two logs, and colonization was transient as the ruvC mutant was spontaneously cleared 1 to 3 months after inoculation (310).

Other less well-characterized loci also have been identified that, in the future, may become established pathogenic factors. Shibayama and colleagues (311) identified an apoptosis-inducing H. pylori protein that also exhibits γ-glutamyl transpeptidase-like activity. Ando and colleagues (312) identified a novel gene, hrgA, that replaces a previously identified H. pylori restriction endonuclease. Although the function of hrgA remains speculative, its presence is more common among H. pylori strains isolated from patients with gastric cancer than from subjects with gastritis alone (312). Another H. pylori locus, JHP0947, was reported to be more frequently present in duodenal ulcer and gastric cancer strains than in strains harvested from patients without these lesions (313). If these genes are consistently found to be related to distinct disease outcomes, they may represent novel molecular epidemiologic markers, which could identify colonized individuals at increased risk for clinical sequelae, who then might be considered for eradication therapy.

HUMAN GENETIC POLYMORPHISMS THAT INFLUENCE THE PROPENSITY TOWARD DEVELOPMENT OF DISEASE Although H. pylori strain-specific constituents clearly influence disease outcome, these factors are not absolute determinants of virulence. Most individuals colonized with disease-associated H. pylori strains remain asymptomatic. Furthermore, H. pylori cag+ toxigenic strains are related to both duodenal ulcer disease and distal gastric cancer, two mutually exclusive disease outcomes (314). The inability of bacterial virulence components to account completely for pathologic outcomes has highlighted the need to explore host factors that may influence disease, particularly gastric cancer. The critical components that regulate the H. pylori– induced cascade to carcinogenesis are: (1) presence of the bacterium; (2) the concomitant inflammatory response; and (3) a reduction in acid secretion, because inflammation that involves the acid-secreting gastric corpus increases the risk for development of gastric adenocarcinoma by inducing hypochlorhydria and atrophic gastritis (23,315). A host effector molecule that interacts with all three of these parameters therefore would represent an ideal candidate to investigate. The Th1 cytokine IL-1β, a pleiotropic proinflammatory molecule that is increased within the gastric mucosa of individuals with H. pylori infection, robustly satisfies these criteria (316). IL-1β both amplifies host inflammatory responses and potently inhibits gastric acid secretion by parietal cells (317). Furthermore, the IL-1β gene cluster, consisting of IL-1β and IL-1RA (encoding the naturally occurring IL-1β receptor antagonist), contains a number of functionally relevant polymorphisms that are associated with either increased or decreased IL-1β production, and this has facilitated case–control studies that relate host genotypes and disease. The seminal report in this series was published by El-Omar and colleagues (318), who demonstrated that individuals colonized with H. pylori with

MECHANISMS OF HELICOBACTER PYLORI–INDUCED GASTRIC INFLAMMATION / 1105 “high-expression” IL-1β promoter region polymorphisms have a significantly increased risk for hypochlorhydria, gastric atrophy, and distal gastric adenocarcinoma than individuals with genotypes that limit IL-1β expression. These relationships were present only among individuals colonized with H. pylori emphasizing the importance of host–environment interactions and inflammation in the progression to gastric cancer (Table 44-2). These results have now been replicated in geographically distinct regions of the world (319,320). For example, Furuta and colleagues (320) showed that H. pylori–infected Japanese subjects with proinflammatory IL-1β genotypes had the greatest atrophy and gastritis scores and the highest gastric juice pH values. Within the H. pylori–negative group, polymorphisms within the IL-1 locus had no effect on these parameters (320). These associations also mirror alterations in mucosal IL-1β production induced by H. pylori because gastric tissue levels of this cytokine are significantly greater in colonized patients possessing high- versus low-expression IL-1β polymorphisms, and increased IL-1β levels are significantly related to the intensity of gastric inflammation and atrophy (321). Rodent models also support this paradigm; in Mongolian gerbils and mice infected with H. pylori, gastric mucosal IL-1β levels increase 6 to 12 weeks after challenge, and in gerbils, this is accompanied by a reciprocal decrease in gastric acid output (322,323). Administration of recombinant IL-1 receptor antagonist to H. pylori–infected gerbils normalizes acid outputs (322), thus implicating IL-1β as a pivotal modulator of acid secretion within inflamed mucosa. Because IL-1β is the most powerful known inhibitor of acid secretion, is profoundly proinflammatory, is up-regulated by H. pylori, and is regulated by a promoter with informative polymorphisms, this molecule is likely to be of substantial importance in regulating the development of gastric cancer (see Table 44-2). Although IL-1β appears to be the ideal host candidate, other molecules are legitimate targets. For example, TNF-α

TABLE 44-2. Human genetic polymorphisms and gastric carcinogenesis

Gene

Function

IL-1β

Induction of cytokines; inhibition of acid secretion IL-1 receptor antagonist Induction of cytokines; inhibition of acid secretion Chemokine Inhibition of proinflammatory cytokine secretion

IL-1RA TNF-α IL-8 IL-10

Allele associated with increased cancer risk −31 C/C −511 T/T 2/2 −308 A/A −251 AT/AA −592 ATA/ATA −819 ATA/ATA −1082 ATA/ATA

IL, interleukin; TNF-α, tumor necrosis factor-α.

is a proinflammatory acid-suppressive cytokine that is increased within H. pylori–colonized mucosa (124). TNF-α polymorphisms that increase TNF-α expression have now been associated with increased risk for gastric cancer and its precursors (see Table 44-2) (324). Smith and colleagues (325) reported that polymorphisms within the IL-8 promoter that increase IL-8 expression also augment the risk for atrophic gastritis among subjects infected with H. pylori (see Table 44-2). Conversely, polymorphisms that reduce the production of anti-inflammatory cytokines may similarly increase the risk for gastric cancer, and El-Omar and colleagues (324) demonstrated that “low-expression” polymorphisms within the locus controlling expression of the anti-inflammatory cytokine IL-10 are associated with an enhanced risk for distal gastric cancer (see Table 44-2). This group also examined the combinatorial effect of polymorphisms within immune response genes on cancer risk and found that the relative risk for distal gastric cancer increased progressively with an increasing number of proinflammatory polymorphisms to the point that 3 polymorphisms increased the risk for cancer 27-fold greater than baseline (324). An important question raised by studies focused on immune response gene polymorphisms is whether H. pylori strain characteristics can further augment cancer risk exerted by host genotypes. To directly address this, Figueiredo and colleagues (326) stratified subjects infected with H. pylori on the basis of both high-expression IL-1β polymorphisms and virulence genotype of their infecting H. pylori strains. Odds ratios for distal gastric cancer were greatest in those subjects with high-risk host and bacterial genotypes; in subjects with high-expression IL-1β alleles that were colonized by H. pylori vacA s1-type strains, the relative risk for gastric cancer was 87-fold greater than baseline (326), indicating that interactions between specific host and microbial characteristics are biologically significant for the development of gastric cancer, and that combined bacterial/host genotyping determination may identify individuals at extremely high risk for gastric cancer. From these case–control studies in human populations, it is apparent that H. pylori are able to send and receive signals from their hosts, allowing host and bacteria to become linked in a dynamic equilibrium (23,327). The equilibrium is likely different for each colonized individual, as determined by both host and bacterial characteristics, and may explain why certain H. pylori strains augment the risk for inflammation-induced carcinogenesis. For example, H. pylori cag+ strains induce an intense inflammatory response that involves production of IL-8. This leads to increased production of IL-8, IL-1β, and TNF-α that not only can amplify the mucosal inflammatory response, but also may inhibit acid production, especially in hosts with polymorphisms that permit high expression levels of these molecules. Although the host genotype remains stable over time, cag+ and cag− strains can coexist and recombine within the same host; thus, the percentage of cag+ strains is likely to vary over time, which will affect the amplitude of this equilibrium. An augmented inflammatory response induced by cag+ strains

1106 / CHAPTER 44 in the gastric body leading to decreased acid production can also permit overgrowth of pH-sensitive bacteria, conversion of ingested N-nitrosamines to nitrites, and an increased risk for gastric cancer.

CONCLUSION Regulation of gastric inflammation that develops in response to H. pylori is governed by levels of host-bacteria equilibria that are not found during interactions with acute pathogens. Because of the substantial morbidity and mortality associated with H. pylori–induced diseases, considerable efforts have focused on delineating the precise mechanisms by which this pathogen induces gastric inflammation. The excitement for future research efforts within this field is palpable because analytic tools now exist, including genome sequences (H. pylori and human), measurable phenotypes (CagA seropositivity), and practical animal models, to discern the fundamental biological basis of H. pylori–associated diseases. It is important to gain more insight into the pathogenesis of H. pylori–induced peptic ulcer disease and gastric cancer, not only to develop more effective treatments for these disease states, but also because it might serve as a paradigm for the role of chronic inflammation in the genesis of other clinical sequelae within the gastrointestinal tract.

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1108 / CHAPTER 44 79.

80.

81. 82.

83. 84.

85.

86.

87. 88. 89.

90. 91. 92. 93.

94. 95. 96. 97.

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1114 / CHAPTER 44 interleukin 1beta production in Helicobacter pylori infection. Gastroenterology 2002;123:1793–1803. 322. Takashima M, Furuta T, Hanai H, Sugimura H, Kaneko E. Effects of Helicobacter pylori infection on gastric acid secretion and serum gastrin levels in Mongolian gerbils. Gut 2001;48:765–773. 323. Fox JG, Wang TC, Rogers AB, Poutahidis T, Ge Z, Taylor N, Dangler CA, Israel DA, Krishna U, Gaus K, Peek RM Jr. Host and microbial constituents influence Helicobacter pylori-induced cancer in a murine model of hypergastrinemia. Gastroenterology 2003;124: 1879–1890. 324. El-Omar EM, Rabkin CS, Gammon MD, Vaughan TL, Risch HA, Schoenberg JB, Stanford JL, Mayne ST, Goedert J, Blot WJ, Fraumeni JF Jr, Chow WH. Increased risk of noncardia gastric cancer associated with proinflammatory cytokine gene polymorphisms. Gastroenterology 2003;124:1193–1201.

325. Smith MG, Hold GL, Rabkin CS, Chow WH, McColl KEL, PerezPerez GI, Mowat A, El-Omar EM. The IL-8-251 promoter polymorphism is associated with high IL-8 production, severe inflammation and increased risk of pre-malignant changes in H. pylori-positive subjects. Gastroenterology 2004;126:A23. 326. Figueiredo C, Machado JC, Pharoah P, Seruca R, Sousa S, Carvalho R, Capelinha AF, Quint W, Caldas C, van Doorn LJ, Carneiro F, Sobrinho-Simoes M. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma. J Natl Cancer Inst 2002;94:1680–1687. 327. Kirschner DE, Blaser MJ. The dynamics of Helicobacter pylori infection of the human stomach. J Theor Biol 1995;176:281–290.

CHAPTER

45

Mechanisms and Consequences of Intestinal Inflammation Wallace K. MacNaughton Intestinal Inflammation, 1115 Overview, 1115 Initiators and Mediators of Acute Intestinal Inflammation,1115 Resolution Phase of Inflammation, 1121 Chronic Inflammation, 1121 Characterizing Inflammatory Disorders of the Intestine, 1122 Summary, 1123

Effects of Inflammation on Intestinal Function, 1124 Mucosal Dysfunction, 1124 Motor Disturbances, 1128 Summary, 1130 Future Directions for Research, 1130 References, 1130

INTESTINAL INFLAMMATION

pathophysiology that characterizes inflammatory diseases of the intestine. As originally described by Celsus in the first century (1), inflammation is characterized by the cardinal signs of calor (heat), rubor (redness), dolor (pain), and tumor (swelling). To this list we can also add loss of function, which is another hallmark feature of inflammation and one clearly evident in various facets of intestinal function during and after the inflammatory response. The inflammatory response can be divided into the acute, resolution, and chronic phases. The acute phase of the inflammatory response is triggered by infection or injury and is characterized by leukocyte recruitment, changes in vascular permeability, and killing of the invading pathogen. In addition to innate immunity, adaptive immune processes are activated. The acute phase is followed by the resolution phase in which the stimuli for acute inflammation are actively countered and the stage is set for repair. In some instances, the resolution phase fails and chronic inflammation develops, with excessive deposition of extracellular matrix and resultant fibrosis.

Overview Inflammatory conditions of the intestine comprise a significant proportion of patient visits to gastroenterologists. In many cases, the underlying causes of intestinal inflammation are not well understood and treatment is difficult. In addition, the effects of inflammation on intestinal function are varied and profound and can be a source of symptom generation and perpetuation of the inflammatory state. This chapter reviews intestinal inflammation in terms of cause and pathology. Clinical aspects of diagnosis and treatment are not discussed here because these topics are dealt with in other texts. Other chapters in this section also provide more in-depth information on various facets of the inflammatory response, including innate immunity, adaptive immunity, immune cell trafficking, and pathogen–host interactions. The main focus of this chapter is how inflammation affects intestinal form and function, and how this results in the

W. K. MacNaughton: Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Initiators and Mediators of Acute Intestinal Inflammation The intestinal mucosa sits at the interface between the external environment and the body. Thus, as with other mucosae,

1115

1116 / CHAPTER 45 it plays a vital role as a barrier regulating movement across this interface. However, the barrier function of the intestinal mucosa is even more critical than that of other mucosal surfaces, because of the aggressive nature of the external environment against which it must defend. The epithelium lining the mucosa must sense and, when appropriate, respond to bacteria and their products, other pathogens, food antigens, drugs, and other material that may be ingested. In the case of intestinal inflammation, pathogens and commensal bacteria and antigens represent the triggers, whereas epithelial and dendritic cells are the sensors that respond to these triggers. Detecting Luminal Triggers of Inflammation Toll-like Receptors The toll-like receptors (TLRs) are a family of patternrecognition receptors that can be expressed by intestinal epithelial cells and that respond to a wide range of bacterial products. Each TLR responds to a specific type of pathogenassociated molecular pattern (PAMP). For example, TLR4 recognizes bacterial lipopolysaccharide (LPS), and TLR5 responds to flagellin (2,3). The majority of TLRs signal through a pathway that involves association of the intracellular domain of the TLR with the signaling protein MyD88. Most of these signaling pathways converge on tumor necrosis factor receptor–associated factor 6 (TRAF6; directly or via interleukin-1 receptor–associated kinase [IRAK]) to activate the transcription factors nuclear factor-κB (NF-κB) or interferon regulatory factor 3 (IRF-3) (3). There is evidence to suggest that the TLR signaling pathways that require IRAK interaction with TRAF6 stimulate an “early” NF-κB response that is involved in the production of inflammatory cytokines, whereas those TLR signaling pathways that are IRAK independent stimulate a “later phase” NF-κB response that, together with IRF-3, mediates interferon-β (IFN-β) expression (3). The signaling cascades downstream of TLR activation are extremely complex, and thus are beyond the scope of this chapter (see reviews by Abreu and colleagues [2], Takeda and Akira [3], and Haller and Jobin [4] for more detailed discussions). There is also a TLR-independent group of PAMPrecognition proteins, the nucleotide-binding oligomerization domain (NOD) peptides, which can recognize cytoplasmic bacterial peptidoglycans to initiate NF-κB signaling. This is of relevance to inflammatory bowel diseases (IBDs) such as Crohn’s disease, which has been associated with mutations in the NOD2 gene (see Genetic Factors section later in this chapter) (5,6). Although much attention has focused on epithelially expressed TLRs and NODs, other cells important in intestinal inflammation express TLRs. Important among these are dendritic cells, which are key sensors of the gut lumen and initiators of the adaptive immune response that express a variety of different TLRs (7). In addition, macrophages express TLRs, particularly TLR-4, the LPS receptor, which on activation stimulates production of the chemokine macrophage inhibitory protein-2 (MIP-2) (8).

Dendritic Cells Dendritic cells are critical sentinel cells of the intestinal mucosa. They can gain access to luminal antigens in a number of ways including via M cells, by extending processes between epithelial cells, by sensing antigens trafficked through epithelial cells, and by taking up apoptotic epithelial cells (7). They use an array of surface receptors (TLRs and C-type lectins) to sample luminal contents at Peyer’s patches and other mucosal sites, and act as antigen-presenting cells to influence T-lymphocyte function. Dendritic cells that initially take up antigen are considered immature cells and subsequently undergo maturation and migration to the lymph nodes draining the gut. From this pool, they can migrate throughout the gut, under the control of numerous chemokines acting on receptors differentially expressed on various dendritic cell subpopulations (9). As they undergo maturation, dendritic cells up-regulate their expression of major histocompatibility complex (MHC) class II molecules, thereby becoming potent activators of T cells. As such, they are key players in adaptive immunity and are central to the balance between immunity and tolerance. Oral tolerance is a critical feature of the mucosal immune system, allowing it to guard against pathogenic infection while at the same time suppressing inappropriate immune and/or inflammatory responses to normal food antigens and commensal bacteria. Many factors are involved in oral tolerance, but it is becoming clear that the ability of dendritic cells to drive T-cell maturation in the direction of Tr1 (IL-10–producing) and T-helper cell type 3 (Th3; transforming growth factor-β [TGF-β]–producing) phenotypes is important (9). Altering dendritic cell subpopulations may change patterns of T-cell activation, which has been suggested as one way in which dendritic cells may be involved in the development of IBD (7). Similarly, differential migration patterns may also play a role in the switch from tolerance to immunity (10). Response to Luminal Triggers of Inflammation: Transcription Factors Numerous factors are involved in the transduction of a signal initiated at the cell surface to the response of gene transcription. This is certainly true in the case of intestinal inflammation where NF-κB, signal transducers and activators of transcription (STATs), Akt/phosphatidylinositol 3-kinase, and others have been shown to play a role. A consideration of two of these pathways, NF-κB and STATs, is warranted, because these transcription factors are important in the alteration of cellular functions that characterize the pathophysiologic consequences of intestinal inflammation (see Effects of Inflammation on Intestinal Function later in this chapter). Nuclear Factor kB It has become clear that NF-κB is the transcription factor that plays the most predominant and widest variety of roles

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1117

Ligand

TNF receptor Toll-like receptor IL-1 receptor

NEMO Receptor-dependent signaling IKKα/β

IKKα/β IκB

Proteasome

p65 p50 Phosphate Ubiquitin Transcription

FIG. 45-1. The classical pathway of nuclear factor-κB (NF-κB) signaling. In the unstimulated cell, the NF-κB Rel family proteins p65 and p50 exist as a dimer bound to an inhibitory protein, IκB. Also in the cytoplasm is a dimer of the IκB kinase (IKK) α and β subunits, bound to the regulatory subunit, NF-κB essential modifier (NEMO). On receptor activation, IKKβ is phosphorylated by a receptordependent mechanism. This activates the kinase, which, in turn, phosphorylates IκB, targeting it for ubiquitination and subsequent degradation in the proteasome. The unbound p65/p50 dimer translocates to the nucleus, where it binds to NF-κB binding sequences on gene promoters to initiate gene transcription. IL-1, interleukin-1; TNF, tumor necrosis factor.

in intestinal inflammation. The complexities of the NF-κB system have been reviewed in detail elsewhere (11). The classical pathway of NF-κB activation occurs as depicted in Figure 45-1. In the unstimulated cell, the NF-κB Rel family proteins p65 and p50 exist as a dimer bound to an inhibitory protein, IκB, of which there are several subtypes. Also in the cytoplasm is a dimer of the IκB kinase (IKK) α and β subunits, which is bound to the regulatory subunit, NF-κB essential modifier (NEMO). On activation of a cell-surface receptor, IKKβ is phosphorylated, thus activating the kinase; this, in turn, phosphorylates IκB, targeting it for polyubiquitination and subsequent degradation in the proteasome. Numerous receptors are coupled to IKK activation, including the tumor necrosis factor (TNF) receptors 1 and 2, the IL-1 receptor, the TLRs, and many others. Phosphorylation of IKK is to a large degree dependent on the receptor activated, and a plethora of intermediary kinases and coregulatory proteins are involved (11). After dissociation from IκB, the p65/p50 dimer translocates to the nucleus, where it binds to NF-κB binding sequences on gene promoters to initiate gene transcription. There is a balance between the amount of NF-κB that exists in the nucleus and in the cytoplasm.

The tight regulation of IκB phosphorylation, through the IKKs, controls this balance. The alternate pathway of NF-κB signaling involves a dimer of p100 and Rel-B. IκB is not bound to this dimer. After activation by NF-κB–inducing kinase (NIK), a dimer of IKKα phosphorylates p100, triggering its degradation to p52. The resultant p52-Rel-B dimer translocates to the nucleus, where it binds to recognition sites on gene promoters to initiate transcription (11). The lymphotoxin-β receptor and CD40 activate NF-κB through the alternate pathway, although these receptors can also signal through the classical pathway. Signal Transducers and Activators of Transcription STATs are important mediators of cytokine-induced gene transcription (12). There are seven members of the STAT family in mammalian systems: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. STATs, particularly STAT1, are best known as the proteins associated with signaling after activation of the IFN receptor. Other cytokines activate other members of the STAT family.

1118 / CHAPTER 45 INF receptor Ligand

Jak

IL-6 receptor IL-10 receptor

Jak

STAT

Dimerization

Phosphate SOCS

Transcription

FIG. 45-2. Signal transducer and activator of transcription (STAT) signaling. Binding of specific cytokines to their cognate receptors recruits the Janus kinases (Jaks). These are phosphorylated and subsequently act as tyrosine kinases to phosphorylate the cytokine receptor. STATs bind to the phosphorylated region and are in turn phosphorylated. After activation, activated STATs dimerize, translocate to the nucleus, and stimulate transcription of genes involved in the immune functions ascribed to their respective cytokines. STAT signaling is turned off by expression of inhibitory proteins, including suppressors of cytokine signaling (SOCs), most of which bind to and inactivate Jak to inhibit STAT signaling. IL, interleukin; INF, interferon.

For example, STAT3 can be activated by IL-6, IL-10, and IFN-α/β; STAT4 is activated by IL-12 and IL-23, and STAT-6 is activated by IL-4 and IL-13 (12). Figure 45-2 depicts STAT activation. On binding by its cytokine, the receptor recruits the Janus kinases (Jaks), which are phosphorylated and subsequently act as tyrosine kinases to phosphorylate the receptor. STATs bind to the phosphorylated sequence and are, in turn, themselves phosphorylated. Before activation, STATs exist in the cytoplasm as monomers, but after activation, they dimerize, translocate to the nucleus, and stimulate transcription of genes involved in the immune functions ascribed to their respective cytokines (12). STAT signaling is turned off by expression of inhibitory proteins, including suppressors of cytokine signaling (SOCs), most of which bind to and inactivate Jak to inhibit STAT signaling (13). STATs play an important role in intestinal inflammation, particularly because of their central role in mediating signaling in response to IFN-γ and IL-6 (14). STAT1 is overexpressed in ulcerative colitis and some populations of T cells in ulcerative colitis, and patients with Crohn’s disease express high levels of STAT3 (14). STAT4 is important in Th1-mediated inflammation.

Effectors of the Response to Luminal Triggers of Inflammation Chemokines and Cytokines The role of chemokines and cytokines in the development of intestinal inflammation has been a topic of intense study for the past few decades, in terms of understanding their roles both in the inflammatory process and in the development novel therapeutic strategies. The roles of chemokines and cytokines in innate and adaptive immunity and in the host response to pathogens are discussed in detail in Chapters 42, 44, 46, and 47. Thus, this section summarizes the involvement of these mediators in the inflammatory response in general, to set the stage for subsequent discussion of how they alter function in the inflamed gut (see Effects of Inflammation on Intestinal Function later in this chapter). Chemokines are peptide mediators involved in the recruitment and trafficking of inflammatory cells to sites of tissue invasion, infection, or injury. Chemokines are characterized chemically by having pairs of cysteine residues, which may be interposed with one or three other amino acids. Hence, they are classified as C, CC, CXC, or CX3C, with their corresponding receptor having a similar designation followed

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1119 by “R” to denote receptor (15). As in most tissues, expression of various chemokines by intestinal cells, particularly those of the epithelium, is key to the development of an inflammatory response (16). Because there is mounting evidence of a luminal factor as a triggering event in the development of several inflammatory diseases of the intestine, much attention has focused on the epithelium as an integral player (17). IL-8 (CXCL-8) has been emphasized as the chemokine most involved with the epithelial response to luminal factors and subsequent leukocyte migration to the mucosa (18). Other epithelially derived chemokines implicated in inflammatory cell infiltration to the mucosa include Gro-α and ENA 78, which also attract neutrophils, and monocyte chemoattractant protein-1 (MCP-1) and MIP-1β, which are chemotactic for macrophages (19). A feature of some inflammatory diseases, including ulcerative colitis, is the formation of crypt abscesses, which are composed of neutrophils that have migrated across the epithelium and into the crypt lumen. Several epithelially expressed adhesion molecules, including JAM-C and CD47 (20), and apically secreted chemotactic factors such as hepoxilin A3 (21) facilitate transepithelial neutrophil migration from the lamina propria to the lumen. Figure 45-3 depicts an overview of these processes. Cytokines are protein messengers that affect the function of immune and inflammatory cells. The list of cytokines that

play a role in intestinal inflammation, through either their anti-inflammatory or proinflammatory effects, is growing rapidly, and a complete survey is beyond the scope of this chapter. Most intense study has focused on the cytokines involved in the CD4+ T-helper cell subsets predominating in different types of intestinal inflammation. Normally, T cells respond to antigen with the production of IFN-γ, a proinflammatory cytokine. This is countered by T regulatory cells producing the anti-inflammatory cytokines, IL-10 and TGF-β. The currently accepted paradigm for intestinal inflammation is based on the predominance of two different T-cell phenotypes, Th1 and Th2. In the Th1 response, dendritic cells or macrophages detect antigen and secrete IL-12, which causes T cells to secrete IFN-γ and TNF-α that, in turn, stimulate resident or recruited macrophages to produce the proinflammatory cytokines IL-1β, TNF-α, and IL-6 (22,23). This type of response is characteristic of diseases such as Crohn’s disease. In the Th2 response, activated T cells produce IL-4, IL-5, and IL-13, but not IFN-γ. These responses are important in dealing with parasite infections. The Th1/Th2 paradigm has been established primarily from studies in mice, and differences in cytokine profiles may exist in human disease. Furthermore, some animal models demonstrate a mixed Th1 and Th2 response, further complicating our understanding of how these cytokines participate in intestinal inflammation (24).

Stimulus

Hepoxilin A3 gradient Signal transduction

Epithelially expressed adhesion molecules (JAM, CD47)

NF-κB Endothelially expressed adhesion molecules (selectins, ICAM-1/2, VCAM-1, MadCAM) IL-8 Gradient

IL-8

Leukocyte rolling, adherence, diapedesis

FIG. 45-3. Simplified view of the initiation of acute intestinal inflammation. A luminal stimulus (such as bacteria or bacterial products) triggers epithelial intracellular signaling pathways leading to the transcription and translation of chemokines such as interleukin-8 (IL-8), which initiate the processes whereby circulating leukocytes interact with adhesion molecules expressed by the vascular endothelium and migrate into the lamina propria. Leukocytes such as neutrophils can, in turn, interact with epithelially expressed adhesion molecules and migrate toward a luminally directed chemotactic mediator, likely hepoxilin A3. It is through this mechanism that crypt abscesses can form in diseases such as ulcerative colitis. ICAM-1/2, intercellular adhesion molecule-1 and -2; MadCAM, mucosal addressin cell adhesion molecule-1; NF-κB, nuclear factor-κB; VCAM-1, vascular cell adhesion molecule-1.

1120 / CHAPTER 45 Neutrophil Products The primary function of neutrophils is to destroy invading pathogens, and they produce an arsenal of mediators to accomplish this task. Many neutrophil products are relatively indiscriminate in terms of their target, and thus surrounding cells, as well as the invading pathogen, may be damaged when activated neutrophils are recruited to sites of infection or injury. Azurophilic granules within the neutrophil store a variety of serine proteases, including human leukocyte elastase, cathepsin G, and proteinase 3. These enzymes act predominantly within the phagosome to destroy endocytosed microorganisms, but there is evidence that they can also be released into the extracellular milieu where they can degrade matrix. In addition, neutrophil serine proteases participate in the inflammatory response by activating cytokines and growth factors (25). For example, proteinase 3 can activate TNF-α, IL-1β, and IL-8 (25). Although these cytokines have their own selective activating enzymes, release of proteinase 3 from neutrophils can augment this activity. In addition, elastase is known to activate the epidermal growth factor receptor (via liberation of the epidermal growth factor receptor ligand, TGF-α) (26), and possibly also TLR4 (27), and can “disarm” and thus inactivate protease-activated receptor 2 (PAR2) (28). Cathepsin G is an activator of PAR4 (29). Whether all of these activities of neutrophil serine proteases participate in intestinal inflammation has not been determined. In addition to proteases, neutrophils are a rich source of reactive oxidant species that can have deleterious effects on cells. The predominant source of reactive oxidant species is the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (30), which produces superoxide anion (O2.−). This is a highly reactive free radical that can react with other molecules to produce further, potentially harmful free radical oxidants such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and the highly reactive hydroxyl radical (OH.). Hydroxyl radical production is particularly robust if catalyzed by metals such as iron, which is present as heme-Fe in the case of microvascular hemorrhage at sites of injury (31). If sufficient reactive oxygen species are produced, the normal cellular antioxidant defenses (superoxide dismutase, catalase, glutathione [GSH] peroxidase) are overwhelmed and tissue damage occurs. Reduction of oxidative stress under these conditions has drawn attention as a therapeutic target in inflammatory diseases of the gut, particularly IBD and radiation enteritis (32–34).

Arachidonic Acid Metabolites Arachidonic acid can be released from phospholipids in the plasma membrane through the action of phospholipase A2. It can then be metabolized by the cyclooxygenase enzymes, COX-1 or COX-2, to form prostaglandins and thromboxanes. COX-1 is constitutively expressed, whereas COX-2 is induced by inflammatory cytokines. Prostaglandins have

multiple roles, both physiologic and pathologic. It was previously thought that COX-1 produced “cytoprotective” prostaglandins, whereas COX-2 produced inflammatory prostaglandins. This dogma is breaking down because it is now known that COX-1 can participate in gastrointestinal inflammation (35) and COX-2 inhibitors can exacerbate colitis (36). The role of COX-2–derived prostanoids is discussed later in terms of the resolution of inflammation (see Resolution Phase of Inflammation section later in this chapter) and their role in epithelial dysfunction in inflammation (see Hyporesponsiveness to Secretagogues section later in this chapter). Thromboxanes are involved in platelet function, specifically aggregation, and microvascular dysfunction during inflammation. They appear to play a role in intestinal inflammation, because specific thromboxane receptor antagonists ameliorate inflammation in animal models of colitis (37,38). Similar to prostanoids, leukotrienes are synthesized from arachidonic acid. Leukotriene synthesis is catalyzed by 5-lipoxygenase, with subsequent enzymatic and nonenzymatic reactions responsible for the production of leukotriene B4 (LTB4) and the peptide-leukotrienes, LTC4 and LTD4. LTB4 is a potent chemotactic agent for neutrophils and eosinophils (39). Leukotrienes may be involved in acute intestinal inflammation, as shown in animal models of disease where 5-lipoxygenase inhibitors and leukotriene receptor antagonists reduce inflammation (40,41). Conversely, inhibition of leukotriene synthesis did not reduce inflammation in a reactivation model of relapsing disease (42). Thus, the importance of leukotrienes in intestinal inflammation has not been firmly established. Nitric Oxide and Its Metabolites Nitric oxide (NO) is a free radical gas formed from the enzyme-catalyzed reaction of L-arginine with molecular oxygen. Three isoforms of NO synthase (NOS) have been described. Two of these, endothelial NOS and neuronal NOS, are constitutively expressed in vascular and neural tissues, respectively, and their activities are regulated by intracellular calcium. Inducible NOS (iNOS) is expressed in response to inflammatory mediators, although it may be constitutively expressed in some mucosal cells. The activity of iNOS is calcium-independent and the NO it produces plays a bacteriostatic role in host defense. NO had been variously described as beneficial or injurious toward mucosal integrity (43,44). The expression of the inducible isoform of NOS has been described in both ulcerative colitis and Crohn’s disease (45,46). Studies have been conducted in animal models in an attempt to determine if iNOS inhibition could promote the resolution of inflammation, but with only limited success (47,48). The extent of conflicting data on whether NO is proinflammatory or antiinflammatory has led to the suggestion that NO protects against acute inflammation, but may exacerbate chronic inflammation (31,44). Nevertheless, there are solid data pointing to a role for iNOS-derived NO in some aspects of mucosal

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1121 pathophysiology, and barrier function in particular (see discussion in Barrier Dysfunction section later in this chapter). The mechanism whereby NO may contribute to intestinal inflammation is unclear. NO is a free radical that can react with superoxide anion to produce peroxynitrite; thus, it had been thought that this reactive nitrogen species could nitrosylate tyrosine residues on proteins, and thereby contribute to inflammation (49). However, the evidence that peroxynitrite is produced in sufficient quantities in vivo to participate in the inflammatory response is not strong (31,50). It is more likely that NO triggers a cascade of inflammatory cytokines and signaling events, because the intestinal inflammation that accompanies hemorrhagic shock is reduced by an iNOS inhibitor and in iNOS knockout mice, through a mechanism that includes down-regulation of NF-κB and STAT3, with subsequent decreases in IL-6 among other inflammatory mediators (51). Proteases and Protease-Activated Receptors It has become recognized that proteases may function as important signaling molecules, in addition to their roles in protein metabolism. In particular, several serine proteases have been shown to induce cellular responses through their interactions with PARs. PARs are G protein–coupled receptors that are activated when the extracellular N terminus is cleaved, revealing a new N terminus that acts as a “tethered ligand.” This, in turn, binds to another extracellular domain and activates the receptor. To date, four PARs have been identified: PAR1 is the prototypical thrombin receptor, PAR2 is activated by trypsin and tryptase, and PAR3 and PAR4 also are activated by thrombin. There is interest in the role of PARs in intestinal function because of the prevalence of serine proteases in the gut, in immune cells resident in the intestinal mucosa, and in the microcirculation. PARs are distributed throughout the gastrointestinal tract, and their activation has a number of physiologic effects including ion transport, smooth muscle activity, and neuronal activity (52). PARs play an important role in intestinal inflammation. PAR1 expression is increased in IBD, and PAR1 activation exacerbates colitis in a mouse model (53). In addition, PAR1 activation results in COX-2 induction and prostaglandin E2 (PGE2) synthesis by colonic myofibroblasts (54). Activation of PAR1 on sensory afferent nerves is associated with increased plasma extravasation through the release of substance P (55), and PAR1 has been postulated to play a role in the development of fibrosis in radiation enteritis (56). Intracolonic administration of a PAR2-activating peptide results in the development of colitis in a mouse model (57), which may be caused by neurogenic inflammation (58). The role of PAR2 in intestinal inflammation is complicated somewhat by the observation that, in some experimental systems, activation of PAR1 may also confer protection against the development of colitis (59). PAR4, which is functionally expressed in rat colon (60), has also been shown to play a role in the development of inflammation (61,62).

Resolution Phase of Inflammation The resolution of acute inflammation traditionally has been considered to be a passive event brought about by the cessation or removal of inflammatory stimuli. We now know that the resolution phase is an active process characterized by the synthesis and release of specific anti-inflammatory compounds (63). Specifically, these include cyclopentenone prostaglandins, particularly 15-deoxy-∆12,14-PGJ2, lipoxins, NF-κB, mediators of apoptosis, and annexin-1 (63). In an experimental model of colitis in rats, inflammation was attenuated by the intraperitoneal administration of 15-deoxy-∆12,14PGJ2 (64), which is known to bind to the peroxisome proliferator-activated receptor γ (PPAR-γ) (65,66). PPAR-γ has been shown to have anti-inflammatory effects in animal models of intestinal inflammation (67). In addition to its effect on PPAR-γ, 15-deoxy-∆12,14-PGJ2 has anti-inflammatory effects that are independent of PPAR-γ, including interactions with prostaglandin receptors, direct inhibition of NF-κB, and inhibition of IκB kinase (68). Lipoxin A4 (LXA4) is a metabolite of arachidonic acid that is formed by a number of tissue- and cell-dependent pathways (63,69). LXA4, as well as its metabolite that is formed after the covalent binding of aspirin to COX-2 (the so-called aspirin-triggered 15-epi-LXA4 [ATL]), has potent anti-inflammatory properties (70). Several mechanisms are involved. Stable analogues of LXA4 can prevent TNF-α–induced IL-8 release from human intestinal mucosa (71) and can block leukocyte rolling and adhesion to the vascular endothelium (72). LXA4 also acts to inhibit neutrophil migration through epithelial monolayers (73). The actions of LXA4 are mediated through a specific G protein–coupled receptor, ALXR, which is homologous to the formyl peptide receptor-like 1 (FPRL-1) (74). Apoptosis, or programmed cell death, is considered to be a component of the resolution phase of inflammation, because it is an important mechanism for the removal of inflammatory cells from the site of injury. In particular, apoptosis of neutrophils is important in the resolution of acute inflammation (75). This may also underlie the effects of some anti-inflammatory agents such as the azithromycinlike antibiotics (76). However, apoptosis also has been associated with inflammation-induced barrier dysfunction (see Epithelial Cell Apoptosis section later in this chapter); thus, under some circumstances, it may be involved with the perpetuation, as opposed to the resolution, of inflammation.

Chronic Inflammation The progression from acute to chronic inflammation may be considered a failure of the resolution phase. However, the circumstances under which resolution fails and the inflammatory condition becomes chronic are unclear. Macrophages and lymphocytes, particularly CD4+ T lymphocytes, are the key cellular players in the development of chronic inflammation, through the generation of specific cytokines that act

1122 / CHAPTER 45 to perpetuate inflammation and direct tissue remodeling. Chronic intestinal inflammation, particularly in Crohn’s disease, is characterized by an increase in fibrosis in the submucosal and muscularis layers of the bowel wall. Fibrosis and the associated bowel wall thickening can lead to stricture formation and obstruction, which are common manifestations of chronic IBD. There is a growing body of evidence that implicates prolonged expression of TGF-β in development of the chronic fibrotic response. This cytokine binds to the heterodimeric TGF-β receptor, causing phosphorylation and the initiation of a signaling pathway involving members of the SMAD protein family. Cytosolic SMAD 2/3 are phosphorylated and combine with SMAD 4 to act as a transcription factor for genes encoding proteins involved in cellular proliferation, differentiation, and matrix deposition (77). SMAD 7 inhibits SMAD 2/3 phosphorylation, thus preventing TGF-β–induced gene transcription (78). TGF-β is an important regulator of mucosal immunity and is anti-inflammatory. TGF-β is significantly increased in colonic mucosa from patients with IBD (79). Mice deficient in SMAD 3 show a number of abnormalities, including a decrease in neutrophil chemotaxis in response to TGF-β, and an increase in mucosal bacterial infection and abscess formation (80). Good evidence implicates TGF-β in the fibrosis associated with chronic intestinal inflammation, including that which characterizes IBD (81) and radiation enteritis (82–84). The effect of TGF-β on the deposition of type III collagen is greater in fibroblasts from regions of stricture formation in patients with Crohn’s disease (85). Other cytokines, in addition to TGF-β, are becoming recognized as important participants in chronic inflammation. For example, IL-18 has distinct, differential roles in acute and chronic inflammation in the intestine. Acutely, IL-18 is produced in epithelial cells and appears to downregulate the production of inflammatory cytokines, whereas in chronic inflammatory conditions such as Crohn’s disease, IL-18 is produced by macrophages and dendritic cells (86) and promotes a Th1-type immune response (87). Blocking IL-18 reduces the inflammation associated with administration of dextran sodium sulfate (DSS) to mice (88), suggesting that it plays a key role. Nevertheless, blocking IL-18 in the CD45RBhighCD4+ severe combined immunodeficient (SCID) mouse (a model of chronic colonic inflammation caused by dysregulation of regulatory T cells) did not reduce the development of disease (89). Thus, IL-18 may be a key cytokine of chronic inflammation in some models of inflammation, but its role in chronic intestinal inflammation in humans has yet to be determined.

Characterizing Inflammatory Disorders of the Intestine

inflammatory diseases of unclear cause and for which there is no cure, other than surgical resection of inflamed colon in the case of ulcerative colitis. Crohn’s disease was first described as a distinct clinical entity in 1932 (90). It may occur anywhere along the gastrointestinal tract and is characterized by transmural inflammation. The mucosa has a characteristic “cobblestone” appearance with regions of inflammation interrupted by areas of normal mucosa. The major symptoms are pain, diarrhea, and weight loss. In chronic phases of the disease, fibrosis may lead to obstruction, perforation, or fistulization to other regions of the gut or to other organs. In some instances, perianal fistulae may form from the rectum to the skin. Ulcerative colitis, as the name suggests, is restricted to the colon. What is now considered as ulcerative colitis was first described by Wilks in 1888 (91). It involves only the mucosa, but may cause widespread loss of colonic mucosal surface area, with resultant loss of absorption and barrier function. Pain, bleeding, and diarrhea are key features of ulcerative colitis. In some cases of colonic inflammation, a clear diagnosis of either Crohn’s disease or ulcerative colitis cannot be made. The term indeterminate colitis is applied as a clinical pathologic diagnosis (92). Often, this diagnosis is temporary, as further investigation may provide additional evidence on which a reclassification to Crohn’s disease or ulcerative colitis may be made. Age at onset for indeterminate colitis is usually earlier than for ulcerative colitis, and the disease may be more extensive and have a more severe clinical course (92). Radiation Enteritis Radiation enteritis occurs after exposure of the gut to ionizing radiation at doses that damage the intestinal epithelium, usually as a result of abdominopelvic radiotherapy. Acute radiation injury is initiated by the production of free radicals when radiation interacts with molecular constituents of the cell, in particular, the formation of hydroxyl radical after interaction of radioactive particles with water (93). Free radicals cause single- or double-stranded DNA breaks that, in rapidly dividing tissues such as the intestinal epithelium, trigger apoptosis. Thus, crypt epithelial cell loss and compromised barrier function constitute the precipitating events in radiation enteritis. It has been recognized that radiationinduced endothelial damage also plays a critical role in the development of acute radiation enteritis (94). These initiating events lead to increased vascular permeability and leukocyte infiltration characteristic of acute inflammation (84). The acute injury is followed by resolution or, alternatively, may lead to a chronic, TGF-β–mediated fibrosis (82,93). Chronic radiation enteritis is associated with impaired motility, stricture and obstruction, and diarrhea and pain.

Clinical Manifestations of Intestinal Inflammation Inflammatory Bowel Disease

Microscopic Colitis

IBD is composed of two distinct entities, Crohn’s disease and ulcerative colitis. Both are chronic remitting and relapsing

First recognized as a clinical entity in the 1970s (95), microscopic colitis comprises collagenous and lymphocytic colitides

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1123 and is a major cause of chronic diarrhea (96). Often mistaken for diarrhea-predominant irritable bowel syndrome, it presents with a relatively normal mucosa on gross examination (97). Biopsy shows an increase in intraepithelial lymphocytes and an inflammatory cell influx into the lamina propria. In contrast to IBD, the crypt architecture is preserved. In collagenous colitis, there is a thick band of subepithelial collagen that is absent in lymphocytic colitis (97). The disease is relatively benign, other than quality-of-life issues associated with the chronic, watery diarrhea. The diarrhea is predominantly caused by sodium malabsorption (98). The cause of microscopic colitis is unknown, but an association with IBD has been postulated in some cases (96). Infectious Enteritis and Colitis Other inflammatory diseases of the intestine include those caused by specific pathogenic bacteria, including enterohemorrhagic Escherichia coli, Clostridium difficile, and Salmonella spp, among others. Both invasive and noninvasive pathogens can stimulate an inflammatory response. Although many of these infections are self-limiting, others may be persistent. For example, C. difficile infection, an increasingly prevalent opportunistic infection associated with the use of broad-spectrum antibiotics, can lead to the development of pseudomembranous colitis (99). Most intestinal inflammatory conditions occurring as a result of infection with specific pathogens are associated with compromised barrier function, which occurs through a variety of mechanisms (18). These mechanisms are discussed later in this chapter (see Tight Junctions). Drug-Induced Colitis Several drugs have been associated with the development of intestinal inflammation (100). Predominant among these are chemotherapeutic agents, nonsteroidal anti-inflammatory drugs, and antibiotics (101,102). Numerous case reports associate other drugs with the development of colitis, but most of these fail to establish a direct cause-and-effect relation. Factors Contributing to the Development of Intestinal Inflammation Genetic Factors There is now ample evidence for a genetic component to the development of IBD. First, there is familial clustering of IBD, particularly among first-degree relatives (103). Indeed, this is one of the greatest risk factors for IBD. Both members of a pair of identical twins are much more likely to have IBD than both members of a pair of fraternal twins. Aside from familial aggregation, there is an association of IBD among racial groups (e.g., Ashkenazi Jews) (104). More recently, linkage studies have identified a number of genetic loci associated with increased susceptibility to the development of IBD (105) (see Toll-like Receptors section earlier in this chapter). Several disease genes have now been described,

including NOD2 (5,6), SLC22A4 and SLC22A5 (106), and DGL5 (107). The exact role of these genes in IBD is unclear, but each is associated with some facet of epithelial structure or function. NOD2, also called CARD-15, is an intracellular pattern-recognition molecule related to the TLRs (105). SLC22A4 and SLC22A5 encode for proteins in the organic cation transport gene family, OCTN (106), and DGL5 is a scaffolding protein that plays a role in maintaining epithelial integrity (107). Environmental Factors Although genetic factors are clearly linked to some inflammatory diseases of the gut, particularly Crohn’s disease, there is also strong evidence that environmental factors are key contributors to the development of inflammation. In IBD, loss of immune tolerance to an “environmental factor,” perhaps commensal bacteria in the gut lumen, is thought to trigger an acute, uncontrolled inflammatory response in genetically susceptible individuals. Thus, microbe–host interactions and innate and adaptive immune responses are key contributors to the development of intestinal inflammation and are covered in detail in Chapters 42, 43, 44, 46, and 47. In addition, stress, diet, some drugs, socioeconomic conditions, and smoking may interact with genetic factors and the mucosal immune system to trigger or exacerbate inflammation (108–111). The greatest evidence for the involvement of environmental factors in the development of some types of intestinal inflammation comes from studies in genetically modified mice that develop intestinal inflammation resembling IBD (112–114). In almost all of these models, a well-developed enteric microflora is required for the development of intestinal inflammation. For example, IL-10 knockout mice raised under conventional conditions experience development of enterocolitis characterized by mucosal hyperplasia, ulceration, and crypt abscesses (115). Mice raised in specific pathogen-free conditions experienced a milder, less extensive colitis, and germ-free mice had no disease at all. Recolonization of germ-free IL-10 knockout mice with specific strains of bacteria experienced development of only mild colitis, suggesting that the more extensive microflora that develops under conventional conditions plays a role in determining the severity of colitis in this model (116). Similarly, SCID mice that are given CD4+CD45RBhigh regulatory T lymphocytes from naive wild-type mice experience development of colitis (117), but only when raised under conventional conditions (118). In addition, when the wild-type T regulatory cell donor animals are raised in germ-free conditions, the SCID recipients experience less severe colitis, suggesting that T regulatory cell priming by bacteria is important in the development of disease (118). Summary The mechanisms underlying the development of intestinal inflammation are extremely complex. Genetic factors influence how the mucosal innate and adaptive immune systems

1124 / CHAPTER 45 respond to the commensal microflora. In addition, different triggers of inflammation may stimulate diverse pathways that provoke a range of inflammatory diseases. Regardless of the details as to how the inflammatory response develops, there are common manifestations of the effect of inflammation on intestinal function. The following section addresses the impact of inflammation on intestinal physiology.

EFFECTS OF INFLAMMATION ON INTESTINAL FUNCTION The development of intestinal inflammation has important implications for intestinal function. In general, this is because of two factors. First, inflammation causes changes to the intestinal architecture such that normal functions are impaired. For example, fibrosis during chronic inflammation can restrict normal smooth muscle contraction, and hence motility. Second, inflammatory mediators can profoundly alter normal cellular physiology. The inability of secretory epithelial cells to respond to secretagogues during the inflammatory response is an example. This section details the mechanisms whereby the “form and function” relationships responsible for normal intestinal function are altered during and after the inflammatory response.

Mucosal Dysfunction Disruption of Control Systems Sensory Nerves Because of the important role the enteric nervous system plays in the control of mucosal physiology, particularly blood flow and epithelial function, inflammation-induced changes in submucosal neuronal structure could have important implications. Intrinsic and extrinsic sensory nerves also regulate mucosal function through long and short reflex loops and through local release of neurotransmitters (119). Mechanical or chemical perturbation of the gut can activate enterochromaffin cells to release 5-hydroxytryptamine (5-HT). This then activates intrinsic sensory nerves to stimulate epithelial ion transport (120). In a guinea pig model of ileitis induced by trinitrobenzenesulfonic acid, the number of enterochromaffin cells increased within 7 days and the amount of 5-HT released after bile acid stimulation of the mucosa was increased at 3 and 7 days (121). Interestingly, mucosal expression of the serotonin reuptake transporter was decreased at 3 and 7 days after induction of ileitis. The increased mucosal 5-HT could lead to down-regulation of 5-HT receptors, resulting in an overall decrease in sensory afferent input to submucosal secretomotor neurons (121). Activation of extrinsic sensory afferent nerves in guinea pig ileum by the in vitro application of capsaicin, the pungent ingredient of hot peppers that activates the transient receptor potential vanilloid receptor 1 (TRPV1), leads to increased

chloride secretion through the activation of submucosal secretomotor neurons (122). This occurs partly through the activation of neuronal neurokinin-1 (NK1) receptors (123). In the guinea pig model of ileitis discussed earlier, the in vitro secretory response to capsaicin is significantly diminished early (i.e., in the first 24 hours) in the inflammatory response (124), before the general hyporesponsiveness to secretagogues that develops in the epithelium in subsequent days (see Nerve/Mast Cell Interactions section later in this chapter). This early loss of local sensory afferent control may be caused by rapid depletion of neurotransmitters at the onset of the inflammatory response (125). Submucosal Secretomotor Neurons Two populations of submucosal secretomotor neurons, containing vasoactive intestinal peptide (VIP) or acetylcholine, release their neurotransmitters to stimulate epithelial secretion of chloride ion by cyclic adenosine monophosphate (cAMP)– and calcium-dependent pathways, respectively. In a guinea pig model of colitis, these neurons receive larger synaptic potentials and receive more fast excitatory postsynaptic potentials (EPSPs) than do neurons in control colons, suggesting that these neurons release more neurotransmitters during inflammation (126). This could potentially lead either to increased secretion, because of the greater amount of secretagogue released, or decreased secretion if the increased amount of neurotransmitters caused receptor desensitization. More studies are needed to clarify the physiologic significance of these changes in synaptic sensitivity. It also has been shown that secretomotor neurons in the mouse colonic submucosa express PAR1, and that its activation leads to a suppression of subsequent response, in terms of ion transport, to electrical field stimulation (127). This suggests that during inflammation, when vascular leakage may allow activated thrombin to gain access to the intestinal submucosal and mucosa, neurally evoked secretion may be down-regulated. Nerve/Mast Cell Interactions Nerves and mast cells are closely apposed to each other in the intestinal mucosa (128,129), and nerve/mast cell interactions regulate mucosal functions, including blood flow (130) and ion transport (131). Mast-cell degranulation releases mediators that can evoke local inflammatory responses, either through direct effects on the surrounding tissue or through interactions with effector cells such as enteric nerves. Indeed, mast-cell degranulation has been suggested to participate in IBD (132). Mast cells participated in mucosal barrier dysfunction in a rat model of colitis (133). Mast cells isolated from inflamed tissue show a greater mediator release than mast cells from uninflamed tissue (132,134). Enhanced mast-cell degranulation has been implicated in the reduced ion secretory response to immunoglobulin E (IgE) in segments of gut from patients with IBD (135).

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1125 Barrier Dysfunction A recurring theme in this chapter has been that many forms of intestinal inflammation result from luminal factors that gain access to the mucosa. Thus, maintaining a proper barrier is key to preventing the onset of inflammation. Furthermore, inflammation itself can significantly affect the epithelial barrier, through the death of epithelial cells by necrosis or apoptosis or through altering those structural features, including tight junctions, or physiologic processes that help to maintain barrier function.

Tight Junctions Transepithelial movement of solutes can occur through cells, via the transcellular route usually mediated by transport proteins, or between cells, via the paracellular route. The tight junction exists as a regulated impediment to the movement of material via the paracellular route. It is a complex structure consisting of intercellular bridging proteins, the claudins and occludin, bound to zonula occludens proteins (ZO-1, ZO-2, or ZO-3). ZO-1, in turn, is bound to the actin cytoskeleton (136,137). Various protein kinases can phosphorylate tight junction proteins, particularly ZO-1, to regulate their function. In addition, myosin light chain kinase (MLCK) can phosphorylate MLC, which causes actin remodeling and ultimately changes the integrity of the tight junction itself. Many pathogens compromise epithelial barrier function by altering the regulation of tight junction structure. For example, enteropathogenic E. coli activates MLCK to cause actin filament shortening to disrupt the tight junction and to cause occludin dephosphorylation, resulting in its movement into the cytosol; both of these events are associated with a decrease in transepithelial resistance (18). Toxins A and B from C. difficile activate protein kinase C to subsequently inactivate the Rho family proteins, Rho, Rac, and Cdc42, resulting in actin disorganization and loss of tight junction resistance (18). The intestinal pathogen, Giardia lamblia, also causes an increase in epithelial permeability by disruption of tight junctional proteins (138). Initiation of the inflammatory response has an impact on tight junction integrity. Several cytokines are known to modulate tight junction proteins to affect epithelial permeability. Both TNF-α and IFN-γ disrupt the tight junction via activation of MLCK (139). The effect of these two cytokines appears to be synergistic; in Caco-2 epithelial cell monolayers, priming with IFN-γ permitted TNF-α–induced activation of MLCK, phosphorylation of MLC, and disruption of the tight junction. Interestingly, the effects of TNF-α were NF-κB independent (140). Enzymes induced by inflammatory cytokines, such as iNOS and COX-2, may also produce mediators that exert effects on epithelial barrier function in response to bacterial infection. In studies using human intestinal epithelial cell lines capable of iNOS and COX-2 expression, enteroinvasive bacterial species including E. coli 029:NM or Salmonella dublin

caused a significant decrease in the transepithelial resistance across the cell monolayers that was blocked by iNOS and COX-2 inhibitors (141). Interestingly, T84 cells, an epithelial cell line that exhibits less consistent expression of iNOS and COX-2 (142), did not respond to infection with a change in barrier function. These studies did not address effects of invading pathogens, iNOS or COX-2 on the structure of the tight junction itself. However, others have shown that NO, produced from iNOS expressed after exposure to cytokines such as IFN-γ, can alter the expression and organization of tight junction proteins (143). Indeed, bacterial LPS causes ZO-1 disruption that is dependent on iNOS-derived NO, and that is alleviated by iNOS inhibition (143). It has been shown that luminal proteases may also selectively regulate the phosphorylation state of tight junctional proteins. PAR1 activation results in mislocalization of ZO-1, which, in concert with stimulated apoptosis, increases epithelial permeability in a process dependent on caspase-3, tyrosine kinase activity, and MLCK (144). Similarly, PAR2 activation can increase epithelial permeability in an MLCKdependent manner (145). Epithelial Cell Apoptosis As alluded to earlier, apoptosis of epithelial cells may contribute to barrier dysfunction in inflammation. Apoptosis is a physiologic mechanism to eliminate old cells and control cell numbers in the proliferative zone of the crypt (146). The mechanisms of intestinal epithelial apoptosis have been reviewed (147), and several cytokines, including TNF-α and IFN-γ, have been shown to play a role, even in physiologic epithelial apoptosis. Certainly in the case of inflammation, including that caused by bacterial infection, TNF-α, IL-1β, IFN-γ, FAS, and NO have been implicated in increased rates of apoptosis (147). Although the inducers of apoptosis in response to inflammation have been described, the relative contribution of apoptosis to overall barrier dysfunction is less clear. It has been suggested that apoptosis may have little impact on overall barrier function, and even in inflammation, increased rates of apoptosis may not have a significant effect. In epithelial cell monolayers, induction of apoptosis by crosslinking the Fas receptor with an anti-Fas antibody resulted in a decrease in transepithelial resistance and an increase in permeability. However, E cadherin–associated junctions were maintained, and there was evidence of resealing of apoptosis-induced epithelial gaps, suggesting that the effect was transient (148). However, in SCBN intestinal epithelial cells, exposure to lysates from Giardia or to activators of PAR1 induced an apoptotic response that was associated with a significant and sustained increase in permeability (138,144). The change in permeability was caused by apoptosis shown by the fact that the permeability defect could be prevented by blocking apoptosis with a caspase-3 inhibitor (138,144). Studies in in vivo models of intestinal inflammation have also implicated apoptosis as a factor contributing to impaired

1126 / CHAPTER 45 barrier function. In rats exposed to the apoptosis inducer doxorubicin, epithelial cell apoptosis was associated with increased epithelial permeability (149). Intestinal epithelial apoptosis also was induced in mouse models by several cytokines, including TNF-α, IFN-γ, and IL-12 (150). However, because these cytokines were also associated with necrotic cell death in this study, determining the relative contribution of apoptosis to barrier dysfunction is difficult. This issue has been dealt with in a study in which conductance scanning, a technique that measures changes in current density over the epithelium at a single-cell level (151), was used to determine the effects of focal apoptosis on overall permeability. This study showed that approximately half of the TNF-α–induced increase in permeability was caused by apoptosis, whereas the rest was caused by changes in tight junction structure in areas in which apoptosis was not occurring (151). The same technique was used to assess apoptosis-associated permeability in segments of inflamed colon from patients with ulcerative colitis. Severe colitis that was associated with visible lesions showed a high surface conductance as expected, but areas of milder inflammation exhibited focal apoptosis associated with high local conductance (152). Overall, the data suggest that the increased epithelial cell apoptosis observed in inflammation can have a significant impact on epithelial barrier function. Taken together, increased permeability caused by inflammation may be important for perpetuating the inflammatory response, by allowing more luminal contents to gain access to immune cells in the mucosa. This would lead to increased production of inflammatory cytokines that, in turn, can further disrupt the barrier properties of the epithelium. Understanding apoptosis and tight junction structure and regulation could provide a useful avenue for development of new therapies. Hyporesponsiveness to Secretagogues The ability of some epithelial cells to secrete chloride and water is considered another facet of epithelial barrier function. Infection with an enteric pathogen can stimulate secretion that, when combined with increased motility, serves to flush away the offending pathogen (153). In addition, inhibition of secretion or rendering the secretory epithelium unresponsive to secretagogues can lead to increased translocation of commensal bacteria, as detailed later, suggesting that secretion is an important component of the mucosal barrier even in the absence of a specific pathogen (154). NO has both secretory and inhibitory effects on chloride secretion by the intestinal epithelium. Exogenous NO initially stimulates secretion in several animal models in vitro, including guinea pig and rat, and in human tissue studied in vitro (155,156). NO also has been proposed as a mediator of chemically induced diarrhea in animal models (157–159). In contrast, it has been known for some time that endogenous NO can inhibit chloride secretion. Inhibition of endogenous NOS by intravenous infusion of the NOS inhibitor, L-NAME, enhances ileal secretion caused by cholera toxin,

E. coli heat-stable enterotoxin, 5-HT, or PGE2 in the rat small intestine (160,161). iNOS-derived NO appears to play a role in the hyporesponsiveness to secretagogues observed in both acute and chronic inflammation. In the 2,4,6-trinitrobenzenesulfonic acid (TNBS) model of colitis in mice, decreased response to the cAMP-dependent secretagogue forskolin was reversed by acute in vivo treatment with the iNOS inhibitor, L-NIL, or when colitis was induced in iNOS-deficient mice (162). Similarly, iNOS-derived NO was responsible for hyporesponsiveness to forskolin in a mouse model of acute radiation enteritis (163). Interestingly, in a model in which rats were studied 6 weeks after a bout of colitis, when most parameters of inflammation had returned to normal, an NO-dependent suppression of cAMP-mediated chloride secretion persisted (164). This was associated with an increase in bacterial translocation, and when secretory function was restored by blocking iNOS with a selective inhibitor, bacterial translocation was inhibited (154). The mechanism whereby NO inhibits cAMP-dependent chloride transport is unclear. However, it has been shown that adenylate cyclase isoforms 5 and 6 are inhibited by NO (165). Indeed, these isoforms are expressed in intestinal epithelium and are targets for iNOS-derived NO in the mouse radiation enteritis model (166). Figure 45-4 depicts the relationships among inflammation, iNOS, and cAMPdependent chloride secretion. Calcium-dependent epithelial chloride transport also has been shown to be decreased in models of inflammation, but in most animals models where this has been studied, the effect appears to be independent of NO. In a mouse model of acute colitis (167) and a rat model of chronic colitis (164), the decreased responsiveness of the colon to the cholinergic agonist carbachol in vitro was not reversed by NOS inhibition. Similarly, exposure to an NO donor did not cause an acute reduction in the in vitro secretory response of mouse colon to carbachol (166). In colons from mice with colitis induced by ingestion of DSS, exposure to carbachol caused a decrease in short-circuit current, rather than the characteristic increase associated with chloride secretion (168). This effect was blocked with the neurotoxin, tetrodotoxin (TTX), and the ganglionic blocker hexamethonium, and it was suggested to involve an unmasking of nicotinic receptors that promote chloride absorption (168). Interestingly, and in contrast with other studies where iNOS is considered to be epithelial, this group showed that NO was likely derived from iNOS expressed in either enteric nerves or glial cells (169). Another mediator that may inhibit intestinal epithelial ion transport to compromise mucosal barrier function is IFN-γ. Exposure of T84 cells to IFN-γ resulted in a decreased response to several chloride secretagogues through downregulation of a number of key transport proteins, including the Na+-K+-2Cl− cotransporter, NKCC1 (162). In addition, T84 cells cocultured with monocytes and activated T cells, or cocultured with monocytes in the presence of conditioned media from activated T cells, displayed decreased secretory responses to forskolin and carbachol. The effect could be prevented by application of a neutralizing antibody to

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1127 Cl−

CFTR

P

Lumen

PKA TJ cAMP AC5/6 L-arginine

NO

and PGD2 synthesis are increased (179). This accounted for the observed postinflammatory decrease in the responsiveness to secretagogues because the hyporesponsiveness was reversed by treatment of the colonic tissue in vitro with a selective COX-2 inhibitor, but not by treatment with a selective COX-1 inhibitor (179). Furthermore, the effect was mimicked by a metabolite of PGD2, 15-deoxy-∆12-14PGJ2 (179). In these experiments, the effect was likely at the level of the epithelial cell because both PGD2 and 15-deoxy∆12-14PGJ2 blocked the effect of a phosphodiesterase inhibitor (179), which stimulates secretion by increasing cAMP in several model systems (180) including colonic epithelial cell cultures (181).

iNOS Secretagogue

Epithelial Digestive and Nutrient Transport Dysfunction Digestive Dysfunction

AC4?

Inflammation

FIG. 45-4. Inhibition of cyclic adenosine monophosphate (cAMP)–dependent chloride secretion by inducible nitric oxide synthase (iNOS)–derived nitric oxide. Secretagogues bind to receptors coupled to different isoforms of adenylate cyclase (AC), all of which can stimulate chloride secretion through activation of the cystic fibrosis transmembrane conductance regulator (CFTR), in a cAMP- and protein kinase A (PKA)–dependent manner. During or after a bout of inflammation, iNOS expression is increased and NO acts to inhibit AC 5/6 to prevent cAMP-dependent chloride transport. TJ, tight junction.

IFN-γ (170). The suppressive effect of IFN-γ may be because of altered expression of ion transporters. In human fetal small-intestinal xenografts growing in SCID mice, IFN-γ decreased the expression of NKCC1 and the α subunit of Na+-K+-ATPase (171). In those studies, the levels of the calcium-activated Cl− channel and cystic fibrosis transmembrane conductance regulator were unaltered, which is contrast with other in vitro studies that showed a downregulation of chloride channels after exposure to IFN-γ (162). Finally, cyclooxygenase products of arachidonic acid metabolism may participate in inflammation-induced suppression of epithelial secretory function. The products of the catalysis of arachidonic acid by cyclooxygenase differ depending on the predominant cyclooxygenase in the tissue. COX-2 preferentially produces PGD2 (172). PGD2 has been shown to inhibit short-circuit current responses in rat (173,174) and dog (175) colon in vitro. Ion flux experiments in rat colon demonstrated a combined inhibition of chloride secretion and sodium-chloride absorption (176) occurring as a result of decreased release of acetylcholine from cholinergic secretomotor neurons (174). In the dog, the effect is likely mediated by a metabolite of PGD2 (177) acting at an atypical DP receptor on the epithelial cell (178). It has been shown that, at 6 weeks after a bout of experimental colitis induced by TNBS in the rat, colonic mucosal COX-2 levels

Impaired digestive function during intestinal inflammation could have significant consequences for nutritional status, particularly in chronic diseases of the small intestine, such as Crohn’s disease. Deficits in digestive function could be ascribed to a loss of brush-border enzyme activity caused by decreased functional surface area of the small intestine. However, the recent literature is surprisingly sparse regarding the effects of inflammation on digestive enzyme activities. In earlier studies of rats infected with the nematode Nippostrongylus brasiliensis, villous atrophy was associated with decreased activities of digestive enzymes (182). In rabbits infected with Yersinia enterocolitica, decreased brush-border enzyme activities were associated with decreased brush-border surface area (183). The effect was more persistent in the jejunum and more transient in the ileum. Exposure of the rat small intestine to ionizing radiation causes a decrease in brush-border peptidases secondary to release of the enzymes into the lumen, where they retain their activity (184). In studies of human biopsies, disaccharidase activity was decreased in inflamed proximal duodenum, with the degree of deficit being proportional to the severity of inflammation (185). In ileal Crohn’s disease, activities of lysophospholipase and disaccharidases were decreased in inflamed regions and, interestingly, also in noninvolved areas of ileum, albeit to a lesser extent (186). More recent studies have begun to investigate the mechanisms underlying inflammation-induced changes in the expression of digestive enzymes. In the human intestinal epithelial cell line Caco-2, IL-6 and IFN-γ both cause a decrease in sucrase-isomaltase gene expression, whereas TNF-α caused an increase and IL-1β had no effect (187). This was not a generalized effect on all digestive enzymes because expression of lactase was not affected by these cytokines. Decreases in sucrase-isomaltase expression were also observed in Crohn’s disease (187). More studies are warranted to further examine the mechanisms underlying altered digestive enzyme expression in inflamed gut and to determine the importance of this functional deficit in the nutrient malabsorption characteristic of chronic inflammatory intestinal diseases.

1128 / CHAPTER 45 Altered Nutrient Absorption Weight loss is a serious symptom of Crohn’s disease. This is partly caused by anorexia during flares of inflammation, but it is also attributable to malabsorption when large areas of small intestine are involved. Indeed, in an experimental model of jejunitis induced by infection of gerbils with Giardia lamblia, decreased glucose and fluid absorption was due to decreased brush-border surface area caused by a reduction in the length of microvilli on epithelial cells (188). A decreased surface area for digestion and absorption is an obvious contributor to malabsorption in Crohn’s disease, but there is also evidence for altered function of absorptive processes in inflamed gut. Glucose absorption occurs through the sodium-glucose cotransporter, SGLT-1. A rabbit model of ileal inflammation induced by Eimeria magna was associated with a reduction in the number of transporters in the brush-border membrane (189), an effect that was reduced by pretreatment with the corticosteroid, methylprednisolone (190). In contrast with SGLT-1, there is evidence that expression of the oligopeptide transporter Pept-1 may be enhanced in inflamed small intestine (191), although the significance of this is unclear.

Motor Disturbances It has long been known that intestinal inflammation significantly disrupts normal patterns of motility in the gastrointestinal tract. Because the inflammation associated with diseases such as Crohn’s disease is transmural, all levels of motor control, from perception to neural regulation to smooth muscle cell function, could potentially be affected. Not surprisingly, the effects of inflammation on intestinal motor function are complex and the literature contradictory. Disruption of Control Systems Interstitial Cells of Cajal Control of basal smooth muscle activity is regulated by the interstitial cells of Cajal (ICCs), which act as the pacemakers of the gut. ICC structure and function are altered in models of inflammation. For example, in jejunum of rats infected with the nematode N. brasiliensis, ICCs in the deep muscular plexus were altered during inflammation (192). The effect was persistent, because ICCs were absent 30 days after infection (192). Ultrastructural studies in inflamed canine colon and mouse small intestine showed reduced ICC density, damage to the ICC processes and contacts with smooth muscle cells, and cytoplasmic vacuolization (193–195). Changes in ICC function were associated with contact with immune cells (194). Physiologically, disruption of the ICCs in mouse small intestine infected with Trichinella spiralis, which is characterized by an acute inflammatory reaction in association with worm expulsion (perhaps caused by the presence of cytokines such as IL-1β, IL-6, or TNF-α) (196), was associated with abnormal basal

electrical rhythm characterized by a loss of synchronization of the pacemaker activity of the cells (195). This resulted in a motor pattern characterized by periods of quiescence interrupted by periods of both retrograde and anterograde peristaltic activity (195). In T. spiralis–induced inflammation, ICCs recovered by day 40 after infection (194). Taken together, these data suggest that inflammation-induced changes to ICC structure and function play an important role in the motor dysfunction observed in inflamed intestine, not just acutely, but during the postinflammation period as well. Enteric Nervous System As described elsewhere, the myenteric plexus of the enteric nervous system lies between the circular and longitudinal layers of the muscularis externa and is the main neuronal regulator of intestinal motor function. The rhythmicity of intestinal motor function is tied to the intrinsic oscillating transmembrane potential of the ICCs and is propagated among smooth muscle cells via membrane gap junctions. Enteric nerves serve to alter smooth muscle membrane potentials, either to depolarize or hyperpolarize, such that oscillating changes in potential become closer to or further from the threshold at which spike potentials, as well as subsequent muscle cell contraction, occur. Mechanical activities such as distention can activate an enterochromaffin cell–sensory afferent nerve pathway that projects to the myenteric plexus to trigger either ascending or descending interneurons, which, in turn, activate excitatory or inhibitory motor neurons. A growing body of knowledge is accumulating, based primarily on work in animal models, to suggest that inflammation alters the structure and function of enteric nerves, and hence motor function (197). Interestingly, some of the changes observed are relatively subtle, even in severe inflammation, perhaps because the enteric ganglia may be immune-privileged sites (198) or because, in most animal models, the period of inflammation is relatively brief (197). Interestingly, structural changes in the enteric nervous system for human IBD are more pronounced (197). Inflammation and specific inflammatory mediators have been shown to alter the excitability of myenteric neurons. Noncholinergic neuronal activity was enhanced in rabbit ileitis induced by administration of ricin, with further data supporting a role for increased NO-mediated inhibition of motor activity (199). In guinea pig ileum, exogenous application of PGE2 acts as an excitatory neural modulator, causing a slow depolarization of both AH- and S-type neurons (200). In a guinea pig model of distal colonic inflammation induced by TNBS, up-regulation of COX-2 in the myenteric plexus was associated with an enhanced excitability of AH-type neurons, together with decreased propulsive activity as assessed in vitro (201). Inhibition of COX-2 did not reverse the increase in mucosal 5-HT or the enhanced fast EPSPs observed in AH- and S-type neurons in this model, but significantly reversed the inflammation-associated alterations in the electrical properties of AH neurons and normalized the proportion of S-type neurons exhibiting slow EPSPs (201).

MECHANISMS AND CONSEQUENCES OF INTESTINAL INFLAMMATION / 1129 The function of enteric nerves, including those regulating motility, is modulated by neuronal opioid receptors expressed on neurons (202). Opioid agonists significantly decrease motility, which contributes significantly to the gastrointestinal side effects of these compounds (203). Interestingly, inflammation of mouse jejunum caused an up-regulation of µ-opioid receptors in myenteric, but not submucosal, neurons (204). This inflammation-induced increase in opioid receptor expression may underlie the enhanced antimotility effect of opioids observed in inflamed gut (205). Altered myenteric neuronal function during intestinal inflammation also has been demonstrated for neurotransmitter release. The release of norepinephrine from myenteric plexus preparations in vitro is suppressed in rats infected with T. spiralis (206,207). The mechanism of altered neurotransmitter release during inflammation is unclear, although changes in the expression of synaptic proteins may be involved. In hapten-induced colitis in rats, the expression of the synaptic vesicle protein, neuronal calcium sensor-1 (NCS-1), was suppressed during the acute phase of inflammation (2–4 days), but returned to normal by 16 days (208). Alternatively, changes in postrelease neurotransmitter processing could be involved in myenteric dysfunction in inflammation. For example, in the T. spiralis infection model, although inflammation was associated with an accelerated rate of 3H-choline uptake into smooth muscle/myenteric plexus preparations, there was a significant decrease in acetylcholinesterase activity by day 6 of the infection (209), suggesting a disruption of acetylcholine reprocessing that could contribute to altered neuronal control of motor function during inflammation.

neurotransmitters. Neurokinin receptor expression is altered in inflammation and may contribute to the altered actions of tachykinins in inflamed gut. In the N. brasiliensis model, both NK1 and NK2 receptors were decreased at 14 days, but recovered by 30 days after infection (192). However, an earlier study in both Crohn’s disease and ulcerative colitis failed to demonstrate any inflammation-associated changes in expression of receptors for other neuropeptides localized to sensory afferent neurons, including receptors to bombesin, CGRP, cholecystokinin, galanin, glutamate, substance P, and VIP (214). Finally, inflammation may also alter intestinal afferents at extraintestinal sites. For example, dorsal root ganglion neurons are activated during intestinal inflammation. Ileitis is associated with a reduction in the activity of outwardly rectifying K+ currents and an increase in TTX-resistant Na+ currents, suggesting that increased excitability of these neurons in inflammation is caused by effects at multiple ion channels (215). Although the mechanism of increased TTXresistant Na+ channel activity remains unclear, it is known that PGE2 can activate these channels in dorsal root ganglion neurons grown in culture (216). In addition to altered ion channel function in dorsal root ganglion neurons, neurokinin receptors may be altered in these regions during intestinal inflammation. For example, NK1 receptor down-regulation was observed after T. spiralis–induced inflammation in the rat (217). Neurotransmitter expression also is altered in dorsal root ganglion neurons during inflammation. CGRP expression is increased in response to exposure of the rat ileum to C. difficile toxin A (218). In contrast, other studies have shown that the levels of substance P in dorsal root ganglion are unchanged (217).

Extrinsic Primary Afferent Nerves

Enteric Glia

In addition to changes in myenteric motor neurons during inflammation, alterations in sensory afferent nerves could contribute to inflammation-induced motor disruption. Both spinal and vagal extrinsic sensory afferents are affected by inflammation. Spinal afferents express the neuropeptides substance P and calcitonin gene–relate peptide (CGRP), have their cell bodies located in the dorsal horn of the spinal cord, and are sensitive to capsaicin. Capsaicin-sensitive afferents appear to contribute to inflammation-induced ileal dysmotility through the release of tachykinins (199), which may subsequently trigger the release of inflammatory mediators including LTC4 and PGE2 (210). However, conflicting data exist with respect to how inflammation changes the levels of substance P. For example, substance P levels are increased in jejunal myenteric plexus of rats infected with T. spiralis (211), but not in the ileum of TNBS-inflamed guinea pigs (212). Muscularis substance P content was chronically decreased in the N. brasiliensis model of jejunal inflammation in the rat (192). CGRP in sensory neuronal fibers is also decreased acutely during experimental colonic inflammation (213). Altered motor function in inflammation may also relate to changes in the expression of receptors for sensory

Enteric glial cells, analogous to astrocytes in the central nervous system, provide critical support to enteric nerves and may contribute to changes in enteric neural structure and function in inflammation. Glial cells can be activated in models of inflammation and may contribute to the inflammatory response by presenting antigens and producing cytokines (219). We are only beginning to appreciate the role that enteric glia may play in inflammation-induced neuronal and, possibly, motor function. Change in Smooth Muscle Phenotype in Inflammation Intestinal smooth muscle cells have been shown to be active participants in the inflammatory response, and thus may contribute to their own dysfunction in inflammatory diseases of the gut. In rats, in vitro smooth muscle preparations have been shown to produce several cytokines, including IL-1α, IL-1β, IL-6, and TNF-α during the inflammatory phase of T. spiralis infection (196). In isolated human smooth muscle cells, exposure to conditioned media from C. difficile cultures stimulated IL-6 production (220). More recent studies have shown that smooth muscle preparations exposed to IL-1β, TNF-α, or IFN-γ respond with an NF-κB–dependent

1130 / CHAPTER 45 increase in production of IL-1β (thus demonstrating an amplification effect), IL-6, IL-8, RANTES, and COX-2 (221). Other signaling pathways, including Src and mitogenactivated protein kinases, were implicated, suggesting a complex pathway of induction (221). Similarly, exposure of rats to abdominal ionizing radiation stimulates NF-κB– dependent induction of IL-1β, IL-6, and TNF-α from the muscularis (222). Importantly, the induction of these cytokines was associated with a reduction in the production of IL-10 (222). The production of cytokines by intestinal smooth muscle cells can be modulated by other cell types, including enteric neurons. Smooth muscle production of IL-6 in response to IL-1β is enhanced by VIP, CGRP, and norepinephrine, possibly through increases in cAMP (223). The contribution of smooth muscle cell–derived cytokines to either the inflammatory response itself or to motor dysfunction during inflammation is unclear. Exposure of isolated murine intestinal smooth muscle cells to inflammatory stimuli that in other studies caused cytokine production resulted in the expression of MHC class II and intercellular adhesion molecule-1, and stimulated T-cell proliferation in culture (224). The importance of this action for human disease is unknown. Cytokines elaborated by smooth muscle cells during inflammation, particularly IL-6, can decrease the contractility of smooth muscle and can alter neurotransmission (225). The mechanism whereby smooth muscle cell contractility is decreased in inflammation is being described. Patchclamp experiments in smooth muscle cells isolated from DSS-inflamed mouse colon showed enhanced activation of ATP-sensitive K+ channels (226). The change in the activity of ATP-sensitive K+ channels may be caused by altered subunit expression, because in the DSS colitis model in mouse, smooth muscle expression of the Kir6.1 subunit is increased, whereas the SUR2B is decreased (227).

SUMMARY Intestinal inflammation has profound effects on all aspects of gut physiology. Impaired barrier function as a result of inflammation can lead to increased transepithelial movement of luminal elements to the lamina propria, where they may further stimulate immune responses to perpetuate the inflammation. Breaking this cycle of permeability and immune activation will likely be central to the development of therapies for inflammatory diseases of the gut.

Future Directions for Research Great strides have been made in our understanding of the mechanisms and consequences of intestinal inflammation. Nevertheless, the incidence of conditions such as Crohn’s disease continues to climb in Western societies, and both Crohn’s disease and ulcerative colitis remain without a cure. IBD, radiation enteritis, and other inflammatory diseases

likewise are difficult to treat, particularly in their chronic stages. As our understanding of the genetics of complex diseases such as IBD develops, we will be able to identify subpopulations of patients with different underlying causes of their disease, and our growing repertoire of genetically based murine models of intestinal disease will help us to better understand the multiplicity of causative factors for these disorders. Therefore, with an improved understanding of the genetic basis for disease, of the physiologic ramifications of the genetic predisposition, and how our gut microflora takes advantage of immunologically altered function, we will be able to identify, target, and more successfully treat specific patient populations.

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46

Recruitment of Inflammatory and Immune Cells in the Gut: Physiology and Pathophysiology D. Neil Granger, Christopher G. Kevil, and Matthew B. Grisham Adhesion Molecules, 1138 Selectins, 1138 Immunoglobulin Superfamily, 1140 Integrins, 1141 Cadherins, 1142 Platelet Glycoproteins, 1142 Regulation of Blood Cell–Endothelial Cell Interactions in Nonlymphoid Tissues, 1143 Adhesion Molecules, 1143 Products of Endothelial Cell Activation, 1145 Auxiliary Cells, 1146 Gut-Associated Lymphoid Tissue and Intestinal Immunity, 1148

Lymphocyte Homing and Activation in Gut-Associated Lymphoid Tissue, 1149 Leukocyte Movement through the Interstitium, 1151 Adhesion Molecule Regulation of Migration, 1151 Chemoattractant Regulation of Migration, 1152 Leukocyte Trafficking during Acute Inflammation, 1153 Oxidative Stress–Dependent Models of Inflammation, 1154 Bacterial Toxins, 1154 Leukocyte Trafficking during Chronic Gut Inflammation, 1155 References, 1157

The recruitment of leukocyte to sites of inflammation, infection, or tissue injury is a complex process that involves a tightly coordinated sequence of adhesive interactions between leukocytes and the walls of microscopic blood vessels. These adhesive interactions are regulated by a variety of molecules that are expressed on (e.g., adhesion glycoproteins) or released by (chemokines and nitric oxide [NO]) activated leukocytes, endothelial cells, and different auxiliary cells (e.g., mast cells, macrophages, and platelets). Although leukocyte recruitment in most tissues is generally associated with pathology, the gastrointestinal (GI) tract is considered normally to exist in a state of controlled inflammation, wherein there is a

constant trafficking of leukocytes in both lymphoid and nonlymphoid regions of the mucosa. However, many GI diseases and disorders are characterized by a more pronounced and persistent recruitment of leukocytes into gut tissue, coupled with dysregulated activation of the infiltrating cells, which ultimately contributes to tissue injury and organ dysfunction. Consequently, the factors regulating leukocyte recruitment in the GI tract have been studied extensively in an effort to develop novel strategies for treatment of GI inflammation. This chapter provides an overview of the factors that regulate the recruitment and activation of inflammatory and immune cells under conditions of acute and chronic GI inflammation. After a review of the adhesion glycoproteins that mediate leukocyte adhesion to vascular endothelial cells, this review (1) focuses on the factors that regulate blood cell–endothelial cell interactions in nonlymphoid tissues, (2) provides a brief overview of gut-associated lymphoid tissue (GALT) and intestinal immunity, (3) addresses the mechanisms that determine leukocyte movement through the interstitium of inflamed tissue, and (4) discusses the role of leukocyte trafficking in experimental models of acute and chronic GI inflammation.

D. N. Granger and M. B. Grisham: Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130. C. G. Kevil: Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1137

1138 / CHAPTER 46 ADHESION MOLECULES Cell–cell adhesion can be accomplished through homotypic or heterotypic protein interactions, namely, receptor–ligand interactions. Of the diverse groups of cell adhesion molecules (CAMs), glycoproteins of the selectin, integrin, cadherin, and immunoglobulin families have all been implicated in the recruitment of leukocytes. Table 46-1 lists the adhesion molecules that are known to mediate specific adhesive interactions between leukocytes and vascular endothelial cells in the GI tract, and includes the respective ligands and tissue expression of each molecule. Figure 46-1 illustrates the

multistep paradigm of leukocyte recruitment in inflamed tissue and identifies the adhesion molecules involved in each step of the recruitment process. The following section addresses the specific functions of various adhesion molecules that mediate leukocyte trafficking.

Selectins The selectins were first discovered through the use of monoclonal antibodies that inhibited leukocyte homing (L-selectin) and leukocyte binding (E-selectin) to endothelial cells (1,2).

TABLE 46-1. Adhesion molecules involved in recruitment of leukocytes in inflamed tissue Adhesion molecule

Primary tissue expression

Major ligands

Primary function

P-selectin

Endothelial cells, platelets

PSGL-1, sialylated Lewis X

E-selectin L-selectin

Endothelial cells Neutrophils, monocytes, most lymphocytes Endothelial and epithelial cells, lymphocytes, fibroblasts, and others

ESL-1, sialylated Lewis X CD34, GLYCAM-1, MadCAM-1

Leukocyte capture and rolling, thrombosis Leukocyte rolling and firm adhesion Leukocyte rolling and homing

ICAM-2

Endothelial cells, lymphocytes

LFA-1

ICAM-3

Neutrophils, monocytes, lymphocytes Endothelial cells

LFA-1

ICAM-1

VCAM-1 PECAM-1 MadCAM JAM LFA-1 Mac-1 VLA-1 VLA-2 VLA-3 VLA-4 VLA-5

Endothelial cells, neutrophils, platelets, lymphocytes Endothelial cells Endothelial and epithelial cells, neutrophils, monocytes, platelets Neutrophils, monocytes, lymphocytes, most other leukocytes Neutrophils, monocytes, platelets Neutrophils, lymphocytes, most other leukocytes Neutrophils, lymphocytes, most other leukocytes Neutrophils, lymphocytes, most other leukocytes Neutrophils, lymphocytes, most other leukocytes

E-cadherin GPIb-IX-V

Neutrophils, lymphocytes, most other leukocytes Epithelial cells Platelets

GPIIa-IIIb vWF

Platelets Platelets

LFA-1, Mac-1, p150/95, fibrinogen, fibronectin (connecting segment-1)

VLA-4 PECAM-1

Leukocyte adhesion and extravasation, leukocyte and endothelial cell motility, cell activation Leukocyte adhesion and extravasation, cell activation Leukocyte adhesion, cell activation Leukocyte adhesion and extravasation, cell activation Leukocyte extravasation

L-selectin, α4β7 JAM, LFA-1, Mac-1, VLA-4

Leukocyte homing and adhesion Cell–cell adhesion, regulation of polarity, cell motility

ICAM-1, -2, -3, JAM ICAM-1, JAM, Factor X, iC3b, fibrinogen Collagen, laminin

Leukocyte adhesion and extravasation, cell activation, cell migration Leukocyte adhesion and extravasation, cell activation Cell–matrix adhesion

Collagen, laminin

Cell–matrix adhesion

Fibronectin, collagen, laminin

Cell–matrix adhesion

VCAM-1, fibronectin

Leukocyte rolling, adhesion, extravasation, cell–matrix adhesion Cell–matrix adhesion

Fibronectin E-cadherin, αEβ7 vWF, Mac-1, thrombin, P-selectin vWF, fibrinogen GPIIa-IIIb, collagen, P-selectin, GPIb

Leukocyte adhesion Platelet adhesion, thrombosis Platelet adhesion, thrombosis Platelet adhesion, thrombosis

ESL-1, E-selectin ligand 1; GP, glycoprotein; ICAM, intercellular adhesion molecule; JAM, junctional adhesion molecule; LFA-1, lymphocyte function associated antigen-1; MadCAM-1, mucosal addressin cell adhesion molecule-1; PECAM, platelet endothelial cell adhesion molecule; PSGL-1, P-selectin glycoprotein ligand-1; VCAM, vascular cell adhesion molecule; VLA-4, very late antigen 4; vWF, von Willebrand factor.

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1139 Leukocyte capture

Rolling adhesion - Selectins - VLA-4 - PNad - GLYCAM-1 - MadCAM-1

Firm adhesion Leukocyte activation - Chemokines

- ICAM-1, -2 - VCAM-1 - MadCAM-1 - LFA-1 - Mac-1 - VLA-4

Extravasation - ICAM-1 - VCAM-1 - PECAM-1 - JAM

Proteoglycans

Endothelial activation Chemokines

Matrix transmigration

Tissue inflammatory mediators

FIG. 46-1. The multistep process of leukocyte recruitment. Stimulation of leukocyte recruitment involves three major steps: leukocyte rolling, firm adhesion, and extravasation. Tissue inflammatory mediators, including cytokines and chemokines, are important in initiating these cellular responses through increased gene expression of various adhesion molecules and functional changes in adhesion molecule affinity/avidity. Adhesion molecules, cytokines, and chemokines also play important roles in regulating leukocyte motility through extracellular matrix, thus facilitating immune cell migration through tissue interstitium. ICAM, intercellular adhesion molecule; JAM, junctional adhesion molecule; MadCAM-1, mucosal addressin cell adhesion molecule-1; PECAM, platelet endothelial cell adhesion molecule; PNAd, peripheral node addressin; VCAM, vascular cell adhesion molecule; VLA-4, very late antigen 4.

P-selectin also was found as a membrane protein within platelet granules with an unknown function, and later was identified in endothelial cells (3,4). It is now known that the selectins are critically important for the low-affinity adhesive interactions that are manifested as leukocyte rolling in inflamed microvessels. The selectin proteins consist of a short cytoplasmic domain, a transmembrane domain, a series of short consensus repeat domains (SCR domains), a single epidermal growth factor (EGF)–like domain, and a Ca2+-dependent lectin binding domain at the amino terminus. It is believed that the SCR domains may be involved in maintaining protein structure and may enhance ligand binding. The EGF-like domain is quite homologous among all selectins, and it appears to contribute to cell adhesion. The most critical domain for selectin function is the Ca2+-dependent lectin domain. Ca2+ binding induces a conformational change within this domain, which can facilitate ligand binding. P-selectin expression is exhibited by platelets and endothelial cells, whereas E-selectin expression is observed

only in endothelium. Both E- and P-selectin facilitate leukocyte rolling on the endothelial cell surface, and P-selectin is also used for platelet adhesion. P-selectin is stored in preformed pools within platelet α-granules and Weibel– Palade bodies of endothelial cells. Inflammatory activation of both platelets and endothelium results in a rapid mobilization of P-selectin to the cell surface within a matter of minutes. Endothelial P-selectin is important for leukocyte and platelet rolling on stimulated endothelium (5–7). The expression of both E- and P-selectin is induced transcriptionally by several inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), or bacterial lipopolysaccharide (LPS). Together, these proteins facilitate leukocyte rolling, the initial step in leukocyte recruitment from the vascular lumen to the extravascular tissue. The role of selectins in leukocyte recruitment depends on the type of inflammatory stimuli, the time period of inflammation (acute vs chronic), and the organ studied. However, most studies suggests that P-selectin is important for immediate leukocyte rolling in response to inflammatory stimuli

1140 / CHAPTER 46 and trauma, and that E-selectin plays a more important role during longer periods of inflammation (>4 hours) and may also mediate the firm adhesion of leukocytes. The initial studies of P-selectin knockout mice showed that loss of this molecule results in a decrease in early leukocyte rolling in response to trauma and inflammatory stimuli, increased leukocyte rolling velocities during longer periods of inflammation (>1 hour), and decreased leukocyte recruitment into the peritoneum over a 4-hour period (8,9). Interestingly, the cytoplasmic domain of P-selectin is important for proper targeting to endothelial cell Weibel–Palade bodies in vivo, and mice containing P-selectin cytoplasmic domain deletions demonstrate many of the phenotypic features of P-selectin knockout mice (10). Although the importance of P-selectin in inflammation has been well identified, the exact role of E-selectin has remained somewhat elusive. This may be because of the manner in which both of the molecules are regulated and expressed. As mentioned earlier, P-selectin is synthesized and stored in Weibel–Palade bodies, which can be mobilized within minutes after an inflammatory response. In contrast, preformed pools of E-selectin do not exist, and induction of this molecule requires transcriptional activation. However, these same signaling mechanisms also induce transcription of the P-selectin gene. Therefore, an increase in abundance of both selectin molecules can be observed simultaneously during a given inflammatory response. This concept has led to the realization that there can be considerable overlap of function between the molecules (11). Careful examination of E-selectin mutant mice has shown that this molecule is important for leukocyte slow rolling and mediates increased leukocyte transit times, thereby allowing adequate leukocyte sampling of the endothelial cell surface, thus facilitating interaction between cell types (12). This notion is further supported by the observation that loss of E-selectin results in decreased firm adhesion of leukocytes and may be necessary for proper integration of signals involved in leukocyte arrest (13,14). L-selectin expression is observed on most leukocytes including lymphocytes, granulocytes, and monocytes. L-selectin has been shown to be an important mediator of lymphocyte homing to peripheral lymph nodes and Peyer’s patches in response to inflammatory stimuli (2,15–17). Engagement of ligands, such as peripheral node addressin (PNAd) or mucosal adressin cell adhesion molecule (MAdCAM), stimulates L-selectin–mediated outside-in signaling that can promote additional adhesion molecule interactions such as β2 integrin–ligand association (18–20). Importantly, L-selectin is proteolytically cleaved from the leukocyte surface after activation, which has become a useful indicator of immune cell activation (21,22).

Immunoglobulin Superfamily Members of the immunoglobulin superfamily share a similar structural motif, with immunoglobulin-like extracellular domains that are responsible for cell adhesion. These molecules

are important for normal immune surveillance, leukocyte adhesion, and leukocyte emigration. The proteins involved in these functions are intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, and -3), vascular cell adhesion molecule (VCAM), platelet endothelial cell adhesion molecule (PECAM), MAdCAM, and junctional adhesion molecule (JAM). ICAM-1 originally was discovered through studies using blocking monoclonal antibodies against the integrin LFA-1 (CD11a/CD18) (23). ICAM-1 also binds to the integrins Mac-1 (CD11b/CD18) and p150/95 (CD11c/CD18). ICAM-1 is constitutively expressed at low levels and is induced transcriptionally by several inflammatory mediators. ICAM-1– ligand interactions are involved in the firm adhesion and emigration of leukocytes through engagement with the endothelium, and they also are involved in leukocyte– leukocyte aggregation and activation. These cellular events are facilitated by ICAM-1–mediated outside-in signaling through integrin binding. Cross-linking of ICAM-1 has been shown to stimulate many signal transduction cascades, such as mitogen-activated protein kinase and src kinase pathways (24–26). ICAM-1 expression may also regulate endothelial cell glutathione levels that can modulate inflammatory responses through a redox-dependent mechanism (27). Given the diverse functions of ICAM-1 during an immune response, the precise role of this molecule in mediating leukocyte–endothelial cell interactions has been examined. Interestingly, the loss of ICAM-1 specifically influences the adhesion of certain leukocyte types in a stimulus-dependent manner. For example, loss of ICAM-1 decreases eosinophil adhesion in mesenteric venules in response to ragweed allergen challenge (28,29). However, TNF-α stimulation of neutrophil adhesion in ICAM-1 mutant mice has shown that this protein is either not required for adhesion or is redundant with other adhesion molecules (14). Closer examination of this finding suggests that ICAM-1 is required for chemoattractant-mediated leukocyte adhesion in resting venules, but not TNF-α–stimulated venules (30). In vitro studies using endothelial cells from ICAM-1–deficient mice have shown that this molecule is important for monocyte firm adhesion and T-cell emigration across endothelial monolayers (31–34). Lastly, studies have shown that ICAM-1 is necessary for optimal selectin-mediated rolling, in that leukocyte rolling velocities on TNF-α–stimulated vessels and endothelium were faster compared with wild-type control animals (35,36). Collectively, these findings demonstrate that the role of ICAM-1 in inflammation is multifaceted, and that many or all of these functional characteristics may be important in leukocyte–endothelial cell interactions. ICAM-2 is constitutively expressed on the cell surface and was primarily described in endothelial cells (37). The structure of ICAM-1 and -2 are similar in that they consist of a cytoplasmic domain, a transmembrane domain, and immunoglobulin-like regions. However, they differ in the number of extracellular immunoglobulin-like domains in that ICAM-1 contains five immunoglobulin domains compared with two for ICAM-2. Unlike ICAM-1, ICAM-2

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1141 is not inducible by inflammatory mediators, suggesting that ICAM-2 is important for normal immune surveillance. Interestingly, gene-targeted loss of ICAM-2 does not alter lymphocyte homing or development of leukocytes (38). However, ICAM-2 deficiency reduces eosinophil transmigration across endothelial monolayers and prolongs allergic eosinophil accumulation in certain organs. ICAM-3 is expressed on all resting leukocytes and also contains five immunoglobulin domains similar to ICAM-1 (39,40). ICAM-3 is important for leukocyte aggregation and activation through its interaction with the counterligand LFA-1 (41,42). VCAM-1 is a cytokine-inducible surface molecule that mediates adhesion of different leukocyte populations, such as lymphocytes, monocytes, and eosinophils (43–45). VCAM-1 was identified through direct expression techniques using a cell–cell adhesion-based screening procedure with cytokine-induced complementary DNA genes. Candidate genes were transfected into epithelial cells and examined for adhesion to lymphocytic cell lines. Since this characterization, VCAM-1 has been shown to be involved in both acute and chronic inflammation, and it is expressed primarily in endothelial cells. VCAM-1 binds α4β1 (very late antigen 4 [VLA-4]), and possibly α4β7 integrin, and participates in the firm adhesion and emigration of leukocytes. VCAM-1–ligand interactions are largely mediated by domain 1 and possibly domain 4. VCAM-1 also appears to bind the α9β1 integrin of neutrophils and facilitates neutrophil migration across endothelial cells (46). PECAM-1 contains six immunoglobulin domains, a transmembrane domain, and a cytoplasmic tail that can be differentially phosphorylated and palmitoylated (47). Modification of the cytoplasmic tail facilitates outside-in signaling that can influence endothelial cell function (e.g., angiogenesis or cell survival) and leukocyte recruitment. PECAM-1 preferentially localizes at intercellular junctions of endothelial cells, suggesting a role in leukocyte emigration. Consistent with this hypothesis, blocking antibodies against PECAM-1 on endothelium can prevent leukocyte emigration across endothelial monolayers (48,49). PECAM-1 also is expressed on other cell types including platelets, monocytes, neutrophils, natural killer cells, and certain T-cell subsets, which may facilitate adhesion and transmigration through homotypic binding to endothelial PECAM-1. MAdCAM-1 was identified through monoclonal antibody (MAb) screening studies that showed discreet staining of high endothelial venules (HEVs) of mucosal lymph nodes (Peyer’s patches) (50). Additional studies of MAdCAM-1 expression identified constitutive expression in tissues involved in the mucosal immune system, as well as inducible expression by proinflammatory mediators (e.g., TNF-α) (51). As suggested by its tissue distribution, MAdCAM-1 mediates lymphocyte homing by directing adhesion and emigration across HEVs of mucosal tissue. MAdCAM-1 contains three extracellular immunoglobulin domains, with a mucin-like region between domains 2 and 3 (52,53). Primary ligands for MAdCAM-1 include L-selectin and α4β7 integrin, which bind to immunoglobulin domain 1 (17).

The most recently reported immunoglobulin superfamily adhesion molecule is JAM. Three different JAMs have been described in detail and are known as JAM-A, -B, and -C (54). JAM expression is observed in endothelial and epithelial cells, leukocytes, and platelets, and the molecule consists of two extracellular immunoglobulin domains and a cytoplasmic tail that differentially associates with multiple signaling and cytoskeletal-associated proteins (55). The original identification of JAM (JAM-A) indicated junctional localization in endothelial cells that participated in monocyte transmigration (56). Studies also demonstrate junctional localization of JAM in epithelial cells; also, members of the JAM subfamily homotypically bind other JAM molecules, as well as other heterotypic ligands (54,57–59). Specific heterotypic ligands for the JAM subfamily include the integrins LFA-1 (αLβ2), Mac-1 (αMβ2), and VLA-4 (α4β1), suggesting an important role for these molecules in regulating leukocyte recruitment.

Integrins The integrin family of adhesion molecules consists of a wide array of heterodimeric proteins produced through various combinations of 17 α and 8 β subunit genes. Figure 46-2 illustrates the molecular associations between different α and β subunits. These proteins participate in cell–cell and cell–matrix adhesion and have been shown to play important roles in regulating intracellular signal transduction and cytoskeletal organization of all cell types. The α and β subunits are encoded by separate genes that do not necessarily show coordinate transcriptional regulation. As such, several α protein chains interact noncovalently with multiple β protein chains, yielding a diverse array of heterodimer pairings. Several integrin ligands have been described and largely include members of the immunoglobulin superfamily and extracellular matrix (ECM) proteins. The β2 integrins are heterodimeric proteins that consist of a common β subunit (CD18) noncovalently attached to different α subunits (CD11a-d) (60). The β2 integrin family, or CD18 integrins, consists of four different adhesion proteins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150/95 (CD11c/CD18), and CD11d/CD18. Expression of the β2 integrins is limited to leukocytes and platelets, which suggests their importance for immune responses. Members of the immunoglobulin superfamily are major ligands for the β2 integrins, including ICAM-1, -2, and -3, and members of the JAM family of proteins (55,61,62). Integrin-mediated cell adhesion is controlled through multiple mechanisms, including alterations in surface density and changes in receptor affinity. The important role for β2 integrin–ligand interactions in inflammatory and immune processes is illustrated by several studies using gene-targeted mutant mice lacking CD18 that show attenuated inflammatory responses under various conditions (62,63). Data from numerous studies indicate that the β2 integrins are critical mediators of leukocyte adhesion and emigration, costimulation and activation, and leukocyte-mediated tissue damage. Studies also

1142 / CHAPTER 46 B

A β8 β6 β5

α2

α3

α4

β7

αE

Cation binding region

α1

β1

αv α9

α8

α5

α7

β3

α6

β4

β2

αIIb αL

αM

αX

αD Cytoskeleton binding region

FIG. 46-2. Integrin subunit interactions and molecular properties. (A) The various heterodimeric associations of α and β subunit associations. (B) Important molecular features of integrin subunits, including the cationic binding feature of the α subunit and the cytoplasmic cytoskeletal binding region of the β subunit.

suggest that the β2 integrins play important pathophysiologic roles during autoimmune disease (64,65). Thus, β2 integrin adhesion molecules play a key role in facilitating pathologic leukocyte adhesion and transendothelial migration, which is an important prerequisite for subsequent tissue injury. The α4 integrins, α4β1 (VLA-4) and α4β7, are primarily expressed on lymphocytes, but they may also be found in granulocytes and monocytes. These adhesion molecules are unique in that they can participate in all of the steps necessary for leukocyte recruitment such as tethering, rolling, and firm adhesion (66). VCAM-1 ligand binding occurs with both molecules, whereas MAdCAM is the primary ligand for α4β7 (17,67). Through these interactions, α4β7 facilitates lymphocyte homing to the mesenteric lymph nodes (MLNs) and Peyer’s patches, whereas VLA-4 plays a role in facilitating immune cell recruitment in the blood circulation. VLA-4 may also play an important role in autoimmune responses because antibody blockade of the molecule protects in several autoimmune disease models (68–70).

Cadherins The cadherin family of adhesion proteins is typically identified as surface proteins located in the adherens junction or desmosome with specific calcium requirements for homotypic interaction (71,72). The cadherin proteins contain three general structural regions including a cytoplasmic domain, a transmembrane domain, and extracellular cadherin repeat domains that are structurally similar to immunoglobulin domains. The amino-terminal end of the extracellular region contains the HAV domain that is primarily involved in homotypic binding. Cadherin molecules provide structural

support to adherens junctions and desmosomes through binding catenins, which link to the actin cytoskeleton. Moreover, cadherins influence signal pathways and gene expression through differential association with various catenins (e.g., β- or γ-catenin) (73,74). The cadherins have been divided into classical and nonclassical groups (71,75). A primary function of nearly all cadherins is the regulation of homotypic adhesion of several cell types such as epithelial, endothelial, neuronal, and other types (76,77). The homotypic adhesion of cells typically involves one predominant cadherin molecule; however, different cadherin molecules may also be expressed in the same cell. For example, the lateral epithelial adherens junction is maintained by homotypic E-cadherin interactions, yet both N- and P-cadherin may also be expressed (78). Reports indicate that cadherins may bind other adhesion molecules in a heterotypic manner. T-lymphocyte adhesion to epithelial cells has been reported to occur through T-cell αEβ7 integrin interactions with epithelial E-cadherin (79,80). The pathologic importance of this heterotypic interaction between E-cadherin and β7 integrin could be minimal, however, because genetic loss of β7 integrin does not prevent spontaneous colitis in IL-2–deficient mice and only delays the development of experimental colitis in the CD4+CD45RBhigh transfer model (81).

Platelet Glycoproteins An important role for platelet adhesion to the endothelium or subendothelial matrix has emerged in several models of inflammatory disease, including GI inflammation. Platelets contain several unique adhesion molecules that facilitate their binding to vascular endothelium and other platelets.

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1143

REGULATION OF BLOOD CELL–ENDOTHELIAL CELL INTERACTIONS IN NONLYMPHOID TISSUES

adhesion glycoproteins such as PECAM-1, MAdCAM-1, or JAM-A (91,92). Although immunohistology has rather consistently demonstrated differences in the intensity of endothelial CAM expression among microvessels within a tissue, this method has not allowed for quantitative analyses of interorgan differences in CAM expression. An approach that has been developed to overcome many of the limitations of immunohistochemistry for quantification of endothelial CAM expression involves monitoring the accumulation of a radiolabeled MAb that is directed against a specific endothelial CAM (93–96). The dual radiolabeled MAb technique has provided novel insights into the magnitude and kinetics of endothelial CAM expression in the GI tract and other vascular beds. Figure 46-3 illustrates the relative magnitude of basal and induced (by either LPS or TNF-α) expression of different endothelial CAMs in the murine intestinal vasculature. The figure illustrates that members of the immunoglobulin supergene family exhibit a high level of constitutive expression in the intestine, and that substantial

Endothelial expression

The GPIb-IX-V complex of glycoproteins facilitates platelet adhesion through binding counterligands P-selectin, von Willebrand factor (vWF), and Mac-1 (82,83). GPIb-IX-V– dependent adhesion is important for platelet adherence under high shear stress to matrix vWF (e.g., denudation of arterial endothelium), bacterial endotoxin–mediated adhesion to the microvasculature, and platelet recruitment to mesenteric venules activated with histamine (84–87). Likewise, platelet GPIIa-IIIb (also classified as integrin αIIbβ3) mediates platelet adhesion through ligand binding to other GPIIa-IIIb molecules, ICAM-1 via fibrinogen bridging, Mac-1, and αvβ3 integrin. GPIIa-IIIb is important for platelet adhesion to venular endothelial cells experiencing low shear stress and binds to fibrinogen linked to ICAM-1 in venules of postischemic intestine and liver (88–90). Both the GPIb-IX-V and GPIIa-IIIb glycoproteins participate in intracellular signaling processes with GPIb-IX-V demonstrating outsidein and inside-out signaling, whereas GPIIa-IIIb undergoes inside-out activation (83). The glycoprotein vWF is released from platelet α granules, as well as endothelial cell Weibel–Palade bodies, to facilitate platelet binding to the vessel wall and to other platelets. These adhesive interactions are aided by platelet P-selectin interactions with PSGL-1 (P-selectin glycoprotein ligand-1) expressed on vacular endothelial cells or leukocytes.

P-selectin

P-selectin

Adhesion Molecules E-selectin 2

4

A

6 8 Time (hours)

10

12

PECAM MAdCAM-1

Endothelial expression

The recruitment of leukocytes to sites of inflammation in the GI tract is an orderly and well-regulated process that allows for an accumulation of inflammatory cells in affected tissue that is both rapid and sustained. A multistep model of leukocyte binding to vascular endothelium (see Fig. 46-1) has been universally adopted to explain the sequence and time course of involvement of different adhesion molecules in the recruitment of leukocytes in inflamed tissue. The initial event in this process is a weak adhesive interaction that results in leukocyte rolling along the endothelial lining of blood vessels. This interaction, which can occur within minutes of an inflammatory challenge, is largely mediated by the selectins. The transiently bound (or tethered) leukocytes are exposed to low concentrations of chemoattractants/ inflammatory mediators that can result in leukocyte activation, which rapidly (within minutes) leads to an increased expression or activation, or both, of integrins on the rolling leukocytes. The leukocyte integrins (e.g., CD11/CD18) can bind to counterreceptors that are constitutively expressed on the surface of endothelial cells (e.g., ICAM-1), thereby allowing leukocytes to become firmly attached to the endothelium and remain stationary. The subsequent transendothelial migration of leukocytes into the interstitial compartment involves the participation of additional

ICAM-1 VCAM-1 ICAM-1

2

B

4

6

8 10 12 14 16 18 20 22 24 Time (hours)

FIG. 46-3. Kinetics of endothelial cell adhesion molecule expression in the mouse intestine after challenge with cytokines or endotoxin. Also shown is the response of P-selectin expression to histamine challenge (51,95–98). ICAM, intercellular adhesion molecule; MadCAM-1, mucosal addressin cell adhesion molecule-1; PECAM, platelet endothelial cell adhesion molecule; VCAM, vascular cell adhesion molecule.

1144 / CHAPTER 46 sustained increases in expression can be demonstrated for ICAM-1, VCAM-1, and MAdCAM-1. However, the high basal levels of PECAM-1 and ICAM-2 appear to remain unchanged in the intestine even after stimulation with either LPS or TNF-α. These responses for the immunoglobulin supergene family are in contrast with those seen for the selectins, which exhibit a much lower level of basal expression with more profound (yet transient) increases in expression after endothelial cell activation (51,93–99). Significant constitutive expression of P-selectin can be demonstrated in the intestines of wild-type mice, but not in mice that are genetically deficient in P-selectin (95). The high constitutive levels of P-selectin and other endothelial CAMs (Table 46-2) in the gut are consistent with the view that this organ is normally in a state of controlled inflammation. Transcription-dependent up-regulation of both P- and E-selectin can be induced by cytokines and LPS (see Fig. 46-3), with P-selectin expression reaching a maximum between 4 and 8 hours and E-selectin requiring 3 to 5 hours for peak expression (95). The increment in selectin expression associated with transcription generally exceeds 10-fold. Histamine also significantly increases the cell-surface expression of P-selectin, but not E-selectin, in the GI tract and most vascular beds (95). However, unlike the transcriptiondependent response, histamine produces approximately a twofold to fourfold increase in P-selectin expression on endothelial cells within 5 minutes and the adhesion molecule remains increased for as long as 1 hour. The rapid expression of P-selectin after histamine likely represents mobilization of a preformed pool of the adhesion molecule from Weibel–Palade bodies. The brain vasculature is unresponsive to histamine, whereas the stomach responds most vigorously to the autacoid, followed by the heart, lung, and intestine. The histamine-induced P-selectin expression in all vascular beds is inhibited by a histamine H1 (but not an H2) receptor antagonist, indicating that histamine engagement of H1 receptors on endothelial cells results in the mobilization of preformed P-selectin to the endothelial cell surface (95). Direct visualization of the microvasculature in GI tissues that are either acutely or chronically inflamed has revealed that leukocytes selectively bind to endothelial cells that line postcapillary venules. Leukocyte sequestration in capillaries can occur during GI inflammation, but steric hindrance of

less deformable circulating leukocytes within long, narrow capillaries, rather than cell–cell interactions mediated by adhesion molecules, often is offered as an explanation for this response (99). Endothelial cell swelling within capillaries, as well as compression of the capillary lumen by an increased interstitial fluid pressure caused by accumulation of edema fluid, also has been implicated in the entrapment of leukocytes within capillaries of inflamed tissues (100). However, it is the endothelial cells that line the walls of postcapillary venules that appear to sustain most of the leukocyte trafficking that occurs in inflamed tissues. In rat mesenteric microcirculation, for example, 39% of all leukocytes passing through venules are rolling, whereas only 0.6% of leukocytes roll in the upstream arterioles (101,102). Relatively greater shear rates in arterioles and higher endothelial CAM expression in venules are two explanations that are most often provided for the differences in leukocyte–endothelial cell adhesion between these two vascular segments. Shear forces generated by the movement of blood within the microvasculature are generally greater in arterioles than in downstream venules. For example, a 30-µm-diameter venule in rat mesenteric is likely to exhibit a resting shear rate that is half that of an arteriole of comparable diameter. Because wall shear stress or shear rate represent antiadhesion forces that oppose the proadhesive forces generated by CAMs, vessels with a low spontaneous shear rate (low blood flow) would tend to exhibit more leukocyte adhesion than vessels with high shear rates. For this reason, it has been proposed that leukocytes rarely roll and adhere in arterioles because the greater shear forces exceed the adhesive force in these vessels. Based on this proposal, one might predict that reductions in arteriolar shear rate to levels experienced by venules should promote leukocyte adhesion in arterioles. However, this is not the case, because when cat mesenteric arterioles and venules of the same size are exposed to the same range of shear rates (100–1250 sec−1), venules exhibit far more leukocyte rolling and adherence than arterioles (103). Studies using retrograde perfusion of the microcirculation have also provided some insight into the role of shear rate in arteriolar-venous differences in leukocyte– endothelial cell adhesion. In the mesentery, retrograde perfusion is associated with a reduced flux of rolling leukocytes in venules and increased leukocyte rolling in arterioles (104).

TABLE 46-2. Regional differences in the constitutive expression of endothelial cell adhesion molecules Endothelial CAM Expression (molecules × 106 per cm2) Organ Small intestine Lung Heart Muscle Brain

P-selectin

E-selectin

ICAM-1

VCAM-1

254.9 26.6 5.7 10.2 4.8

87.8 1.10 46.5 0.0 1.5

171.0 268.1 92.5 89.0 9.0

32.7 23.7 81.8 35.0 42.9

Data derived from Eppihimer and colleagues (95,97), Henninger and colleagues (96), Langley and colleagues (98). CAM, cell adhesion molecule; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule.

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1145 However, more leukocytes still rolled in venules during normograde perfusion than rolled in arterioles during retrograde flow. These observations indicate that hemodynamic differences between arterioles and venules cannot explain the predilection for leukocyte rolling and adherence in venules, and that a more probable explanation for the greater adhesive interactions between leukocytes and venular endothelium is that the counterreceptors (ligands) for leukocyte adhesion molecules are more densely concentrated on venular endothelium. Immunohistochemical analyses of CAM expression in the microcirculation have yielded results that consistently show a preferential expression of endothelial CAMs in postcapillary venules. Endothelial CAMs such as ICAM-1, VCAM-1, E-selectin, and P-selectin can be detected on the surface of activated endothelial cells in arterioles and occasionally capillaries; however, the density of these adhesion molecules is far greater on venular endothelium (105). Although immunohistochemical localization of endothelial CAMs has clearly demonstrated preferential distribution of these adhesion glycoproteins in venules, this approach has yielded relatively little quantitative information concerning the density of CAM expression within vascular beds. However, laser confocal microscopy has been used successfully to quantify the constitutive expression of ICAM-1 in arterioles, capillaries, and venules in the mesentery and liver of rats, using an FITC-labeled anti-rat MAb (106). In the mesentery, venules exhibit a 10-fold higher density of ICAM-1 than in the upstream arterioles and capillaries. Although the level of ICAM-1 expression in liver venules is comparable with that detected in mesenteric venules, there is no capillary-venule expression gradient of ICAM-1 within the liver microcirculation. Laser confocal microscopy also has revealed heterogeneity of ICAM-1 expression within different sized venules of the rat mesentery (Fig. 46-4). Venules with diameters of 25 µm appear to exhibit the greatest density of ICAM-1 on the endothelial cell surface, whereas 15-µm and 35- to 40-µmdiameter venules exhibit the lowest constitutive expression of the adhesion molecule (106). This venule size-dependent

Apparent ICAM-1 conc. (µM)

1.0

Mesentery 0.5

0

15

20 25 30 Venular diameter (µM)

35

40

FIG. 46-4. Laser confocal microscopic determinations of endothelial intercellular adhesion molecule (ICAM-1) expression in different sized venules of rat mesentery (106).

distribution of ICAM-1 is consistent with functional evidence demonstrating that 25- to 30-µm-diameter venules sustain the most intense leukocyte adhesion responses to proinflammatory mediators (107). Whether other endothelial CAMs exhibit a similar size-dependent distribution within postcapillary venules remains undetermined.

Products of Endothelial Cell Activation Bacterial toxins, as well as a variety of cytokines produced by macrophages and mast cells that normally reside in the GI tract, can engage with specific receptors on endothelial cells to elicit an increased expression of CAMs. Inducible gene expression, mediated by transcription factors, appears to be a key regulatory mechanism that allows endothelial cells to increase the synthesis and surface expression of the CAMs that regulate leukocyte trafficking (91,92). Of the many transcription factors that have been identified and characterized to date, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) appear to be most relevant to the regulation of endothelial CAMs. LPS, cytokines, and oxidative stress can result in the activation of both NF-κB and AP-1 in endothelial cells, and there is evidence that clearly links the activation of these transcription factors with the increased CAM expression caused by the well-established inducing factors. Binding sites for NF-κB have been identified in the promoter regions of the genes for E-selectin, VCAM-1, MAdCAM-1, and ICAM-1, whereas AP-1 has a binding site on the promoter region of the ICAM-1 gene (92,108–110). Reactive oxygen species (ROS) are products of endothelial cell activation that are frequently implicated in the regulation of endothelial CAMs and show a strong linkage to both NF-κB and AP-1. ROS are capable of eliciting a rapid, transient as well as a slow, prolonged increase in leukocyte– endothelial cell adhesion. The rapid response may be explained by the ability of ROS to induce the production of a variety of inflammatory mediators (e.g., platelet-activating factor [PAF], C5a, and LTB4) that can elicit the rapid up-regulation/activation of β2 integrins on leukocytes, which, in turn, can rapidly initiate the firm adhesion of leukocytes to endothelial cells. ROS rapidly induce the activation of phospholipase A2 (PLA2) in endothelial cells, leading to the generation of PAF and arachidonic acid metabolites. When endothelial cell–derived hydrogen peroxide reacts with plasma, a C5a-like chemotactic factor is generated through the hydrolysis of C5. The ability of H2O2 to activate complement and generate a chemotactic agent in plasma is enhanced by the presence of catalytically active iron (e.g., Fe EDTA and hemoglobin), suggesting that the process of complement activation is not brought about by H2O2 per se, but by an oxidant derived from H2O2 (109,110). ROS can also induce a rapid, transient (transcriptionindependent) and slow, prolonged increase in the expression of endothelial CAMs. The rapid response reflects the ability of ROS to mobilize the preformed pool of P-selectin from Weibel–Palade bodies to the endothelial cell surface.

1146 / CHAPTER 46 Both in vitro and in vivo experiments support a role for a ROS-dependent, rapid up-regulation of P-selectin on vascular endothelial cells. Exposure of mesenteric venules to a superoxide-generating system results in an enhanced recruitment of rolling leukocytes, which is prevented in animals receiving a P-selectin–specific MAb. Similarly, exposure of monolayers of cultured endothelial cells to an oxidative stress (e.g., anoxia-reoxygenation) results in the rapid surface expression of P-selectin, a response that is significantly blunted by catalase or allopurinol, suggesting that xanthine oxidasederived oxidants mediate the rapid P-selectin mobilization (92,108–110). The slow and prolonged increase in endothelial CAM expression induced by ROS reflects the ability of these endothelial cell products to activate the oxidant-sensitive transcription factors NF-κB and AP-1. Antioxidants and ironchelators have been shown to interfere with the activation of these transcription factors in endothelial cells and to lead to an attenuated up-regulation of endothelial CAMs (e.g., VCAM-1 and E-selectin) in response to a variety of stimuli, including cytokines, LPS, and exogenous oxidants. The CAM biosynthesis initiated by ROS through activation of NF-κB and AP-1 leads to a more intense and prolonged expression of CAMs on the endothelial cell surface, which serves to sustain leukocyte recruitment into inflamed tissue for a period of hours to days (92,108–110). Endothelial cell–derived NO has also been implicated as a modulator of leukocyte–endothelial cell adhesion in the GI tract. Inhibition of NO synthase with L-arginine analogues (e.g., nitro-L-arginine methyl ester) rapidly induces an inflammatory response that is characterized by the recruitment of rolling, adherent, and emigrating leukocytes in mesenteric venules, the formation of platelet-leukocyte aggregates, and the degranulation of mast cells in the perivenular interstitium. NOS inhibition is also accompanied by an oxidative stress in venular endothelium and the perivenular mast cells. Enhanced leukocyte adhesion in postcapillary venules has also been demonstrated in endothelial NOS-deficient mice, compared with their wild-type counterparts. These observations have led to the proposal that NO is an endogenous inhibitor of leukocyte–endothelial cell adhesion. One mechanism that is frequently offered to explain the antiadhesion properties of NO relates to the ability of this molecule to rapidly interact with and decompose superoxide. Because superoxide reacts with NO three times faster than superoxide dismutase (SOD), it has been proposed that NO serves as a physiologic scavenger of superoxide. The possibility that NO exerts its antiadhesive properties through this mechanism is supported by the observation that NOS inhibition elicits an oxidative stress in venular endothelium and that NO donating agents (e.g., NONOates) are only effective in inhibiting leukocyte–endothelial cell adhesion in models of inflammation wherein SOD also exerts an antiadhesion effect. NO donors are also known to inhibit several of the other inflammatory responses that have been attributed to superoxide-mediated signaling and PLA2 activation, including the rapid mobilization of P-selectin in activated endothelial

cells, transcription-dependent endothelial CAM expression, mast-cell degranulation, and platelet–leukocyte aggregation (92,109–112). Although NO and superoxide per se are often ascribed antiinflammatory and proinflammatory roles, respectively, the reactive nitrogen oxide species that are produced by their chemical interaction can yield either phenotype (anti-inflammatory or proinflammatory), depending on whether there is net oxidation or nitrosation of specific molecular targets that regulate the inflammatory response. These observations have led to the concept that a critical determinant of the adhesive phenotype (proinflammatory or anti-inflammatory) assumed by venular endothelium is the existing balance between ROS and NO (113). Under normal conditions, the balance between NO and ROS favors an antiadhesive phenotype, because NO chemistry predominates as a result of the approximately 1000-fold greater production of NO compared with superoxide (Fig. 46-5A). The excess NO yields an antiadhesive phenotype through sustained inhibition (related to targetspecific nitrosation) of transcription factor activation and endothelial CAM production/expression. The excess NO also favors cyclic guanosine monophosphate–mediated, transcription-independent signaling that tends to promote an antiinflammatory and antithrombogenic phenotype in endothelial cells. However, during inflammation (Fig. 46-5B), the flux of superoxide relative to NO increases such that ROS-dependent mechanisms predominate. Under these conditions, ROSmediated transcription-dependent and -independent processes are initiated that favor the following: (1) the production of inflammatory mediators that activate β2 integrins on leukocytes; (2) the mobilization of preformed pools of P-selectin; and (3) the transcription-dependent up-regulation of different endothelial CAMs. These changes promote both the rapid and sustained leukocyte–endothelial cell adhesion that accompany inflammation.

Auxiliary Cells Although endothelial cells remain a major focal point in the search for the source(s) of mediators that elicit and amplify leukocyte–endothelial cell adhesion during inflammation, there is growing evidence that other cell populations also contribute to this component of the inflammatory response (114,115). Mast cells, macrophages, and platelets are good examples of auxiliary cells that are in proximity to venular endothelial cells and can produce a variety of inflammatory mediators (e.g., PAF and ROS) and cytokines that are known to promote leukocyte adhesion. Mast cell products have been implicated in the leukocyte–endothelial cell adhesion in different experimental models of GI inflammation (26). Support for this view is provided by reports describing increased numbers of activated or degranulated mast cells, or both, in proximity to postcapillary venules. Further support is given by results obtained from experiments using agents that either degranulate (compound 48/80) or stabilize (ketotifen) mast cells or that antagonize

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FIG. 46-5. A critical determinant of the adhesive phenotype (proinflammatory or anti-inflammatory) assumed by venular endothelial cells is the existing balance between reactive oxygen species (ROS) and nitric oxide (NO). Under normal conditions (A), the balance between NO and ROS favors an anti-inflammatory phenotype because NO chemistry predominates. The excess NO yields an antiinflammatory phenotype through sustained inhibition (related to target-specific nitrosation) of transcription factor activation, and cyclic guanosine monophosphate (cGMP)–mediated, transcription-independent signaling. During inflammation (B), the balance between NO and ROS is shifted toward the latter species, either as a result of a reduction in NO biosynthesis, inactivation of NO by O2•−, or both. In this instance, the flux of O2•− relative to NO increases such that ROS-dependent mechanisms predominate and NO-dependent mechanisms are rendered inactive. ROS (and possibly reactive nitrogen oxide species [RNOS])-mediated transcription-dependent and -independent processes then promote a proinflammatory phenotype, the intensity of which not only depends on the relative fluxes of NO and O2•−, but also on the specific RNOS formed (113).

mediators that are relatively unique to mast cells (antihistamines). Relatively little information is available concerning the role of macrophages in the regulation of leukocyte– endothelial cell adhesion in the intact GI tract, largely as a result of the limited experimental strategies that are available to selectively manipulate the numbers and activity of these resident phagocytic cells in GI tissues (114). A growing number of reports indicate that the development of a proinflammatory phenotype in the microvasculature is frequently accompanied by a prothrombogenic phenotype, which likely reflects molecular and chemical changes in endothelial cells that create a hyperadhesive surface for both platelets and leukocytes (116). This link between hemostasis and inflammation appears to have important pathophysiologic consequences, with platelets increasing the intensity of the inflammatory response and exacerbating microvascular dysfunction and tissue injury. Several experimental models of GI inflammation are associated with the formation of

platelet–leukocyte aggregates, the adhesion of platelets directly onto endothelial cells, and the attachment of platelets to already adherent leukocytes. There is mounting evidence that adherent leukocytes represent the major substrate for binding of platelets to the walls of inflamed microvessels (117–119). Platelet–leukocyte adhesion can be mediated by different ligand–receptor interactions, including P-selectin (platelet)–PSGL-1 (leukocyte) and GPIbα (platelet)– CD11b/CD18 (leukocytes) interactions. Once platelets attach to adherent leukocytes, the capacity of the latter cells to produce superoxide is dramatically increased, suggesting that diffusible molecules liberated by the attached platelets and/or signaling mechanisms that are activated as a result of this heterotypic cell adhesion greatly enhance the activation state of the leukocyte (116). Notably, roughly half of the leukocytes that adhere in inflamed intestinal venules are platelet bearing and the other half are platelet free, suggesting that the subpopulation of adherent leukocytes that sustain

1148 / CHAPTER 46 platelet adhesion may achieve a greater state of activation that favors platelet adhesion. Because neutrophil-derived superoxide has also been implicated in the modulation of platelet adhesion in intestinal venules (120), an enhanced production of this ROS by the platelet-bearing leukocytes may account for the preferential binding to a subset of adherent leukocytes. Because the nature, underlying mechanisms, and pathophysiologic importance of the platelet–vessel wall interactions that accompany inflammation remain poorly understood, additional work is needed to address this phenomenon.

GUT-ASSOCIATED LYMPHOID TISSUE AND INTESTINAL IMMUNITY The intestinal mucosal immune system encounters more antigen than any other tissue in the body; consequently, it represents the largest and most complex component of the immune system. Fortunately, the intestinal immune system has evolved efficient mechanisms to distinguish potentially pathogenic bacteria, parasites, and viruses from harmless dietary proteins and commensal bacteria. In addition to being able to respond to specific antigens, the mucosal immune system is able to choose the appropriate effector functions necessary to deal with each pathogen. Specific subsets of lymphocytes play a major role in mediating and regulating these effector functions in vivo and are called T lymphocytes or T cells. The three major effector T-cell populations that protect our bodies from invading pathogens include CD4+ T helper cell 1 (Th1), CD4+ T helper cell 2 (Th2), and cytotoxic CD8+ cells (121). Although murine and human CD4+ T cells may be divided into several additional subsets, the two best characterized subsets, based on their patterns of cytokine production, are the Th1 and Th2 subsets (122,123). The major function of these CD4+ T cells is to secrete cytokines that activate other cells of the immune system, thus “helping” other cells to achieve their full effector cell function. Activated Th1 cells secrete IL-2, interferon-γ (IFN-γ), and lymphotoxin-α (LT-α), whereas Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. There is now good evidence demonstrating that macrophage or antigen-presenting cell (APC)–derived IL-12 is crucial for inducing Th1 differentiation, whereas IL-4 is required to promote the differentiation of Th2 cells (122,123). Th1 cells are involved in cell-mediated immune responses against certain infectious agents such as intracellular bacteria, fungi, and protozoa. This protective immune response involves not only activation of Th1 cells and the subsequent release of their cytokines, but also involves Th1 cytokine-mediated activation of interstitial macrophages and other phagocytic leukocytes to release additional antimicrobial and proinflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-8, IL-12, and IL-18. In contrast, Th2-type responses are required for humoral immune responses (i.e., antibody production), as well as effective protection against extracellular pathogens, such as helminths. The cytotoxic CD8+ T cells

are responsible for killing virus-infected cells, as well as tumor cells. Once activated, these cells develop membrane cytosolic granules that contain proteins (granzyme, perforin) that mediate cell killing. To mount effective immune responses to potentially harmful enteric microorganisms or to maintain tolerance to commensal bacteria and food antigens, the intestinal tract has evolved a complex network of lymphoid tissue within the gut that is involved in antigen transport, processing, and presentation to T cells within the intestinal tissue. This gutassociated lymphoid tissue (GALT) is composed of several discrete inductive and effector sites that include the macroscopically visible Peyer’s patches and MLNs, as well as microscopic lymphoid aggregates that are scattered throughout the wall of the small and large intestine (Fig. 46-6) (124). The Peyer’s patches are “nodules” of lymphoid aggregates immediately underlying the epithelium and are found throughout the entire length of the small intestine. These macroscopically visible structures consist of collections of large numbers of T cells separated by well-defined B-cell follicles. In addition, Peyer’s patches are separated from the intestinal lumen by a single layer of columnar epithelial cells called follicleassociated epithelium, or FAE. The space immediately underlying the FAE is referred to as the subepithelial dome (SED). The FAE can be distinguished from the epithelial cells that cover intestinal villi by its less pronounced brush border, reduced levels of digestive enzymes, and the large number of infiltrated lymphocytes (both T and B cells) and antigenpresenting dendric cells. The most characteristic feature of the FAE is the presence of microfold (M) cells (see Fig. 46-6). These cells are specialized enterocytes that contain neither surface microvilli nor a layer of mucus that covers all other epithelial cells. The M cells play a critical role in binding invasive microorganisms and particulate antigens, as well as transport of luminal antigens to the SED. The MLNs are the largest lymph nodes in the body and function to collect and concentrate antigens draining the intestinal mucosa. Naive T cells continuously recirculate from the blood into the MLNs in search of their cognate antigen. The first step in mounting an effective immune response against enteric microorganisms is the uptake and transport of enteric antigens from the lumen to the Peyer’s patches, gut interstitium, and MLNs draining the intestine. Transfer of enteric antigens may occur by at least three different pathways. The first and best-characterized pathway involves the uptake and transfer of antigen by the M cells from the lumen to the underlying SED region of the Peyer’s patches (124). There, the antigen is endocytosed by APCs called dendritic cells (DCs), followed by migration of the DCs to the B- and T-cell regions of the Peyer’s patches. Interaction of antigenloaded DCs with the different B- and T-cell populations activates/primes these cells to undergo immunoglobulin class switching from expression of IgG to IgA for B cells, and T-cell polarization to different Th subsets. Activated lymphocytes within the Peyer’s patches may then migrate to the MLNs via lymphatic vessels, where they may reside for some time during which they differentiate further before entering the

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Antigen

Antigen

M cell DC

LUMEN SED

DC

Afferent lymphatics

Free antigen

Lamina propria

Peyer's patch Crypt

Blood drainage

Afferent lymphatic Mesenteric lymph node

Antigenloaded DC

Peripheral lymph node / spleen

GUT

Efferent lymphatic

FIG. 46-6. Gut-associated lymphoid tissue and intestinal immune responses. Enteric antigens enter the gut interstitium via M cells to the underlying subepithelial dome (SED) region of the Peyer’s patch containing naive T cells and B-cell follicles (BCFs). There, the antigens are endocytosed, processed, and presented by dendritic cells (DCs) to naive T cells to initiate T-cell activation. Alternatively, antigenloaded DCs may migrate to the mesenteric lymph nodes (MLNs) via the afferent lymphatics draining the bowel where they present antigen to naive T cells within the lymphoid tissue. Once activated, T cells will leave the lymphoid tissue via the efferent lymphatics and enter the circulation, ultimately migrating back into the gut lamina propria. In addition to M-cell–mediated antigen uptake, enteric antigens may gain access to the intestinal interstitium by paracellular diffusion between the epithelium or by direct endocytosis of luminal antigens by basolateral DCs extending pseudopodia between epithelial cells and into lumen, or by both ways. Antigen-loaded DCs may migrate to the MLNs via the afferent lymphatics draining the gut, where they interact with and activate naive T cells. Alternatively, free antigen may enter the circulation and be transported to peripheral lymph nodes and spleen, where DCs in these tissues process and present antigen to peripheral T cells (modified from 124).

blood circulation via the thoracic duct. A second pathway by which luminal antigens may be transported to the gut interstitium or MLNs, or both, is by direct endocytosis of luminal antigens by DCs located in the basolateral area of the villi epithelium. In vitro and in vivo studies by Rescigno and colleagues (125,126) have demonstrated that DCs may migrate to the basolateral space between epithelial cells where they send out their pseudopodia apically/luminally between epithelial cells to “sample” the luminal environment. The authors proposed that these APCs can then migrate to the MLNs via the lymphatics, where they present their antigen to naive T cells. Finally, luminal antigens may gain access to the intestinal tissue by simple paracellular diffusion processes from where these antigens may gain access to the systemic

circulation and other lymphoid tissue such as the spleen, or they may be endocytosed by interstitial DCs, or both. As described earlier, antigen-loaded DCs will most likely migrate to the MLNs, where they will interact with naive T cells.

LYMPHOCYTE HOMING AND ACTIVATION IN GUT-ASSOCIATED LYMPHOID TISSUE Once antigen has been endocytosed and transported to the GALT by the DCs, it must be processed and presented by the DCs to antigen-inexperienced (naive) T lymphocytes for effector T-cell function to be initiated. Naive T cells have but one objective: to continuously traffic between the bloodstream

1150 / CHAPTER 46 and the secondary lymphoid tissue (e.g., GALT) in search of their cognate antigen. A great deal of information has been generated describing the mechanisms responsible for naive T-cell homing to Peyer’s patches. The homing or migration of naive T cells from the blood into the Peyer’s patches is thought to occur exclusively by way of HEVs, which are composed of specialized postcapillary venular endothelial cells in the interfollicular area of the Peyer’s patches. Naive T cells initiate their migration out the circulation by first rolling along the HEV, which is mediated by the interaction between lymphocyte-associated L-selectin and MAdCAM-1 expressed on the surface of HEVs. Although this interaction mediates most of the rolling, approximately 30% of the naive T cells may also use α4β7 to bind to MAdCAM-1, thereby providing additional tethering capacity for these cells (127–129). The chemokines (B-lymphocyte chemoattractant [BLC], secondary lymphoid tissue chemokine [SLC], Epstein–Barr virus–induced gene-1 ligand chemokine [ELC]) presented on the luminal surface of the HEV activate LFA-1 and α4β7 on naive T cells to promote firm adhesion (127–129). The lymphocyte-associated chemokine receptors CCR5 (for BLC) and CCR7 (for SLC and ELC) play important roles in this process (129). Notably, high levels of SLC are present on the HEV located in the interfollicular sections of the HEV where these vessels cross T-cell areas of Peyer’s patches; thus, only naive T cells will home to these areas of the Peyer’s patches (128,129). Naive T-cell homing to MLNs is less well defined. The MLNs are unique with respect to the distribution of the different adhesion molecules. For example, some HEV may express either MAdCAM-1 or PNAd, whereas other HEVs may express both adhesion molecules, giving the MLNs redundant pathways for lymphocyte homing (128,129). T-cell associated L-selectin will bind to both MAdCAM-1 and PNAd. Although there is ample evidence demonstrating that MLNs are absolutely required for the induction of mucosal immunity and tolerance, there is a growing body of evidence to suggest that Peyer’s patches may not be critical for mounting immune responses (124,127,128). Once the naive T cell enters the MLN, it may encounter its specific (cognate) antigen presented on the surface of DCs in association with major histocompatability complex class II (MHC II) for CD4+ T cells or MHC I for CD8+ T cells. In a process that is not well understood, the T cell will move into close proximity of the DCs, ultimately binding to the MHC-Ag complex via its T-cell receptor (TCR) complex, thereby initiating cell activation. During this local activation process, the T cell will proliferate, shed its L-selectin, and enhance surface expression of certain adhesion molecules such as LFA-1, α4β7, VLA-4 (α4β1), VLA-5, VLA-6, and CD44 (121,124,127–129). The progeny of activated T-cells will differentiate into effector or memory cells. Activation and differentiation of naive CD4+ T cells into Th1 and Th2 effector cells are important for activating macrophages and B cells, respectively. Activation of naive CD8+ T cells results in the production of cytolytic T lymphocytes (CTLs) capable of killing tumor cells and virus-infected cells that express

certain MHC I antigens. Unlike effector cells, memory cells may exist for long periods (20 years) in the absence of antigen (121). This population of lymphocytes is responsible for the enhanced or accelerated immune response after antigen challenge. After the initial activation or priming of T cells within secondary lymphoid tissue, effector or memory cells reenter the circulation via the efferent lymphatics in search of their cognate antigens. On returning to the blood circulation, activated T cells will no longer efficiently home to lymphoid tissue because of loss of surface L-selectin. Rather, these cells will preferentially home to tissues or sites of infection/ inflammation that contain the original antigen (e.g., the gut). This new pattern of homing is mediated by the interaction between lymphocyte-associated VLA-4, LFA-1, and CD44 with different endothelial cell adhesion molecules including MAdCAM-1, VCAM-1, ICAM-1 and ICAM-2, and hyaluronate (129). Thus, effector and memory T cells possess a different homing pattern than do naive cells. The mechanisms responsible for recruitment of T cells to specific tissue after antigen stimulation within the MLNs are not known with certainty. It is thought that effector T cells are exposed to specific signals that result from subtle differences in antigen–T-cell interactions, as well as the local environment of the lymphoid tissue, which imprint on these cells tissue specificity (129). For example, it is well appreciated that T cells activated within MLNs express high levels of α4β7, as well as the chemokine receptor CCR9, which can bind to TECK, a ligand that is constitutively expressed on small-bowel endothelial cells, and to constitutively expressed MAdCAM-1 (129). Once an effector T cell has been recruited to the gut, it may reencounter its specific antigen via the specific binding of its TCR to antigen bound by the MHC I or II complex expressed on a wider range of APCs, including macrophages and B cells, as well as DCs (Fig. 46-7). In fact, macrophages and B cells are more efficient activators of effector T cells than are DCs and do not require costimulation, which was required in the MLNs. Thus, macrophages and B cells are most likely the most important APCs for effector cell– memory cell activation in the tissue. This second antigenspecific interaction further activates lymphocytes to synthesize and release large amounts of IFN-γ and IL-2. IL-2 promotes the clonal expansion of T cells and enhances the function of Th and B cells, whereas IFN-γ interacts with and activates APCs and macrophages to produce additional IL-12 (130). IL-12 acts as a positive feedback mediator that continuously activates T cells to produce IFN-γ and other Th1 cytokines. IFN-γ can then activate endothelial cells and enhance endothelial CAM expression in postcapillary venules. In addition, IFN-γ–activated macrophages produce large amounts of proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-8, IL-12, and IL-18, as well as reactive oxygen and nitrogen metabolites (e.g., superoxide, hydrogen peroxide, and NO) (see Fig. 46-7). All of these mediators are thought to be important for the activation of microvascular endothelial cells and for the recruitment and activation of

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ROS NO IL-12 IFN-γ

ROS IL-1β TNFα

Effector T-cell

NO

TNFα

FIG. 46-7. Effector T-cell–dependent activation of macrophages, endothelial cells, and neutrophils. After activation and polarization of CD4+ to their T helper cell 1 (Th1) phenotype within the secondary lymphoid tissue, these cells will enter the circulation where they will migrate to the gut interstitium. Once extravasated, the effector T cells will reencounter their enteric antigen presented by antigenpresenting cells (APCs) such as B cells or macrophages, or both, and produce large amounts of tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ). APCs will, in turn, release substantial amounts of interleukin-12 (IL-12) as well. These cytokines may activate the endothelium to increase expression of different adhesion molecules, as well as activate interstitial macrophages (Mø) to release additional proinflammatory mediators including reactive oxygen species (ROS), nitric oxide (NO), IL-1β, and TNF-α. The net result of these interactions is the recruitment of phagocytic leukocytes such as polymorphonuclear leukocytes, monocytes, and additional lymphocytes into the interstitium where they engulf and destroy invading microorganisms.

phagocytic leukocytes such as polymorphonuclear leukocytes (PMNs) and monocytes/macrophages, thereby allowing for the efficient destruction of the invading pathogens (see Fig. 46-2). Failure to down-regulate the production of these proinflammatory cytokines may lead to inflammatory tissue injury.

organization, and modulation of various signal transduction pathways. Numerous studies have addressed the cellular mechanisms that allow for leukocyte migration through the ECM and interstitium, with adhesion molecules, chemokines, and cytokines serving as the key modulators of this process.

Adhesion Molecule Regulation of Migration

LEUKOCYTE MOVEMENT THROUGH THE INTERSTITIUM Leukocyte homing is initiated in the microvasculature by a process that involves cell capture, adhesion, and emigration. However, after extravasation of the leukocytes another equally important process occurs, that is, leukocyte migration through the interstitium. This process involves several cellular responses including differential regulation of cell adhesion to ECM, chemosensation of directional chemical and protein gradients, regulation of intracellular cytoskeletal

Immune cell migration through tissues, such as that seen during inflammation, involves dynamic leukocyte responses combined with differential activation of several molecular pathways. Numerous environmental signals, such as adhesion molecules, chemoattractants, ECM proteins, and mechanical forces are integrated in the cell to generate specific cellular responses. Leukocyte migration is highly adhesion dependent and occurs through coupling actinbased protrusion and contraction to the dynamic formation and disassembly of cell-ECM adhesions. Both the formation

1152 / CHAPTER 46 of new matrix contacts and the generation of tractional forces are mediated through integrins (131). Altering the adhesive state of the cell induces a biphasic effect on cell migration, with low levels of adhesion insufficient to support tractional forces and high levels of adhesion preventing cellular displacement. Integrin adhesion to ECM components, such as collagens, fibronectin, and laminins, is largely responsible for neutrophil movement through the interstitium. Members of the β1 integrin family (VLA members) are primarily responsible for neutrophil adhesion to matrix, with no appreciable role for β2 integrins (132,133). Interestingly, β1 expression on circulating leukocyte populations is low in comparison with β2 integrins. However, β2 integrin activation mediated by binding to its ligand, when concomitant with extravasation, can quickly increase β1 integrin surface localization on neutrophils (118). Consistent with increased expression of β1 integrins, antibody blockade or genetic deletion of β2 integrin does not alter neutrophil migration through the interstitium or adhesion to matrix proteins, suggesting that additional mechanisms exist to increase β1 expression and function (135,136). Together, these reports suggest that neutrophil extravasation involves conversion from β2- to β1-integrin–mediated cell motility, which is regulated by β2-integrin–ligand interactions and subsequent engagement of the endothelial cell basement membrane. Additional regulation of neutrophil migration through the interstitium occurs through differential integrin α chains. Different heterodimeric α integrin chain (α1-6) association with the β1 chain significantly alters neutrophil motility through different extracellular matrices. For example, α2β1 has been reported to play a primary role in neutrophil chemotaxis through extravascular tissue, such as the mesentery, largely through interactions with collagen (133). Interestingly, neutrophil migration through collagen gels supplemented with fibronectin or tenascin is substantially diminished compared with collagen alone (137,138). Closer examination of this response showed that antibody inhibition of α4β1 and α5β1 integrins restores neutrophil motility through combined matrices similar to that seen through collagen alone, suggesting that differential integrin engagement with various matrix components regulates the overall adhesive state of the neutrophil (132,137). Together, these studies suggest an important role for integrin-regulated haptotaxis that may be involved in controlling neutrophil tissue localization. T-cell migration through the interstitium can use β1 integrins and other unknown mechanisms (132,139,140). However, unique molecular mechanisms regulating T-cell motility also exist (141,142). T-cell engagement of CD3 or CD3/CD2 can significantly enhance β1-integrin–dependent matrix adhesion through activation of multiple signaling pathways that could alter integrin affinity/avidity and cytoskeletal interactions (140,143,144). Specifically, CD3-TCR stimulation results in Lck/Zap-70 kinase activation, which increases phosphatidylinositol-3 kinase activity. This enhances Itk tyrosine kinase membrane localization where it can be phosphorylated by Lck to alter actin polymerization,

thus modulating β1-integrin function. T-cell migration is also unique in that the β2 integrin LFA-1 (αLβ2) is involved in enhancing T-cell motility. LFA-1 is ubiquitously expressed on all T cells and plays an important role in homing, recruitment, and immunologic synapse formation (145,146). However, several studies have shown that LFA-1 activation with antibodies or ligand (e.g., ICAM-1) enhances T-cell motility on several ECM proteins including fibronectin, laminin, and collagen IV (147,148). LFA-1–enhanced T-cell motility also involves activation of intracellular signals including calmodulin/MLCK, PKC-β1, and RhoA/ROCK pathways (146,147). These reports demonstrate that T-cell motility is highly dynamic and occurs in such a manner as to effectively survey tissue for antigen. Memory T cells isolated from tissue microenvironments rather than peripheral blood also display differential integrin expression, which alters cell-ECM adhesion. A study has examined integrin expression profiles and adhesion to native ECM or its components between lamina propria T cells or peripheral blood T cells that were cultured and activated in vitro (149). Several important findings were reported in this study. First, peripheral T cells showed less adhesion to ECM compared with lamina propria T cells. Second, peripheral lymphocytes displayed preferential binding to fibronectin, whereas lamina propria lymphocytes preferred collagen. Third, the differential matrix binding was associated with a greater percentage of lamina propria T cells expressing VLA α integrin chains 1, 2, 3, and 5 that was not associated with differences in subunit surface expression on an individual cell basis. Lastly, long-term IL-2 expansion of peripheral blood lymphocytes cultured on matrix proteins generated surface integrin expression and adhesion characteristics similar to that of lamina propria lymphocytes. These findings suggest that local tissue matrix environments can further differentiate recruited T lymphocytes to a highly adhesive phenotype conducive for long-term sequestration in tissue. Monocyte motility in the interstitium also involves α1β1 (VLA-1). Studies using antibody blockade or gene-targeted ablation of the α1 integrin chain demonstrate significant protection against dextran sodium sulfate (DSS) or 2,4,6trinitrobenzene sulfonic acid (TNBS)–induced colitis (150,151). Interestingly, inhibition of α1 function preferentially inhibited monocyte infiltration into the interstitium that was not associated with decreased immune cell recruitment as determined by intravital microscopy (150). These reports clearly demonstrate that α1β1 expression is important for monocyte adhesion to ECM and subsequent movement through the interstitium. Together, all of the abovementioned studies suggest that single or multiple inhibition of the association of VLA proteins with ECM represents a unique therapeutic approach for inflammatory disorders.

Chemoattractant Regulation of Migration The process of chemosensation of leukocytes during an inflammatory response is essential to ensure optimal recruitment of specific leukocyte types to discrete tissue sites

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1153 and organs. Numerous molecules have been described that facilitate directional leukocyte migration including chemokines, cytokines, and other biochemical mediators (e.g., fMLP, PAF, complement products, arachidonic acid metabolites, and others). Research suggests that chemokines play a critical role in regulating leukocyte recruitment from the vasculature and their subsequent migration through tissue. Chemokines are small heparin-binding proteins that have been defined based on amino acid composition of a conserved tetra-cysteine motif, resulting in two major subclasses, CXC (separation by a nonconserved amino acid) or CC (adjacent cysteine location), together with three other homologous molecules of differing motifs (152). Forty-three distinct chemokines currently have been identified, and these chemokines bind to 19 receptors in promiscuous fashion. Chemokine receptors are surface G protein–coupled receptors that contain seven membrane-spanning domains and activate downstream G-protein signal cascades. Chemokines avidly bind glycosaminoglycans associated with cells or matrix proteins and are readily diffusible because of their small size (7–15 kDa). Chemokines may also be transported through or around microvascular endothelium, thus further identifying discrete regions for leukocyte recruitment (153). Collectively, these structural and biochemical features contribute to the ability of chemokines to mediate directional leukocyte recruitment and migration. Several chemokines have been implicated in dysregulated immune responses of the GI system. In patients with active ulcerative colitis (UC) or Crohn’s disease, increased expression of chemokines CXCL5, CXCL8, CXCL10, CCL2, CCL5, CCL6, and CCL10 has been reported (154,155). Likewise, increased chemokine receptor expression of CXCR1 for CXCL8 and CXCR3 for CXCL10 is also observed in colon tissue from patients with inflammatory bowel disease (IBD). An important pathologic role for chemokines has further been demonstrated in animal models of experimental colitis. Increased expression of chemokines CXCL1, CCL2, CCL3, and CCL5 has been reported in different colitis models (154). Consistent with these observations, gene-targeted deletion of CCR2, CCR5, or CCR6 attenuates experimental colitis, suggesting that enhanced chemokine expression and release elicits recruitment of leukocytes bearing specific chemokine receptors (156,157). Together, these findings demonstrate an important pathologic role for chemokines and their receptors in GI disease. Chemokines can facilitate leukocyte recruitment and migration through alterations of adhesion molecule function or cellular location. For example, multiple chemokines including CXCL8, CXCL9, CXCL10, CXCL12, CCL2, CCL11, CCL17, CCL19, CCL21, and CCL22 have all been reported to stimulate rapid, integrin-mediated leukocyte firm adhesion under hydrodynamic flow conditions (158). Chemokine stimulation enhances integrin-mediated adhesion by changing the state of integrin activation (conversion from a “closed” nonbinding conformation to an “open” high-binding state), thus increasing its affinity for ligand, or it may alter integrin cell-surface location into discrete clusters, thereby

increasing avidity for ligand (159,160). Both of these molecular events may also occur simultaneously in the leukocyte, thus explaining the rapidity and avidity of chemokinemediated leukocyte adhesion (161). Chemokine activation of leukocytes also increases adhesion to ECM proteins through similar mechanisms and stimulates robust migration through ECM (162–166). However, chemokines are but one component of the chemoattractant inflammatory milieu, and how these molecules act in concert with other cytokines to stimulate tissue infiltration of leukocytes is an active area of investigation. Like chemokines, cytokines may also bind to matrix proteins and establish a “chemoattractant trail” for leukocytes to follow (167). For example, TNF-α binds to the N-terminal domain of fibronectin, which enhances T-cell binding to fibronectin in a β1-integrin–dependent manner (168). Similarly, transforming growth factor-β (TGF-β) modulates leukocyte adhesion to and migration through matrix proteins (169,170). Preliminary studies have addressed how combinatorial gradients of cytokines and chemokines affect leukocyte migration through ECM. Experiments using threedimensional extracellular matrices containing cytokine (e.g., TNF-α or IL-2) and/or chemokine (e.g., CXCL12 or CCL5) gradients show that cytokine stimulation inhibits lymphocyte chemotaxis because of enhanced integrin adhesion to matrix proteins (171). Importantly, the adhesion response stimulated from combined cytokine/chemokine stimulation is substantially greater than cytokine-mediated adhesion alone, suggesting a cooperative effect of these chemoattractants in enhancing cell–matrix interaction. Interestingly, the effects of combined chemokine treatment may actually antagonize cellular responses from one to another, including decreased chemotactic responses and adhesion to matrix (172). These observations indicate that biophysical regulation of leukocyte migration through tissue is multifactorial and suggest that differential leukocyte recruitment during various inflammatory responses involves several members of the cytokine and chemokine family.

LEUKOCYTE TRAFFICKING DURING ACUTE INFLAMMATION Leukocytes have been implicated in the pathogenesis of a variety of GI diseases that are characterized as either an acute or chronic inflammatory condition. This section focuses on some acute inflammatory conditions of the GI tract in which the dominant population of recruited leukocytes is neutrophils. The trafficking of leukocytes has been characterized using intravital microscopy in several experimental models of acute GI inflammation, including ischemia– reperfusion (I/R), nonsteroidal anti-inflammatory drug– induced ulcers, radiation enteritis, ethanol-induced ulcers, endotoxemia, Helicobacter pylori infection, and Clostridium difficile colitis (92,100,109,113,115). The data collected from these models indicate that they all share features that are characteristic of an inflammatory phenotype, including

1154 / CHAPTER 46 the rolling, firm adhesion and emigration of neutrophils, oxidative stress, platelet–leukocyte adhesion/aggregation, increased vascular permeability, and interstitial edema. Furthermore, there is evidence for neutrophil-mediated tissue injury in all of these experimental models. The factors that appear to distinguish these models from one another are the inciting events that initiate the inflammatory response and the molecular and biochemical mechanisms that underlie the recruitment or activation, or both, of the tissue injury– inducing leukocytes. The models that are discussed later exemplify the diversity of inciting factors and mechanisms that contribute to the trafficking of leukocytes in acute GI inflammation.

Oxidative Stress–Dependent Models of Inflammation I/R and abdominal irradiation are two distinct inciting factors that can produce an acute intense inflammatory response in the GI tract. A major component of the inflammatory response elicited by both stimuli results from an increased production of reactive oxygen metabolites (ROS) resulting from effects of the oxidative stress on leukocyte recruitment and activation. The enhanced ROS production occurs in microvascular endothelial cells, particularly in venules, suggesting that these cells assume an activated state. The increased endothelial cell ROS production induced by I/R occurs within seconds to minutes after the restoration of blood flow to an ischemic tissue and is largely generated by the enzyme xanthine oxidase (173,174). As neutrophils are recruited into venules of the postischemic tissue, a second, more intense oxidative stress results from the ROS production by adherent and emigrating leukocytes. Neutrophil adhesion in postischemic venules can occur within a few minutes after the onset of reperfusion, but this accumulation of adherent cells increases over the next few hours, thereby accounting for the slow progressive increase in the intensity of the oxidative stress experienced by the vessel walls. With abdominal radiation, however, the oxidative stress exhibited by intestinal venules appears to parallel the accumulation of adherent and emigrating leukocytes (175,176). The leukocyte dependency of the accelerated ROS production is most pronounced at 6 hours after irradiation, and the oxidative stress detected at 2 hours is only partially dependent on adherent leukocytes. This is supported by that inhibition of the later leukocyte adhesion response with an anti-CD18 MAb eliminates the corresponding oxidative stress, whereas the oxidative stress is only partially blunted at 2 hours. In contrast with what is seen after I/R, neither the leukocyteindependent or -dependent components of the oxidative stress are attenuated by the xanthine oxidase inhibitor allopurinol (175,177). Both I/R and abdominal radiation elicit the recruitment of rolling leukocytes in venules that can be blocked by P-selectin immunoneutralization. Similarly, both models have been shown to result in an increased expression of P-selectin in the gut microvasculature. Increased E-selectin expression

and a blunted neutrophil recruitment response after E-selectin immunoblockade have been demonstrated in the gut after I/R, but the contribution of this adhesion molecule to radiationinduced inflammation has not yet been evaluated. The firm adhesion of leukocytes in both models appears to involve an interaction between CD11/CD18 on leukocytes with constitutively expressed ICAM-1 on endothelial cells (92). If this firm adhesion is blocked by treatment with either a CD18or ICAM-1–specific MAb, the increased vascular permeability that results from I/R or abdominal irradiation is prevented. Although a genetic deficiency or immunoblockade of P-selectin has been shown to protect the intestine against histologic mucosal damage after I/R, no such protection is observed after abdominal radiation (176,178). Although oxidative stress appears to be a major factor in the initiation and progression of leukocyte recruitment in I/R- and abdominal irradiation-induced inflammation, the involvement of ROS has been more clearly delineated when I/R is the inciting stimulus. A variety of ROS-directed interventions have been shown significantly to diminish the leukocyte trafficking that occurs in the intestinal vasculature after I/R (173,174). Antioxidant enzymes such as SOD and catalase, iron-chelators such as desferrioxamine, and xanthine oxidase inhibitors such as allopurinol are all capable of blunting the leukocyte recruitment response whether administered before or immediately after the onset of reperfusion. SOD1 transgenic mice (that overexpress CuZn SOD) show an attenuated I/R-induced up-regulation of P- and E-selectin. A similar attenuation of I/R-induced E-selectin expression is noted in mice treated with an inhibitor of NF-κB activation, supporting a role for this transcription factor as the link between oxidative stress and the increased biosynthesis/expression of E-selectin after I/R (109,178). There are several lines of evidence that support a role for NO in I/R-induced leukocyte trafficking (179). NO donors appear to be as effective as SOD in attenuating I/R-induced leukocyte rolling and adhesion. Nonselective inhibitors of NOS (e.g., L-NAME) induce a similar endothelial cell phenotype as seen after I/R, including oxidative stress and leukocyte–endothelial cell adhesion. The observation that NOS inhibition does not induce further increases in leukocyte adhesion after I/R is consistent with the view that the bioavailability of NO after reperfusion in untreated animals is already limited to inactivation by the large fluxes of superoxide produced in the postischemic vasculature. Hence, I/R (and possibly abdominal irradiation) appears to be an excellent example of increased leukocyte trafficking that results from an imbalance between superoxide and NO fluxes through mechanisms depicted in Figure 46-6.

Bacterial Toxins Translocation of enteric bacteria or bacterial products (e.g., LPS), or both, from the gut lumen to the mucosal interstitium is a potential mechanism for the initiation of leukocyte trafficking in the GI tract. LPS or other bacterial products

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1155 that gain access into the interstitium can interact with receptors on mucosal macrophages to stimulate the production and release of cytokines that can act on vascular endothelium to enhance CAM expression and promote leukocyte adhesion. It has been demonstrated that the constitutive expression of ICAM-1 in the GI vasculature of germfree mice is significantly lower than in their conventional counterparts, suggesting that indigenous GI microflora is responsible for a significant proportion of the basal ICAM-1 expression detected in the GI tract (180). This raises the possibility that enteric bacteria may also contribute to the increased CAM expression and leukocyte recruitment that is observed in GI diseases that are accompanied by mucosal injury. However, in a murine model of I/R injury, this does not appear to be the case, because the I/R-induced increase in E-selectin expression does not differ between LPS-sensitive and -insensitive mice (181). A similar finding has been reported for mice that are subjected to cecal ligation and puncture (a model of sepsis), where no differences in the E-selectin up-regulation responses are noted between LPSsensitive and -insensitive mice (182). Two models of GI inflammation for which there is clear evidence of bacterial toxin involvement in the initiation of leukocyte activation and recruitment are H. pylori gastritis and C. difficile colitis. Soluble extracts of both H. pylori and C. difficile can elicit an inflammatory response when applied directly to the mesenteric microcirculation (183,184). Both low-molecular-weight nonprotein substances and highmolecular-weight protein fractions of water-soluble extracts of H. pylori have been shown to elicit an increased expression of different CAMs on monolayers of cultured endothelial cells (185). The same water extract of H. pylori has been shown to increase the adhesion and emigration of leukocytes, to promote leukocyte–platelet aggregation, and to increase vascular permeability in mesenteric venules and elicit the degranulation of perivenular mast cells (115,183). Both in vitro and in vivo models of H. pylori–induced inflammation implicate CD11/CD18 interactions with ICAM-1 in the neutrophil adhesion response, whereas the leukocyte– platelet aggregation appears to be largely mediated by P-selectin expressed on the activated platelets interacting with PSGL-1 on neutrophils. Immunoblockade of either CD18 or ICAM-1 diminishes the H. pylori–induced vascular permeability response, suggesting that the adherent leukocytes are responsible for this manifestation of endothelial cell dysfunction (115,183). C. difficile is the principal agent that is responsible for antibiotic-associated pseudomembranous colitis in humans and experimental animals. This bacterium produces two protein exotoxins (A and B) that have different biological properties, but only one (toxin A) of which is responsible for the inflammatory responses (92). Exposure of the mesenteric microcirculation to C. difficile toxin A elicits an inflammatory response that is nearly identical to that observed with the water extract of H. pylori, that is, leukocyte adhesion and emigration, increased vascular permeability, leukocyte–platelet aggregation, and mast-cell degranulation.

Furthermore, the leukocyte adhesion response is mediated by CD11/CD18–ICAM-1 interactions (184). A shared feature of the microvascular responses to the products of H. pylori and C. difficile toxin is degranulation of perivenular mast cells. In both models of inflammation, this mast-cell response makes an important contribution to the overall inflammatory response because mast stabilizers not only blunt the leukocyte recruitment, but also protect against the endothelial barrier dysfunction (increased vascular permeability) (183,184). Although mast-cell degranulation appears to be a critical early step in both inflammatory responses, the mast-cell–derived substances that mediate these effects appears to differ between the two stimuli. In C. difficile toxin A–mediated leukocyte recruitment and microvascular dysfunction, histamine appears to play a major role because treatment with either diamine oxidase (histaminase) or an H1 receptor antagonist is as effective as lodoxamide (a mastcell stabilizer). However, PAF appears to be the major mastcell–derived product that contributes to H. pylori–induced leukocyte adhesion and increased vascular permeability. Support for this contention is provided by the observation that PAF receptor antagonists are as effective in blunting the H. pylori–induced responses as the mast-cell stabilizer ketotifen. PAF is known to be a potent inducer of leukocyte adhesion and can increase vascular permeability in both a leukocyte-dependent and a leukocyte-independent manner (92,115,183,184).

LEUKOCYTE TRAFFICKING DURING CHRONIC GUT INFLAMMATION The potential for the immune system to produce local and systemic tissue injury suggests that the immune system has ways to efficiently regulate immune responses to both pathogenic and harmless antigens. This suppressive or regulatory activity is accomplished by additional subsets of T cells. Although several different populations of regulatory T cells (Tregs) have been identified, the best characterized of the naturally occurring regulatory cells is the CD4+CD25+ subset. These Tregs are capable of inhibiting a variety of experimental autoimmune diseases including chronic colitis (186–188). In addition to these CD4+ Tregs, studies have identified certain CD8+ T cells that are part of the intraepithelial lymphocyte compartment in the gut and are capable of attenuating chronic colitis in vivo (189,190). Furthermore, work by Mizoguchi and Bhan suggests that, in some situations, B cells may function as regulatory cells and attenuate chronic colitis in one mouse model (191–193). It is becoming increasingly appreciated that IL-10 and TGF-β are required for the regulatory function of Treg and B cells in vivo, and that both of these regulatory cytokines play important roles in regulating tissue inflammation and injury in different models of IBD (186–188). These cytokines inhibit Th1 activation, as well as Th1-dependent activation of tissue macrophages. Indeed, there is growing acceptance that chronic gut inflammation results from a dysregulated

1156 / CHAPTER 46 immune response to components of the normal gut flora (187,188). Failure to properly regulate intestinal immune responses to enteric antigens is thought to promote chronic gut inflammation and may be important in the pathophysiology of the IBDs (Crohn’s disease and UC). Sustained overproduction of Th1- and macrophage-derived cytokines such as TNF-α, IL-1β, IFN-γ, LT-α, and IL-12 activate postcapillary venular endothelial cells and enhance the expression of endothelial CAMs and chemokines not normally present or functional in the healthy bowel, resulting in massive leukocyte adhesion and extravasation. Understanding the complex leukocyte– endothelial cell interactions in chronic gut inflammation may provide new insights into the pathophysiologic mechanisms responsible for inflammatory tissue injury and organ dysfunction in IBD. The selectins have been implicated in the tethering and subsequent rolling of the cells along the postcapillary endothelium. Koizumi and colleagues (194) and Nakamura and colleagues (195) have demonstrated that E-selectin is significantly up-regulated on endothelial cells in mucosal biopsies obtained from patients with active (but not quiescent) UC or Crohn’s colitis. We have demonstrated that colonic E-selectin expression is significantly enhanced with the onset of chronic colitis in severe combined immunodeficiency disease (SCID) mice reconstituted with naive T cells or in colons of IL-10–deficient (IL-10−/−) mice with colitis (196–199). Intervention studies using two different antibodies to E-selectin were not effective at attenuating the spontaneous colitis that develops in the nonhuman primates, cottontop tamarins, suggesting that E-selectin–dependent leukocyte adhesion may not represent an important interaction to sustain the inflammatory response in this model of colitis (200). Nakamura and colleagues (195) and Schurmann and colleagues (201) have shown that P-selectin is up-regulated in venules of mucosal biopsies obtained from patients with active IBD. As with earlier work using antibodies to E-selectin, studies addressing the importance of P- and E-selectin in experimental colitis using mice genetically deficient in these adhesion molecules have also proved rather disappointing (202). In these studies, investigators found that colitis induced by acetic acid or TNBS in ethanol was either not attenuated or enhanced in mice deficient in P- selectin, E-selectin, or both (202). Interpretation of these studies in the context of human IBD is difficult because neither the acetic acid nor the TNBS/ethanol model is a T-cell–dependent model, and thus their use as clinically relevant models is questionable. PMNs, monocytes, and lymphocytes adhere to venular endothelium by virtue of the strong adhesive interactions between β2 integrins (CD11/CD18) on these leukocytes and ICAM-1 on endothelial cells. Although it has been suggested that CD18-dependent adhesion is essential for mediating much of the tissue injury and dysfunction during acute flares of colitis, reports by D’Agata and colleagues (203) and Uzel and colleagues (204) suggest that this concept may need to be reexamined. They report a case of CD18 deficiency characterized by chronic ileocolitis. Bone marrow

transplantation completely resolved the intestinal inflammation, suggesting that marrow-derived leukocyte dysfunction may contribute to disease. These data are similar to those reported demonstrating that four of five patients with Crohn’s disease given allogenic marrow transplantation remained disease free for several years after transplantation (205), and they support the concept that Crohn’s disease may arise from alterations in leukocyte function. Several different reports have demonstrated enhanced staining for ICAM-1 on mucosal mononuclear or endothelial cells in biopsies obtained from patients with active UC and Crohn’s disease (195,206,207). We have shown that colonic ICAM-1 (but not ICAM-2) expression is significantly enhanced in colitic SCID mice reconstituted with CD45RBhigh T cells and in the colons of IL-10−/− mice with active enterocolitis (196–199,208). Infusion of an antisense oligonucleotide or antibodies directed against ICAM-1 produced clinical improvement in mouse models of colitis or ileitis and in steroid-resistant patients with Crohn’s disease, respectively (209–211), although larger controlled trials were negative (212,213). The ineffectiveness of ICAM-1 blockade in the above-mentioned studies would appear to rule out a major role for LFA-1 (or CD11b/CD18) in the pathogenesis of IBD. However, LFA-1 also interacts with other ligands such as ICAM-2 (214). This counterreceptor for LFA-1 is actually a truncated form of ICAM-1 that is constitutively expressed at a 10-fold greater level on venular endothelium than is ICAM-1 (214,215). In addition, ICAM-2 expression does not change on stimulation of the endothelium, and the expression is constitutive in models of IBD (196,198,208). Furthermore, lymphocyte adhesion to human umbilical vein endothelial cells (HUVECs) has been shown to be mediated by ICAM-2 to a greater extent than by ICAM-1 (214). Furthermore, we have demonstrated that chronic colitis fails to develop in recombinase-activating gene-1 (RAG-1)– deficient mice injected with naive T cells genetically devoid of LFA-1, whereas transfer of wild-type T cells induces severe colitis within 6 to 8 weeks after transfer (216). Taken together, these studies suggest that LFA-1 and ICAM-1 and/or ICAM-2 may play a more important role in IBD than was originally thought. PMN-endothelial cell interactions predominate in acute flares of IBD, whereas lymphocyte, monocyte, and in some cases, eosinophil interactions with the microvascular endothelium are more prevalent during the chronic stages of gut inflammation. Studies from our laboratory demonstrate a significant enhancement in colonic VCAM-1 expression with the onset of chronic colitis in the SCID/CD45RBhigh or IL-10−/− models of chronic colitis (196–198,208,217). Although some studies have demonstrated that VCAM-1 blockade attenuates DSS-induced colitis (218), several other laboratories have failed to consistently demonstrate enhanced expression of VCAM-1 in biopsies obtained from patients with active colitis (194,195,206). These observations are somewhat surprising in view of two reports demonstrating that primary cultures of microvascular endothelial cells isolated from human intestine and colon respond to different proinflammatory cytokines with enhanced surface

RECRUITMENT OF INFLAMMATORY AND IMMUNE CELLS IN THE GUT: PHYSIOLOGY AND PATHOPHYSIOLOGY / 1157 expression of VCAM-1 (196,198). The reasons for these differences between experimental and human IBD are not known, but may represent the inherent variability associated with immunohistochemistry compared with the objective, more quantitative method used to measure endothelial CAM expression in vivo (196–198). Alternatively, the quality of the antibodies used for the immunolocalization studies may also represent an important determinant for accurate determination of VCAM-1. Lymphocytes possess an additional ligand/counterreceptor pair that is important in cell–cell adhesion, signaling, trafficking, and regulation of the immune responses in mucosal tissues, especially in the GI tract (221,222) MAdCAM-1 is a member of the immunoglobulin supergene family that is expressed on GI mucosal endothelial cells and high endothelial cells in lymph nodes and Peyer’s patches and is involved in the selective homing of lymphocytes to mucosal tissue (221,222). Surface expression of MAdCAM-1 is induced by certain Th1 or macrophage-derived cytokines and bacterial products and may last for several days (51). Its lymphocyte-associated counterreceptor, α4β7, is found primarily on a subpopulation of memory/activated CD4+ T lymphocytes involved in mucosal immunity. Briskin and colleagues (50) report that MAdCAM-1 expression on venular endothelial cells in the lamina propria of the gut is enhanced in inflammatory foci in biopsies obtained from patients with active UC or Crohn’s disease. Colonic and cecal (but not ileal) MAdCAM-1 expression increases dramatically (11-fold) with the onset of colitis in the SCID/ CD45RBhigh and IL-10−/− models of colitis (51,196–198, 208). Studies from our laboratory have demonstrated that immunoneutralization of MAdCAM-1 (but not ICAM-1 or VCAM-1) profoundly reduced the adhesion of CD4+ T cells in inflamed postcapillary venules of mice with chronic colitis (79). Several additional studies using low-molecular-weight antagonists or immunoneutralizing monoclonal antibodies to either α4β7 or MAdCAM-1 demonstrate that the α4β7/ MAdCAM-1 interaction plays an important role in the pathophysiology of three different experimental models of colitis (79,223–226). MAdCAM-1 represents a potentially important therapeutic target for the treatment of IBD. Studies using antibodies to α4, which is directed against VLA-4 (α4β1) and α4β7 in patients with Crohn’s disease, demonstrated significant clinical efficacy and may represent a new therapeutic strategy for the treatment of IBD (227,228). PECAM-1, which is preferentially distributed between endothelial cells (intercellular junctions), appears to play an important role in the extravasation of leukocytes. A report by Shuermann and colleagues (229) demonstrates a significant increase in PECAM expression on venules in mucosal biopsies obtained from patients with active UC. In other studies, JAM-1 has been found to be critical for both PMN and T-cell transendothelial cell migration (230). The ECM-rich environment of perivascular tissue is thought to be an important site where certain human leukocytes undergo differentiation (e.g., monocytes) and activation (neutrophils, monocytes, and lymphocytes) on extravascular

migration. The α1β1 integrin is a major cell-surface receptor for collagens with a preference for type IV collagen. The presumed role of the ECM environment in the inflammatory process, and the fact that chronically activated immune cells can express α1β1, has led to the hypothesis that α1β1 integrin may contribute to the pathophysiology of intestinal inflammation. In a series of studies, we have found that treatment of mice with acute self-limiting colitis induced by DSS with a blocking anti-α1 MAb resulted in significant attenuation of colitis, and that similar protection was conferred to DSSexposed mice lacking α1β1 integrin (150). Blockade of α1β1 also was associated with decreased mucosal inflammatory cell infiltrate and cytokine production. It was shown that both the induction of DSS-induced colitis and α1-mediated inhibition of colitis occurred independently of lymphocytes and identified the monocyte as a key α1β1-expressing cell involved in the development of colitis in this model. Additional studies using naive T cells deficient in α1β1 demonstrated that T-cell–associated α1β1 is important for initiation or perpetuation, or both, of chronic colitis induced in immunodeficient recipient mice (231). Given that α1β1 represents a major collagen-binding integrin expressed on leukocytes, these findings highlight the important role of ECM-leukocyte interactions in the pathogenesis of colitis, with particular relevance to monocytes.

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214. Xu H, Gonzalo JA, St Pierre Y, Williams IR, Kupper TS, Cotran RS, Springer TA, Gutierrez-Ramos JC. Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice. J Exp Med 1994;80:95–109. 215. de Fougerolles AR, Stacker SA, Schwarting R, Springer TA. Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1. J Exp Med 1991;174:253–267. 216. Pavlick KP, Ostanin D, Laroux FS, Gray L, Kevil C, Grisham MB. LFA-1 Deficient T-cells fail to induce chronic colitis in an immunebased mouse model. Gastroenterology 2004;126:A-417 (Abstract). 217. Conner EM, Brand S, Davis JM, Laroux FS, Palombella VJ, Fuseler JW, Kang DY, Wolf RE, Grisham MB. Proteasome inhibition attenuates nitric oxide synthase expression, VCAM-1 transcription and the development of chronic colitis. J Pharmacol Exp Ther 1997;282: 1615–1622. 218. Soriano A, Salas A, Salas A, Sans M, Gironella M, Elena M, Anderson DC, Pique JM, Panes J. VCAM-1, but not ICAM-1 or MAdCAM-1, immunoblockade ameliorates DSS-induced colitis in mice. Lab Invest 2000;80:1541–1551. 219. Binion DG, West GA, Ina K, Ziats NP, Emancipator SN, Fiocchi C. Enhanced leukocyte binding by intestinal microvascular endothelial cells in inflammatory bowel disease. Gastroenterology 1997;112: 1895–1907. 220. Haraldsen G, Kvale D, Lien B, Farstad IN, Brandtzaeg P. Cytokineregulated expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human microvascular endothelial cells. J Immunol 1996;156: 2558–2565. 221. Butcher EC, Picker LJ. Lymphocyte homing and homeostasis. Science 1996;272:60–66. 222. Butcher EC, Williams M, Youngman K, Rott L, Briskin M. Lymphocyte trafficking and regional immunity. Adv Immunol 1999; 72:209–253. 223. Picarella D, Hurlbut P, Rottman J, Shi X, Butcher E, Ringler DJ. Monoclonal antibodies specific for beta 7 integrin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) reduce inflammation in the colon of scid mice reconstituted with CD45RBhigh CD4+ T cells. J Immunol 1997;158:2099–2106. 224. Viney JL, Jones S, Chiu HH, Lagrimas B, Renz ME, Presta LG, Jackson D, Hillan KJ, Lew S, Fong S. Mucosal addressin cell adhesion molecule-1: a structural and functional analysis demarcates the integrin binding motif. J Immunol 1996;157:2488–2497. 225. Hesterberg PE, Winsor-Hines D, Briskin MJ, Soler-Ferran D, Merrill C, Mackay CR, Newman W, Ringler DJ. Rapid resolution of chronic colitis in the cotton-top tamarin with an antibody to a gut-homing integrin alpha 4 beta 7. Gastroenterology 1996;111:1373–1380. 226. Hokari R, Kato S, Matsuzaki K, Iwai A, Kawaguchi A, Nagao S, Miyahara T, Itoh K, Sekizuka E, Nagata H, et al. Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats. Clin Exp Immunol 2001;126:259–265. 227. Ghosh S, Goldin E, Gordon FH, Malchow HA, Rask-Madsen J, Rutgeerts P, Vyhnalek P, Zadorova Z, Palmer T, Donoghue S. Natalizumab for active Crohn’s disease. N Engl J Med 2003;348: 24–32. 228. Gordon FH, Lai CW, Hamilton MI, Allison MC, Srivastava ED, Fouweather MG, Donoghue S, Greenlees C, Subhani J, Amlot PL, et al. A randomized placebo-controlled trial of a humanized monoclonal antibody to alpha4 integrin in active Crohn’s disease. Gastroenterology 2001;121:268–274. 229. Schuermann GM, Aber-Bishop AE, Facer P, Lee JC, Rampton DS, Dore CJ, Polak JM. Altered expression of cell adhesion molecules in uninvolved gut in inflammatory bowel disease. Clin Exp Immunol 1993;94:341–347. 230. Ostermann G, Weber KS, Zernecke A, Schroder A, Weber C. JAM-1 is a ligand of the beta(2) integrin LFA-1 involved in transendothelial migration of leukocytes. Nat Immunol 2002;3:151–158. 231. Pavlick KP, Krieglstein CF, Laroux FS, Gray L, Granger DN, Grisham MB. Importance of the alpha-1-beta-1 integrin in acute and chronic intestinal inflammation. Gastroenterology 2003;124:A120 (Abstract).

CHAPTER

47

Physiology of Host–Pathogen Interactions Kim Hodges, V. K. Viswanathan, and Gail Hecht Toxin-Mediated Effects on Ion Secretion, 1165 Vibrio cholerae, 1165 Vibrio parahemolyticus, 1169 Escherichia coli, 1170 Helicobacter pylori, 1170 Rotavirus, 1171 Absorption: Enteropathogenic Escherichia coli, 1171 Indirect Effects on Ion Secretion, 1172 Clostridium difficile, Inflammation, and Neuropeptides, 1172 Cyclooxygenase-2/Nitric Oxide/Prostaglandin E–Mediated Cl− Secretion, 1172 Cryptdins, 1172 Galanin-1 Receptor, 1173 5′-Adenosine Monophosphate, 1173

Barrier Function and Cytotoxicity, 1173 Vibrio cholerae, 1173 Escherichia coli, 1174 Shigella flexneri, 1176 Bacteroides fragilis, 1176 Clostridium, 1176 Infection-Mediated Barrier Changes, 1178 Helicobacter pylori, 1178 Escherichia coli, 1179 Shigella, 1180 Salmonella enterica, 1181 Rotavirus, 1181 Junctions as Pathogen Receptors, 1181 Summary, 1182 References, 1182

The human gastrointestinal tract is colonized with billions of bacteria composed of a multitude of species, each with varying needs and adaptations that alter its interaction with the host. The majority of colonizing species are, at worst, benign passengers taking advantage of the rich nutrient environment provided by the human host. At best, the commensal flora is essential for full development of the immune system, assists in digestion, and likely provides a level of protection against pathogenic intruders. Despite the beneficial effects associated with these inhabitants, sheer numbers often evoke an image of bacteria freely proliferating throughout the intestinal lumen. This image could not be further from the truth. Instead, the gastrointestinal tract is highly ordered with the colon harboring abundant bacteria, whereas other regions, such as the small intestine, are virtually sterile. In these regions, even nonpathogenic bacteria

are capable of altering the host physiology to an extent that diarrheal disease can ensue (1). In the healthy human host, more elaborate mechanisms typically are required to perturb the standard function of the digestive system. Such mechanisms have been acquired by pathogenic bacteria in the form of phages, plasmids, and pathogenicity islands. These segments of genetic information provide the blueprints for everything from toxins designed to act independently to elaborate multiprotein structures designed to allow attachment to a eukaryotic cell and direct injection of effector molecules into the host cytosol. The end result of years of competitive evolution is a series of elegant, but sometimes frighteningly powerful, molecules that enable pathogens to overcome a variety of challenges in the host environment. In the course of ensuring their own survival and spread to other hosts, these pathogens often produce a series of undesirable outcomes within the colonized host. Of primary concern in the gastrointestinal tract are alterations in ion transport and fluid balance and disruption of tight junctions leading to increased paracellular permeability. These topics are covered in detail in this chapter. A number of tables are provided to clarify each section. Table 47-1 consists of effects on secretion,

K. Hodges, V. K. Viswanathan, and G. Hecht: Section of Digestive Diseases and Nutrition, University of Illinois, Chicago, Illinois 60612. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1163

1164 / CHAPTER 47 TABLE 47-1. Alteration of secretion by various pathogens Organism/effector molecule

Vibrio cholerae 1. Cholera toxin 2. Accessory cholera toxin (ACE) 3. V. cholerae cytolysin (VCC) 4. NAG-stable toxin

Vibrio parahemolyticus 1. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) Escherichia coli 1. Heat-stable enterotoxin (STa) 2. Enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1) 3. Heat labile enterotoxins (LTs): LTI LTIIa LTIIb

Helicobacter pylori Vacuolating toxin (VacA) Rotavirus Nonstructural protein 4 (NSP4) Enteropathogenic E. coli Type III secreted protein(s)? Indirect Effects on Ion Secretion 1. Inflammation 1. Clostridium difficile toxins A and B (TcdA and TcdB)

2. COX-2, nitric oxide, prostaglandins Salmonella dublin and Enteroinvasive E. coli 3. Cryptdins LPS and LTA of various pathogens 4. Galanin and galanin receptor EPEC, EHEC, ETEC, Salmonella, Shigella 5. 5′′-AMP LPS, FMLP, or PMA

Site/mechanism of action Binds GM1, enters cells and activates adenylate cyclase, increases cAMP Increase in Ca2+ levels Pore-forming toxin Binds and activates guanylyl cyclase, increases cGMP, promotes Ca2+ release and PKCα activation

Net result CFTR-dependent chloride secretion CLCA-dependent chloride secretion Secretion via an anion-selective channel Vacuole formation PKG II/PKA II-dependent activation of CFTR

Increase of Ca2+ PKC activation

CLCA-dependent chloride secretion

Bind and activate guanylyl cyclase, increase cGMP Ca2+ release and PKCα activation Bind to: GM1 GD1b GD1a Activate adenylate cyclase, increase cAMP

PKG II/PKA II-dependent activation of CFTR

Pore-forming toxin

Secretion via an anion-selective channel

Increase in Ca2+ levels Inhibits SGLT1

CLCA-dependent chloride secretion

Activation of NHE1 and NHE2 Inhibition of NHE3

Net increase in sodium uptake

p38 activation and production of proinflammatory molecules (IL-8, MIP-2, IFN-γ) (glucosylation of Rho/Rac/Cdc42)

Neutrophil infiltration, leading to fluid secretion Production of neuropeptides (substance P) that promote fluid secretion

Nitric oxide synthase (iNOS) activation, COX-2 activation; increase in cGMP and cAMP

CFTR and NKCC1 activation

Mechanism unknown

Chloride secretion

NF-κB activation leads to increase in galanin-1 receptor

Increased short circuit current

Conversion of 5′-AMP to adenosine; interaction of adenosine with A2b receptor

Increased short circuit current

CFTR-dependent chloride secretion

cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; cGMP, cyclic guanosine monophosphate; CLCA, Ca2+-activated Cl− channel; COX-2, cyclooxygenase-2; EHEC, enterohemorrhagic E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic Escherichia coli; FMLP, N-formyl methionyl leucyl phenylalanine; IFN-γ, interferon-γ; IL-8, interleukin-8; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; LTA, lipoteichoic acid; NF-κB, nuclear factor-κB; NKCC1, Na+-K+-2Cl− cotransporter; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; SGLT1, sodium-dependent glucose transporter 1.

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1165 whereas Table 47-2 contains effects on tight junctions, adherens junctions, and cytotoxicity. Finally, Table 47-3 lists inhibitors used in the various studies referenced in this chapter.

TOXIN-MEDIATED EFFECTS ON ION SECRETION Vibrio cholerae Vibrio cholerae is the causative agent of cholera. Cholera is perhaps the most dramatic of the diarrheal diseases, causing copious amounts of fluid production primarily because of the presence of cholera toxin (CT). Perhaps the first meaningful discovery regarding cholera was the association of the disease with the consumption of tainted water. Careful observations and mapping over several cholera epidemics in the 1850s led John Snow to theorize that the agent of cholera was carried in water rather than through the air, as was thought by other contemporaries (2). An independent study around the same time by Fillippo Pacini microscopically identified a comma-shaped bacterium as the putative disease agent (3). However, this finding was not widely disseminated, and thus it was possible for Robert Koch in 1884 to rediscover the causative agent of cholera and to take the additional step of isolating a pure culture (3). Since that time, a number of scientists have followed in these historic footsteps, and the mechanisms of disease for V. cholerae have become the best characterized of all enteric pathogens. Cholera Toxin Although V. cholerae secretes a number of toxins, the most widely recognized and perhaps the most significant in terms of disease is CT. CT was beginning to be characterized in the 1960s. Initial experiments involved bacterial lysates or filtered culture supernatants of V. cholerae that were found to inhibit sodium uptake (4). During attempts to purify the active toxin, the authors inadvertently discovered the two-component nature of CT. Initial preparation and dialysis allowed for retention of one component in the dialysis bag and another component in the surrounding media. Neither component was functional alone, but when combined, the putative CT was reformed (4). In later studies, the same group used antibody against CT to identify the components of the toxin at a more purified level (5). They found what they referred to as choleragen, a single active unit, and choleragenoid, an inactive five-component protein that also reacted to the antibody. Through further studies, including the ultimate crystallization of the toxin, it became increasingly clear that CT contains a single A subunit consisting of 2 peptides, A1 and A2 (22 and 5 kDa, respectively), joined by a disulfide bond and 5 B (11 kDa) subunits arranged in a ringlike structure (6,7). The choleragen, previously described by Finkelstein and LoSpalluto (5), represents the intact CT, whereas the inactive choleragenoid consists of the

pentameric B subunit ring. The A subunit is itself the active portion of the molecule, specifically the A1 peptide; however, the B subunit plays a critical role in binding to the receptor GM1 on the host cell and in the subsequent retrograde transmission through the Golgi (Fig. 47-1) (8). Soon after CT was purified, insights into its mechanism of action were obtained. CT induces high levels of cyclic adenosine monophosphate (cAMP), the product of adenylate cyclase (9,10). High levels of cAMP occur through inhibition of guanosine triphosphate (GTP) hydrolysis via adenosine diphosphate (ADP)-ribosylation of Gs, the regulatory GTPase of adenylate cyclase (11). This results in the constant production of cAMP by adenylate cyclase (11,12). It is the overproduction of cAMP mediated by CT that triggers the Cl− secretion and fluid imbalances typically associated with V. cholerae. At the time that CT was purified and identified immunologically, the nature of individual ion transporters and channels was not clearly understood. However, it was noted that addition of either cAMP or CT to rabbit ileal mucosa produced the same effect, an increase in Cl− secretion and a decrease in Na+ absorption (13,14). Therefore, it was clear even then that alterations in cAMP levels were, in fact, responsible for the altered secretion observed during V. cholerae infection. As additional information regarding individual ion channels and transporters was discovered, a more detailed look at the mechanism of cAMP-induced chloride secretion was possible. It is now known that the Cl− channel targeted by CT is CFTR (cystic fibrosis transmembrane conductance regulator). On production of cAMP, protein kinase A (PKA) is activated and phosphorylates several key serines in the regulatory domain of CFTR (15,16). Phosphorylation of the regulatory domain, together with ATP binding by the two nucleotide-binding domains, results in activation of the channel. The availability of chloride for secretion by CFTR is driven secondarily by Na+,K+-ATPase. The coupled action of the Na+-K+-2Cl− cotransporter (NKCC1), which transports K+, Na+, and 2Cl− into the cell, and the Na+,K+ATPase, which actively secretes Na+ and K+ ions, results in an overabundance of Cl− within the cell. This excess Cl− is rectified through the secretion of Cl− ions. Interestingly, CFTR also plays a role in regulation of other ion channels such as the epithelial sodium channel (ENaC). Activation of CFTR via cAMP results in a decrease in ENaC activity (17,18). Although the nature of the comparably smaller decrease in sodium absorption on stimulation with CT has been largely ignored in favor of studying Cl− secretion, it is likely that down-regulation of absorptive pathways such as ENaC by CFTR is responsible for the decrease in sodium uptake. Inhibition of electroneutral NaCl absorption by cAMP is likely also critical in cholera pathogenesis. Because of extensive research over past few decades, CT has become one of the best-understood bacterial toxins. Details of the binding and entry of the toxin continue to be elucidated. CT has a unique mechanism for entry into the host cytosol. Although the ganglioside GM1 has been known to be the receptor for CT for many years, the absolute

1166 / CHAPTER 47 TABLE 47-2. Alteration of barrier function by various pathogens Organism/effector molecule

Vibrio cholera 1. RTX toxin 2. Hemagglutinin/protease 3. Zonula occludens toxin (Zot)

Escherichia coli Cytotoxic necrotizing factors (CNF1 and CNF2) Bacteroides fragilis Fragilysin

Clostridium difficile Toxins A and B (CDTA and CDTB)

Clostridium perfringens C. perfringens enterotoxin (CPE)

Clostridium botulinum C. botulinum toxin C3 Helicobacter pylori CagA Enteropathogenic E. coli (EPEC) EspF (other?)

Enterohemorrhagic E. coli (EHEC) EspF/other?

Shigella flexneri Unknown

Salmonella enterica Type III secreted proteins (SopE, SptP, other?)

Rotavirus Nonstructural protein 4 (NSP4), others?

Site/mechanism of action Cross-links actin Occludin degradation ZO-1 relocalization Mimics zonulin

Net result Cell rounding Loss of barrier function Loss of barrier function Loss of barrier function

Deamidation of glu61/63 of Rho family members, leading to constitutive activation

Disruption of MLC, occludin, ZO-1 and JAM-1; internalization of occludin; loss of barrier function

Extracellular protease Relocalization of ZO-1 Degradation of E-cadherin

Tight junction disruption Adherens junction disruption

Glucosylation and inactivation of Rho family proteins; actin stress fiber disorganization and disruption of actomyosin ring; Redistribution of ZO-1 and occludin

Loss of barrier function

Interacts with claudin-3 and -4 Forms a complex, recruits occludin Forms a membrane pore

Disruption of TJ strands, loss of barrier function

ADP-ribosylation of Rho blocks activation; loss of ZO-1 from junctions

Disruption of tight junction and adherens junction

Altered localization of JAM and ZO-1

Disruption of barrier function Ectopic TJ complexes

MLCK activation and MLC phosphorylation Occludin dephosphorylation Occludin/ZO-1 redistribution

Loss of barrier and fence function

Occludin dephosphorylation Occludin/ZO-1 redistribution

Loss of barrier function

Occludin dephosphorylation Occludin/ZO-1/claudin-1 redistribution β-Catenin phosphorylation and redistribution

Loss of barrier function Adherens junction disruption

Altered regulation of Rho family proteins Redistribution of ZO-1 and E-cadherin Actomyosin contraction

Loss of barrier and fence function

Redistribution of ZO-1, claudin-1, and occludin

Loss of barrier function

ADP, adenosine diphosphate; EspF, EPEC secreted protein F; JAM-1, junctional adhesion molecule-1; MLC, myosin light chain; MLCK, myosin light chain kinase; TJ, tight junction.

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1167 TABLE 47-3. Some inhibitors used in the study of ion transport Inhibitor Bumetanide Ouabain DIDS (4,4′-diisothiocyanostilbene-2,2′disulfonic acid) SITS (4-acetamido-4′-isothiocyanostilbene2,2′-disulfonate) Glibenclamide BAPTA-AM (1,2 bis (0-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxymethyl) ester) Dantrolene Brefeldin A Chloroquine Ammonium chloride H-7 (1-[5-isoquinolinesulphonyl]2-methylpiperazine) Staurosporine GGTI N 6-(1-iminoethyl)-lysine (L-NIL), N-nitro-L-arginine-ester (L-NAME) ODQ (1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one) Indomethacin α-β-methylene ADP 8-phenyltheophylline Zincov Nocodazole ML-9 Permeant inhibitor of MLCK (PIK) Calyculin A CP-96,345 CAPE (caffeic acid phenethyl ester) Dexamethasone

Site of action Na+-K+-2Cl− transporter (NKCC1) Na+-K+-ATPase Anion exchange inhibitor Anion exchange inhibitor Cystic fibrosis transmembrane receptor Intracellular calcium chelator Inhibits calcium release from sarcoplasmic reticulum Subset of Sec7-type GTP exchange factors (GEFs) disrupts protein trafficking Prevents endosome acidification Prevents endosome acidification Protein kinase C General protein kinase inhibitor Geranylgeranylation of small G-proteins Inducible nitric oxide synthetase (iNOS) Soluble guanylyl cyclase COX/PGE2 inhibitor Ecto-5′-nucleotidase (CD73) Adenosine receptor Zinc metalloprotease inhibitor Blocks microtubules formation Myosin light chain kinase (MLCK) MLCK (membrane-permeant) Protein phosphatase inhibitor Substance P NF-κB NF-κB

ADP, adenosine diphosphate; COX, cyclooxygenase; ER, endoplasmic reticulum; NF-κB, nuclear factor-κB; PGE2, prostaglandin E2.

requirement for binding to this protein was unknown until recently. GM1 is a glycolipid topped by a branched complex of five sugars including a single sialic acid residue. Each member of the pentameric B subunit ring is capable of binding to the specific carbohydrate moiety of GM1, which leads to an interaction of the five subunits with five separate ganglioside GM1 molecules (19). Surprisingly, the specific interaction of CT with GM1 appears to be critical to toxin internalization. Enterotoxigenic Escherichia coli (ETEC) produce a heat-labile enterotoxin known as LTIIb in which the A subunit functions identically to that of CT, whereas the B subunit preferentially binds to ganglioside GD1a. Chimeras containing the A subunit of CT and the binding subunits of LTIIb were not capable of promoting a secretory response in T84 intestinal epithelial cells despite the availability of GD1a on the surface (8). Reciprocally, LTIIb, which is normally nonfunctional in T84 cells, could be rendered active as a chimera containing the A subunit of LTIIb and the B subunits of CT (8). Ganglioside GM1 clusters within detergent-insoluble membrane microdomains of T84 cells, and this localization appears to be necessary for toxin internalization because β-methyl-cyclodextrin,

a cholesterol chelating agent, prevents CT-mediated Cl− secretion by blocking CT endocytosis (8,20). In human intestinal epithelial cells, lipid raft-mediated endocytosis appears to be the preferred method of entry; however, in other cell types where caveolins are more abundant, caveolae can be used for toxin entry (20–23). Research has shown that CT undergoes retrograde transport from the plasma membrane to the Golgi apparatus, which can be inhibited by brefeldin A (24–26). Reverse transit through the endoplasmic reticulum (ER) occurs using a specific sequence (KDEL) located in the C terminus of the A2 subunit (27,28). During the normal process of protein translation and movement outward to the Golgi, some proteins are targeted specifically for retention within the ER by a similar KDEL sequence (29). If ER-targeted proteins escape the ER and end up in the Golgi, they are returned to their proper ER location through interaction of the KDEL sequence with an integral membrane protein called ERD2, which shuttles between the ER and Golgi (30). Because CT possesses a KDEL sequence, it resembles a lost ER protein and is promptly escorted to the ER by ERD2 (28,31). Although the KDEL motif enhances the process of

1168 / CHAPTER 47 Cholera toxin

Lipid raft

Apical Vesicle

GM1

ERD2 PDI

Golgi

CFTR activation

PKA activation

ER Sec61

A1 Gs.GTP

Gs

NAD

Uncontrolled cAMP production

Gs.GTP ADPR

GTP Basolateral

Adenylate cyclase

FIG. 47-1. Trafficking of cholera toxin (CT) in a polarized epithelial cell. After attachment of the B subunits of CT to the GM1 ganglioside receptors, the holotoxin is trafficked in a retrograde manner via the Golgi to the endoplasmic reticulum (ER). Through the action of the enzyme, protein disulfide isomerase (PDI), the A1 peptide is released from the A2 peptide, and subsequently enters the cytosol in a Sec61-dependent manner. The A2 peptide ADP-ribosylates the Gs subunit of adenylate cyclase and locks it in the active state, resulting in an overproduction of cyclic adenosine monophosphate (cAMP). The ensuing protein kinase A (PKA)–dependent activation of cystic fibrosis transmembrane conductance regulator (CFTR) leads to copious chloride secretion. GTP, guanosine triphosphate. ERD2, endoplasmic reticulum retention defective 2; NAD, β-nicotinamide adenine dinucleotide; ADPR, adenosine diphosphate-ribose.

retrograde transport, it is not entirely necessary for the process because KDEL mutants of CT are still able to elicit some Cl− secretion (28). After arriving in the ER, CT is subject to the actions of protein disulfide isomerase (PDI), which reduces the disulfide bond found between the CT A1 and A2 subunits and assists in the unfolding of the toxin (32). Much in the way that the space shuttle discards spent booster rockets, the CT A1 subunit can now leave behind the B and A2 subunits because they have served their purpose of binding and transport into the ER. The lone A1 peptide now coopts yet another host system, this time one designed to assist improperly folded proteins traffic from the ER to the cytosol, so that they can reach the proteosome and be degraded. This system is referred to as ERAD, or ER-associated protein degradation. The ERAD system uses Sec61, which is normally thought of as an entry channel for newly synthesized proteins. Rather than allowing entry into the ER, the Sec61 complex in conjunction with ERAD allows for the secretion of the CT A1 peptide into the host cytosol (33). Arrival within the cytosol allows for the characteristic interaction with Gαs, production of cAMP, and CFTR-mediated secretion of Cl−. Although CT is well studied and plays an important role in altering host physiology during infection, a number

of other less understood toxins also appear capable of mediating a secretory response. Because toxins are procured by acquisition of foreign DNA through phages, pathogenicity islands, plasmids, and other mobile genetic elements, not all V. cholerae express the same set of toxins. In addition to CT, V. cholerae strains may possess accessory cholera endotoxin (Ace), V. cholerae cytolysin (VCC), or heat-stable enterotoxin (NAG-ST or ST), among others. Toxins such as zonula occludens toxin (Zot) that have secretory effects downstream of alterations in tight junctions are addressed under the heading Barrier Function and Cytotoxicity: Vibriochlerae (Zot). The toxins Ace, VCC, and the heat-stable enterotoxin have more direct effects on secretion either by altering host transport or by the direct formation of an ion channel, as in the case of VCC. Ace Ace was initially discovered as a result of attempts to make a live, attenuated vaccine to prevent cholera. A vaccine strain known as JBK70 was created by deleting the genes encoding both the A and B subunits of CT (34). Although this strain did not promote the severe diarrhea typically associated with cholera, many of the participants in the

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1169 study experienced mild diarrhea (34). This suggested that although CT may be the most potent factor in promoting diarrhea, other toxins exist within the parent El Tor strain (34). By exploring the cholera genome proximal to the CT gene ctx, referred to as the core region, two additional toxins, Ace and Zot, were identified (35,36). In a strain where the core region containing Ace, Zot, and CT was deleted, Ace activity was studied by complementing with an Ace-expressing plasmid (36). Strains expressing Ace were capable of both increasing short-circuit current (Isc), a measure of net ion movement, and fluid accumulation in a rabbit ileal loop model (36). In subsequent in vitro studies using cultured human colonic epithelial T84 cells, purified Ace was able to elicit a similar increase in Isc in a Ca2+ but not cAMP- or cGMP-dependent manner (37). The Ace-mediated increase in Isc could be prevented through the addition of bumetanide or ouabain, which inhibit the basally located NKCC1 and Na+,K+-ATPase, respectively (37). Chelation of Ca2+ with BAPTA-AM (1,2 bis[0-aminophenoxy] ethane-N, N,N′,N′-tetraacetic acid tetra [acetoxymethyl] ester) or addition of dantrolene, a Ca2+ antagonist, also inhibited the Isc response. Removal of Cl− or HCO3− from the Ringer’s solution also prevented an increase in Isc in response to Ace. Because the alterations in Isc were dependent on Cl− and HCO3−, involvement of a disodium 4,4′-diisothiocyanatostilbene-2, 2′-disulfonate (DIDS)–sensitive apical Cl− channel was suspected (37,38). DIDS caused a 50% reduction in Isc, suggesting that secretion is mediated by the subsequently identified DIDS-sensitive Ca2+-activated Cl− channel (CLCA). VCC VCC is a pore-forming toxin that is structurally related to α toxin and leukocidin F from Staphylococcus aureus (39). Although the S. aureus toxins are known to be heptamers, there is contradictory evidence regarding VCC. Although initial biochemical experiments using silver stain suggested that the VCC complex was a pentamer, subsequent electron microscopic studies suggest that the toxin forms a heptamer as seen for α toxin and leukocidin F (40,41). The VCC heptamer has been shown to be an anion-selective channel, partly because of its net positive charge (42). VCC is initially secreted by the bacteria in a procytolysin form that must be activated by a cellular protease. Studies suggest that the pro-VCC initially binds to the host cell membrane where it encounters ADAM-17, a cellular metalloproteinase that cleaves and thereby activates VCC, which can then form an oligomeric pore (43). In addition to alterations in ion permeability, VCC causes a dramatic vacuolation phenotype (44,45). The anion channel activity of VCC is required for vacuolation because DIDS and SITS (4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid [disodium salt]) inhibition of the channel prevents this phenotype and cell death (46). The enlarged vacuoles appear to be derived from a number of cellular compartments including late endosomes or lysosomes and the trans-Golgi network (TGN) based on positive immunostaining with rab7,

LAMP-1, and TGN46 antibodies (46). During the process of vacuole formation, VCC is internalized and associated with vacuolar membranes (46). Although CT is clearly the most important factor in producing cholera-related diarrhea, VCC has also been shown to increase fluid accumulation in the rabbit ileal loop model and in suckling mice (47). Heat-Stable Enterotoxin Heat-stable enterotoxin (NAG-ST or ST) is a 17-amino-acid peptide originally characterized in a nonagglutinable (NAG) strain of V. cholerae, so named because they are not reactive to anti-01 serovar antibodies (48–50). NAG-ST acts in a manner similar to the E. coli toxin heat-stable enterotoxin (STa). NAG-ST induces Ca2+ release from intracellular stores in response to inositol-1,4,5-trisphosphate (IP3), leading to the activation of PKC-α (51,52). The toxin also causes activation of guanylyl cyclase and the production of cGMP (53,54). Most of the work performed with heat-stable toxin has been done with E. coli STa or enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1), and because of its homology, NAG-ST is presumed to act in a similar manner. The E. coli toxins STa and EAST-1 are known to interact directly with membrane-located guanylyl cyclase C (GC-C), and phosphorylation of guanylyl cyclase by PKC-α enhances its activation and production of cGMP (55–57). GC-C receptors mutated to alanine at Ser1029 are no longer capable of an enhanced response to STa with the addition of PMA (phorbol 12-myristate 13-acetate) (56). The activation of PKC-α by NAG-ST and its homology to STa suggest that the two toxins act by the same mechanism, although a direct interaction of NAG-ST with guanylyl cyclase has not been demonstrated. Production of cGMP leads to activation of CFTR through either PKG-II or PKA-II, depending on cell type. Small intestine from PKG-II knockout mice showed reduced fluid accumulation in response to the guanylyl cyclase agonist STa, and Isc was reduced compared with control animals (58). Colon-derived cells such as T84 and Caco-2 also respond to STa through activation of PKA-II (59–61). Vibrio parahemolyticus Thermostable Direct Hemolysin and Thermostable Direct Hemolysin–Related Hemolysin Another member of the Vibrio family, V. parahemolyticus, is predominantly found in brackish coastal waters and causes only a limited number of human infections per year. These infections typically are acquired through the consumption of raw oysters, and during outbreaks, harvest in heavily contaminated oyster beds should be postponed until cooler waters, which are less favorable to V. parahemolyticus growth, arrive. Pathogenic V. parahemolyticus is characterized by production of thermostable direct hemolysin (TDH); however, it should be noted that less than 1% of environmental isolates actually contain the TDH toxin (62). In addition to TDH, V. parahemolyticus produces

1170 / CHAPTER 47 another toxin known as TDH-related hemolysin. TDHrelated hemolysin is similar to TDH, but is incapable of creating the characteristic β-hemolysis on Wagatsuma agar that typically is used for identifying pathogenic isolates of this species (63). TDH and TDH-related hemolysin appear to be structurally and functionally similar; therefore, the better studied TDH is used in this section as an example. There are several variants of the tdh gene that are associated with insertion elements within the V. parahemolyticus chromosome, although tdh2 appears to be primarily responsible for the classic hemolytic phenotype (64). The structure of TDH is not well defined, although the mature toxin functions as a dimer and contains an intramolecular disulfide bond between Cys151 and Cys161 (65). It has been shown to increase intracellular calcium levels and activate PKC, leading to a Cl−-mediated increase in Isc (66). The DIDS and Ca2+ sensitivity of the subsequent Cl− secretion suggest involvement of CLCA (66). Correspondingly, inhibition of CFTR with glibenclamide did not alter TDH-induced increases in Isc (66). Escherichia coli Heat-Stable Enterotoxin and Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 STa and EAST-1 are toxins structurally and functionally similar to the previously discussed NAG-ST of V. cholerae. EAST-1 is found in a broader range of E. coli than the name suggests, but because of its functional similarity and high homology to the better studied STa, only STa is summarized here. E. coli STa consists of either 18 or 19 amino acids, but only residues between cysteines 5 and 17 are critical for toxicity. Crystal structures of this portion suggest that the molecule adopts a hairpin-like structure, stabilized by three intramolecular disulfide bonds (67,68). The receptor for STa is GC-C, which is a single span membrane receptor found most abundantly in the intestine (69). The natural ligands for GC-C are the natriuretic peptides guanylin and uroguanylin, although the binding affinity of these native ligands is substantially lower than that of STa. GC-C−/− mice are protected from STa-mediated secretion in the suckling mouse model (70). Binding of STa to the GC-C receptor leads to cGMP production, which can be further enhanced by subsequent activation of PKC-α. Several studies have suggested that PKC-mediated phosphorylation of Ser1029 of GC-C enhances the effect of STa on cGMP production (55,56). Whereas these studies used PMA to activate PKCs, it was later found that STa itself activates PKC-α (57). Production of cGMP leads to activation of cGMP protein kinase II (PKG-II). PKG-II knockout mice demonstrate no increased fluid accumulation or Isc in response to STa (58). PKG-II is myristated, and therefore membrane associated, and directly phosphorylates CFTR (71). As is the case for CT, there is enhanced Cl− secretion through CFTR. In addition to acting through PKG-II, high levels of cGMP production can cause PKA-II activation leading to CFTR-mediated secretion (59,72). A number of other

pathogens possess heat-stable enterotoxins, including Yersinia enterolitica and Klebsiella pneumoniae, although their mechanisms of action have been less well studied. LTI and LTII In addition to the heat-stable enterotoxins mentioned earlier, ETEC have two heat-labile enterotoxins called LTI and LTII. LTI and LTII resemble CT in both structure and function. LTI and LTII are each composed of an enzymatic A subunit and a pentameric B ring that is responsible for binding to the host cell. The B ring of LTI, like CT, recognizes five separate molecules of ganglioside GM1. LTII is divided into two subtypes with slightly different binding specificities. LTIIa recognizes ganglioside GD1b, whereas LTIIb recognizes ganglioside GD1a (73). These gangliosides differ only slightly in the branched sugar chain attached to the lipid backbone; however, this is sufficient to convey specificity. For example, LTI and LTII ADP-ribosylate the Gαs subunit of adenylate cyclase to elicit cAMP production in much the same way as CT. However, although CT interactions with GM1 mediate cAMP production in T84 cells, there is no cAMP production in response to LTIIb in these cells. In contrast, only LTIIb stimulates cAMP production in mouse Y1 adrenal cells, thus demonstrating the specificity of ganglioside binding (8). As is the case for CT, cAMP activates PKA, which, in turn, phosphorylates CFTR, leading to secretion of Cl− ions. As is the case for CT, LTI and LTII undergo reverse trafficking through the TGN after receptor-mediated endocytosis. The ER-targeting sequence RDEL is present in LTI and LTIIa, whereas LTIIB shares the KDEL sequence with CT. Reversing the RDEL sequence to LEDR reduced cAMP production by 60% in Caco-2 cells (74). Although ER targeting enhances toxin activity, it is not entirely necessary because the toxin remains functional without the appropriate targeting sequence (74). The subsequent reduction of the disulfide bond linking the A1 and A2 peptides by the cellular enzyme PDI is better described for CT; however, it is reasonable to speculate that an equivalent process is responsible for the separation of the toxic A1 peptide from the A2 and B subunit binding and delivery portions. After being freed from the delivery portion of the molecule, the A1 subunit can be secreted by Sec61 into the cytosol where it ADP-ribosylates Gαs, leading to activation of adenylate cyclase, cAMP production, activation of PKA, phosphorylation of CFTR, and Cl− secretion. Helicobacter pylori VacA Although H. pylori typically colonizes the stomach and does not cause diarrheal disease, it is capable of altering cellular secretion. VacA is a vacuolating toxin similar to VCC from V. cholerae as a pore-forming toxin and in its ability to create large vacuoles within the host cell cytoplasm. Although these

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1171 vacuoles have a similar microscopic appearance and both appear to be derived from a late endosomal or early endosomal compartment, they differ in terms of vacuole acidity. VacA-mediated vacuoles are acidic, based on uptake of the weakly basic dye neutral red, and those of VCC are variably acidic or basic. VacA vacuoles stain positive for the late endosomal markers rab7 and H+-ATPase, in addition to the early lysosomal marker Lgp110 (75). Because host colonization dynamics preclude animal studies, work on H. pylori–mediated secretion has been performed using Ussing chambers. Purified VacA increased Isc of Caco-2 cells as early as 40 minutes after application, which was effectively blocked by removing Cl− from the Ringer’s solution, by chelation of Ca2+, or by blocking Cl− channels with NPPB (5-Nitro-2-[3-phenylpropylamino] benzoic acid) (76). This led to the conclusion that the Ca2+-dependent Cl− channel CLCA was involved in the secretion process; however, subsequent studies suggested that VacA, much like VCC, is actually an anion-specific channel capable of altering the permeability of Cl− and HCO3− ions (76,77). NPPB actually inhibits the VacA channel itself, much in the same way that DIDS is capable of blocking VCC activity (78). Ultrastructural analysis suggests that VacA creates a dodecameric pore composed of two stacked hexamers (79). Alterations in Isc occur before the damage associated with cell vacuolation; for VCC, inhibition of the anion-conductive channel prevents the vacuolation phenotype (78). Given that H. pylori colonizes the stomach and not the intestine, it is unclear how altered secretion proves advantageous to the bacterium, or if ion secretion is even altered in vivo. However, it has been suggested that increased bicarbonate secretion may act to help neutralize the low gastric pH in an area adjacent to the pathogen (80). Rotavirus Nonstructural Protein 4 Nonstructural protein 4 (NSP4) of rotavirus is the only widely recognized viral enterotoxin. As a nonstructural protein, NSP4 is not included within the virion particle, but instead requires active infection and protein synthesis, and is therefore consistent with the delayed onset of diarrhea in rotavirus infections. Because of its nonstructural nature and the fact that NSP4 has an ER retention site, it was initially unclear whether the protein could act in a manner consistent with extracellular application; however, studies have shown that a fragment of NSP4 (112–175) secreted by cells during infection is capable of inducing diarrhea in a 6- to 7-day-old mouse (81). NSP4 originally was found to produce diarrhea in neonatal mice during an unrelated attempt to produce antibodies against the NSP4 protein (82). Further characterization of this effect suggested that even a small 21-amino-acid peptide of NSP4 (114–135) is capable of inducing diarrhea in 6- to 7-day-old mice, but not in 18-day-old mice (82). Although the peptide nature of this toxin might suggest comparison with the heat-stable enterotoxins, it is believed to function in an entirely different manner. In the case of the

heat-stable toxins, cGMP is produced, resulting in the activation of CFTR. As a result, CFTR knockout mice are protected from heat-stable enterotoxins; however, NSP4 can still cause diarrhea even in the absence of CFTR (83). NSP4 has been shown to induce a transient increase in Isc believed to be mediated through a calcium-dependent pathway (82). In addition to these alterations in secretion, NSP4 has been found to inhibit sodium-dependent glucose uptake via SGLT1 (sodium-dependent glucose transporter 1) (84). Although the nature of rotavirus-mediated diarrhea is known to be multifactorial, alterations in secretion and absorption are believed to account for the early onset of diarrhea that occurs before disruption of the villous structure.

ABSORPTION: ENTEROPATHOGENIC ESCHERICHIA COLI In all of the cases mentioned so far, alteration of host intestinal ion transport has referred to an electrogenic, channel-mediated secretory response. Enteropathogenic E. coli (EPEC) does not possess any major enterotoxins and does not cause increased anion secretion in an Ussing chamber model (85). However, evidence suggests that electroneutral Na+ and Cl− exchange is altered. As early as 20 minutes after infection, EPEC increases net 5-(N-ethyl-N-isopropyl) amiloride (EIPA)–sensitive Na+ uptake (86). Whereas net uptake is increased, the activities of the three intestinal Na+-H+ exchangers (NHEs) are differentially regulated. The majority of the sodium uptake promoted by EPEC infection is mediated by NHE2, although NHE1 activity is also increased. The activity of NHE3, in contrast, is decreased. Although the increase in sodium uptake is expected to be antidiarrheal and may be compensatory in nature, the decrease in NHE3 activity may play a more important role in the host. NHE3 has been shown to be of primary importance in regulating fluid accumulation in mouse models (87). In addition to alterations in Na+ uptake, there is a similar but opposed decrease in Cl− uptake, suggesting an uncoupling of the apical Na+-H+ and Cl−-OH− exchange (88). More importantly, alterations in Cl− uptake are likely to promote the same effects as Cl− secretion. Because these effects are not toxin mediated, host colonization is likely to play an important role in fluid accumulation in an animal model. Rather than being mediated by a toxin, these effects are caused by the EPEC type III secretion system (TTSS), which allows for tight adherence of the bacteria to host cells and the formation of characteristic attaching and effacing lesions (86). The TTSS is a complex multiprotein system that forms a porelike channel allowing bacterial effector molecules to be translocated directly into the host cytoplasm. Now that a small animal model has been described, ion transport studies on mouse intestinal epithelia infected with deletion mutation strains for specific effector molecules can be performed and potentially will allow activities to be linked to individual effectors (89).

1172 / CHAPTER 47 INDIRECT EFFECTS ON ION SECRETION Clostridium difficile, Inflammation, and Neuropeptides C. difficile toxins promote fluid secretion indirectly through host inflammation. Toxins A and B have multiple effects on host cells. Both toxins A and B catalyze the addition of a glucose moiety to members of the small G-protein family, thus inactivating Rho, Rac, and CDC42. Several morphologic characteristics result from this inactivation (see discussion later in this chapter). In addition to the direct inactivation of small G proteins, a number of proinflammatory cascades are activated, although it has not been shown that glucosylation of Rho or other G proteins is required to initiate inflammation. In fact, inactivation of Rho is expected to prevent inflammation. In the case of the Clostridium botulinum toxin C3-transferase, which ADP-ribosylates Rho, activation of nuclear factor-κB (NF-κB) by bradykinin is blocked (90). Given that C3-transferase acts through inactivation of Rho and prevents activation of NF-κB, a proinflammatory mediator, C. difficile toxins A and B would be expected to similarly prevent inflammation. Toxin B of C. difficile has been shown to prevent c-Jun N-terminal kinase and activator protein-1 activation in response to Neisseria gonorrhoeae infection (91). Therefore, the inflammation caused by toxins A and B may be mediated through some function of the toxin that does not involve Rho. Consistent with a dual mode of action, p38 mitogen-activated protein kinase has been shown to be activated by toxin A within 1 to 2 minutes after exposure, whereas Rho glucosylation cannot be detected for at least 15 minutes (92). Inhibition of p38 with SB203580 completely blocked IL-8 production elicited by toxin A, but not by lipopolysaccharide (LPS). Thus, it would appear that production of the neutrophil attractant IL-8 occurs independently from Rho glucosylation. Neutrophil infiltration is necessary to induce the fluid secretion associated with clostridium toxins, and prevention of this inflammatory cascade by blocking p38 for 30 minutes before toxin A exposure reduced fluid secretion by 74% (92). A number of other studies have shown that various inflammatory mediators such as MIP-2 (macrophage-inflammatory protein-2) and interferon-γ are critical mediators of the host secretory response to toxin A or B, but it is unclear whether Rho-glucosylation is required to activate these inflammatory mediators (93,94). In addition to cytokine release and polymorphonuclear cell (PMN) influx, the enteric nervous system releases neuropeptides including substance P in response to infection, and these have been shown to play an important role in mediating the action of C. difficile toxin A. A chemical antagonist of the substance P receptor (neurokinin-1 [NK-1]) called CP-96,345 is able to block both fluid accumulation and [H3]-mannitol flux in a rat ileal loop model, whereas an inactive antagonist analog CP-96,344 does not (95). Substance P is known to initiate Cl− secretion by intestinal epithelial cells. After toxin A injection into rat ileal loops, neurokinin-1 receptor (NK-1R) messenger RNA (mRNA)

levels increased as assessed by Northern blot, in situ hybridization, and immunohistochemistry (IHC) (96). In a NK-1R−/− mouse ileal loop model, fluid secretion in response to toxin A injection was reduced from a fivefold increase from baseline for wild-type rats to a twofold increase (97). In addition, there is considerably less disruption of the mucosal architecture as assessed by IHC and lower TNF-α production (97). Host neuropeptides also play a role in increasing the Cl− secretion and associated fluid accumulation in other bacterial infections including Shigella flexneri and the opportunistic pathogen Cryptosporidium parvum (98,99).

CYCLOOXYGENASE-2/NITRIC OXIDE/ PROSTAGLANDIN E–MEDIATED Cl− SECRETION Two invasive pathogens, Salmonella dublin and enteroinvasive E. coli (EIAC), cause both an decrease in barrier function as measured by transepithelial resistance (TER) and an earlier increase in Isc (100). The decrease in barrier function and the increase in Isc can be blocked with N6-(1-iminoethyl)-lysine (L-NIL), an inhibitory nitric oxide synthase (iNOS) inhibitor (100). Authors have hypothesized that alterations in Isc may be caused by production of cGMP or cAMP as a result of nitric oxide (NO) production (100). EIAC and S. dublin elicited a cGMP and cAMP response, both of which were dependent on iNOS activity as shown by the loss of cyclic nucleotide accumulation after treatment with N-nitro-L-arginine-ester (L-NAME) (100). However, cAMP, but not cGMP, accumulation was dependent on COX-2 as well, and the iNOS inhibitors L-NIL and L-NAME reduced COX-2 expression levels, suggesting that cAMP production is attributable to COX-2, whereas iNOS is necessary for induction of cGMP production (100). Inhibition of guanylyl cyclase activity with ODQ returned Isc to baseline levels, whereas TER decline remained unaffected (100). In addition, peak Isc in response to S. dublin–conditioned media could be blocked with anti–prostaglandin E2 (PGE2) antibodies (101). These invasive pathogens also increased expression of CFTR and NKCC1, suggesting a multifactorial basis to the observed increase in Isc (100). An independent study showed S. typhimurium–mediated fluid accumulation in a porcine ileal loop model to be inhibited by indomethacin, a COX inhibitor (102).

Cryptdins The human intestine has numerous defense mechanisms to control pathogenic microorganisms. Among these are cryptdins, members of the α-defensin family of pore-forming antimicrobial peptides that are secreted from Paneth cells located in the crypts of the small intestine (103). Secretion of cryptdins occurs in response to both gram-positive and -negative bacteria and their components, including LPS

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1173 and lipoteichoic acid, among others (103). Given the limited bacterial colonization found in the healthy small intestine, a broad host response is reasonable. Although the primary function of cryptdins is antimicrobial activity, studies also implicate the cryptdins in induction of Cl− secretion (104). The six known cryptdins are short peptides (36–37 amino acids) capable of forming pores in bacterial membranes; however, a small subset alters ion permeability in eukaryotic cells (104). Cryptdins 2 and 3 were shown to increase Isc between 50 and 125 minutes after application (104). Cryptdin 3 caused an eightfold increase in Isc, whereas cryptdin 2 increased Isc by only threefold. This increase in Isc was Cl−-dependent and did not require cAMP or cGMP (104). A Cl− secretory response to cryptdin 2 or 3 leads to fluid efflux into the intestinal lumen, which could assist in removing nonadherent or loosely adherent bacteria from the small intestine.

Unstimulated PMN did not provoke this response, and direct neutrophil contact was not required, suggesting that a soluble factor secreted from activated PMN was responsible (107). In addition, the response was inhibitable with bumetanide and was dependent on Cl− (107,108). Initial characterization suggested that this substance was not sensitive to boiling or protease activity and was smaller than 500 Da (107). Later studies using high-performance liquid chromatography and collision-induced dissociation mass spectrometry identified this substance as 5′-AMP (109). 5′-AMP does not act directly as a secretagogue, but instead requires activation by an apical host cell protein CD73 (109). CD73 is a 5′-ectonucleotidase that converts 5′-AMP to adenosine, an active secretagogue (109). Inhibition of CD73 with α-β-methylene-ADP significantly impaired the ability of 5′-AMP, but not adenosine, to increase Isc (109). The adenosine receptor antagonist 8-phenyltheophylline completely inhibited the response to both 5′-AMP and adenosine (109).

Galanin-1 Receptor Galanin is a neuropeptide that promotes Cl− secretion upon binding to its receptor. Galanin-1 receptor (Gal1-R) levels are increased after infection with a wide variety of pathogens including EPEC, ETEC, enterohemorrhagic E. coli (EHEC), Salmonella, and Shigella (105,106). The increase in Gal1-R expression leads to a dramatic increase in the Isc response of T84 monolayers challenged with the neuropeptide galanin. A standard response of T84 monolayers to 1 µM galanin is a fivefold increase in Isc over a matter of minutes; however, monolayers infected with EHEC for 1 hour, cured with gentamicin, and then allowed to recover for 24 hours showed a 20-fold increase in Isc after exposure to the same concentration of ligand (105). The increase in Gal1-R is an NFκB–dependent transcriptional event activated after infection with enteric pathogens. For example, infection with pathogenic E. coli caused an eightfold increase in the amount of Gal1-R mRNA expressed, which could be blocked by the NF-κB inhibitor caffeic acid phenethyl ester or the antiinflammatory NF-κB inhibitor dexamethasone (105). The increase in Gal1-R expression and subsequent Isc response in mouse colonic epithelium was also effectively blocked with dexamethasone (105). Similar results were achieved with Salmonella, despite the twofold greater induction of Gal1-R in response compared with EHEC (106). In addition, GAL1R−/− mice, as well as heterozygotes, showed reduced fluid accumulation in mouse closed loop studies in response to Salmonella and Shigella infection, with or without the application of exogenous galanin-1 (106).

5′-Adenosine Monophosphate Early modeling of the interaction of PMNs with intestinal epithelial cells lead to the discovery that PMN activation with LPS, PMA, or FMLP (N-formyl methionyl leucyl phenylalanine) triggered an increase in Isc in T84 cells (107).

BARRIER FUNCTION AND CYTOTOXICITY Vibrio cholerae RTX V. cholerae produces a number of toxins that affect epithelial barrier function in addition to those known to alter secretion (Fig. 47-2). One of these is the RTX toxin, also referred to as VcRtxA or RtxA, which elicits cell rounding and a loss of barrier. This is mediated in part through cross-linking of actin monomers, with a characteristic laddering of actin seen by Western blot (110). The RTX toxin is unusually large at 484 kDa, although only the 48-kDa actin cross-linking domain (ACD; amino acids 1913–2441) is necessary to mediate actin polymerization (111). Toxins lacking the ACD promote an intermediate phenotype of cell rounding without actin polymerization. T84 monolayers show a marked reduction in resistance without a corresponding increase in Isc in response to RTX expressing V. cholerae, in contrast with RTX mutant control samples that did not decrease TER (112). These studies were performed in a strain background where the HA/P, Ace, Zot, and CT were deleted. In addition, there is a 6.6-fold increase in the permeability to 3000-Da fluorescein-conjugated dextran on infection with the toxinexpressing strain (112). Hemagglutinin/Protease Hemagglutinin/protease or HA/P cleaves a number of substrates including fibronectin, mucin, and lactoferrin, in addition to the nick site necessary for activation of CT (113). There is a 90% decrease in TER in T84 cells infected with a V. cholerae strain deleted for the core region of the CT-containing phage, which includes CT, Zot, Ace, and RtxA (Bah-3) (114). In contrast, an isogenic deletion mutant in hapA

1174 / CHAPTER 47 Zot

Vibrio cholerae

Zonulin receptor HA/P RTX

-1 ZO

ZO-2

Occludin

Actin crosslinking ZO-1

degradation

redistribution β

α γ

Catenins

E-cadherin

Actomyosin ring

FIG. 47-2. Vibrio cholerae produce three toxins that alter barrier function. The RTX protein acts by crossing/lining actin, whereas the hemagglutinin/protease (HA/P) degrades occludin and leads to ZO-1 relocalization. Zonula occludens toxin (Zot) decreases transepithelial resistance (TER) by mimicking an endogenous molecule known as zonulin.

was incapable of reducing resistance (114). Complementation of this mutant fully restored the ability to disrupt barrier function (114). This work represents molecular confirmation of the earlier work by Wu and colleagues (115), which showed that HA/P decreased TER in Madin–Darby canine kidney (MDCK) cells. The earlier study relied on the protease inhibitor Zincov, which inhibits HA/P, as well as FPLC analysis (115). A later study by the same group suggested that the loss of barrier function was caused by both degradation of occludin and relocation of ZO-1 (see Fig. 47-2) (116). HA/P is an extracellular toxin and degrades occludin only at the two extracellular domains, creating two antibodyreactive products of 35 and 50 kDa that differ from those seen with trypsin degradation (116). The degradation of occludin was prevented by the zinc metalloprotease inhibitor Zincov as well (116). Changes in occludin were preceded by the disappearance of ZO-1 from tight junctions; however, ZO-1 was not degraded (116).

cleaved to produce a membrane-bound 33-kDa protein with a 12-kDa peptide (118). The 12-kDa fraction is the active portion of the molecule for altering barrier function, whereas the remainder of the molecule participates in phage formation (118). The smaller active fragment is suspected to be the component originally identified in the smaller than 30-kDa fraction of V. cholerae supernatants (35,119). Zot functions by mimicking a natural host protein referred to as zonulin, which is believed to regulate barrier function (see Fig. 47-2) (120,121). The zonulin receptor is found predominantly in the small intestine, although the colonic cell line Caco-2 also expresses detectable levels (122). Zonulin is thought to play a role in preventing bacterial colonization in the small intestine by promoting fluid fluxes, which help to flush away bacteria (123). Somewhat paradoxically, the same group has found that commensal bacteria also elicit an increase in zonulin, which causes small-intestine barrier disruption (124).

Zot

Escherichia coli

Zot is unusual in that it is actually a structural component of the phage that carries genetic information encoding CT and other V. cholerae toxins. There has been some controversy as to the effects of Zot, because colonic cells such as T84 cells or isolated rabbit colon do not respond to Zot toxin, whereas rabbit ileum responds with a loss of TER and increased paracellular permeability (114,117). Zot is formed as a 44.8-kDa protein that is bound to the Vibrio membrane, and then

Cytotoxic Necrotizing Factors 1 and 2 Some strains of E. coli produce the toxins cytotoxic necrotizing factors 1 and 2 (CNF-1 and CNF-2), which deamidate the glutamine at position 61 or 63 of Rho family proteins (125–127). A glutamic acid at this position prevents GTP hydrolysis leading to a constitutively active form of Rho, Rac, or Cdc42 (125–127). Compared with the inactivation

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1175 of Rho seen with toxin B of C. difficile, activation of Rho with CNF-1 results in a more moderate decrease in TER and barrier function (128). At 4 hours there is a 40% reduction in TER in Caco-2 cells, or a 75% reduction in TER after 24 hours in T84 cells (128,129). In addition, there is a 2.9-fold increase in the permeability of Caco-2 cells to 11,000-Da fluorescein isothiocyanate conjugated (FITC)-dextran after 24 hours (128). Another group showed a 2-fold increase in permeability of 3000 Da FITC-dextran in T84 cells after 6 hours, which increased to a 4-fold increase after 48 hours (129). Treatment with CNF-1 produced a severe alteration in the architecture of the tight junction, with disruption of myosin light chain (MLC), occludin, ZO-1, and junctional adhesion molecule-1 (JAM-1) localization (129). In addition, CNF-1 treatment leads to the internalization of occludin by a number of pathways including caveolae, early endosomes, and recycling endosomes (129). The disruption of these proteins is consistent with the activity of constitutively active RhoA or Cdc42 as opposed to Rac1, which alters only the distribution of claudin1 and -2 (130). CNF-1 recognizes a currently unidentified cellular receptor and is internalized in a clathrin-independent, acid-sensitive process (131). The uptake of CNF-1 is not prevented by an Eps15 mutant, a dominant negative form of dynamin-2, or the SH3-containing fragment of intersectin, which disrupts intersectin–dynamin interaction, all of which effectively block clathrin-mediated endocytosis. In addition, chlorpromazine, which interferes with AP-2 and clathrin assembly, had no effect. Filipin, which blocks caveolin-mediated internalization, and amiloride, which blocks macropinocytosis, also had no effect. Therefore, the toxin is endocytosed by a clathrin/caveolin-independent pathway. After entry into an early endosome, the toxin does not traffic to the TGN or the ER like other toxins such as CT. Levels of brefeldin A that disrupt the Golgi apparatus do not impair the function of CNF-1. However, disruption of microtubules with nocodazole did inhibit CNF-1. Microtubules are necessary for the transition between early and late endosomes, suggesting that CNF-1 requires the transition to a late endosome, but not further progress toward the Golgi apparatus. In these late endosomes, CNF-1 is exposed to the acidic pH that is required for transport across the vesicle membrane. Interestingly, this process can be replicated at the level of the plasma membrane. The CNF-1 toxin could bind, but could not enter HEp-2 cells when exposed to low temperature (4°C) or bafilomycin A. After 4 hours of binding, cells were treated with media at various pH levels for 10 minutes in the presence of bafilomycin A, which disrupts normal trafficking of CNF-1. The temperature was then increased to 37°C to restore membrane fluidity, whereas bafilomycin A remained in the media. Under these conditions, cells treated with pH of 5.2 or less showed the effects of cytoplasmic toxin, whereas those treated with neutral pH showed no effect. Exposure to pH 5.2 allowed the toxin to cross the plasma membrane much in the way that it crossed the late endosomal membrane.

Serine Protease Autotransporters of Enterobacteriaceae There are a number of serine protease autotransporters, or SPATES, present among the different E. coli groups. Perhaps the best studied of these is the plasmid-encoded toxin (Pet) of enteroaggregative E. coli (EAEC). Pet is so named because of its location on the large adherence plasmid pAA2 of EAEC (132). Pet, like other members of the SPATE family, contains a 30-kDa C-terminal portion that forms a β-barrel in the bacterial outer membrane, allowing passage of the toxin into the culture media. Pet is internalized into the eukaryotic cell through a primary interaction with an as yet unidentified cellular receptor. The complete mechanism of intracellular trafficking has not been established; however, inhibitor studies suggest that Pet must traffic to the Golgi apparatus or TGN (133). Inhibition of vesicle acidification with chloroquine or NH4Cl does not block Pet function, suggesting that Pet, unlike CNF-1, does not translocate into the cytoplasm directly from late endosomes. Instead, Pet function can be inhibited with brefeldin A, which disrupts the Golgi apparatus and TGN. The requirement of Pet for the Golgi apparatus or TGN suggests a mechanism similar to CT; however, CT does require translocation through an acidic compartment and possesses a KDEL motif necessary for retrograde translocation that is not found in Pet. Therefore, it is currently unclear how Pet is trafficked through the Golgi. Pet has a number of effects on cells including an increase in Isc associated with fluid accumulation, and cytotoxic effects. In addition to cell rounding and disruption of focal contacts and actin stress fibers, Pet causes the cleavage of spectrin and fodrin, a form of spectrin found in non–red blood cells (134). In both cases, spectrin forms a supportive network just below the plasma membrane; in intestinal epithelial cells, this is most evident at the brush border. Pet caused the degradation of α and β spectrin or fodrin in red blood cell membranes or those of HEp-2 cells, respectively (134). Spectrin degradation yielded a 120-kDa N-terminal fragment similar to that produced during apoptosis. Immunofluorescence showed a severe disruption of fodrin in HEp-2 cells, together with enlargement of the cells caused by swelling. It is currently unclear whether cleavage of fodrin precedes other Petinduced cytotoxicity; however, other substrates for Pet have not been identified. The cytotoxicity associated with Pet is not reversed on removal of the toxin. Even a 10-minute toxin exposure followed by 5 hours of recovery in toxin-free media did not restore treated cells (135). Other types of E. coli also have members of the SPATE family. EspP is a plasmid-encoded SPATE found in EHEC (136). Unlike Pet, which is known for its cytotoxic effects, EspP has been characterized only for its proteolytic activities. EspP degrades pepsin A and factor V, which is involved in coagulation. The degradation of factor V is thought to promote the hemorrhagic phenotype of EHEC. ETEC contain a recently discovered SPATE called EatA. EatA is highly homologous (74% identical, 85% similar) to the SepA SPATE of Shigella and shows a similar specificity for the amino acid sequences AAPM and AAPL (137). In terms of

1176 / CHAPTER 47 pathophysiology, ∆eatA mutants show decreased fluid accumulation in a rabbit ileal loop model at 7 hours, although by 16 hours, ∆eatA mutant fluid accumulation is similar to wild-type ETEC. EspC, found in EPEC, is highly homologous to Pet of EAEC (55% identical, 70% similar) (138). Much like Pettreated cells, HEp-2 cells treated with EspC lose actin stress fibers and undergo fodrin cleavage, an increase in Isc, and cell rounding with membrane blebbing (138). However, there are subtle differences between the two proteins. EspC requires a greater dose and a longer incubation time, hours versus minutes, to elicit the same effects as Pet. In addition, the substrate cleavage is not identical; EspC cleaves fodrin in two sites as opposed to the single site cleavage seen with Pet. Shigella flexneri Serine Protease Autotransporters of Enterobacteriaceae Shigella have several SPATES including SigA, SepA, and Pic. Similar to the E. coli SPATES, those of Shigella are also serine proteases containing a C-terminal domain that forms a pore allowing the remainder of the toxin to translocate through the bacterial outer membrane. SigA is homologous to the EAEC protein Pet, and much like Pet, it is capable of causing severe cytotoxic effects. These include cell detachment associated with rounding and loss of focal adhesions and actin stress fibers (139). SigA mutants cause 30% less fluid accumulation than wild-type strains in a rabbit ileal loop model. Notably, this 30% decrease actually occurs in a strain that harbors another known enterotoxin, ShET1, suggesting that SigA plays a substantial role in fluid accumulation. Another SPATE, SepA, is the most abundant protein secreted into culture supernatant by S. flexneri (140). The proteolytic activity of SepA has been well characterized using artificial peptide substrates; however, the natural substrate for SepA during infection has not yet been identified. The preferred substrates include Phe-Leu-Phe and Val-Pro-Phe peptides, among others that are cleaved with varying degrees of success. Shigella also possesses another SPATE known as Pic (141). Pic has a limited ability to cleave mucin because only purified Pic, and not Picexpressing strains, was capable of clearing zones on agarose plates containing bovine or murine mucin. In addition, Pic appears to prevent activation of the classical complement pathway, although the nature of the affected component has not been determined. Bacteroides fragilis Bacteroides fragilis Toxin or Fragilysin Bacteroides fragilis exists both as a part of normal commensal flora and as pathogenic bacteria expressing a zinc metalloprotease called B. fragilis toxin (BFT) or fragilysin. Purified BFT causes a dose-dependent reduction in TER in HT-29 and MDCK cells with an associated increase in

mannitol flux (142). Treatment with a zinc chelator blocked 90% of the ability of the toxin to increase barrier permeability to mannitol (142). BFT also causes a pronounced loss of ZO-1 localization at the tight junctions by 4 hours, although effects can be seen as early as 2 hours after treatment with 1 µg of the toxin. It is currently unknown whether BFT enters the host cytosol; however, inhibitors of endosomal acidification, chloroquine and NH4Cl, did not block toxin effects, and all known effects are consistent with extracellular protease activity (142,143). In addition to altering tight junctions, BFT also disrupts the adherens junction (143). Purified E-cadherin is partially degraded by 5 nm BFT as quickly as 1 minute after addition (143). BFT produces 28- and 33-kDa fragments of E-cadherin that are further degraded by ATP-dependent cellular proteases, as shown by the use of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (143). The loss of E-cadherin in HT-29 cells can be witnessed by immunofluorescence as early as 30 minutes after BFT treatment, with total degradation apparent by 60 minutes after treatment. Unlike the zinc metalloprotease HA/P of V. cholerae, BFT is incapable of degrading occludin. Wu and colleagues (143) suggested that ZO-1 alterations may be secondary to the breakdown of the adherens junction because of loss of E-cadherin, and that ZO-1 is not itself degraded, but instead simply relocalized. Clostridium Clostridium difficile Toxins A and B C. difficile toxins A and B act to disrupt epithelial barrier function by glucosylating members of the small G-protein family including Rho, Rac, Cdc42 and Rap (Fig. 47-3) (144,145). Toxin A and B deactivate the small G proteins in contrast to the previously described phenotype for E. coli CNF-1 and -2; however, this inactivation produces a far more dramatic phenotype than does activation. Treatment with toxins A and B caused a more than 90% decrease in TER of T84 cells by 2 hours after treatment, although toxin B is more potent and could be used at a 80 ng/ml concentration as opposed to a 240 ng/ml concentration for toxin A (146). Together with this drop in TER, the permeability to [H3] mannitol increased by sixfold (146). Both toxins cause a marked disorganization of actin stress fibers as well as disruption of the apical actomyosin ring. The tight junction protein ZO-1 acquires a granular, almost jagged, appearance with more cytoplasmic staining visible, while ZO-2 is entirely lost from tight junctions. Occludin also dissociates from tight junctions while E-cadherin remains stable suggesting an intact adherens junction. The tight junction proteins ZO-1, ZO-2, and occludin are not degraded as is the case with some of the metalloprotease toxins, but are instead simply relocalized within the cell. The anchoring interaction of ZO-1 to the actin cytoskeleton is disrupted after treatment with toxin B as shown by a decreased ability to co-IP actin and ZO-1 (146). Sucrose density gradients showed that occludin and ZO-1 are lost from detergent-insoluble

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1177 Clostridium botulinum

Clostridium difficile

Clostridium perfringens

CPE

C3

Disruption of junctional strands

Toxin A and B

Pore formation

PKC

ZO

ZO

-1

Rho

ZO-1 redistribution

-1

Rho

Claudin 3 or 4 CPE

Occludin and ZO-1 redistribution

Occludin

Disruption of actomyosin ring

FIG. 47-3. A number of Clostridium species have negative effects on intestinal epithelial barrier function. The Clostridium botulinum toxin C3 adenosine diphosphate (ADP)-ribosylates Rho, preventing interaction with its guanine exchange factor (GEF), leading to ZO-1 relocalization and a reduction in transepithelial resistance (TER). Clostridium difficile toxins A and B glycosylate Rho, leading to its inactivation and subsequent disruption of actin and redistribution of occludin. A loss, but not degradation, of ZO-1 from the tight junction is dependent on protein kinase C (PKC) activation. Clostridium perfringens enterotoxin (CPE) interacts directly with claudin-3 and -4, leading to recruitment of occludin and subsequent pore formation. It is currently unclear whether claudin and occludin contribute directly to membrane pore formation; however, they are part of the complex leading to pore formation.

glycolipid rafts while E-cadherin remains unchanged, consistent with immunofluorescence data (146). Clostridium perfringens Enterotoxin Clostridium perfringens enterotoxin is a unique toxin with two complementary modes of activation. C. perfringens enterotoxin interacts with the tight junction proteins claudin-3 and -4, in addition to forming a pore in the host cell membrane (see Fig. 47-3) (147–149). The C. perfringens enterotoxin binding site in claudin-3 has been localized to the second extracellular loop (150). Subsequent analysis showed that claudin-6, -7, -8, and -14 also are capable of binding C. perfringens enterotoxin (150). C. perfringens enterotoxin initiates its action by binding to claudin-3 or -4 or a hypothetical 45- to 50-kDa receptor and begins formation of a small complex of 90 kDa (151). This small complex is thought to modulate the amphiphilicity of C. perfringens enterotoxin, allowing it to interact with the plasma membrane more directly (151). Although formation of the small complex allows the toxin to associate more closely with the cell membrane, small complex formation is insufficient to mediate pore formation (152). Normally, formation of a large complex of either 160 or 200 kDa occurs after small complex formation, but this process can be inhibited

by incubation at 4°C (152). Formation of the 200-kDa large complex involves the recruitment of occludin from tight junctions (153). Susceptibility to the toxin is highly correlated with the ability to form a small complex; however, occludin-deficient cells that form a small complex are not known; therefore, it cannot be determined whether occludin is necessary for formation of a membrane pore, or whether it is simply brought into the large complex through associations with other proteins (153). During the large complex formation phase, claudin-3 or -4 may actually interact to join the complex with an as yet unidentified 45- to 50-kDa receptor mediating the initial interaction. This scenario is supported by a study of claudin-4, then called C. perfringens enterotoxin-R, which was found in association with the large complex in L929 cells transfected with claudin-4 (149). Unfortunately, small complex formation was not addressed. However, large complex formation was present only in L cells expressing claudin-4, suggesting that it is required for pore formation (149). The pore-forming properties of this toxin are thought to be responsible for its enterotoxic effect; however, there is also an alteration in tight junctions. The recruitment of claudin-3 or -4 and occludin to the large complex suggests that alterations in junctions are tightly linked to changes in membrane permeability. Treatment of Caco-2 cells with

1178 / CHAPTER 47 1 µg/ml C. perfringens enterotoxin applied to the basal compartment for 60 minutes produced subtle changes in actin and occludin localization, whereas 120 minutes of treatment produced a marked loss of occludin and actin from areas just apical to the tight junction itself (154). The effects of C. perfringens enterotoxin at later time points were not determined because of extensive cell lifting at 120 minutes (154). In comparison, ZO-1 remained relatively unchanged throughout infection (154). Other studies show that claudin-4, but not claudin-1, was disrupted and degraded in MDCK cells after 4 hours of treatment with 2.5 µg/ml C. perfringens enterotoxin, although the loss was more apparent at 8 to 24 hours of treatment (155). These effects were reversible because claudin-4 was fully restored by 24 hours after the toxin was washed away (155). Claudin-3 was not assessed, because it does not appear to be expressed in MDCK cells. Basolateral application of C. perfringens enterotoxin caused an 80% decrease in TER, correlated with a 2-fold increase in permeability to 4000 and 10,000 Da FITC-dextran, but not to 40,000 Da FITC-dextran, suggesting that these changes were not caused by a gross disruption of the monolayer, but were instead caused by alterations in paracellular permeability (155). Freeze fracture images showed severe disruption of tight junction strands (155). Some C. perfringens isolates also alter the actin cytoskeleton through expression of iota toxin, which ADP-ribosylates actin on Arg-177, similar to the better described C2 toxin of C. botulinum (156). Clostridium botulinum Toxins C2 and C3 C2 C2 is a two-component toxin made up of two distinct protein subunits, C2I and C2II. The C2II (80-kDa) component acts to bind the cell and deliver the C2I (50-kDa) protein into the targeted cell. C2II binds to a carbohydrate receptor on the host cell, and after protease cleavage, oligomerization occurs, allowing the formation of a heptameric membrane channel (157,158). C2I is internalized through receptormediated endocytosis and requires acidification to exit the endosomal compartment (158). Much like CNF-1, C2 toxin can be persuaded to enter the plasma membrane directly when exposed to an extracellular pH of 5.4 (158). C2 acts by specifically ADP-ribosylating Arg 177 of G-actin, but not F-actin, resulting in disruption of actin filament polymerization both in vivo and in vitro (159–161). Injection of ADP-ribosylated actin in the absence of toxin produces a similar effect (162). C3 C3 toxin of C. botulinum specifically inactivates Rho through ADP-ribosylation of asparagine 41, while leaving the remaining small G proteins functional (163). The Rho protein itself is not rendered inactive because of this modification; instead, ADP-ribosylation prevents activation of Rho by its guanine exchange factor (GEF) (164,165). It does this by causing Rho to remain in the cytosolic compartment

through an interaction with guanine nucleotide dissociation inhibitor (GDI), which prohibits interaction with the membrane-associated GEF (165). CNF-1 activates G proteins in C3-treated cells by allowing binding of GTP, which promotes release from the GDI complex (165–167). The C3 toxin itself does not have a cell binding domain, thus microinjection or other permeabilization techniques often are used to mediate toxin entry. In other cases, the toxin is coupled to the binding domain of another toxin to mediate entry without permeabilization. Both the tight junction and adherens junction are altered by the Rho inactivation produced by C3 toxin (see Fig. 47-3). Treatment of keratinocytes with C3 during a calcium switch process prevents the formation of cadherin mediated cell–cell contacts (168). However, the toxin is also capable of dissociating E-cadherin from junctions that have been formed for 2 to 3 hours (168). Mature adherens junctions of intestinal epithelial cells (Caco-2 or T84), however, do not show alterations in E-cadherin localization after treatment with DC3B, a chimeric toxin composed of the cell binding portion of diphtheria toxin fused to C3 (169). Given that C3 does not have a cell binding domain, it remains unclear how or even if it contributes to the pathogenicity of C. botulinum. After treatment with the C3 toxin, intestinal epithelial cells showed a loss of ZO-1 from the tight junction by 24 hours after treatment; however, it was simply relocalized rather than degraded (169). TER decreased by more than 90% after treatment of cells with 0.5 µg/ml DC3B, and 0.05 µg/ml toxin decreased resistance by ~50% at 24 hours (169). In addition, permeability to 10,000 Da FITC-dextran increased notably with toxin doses greater than 0.5 µg/ml.

INFECTION-MEDIATED BARRIER CHANGES Helicobacter pylori CagA H. pylori inject a protein called CagA via a type IV secretion system. CagA is best known for inducing cellular elongation, also referred to as the hummingbird phenotype. However, studies have shown CagA has additional effects on the tight junctions of epithelial cells. CagA+ H. pylori alter the cellular location of JAM and ZO-1 (170). The ability of CagA to induce cell elongation requires tyrosine phosphorylation at the site EPIYA. A point mutant at this site, CagAEPISA, nevertheless remains competent to alter ZO-1 localization. Ruthenium red staining, together with transmission electron microscopy, suggested that CagA+ bacteria disrupted tight junctions within 3 days of infection. At 10 days of infection, biotinylated albumin could pass from the apical to basal reservoir, although this phenotype is partially attributed to VacA as well. Iodixanol gradients show a shift of ZO-1 and JAM, but not occludin, from the membrane to the soluble fractions, which happens to coincide with CagA localization. Overall, H. pylori CagA has a clear impact on barrier function; whether this is due

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1179

EPEC

ZO-1 disruption

Type III effectors

ZO-1

EspF/?

?

Calmodulin MLC

K P

? P ? Occludin dephosphorylation and redistribution

Myosin II

E-cadherin in

n ate

Myosin light chain phosphorylation

β-c

β-catenin dissociation

Aberrant strand localization

FIG. 47-4. Enteropathogenic E. coli (EPEC) produce a number of changes in an infected host cell. Myosin light chain is phosphorylated leading to contraction of the actomyosin ring. Occludin is dephosphorylated and internalized, as is ZO-1. Tight junction strands become aberrantly located in the lateral membrane. The reduction in TER depends on a functional type III secretion system, and the effector molecule known as EPEC secreted protein F (EspF) has been shown to play a major role in this process. The precise mechanism by which EspF perturbs tight junctions has not been identified. At the adherens junction, β-catenin becomes dissociated from E-cadherin. MLCK, myosin light chain kinase.

specifically to altered localization of ZO-1 or the more dramatic hummingbird phenotype of the cells is unclear. Escherichia coli Enteropathogenic Escherichia coli A number of laboratories have shown that EPEC promote a decline in TER and barrier function in intestinal epithelial cells (171–173). EPEC infection results in the loss of 40% to 60% of barrier function by 6 hours and a 5-fold increase in paracellular permeability. Several factors contribute to the diminution of barrier function (Fig. 47-4). The first of these is activation of myosin light chain kinase (MLCK), which phosphorylates MLC (173). Phosphorylated MLC induces contraction of the perijunctional actinomyosin ring, contributing to the net disruption of barrier function by EPEC (173). As such, EPEC-induced barrier dysfunction as measured by TER can be prevented by the use of an MLCK inhibitor such as ML-9 or a membrane-permeant peptide inhibitor of MLC kinase (PIK) (173,174). PIK mimics the inhibitory domain of MLCK specifically by interacting with the catalytic domain of MLCK (174). Physiologically, the interaction between the MLCK inhibitory domain and the catalytic site is disrupted by calmodulin, rendering

MLCK active. The PIK peptide (RKKYKYRRK) prevents 57% of the loss in barrier function associated with EPEC infection (174). Although MLCK phosphorylation of MLC clearly plays a role in the loss of barrier function, EPEC also induces other alterations in tight junction structure. Occludin is dephosphorylated as early as 30 minutes after EPEC infection and continues to be dephosphorylated for at least 3 hours (175). Immunofluorescence at 3 hours shows a punctate staining of junctional occludin with accumulation in an intracellular compartment (175). These effects, and the decrease in TER, can be prevented by the serine/threonine phosphatase inhibitor calyculin A or can be mimicked by the use of a nonpathogenic E. coli expressing the LEE (locus of enterocyte effacement) pathogenicity island on a plasmid (175). Likewise, a number of mutants that disrupt the TTSS fail to alter localization of occludin, suggesting that the TTSS and a secreted effector molecule are responsible for these alterations in tight junctions (175). In later studies, it was found that EPEC secreted protein F (EspF) mediates changes in TER, as well as occludin movement away from the junctions (176). An IPTG-inducible (Isopropyl β-D-1-thiogalactopyranoside-inducible) espF complement produced a progressive loss in TER (176). A loss in TER also has been associated with activation of ezrin through threonine phosphorylation (177). Dominant negative ezrin expressed in LLC-PK1 cells (porcine proximal

1180 / CHAPTER 47 tubule) prevented 60% of the TER loss seen with EPEC infection, which is comparable with that seen with the MLCK inhibitory peptide PIK (174,177). This dominant negative ezrin-expressing cell line also displayed reduced severity of ZO-1 mislocalization than would otherwise be expected with EPEC infection (177). In addition to alterations in the tight junction, reports indicate modification of the adherens junction in response to outer membrane preparations of EPEC (178). Staining of β-catenin, a cytoskeletal bridging molecule of E-cadherin, becomes diffuse and more cytoplasmic in response to either EPEC infection (3 hours) or bacterial outer membrane preparations (178). These changes appear to be mediated through the activation of PKCα and its increased interaction with E-cadherin as shown by coimmunoprecipitation (178). This phenotype was most dependent on the EAF plasmid, which encodes for bundle-forming pili, although an espB mutant showed a decrease in PKCα activity and β-catenin diffusion as well (178). Enterohemorrhagic Escherichia coli EHEC are closely related to EPEC and share similar trends for disruption of intestinal barrier function. Early studies showed that EHEC induce a 50% decrease in TER after 15 hours of infection (179). Infections with EHEC traditionally were performed overnight with low initial inoculums until it was determined that the bacteria could be made to express virulence factors much sooner by growing to log phase in serum-free cell culture media. In later studies using log phase cultures, the same 50% decline could be seen at 6 hours after infection (180). In side-by-side comparisons with EPEC, EHEC-mediated changes in barrier occurred more slowly and typically reduced TER only half as much as EPEC (180). After compensation for the unequal attachment of EPEC and EHEC, because of the lack of bundle-forming pili in EHEC, the disparities for the kinetics and magnitude of the decreased TER remained (180). Consistent with the decline in TER, there is a loss of barrier function, with a 4-fold increase in [H3]-mannitol flux after 18 hours of infection with low levels of stationary phase EHEC (181). Much like EPEC, EHEC disrupts ZO-1 localization and dephosphorylates and redistributes occludin (179,180). Also much like EPEC, these effects are due, at least in part, to the concerted action of calmodulin and MLCK activation, as well as through PKC activation (179). Considering that the proteins encoded within the LEE region of these two pathogens are highly homologous, the ability of EHEC EspF to reduce TER was assessed. Whereas EHEC espF is able to complement an EPEC ∆espF mutant, an espF mutant in EHEC is fully capable of diminishing barrier function (180). Interestingly, EHEC possess additional genes that are highly homologous to espF, and it was hypothesized that one of these genes could substitute for the LEE-encoded espF (180). One of these genes, designated U-espF for the cryptic prophage on which it resides, was also capable of complementing a ∆espF EPEC mutant for both occludin

disruption and decrease in TER (180). Although a double mutant was not tested, preventing the conclusion that EspF and U-EspF are the only factors responsible for barrier disruption in EHEC, both proteins are clearly sufficient to induce a decrease in TER and disruption of occludin as shown by complementation of the EPEC ∆espF mutant (180). Another group independently identified U-EspF and named it EspFU (182). EspFU was found to play a role in recruiting N-WASP (N-Wiskott-Aldrich syndrome protein) and actin to the EHEC pedestal through a direct interaction with Tir (182). This suggests that in EHEC, there is a relationship between the ability of the bacteria to form an actin pedestal and the subsequent disruption of barrier function. Whether this relationship is direct or indirect remains to be determined.

Shigella Shigella species are capable of intracellular invasion and cell-to-cell spread, the latter requiring E-cadherin (L-CAM) or N-cadherin (183). This originally was demonstrated using two L-cell lines, one of which expressed E-cadherin and another that did not. Although cadherin-transfected cells showed the greatest ability to promote cell-to-cell spread, cadherin-positive cells that were infected and allowed to interact with cadherin-negative cells exhibited a reduced rate of cell-to-cell spread but greater than was seen in cadherin-negative cells alone (183). This suggested that cadherin/cadherin binding was not necessary for cell-to-cell spread, but did enhance this process. In addition to using cadherins, a number of cytoskeletal components that typically support the adherens junction were found to be associated with traversing bacteria. These included α-actinin, vinculin, and α- and β-catenin (183). Later studies showed that IpaC, a type III secreted effector molecule, directly binds to β-catenin (184). Shigella infection leads to tyrosine phosphorylation of β-catenin, which causes alterations in E-cadherin association (184). By 6 hours of infection, dissociation of the E-cadherin complex from α- and β-catenin was evident (184). In addition to effects at the adherens junction, Shigella alter tight junction structure and function as well. ML-7, an MLCK inhibitor, prevented cell-to-cell spread of Shigella, but not another intracellular pathogen, Listeria monocytogenes; however, the activity of this kinase or the phosphorylation of MLC was not studied (185). In a later study, TER was shown to decrease by 70% with 90 minutes after infection with S. flexneri, whether an invasive or noninvasive strain was used (186). Invasive Shigella infection reduced the amount of Triton X-100–insoluble claudin-1 found in apically infected T84 monolayers as soon as 10 minutes after infection, with a return to control levels by 60 minutes (186). In contrast, noninvasive strains did not cause such a dramatic loss; however, levels of Triton X-100–insoluble claudin-1 were diminished at 10 to 30 minutes of infection (186). Neither strain provoked the same response when applied via the basal compartment. By 60 minutes of

PHYSIOLOGY OF HOST–PATHOGEN INTERACTIONS / 1181 infection, both invasive and noninvasive strains showed irregular staining of claudin-1. As is the case for EPEC and EHEC infection, Shigella infection results in dephosphorylation of occludin and loss from the tight junction with a corresponding appearance in cytoplasmic vesicles (186). Although ZO-1 was lost from Triton X-100–insoluble fractions, immunofluorescent staining of ZO-1 in infected cells was not altered compared with control cells. The distribution of ZO-2 and E-cadherin in Triton X-100–soluble and –insoluble fractions also was altered. Perhaps more dramatically, Shigella were visualized by immunofluorescence in the paracellular space between T84 cells, proximal to disruptions in ZO-1. These effects were reproducible among two different S. flexneri strains, although a direct comparison of strain 5 with the previously described serotype 2a showed a slightly reduced ability to reduce TER coupled with an inability to cause alterations in claudin-1 distribution. However, changes in occludin, ZO-1, ZO-2, and E-cadherin were similar (186).

Salmonella enterica S. enterica serovar Typhimurium infection results in a rapid loss of TER beginning as soon as 60 minutes after infection (187). At the same time, there is a marked accumulation of actin, ZO-1, and E-cadherin in a ringlike pattern below adherent bacteria, which tend to localize at the intersection points of the cells rather than across the cell surface (188). In addition to alterations in TER and junctional proteins, the fence function is lost as shown by labeling apical membrane with BODIPY-sphingomyelin and subsequent infection with Salmonella (187). Uninfected cells retain the BODIPY-sphingomyelin apically, whereas infected cells allow passage into lateral membranes (187). Infected cells become notably smaller and contorted compared with adjacent uninfected cells, suggesting contraction of the actinomyosin ring (187). This contraction can be inhibited by the kinase inhibitor staurosporine; however, this inhibitor does not prevent alterations in fence function or TER (187). Instead, staurosporine actually appears to enhance the ability of the bacteria to alter BODIPY migration to the lateral membrane, as well as to reduce TER and to increase inulin fluxes (187). Salmonella produces at least two type III secreted effector molecules, SopE and SptP, that alter regulation of members of the small G-protein family (189,190). It has been hypothesized that small G proteins such as Rac and Rho may contribute to the alterations in epithelial tight junction properties seen with Salmonella infection (187,191). Inhibition of the decrease in TER can be brought about by use of GGTI. This drug inhibits geranylgeranylation of the small G proteins, which is necessary for appropriate membrane localization of active proteins (191). Although this suggests that the small G proteins are involved in barrier alterations, GGTI (N-4-[2(R)-Amino-3mercaptopropyl] amino-2-napthylbenzoyl-(L)-Leucine methyl ester, TFA) also blocks invasion by the strain of Salmonella that was studied (SL1344) (191). The authors suggest that the

50% decrease in invasion is not responsible for the protection seen for TER because lower multiplicity of infections (MOIs), more equivalent to the 50% invasion level, remained effective in reducing TER (191).

Rotavirus NSP4 is the only known viral enterotoxin, and in addition to altering secretion, it has effects on proteins regulating the tight junctions, as well as paracellular permeability (192). The TER of MDCK cells is reduced by 75% after apical, but not basolateral, application of NSP4 (192). NSP4 also increased paracellular permeability to FITC-dextran by fivefold at 46 hours. Toxin treatment was reversible because cells washed after 50 hours of toxin exposure recovered approximately 70% of basal barrier function within 48 hours. In addition, TER development was blocked by NSP4, as was the early recruitment of ZO-1 into the lateral membrane of recently seeded cells (192). However, treatment of 3-day-old cells exhibiting mature junctions with NSP4 did not alter ZO-1 localization compared with control cells treated with the irrelevant viral protein, Vp6 (192). Apical actin levels, in contrast, were subtly decreased after 24 hours of NSP4 treatment. Although these effects were attributed specifically to the enterotoxin NSP4, additional effects on junctions were seen but not characterized for the viral protein responsible. By 10 hours after infection with a rotavirus MOI of 10, claudin-1 shows severe disruption and redistribution to a vesicle-like population located within the cytoplasm (193). Breaks and loss of occludin occurred as early as 10 hours after infection, with more severe disruption by 18 hours (193). Disruption of ZO-1 occurred only when cells were seeded in the presence of virus, in much the same way that NSP4 effectively prevented ZO-1 accumulation without being capable of disrupting preformed junctions containing ZO-1 (192,193).

Junctions as Pathogen Receptors A number of pathogens use proteins from the tight junctions and adherens junctions as receptors. Alternatively, they may use molecules such as integrins that typically are restricted to the basolateral surface. Access to these molecules therefore requires disruption of the fence function of tight junctions, resulting in altered cell polarity. As described previously, claudin-3 and -4 act as a receptor for C. perfringens enterotoxin, and the cell-to-cell spread of Shigella requires E-cadherin (148,149,183). However, some pathogens use junctional molecules for entry. For example, Listeria monocytogenes requires E-cadherin binding to the bacterial surface protein internalin to mediate intracellular invasion, although it is unclear how L. monocytogenes accesses the adherens junction (194). The coxsackie and adenovirus receptor is, as the name suggests, a receptor for coxsackie and adenovirus, and JAM acts as a receptor for reovirus (195,196). This section discusses a few interesting examples

1182 / CHAPTER 47 of the altered polarity associated with access of pathogens to junctional/basolateral receptors in the intestine. Yersinia Yersinia pseudotuberculosis uses a type III secreted effector molecule, YopE, to alter barrier function and promote binding of the bacterial surface protein invasin to β1 integrins (197). Although experiments were not directly performed on Yersinia enterocolitica, it does contain an equivalent plasmid that expresses the Yop/Ysc system. Much like S. enterica, Yersinia adhere predominantly at cell–cell contacts, apparently to low levels of β1 integrins, which are naturally exposed at these apical cell–cell contact regions in MDCK cells (188,197). After 2 hours of infection, greater quantities of β1 integrin are apparent at these apical cell–cell contact regions (197). These changes in fence function occur at the time (2 hours) of the earliest detectable increased permeability to 20 kDa FITC-dextran (197). Permeability to 40 kDa FITC-dextran occurs at 3 hours, whereas permeability to 70 kDa FITC-dextran requires 4 hours. These effects are produced by wild-type Yersinia, but lost in a ∆yopE mutant. YopE is one of the effector molecules secreted via the TTSS. In concert with these changes in barrier and fence function, there are subtle changes in occludin and ZO-1 localization within the lateral membrane (197). By reducing the fence function of MDCK cells, Yersinia are capable of exposing more of their receptor β1 integrin at apical cell–cell junctions (197). Alternatively, recruitment of PMN by FMLP has been shown to cause microdiscontinuities in intestinal epithelia with apical migration of β1 integrin, which also promotes subsequent Yersinia invasion (198). Enteropathogenic Escherichia coli Much like Yersinia, EPEC alter the fence function of tight junctions allowing basolateral proteins to be exposed on the apical surface of the cell (199). This process is presumed to be nonspecific because both the relevant marker, β1 integrin, and the irrelevant basal marker, Na+,K+-ATPase, progressively migrate toward the apical surface throughout the course of an infection as shown by surface biotinylation (199). The interaction of EPEC with β1 integrin is necessary for a complete decrease in resistance compared with controls at both 4 and 6 hours, as shown by the use of a blocking antibody against β1 integrin compared with an irrelevant IgG control. Because of the requirement for the Tir protein in tight adherence of bacteria, a ∆tir mutant is incapable of reducing TER or affecting fence function for β1 migration to the apical surface. However, by EDTA switching the cells and allowing subsequent recovery of TER, the ∆tir mutant becomes competent to reduce resistance in a β1-dependent manner. The EDTA switch allows β1 integrin to distribute evenly throughout the cell membrane, therefore bypassing the need for Tir in inducing alterations in fence function. Apical β1 integrin is presumed

to interact with intimin, which has been described previously as having integrin binding activity (200). An intimin mutant is incapable of reducing TER even after EDTA switch and redistribution of β1 integrin (199). However, the reduction in TER does not appear to be directly caused by intimin/ integrin-mediated signaling, and instead appears to rely on another type III secreted effector, presumably EspF (199). An EPEC ∆espD mutant is defective for type III secretion, but not for intimin expression, yet cannot reduce TER after an EDTA switch (199). Given that β1 integrin also was required for maximal reduction in TER induced by wildtype EPEC, this suggests that at least some portion of wildtype EPEC adherence is mediated by integrins. Therefore, EPEC appear to alter cellular fence function to mediate better cellular attachment, which leads to more effective secretion of type III effector molecules.

SUMMARY The studies described in this chapter lie squarely at the intersection between microbial pathogenesis and intestinal physiology, with both fields benefitting from the interaction. In the case of CT, we have learned that the standard host protein sorting pathway can be reversed to allow for toxin entry into the cytoplasm. This not only helps us better understand protein sorting, but also provides a greater insight into toxin action, which no doubt will assist in creating better treatments specific to CT. Much of our current understanding of intestinal ion transport emerged as a direct result of investigations focused on defining the mechanisms of action of various microbial toxins. Likewise, a number of toxins from Clostridium species have been used as tools to analyze alterations in barrier function. In some cases microbial toxins, such as the heat-stable enterotoxins that are homologs of the host protein guanylin, or Zot that mimics zonulin, prompted the discovery of host systems for transport and barrier regulation. In summary, the intersection of these two fields provides insight into the nature of pathogenmediated disease, as well as normal host physiology, thus allowing advances that might not occur otherwise.

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CHAPTER

48

The Cell Biology of Gastric Acid Secretion Curtis Okamoto, Serhan Karvar, and John G. Forte Morphologic Basis of Hydrochloric Acid Secretion, 1189 Gastric Epithelial Organization, 1189 Parietal Cell Morphology and Ultrastructure, 1191 Membrane Recycling Hypothesis, 1193 Cellular Basis of Hydrochloric Acid Secretion, 1194 H,K-ATPase: The Primary Gastric Proton Pump, 1194 Apical Channel Proteins Directly Supporting Hydrochloric Acid Secretion, 1202 Transport at the Basolateral Membrane, 1203 Homeostatic Requirements, 1203 Na-H and Cl-HCO3 Exchange Pathways, 1203 Na-K-2Cl Cotransport, 1205 Conductance Properties, 1205

Getting the Message to the Parietal Cell, 1205 Methods to Disperse and Isolate Gastric Glands and Cells, 1205 Signal Transduction Underlying Gastric Acid Secretion, 1206 Supporting Membrane Transformations: The Vesicular Trafficking Machinery, 1209 Role of the Cytoskeleton, 1210 Vesicle Trafficking and Protein Sorting Machinery, 1211 References, 1216

The maximally stimulated stomach secretes a fluid of nearly isotonic hydrochloric acid (HCl), that is, 150 mM. The gastric epithelial cell responsible for that secretion is generically known as the oxyntic cell (meaning acid-secreting cell); however, in the mammal, the cell is commonly called the parietal cell because of its histologic location protruding out from the wall of the gastric gland. Secretion of gastric acid is tightly regulated by ligand–receptor binding to effect massive changes in the cytologic structure and metabolism of the parietal cell with the ultimate output of H+, Cl−, and H2O across the apical membrane. This chapter examines the structural and functional basis of HCl secretion. Primary emphasis is directed toward the cell biological mechanisms that operate to transform the parietal cell from a resting, nonsecreting state to its active secretory form.

MORPHOLOGIC BASIS OF HYDROCHLORIC ACID SECRETION

C. Okamoto: Department of Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90033. S. Karvar and J. G. Forte: Department of Molecular and Cell Biology, University of California, Berkeley, California 94720. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Gastric Epithelial Organization The stomach is organized in several concentric tissue layers consisting of: (1) the mucosa, lining the lumen of the stomach; (2) a thin layer of smooth muscle called the muscularis mucosae; (3) the submucosa consisting of connective tissue and blood vessels; (4) the tunica muscularis, composed of several layers of smooth muscle; and (5) the serosal, facing the peritoneal cavity (Fig. 48-1). Gastric digestive juice is secreted by the mucosa as the composite secretion of several types of epithelial cells that make up the densely packed tubular gastric glands. The luminal surface of the mucosa is covered by a layer of columnar epithelial cells, the surface mucous cells, that secrete mucus and an alkaline fluid, which has been proposed to play a role in protecting the surface from direct acid exposure (1,2). The luminal surface is studded with numerous invaginations, or pits, that serve as conduits for secretions from the subadjacent gastric glands. In the mammal, gastric glands are composed of three principal epithelial cell types: chief cells, mucous neck cells, and parietal cells. Chief cells are the principal source of pepsinogen,

1189

1190 / CHAPTER 48 Esophagus

Fundus

Rugae

Cardia

Corpus

Pylorus

Surface mucous cells Duodenum Pyloric antrum

A

Mucous neck cells

Parietal cells Mucosa

Gastric glands

Muscularis mucosae

Endocrine cell

Submucosa

Chief cells Tunica muscularis Serosa

B

C

FIG. 48-1. Stomach and gastric glands. (A) Specific regions of the stomach are indicated. Parietal cell density and acid secretion generally predominate in the corpus, but there are variations among species. (B) A section through the gastric wall indicating gastric glands in the mucosa and the various layers of tissue. (C) Sketch of a gastric gland showing the component cell types of the gastric epithelium. Parietal cells are shown with their basal surfaces projecting from the wall of the gland and extensive canaliculi at their apical surface. Several endocrine, or paracrine, cells also are indicated. (Reproduced from Forte JG. In: Greger R, Windhorst U, eds. Comprehensive human physiology. New York: Springer, 1996;1239–1258, by permission.)

the precursor form of pepsin, and are more abundant toward the base of the glands. Mucous neck cells are small cells located in the region of transition at the base of the pits extending throughout the neck of the glands. Mucous neck cells secrete a mucous glycoprotein that is distinct from that of surface epithelial cells, and they also secrete some pepsinogen. The large acid-secreting parietal cells are found throughout the length of the gland, but tend to be more abundant in the neck region of the gland. Because of their large size and relative abundance, parietal cells represent about 50% to 60% of the mass of the secretory mucosa. Subadjacent to the glandular epithelial cells there are a number of endocrine and paracrine cells that play important regulatory roles in gastric secretory function. In contrast with the cellular specialization of mammals, gastric glands of lower vertebrates (birds, reptiles, amphibians, and fish) have a single epithelial cell that secretes both acid and pepsin, called the oxyntopeptic cell. Gastric epithelial cells are in a constant state of turnover. A population of small pluripotent stem cells located in the

upper neck region of the glands represents the progenitor cells for all gastric epithelial cell types (see Chapter 11). Stem cells that differentiate and migrate up the pit and over the luminal surface cause surface epithelial cells. The same stem cells are the proliferative source of glandular cells, which migrate down the length of the gland. Parietal cells and mucous neck cells differentiate directly from progenitor stem cells, whereas chief cells are derived by further differentiation of mucous neck cells toward the base of the gland. Each of the gastric epithelial cells has a distinct turnover, or lifetime. Studies of 3H-thymidine incorporation into cells of the mouse stomach provide an estimate of average cell turnover times (3,4). Because of their direct luminal exposure, surface epithelial cells have a relatively short life of about 3 days. Mucous neck cells spend about 40 days in the neck region of the gland, and as they migrate to the base, they transform into chief cells that have an average life of about 250 days. Parietal cells have an average turnover time of 71 days. Newly differentiated parietal cells progressively migrate from the neck toward the base of the gland.

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1191 Morphologic and histochemical criteria suggest that functional differences may be associated with age of the parietal cell: The younger, more luminally located cells presumably are more active in HCl secretion than the more basal cells (5).

Parietal Cell Morphology and Ultrastructure The parietal cell has several distinctive morphologic features that are of fundamental significance to function (6–8). The cell is generally pyramidal in shape, with tight junctions surrounding the apex of the pyramid. In the resting, or nonsecreting, cell, there is limited area of apical membrane in direct apposition to the gland lumen, but more extensive luminal contact is provided by distinctive invaginations deep into and throughout the cell, forming a system of tiny canals or secretory canaliculi (Fig. 48-2). The entire apical surface,

including the canaliculi, is lined by short stubby microvilli. Cross section shows an abundance of tubular and vesicular membrane profiles especially prominent in the apical pole of parietal cells, the system of so-called tubulovesicles. Three-dimensional reconstruction indicates that many of the tubulovesicles are, in fact, flattened and more cisternal in shape (9). The H,K-ATPase, which is the primary gastric proton pump, is clearly the most abundant protein in this enormous pool of tubulovesicular membranes. Numerous large mitochondria occupying 30% to 40% of the cellular mass are also characteristic of the parietal cell, which is consistent with the energy requirements and high oxidative capacity of these cells. The most remarkable aspect of parietal cell morphology is its ability to undergo massive morphologic rearrangement associated with stimulation. Although limited by the optics of light microscopy, careful histologic examination by Golgi

L

C

C

FIG. 48-2. Electron micrograph of a parietal cell from a piglet gastric mucosa in the resting, nonsecreting state. The glandular lumen (L) is clearly visible, as are the secretory canaliculi (C) that project from the lumen deep into the cell, constituting the apical surface. The apical surface is covered with numerous short microvilli. Numerous tubular and vesicular membrane profiles (called tubulovesicles) abound in the apical region of the cells. Many mitochondria also are apparent. Scale bar = 1 µm. (Modified from Forte JG, Forte TM, Black JA, Okamoto C, Wolosin JM. Correlation of parietal cell structure and function. J Clin Gastroenterol 1983;5(suppl 1):17–27, by permission.)

1192 / CHAPTER 48 in the late nineteenth century noted the enlargement of canaliculi that occurred in secreting parietal cells (10). Modern electron microscopy has shown that within a few minutes of adding stimulants there are obvious changes in the apical plasma membrane lining the canaliculi (8,11–13). Microvilli grow longer and more elaborate, and the quantity of tubulovesicles in the cytoplasm decreases. The process of apical membrane expansion continues until the steady state of secretory activity is achieved (Fig. 48-3). The maximally stimulated oxyntic cell has an apical membrane surface area that is 5 to 10 times that of the resting cell. Morphometric studies show that membrane surface area is conserved; the large apical plasma membrane expansion is quantitatively matched by the disappearance and decrease in the surface area of cytoplasmic tubulovesicles (11–15). The technique of freeze-fracture microscopy offers an alternative view of parietal cell ultrastructure (14). Here cells are frozen, then cracked open, and a replica of the surface of the fractures is made and examined with an electron microscope. Figure 48-4 provides an image of the fracture face of

a secretagogue-stimulated parietal cell, showing the elaborate microvillar extensions at the apical surface, as well as some remnant tubulovesicles in the cytoplasm. When frozen cells are cracked, one of the most favorable fracture planes is right down the center of cell membranes. The small particles one sees on these membrane faces indicate incumbent membrane proteins; the more particles, the more dense the protein packing. In Figure 48-4, PF refers to the “protoplasmic” face of the particular membrane, and EF refers to the exterior face of that membrane. For the cytoplasmic tubulovesicles, the concave PF is packed full of particles, which represent H,KATPase (see later discussion), whereas the convex EF is relatively particle free. The distribution of particles on the PF and the EF of the apical plasma membrane are the same, except that the PF tends to be convex and the EF is concave because of the eversion of the membrane after tubulovesicle fusion. Interestingly, there are several regions where there are apparent points of fusion between the apical surface and underlying vesicular structures (see Fig. 48-4, arrowheads), as well as an indication of fusion-induced connectivity

FIG. 48-3. Electron micrograph of parietal cell from maximally secreting piglet stomach (histaminestimulated). Apical microvilli within the expanded canaliculi are greatly elongated, whereas tubulovesicles are drastically reduced (cf. Fig. 48-2). The expansion of apical surface appears to be constituted by the tubulovesicular membrane compartment. Scale bar = 1 µm. (Modified from Forte JG, Forte TM, Black JA, Okamoto C, Wolosin JM. Correlation of parietal cell structure and function. J Clin Gastroenterol 1983;5(suppl 1):17–27, by permission.)

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1193

FIG. 48-4. Electron micrograph of a freeze-fracture replica from the canalicular region of parietal cells from piglet mucosa stimulated to secrete acid. Apical microvilli projecting into the gland lumen can be toward the left of the image. There are many tubulovesicles to the right. The fracture planes through the membranes reveal an asymmetry of the membrane-associated particles, which are intrinsic membrane proteins and primarily consist of H,K-ATPase in the case of parietal cells. PF refers to the “protoplasmic” face of the membrane; EF refers to the exterior face of the membrane. The PF is heavily laden with membrane-associated particles for both tubulovesicles and apical microvillar surfaces, whereas the EF in relatively devoid of membrane-associated particles. There are several regions where there are apparent points of fusion between the apical surface and underlying vesicular structures (arrowheads), as well as an indication of fusion-induced connectivity between tubulovesicles (arrows). The inset represents a freeze-fracture image of tubulovesicles that have been isolated from parietal cells and used for H,K-ATPase and proton transport assays. Particle distribution is much the same as for the intact membranes. Scale bar = 0.5 µm. (Modified from Forte JG, Forte TM, Black JA, Okamoto C, Wolosin JM. Correlation of parietal cell structure and function. J Clin Gastroenterol 1983;5(suppl 1):17–27, by permission.)

between tubulovesicles (see Fig. 48-4, arrows). The inset in Figure 48-4 is a freeze-fracture image of tubulovesicles that have been isolated from parietal cells. Particle distribution is about the same as for the intact membranes. Parietal cells retain this “stimulated” morphology and sustain HCl secretion as long as the stimulus is applied and metabolic competence is maintained. Figure 48-5 depicts the general morphologic change in going from rest to maximal secretion. Withdrawal of the secretagogue leads to a collapse of the canalicular spaces, followed by a complex process of reincorporation of the apical surface back into tubulovesicular forms of the resting cell (8,16). The structural remodeling of the recovered cell has been documented, but little is known about the forces and specific membrane resequestration pathways that are involved in the massive recovery.

Membrane Recycling Hypothesis The membrane recycling hypothesis has been proposed as the basis to link the observed morphologic transformations to the functional output of the hydrochloric acid (8,16). In the resting parietal cell, H,K-ATPase is retained in the quiescent state in cytoplasmic tubulovesicles. This has been shown in biochemical analysis of isolated membranes and in intact cells by immunodetection of H,K-ATPase (16–19). Stimulation initiates a series of intracellular activating events that promote the translocation of tubulovesicle membranes through the deployment of recruitment/fusionbased proteins, ultimately expanding the apical canalicular surface with H,K-ATPase–rich membrane and resulting in the secretion of voluminous, isotonic HCl (16,19,20). This chapter

1194 / CHAPTER 48

FIG. 48-5. Stylized representation of parietal cells undergoing the secretory cycle. The drawing summarizes the major membrane transformations at the apical canalicular surface. The cell transitions from the resting state to the maximally secreting state with stimulation, and then back to the resting by recycling of the expanded apical membrane. (Modified from Forte and colleagues [16], by permission.)

examines the experimental evidence to support this membrane recycling model, which also has been called the recruitmentrecycling model of parietal cell activation.

CELLULAR BASIS OF HYDROCHLORIC ACID SECRETION H,K-ATPase: The Primary Gastric Proton Pump Identification of H,K-ATPase as the Gastric Proton Pump Skou’s (21) discovery of a distinctive biochemical entity as the Na+ pump (Na+,K+-ATPase) was a seminal discovery that ignited the search for homologous enzymes to power other transport systems. The biochemical nature of the gastric proton pump was first uncovered through a series of studies that identified unique K+-stimulated ATPase and phosphatase activities in microsomal vesicles that were purified from homogenates of gastric mucosa (22–26). The microsomal

vesicles are, in fact, parietal cell tubulovesicles that are purified on the basis of size and density. In freshly isolated microsomal vesicles from a nonsecreting stomach, K+stimulated ATPase activity also requires K+ ionophores for maximal stimulation; thus, it was concluded that the isolated tubulovesicles have limited K+ permeability and an internal K+ activating site (27,28). The K+-stimulated ATPase was linked to HCl secretion on the basis of its location in the acid-secreting region of all vertebrate stomachs (27) and its coincident expression with the functional onset of HCl secretion in early development (29,30). Functional Transport by H,K-ATPase Through the use of pH electrode and fluorescent dye techniques, investigators linked the K+-stimulated ATPase activity of gastric microsomal vesicles to vectorial H+ and K+ transport: H+ being pumped into the vesicles in exchange for K+ transport out (26,31–33). The pump was shown to be an electroneutral H+ for K+ exchange and became known as

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1195 the H,K-ATPase (32). Figure 48-6 shows a typical record of proton uptake by freshly isolated tubulovesicles, as measured by the accumulation of a fluorescent weak base. There is little ATP-dependent uptake of H+ uptake until the K+ ionophore, such as valinomycin, is added. The rate of valinomycin-induced H+ uptake is dependent on the concentration of K+ and the concentration of a permeant anion, for example, Cl−, in the medium. Making the vesicles leaky to H+

ATP

nig

val

7.5 mM Cl

43 mM

Fluorescence units

79 mM

150 mM

2 min

FIG. 48-6. Typical recording of proton uptake by freshly isolated tubulovesicles, as measured by the accumulation of a fluorescent weak base (acridine orange). When vesicles are quiescent, the acridine orange in the medium fluoresces brightly; when vesicles are activated to accumulate protons, acridine orange is sequestered into the vesicles together with a quenching (diminution) of fluorescence. Vesicles were placed in a medium that contained 150 mM K+ in all cases and variable amounts of Cl−, as indicated (Cl− progressively replaced with isethionate). The experimental test was initiated by adding 1 mM MgATP (ATP). There was little ATPdependent uptake of H+ uptake (i.e., fluorescence quench) until the K+ ionophore valinomycin (val) was added. After val, the proton uptake rate was highly dependent on [Cl−]. Addition of the protonophore nigericin (nig) at the end of the experiment made the vesicles leaky and rapidly dissipated the proton gradient. (Reproduced from Lee and Forte [33], by permission.)

by adding a protonophore immediately dissipates all H+ gradients. The pump-leak model as depicted in Figure 48-7 indicates that ATPase-dependent accumulation of H+ by the vesicle critically depends on the relative permeabilities of the three principal ions: K+, H+, and Cl−. The interrelations between the pumps and leaks of ions could be observed experimentally and predicted mathematically from Nernst–Planck ion flux predictions and Michaelis–Menten enzyme kinetics (34,35). Thus, the H,K-ATPase is the source of gastric H+, operating as a cation exchange ATPase, using the hydrolysis of ATP to transport H+ in exchange for K+, with a stoichiometry of 1 H+:1 K+:1 ATP. Although there have been disagreements on the transport/ATP utilization stoichiometry (36,37), these must be the result of measurements made in vitro, because the enormous transepithelial H+ gradient of 106-fold and fundamental energetic considerations limit the stoichiometry in vivo to 1 H+:1 K+:1 ATP (36,37). Membrane Changes Associated with Hydrochloric Acid Secretion The pump-leak model carefully describes the interrelations among ATP utilization, proton pumping, and ionic dependencies in isolated tubulovesicles, but there was an important factor missing for operating the pump in a functional parietal cell. The tubulovesicle experiments required an exogenous K+ ionophore, for example, valinomycin. What was the equivalent mechanism in vivo? The answer came from experiments contrasting ATPase and pump activities in vesicles prepared from resting and actively secreting stomachs (18,19,38). The first surprise was that H,K-ATPase activity disappeared from the small tubulovesicle fraction and transferred almost quantitatively to larger, denser membrane vesicles, which are called stimulation-associated (SA) vesicles. More importantly, the SA vesicles pump protons with almost all of the same characteristics as resting tubulovesicles, except that they are totally independent of a K+ ionophore. A highlight comparison of ATP-dependent proton uptake in resting tubulovesicles and SA vesicles is compared in Figure 48-8A. In fact, the SA vesicles possess relatively selective ion channels: both K+ channels and Cl− channels (39–41). Comparison of the protein composition of SA vesicles with tubulovesicles demonstrates some additional differences (see Fig. 48-8B). They are both enriched in H,K-ATPase, but several additional protein bands are clearly obvious in SA vesicles, including prominent bands for actin and ezrin (both apical cytoskeletal proteins are discussed later in this chapter), and several other proteins. The data clearly indicate that SA vesicles are derived from the apical plasma membrane of stimulated parietal cells: They are much larger and more structurally complex than tubulovesicles; they contain actin microfilaments and other apical cytoskeletal elements; and they possess K+ and Cl− channels consistent with apical membrane. These observations thus provide the basis for pump operation in vivo, as well as the means to link the enzymatic and transport data with the ultrastructural changes associated with secretory activation. The current model of parietal cell activation and

1196 / CHAPTER 48 net = J pump + J diff JH H H

J diff + H

H+

J pump + H

ATP

P

Cl-

pump K+

J diff + K+

H Vmax [K]i r N ATP + 3 Vi Km + [K]i

FY/ RT J diff = PH+ {[H+]0 – [H+]i eFY / RT} H+ eFY / RT- 1 K+

J

ADP + Pi

pump = JH

J diffCl

K

FY/ RT J diff = PK+ {[K+]0 – [K+]i eFY / RT} K+ eFY / RT- 1 J diff- = FY/ RT PCl- {[Cl-]0 – [Cl-]i eFY / RT} Cl eFY / RT- 1

Cl-

FIG. 48-7. The pump-leak model for gastric microsomal vesicles (isolated tubulovesicles). The vesicles are depicted with an adenosine triphosphate (ATP)–driven proton pump that transports H+ into the vesicles in exchange for K+. Additional paths for the free diffusion (Jdiff, leak) of H+, K+, and Cl− are indicated. Because K+ is a key substrate of the pump, JKdiff is critical to the pump. Likewise, an diff anion must accompany K+ flux into the vesicle; thus, JCl is critical to the pump. (Right) Series of equations describe the net rate of H+ transport (JHnet) as the sum of the pump and leaks. The pump (JHpump) is described in terms of Michaelis–Menten kinetics limited by the internal K+ concentration ([K+]), the Vmax for the enzyme, the Km for K+ (assuming ATP is not rate limiting), and the stoichiometry, N, between transport of H+ and K+. Vi and r and geometry factors for the internal vesicular volume and radius, respectively. The leaks are represented in terms of Nernst–Planck diffusional relations, described individually as Jdiff for the respective ions, where P refers to the permeability coefficient for the particular ion; Y is the membrane potential; and F, R, and T have their usual physicochemical meaning. (Modified from Lee and colleagues [34], by permission.)

HCl secretion is shown in Figure 48-9, placing H,K-ATPase in the context of the membrane recycling hypothesis. In the resting cell, the cytoplasmic compartment abounds in tubulovesicles that are rich in H,K-ATPase, but are functionally inactive because of low K+ (and Cl−) permeability. Secretagogue stimulation of the cell leads to recruitment of tubulovesicles to the apical membrane providing the source for membrane expansion and placing the pump in a functionally relevant position. K+ and Cl− channels in parallel with the proton pump provide the necessary pathways for ionic transfer, with K+ recycling through channel and pump as net H+ and Cl− are secreted together with complementary osmotic flow of water. Removal of secretagogue leads to resequestration of membrane and pumps back into the cytoplasmic compartment and return to the resting state. Exactly how the channels are activated and whether they are also recruited to the apical membrane with stimulation are not certain, but more detail on these channels is provided later in this chapter.

Biochemistry and Structure of the H,K-ATPase Kinetics The gastric H,K-ATPase belongs to the P-type family of transport ATPases, which also includes the ubiquitous plasma membrane Na,K-ATPase and the Ca2+-ATPase that

removes Ca2+ from the cytosol after signaling events. There are also H,K-ATPase isoforms other than gastric, including those performing H+ and K+ homeostasis in mammalian renal tubules and colon, and amphibian urinary bladder (42–44). In addition to a good deal of structural homology, the family of P-type cation transport ATPases shares the characteristic that their transport cycle depends on autophosphorylation, and dephosphorylation (thus designated P-type), of the transport protein itself. The P-type ATPases also exhibit two phenomenologically and structurally discrete conformations, designated E1 and E2, which have distinct kinetic variables, for example, affinities for substrates (45). Thus, P-type ATPases are also called E1-E2 ATPases. The overall catalytic and transport cycle for the H,K-ATPase is made up of a series of partial reactions, as shown schematically in Figure 48-10 (23,46–48). The initial kinase activity is an autophosphorylation step in which the γ-phosphate of ATP is transferred to aspartate residue D387, forming an acyl-phosphate phosphoenzyme intermediate, E-P. The phosphoenzyme has two conformer states, the transition which involves the release of a proton. One conformation (denoted by E1-P in Fig. 48-10) has high reactivity with ADP (and low reactivity with K+), and thus can easily reverse itself; it comprises what is known as the ATP-ADP exchange reaction. Transition to the second conformation, E2-P, is essentially an irreversible step involving the release of a proton to the luminal surface and the binding of luminal K+,

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1197 A

B TV

SA ves val

100

RCOO−

RCOO−

Fluorescence, % maximum

Ise− 80

Ise−

60

NO−3 Br−

CI− NO−3

40 I min

Br− CI−

20 CCCP

CCCP val

FIG. 48-8. Comparison of function and composition of gastric tubulovesicles (TVs) purified from a resting stomach and stimulation-associated vesicles (SA ves) from a maximally stimulated stomach. (A) Proton uptake was measured by the acridine orange quench method (see Fig. 48-6). All solutions contained 150 mM K+ and an equivalent amount of anion as indicated for the individual traces, as well as 1 mM MgATP. The reaction was initiated by adding either TVs or SA vesicles. For TVs, there was little proton uptake until the K+ ionophore valinomycin was added, then rapid uptake occurred with relatively small differences between permeant anions (NO3>Br>Cl), but virtually no uptake for bulky anions, like isethionate (Ise) or methyl sulfate. For SA vesicles, proton uptake did not require K+ ionophore, and the relative anion permeability was Cl>Br>NO3>>Ise. At the end of the experiment, a protonophore (CCCP) was added to dissipate the proton gradient. (B) Coomassiestained gels for TVs and SA vesicles were run at the relatively high protein load of 50 µg/lane. TVs are composed almost exclusively of H,K-ATPase. The α-subunit is a dense band at about 95 kDa, whereas the β-subunit is broad (60–80 kDa) and poorly stained because of the heavy glycosylation. For SA vesicles, the α-subunit of H,K-ATPase is clearly the most dominant protein, but there are several other obvious apical membrane-associated proteins, including actin (Ac) and ezrin (Ez). (Modified from Lee and colleagues [34], by permission.)

that is, K+out. Binding of K+ to the phosphoenzyme participates in formation of E2-P•K+ and promotes another important enzymatic function, the K+-catalyzed dephosphorylation, or phosphatase activity. By this reaction, inorganic phosphate is liberated from the enzyme, which can then resume the phosphorylation cycle and turn over once K+ is released to the cytoplasmic surface, K+in. The scheme in Figure 48-10 indicates that ATP can bind with either the free enzyme (E) or the K+-bound enzyme (E•K). The association of K+ is not a simple, rapidly reversible, ionic bond, but rather is described as an occluded form, similar to that described for Na,KATPase (49,50). It turns out that the affinity of ATP for free E1 is much higher (i.e., low Km) than for E2•K (high Km). Thus, one of the rate-limiting steps in H,K-ATPase turnover is the rate of K+ release from E2•K, which interestingly is promoted by a high ATP concentration (48). Because the

enzyme is embedded in a membrane with distinct sidedness for specific binding sites, the H,K-ATPase cycle provides vectorial transport of ions. Because the concentration of K+ in the cell is relatively high, ATP concentration in the millimolar range is necessary for rapid conversion of E2•K to E1•ATP and high pump turnover. This is the reason for the high sensitivity of H+ secretion to a compromised metabolism or gastric blood flow (e.g., hypoxia), or both. Subunit Structure The H,K-ATPase is made up of two transmembrane subunit peptides, designated α- and β-subunit. The 110-kDa α-subunit contains most of the catalytic activity sites discussed earlier, has 10 membrane-spanning segments, and has an estimated 73% of its mass residing on the cytoplasmic side of the

1198 / CHAPTER 48 ADP + POHK+

H+ ATP

ATP

H+

Stimulation

PH/K Cl-

Low K+ perm.

K+

ADP + POHK+

High perm. KCI

JCI JK

K+ Cl-

ATP

K+

K+

ClH+

ADP + POH-

FIG. 48-9. The current model of parietal cell activation and HCl secretion placing the H,K-ATPase in the context of the membrane recycling hypothesis. The schematic stimulation-associated (SA) vesicle model on the left shows the same representation of the pump as in Figure 48-7, but also includes specific channel proteins, JCl and JK, for the transport of K+ and Cl−; thus, transport is not limited by artificial ionophores. The means for regulating H+ pump activity in vivo is shown on the right. When the pump is contained within the cell in tubulovesicles (TVs), low K+ permeability limits adenosine triphosphate (ATP) utilization and H+ transport. On stimulation, the TVs fuse with the apical surface placing the pump in parallel with high-throughput channels for K+ and Cl−, leading to secretion of HCl and water and the recycling of K+. ADP, adenosine diphosphate. (Modified from Forte JG, Forte TM, Black JA, Okamoto C, Wolosin JM. Correlation of parietal cell structure and function. J Clin Gastroenterol 1983;5(suppl 1):17–27, by permission.)

Catalytic cycle of the H,K-ATPase Lumenal side

Cytoplasmic side E1·ATP

E1-P·ADP

E1-P + ADP H+

ATP

ATP

K+

E2-Pi·K

E2·K

E1 K+

E2-P·K

Pi

FIG. 48-10. The overall catalytic and transport cycle for the H,K-ATPase is made up of a series of partial reactions. E represents the enzyme shown in two conformer states, E1 and E2. Binding with adenosine triphosphate (ATP) and the phosphorylation reaction involves the E1 form. The binding of K+ and dephosphorylation involves the E2 form. The E1 to E2 conversion involves the deposition of H+ and uptake of K+ at the luminal surface. The conversion of E2 to E1 on the cytoplasmic side involves the release of occluded K+, which can be catalyzed by relatively high levels of ATP. ADP, adenosine diphosphate.

membrane (51–53). The 32-kDa β-subunit has only a single transmembrane segment with more than 80% of its peptide mass residing on the extracellular side of the membrane, which also includes seven sites of N-linked glycosylation (54). Although the β-subunit does not directly participate in ATP utilization, it is important for stability and “protection”

of the holoenzyme. For example, precise folding of the enzyme during synthesis and targeting to the appropriate membrane compartment have been attributed to the βsubunit (55–57). Moreover, it has been proposed that the β-subunit may help to provide an answer to the age-old question of why the stomach does not digest itself. A combination

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1199 of extensive, unique, β-subunit glycosylation (58) and bonding forces within the extracellular domain of the enzyme are proposed to offer protection from the acidic and proteolytic conditions on the luminal aspect of the cell (57). The small cytoplasmic tail of the β-subunit also contains a peptide sequence that is important for membrane retrieval and recycling under secretagogue control (59). The protein composition of tubulovesicles from resting stomach is compared with SA vesicle membranes from maximally stimulated stomach in Figure 48-8B. In the highly purified tubulovesicle fraction, the H,K-ATPase is the overwhelmingly predominant protein: the α-subunit is seen as an intensely stained and focused band at about 95 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas the β-subunit runs as a poorly stained and broad band extending from 60 to 80 kDa. The relatively large size, broad distribution, and poor staining of the β-subunit are primarily because of its heavy glycosylation, which interferes with the binding of SDS, as well as Coomassie blue protein stain. Once the oligosaccharides are removed, the deglycosylated β-subunit is nicely focused at its peptide size of ~32 kDa. In SA vesicles, the H,K-ATPase (α-subunit) is also the most prominent band, but several additional protein bands are clearly evident. Some of these are cytoskeletal proteins that are associated with the apical surface, and some may be channel proteins that cannot be definitively identified from these data. Images of the freeze-fracture surfaces of intact or isolated tubulovesicles show an abundance of membrane-associated particles on what is known as the cytoplasmic, or protoplasmic, half (P-face) of the membrane fracture plane (see Fig. 48-4). Because of the predominant H,K-ATPase content of these tubulovesicles, the particles most likely represent the shadowed image of the functional enzyme in the membrane. From a particle size diameter of about 9 nm, and using the simplifying assumption of a spherical, globular shape, this would accommodate a protein volume equivalent to about 340 kDa. Because the size of a single α/β heterodimer, including an oligosaccharide, is ~160 kDa, it would appear that the aggregate unit in the membrane is made up of two heterodimers, that is, (α/β)2, which is consistent with the functional analysis on the size of H,K-ATPase by radiation inactivation analysis (60). However, it is unclear as to what extent the apparent sizes (shapes) may be the result of different E1-E2 conformer states of the enzyme, as has been shown for the Ca2+-ATPase (61). Molecular Structure The oligosaccharides on the β-subunit clearly serve important stabilizing functions, but their very presence and especially their heterogeneity have offered the most significant barrier to forming high-quality crystals of H,K-ATPase suitable for crystallographic analysis. However, Toyoshima and his colleagues (62,63) have succeeded in producing high-resolution structural maps of the Ca2+-ATPase from sarcoplasmic reticulum, which, because of the conserved structural and functional similarities, has been compared

with both Na,K-ATPase (64) and H,K-ATPase (65). As the enzymes are undergoing the catalytic cycle, there are distinctive structural changes associated with the E1 and E2 conformations. Predictably, the motional changes occur in the functional cytoplasmic domains associated with nucleotide binding (N domain), phosphorylation (P domain), and “actuation” (A domain) of the gating mechanism for ion binding and release. There are also E1-E2 motional shifts within the 10 transmembrane segments that serve as a transmembrane channel and predict selective ionic binding sites from the cis or trans side of the membrane, depending on the stage in the turnover cycle. Such structural changes were first predicted from kinetics and low-resolution structural information, but they now can be inferred at the level of specific protein domains, transmembrane segments, and individual amino acids. Sachs and his colleagues (66–68) have used the Ca-ATPase crystallographic data to develop intuitive predictive models of molecular structure for gastric H,KATPase. Figure 48-11 shows a model of the cytoplasmic domain of the gastric H,K-ATPase in the E1 and E2 conformer states. Such models have proved useful to designate and predict the molecular sites of interaction for specific inhibitors of the pump. Inhibitors of H,K-ATPase Identification of the gastric H,K-ATPase provided an important target for development and pharmacologic testing of drugs to regulate and inhibit the acid pump (69–72). Generically, the drugs are called proton pump inhibitors (PPIs), and they have been extremely successful in clinical application and also to study mechanics of pump function. A group of compounds described as substituted benzimidazoles were first suggested as antisecretory drugs via inhibition of H,K-ATPase (73,74). In the late 1980s, one of the substituted benzimidazoles known as omeprazole was launched clinically as an acid secretory antagonist (71,75). Several additional analogues have since been introduced (e.g., lansoprazole, rabeprazole, and pantoprazole), and these drugs are now widely used for treatment of peptic ulcer disease, heartburn, gastroesophageal reflux disease, and Zollinger–Ellison syndrome. Clinical use of the substituted benzimidazoles has virtually eliminated the need for gastric surgery (except for gastric carcinoma) or vagotomy that was so common before 1990. Figure 48-12A shows the structure of a typical substituted benzimidazole PPI, both in its neutral inactive form and in the acid-activated state. The generic molecule consists of a benzimidazole ring connected to a pyridine ring via a sulfoxide-containing chain; substitutions at the various R positions contribute to the specific differences among the PPIs (72,76). For example, omeprazole is substituted on the benzimidazole ring with methyl groups at R3 and R5, and with a methoxy group at R4; there is also a methoxy on R5 of the pyridine. Figure 48-12B depicts a series of chemical reactions, ultimately leading to inhibition of H,K-ATPase. In their neutral form, substituted benzimidazoles are highly membrane permeable; therefore, the commercial drugs are

1200 / CHAPTER 48 Cytoplasmic side N domain

A domain P domain

K+

Lumenal side E1 Form

E2 Form

FIG. 48-11. Structural model of the gastric H,K-ATPase α-subunit as it changes conformation in going from the E1 to the E2 form. The model was developed by Sachs and his colleagues based on homology with the Ca2+-ATPase of sarcoplasmic reticulum. The model is oriented vertically with respect to the plane of the lipidic membrane and illustrates the juxtaposition of the nucleotide binding (N), actuator (A), and phosphorylation (P) domains on the cytoplasmic side. There are 10 transmembrane segments (TM) within the membrane stalk connecting to the relatively minimal protein exposure on the luminal side of the membrane. Note the rather large conformational change as the N, P, and A domains approach one another in transforming from E1 to E2; at the same time there are only subtle changes in the TM. Also shown are possible cation binding sites and sites for the reaction of proton pump inhibitors at Cys813 and Cys822 with the TM. (Modified from Munson and colleagues [68], by permission.)

coated for “protection” from gastric acid so that they reach the intestine for ready absorption into the blood. The reason the drugs are targeted to the stomach is based on their structure as a weak base; therefore, they are protonated by acid and actually “trapped” and efficiently accumulated within highly acidic spaces of secretory canaliculi. Now, exposure to acid within the canaliculus catalyzes activation of the drugs to a sulfonamide, which is the form of the drug that reacts with cysteine sulfhydryl groups, in this case, Src homology (SH) groups on the luminal facing aspect of the H,K-ATPase, thus inactivating the pump (72). The disulfide bond formation is a covalent modification, but it can be reversed in vitro by strong reducing compounds. The most likely site and common of reaction is Cys813, although certain PPIs (e.g., pantoprazole) may also bind to Cys822 (68,77,78). There remains, however, some debate whether and to what extent the disulfide can be reduced and reversed in vivo, or whether H,K-ATPase must be replaced by de novo synthesis of the pumps. The latter may also be a function of

the specific PPI (77) and some additional chemical reactions not discussed in this chapter (79). So, why are the PPIs so specific for the gastric proton pump? In fact, the “specificity” is more a function of simple chemistry and local environment than high degree of binding affinity, as is usually the case with receptor blockers. First, the drugs are relatively stable, innocuous, and membrane permeable in their neutral from. This suggests there would be accessibility to all cells, but as weak bases they are protonated and trapped in acidic spaces, and there is no more acidic space than the parietal cell canaliculus. Thus, there is the great power of acidic space accumulation. Second, the PPIs require acid to be activated to a reactive form, acid that is provided by pump in the canalicular space. Finally, the activated drug requires a vicinal SH group for reaction, and within extracellular spaces, the cysteine-SH often are tied up in disulfide linkage. (For example, the β-subunit of H,K-ATPase has six extracellular cysteine-SH linked as three disulfide bonds, and thus are totally nonreactive.) However, there are at

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1201 A H N

R5

N

O

+H

R4

+ −H

S N

H N

+

R5

N H

R3 1

+

N

S O

2 + −H

H N

H N

+H2O

+

N

+

N

−H2O

N

+

+H

N

S 4

SOH

3

B Secretory canaliculus HS-Omep-R H+ 4 H+ ep-R + H

3

H+

K+

-S-S

2

-O m

O=S-Omep-R H+

O=S-Omep-R

HKpump

K+

1

Cytoplasm O=S-Omep-R

Blood

FIG. 48-12. Structure and function of a substituted benzimidazole proton pump inhibitor (PPI). (A) The generic molecule in neutral solution (1) consists of a benzimidazole ring connected to a pyridine ring via a sulfoxide-containing chain; substitutions at the various R positions contribute to the specific differences among the PPIs. In the presence of acid, the benzimidazole ring becomes protonated (2), and eventually a further rearrangement, which is also an acid activation, leading to formation of the sulfenic acid form (3) with a loss of water to the sulfonamide (4), which is most highly reactive with cysteine sulfhydryls on the H,K-ATPase. (B) A scheme for the reaction of PPIs in vivo. At neutral pH in the bloodstream, PPIs such as omeprazole are weak bases that cross plasma membranes and enter into (1) acidic spaces (e.g., parietal cell canaliculi). There they are trapped by protonation (2) and converted into an active form by acid (3), leading to high reactivity with surface src homology groups (4), for example, on the H,K-ATPase.

1202 / CHAPTER 48 least two cysteine-SH available on the α-subunit for reaction with PPIs and functional inactivation of the enzyme (77,80,81). Alternative types of PPIs are known functionally as K+ site competitors. There appear to be several classes of these compounds, as exemplified by SCH-28080 (82) and AHR9294 (83), which can be characterized as hydrophobic cations that compete with K+, and unlike omeprazole, they are rapidly reversible. Because of unwanted side effects, these drugs are not yet clinically useful. However, these reagents have already proved valuable for insight into pump mechanics, and future developments may find analogues that offer effective modulation of the proton pump in patients. AHR-9294 competes directly with K+ in binding to the E2-P form of the H,K-ATPase, preventing dephosphorylation of the enzyme (83). Studies with SCH-28080 suggest a means to characterize the nature of the K+ channel within the pump (66,84).

Apical Channel Proteins Directly Supporting Hydrochloric Acid Secretion Physiologists generally accept H,K-ATPase as the principal gastric proton pump, operating as an electroneutral one-forone H+ for K+ exchange pump. However, this explanation leaves questions about the regulation of the system, and it is superficially inconsistent with many studies on intact gastric mucosal preparations. Quantitative models of pump operation demand that channels for K+ and Cl− be available for efficient HCl secretion (34), and electroneutral exchange must be coupled to some conductive pathway to account for polarization phenomena noted in intact tissue. One of the current challenges in gastric secretory physiology is the identification and characterization of the K+ and Cl− efflux pathways across the apical membrane that must accompany operation of the H+-K+ pump. Studies on intact preparations demonstrated that the gastric epithelium could generate a current of H+, leading to an electrogenic theory of HCl secretion (85,86). Electric current opposing HCl secretion (mucosa to serosa) diminished H+ output, whereas enhancing Cl− flux, and current passed in the direction of HCl secretion (serosa to mucosa) enhanced H+ output and reduced net Cl− flux. Furthermore, in the absence of Cl− in the bathing solutions, the electric current was directly equivalent to the flux (current) of H+ (87). These observations cannot be accounted for by an electroneutral exchange pump; any comprehensive mechanism of the acid secretory process must be consistent with and provide rational explanation for the observations. In fact, the observation cited earlier that SA vesicles have conductive pathways for Cl− and K+ in parallel with the proton pump offers the means by which an electroneutral H+ for K+ exchange pump can provide electrogenic HCl secretory activity (88,89). The simplified scheme shown in Figure 48-13 places the K+ and Cl− conductive pathways in parallel with an electroneutral H,K-ATPase; thus, the entire system is electrogenic and can be expressed as a mathematical model (34).

Cytoplasm

Lumen H+

ATP K+

K+

Cl-

-

+

+

-

Cl-

FIG. 48-13. Simplified scheme showing that an electroneutral, 1 for 1, H+ for K+ exchange pump can provide an overall electrogenic activity when coupled in parallel with K+ and Cl− conductive pathways. The K+ channel provides a path for K+ to enter the secretory lumen of the parietal cell and be recycled back to the cytoplasm as a substrate for the H+-K+ pump exchange. A Cl− channel provides a means for Cl− to enter the secretory lumen, representing a counter ion for K+ current across the apical membrane and balancing the charge for electroneutral HCl secretion. In the absence of Cl−, the apical membrane becomes a potential electrogenic H+ pump. Current passed from serosa to mucosa would be carried by K+ into the canalicular lumen; resulting delivery of substrate to the H+-K+ exchange pump would deliver a “current” of H+. Without external current, the K+ channel together with the H+-K+ exchange pump provide an equivalent electromotive force. ATP, adenosine triphosphate. (Reproduced from Forte [89], by permission.)

The proposed K+ channel provides a path for K+ to enter the secretory lumen of the parietal cell, where it is recycled back to the cytoplasm as a substrate for the H+-K+ pump exchange. A Cl− channel provides a means for Cl− to enter the secretory lumen, representing a counter ion for K+ current across the apical membrane and balancing the charge for net HCl secretion. In the absence of Cl−, the apical membrane becomes a potential electrogenic H+ pump. Current passed from serosa to mucosa would be carried by K+, and the resulting delivery of substrate to the H+-K+ exchange pump would deliver a current of H+. Without external current the K+ channel together with the H+-K+ exchange pump provide an equivalent electromotive force. Thus, the existence of K+ and Cl− channels in the stimulated apical membrane, as implied from functional studies of the SA membranes (40,41), account for observations on purified gastric vesicles, as well as electrophysiologic data

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1203 in situ. It remains to specifically identify the K+ and Cl− channel proteins, as well as to discover how they are regulated with respect to acid secretion. A ClC-2–type Cl− channel, capable of functioning at low extra-cellular pH, has been identified in H, K-ATPase–rich membrane vesicles (90). These ClC-2-type channels are regulated by protein kinase A (PKA) and low extracellular pH, which is consistent with regulation of gastric HCl secretion by cyclic adenosine monophosphate (cAMP). In addition, a possible Cl− channel regulator called parchorin, with structural homologies to the family of chloride intracellular channels, has been implicated in gastric Cl− and water transport (91,92). Identification of the K+ channel has been somewhat more controversial. At least three candidate channels for the apical K+ conductance associated with acid secretion have been identified. The KCNQ1 K+ channel is abundantly expressed and colocalizes with H,K-ATPase in human and mouse gastric mucosa (this was originally called the cardiac K+ channel, KvLQT1, because of mutations known to be associated with hereditary long QT wave syndrome) (93,94). Inhibition of acid secretion by a putative inhibitor of KCNQ1 in several model systems prompted a proposed role for KCNQ1 and its functional subunits as the critical K+ channel at the apical membrane of the parietal cell. Several members of the inwardly rectifying K+ channel (Kir) family, including Kir2.1, Kir4.1, Kir4.2, and Kir7.2, are also expressed in gastric tissue (95,96). Both Kir4.1 (96) and Kir2.1 (95) have been localized by immunocytochemistry to the apical microvillar regions of parietal cells, a region consistent with an acid-related channel. Kir 2.1 is present in membrane vesicles isolated from stomach, and channel properties measured when these H,K-ATPase–rich membranes were incorporated into planar lipid bilayers suggested an increased open probability for membranes derived from stimulated, compared with resting, stomach (95). Treatment of resting vesicles with PKA or reduction of pH tended to increase open probability of incorporated channels toward that of stimulated vesicles, thus prompting a possibility for regulation, analogous to gastric ClC-2 channels. In addition to the above K+ channels, there have been suggestions that a ROMK channel (renal outer medullary K+ channel) functionally participates as the apical channel in parietal cells. It is difficult to draw a conclusion from these many K+ channel candidates. Currently, we cannot adopt, or even exclude, any of the candidates as the essential operator in apical K+ recycling to coordinate with H,K-ATPase–mediated acid secretion. They each have been shown to be expressed in parietal cells (albeit not always in the same animal species) and at locations consistent with functional participation in the secretory process. They have the interesting property of sustained activity, and possibly activation, at low pH necessary for participation in acid secretion. Patch-clamp studies on basolateral membrane of gastric acid–secreting cells have demonstrated a variety of K+ channels, each with distinctive channel properties (97,98). It is not unreasonable to suspect that more than one apical K+ channel may be operating to serve the acid secretory function. The problem is now clearly

focused, and further work amplifying the signature channel properties of the apical membrane surely will clarify these questions for the parietal cell.

TRANSPORT AT THE BASOLATERAL MEMBRANE Homeostatic Requirements There is voluminous secretion of HCl as the gastric juice is produced at the apical surface of the oxyntic cell. This requires that an equivalent amount of base is delivered by the cell into the blood. Consistent with the secretory model proposed earlier, gastric juice also typically contains concentrations of K+ that exceed plasma [K+] by twofold or more. These secretory activities clearly impose a need for mechanisms for ion uptake at the basolateral cell membrane for homeostatic maintenance of intracellular pH (pHi) and ion balance. Minimal requirements will include a capacity for K+ and Cl− uptake and the extrusion of base sufficient to cope with the change in levels associated with transitions to and from the secretory state. On the basis of electrophysiologic studies in isolated frog gastric mucosa, Rehm (85) was the first to conclude that the basolateral surface was endowed with an electroneutral Cl-HCO3 exchanger. The uptake of K+ at the basolateral membrane is attributable to the activity of the Na,K-ATPase, or Na+ pump. Basolateral K+ uptake leads to increased cellular [K+] with respect to the extracellular milieu, and given the exchange nature of the Na+ pump, maintenance of the K+ gradient requires intracellular Na+. Inhibition of the Na,K-ATPase by ouabain or elimination of Na+ from the serosal bathing solution leads to relatively rapid diminution of Cl− secretion, as well as slower loss of cellular K+ together with reduced H+ secretion (99,100). From these types of studies with intact mucosa, it was reasoned that the source of cellular Na+ to sustain K+ gradients and secretory activity was provided by basolateral NaCl uptake mechanisms (100). The next section describes details of the basolateral transporters in the parietal cell; Figure 48-14 summarizes these transporters.

Na-H and Cl-HCO3 Exchange Pathways Studies with isolated gastric glands and probes to measure pHi have demonstrated that NaCl uptake is based on independent electroneutral Na-H and Cl-HCO3 exchangers (101–103). When parietal cells were depleted of K+ and loaded with Na+, and then resuspended into Na+-free media, pHi decreased. Conversely, when cells were first acid loaded, the rate of pHi recovery was promoted by the presence of Na+ in the extracellular medium, but not by K+ or impermeant cations. These changes in pHi, in response to changes in Na+ concentration, were blocked, or slowed down, by amiloride, an inhibitor of Na-H antiport. After blockade by amiloride, the changes in pHi could be mimicked by the exogenous addition of a Na-H

1204 / CHAPTER 48 BL AP

Ca2+

K+

K+

CO-3 + OHPNaK

Na+ O2 +

2O

H+ HCO-3

ClK+

Na+

ATP

Na+ Exc Exa

HCO-3

Ki + ADP

PHK

K+ K+

H+ HCO-3 Cl-

CoNKCC

H+

Cl-

Cl-

K+ Na+

FIG. 48-14. The operation of ion transporters on the basolateral membrane of the parietal cell. The basolateral membrane (BL) is indicated on the left including a sodium pump PNa/K, a Na+ for H+ cation exchanger (Exc), a Cl− for HCO3− anion exchanger (Exa), and a Na+-K+-2Cl− cotransporter (CoNKCC). There is also an important basolateral K+ channel conductance with the suggestive possibility that it may be modified by a regulated Ca2+ channel. For completeness, the apical membrane (AP) is also shown, including the proton pump (PHK) and K+ and Cl− conductivities, indicating the activated secretory state. Intracellular base that is produced by the PHK is eliminated via the Exa at the BL. The Cl− of the HCl secretory product is provided by the Exa and the CoNKCC at the BL.

exchange ionophore. Similar kinds of experiments support the existence of a Cl-HCO3 exchange activity (101,103). For example, replacement of extracellular Cl− by impermeant anions induced an increase in pHi; this increase was rapidly reversed on reintroduction of Cl−. The Cl−–dependent changes in pHi were abolished by treatment with stilbene disulfonates, and then were reestablished by introduction of “artificial” Cl-OH exchanger by adding tributyl-tin. The Cl-HCO3 anion exchange isoform known as AE2 is expressed at high levels on the basolateral membrane of parietal cells (104,105), and AE2, subtype b, appears to be the predominant subtype (106). Studies using AE2 knockout mice show that ablation of the AE2 gene results in achlorhydria, clearly indicating that AE2 plays a central role in acid secretion (107). Parietal cells of the AE2 null mice also exhibited a severe deficiency in the development of secretory canaliculi, although AE2 expression per se occurs at the basolateral membrane. Certain functional properties of basolateral Cl-HCO3 exchange in parietal cells differ from the known functional properties of AE2 (108,109), raising the possibility that other basolateral Cl-HCO3 exchanger(s) may be present to play a role in bicarbonate exit. A Cl-HCO3 exchanger belonging to the SLC26A family, which include at least 10 distinct genes expressed variably in kidney, intestinal cells, and

testis, has been identified in parietal cells (110). One family member, known as SLC26A7, has been shown to be expressed in kidney and stomach, with most prominent expression on the basolateral membrane of parietal cells. It would be of interest to ascertain the extent to which SLC27A7 participates in acid secretion and the exchange of intracellular HCO3− for extracellular Cl−. Three Na-H exchanger isoforms (NHE1, NHE2, and NHE4) are expressed in parietal cells, and therefore could contribute to coupled Na-H and Cl-HCO3 exchange at the basolateral membrane. NHE1 is expressed at relatively low levels in parietal cells (111), and analyses of NHE1 knockout mice indicated that this isoform is not essential for acid secretion (112). Mice having a knockout of the NHE2 gene exhibited a progressive loss of parietal cells and became achlorhydric as they aged; however, gastric secretions in young NHE2 knockouts had normal acidity (113). Thus, NHE2 seems to be essential for parietal cell survival, but does not appear to be involved directly in gastric acid secretion. The NHE4 isoform is most abundant in parietal cells and functionally appears to be more sensitive to hyperosmolarity than to pHi; thus, it has been proposed to be more important in the regulation of parietal cell volume than pHi (111). Studies with the NHE4 knockout mice demonstrated that hypochlorhydria paralleled the loss of much of the Na-H

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1205 exchange activity (114), including major histologic abnormalities such as sharply reduced numbers of parietal cells, limited development of canalicular membranes, and a virtual absence of tubulovesicles. Thus, the Na-H and Cl-HCO3 exchange, involving NHE4 and AE2, with secondary coupling to the Na,K-ATPase is the predominant mechanism for electrolyte uptake and maintenance of cell volume and membrane potential during gastric acid secretion. The Na-H exchangers are also important for gastric epithelial cell differentiation and development of secretory canaliculi and tubulovesicular membranes in the parietal cell.

Na-K-2Cl Cotransport Additional transporters are present in the basolateral membranes. The Na+-K+-2Cl− (NKCC1) cotransporter is expressed in several gastric mucosal cells, including parietal cells (115,116). NKCC1 cotransporters move Na+, K+, and Cl−, with water following osmotically, into the cell. The NKCC1 isoform is present in the basolateral membrane of the parietal cell, where, coupled to the Na+ pump, it is thought to function in the maintenance of intracellular Cl− and K+ concentrations and cell volume. It has been proposed that NKCC1 participates in HCl secretion from studies showing that HCl secretion is dependent on serosal Na+ and Cl− and is blocked by Na-K2Cl cotransport inhibitors such as furosemide and bumetanide (116). However, in adult mice for which the gene responsible for NKCC1 expression was knocked out (Nkcc1−/−), secretion of gastric acid stomach was normal, with generally no apparent histologic abnormalities in parietal cells or the stomach (117). Interestingly, McDaniel and Lytle (115) have reported abundant immunohistochemical staining of NKCC1 in rat stomach; however, it was predominantly distributed to parietal cells located in the lower half of the gastric glands. Parietal cells in the upper half of the rat glands displayed prominent AE2 staining. These data suggest there may be multiple ways to satisfy the K+ and Cl− requirements for parietal cell function and HCl secretion. The AE2 exchanger may be more important in supporting the proton transport activity of the nascent, more active, secretory cells in the upper portion of the gland, whereas the NKCC1 cotransporter may be more functionally active for active chloride transport and nonacidic secretion in the lower regions of the gland.

Conductance Properties The conductance of the basolateral membrane is dominated by a K+ pathway. This conclusion is based on a number of observations, including the effect of changes in nutrient K+ on transepithelial potential difference (118), a negative intracellular potential in keeping with the direction of the K+ gradient (119), and the demonstration of K+ conductance in isolated basolateral membrane vesicles (103). However, determination

of exact conductance ratios for the principal ions, Na+, K+, and Cl−, remains elusive, although morphologic observations suggest that the conductance of K+ increases in the stimulated state. The basolateral membrane is also endowed with a carbachol-induced Ca2+ permeability pathway. In isolated canine parietal cells exposed to cholinergic agonists, free cytosolic Ca2+ was observed to increase when Ca2+ was present in the extracellular medium and decrease when Ca2+ was absent (120). The response to carbachol was brisk, and it was blocked by atropine and Ca-channel blockers, suggesting a direct link between the muscarinic receptors and the cation channel. From their studies with isolated parietal cells, gastric glands, and isolated membrane vesicles, Muallem and Sachs (120) concluded that a calmodulin-dependent Ca-ATPase was also present in the basolateral membrane. This Ca2+ pump, and not Na-Ca exchange, was proposed to be responsible for the efflux of Ca2+ in the steady state. In view of some pharmacologic similarities and current information from a variety of cellular systems, it is tempting to speculate that the increased K+ conductance observed with cell stimulation reflects the existence of Ca-dependent K+ channel in the basolateral membrane.

GETTING THE MESSAGE TO THE PARIETAL CELL The secretion of gastric acid is under complex neural and hormonal regulatory control linked to the quantity and quality of food intake into the alimentary tract. Acid secretion by the parietal cell per se is triggered by neural, paracrine, and endocrine stimuli. See Chapter 49 for a more complete description of how those processes are integrated with a meal to activate parietal cell secretion. There are well-defined activating receptors on the basolateral membrane of parietal cells, for example, histamine H2-type receptors and acetylcholine M3-type receptors. Gastrin is a principal hormonal mediator of gastric acid in vivo, but the majority of its effects on activating parietal cells is via an intermediary basal cell, known as the ECL (enterochromaffin-like cell), which responds to circulating gastrin levels by releasing histamine for a paracrine-like action on parietal cell H2 receptors.

Methods to Disperse and Isolate Gastric Glands and Cells Like most cellular processes, understanding of acid secretion has been greatly facilitated by the techniques of tissue, cell, and membrane isolation. The work on parietal cell receptor identification and the intracellular signaling pathways depends heavily on the following methods: (1) gastric gland isolation, developed by Berglindh and Obrink (121); (2) isolating and separating parietal cells, developed by Soll (122); and (3) developing primary cultures of parietal cells, presented by Chew and her colleagues (123,124).

1206 / CHAPTER 48 These various preparations have proved valuable for providing direct access to specific experimental questions, especially because methods are available to measure acid secretory and metabolic activities of the respective preparations. Relatively intact individual gastric glands are produced via a two-stage process: an initial high-pressure perfusion of the gastric tissue in situ, followed by enzyme digestion to disperse cells and glands from the fundic mucosa. The milder the enzyme treatment, the more intact the glands. When one wishes to disperse individual epithelial cells, harsher enzymes are used, with or without a Ca2+ chelation step to separate junctional contacts. Good reviews and detailed protocols are available in the literature (121,125). There is some variability among species with respect to how readily glands are produced and mucosal cells are dispersed. Rabbit mucosa has been the tissue of choice for gland preparations, whereas rat, mouse, guinea pig, and others require more aggressive techniques. The reasons for tissue differences are uncertain, but may be related to the nature and degree of connective tissue investiture within the lamina propria. However, there is reason for caution because excessive treatment with enzymes and Ca2+ chelation may destroy surface receptors and be deleterious to cell function, requiring critical interpretation of findings. Isolated glands contain the full complement of gastric epithelial cells, but the predominance of parietal cells, comprising nearly 50% of the cellular mass, and the ability to specifically monitor acid secretion by the technique of weak base accumulation, pioneered by Berglindh (126), offer ready access to parietal cell function. Secretion of acid creates highly acidic spaces within glands; weak bases, such as aminopyrine or acridine orange, penetrate into the acid spaces as uncharged species where they pick up a proton and are trapped by charge. Thus, the accumulation of radioactiveor fluorescent-labeled weak base provides a quantitative index of acid secretion. Investigators also have monitored parietal cell responses via the changes in oxidative metabolism (increased O2 uptake or CO2 output) associated with the enormous energy demand for the secretion of HCl (125). In the case of dispersed gastric epithelial cells, a variety of centrifugation techniques have been devised to purify parietal cells based on their larger size and lower density (123). Isolated parietal cells may be studied immediately after isolation using methods similar to glands (e.g., weak base accumulation, oxidative metabolism), or they can be maintained in primary cell culture for up to 1 week with full functional responsiveness, which also can be conveniently assessed with a microscope by morphologic criteria. Unfortunately, an immortal parietal cell line with the acid secretory phenotype has not yet been developed.

Signal Transduction Underlying Gastric Acid Secretion Several intracellular signaling pathways have been suggested to play a role in parietal cell activation, including PKA,

protein kinase C (PKC), Ca2+-calmodulin (CaM) kinase II, phosphatidylinositol 3-kinase (PI3K), and several other downstream kinases. Histaminergic stimulation is by far the most potent activation pathway observed for the stimulation of gastric acid secretion in vitro. Histamine H2 receptors are G protein–coupled receptors functionally linked to adenylate cyclase for the production of messenger cAMP. Stimulation by gastrin and by neural pathways are of paramount importance in vivo, but the magnitude of gastrinergic and cholinergic stimulation for many species is much reduced in isolated glands and cells, most likely because of the lack of interface with histamine-releasing ECL cells (127). Isolated canine parietal cells appear to be an exception, tending to be more responsive to cholinergic stimuli than to histamine (128). This review first focuses on signaling events underlying cAMP-mediated stimulation and the PKA stimulation pathway, and then moves on to a discussion of cholinergic pathways, as well as an overview of other kinase pathways. Cyclic Adenosine Monophosphate–Mediated Protein Kinase A Activation and Protein Phosphorylation Chew and colleagues (129) used isolated gastric glands to show that histamine increases intracellular levels of cAMP, leading to activation of PKA (130), and specifically type I cAMP-dependent protein kinase (131). Activated PKA initiates a cascade of phosphorylation events through a series of downstream effectors. Collectively, these protein phosphorylations trigger membrane and cytoskeletal rearrangements within the parietal cell supporting the morphologic rearrangements, as well as increase electrical conductivity across the gastric epithelium, likely by increasing the number of functional ionic channels (128). Early work on the activation of acid secretion simply traced the phosphorylation of proteins that occurred concomitant with stimulation of parietal cells and identified candidates by molecular size (132–135). Our laboratory first identified an 80-kDa protein that we now know as the membrane-cytoskeletal linker protein ezrin (136,137) and a 120-kDa protein now called parchorin, which has been implicated in Cl− and water transport (91,92). Chew and her colleagues identified 40-kDa Lasp-1 (124,135), 66-kDa coroninse (138), and CSPP-28, a calcium-sensitive phosphoprotein of 28 kDa, (139) as phosphoproteins associated with secretory activation. Several permeabilized models offer useful information on parietal cell function because they provide access for activators, intermediates, and inhibitors directly into the cytosol. Early methods using electroporation (140) and digitonin (140–142) to permeabilize gastric glandular cells demonstrated the importance of ATP, but they were limited because cells could not be transformed from the resting to secreting state by secretagogues or cAMP. The use of bacterial toxins that partition into plasma membranes to form pores eliminated this problem. For example, gastric glands permeabilized with α toxin purified from Staphylococcus aureus can be triggered by cAMP to effect the resting-to-secreting transition that is correlated with phosphorylation, from 32P-ATP, of a dozen

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1207 phosphoproteins (143), several of which are similar to those observed for intact glands. The narrow pore size generated by α toxin (~3 nm diameter) allows passage of only small molecules such as nucleotides, whereas access to large peptides and macromolecules is required for biochemical reconstitution of parietal cell activation. Permeabilized systems have been developed for introducing larger molecules while retaining the restingto-secreting transition, for example, in response to cAMP. These models have used toxins such as Streptococcal streptolysin O (SLO) or mild detergents such as β-escin to permeabilize gastric glands. Both systems have a good functional response to cAMP; therefore, they can be used to assess pathways operating downstream of the cyclic nucleotide, for example, testing the response to peptides that block specific protein kinases (144). Inhibitory peptides for PKA and myosin light chain kinase (MLCK) inhibited glandular secretory response, indicating important roles for these kinases in parietal cell secretion, but not for CaM kinase or PKC. In addition, attenuation of cAMP-stimulated parietal cell secretion by peptides that inhibit Arf (adenosine ribosylation factor) suggested that Arf may be involved in the parietal cell activation pathway. The SLO-permeabilized gland model has proved useful to track the incorporation of fluorescent-labeled proteins (such as actin) into the endogenous protein pools, as well as to test the action of several syntaxin isoforms on the cAMP-activated secretory response (see Identification of a Role for Soluble N-ethylmaleimide– Sensitive Factor Attachment Proteins section later in this chapter). An alternative approach to studying signaling pathways and downstream effectors has used the more aggressively permeabilized model system, for example, digitonin, to deplete cytoplasmic constituents and subsequently test for those components that restore functional activity. Using digitonin-permeabilized gastric glands, Akagi and colleagues (145) reconstituted parietal cell secretion by adding back cytosol and fractions derived from cytosol. Cytosol derived from brain effectively restored secretion, and its secretory potency was only mildly influenced by cAMP or PKA inhibitors, but completely abolished by SM-1, an inhibitor of MLCK. Thus, brain cytosol contains stimulatory factors downstream of PKA. In contrast, assuming the specificity of SM-1, a role for MLCK in acid secretion is clearly implicated, which is consistent with other observations (146). It is of special interest that raw cytosol derived from gastric tissue did not stimulate secretion, because it contained highmolecular-weight components (~200 kDa) that potently inhibited acid secretion, as well as low-molecular-weight components (<30 kDa) that stimulated secretion (145). One of the important cytosolic activators was identified as phosphatidylinositol transfer protein, but another essential smaller molecule(s) could not be identified. Interestingly, the inhibitory activity in gastric cytosol was increased when gastric glands were stimulated. These data suggest a regulatory mechanism that includes activating signaling pathways and inhibitory molecules that modulate secretory activity, possibly

by translocation and redistribution between membrane and cytosol. Perhaps more importantly, this reconstitution model promises to be of importance in the future identification and function of critical components necessary for parietal cell activation. Lasp-1 is an actin-binding protein that was initially identified in parietal cells as a 40-kDa phosphoprotein related to cAMP-mediated activation of acid secretion, with PKA-dependent phosphorylation sites localized to S99 and S146 (135,147). In gastric glands stimulated by the cAMPdependent pathway, Lasp-1 redistributed from the basolateral membrane to the apical canalicular membrane of parietal cells (147,148). Lasp-1 is also selectively expressed in many ion-transporting epithelial cells, such as pancreatic and salivary duct cells, and in certain F-actin–rich cells in the distal tubule/collecting duct. In vitro studies demonstrate that Lasp-1 binds to filamentous (F), but not monomeric (G), actin in a phosphorylation-dependent manner (147). The selective phosphorylation-dependent regulation of Lasp-1 in F-actin–rich epithelial cells and the recruitment of Lasp-1 to cellular regions associated with dynamic actin turnover suggest that this protein may play an integral and specific role in cytoskeletal/membrane-based cellular activities underlying acid secretion. Parchorin was first identified by SDS-PAGE as a 120-kDa phosphoprotein associated with histamine/cAMP stimulation of acid secretion (134), and the cDNA sequence of parchorin has been cloned, having an actual molecular mass of 65 kDa (92). Its anomalous migration on gels is caused by an unusually high content of acidic amino acids. Parchorin is a novel protein that has significant homology to the family of chloride intracellular channels and has been localized to a variety of epithelial tissues that are active in the transport of salt and water, especially choroid plexus, salivary glands, lacrimal glands, in addition to the parietal cell (91,92). The endogenous parietal cell parchorin is present in the cytosol and relocated to the apical plasma membrane on histamine stimulation, which is reminiscent of the translocation of H,K-ATPase in response to stimulation. When GFPparchorin was exogenously expressed in kidney cells, it also appeared in the cytosol and was relocated to the plasma membrane when Cl− efflux from the cells was triggered. Interestingly, parchorin was copurified with a new type of kinase activity (149), suggesting that there may be some functional link between that kinase and the phosphorylated form of parchorin. Cholinergic Pathways of Activation Parietal cell activation by cholinergic agents acting on muscarinic M3 receptors clearly involves an increase of intracellular Ca2+ ([Ca2+]i). Within 4 to 6 seconds after adding carbachol to gastric glands, there is a rapid spike in parietal cell [Ca2+]i, up to 0.15 to 0.5 µM, followed by a lower level, sustained increase of [Ca2+] i that persists during the resulting stimulation of acid secretion (150,151). The secretogogue-dependent changes of [Ca2+]i draw from an

1208 / CHAPTER 48 intracellular store of bound Ca2+ and are independent of extracellular Ca2+, unless the stores are first depleted (152,153). If the increase in [Ca2+]i is buffered by chelating agents, the acid secretory response to carbachol is blocked, but subsequent stimulation by histamine or forskolin will occur (154). Intracellular Ca2+ stores are relatively easily depleted by cholinergic stimulation in a Ca2+-free medium; however, the stores are readily replaced by extracellular Ca2+ through a La3+-sensitive pathway (153). The carbachol-induced increase in [Ca2+]i has been correlated with an increase in parietal cell inositol-1,4,5trisphosphate (IP3), thus implicating IP3 as a participating messenger from the M3 receptor to the internal Ca2+ stores (150). Several isozymes of PKC are expressed in parietal cells with some, particularly PKCε, being localized relatively close to filamentous actin at the apical canalicular membrane (155). However, a role for PKC in cholinergic activation has been controversial, especially because PKC activators, such as phorbol esters (e.g., TPA), have been observed to inhibit or activate both carbachol- and histamine-stimulated acid secretion (156–159). Moreover, inhibitors of PKC, such as Ro 31-8220, were found to potentiate both carbachol- and histamine-mediated acid secretion and to cause a dosedependent decrease in the phosphorylation of the cytoskeletalrelated proteins ezrin and coroninse (138,155). These data, which are consistent with permeabilized gland data, suggest a negative modulatory role for PKC in acid secretion. Several effectors downstream of the cholinergic-induced increase of [Ca2+]i have been suggested. CaM kinase is one such effector. The pharmacologic inhibitor of CaM kinase II, KN-62, blocked carbachol-mediated acid secretion, but it did not alter the increase in cytosolic Ca2+ caused by carbachol (160). Also, KN-62 was without effect on histamine- or forskolin-mediated secretory responses (161). The calcium-sensitive phosphoprotein of 28 kDa, known as CSPP28, is rapidly phosphorylated in intact parietal cells in response to an increase in [Ca2+]i produced by either cholinergic agonists or calcium ionophores (139). In vitro studies show that recombinant CSPP28 is phosphorylated both by crude parietal cell homogenate and purified CaM kinase II in a calcium/calmodulin-dependent manner (139). Thus, CSPP28 is closely tied to a calcium-mediated phosphorylation. It will be of great interest to ascertain the nature of CaM kinase–mediated phosphorylation on CSPP28, and whether such modification regulates the function of CSPP28 in the activation process. Carbachol also has been shown to activate a whole variety of kinases via Ca2+- and PKC-dependent pathways in the canine parietal cell system (162,163). These include IκB kinase (164), which is an important transcription factor and regulator of numerous genes modulating the inflammatory response (165,166), as well as mitogen-activated protein kinase, extracellular signal-regulated protein kinases (ERKs), and the c-Jun NH2-terminal protein kinases. Many of these kinases participate in pathways signaling broad cellular functions of growth, differentiation, and secretion (167–170). However, the specific role they play in the acid secretory

activation pathway remains to be clarified, especially because specific inhibitors of the various kinases reportedly produce both inhibitory and enhancing effects (171). Collectively, these data demonstrate that M3 muscarinic stimulation of gastric parietal cells activates extensive interrelation of signaling elements. It remains to be established which elements are directly involved in the activation of parietal cell secretion, and which may be modulatory or inhibitory to the activation. Specific downstream effector proteins, as opposed to secondary and tertiary cellular response to metabolic load, also remain to be identified. Modulation by Epidermal Growth Factor and Transforming Growth Factor-a Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) are well known to inhibit gastric acid secretion in vivo and in vitro. Because of their potent secretory inhibition and ability to promote cell proliferation and repair, these agents have long been suggested as potential antiulcer agents (172). Both growth factors bind to the same EGF/TGF-α receptor (173), frequently linked to tyrosine kinase activation pathways. A number of groups have studied the mechanisms underlying the actions of EGF and TGF-α on acid secretion using preparations of isolated gastric glands, isolated parietal cells, and primary parietal cell cultures. Chew and colleagues (174) categorized two apparently opposed responses: (1) the acute inhibition of histamine- and carbachol-mediated acid secretion as seen by many others, and (2) a novel enhancement of acid secretory function with chronic exposure to the growth factors. Let us examine the mechanism for the acute inhibitory effects on the histamine/cAMP pathway. EGF (TGF-α) inhibits histamine- or forskolin-mediated acid secretion, but not secretion mediated by dibutyryl cAMP (dbcAMP), suggesting that the EGF effect is upstream of cAMP formation (175). The EGF/TGF-α receptor, and possibly a downstream protein tyrosine kinase (PTK) pathway, must be involved because EGF had no effect in mice with a defective mutation in the EGF receptor (173). Furthermore, the use of the tyrosine kinase inhibitor genistein totally reverses the EGF/TGF-α inhibition of histamine-stimulated acid secretion (161). Currently, these data are consistent with the cAMP pathway of activation via an ultimate PKA substrate(s), and some modulation or inhibition by an antagonistic PTK substrate. Further analysis of the system indicated that, independent of added EGF, inhibition of PTK by genistein also has a profound potentiating effect on acid secretion stimulated by histamine or forskolin (160,174), but no significant effect on dbcAMP-mediated secretion or on the maximum level of secretion (160). We could interpret these data in a more general scheme, such as shown in Figure 48-15, that proposes some basal PTK level in parietal cells, possibly with the same antagonistic tyrosine phosphoprotein. Inhibition of basal PTK activity would remove the antagonism, allowing a given intracellular cAMP level to produce sufficient PKAactivated substrate for maximum acid secretion, thus providing

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1209

ATP

AC Histamine

PDE AMP cAMP

Gs H2

i-PKA a-PKA

Prot-P1

cell activation; memb recruitment P2-Prot-P1

Prot PTK EGF/ TGF-a

EGFR

H/K

HCI Secretion

KA

P2-Prot

a-P

PTK Protein kinase C

ACh

M3 PIP2

PLC

DAG IP3

Ca/CAM kinase Ca2+

Ca2+

FIG. 48-15. Scheme depicting the histamine/cyclic adenosine monophosphate (cAMP) pathway of parietal cell activation and the possible antagonistic effects of epidermal growth factor (EGF) (and transforming growth factor-α [TGF-α]) via protein tyrosine kinase (PTK). The basic hypothesis is that protein kinase A (PKA) and PTK are competing for the phosphorylation a pool of important activating proteins (Prot). The kinases lead to different sites of phosphorylation, for example, Prot-P1 being an activated phosphorylation site and P2-Prot representing an inhibitory form; however, high levels of cAMP and consequent high activity conversion of inactive PKA (i-PKA) to activated PKA (a-PKA), can phosphorylate P2-Prot into an active P2-Prot-P1 form. Histamine released from local paracrine cells operates through a G protein (Gs)–coupled H2 receptor to activate adenylyl cyclase (AC), which produces cAMP to convert i-PKA to a-PKA. Cellular levels of cAMP are controlled via the AC synthetic path and degradation by phosphodiesterase (PDE). The path for up-regulating PTK level via EGF receptor (EGFR) is indicated. A cholinergic-mediated M3 receptor path for potentiating parietal cell activation via Ca2+ and protein kinase C (PKC) also is indicated. ACh, acetylcholine; ATP, adenosine triphosphate; CaM, Ca2+-calmodulin; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate.

the potentiating effects for histamine and forskolin. In contrast, EGF/TGF-α would up-regulate the antagonistic PTK substrate, reducing the effectiveness of the PKA-activated substrate for all but maximum cAMP levels. Interestingly, the model allows for the final mediating product to be a substrate for both the PKA and PTK pathways; for example, ezrin is one such protein that has potential for heteromorphic activities. Notably, without further modification, the model does not account for inhibition of carbachol-mediated acid secretion by EGF (174), but there may be cooperation (“cross talk”) between the carbachol/Ca2+ and cAMP/PKA pathways. Chew and her colleagues (174) proposed that the chronic enhancing effects of EGF on parietal cell function operate via classical EGF/TGF-α receptor–associated tyrosine kinases. Enhanced parietal cell secretory activity in response to EGF or carbachol has been correlated with increased activity of ERKs: ERK-1 and ERK-2 in rabbit cells (176) and ERK-2 in canine cells (162,163). Increased ERK activity stimulated by EGF appeared coupled to the expression of H,K-ATPase α-subunit (177), which was suggested as the reason for the slightly enhanced secretory activity associated with ERK activation, and may even form the basis for the long-term stimulatory effects of EGF. The serine-threonine

protein kinase Akt appears to be yet another element in the EGF activation cascade leading to increased H,K-ATPase expression in canine parietal cells (178).

SUPPORTING MEMBRANE TRANSFORMATIONS: THE VESICULAR TRAFFICKING MACHINERY The extensive morphologic transformations occurring during the secretory cycle of the parietal cell suggests that proteins that regulate vesicle trafficking and the actin cytoskeleton are involved in this process. Specifically, these two classes of proteins must coordinate their activities to regulate vectorial trafficking of the H,K-ATPase, and perhaps other membrane proteins, throughout the secretory cycle, namely, during their exocytosis and endocytosis. Despite the highly specialized morphology and function of parietal cells, recent characterization of its cellular machinery has clearly shown that components of this machinery are more similar, rather than dissimilar, to that present in many, if not all, other cell types. The following sections describe the identification and characterization of these proteins in parietal cells.

1210 / CHAPTER 48 Role of the Cytoskeleton Actin The actin family of proteins is a major constituent of the contractile machinery of muscle cells and regulates cell shape and motility of nonmuscle cells. There is growing evidence that actin and associated proteins play a major regulatory role in protein trafficking and intracellular motility of membrane-bound organelles (179–183). The microvilli lining the canalicular membrane are lined by regularly spaced, radially distributed actin microfilaments composed predominantly of the β-actin isoform (184). In the resting state, the microvilli are short and stubby, and the microfilaments lining the microvilli extend into the cytoplasm for one or more micrometers. On stimulation and the incorporation of tubulovesicular membranes into the canalicular membrane, the microvilli become greatly elongated, and there is an apparent elongation of microfilaments lining the microvilli. When HCl secretion ceases on removal of the secretagogue, the earliest morphologic changes in the stimulated-to-resting transition are observed in the microvilli: They appear to collapse as the microfilaments appear to become fragmented and severed (8,16). Thus, changes in the state of filamentous actin (F-actin) appear to occur concomitantly with various stages of the secretory cycle, and these changes may regulate HCl secretion. Results from experiments with drugs that disrupt that actin cytoskeleton suggest that intact actin microfilaments are required for HCl secretion (185–187), but the mechanism for the apparent elongation of microfilaments and microvilli on stimulation of the parietal cell does not appear to involve de novo polymerization of F-actin from monomeric actin (188). Candidate proteins for regulating actin function with respect to secretion are likely to be found in the family of actin-binding proteins that regulate the stabilization of preexisting small polymers of actin, their incorporation into preexisting microfilaments, and those proteins that regulate the stability of microfilaments (189). Actin-Binding Proteins and Actin-Dependent Motors Several proteins that are known to bind to F-actin in vitro and that, in other cell types, are associated with intracellular regions rich in F-actin, such as microvilli, or regions of active actin remodeling and dynamics, such as in membrane ruffles and lamellipodia, have been identified in parietal cells: ezrin (136,137), spectrin and ankyrin (190,191), LIMand SH3-domain–containing protein (Lasp-1) (148,192), coronin (138), and developmentally regulated brain protein (drebrin) (193). None of these proteins is unique to parietal cells, underscoring the commonality of such cellular machinery, and much of what is known about the function of these proteins comes from other systems. However, in parietal cells, all of these proteins appear to be expressed at the canalicular membrane, placing them in a location where they would be predicted to regulate the actin cytoskeleton during the secretory cycle. Ezrin is the only protein among

these that is predominantly distributed to the canaliculus, where it colocalizes at the ultrastructural level with F-actin in the microvilli (194). All the other proteins appear to be expressed also at the basolateral membrane, which is characterized by basal infoldings that are lined with F-actin. The localization of all of these proteins at the canalicular and basolateral membranes suggests that they may play a general role in the regulation of F-actin structures in the parietal cell. In fact, in other cell types, these proteins localize to actinrich structures and to regions of active actin remodeling. Ezrin and Lasp-1 have been localized to actin-rich protrusive regions such as microvilli, filopodia, lamellipodia, and neuronal dendritic spines (147,195–199). In fibroblasts, Lasp-1 has been shown to be localized to focal adhesions (198,200). In neuronal cells, drebrin has been localized to neurites, which are similar morphologically to filopodia, in addition to being localized to the submembranous actin cytoskeleton (201,202). Coronin has one of the more intriguing localizations in nonparietal cells; it is localized to actinrich phagocytic cups and lamellipodia in amoeba and lymphocytes (203). Given the massive recycling of membranes that occurs in parietal cells during the secretory cycle, perhaps coronin participates in a phagocytic-like function during the early stages of canalicular membrane recycling. Commonalities are likely to emerge regarding the function of ezrin, Lasp-1, coronin, and drebrin in other cells and how they function in parietal cells. All of the proteins mentioned earlier also are multidomaincontaining proteins, thereby providing sites for interaction with diverse other proteins and being able to function as molecular scaffolds. Ankyrin and spectrin are the prototypical scaffolding proteins, with clear examples of these two proteins linking transmembrane proteins to the actin cytoskeleton (204–206). Ezrin binds to the regulatory RII subunit of PKA in parietal cells (207); in other cell types, ezrin binds to transmembrane proteins, other signaling molecules, or other scaffolding molecules, thereby serving as a putative link among transmembrane proteins, the actin cytoskeleton, and signaling pathways (196). Lasp-1 has been shown to bind to dynamin, a protein that regulates vesicle budding and the actin cytoskeleton (208), and to zyxin, a component of focal adhesions (200). Ezrin, Lasp-1, and coronin are of additional particular interest because they are phosphorylated on serine or threonine residues on the stimulation of parietal cells with agents that activate intracellular signaling pathways involving cAMP-dependent PKA or Ca2+-dependent PKC, thus providing links from signaling pathways to the actin cytoskeleton (138,209,210). A role in such a link was observed for Lasp-1 in which mutation of the serines that are phosphorylated in Lasp-1 by PKA and expression of this mutated Lasp-1 in cultured parietal cells blocks SA membrane transformations (147). Continued research into the role that each of these actin-binding proteins plays in the intersection of signaling pathways and the actin cytoskeleton in the parietal cell will likely yield exciting results. Myosin II is currently the only member of the myosin family of force-generating, actin-based molecular motors to

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1211 be identified in parietal cells. Its activity appears to be stimulated on activation of parietal cells maintained in primary culture, and it participates in dynamics of lamellipodia at the basolateral membrane (211). However, because of its association with the basolateral membrane, myosin II would not be predicted to regulate actin dynamics at the apical membrane. In contrast, regulation of actin and membrane dynamics at the basolateral membrane may be important in vivo, by perhaps regulating the activity of basolateral membrane transporters or signaling receptors. In addition, it appears likely that other myosins are present in the parietal cell and may play a role in regulating actin dynamics at the canalicular membrane. The possible roles of these actin-binding

proteins in H,K-ATPase recycling and apical membrane dynamics is illustrated in Figure 48-16 for secretion and Figure 48-17 for membrane retrieval. Vesicle Trafficking and Protein Sorting Machinery Rab11 and Rab25 Rab proteins are a family of small Ras-like GTPases that regulate membrane trafficking along the exocytotic and endocytotic pathways in all eukaryotic cells, from yeast to human (212). Some 60 members currently have been identified in mammalian cells, many of which display distinctive

FIG. 48-16. Stimulus–secretion coupling. On stimulation, tubulovesicles (i.e., recycling or “storage” endosomes) are translocated to the canalicular membrane, where they undergo soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE)–dependent fusion. All of the transporters and channels required for HCl secretion are thereby placed in parallel at the canalicular membrane. The small GTPases Rab11 and Arf6 also translocate to the canalicular membrane. Dramatic changes in the actin cytoskeleton also occur on stimulation; in particular, there is an apparent elongation of the microvilli, presumably because of incorporation of additional membrane resulting from tubulovesicular fusion. The elongation of microvilli must be accompanied by growth of actin filaments lining the microvilli and lateral attachment of microfilaments to the microvillar membrane. Shown are the actin-associated proteins that are localized to the stimulated canalicular membrane (for simplicity, only a couple of microvilli are replete with such proteins). Some show an increased localization to the canalicular membrane on stimulation, such as Lasp-1 and spectrin. Others, such as drebrin and ezrin, appear to be associated with the canalicular membrane in both resting and stimulated cells. Lasp-1, drebrin, and ezrin have been localized to microvilli and filopodia in other cell types and may be involved in the regulation of microvilli in parietal cells as a consequence of their association with or structural modification of F-actin, or both. Ezrin and spectrin may function as F-actin, microvillar, membrane anchoring and scaffolding proteins in parietal cells. Yet to be characterized in the parietal cell are proteins that regulate actin polymerization and perhaps bundling of actin microfilaments. cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; VAMP, vesicleassociated membrane protein.

1212 / CHAPTER 48 A

B

FIG. 48-17. Membrane retrieval from the canalicular membrane after removal of stimulus. (A) Rapidly after the removal of stimulus, microvilli are observed to collapse and F-actin within the microvilli become fragmented. Drebrin may effect actin fragmentation through activation of its actindepolymerizing factor (ADF)/cofilin domain. Coronin and clathrin may be recruited to the canalicular membrane. (B) Once the actin cytoskeleton begins to fragment, coronin and clathrin would then regulate large-scale, phagocytic-like internalization of canalicular membrane, retrieving massive amounts of H,K-ATPase and other proteins. The endocytic membrane structures may be curved, discoidal, or cup shaped (the endocytosed luminal contents are slightly shaded). Curved or discoidal endocytic membrane may then undergo homotypic fusion to produce larger tubulovesicles, cisternae, or cup-shaped membranes. When such cup-shaped endocytic membranes are sectioned in the plane shown, it may produce a pentilaminar membrane profile, as shown. Such structures are commonly observed in ultrastructural studies of the retrieval phase of the secretory cycle. VAMP, vesicle-associated membrane protein.

intracellular distributions among the various exocytotic and endocytotic organelles and regulate anterograde or retrograde membrane trafficking by these organelles. For example, Rab1 and Rab2 are associated with the endoplasmic reticulumcis-Golgi network, Rab6 and Rab8 are associated with the Golgi and trans-Golgi network, and Rab4 and Rab5 are associated with early endosomes. As with all other members of the small Ras-like GTPase family, the activity of Rabs, and therefore the regulation of their effector function, is regulated by their cycling between the activated, GTP-bound state and the inactive, GDP-bound state. This cycle is regulated by the intrinsic GTPase activity of the Rabs, which can be

modulated by the interaction of the Rab protein with other proteins possessing the following activities: GTPase-activating proteins; guanine nucleotide exchange factors, which are involved in the exchange of GDP for GTP; and GDP dissociation inhibitors. The regulation of the GTP-bound state will, in turn, regulate the effector function of the Rab in membrane trafficking by any of the following mechanisms: (1) to recruit vesicle coat proteins (see later) to membranes to facilitate incorporation of cargo into a nascent vesicle and to provide the structural features that deform a planar membrane into a spherical vesicle; (2) to recruit effector machinery for membrane fusion; (3) to recruit cytoskeletal machinery for

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1213 organelle motility and protein trafficking; or (4) to bind to cargo protein directly and regulate their trafficking by vesicle coat proteins. Although the parietal cell is sure to have some level of many of the ubiquitous Rab proteins such as Rab2 and Rab5, the major Rab proteins characterized in parietal cells are Rab11 and Rab25 (213–216). Rab11 was the first Rab identified in parietal cells. It was shown to be localized to the tubulovesicular compartment (213,216,217), and it translocates together with the H,K-ATPase to the canalicular membrane on stimulation of parietal cells, implying a functional association between the two proteins (218). The functional association between the two proteins was confirmed by expression of a dominant-negative form of Rab11 in primary cultures of parietal cells that blocked H,K-ATPase translocation and acid secretion (219). Characterization of Rab11 in other cell types confirmed that Rab11 is ubiquitous and that it is associated with endosomal compartments that regulate recycling of membrane proteins (220). In particular, in epithelial cells, Rab11 appears to be a marker for apical endosomal recycling compartments and regulates recycling at the apical membrane, which is consistent with its function in parietal cells (221–223). In addition, a number of Rab11-interacting proteins have now been identified in epithelial and nonepithelial cells, and many of these interacting proteins have been shown also to regulate the recycling of membrane proteins (223–227). The best-characterized of these proteins is myosin Vb, which would link Rab11 activity with organelle motility or protein kinesis, or both; however, its presence in parietal cells has not been confirmed (225). In contrast, Rab11 family interacting proteins have been identified in parietal cells and have been found to be associated with the H,KATPase, but their possible role in H,K-ATPase trafficking is less clear (224). The other Rab identified in parietal cells is Rab25 (213, 214). Like Rab11, Rab25 is associated with the tubulovesicular compartment. When assayed in a heterologous expression system, it too regulated recycling from an apical endosomal compartment, although it appeared to be a negative regulator of recycling (221,222). As with other Rabs, characterization of their functions will depend on characterization of their effector protein complexes. Identification of a Role for Soluble N-ethylmaleimide–Sensitive Factor Attachment Proteins A group of proteins called soluble N-ethylmaleimide– sensitive factor attachment protein receptors (SNAREs) are highly conserved integral membrane proteins present in all eukaryotic cells that regulate the fusion of transport vesicles that are derived from a donor organelle with their target membrane or organelle and are thought to mediate, in part, the specificity of such fusion events (228–230). With respect to this section, for simplicity, SNAREs present in the transport vesicle are designated as v-SNAREs, and those present in target membranes are designated as t-SNAREs, with v- and t-SNAREs representing cognate pairs or multimers to

mediate specific fusion events as a consequence of their interactions. Also, consistent with these designations, many SNARE proteins have distinctive steady-state subcellular localizations. The core components of the so-called SNARE complex are N-ethylmaleimide–sensitive factor (NSF), the soluble NSF attachment proteins (SNAPs), and SNAREs. Currently, a number of SNARE proteins have been identified in parietal cells: four isoforms of syntaxin (1–4) (231), synaptosomal associated protein 25 (SNAP-25) (231), and vesicle-associated membrane protein 2 (VAMP-2) (213,231). In addition, NSF, a protein that is thought to regulate SNARE-SNARE interactions, has been identified in parietal cells (232). With respect to the characterization of the cellular secretory machinery in the parietal cell and support for the fusion-dependent hypothesis of H,K-ATPase and tubulovesicle recruitment to the canalicular membrane, the discovery and characterization of SNARE proteins in the parietal cell are among the most significant advances. The adenoviral-based transduction of parietal cells in primary culture for the heterologous expression of proteins has proved useful in providing functional evidence for the involvement of SNARE proteins in H,K-ATPase translocation and tubulovesicle fusion (233,234). Fluorescent-tagged constructs were used to show that, in resting parietal cells, VAMP-2 is localized to tubulovesicles, whereas SNAP-25 is localized to the canalicular membrane, consistent with their function as v-SNARE and t-SNARE, respectively. Moreover, after stimulation there was a dynamic redistribution of VAMP-2 from cytoplasmic tubulovesicles to the apical plasma membrane, there colocalizing with SNAP-25 and H,KATPase. A functional role for SNAP-25 in acid secretion was confirmed by the expression of a dominant-negative C-terminal deletion mutant, SNAP-25 (∆181-226), to inhibit the acid secretory response. In addition, the involvement of VAMP-2 is supported by the observation that tetanus toxin, which specifically cleaves VAMP-2, blocks HCl secretion in the permeabilized gastric gland system. A functional role for another SNARE protein, syntaxin 3 (192), is implied by the reported dose-dependent inhibition of acid secretion when the soluble form of recombinant syntaxin 3 (i.e., lacking the membrane-spanning domain) was added to permeabilized gastric glands, presumably by competing with endogenous syntaxin 3 that is bound to H,K-ATPase–rich tubulovesicles. Again, the presence of these typical SNAREs in the parietal cell underscores the commonality of vesicle trafficking machinery involved in the process of HCl secretion. Although these data emphasize commonalities, there are possible parietal cell specializations that should be mentioned. It is highly unusual for a cell to express both the syntaxin 1 and syntaxin 3 isoforms. Syntaxin 1 is expressed almost exclusively in neuronal cells, typically on presynaptic terminal plasma membranes, where it operates as a typical t-SNARE. In contrast, epithelial cells generally lack syntaxin 1, thus syntaxin 3 plays the analogous trafficking role at the apical plasma membrane or recycling granule and vesicle membranes, or both. So, why would both syntaxin 1 and 3 be expressed in the parietal cell? It is likely that the massive membrane recruitment associated

1214 / CHAPTER 48 with parietal cell activation requires heterotypic fusion between tubulovesicle membrane and target plasma membrane, as well as homotypic fusion among tubulovesicles themselves. Such a circumstance might be facilitated by some preferred distribution of syntaxin 1 and 3 isoforms in selected membrane compartments. That this might be the case is suggested by studies showing that syntaxin 1 copurifies with VAMP-2 on H,K-ATPase–rich tubulovesicles (213,231), and in resting parietal cells they are broadly distributed throughout the cytoplasm, which is consistent with tubulovesicle distribution. In contrast, syntaxin 1 and SNAP-25 are localized to the apical plasma membrane. With the initial characterization of the fusion machinery, several questions remain outstanding with respect to the fusion process that regulates HCl secretion. Important among them is the mode of fusion. That is, one possible model is that each tubulovesicle fuses with the canalicular membrane in exclusive heterotypic fusion events. The other model would propose that many tubulovesicles fuse with each other in homotypic fusion events and only a few heterotypic fusion events with the canalicular membrane are necessary to form the elaborated apical membrane of stimulated parietal cells. Although both models must incorporate heterotypic fusion events, evidence of homotypic fusion events was provided by in vitro fusion assays with purified tubulovesicles (235,236). These studies suggested that the process was protein-mediated, required cytosol, divalent cations (e.g., Ca2+), and/or ATP. Although the in vivo relevance of the in vitro fusion assay remains to be established, such an in vitro system will be an important one to characterize further the cofactors that regulate homotypic tubulovesicular fusion. Another issue is the identification of proteins that work upstream of SNARE proteins, the so-called tethering proteins that work at longer molecular distances than do the SNAREs (230,237). Thus, the tethering complexes impart another level of specificity in the recognition and engagement of a vesicle with its target membrane. The final issue is how the entire fusion complex is quiescent in unstimulated cells and how it becomes activated in stimulated cells. Clathrin and Clathrin Adaptors A current paradigm of vesicle formation has the incorporation of membrane-bound cargo into a nascent vesicle coupled to the formation of a multiprotein complex known as a “coat” (238–242). The formation of the coat serves two purposes: (1) to help recruit membrane-bound cargo into the nascent vesicle; and (2) to facilitate the physical deformation into a spherical vesicle or tubular membrane carrier. Given the abundance of H,K-ATPase as putative cargo to be ferried about in the parietal cell, these cells might be expected to express significant quantities of cellular machinery for such purposes as well. In particular, such coat proteins might be expected to participate in the endocytosis of canalicular membrane, H,K-ATPase, and v-SNAREs such as syntaxin 3 and VAMP-2 on initiation of the recycling phase of the parietal secretory cycle, as the tubulovesicular system is reestablished.

It may also participate in other constitutive trafficking pathways in resting parietal cells, as described later. Clathrin and its associated proteins, the clathrin family of adaptors, are the best-characterized family of vesicular coat proteins and are one of the three families of coat proteins conserved from yeast to human (242). The most abundant constituents of a typical clathrin coat are the heavy and light chains of clathrin and one of the four members of the clathrin adaptor protein family, AP-1, AP-2, AP-3, and AP-4 (238,239,242,243). The adaptors are heterotetrameric protein complexes that have in common the properties of one of the subunits possessing a cargo selection function resulting from interaction with sorting motifs on membrane cargo proteins (243,244), and another being responsible for binding to and therefore helping to assemble clathrin heavy chain into its distinctive lattices, composed of alternating hexagons and pentagons. These, in turn, ultimately would form a cage around a nascent vesicle. In mammalian cells, the two major sites of clathrin-mediated intracellular trafficking of cargo occur from the plasma membrane to endosomes, mediated by AP-2, and from the trans-Golgi network to endosomes, mediated by AP-1, AP-3, and AP-4 (245). Clathrin, AP-1, and AP-2 have been identified and localized in parietal cells (246,247). Clathrin was clearly localized to the canalicular membrane and to tubulovesicles in resting parietal cells by immunofluorescence and immunoelectron microscopy (247). In addition, numerous, clearly morphologically identifiable clathrin coats were observed for the first time at the ultrastructural level on canalicular and tubulovesicular membranes in parietal cells fixed by high-pressure freezing (194). Moreover, the AP-2 adaptor was predictably immunolocalized to canalicular membranes, but surprisingly, AP-1 was localized to tubulovesicular membranes, representing one of the first demonstrations of a non-Golgi localization of AP-1 (246,247). Indirect evidence has been provided that the H,K-ATPase may interact with the AP-1 adaptor (246), although it is likely that other membrane proteins also will be found to interact with clathrin and clathrin adaptors. Finally, the stomach is one of the few organs from which clathrin has been purified biochemically, suggesting that it is relatively abundant in gastric epithelial cells. In addition, the number of proteins found to interact with clathrin and clathrin adaptors has been rapidly expanding (238,241,242,248,249). Most of these proteins are present in substoichiometric amounts relative to clathrin and clathrin adaptors, and some of these proteins appear to function as adaptors as well, either interacting with cargo, clathrin and/or clathrin adaptors, membrane lipids, the cytoskeleton, and/or molecules in intracellular signaling pathways. These proteins are also likely to exist in parietal cells to provide an additional, critical level of regulation of vesicle trafficking and membrane protein sorting for linking clathrin-based machinery to cargo, the cytoskeleton, and/or signaling molecules, links that are essential for the integration of vesicle trafficking with intracellular signaling and the cytoskeleton. The presence of clathrin at various intracellular sites in the parietal cell suggests that putative cargo molecules, such as

THE CELL BIOLOGY OF GASTRIC ACID SECRETION / 1215 the H,K-ATPase, possess sorting motifs to interact with clathrin adaptors. Evidence for an internalization motif in H,K-β was provided by a transgenic mouse model, in which a cytoplasmic tyrosine residue within a putative consensus motif in H,K-β was mutated to an alanine (250). These mice experienced development of histologically observable pathologies consistent with a hypersecretory phenotype, supporting the possibility that the H,K-ATPase could not be endocytosed. The H,K-ATPase may also interact with other membrane proteins to regulate its trafficking. It has been shown to interact with CD63, a membrane protein with a steady-state distribution in fibroblast cells that is in the endosomal-lysosomal system, and is present in tubulovesicles (251). In fibroblasts, the trafficking of CD63 is regulated by a cytoplasmic tyrosine-based motif, and when coexpressed with H,K-β, changes the subcellular distribution of H,K-β, from a predominantly cell-surface distribution to an intracellular one. This distribution is dependent on an intact tyrosinebased motif in CD63. However, the physiologic role of CD63 in H,K-ATPase trafficking has not been established.

The role that clathrin plays in the regulation of intracellular trafficking in the parietal cell is not yet characterized. However, the observation that numerous clathrin-coated membranes are present on canalicular and tubulovesicular membranes in resting parietal cells suggests that even in the resting state, there is active trafficking between these two membrane domains. It also suggests that regulation of HCl secretion may involve the regulation of the relative rates of exocytosis and endocytosis, rather than a simple “on-off ” process. It is also consistent with a model in which, on the cessation of HCl secretion, there is a massive, phagocyticlike process resulting in the internalization of much of the canalicular membrane, and therefore most of the H,KATPase, and that the clathrin-based machinery spends the time between secretory phases in a proofreading function. For example, residual H,K-ATPase remaining at the canalicular membrane after phagocytosis will be internalized via AP-2 and clathrin, and t-SNAREs that were internalized will be recycled via AP-1 and clathrin. Figure 48-18 presents a model incorporating these possibilities.

FIG. 48-18. Clathrin-dependent trafficking in resting parietal cells. Clathrin-coated membranes are localized to the canalicular membrane and to tubulovesicular membranes. In addition, clathrin-coated vesicles are frequently observed beneath the canalicular membrane. F-actin is shown lining the microvilli and in association with a clathrin-coated vesicle forming the canalicular membrane. The assignment of membrane identities, such as “endocytic vesicle” and “recycling endosome,” are speculative, as are some of the membrane proteins that may be found associated with each. The trafficking patterns are also speculative, representing primarily “proofreading” or “degradative” pathways in the resting cell. In the proofreading pathway, canalicular membrane proteins that may have been internalized on cessation of parietal cell secretion, such as syntaxin 1, would be recycled to the apical membrane. Concomitantly, tubulovesicular membrane proteins left at the canalicular membrane, such as the H,K-ATPase and vesicle-associated membrane protein 2 (VAMP-2), are endocytosed and sorted to recycling or “storage” endosomes. K+ and Cl− channels required for HCl secretion may also be sorted in such fashion. The parietal cell would be actively engaged in these processes between secretory periods. In the degradative pathway, internalized membrane proteins are sorted to multivesicular bodies and lysosomes. SNAP, soluble N-ethylmaleimide–sensitive factor attachment protein.

1216 / CHAPTER 48 Dynamin Dynamin is a large GTPase with activity that regulates vesicle fission (252). It polymerizes around the neck of the budding vesicle, but the precise mechanism by which dynamin catalyzes vesicle fission remains unresolved. It might act as a mechanochemical enzyme, in which GTP hydrolysis is coupled to conformational changes that would result in the separation of the vesicle from the donor membrane. Alternatively, it may act as a classical GTPase, in which other effector, vesicle-scission proteins are recruited to the neck of the budding vesicle when dynamin is in the GTP-bound form. Dynamin also regulates the formation of F-actin–rich structures at the plasma membrane, such as lamellipodia, podosomes, and actin comet tails, apparently independently of its regulation of endocytosis (253). In addition to its GTPase domain and an intramolecular GTPase-activating (effector) domain, dynamin contains a pleckstrin homology domain for interaction with phosphatidylinositol lipids and a carboxy-terminal proline-arginine–rich domain (PRD) for interaction with proteins containing SH3 domains. Numerous SH3 domain–containing proteins that bind to the PRD of dynamin have been identified, and their interaction with dynamin regulates endocytosis or the actin cytoskeleton, or both. In fact, as mentioned earlier, Lasp-1, which contains a carboxy-terminal SH3 domain, binds to the PRD of dynamin in vitro (208), raising the intriguing possibility that Lasp-1 may regulate dynamin function or vice versa. Dynamin has been identified in parietal cells (218,247). It appears to be expressed at significantly greater levels in parietal cells compared with nonparietal cells (247). It is concentrated at the canalicular membrane in resting parietal cells and colocalized with clathrin and F-actin, a distribution consistent with its known regulatory function in endocytosis or the F-actin cytoskeleton, or both. However, the function of dynamin in the regulation of H,K-ATPase recycling has yet to be characterized, with either of its functions as a regulator of endocytosis or the actin cytoskeleton as possibilities. Other GTPases Arfs are another family of small, Ras-like GTPases. There is clear evidence that Arf1 and Arf6 regulate membrane trafficking at the Golgi, endosomes, and plasma membrane (254–256). In addition, Arf6 regulates F-actin–based processes, such as neurite outgrowths (257,258). Arf 6 is expressed in parietal cells, and at significantly greater levels than in nonparietal cells (259). The intracellular distribution of Arf6 changes from predominantly cytoplasmic to predominantly canalicular on stimulation of the parietal cell, coinciding with the change in distribution of H,K-ATPase. Moreover, in permeabilized parietal cells, the intracellular distribution of Arf6 among cytosol, tubulovesicles, and canalicular membrane can be altered by altering the cycling of Arf6 through its GTPase cycle, with the GTP-bound form associating with membranes. Adenoviral-mediated overexpression of a GTPase-defective mutant of Arf6 in primary

cultures of gastric glands results in an inhibition of HCl secretion. One model for Arf6 regulation of HCl secretion consistent with these data is that, in the GTP-bound form, Arf6 associates with tubulovesicles to prevent fusion, but may translocate with tubulovesicles to the canalicular membrane. On GTP hydrolysis, Arf6 may catalyze fusion, and then dissociate from membranes to the cytosol. Similar to the situation with Rab proteins, identification and characterization of Arf6interacting proteins and effectors will provide important keys in the characterization of Arf6 function in HCl secretion. Significant progress has been made in the characterization of the subcellular machinery involved in the regulation of membrane transformations occurring during the secretory cycle of the parietal cell. Clearly, the components characterized thus far support the notion that the machinery in this specialized cell is more similar to, rather than distinct from, that present in all cells. As additional proteins are characterized, the expectation is that they will also be expressed in other cell types. The well-characterized function and secretory cycle of the parietal cell provides a framework with which to characterize the role of these proteins in the regulation of H,K-ATPase trafficking and HCl secretion. In addition, the relatively high endogenous level of expression of many of these proteins may provide a unique opportunity to characterize the role of these proteins in a cell biological context.

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CHAPTER

49

Regulation of Gastric Acid Secretion Arthur Shulkes, Graham S. Baldwin, and Andrew S. Giraud Functional Anatomy, 1223 Structure, 1223 Innervation, 1225 Vasculature, 1225 Regulation of Gastric Acid Secretion, 1225 Cell Biology, 1225 Integrated Control of Gastric Acid Secretion, 1238

Disorders of Gastric Acid Secretion, 1246 Increased Gastric Acid Secretion, 1246 Decreased Gastric Acid Secretion, 1246 References, 1247

It was not until the seventeenth century that physicians began to accept that the stomach contained acid and that the acid aided in the digestion of food (1). In the early nineteenth century, the English physician William Prout demonstrated that hydrochloric acid (HCl) was the major component of gastric juice (2). In 1930, Dodds and Robertson (3) showed that HCl was the only acid produced by the stomach, and that the other detected acids such as lactic acid were the product of fermentation. Studies on the regulation of gastric acid secretion were most famously described by Beaumont (4) from observations on a patient with a gastric fistula. The regulatory factors gastrin (5), histamine (6), and vagally released acetylcholine (1) were described in the early part of the twentieth century, and the characteristics of the gastric H+,K+-ATPase required to drive the secretion of HCl (7) were elucidated in the latter part of the twentieth century. Figure 49-1 presents a current view on the regulation of gastric acid secretion and should be consulted throughout the reading of this chapter.

FUNCTIONAL ANATOMY

A. Shulkes, G. S. Baldwin: Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia. A. S. Giraud: Department of Medicine, University of Melbourne, Western Hospital, Footscray 3011, Australia.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Structure Functionally, the gastric mucosa is divided into the proximal oxyntic gland area and the distal antral or pyloric gland area (8). The oxyntic mucosa comprises the proximal 80% of the stomach (body and fundus) and secretes acid, whereas the distal part (antrum) synthesizes, stores, and secretes gastrin. The gastric mucosal surface is lined with surface mucous cells that secrete the mucus that protects the mucosa against acid, pepsin, ingested substances, and pathogens. The functional units of the stomach are the gastric glands (Fig. 49-2), which access the mucosal surface via gastric pits that collect the secretions from four to five glands. The top of the glands contains mucous surface cells and mucous neck cells. The predominant cell type lining the gastric glands is the parietal cell, which secretes HCl. The next most abundant cell type is the pepsinogen-secreting chief cell (8,9). The somatostatincontaining D cell is an important component, and these cells have long processes that surround the parietal cell and mediate the inhibitory paracrine effect of somatostatin on gastric acid secretion (10). Other endocrine cells defined by their product are also present, for example, the serotonincontaining enterochromaffin cell. Histamine-containing enterochromaffin-like (ECL) cells are located in the basal part of the gastric glands (11).

1223

1224 / CHAPTER 49 Stress Sensory stimuli

Vagus N.

Sympa th

c N. eti Gastric pit

Oxyntic gland

Antral gland

ACh

ACh

CCKB

M

D

M CCKA

M

VPAC1

Lumen

P M3

M

CCKB

SST2

ECL PAC1

M

Gal1

SST2

M

GRPR

GRP

D

CCKB Y1

M

G

M

H+

Lumen pH

H2

Phe, Trp, Tyr pH

ACh

SMS Gastrin

PYY Galanin

ENS

ENS

VIP CCK

Histamine

SMS

FIG. 49-1. Cephalic (neural) and peripheral (gastric and intestinal) endocrine and paracrine regulation of gastric acid secretion. ACh, acetylcholine; CCKA and CCKB (alternative nomenclature CCK-1 and CCK-2, respectively), cholecystokinin type A and B receptors; D, D cell; ECL, enterochromaffinlike; ENS, enteric nervous system; G, G cell; Gal1, galanin type 1 receptor; GRP, gastrin-releasing peptide; GRPR, GRP receptor; H2, histamine type 2 receptor; M cell, mucous cell; M, muscarinic receptor; M3, muscarinic receptor type 3; P, parietal cell; PACAP, pituitary adenylyl cyclase–activating polypeptide; PYY, peptide YY; SMS, somatostatin; SST2, somatostatin type 2 receptor; VIP, vasoactive intestinal polypeptide; Y1, PYY type 1 receptor. Lines depicting inhibitory effects are fall in pH, SMS, PYY, ACh on the D cell in the oxyntic gland. The remaining lines indicate stimulatory effects. (Reproduced from Wank SA. PACAP upsets stomach theory. J Clin Invest 1999;104:1341–1342, by permission.)

REGULATION OF GASTRIC ACID SECRETION / 1225 Gastric gland

MSC

Thus, agonists such as histamine, gastrin, and acetylcholine increase blood flow, whereas inhibitors of secretion (catecholamines, atropine, and somatostatin) decrease blood flow. The mechanism may be either via direct action on the vasculature or via the changes consequential to the release of vasoactive metabolites during an alteration in secretion.

MNC

REGULATION OF GASTRIC ACID SECRETION ECL cell

Cell Biology D cell

Parietal Cell PC

CC

FIG. 49-2. Schematic representation of an oxyntic (gastric) gland. Mucous surface cells (MSC), mucous neck cells (MNC), enterochromaffin-like cells (ECL), somatostatincontaining D cells (D cell), parietal cells (PC), and chief cells (CC) are shown. (Reproduced from Lloyd and Debas [443], by permission.)

Innervation A detailed functional description of the innervation of the gastrointestinal tract is found in Chapters 21, 24, and 25. Extrinsic innervation of the stomach is by both the parasympathetic (vagus) and the sympathetic nervous systems. Both afferent and efferent traffic is carried. The majority of the vagal fibers and 20% of the splanchnic nerve fibers are afferent. Afferent fibers respond to changes in gastric distension via mechanoreceptors and gastric contents via chemoreceptors and transmit the information to central sensory nuclei that initiate gastric reflexes. The enteric nervous system also initiates local reflexes, and these can act in concert with the centrally integrated reflexes. Activation of the vagal efferents increases gastric secretion, motility, and blood flow, whereas sympathetic stimulation tends to decrease gastric secretion and gastric blood flow (12).

Vasculature The anatomy and regulation of gastrointestinal blood flow are described in Chapters 31 and 63. Blood supply to the gastric mucosa is from branches of the celiac artery (in particular, the common hepatic, left gastric, and splenic arteries). Blood flow tends to increase in proportion to gastric secretion.

The principal function of the parietal cell is to secrete HCl, which serves to sterilize the stomach lumen, thereby preventing, for the most part, bacterial colonization, as well as providing a permissive environment for activation of the zymogenic form of the protease pepsin. That HCl secretion is an active, energy-dependent process explains the large numbers of mitochondria in parietal cells. Indeed, the parietal cell has the largest content of mitochondria of any mammalian cell, and most mitochondrially generated ATP is used for acid secretion (7). The detailed cell biology of the parietal cell is described in Chapter 48. HCl is formed by the actions of H+,K+-ATPase, which is a specialized membrane-bound proton pump on the apical membrane of the parietal cell. The proton pump is a α,β heterodimer composed of a catalytic α-subunit of 1033 amino acids and a smaller, glycosylated β-subunit of 290 amino acids (13). The proton pump can secrete H+ against a concentration gradient greater than 106. H+ ions are secreted by the proton pump in exchange for K+ ions, and for each proton that is secreted, an intracellular OH− is generated. The OH− is converted to HCO3− by carbonic anhydrase and is exchanged for Cl− at the basolateral membrane. Activation of the parietal cell by neural, paracrine, and endocrine pathways that converge on receptors in the basolateral membrane results in the secretion of HCl into the stomach. Stimulation of secretion involves a profound morphologic transformation with the tubulovesicular membranes decreasing and the development of an intracellular canalicular system (14). On stimulation, the proton pump translocates from the tubulovesicles to the membranes of the secretory canaliculus (7,14). Enterochromaffin-like Cell This section describes the morphology of the ECL cell and its role in regulating gastric acid secretion. The actions of neuronal, endocrine, or paracrine stimulators or inhibitors of ECL cell activation, as well as of factors that regulate ECL cell growth, are described. Next, the main secretory products of the ECL cell are described with special emphasis on the synthesis and secretion of histamine, the primary acid secretagogue. Finally, the modulatory effect of Helicobacter pylori infection on ECL function is reviewed briefly.

1226 / CHAPTER 49 A number of detailed reviews of ECL cell structure and function have been published since Hakanson and colleagues’ (15) comprehensive chapter devoted to this cell type appeared in the previous edition of this textbook. These specialist reviews relate to regulation of ECL function by gastrin (16–18), histamine control of acid secretion (19), and ECL physiology (20–23), and a 1998 issue of the Yale Journal of Biological Medicine (Volume 71, Issue 3-4) was devoted to the biology of ECL cells. Enterochromaffin-like Cell Distribution and Morphology ECL cells were first recognized as discrete, nonargentaffin, argyrophil-positive cells of the oxyntic (fundic) stomach nearly 60 years ago (24). The name ECL cell was coined to distinguish these cells from the morphologically similar enterochromaffin cells (25). Numerically, ECL cells are the principal endocrine cell type of the stomach. They constitute 65% and 35% of the total oxyntic endocrine population in rats and in humans, respectively (26). They are “closed-type” endocrine cells because their cytoplasmic processes do not access the gastric gland lumen (27), indicating an involvement in paracrine and endocrine regulation of gastric function (28). ECL cells

have been found in all mammalian species examined, as well as in nonmammalian vertebrates and protochordates (29). This phylogenetic conservation emphasizes their importance in regulating gastric acid secretion. Apart from their characteristic silver-staining profile (argyrophil-positive, argentaffin-negative), ECL cells can be detected by immunohistochemical and immunofluorescent staining of specific markers including histamine and its synthetic enzyme histidine decarboxylase (HDC), as well as the secreted products chromogranin A and pancreastatin, and molecules characteristic of the secretory apparatus including vesicular monoamine transporter type 2 (VMAT2) (30). Ultrastructurally, ECL cells display a unique mix of variably sized and electron-opaque secretory granules and vesicles, as demonstrated in Figure 49-3 (31). Vesicles are numerous and heterogeneous in size, ranging from 200 to 600 µM, and may contain an eccentric granular core (32,33). The electron-lucent region of such vesicles has been shown to store histamine (34), although in hypergastrinemic rats treated with lansoprazole, immunoelectron microscopy localized histamine to both granule cores and secretory vesicles (35). Other profiles show vesicles containing electrondense granular or filamentous material detached from the vesicle membrane, or small, dense granules. Pancreastatin has

FIG. 49-3. Electrophotomicrograph of an enterochromaffin-like (ECL) cell. The ECL cell is surrounded by other epithelial cells. The ECL cell displays typical secretory granule ultrastructure including large electron-lucent vacuoles (long arrows) and small, dense-cored granules (short arrows), as well as prominent lipofuscin bodies (L). BL, basal lamina. Original magnification ×10,400. (Reproduced from Giraud and colleagues [31], by permission.)

REGULATION OF GASTRIC ACID SECRETION / 1227 H

mac

Epi

Ca2+ +

PGE1,2

β-adr

H

+

EP3 HDC

Ca2+

IL-1β −

IL-1β IL-1r

VMAT2

ChA

−

+

Gastrocalcin + FGF-r

bFGF +

EGF-r

PACAP

+ +?

PAC1

− −?

−

TGFα ECL cell Y1

SS SS

PYY

Reg 1α

SST2 D cell

PYY

CCK2 G

Gal-r1 Gal

G

Reg1-r ?

Reg 1α

FIG. 49-4. Regulation of enterochromaffin-like (ECL) cell activation. The inhibitory and stimulatory inputs by endocrine, neuronal, autocrine, and paracrine regulators of ECL cell activation, as well as local and endocrine action of ECL secretory products are shown. Physiologically characterized stimulatory inputs include gastrin (endocrine), and pituitary adenylyl cyclase–activating polypeptide (PACAP) (neuronal). Inhibitory inputs include somatostatin (paracrine), galanin (neuronal), interleukin-1β (IL-1β) (paracrine), and prostaglandins (paracrine/autocrine). Epinephrine and norepinephrine acting via β-adrenergic receptors (β-adr) augment histamine release. Peptide YY (PYY) inhibitory and transforming growth factor-α (TGF-α) stimulatory regulation have not been fully characterized. Basic fibroblast growth factor (bFGF) and TGF-α act as autocrine growth factors, and regenerating protein 1α (Reg 1α) acts as a local oxyntic stomach mitogen. Gastrocalcin may be an endocrine factor mobilizing calcium uptake into bone. ChA, chromogranin A; CCK-2, cholecystokinin 2 receptor; Epi, epinephrine; G, gastrin; Gal, galanin; Gal-r1, galanin receptor 1; H, histamine; HDC, histidine decarboxylase; Mac, macrophage; PGE, prostaglandin E; PYY, peptide tyrosine tyrosine; Y1, peptide tyrosine tyrosine receptor 1; Reg1-r, regenerating protein 1 α receptor; SS, somatostatin; SST2, somatostatin type 2 receptor; VMAT2, vesicular monoamine transporter 2.

been reported to localize to both dense granules and secretory vesicles (34), whereas its precursor chromogranin A is localized to both granular and nongranular subcellular compartments (36). Lysosomal degradation of vacuoles causing prominent lipofuscin bodies is common in stimulated ECL cells (30) (see Fig. 49-3). Regulation of Enterochromaffin-like Cell Activation and Growth ECL cell activation may be defined as the actions of regulatory factors that stimulate or inhibit histamine synthesis or secretion; the release of chromogranin A and its proteolytic product pancreastatin also are relevant, but histamine is the only product from the ECL cell that directly stimulates acid

secretion by the parietal cell. The regulation of ECL cell activation and growth is summarized diagrammatically in Figure 49-4. Regulators of ECL cells are considered in turn in the following sections. Gastrin. The proliferation and activation of the ECL cell is driven primarily by circulating gastrin after binding and initiation of signal transduction via the cholecystokinin 2 (CCK-2) receptor (37). Early studies in which circulating gastrin level was increased by gastrin infusion, partial fundectomy, antral isolation from acid, inhibition of acid secretion by H2 receptor antagonists, or proton pump inhibitors, demonstrated a stimulatory effect on ECL cell hypertrophy (38) and hyperplasia (39–45). More recent data supporting this conclusion and clarifying the mechanisms by which stimulation occurs have been derived using

1228 / CHAPTER 49 specific gastrin receptor antagonists (46), mutant mice that lack gastrin (47–50) or the gastrin (CCK-2) receptor (51,52), as well as gastrin-overexpressing transgenic mice (53–55; see also review by Samuelson and Hinkle [56] and Chapter 51). Thus, gastrin loss by homologous recombination results in oxyntic atrophy and impaired acid secretion caused by reduced numbers of ECL cells (47) or loss of ECL cell function in terms of HDC activity and histamine production, chromogranin A expression, and impairment of secretory vesicle production (48). Similar outcomes are observed in CCK-2 receptor null mice (51,52). Conversely, the phenotypic outcomes of gastrin infusion or H2 receptor blockade (i.e., ECL cell hyperplasia and hypertrophy) are recapitulated in transgenic mice that overexpress gastrin because of chronic expression of a human gastrin transgene (53,55), or mice that are hypochlorhydric and hypergastrinemic either because of a null mutation of the H2 receptor (57,58) or of HDC (59). These observations confirm the pivotal role that gastrin plays in stimulating ECL cell growth and secretory activity. Somatostatin. Somatostatin is a potent inhibitor of gastric acid secretion. Inhibition is accomplished in part by directly and indirectly suppressing ECL cell activity (60). A longacting release formulation of the somatostatin analog octreotide was shown to inhibit both basal- and gastrinstimulated ECL cell function (in terms of a reduction of chromogranin A and HDC messenger RNA [mRNA], ECL cell density, and plasma histamine) in the rat (61). Highly enriched isolated rat ECL cells have been used to demonstrate that picomolar concentrations of somatostatin effectively inhibit pituitary adenylyl cyclase–activating polypeptide (PACAP) and gastrin stimulation of ECL cell histamine and pancreastatin secretion (62). Currently, the true physiologic relevance of somatostatin regulation of ECL cell function in species other than the rat has not been clarified. However, the proximity of paracrine D and ECL cells in many species and the demonstration of somatostatin 2 (SST2) receptors on ECL cells (63,64) make a physiologic role for somatostatin likely. Pituitary Adenylyl Cyclase–Activating Polypeptide. PACAP is a neuropeptide and member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family that is expressed by cell bodies of the myenteric plexus of the stomach that project to both the muscle and mucosa (65,66). There are two bioactive forms of PACAP, 27 and 38 residues (67). PACAP acting through the PAC1 (PACAP-specific) receptor can mobilize ECL cell histamine at picomolar concentrations in isolated cell preparations (62) and can stimulate HDC promoter activity leading to increased histamine synthesis (68) and stimulation of gastric acid secretion. In addition, PACAP stimulates ECL cell proliferation more potently than gastrin (68,69). Peptide YY. Peptide YY (PYY) is a member of the pancreatic polypeptide/neuropeptide Y (PP/NPY) family. The bioactive peptide PYY36 was initially isolated from the porcine duodenum (70). PYY36 acts as an endocrine inhibitor of gastric acid secretion in a variety of species (summarized by Zeng and colleagues [71]) because it is released into the

peripheral circulation in response to a meal or intestinal infusion of amino acids (72,73). In a comprehensive study in the rat, a direct inhibitory effect of PYY on the isolated ECL cell, mediated by activation of the Y1 receptor, was demonstrated (71). PYY effectively inhibited gastrin-stimulated histamine release and calcium entry and signaling at nanomolar concentrations and in a pertussis toxin–sensitive fashion suggesting Gi,o-protein coupling. This inhibition was later shown to be mediated by inhibition of L-type Ca2+ channels (74). Although Y1 receptors are expressed by ECL cells, studies using highly purified (~90%), stabilized ECL cell cultures (62,75) and implanted perfused microdialysis probes in conscious rats (76) have suggested that exogenously applied PYY is effective in transiently mobilizing histamine in vivo, but is weak or ineffective in vitro (62,75). Until these discrepancies can be resolved, the physiologic significance of a direct effect of PYY on ECL cell activation remains unclear. Galanin. Galanin is a 29-amino-acid peptide first isolated from the porcine intestine (77) and subsequently shown to be expressed in both central and peripheral neurons (78,79). The GalR1 receptor complementary (cDNA) has been cloned from the human oxyntic stomach (80) and specific GalR1 mRNA detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in highly purified rat ECL cell preparations (81). Galanin has been shown by several groups to inhibit stimulated gastric acid secretion in the rat (82,83), dog (84), and human (85). These actions are likely to be both indirectly via inhibition of gastrin release from antral G cells (86–88) and directly on the rat ECL cell, because galanin at physiological concentrations inhibited both basal- and gastrin-stimulated histamine release (62,75,81), and its agonist activity could be blocked with the specific GalR1 receptor antagonist galantide (81). The direct effect of galanin occurs in a neuroendocrine or paracrine fashion because galaninpositive nerve terminals are localized in close proximity to ECL cells in the rat stomach (89). These inhibitory actions of galanin on the ECL cell have been reproduced in vivo using implanted, perfused microdialysis probes (76). Transforming Growth Factor-α. Transforming growth factor-α (TGF-α) is expressed by gastric parietal cells of several species (90–92) and has an autocrine inhibitory effect on gastric acid secretion (93). Its synthesis is stimulated during the early phase of mucosal repair after experimentally induced ulceration (94). TGF-α accelerates mucosal ulcer healing by increasing cell migration (restitution) and epithelial cell proliferation after binding the epidermal growth factor (EGF) receptor (95). Isolated ECL cells express the EGF receptor, and paradoxically, TGF-α has been reported to stimulate expression of HDC, VMAT2, and histamine release in vivo and in vitro (94) and to inhibit cytokineinduced ECL cell apoptosis (96). Further in vivo studies comparing the temporal expression of TGF-α with ECL cell activation markers, acid secretion, and mucosal repair status are required to shed light on the physiologic role of TGF-α.

REGULATION OF GASTRIC ACID SECRETION / 1229 Interleukin-1β. Interleukin-1β (IL-1β is a proinflammatory cytokine that is produced in the gut and elsewhere by macrophages and monocytes during conditions of local inflammation induced by epithelial damage or H. pylori infection. IL-1β has been reported to inhibit gastric acid secretion in the rat (97). Because the IL-1β receptor is expressed by isolated rat ECL cells (98), and IL-1β itself can inhibit histamine synthesis and gastrin-stimulated histamine secretion (96,99) and the histamine-synthesizing enzyme, HDC (99), this cytokine may be important locally in limiting acid secretion during mucosal repair. Prostaglandins. Prostaglandins are local regulators produced by the actions of the cyclooxygenase enzymes on arachidonic acid. Although many cell types can synthesize prostaglandins, macrophages are an important source in numerous tissues including the stomach. It has been known for many years that exogenously applied prostaglandins effectively inhibit gastric acid secretion (100) apart from their well-known cytoprotective effects. Both the parietal (101,102) and ECL cells (103,104) are targets for prostaglandin action, which may have an autocrine component because both cell types also synthesize prostaglandins in appreciable amounts (104,105). Both prostaglandin E1 (PGE1) and PGE2 and their analogs effectively inhibit gastrin- or PACAP-stimulated histamine (104) and pancreastatin (103) release from isolated rat ECL cells at nanomolar to picomolar concentrations with little effect on basal secretion. Although small numbers of D cells were present in ECL cell cultures, the inhibitory effect was independent of somatostatin because application of neutralizing antisomatostatin antisera had no effect on prostaglandin-induced inhibition (103). The rank order of inhibitory potency of various prostaglandins and analogs suggests action through EP3 receptors (103). Both EP2 and EP3 receptor mRNA and 3H-PGE2 binding sites have been demonstrated on membranes from ECL cell carcinoids from the African rodent Mastomys natalensis (106). Epinephrine and Norepinephrine. Epinephrine and norepinephrine bind β-adrenergic receptors, thereby regulating cyclic adenosine monophosphate (cAMP)–dependent signaling in target cells. Epinephrine and norepinephrine have been reported to stimulate ECL cells to release histamine in vivo (76) independently of HDC activation (107). Epinephrine or isoproterenol acting via β2-adrenergic receptors produce similar effects in vitro with isolated ECL cell preparations (62,98,108). Secretory Products of the Enterochromaffin-like Cell: Histamine Comprehensive reviews of the physiology of histamine synthesis, secretion, and influence on acid secretion have been published in the late 1990s and early 2000s (19,20,22,23,109). Synthesis. Histamine is an amine neurotransmitter in neurons and a paracrine regulatory product of the ECL cell. ECL cells of all mammalian species studied in detail are the main sources of gastric histamine (11,23), the prime agonist mediating gastric acid secretion (110). Gastric mast cells

also synthesize histamine; however, this pool is not regulated by gastrin and does not affect gastric acid secretion (59). Individual ECL cells store ~3 to 10 pg histamine, and synthesis is both rapid and finely tuned to maintain cellular stores (98). Histamine is synthesized by decarboxylation of L-histidine by HDC (111). The observation that L-histidine uptake is blocked by specific HDC inhibition (112), but not by cycloheximide (a protein synthesis inhibitor), suggests that histidine uptake is destined for histamine, not protein, synthesis (23). HDC is a cytoplasmic enzyme (113), and histamine synthesis occurs in the cytoplasm before transport and packaging into secretory granules. The cDNA of HDC has been cloned from the rat (114), mouse (115), and human (116). The enzyme can be activated rapidly by numerous stimuli including feeding, vagal stimulation (23,111), TGF-α (94), gastrin (117), and PACAP (68). Gastrin and to a lesser extent PACAP are probably the most physiologically relevant local regulators of HDC expression, and although both enhance HDC transcription, they do so via distinct mechanisms (68). Gastrin has been shown to stimulate HDC transcription by activating one or more of three separate gastrin response elements (118,119) linked to mitogen-activated protein kinase (MAPK) signaling pathways (118). Kruppel-like factor 4 has been shown to bind gastrin response elements to repress HDC expression (120). Other gastrin-linked response elements in the HDC promoter are linked to transcription factors including activator protein-1 (AP-1) (121), GATA4, and GATA6 (122). In addition to regulating HDC transcription, gastrin can also stabilize HDC enzyme isoforms by interacting with PEST domains to prevent HDC degradation at the proteasome (123). PACAP, in contrast, regulates the HDC promoter via a discrete response element linked to adenyl cyclase and phospholipase C–dependent signaling pathways (68). The importance of HDC in regulating histamine synthesis in the ECL cell and ultimately gastric acid secretion is underscored by the diversity of endocrine, paracrine, and neural pathways regulating its expression, as well as the informative phenotype of the HDC-deficient mouse (59,124–127). When fasted or kept on a histamine-free diet, these mice were deficient in histamine and showed depressed basal acid secretion and increased intragastric pH (59,125). In addition, HDC-deficient mice were hypergastrinemic (59,126,127), resistant to exogenous gastrin, and partially or completely resistant to carbachol-stimulated parietal cell activation (125–127). ECL cell granule contents are acidic as determined by acridine orange staining (128). A V-type ATPase (129) regulates granule acidity by establishing a proton gradient with histamine and chromogranin A accumulating in granules in exchange for the counter ion H+ (98,130) and finally being transported into the granule by the vesicular monoamine transporter VMAT2 (130). VMAT2 has been cloned from brain or adrenal gland of a number of mammalian species including the rat (131), mouse (132), human (133), and cow (134). In rat oxyntic stomach, a single 4-kb mRNA species

1230 / CHAPTER 49 corresponding in size to brain VMAT2 has been detected and the corresponding cDNA cloned (135). Apart from the localization of the VMAT mRNA and protein (136–138), there is now good functional evidence that VMAT2 is the histamine transporter in the ECL cell. For example, several groups have demonstrated that gastrin induces the promoter activity and expression of VMAT2 in gastric ECL cells in a cAMP-dependent manner, although the exact role of SP1 elements in gastrin activation is in dispute (139,140). In addition, reserpine-induced depletion of histamine uptake into rat ECL cells inhibits VMAT2 activity; it also inhibits HDC activity and prevents gastrin activation (141). Secretion. Histamine is secreted from the ECL cell in response to receptor binding by ligand, followed by calcium influx and exocytosis of histamine into the extracellular environment (20,109,142). After secretagogue binding, secretion depends on both extracellular Ca2+ influx and also Ca2+ release from cytoplasmic stores (109). In the case of gastrin, Ca2+ influx occurs through voltage-dependent L-type and N-type Ca2+ channels, whereas for PACAP, influx is via L-type and receptor-operated Ca2+ channels (143). Studies in isolated ECL cells have shown that gastrininduced histamine secretion is characterized by a biphasic Ca2+ response with a sharp increase in intracellular Ca2+ followed by a decline and plateau at a lower concentration that is maintained by an influx of extracellular Ca2+. This plateau phase is important for secretion to proceed because the addition of calcium chelating agents or a calcium channel blocker at this time blocks histamine release (20,98). PACAP also induces a biphasic calcium signal associated with histamine release (144), suggesting a common secretory mechanism for histamine secretion driven by either endocrine or neuronal agonists. The process of exocytosis is highly regulated and is mediated by the N-ethylmaleimide–sensitive factor attachment (SNARE) proteins (145). The v- (vesicle) and t-type (target membrane) SNARE proteins synaptotagmin, synaptophysin, synaptobrevin (VAMP), and syntaxin and synaptosomal associated protein 25 (SNAP-25) have been localized immunochemically to rat and human ECL cells (142,146,147). The exocytotic process appears to be common to most cell types and initially involves synaptotagmin I, a secretory vesicle membrane protein that senses and binds calcium (Ca2+), and mobilization of several plasmalemma proteins including synaptobrevin, syntaxin, and SNAP-25, which complex (148) and then form a fusion pore (145,149). All these proteins originally were characterized in other secretory systems (e.g., see reviews by Ungar and Hughson [145] and Sudhof [149]) and subsequently have been shown to be present in ECL cells (109,142). Once secretion has occurred, vesicle membranes are likely recycled because the ratio of electronlucent/electron-dense microvesicles is increased (34,150). The proteins dynamins 1, 2, and 3 and amphiphysins 1 and 2, which facilitate membrane recycling, have been demonstrated to be synthesized and expressed in ECL cells (151). Receptors and Action on Cellular Targets. Histamine binds H2 receptors on the parietal cell to evoke gastric acid secretion.

The Cellular Regulation section later in this chapter details the receptors involved and the regulation of this process. Secretory Products of the Enterochromaffin-like Cell: Proteins and Peptides The heterogeneous structure of the granules and vesicles of the ECL cell cytoplasm, as well as the multiplicity of regulators governing ECL cell function, indicate that numerous secretory products are likely to be produced by these cells. Although histamine is the most important physiologically and best understood for its effector function in regulating gastric acid secretion by the parietal cell, numerous peptide products also have been localized to ECL cells, and once secreted, are known to impact gastric function. These are summarized diagrammatically in Figure 49-4 and described below. Chromogranin A. Chromogranin A is an acidic, secretory protein (152) found in many (neuro) endocrine cells (153). In ECL cells, it is stored in both dense core secretory granules (154) and in a nongranular form (36). Like HDC and VMAT2, transcription of chromogranin A is regulated by gastrin (155). Chromagranin A is cosecreted with regulatory peptides and histamine and is thought to function as a facilitator of secretory granule packaging and as a chaperone in the secretory protein sorting pathway (156–159). There is debate whether chromogranin A acts as a master on/off switch for secretory granule biogenesis or as an assembly factor for producing functional granules (157,160). The separate and accepted function of chromogranin A is as a precursor for numerous bioactive peptide cleavage products (161). Chromogranin A has multiple prohormone convertase (PC) processing sites (162). Both prohormone convertase 1 (PC1) and carboxypeptidase E have been identified in ECL cells (147), suggesting that posttranslational processing is likely to generate a spectrum of bioactive products (163). The best candidates for physiologically relevant regulatory peptides include chromascin, parastatin, catestatin, the vasostatins, WE-14, and pancreastatin (153); only the last two have been convincingly characterized as ECL cell secretory products. Pancreastatin. Pancreastatin is a bioactive proteolytic cleavage product of chromogranin A (164), which like its precursor, occurs in many endocrine cells. It is secreted from ECL cells in response to gastrin (165) or PACAP stimulation. ECL cell–derived pancreastatin accounts for most of the measurable circulating peptide (166). Pancreastatin has been shown to inhibit acid secretion by the parietal cell (167), insulin and amylase release from the pancreas (164,168), and parathyroid hormone release (169,170). Pancreastatin binding activity has been purified from rat liver membranes (171), and the pancreastatin-binding protein is thought to be G protein–coupled receptor (170). WE-14. WE-14 is a peptide derived from proteolytic cleavage of chromogranin A in different chromaffin cells including the ECL cells of the oxyntic stomach (172). The observation that rats treated with reserpine, which irreversibly blocks histamine uptake (and therefore inhibits secretion),

REGULATION OF GASTRIC ACID SECRETION / 1231 showed a marked increase in WE-14 staining mainly in ECL cells of the proximal stomach suggests stimulation of posttranslational processing of chromogranin A or cellular accumulation of WE-14 (173). Currently, the physiologic role for WE-14 and many of the other chromogranin A–derived processing products remains unknown. Regenerating Protein 1α. Regenerating protein 1α (Reg 1α) is one of a family of six peptides with overlapping distribution and functions in the gastrointestinal tract. It was originally cloned from the pancreas, where it acts as a local mitogen and promoter of repair (174). Subsequently, Reg 1α was found to be expressed in the stomach (175) and localized to ECL cells from which it is secreted. Reg 1α acts as a growth factor for other gastric epithelial cells (176,177) and as a differentiation factor for gastric progenitor cells including those in the parietal cell lineage (178). Reg 1α is regulated by gastrin (177,179) and also by IL-6 in a signal transducer and activator of transcription 3 (STAT3)–dependent fashion (180). Reg 1α overexpression is associated with gastric tumorigenesis in the mouse (181), and mutations in the Reg 1α gene that prevent secretion are associated with ECL cell carcinoma (179). Because Reg 1α is a potent mitogen that is positively regulated by gastrin and has numerous epithelial targets (182), it may be an important oxyntic growth factor particularly under hypergastrinemic conditions. Transforming Growth Factor-α. TGF-α mRNA (183) and protein have been detected by immunohistochemistry (96) in both normal rat ECL cells and in hyperplastic ECL cells from Mastomys after chronic loxtidine treatment (183). In M. natalensis ECL cells, TGF-α mRNA increased as hyperplasia developed. Because these cells express the EGF receptor and TGF-α treatment results in ECL cell proliferation (183), TGF-α probably acts as an autocrine growth factor for the ECL cells. Gastrocalcin. Gastrocalcin (184) is the name given to a yet to be isolated or cloned osteotropic factor(s) produced by ECL cells. Gastrocalcin can inhibit circulating calcium and mobilize calcium in bone and fibroblasts (185). This effect can be mimicked by fundectomy and is stimulated by gastrin, presumably by releasing gastrocalcin from ECL cells. Evidence supporting the existence of such a peptide factor includes the stimulation of Ca2+ in osteoblast cell lines by ECL cell granule/vesicle extracts, the activation of Ca2+-induced signaling in response to granule/vesicle extracts, and the abolition of activity after aminopeptidase proteolysis (185,186). The putative gastrocalcin is distinct from ghrelin because the latter does not mobilize Ca2+ (186) and acts independently of gastrin (187). Basic Fibroblast Growth Factor. Basic fibroblast growth factor (bFGF) is a heparin-binding protein that acts as a mitogen in many tissues. bFGF mRNA has been detected in human ECL cell tumors (188) and bFGF protein is found in normal ECL cells (28). Because bFGF can stimulate ECL cell proliferation and inhibit apoptosis, and because ECL cells express the bFGF receptor (96), bFGF, like TGF-α, may act as an autocrine growth factor for ECL cells and also promote their longevity.

Helicobacter pylori Infection and Enterochromaffin-like Cell Function It is well established that H. pylori infection of the stomach has a major impact on all aspects of gastric physiology including induction of inflammation, ulceration, and neoplasia. H. pylori–induced oxyntic gland atrophy leads to a reduction in parietal cell mass and acid secretion, with hypergastrinemia inducing ECL cell hyperplasia (189). Although ECL cells are without luminal contacts and are positioned in the gastric glands so that paracrine effects are optimized, a number of mainly in vitro studies have demonstrated that H. pylori–derived molecules can potently regulate ECL cell function. The physiologic relevance of these effects is unclear. However, they may be important in the context of gastric mucosal ulceration or by virtue of an indirect effect via another mucosal cell. One example of such regulation comes from the finding that H. pylori is associated with the production of Nα-methyl-histamine (190), an agonist at histamine H2 (191), H3, and H4 receptors (192). Histamine activation of pharmacologically identified H3 receptors (but not defined in terms of protein or mRNA/cDNA sequence) on ECL cells has been shown to inhibit HDC and histamine secretion (98,193). Reduced histamine secretion from the ECL cell might be expected to reduce acid secretion. However, the situation is complex because Nα-methyl-histamine also has been reported to stimulate acid secretion in human stomach (194) and by rabbit parietal cells via H2 receptors (195), and H. pylori infection is associated with increased circulating gastrin, an ECL cell activator. The production of an ECL cell activation inhibitor such as Nα-methyl-histamine by H. pylori may confer a distinct survival advantage particularly early in the infection cycle; however, the means by which its actions might be targeted to the ECL cell rather than the parietal cell remain to be defined. In contrast with the effects of H. pylori on ECL cell inhibition, other products of the bacterium activate ECL cells. Thus, H. pylori lipopolysaccharide dose-dependently stimulated histamine release from isolated ECL cells via its receptor CD14 (196), an effect that was inhibited by somatostatin but not by a CCK-2 receptor antagonist (197). In addition, an H. pylori–derived soluble factor that activates the HDC promoter downstream of a G protein–coupled receptor and the β-Raf/Rap1/mitrogen activated protein kinase kinase (MEK)/extracellular signal–regulated kinase (ERK) pathway also has been described (198). Cellular Regulation This section describes the receptors that are involved in the regulation of gastric acid secretion. The ligand-binding properties and structure of all known subtypes of each receptor are compared first. Detailed discussion of receptor signaling then is confined to those subtypes known to be present in gastric D, ECL, G, and parietal cells. Chapter 48 provides a comprehensive presentation of signal transduction pathways.

1232 / CHAPTER 49 Acetylcholine Receptors Ligand Binding. Acetylcholine receptors fall into two broad groups: the acetylcholine-gated cation channels stimulated by nicotine, and the seven-transmembrane domain receptors stimulated by muscarine (199). The latter group of muscarinic acetylcholine receptors contains five distinct members M1 to M5 (199). The affinity of all muscarinic acetylcholine receptors for carbachol is similar, with Ki values ranging from 31 µM for the M5 receptor to 346 µM for the M1 receptor, when expressed in baculovirus-infected insect cells (200). No single antagonist has high selectivity for any one muscarinic acetylcholine receptor subtype (199). For example, the Ki values for the “M1-selective” antagonist pirenzepine on the cloned human receptors expressed in CHO cells are 6.3, 224, 138, 37, and 89 nM for the M1 to M5 receptors, respectively (201). The corresponding values for the “M3-selective” antagonist darifenacin are 13, 77, 2.0, 22, and 5.4 nM, respectively (202). One promising development in the field of selective antagonists has been the purification of snake toxins (203). The most selective example is the MT7 toxin from Eastern green mamba venom (Dendraspis angusticeps), which recognizes only the M1 receptor (Ki for M1, 158 pM; Ki for M2-M5, >1 µM) (199). Structure. All five classes of muscarinic acetylcholine receptors belong to the seven-transmembrane domain receptor family. The M1 and M2 receptor cDNAs were both cloned in 1986 from porcine cerebral (204) and porcine cardiac (205) cDNA libraries, respectively. The rat M3, M4 (206), and M5 (207) receptor cDNAs were then isolated. Interestingly, the five receptors are not closely related in overall sequence, with the percentage identity to the M1 subtype being 44%, 55%, 46%, and 55% for the M2 to M5 subtypes, respectively. Most of the differences occur in the third intracellular loop. In contrast, species comparisons indicate significant similarities in M3 receptor sequences,

with the percentage identity between pig (96%), cow (95%), rat (92%), chicken (87%), and human shown in brackets (199). Sequences are also available from gorilla, chimpanzee, orangutan, mouse, chicken, and Caenorhabditis elegans. Because the genes encoding muscarinic acetylcholine receptors lack introns, splice variants are not expected. The molecular mass of the M1 receptor purified from porcine cerebral membranes was 70 kDa (208), indicating extensive glycosylation (Table 49-1). Gastric Expression. The muscarinic acetylcholine receptor agonist carbachol stimulates acid secretion via a direct effect on the parietal cell (209,210). Studies with muscarinic antagonists suggested that only the M3 subtype was involved (211–213). This conclusion was confirmed by PCR of only M3 receptor fragments with primers designed for all subtypes (213), and by the impairment of acid secretion in response to carbachol observed in M3 receptor–deficient mice (214,215). The identity of the subtype responsible for the residual 30% acid output in M3 receptor–deficient mice could be determined by study of crosses between M3 receptor– deficient mice and strains each lacking one of the other four receptor subtypes (215). Early reports suggested that carbachol treatment of gastric ECL cells resulted in histamine release (98). More recent studies with well-characterized preparations of rat gastric ECL cells (62), or by microdialysis in vivo (76), have not indicated any increase in histamine release in response to acetylcholine or carbachol. The observation that both H2 receptor–deficient (57) and HDC-deficient mice (125,126) retained some carbachol-stimulated acid secretion is consistent with the conclusion that gastric ECL cells lack functional acetylcholine receptors. Although several functional studies have suggested that gastric D cells express M3 receptors, the response depends on the species. In cultured canine fundic D cells, carbachol treatment inhibited pentagastrin-stimulated somatostatin

TABLE 49-1. Physical characteristics of human receptor subtypes that control acid secretion Receptor Acetylcholine Ca2+-sensing Gastrin GRP Histamine PACAP Somatostatin a The

Subtype M3 — CCK-1 CCK-2 GRP H2 H3 PAC1 VPAC1 SSTR2a SSTR2b

Sizea (amino acids) 460 1078 428 447 384 359 445 468 457 369b 356b

Predicted mass (Da) 51,420 120,673 47,841 48,419 43,198 40,098 48,671 53,313 51,547 41,332 40,007

Observed mass (Da) 70,000 220,000 85-95,000 74,000 — 59,000 70,000 60-65,000 63,000 72,000 —

References 208 234 249 251 333 334 357,358 520 368

sizes given are for the receptor precursors. In the case of the bovine PAC1 receptor N-terminal amino acid sequencing of the purified receptor indicates that the first 20 amino acids of the precursor are removed as a signal sequence. The calculated mass of the mature unglycosylated protein is 51,357. b The SSTR2a and SSTR2b splice variants are identical in sequence for the first 332 amino acids. CCK, cholecystokinin; GRP, gastrin-releasing peptide; PACAP, pituitary adenylyl cyclase–activating polypeptide; PAC1, type 1 PACAP receptor; SSTR2, type 2 somatostatin receptor; VPAC1, vasoactive intestinal peptide receptor 1.

REGULATION OF GASTRIC ACID SECRETION / 1233 release (216,217). However, in human antral D cells, carbachol treatment stimulated somatostatin release, and the effect was blocked by an M3 antagonist (218). The presence of muscarinic acetylcholine receptor on gastric antral G cells has also been a controversial issue. Carbachol has been shown to stimulate gastrin release from populations of rabbit and canine antral cells enriched in G cells, and antagonist studies have indicated that the receptor involved was of the M3 subtype (219,220). However, imaging of intracellular calcium did not show any response in enriched human G cells after treatment with methacholine (221). Signaling. In many cell types, agonist activation of the M3 muscarinic receptor activates phospholipase C, which hydrolyzes phosphatidyliositol-4,5-bisphosphate to inositol triphosphate and diacylglycerol (222). These breakdown products, in turn, mobilize Ca2+ from intracellular stores and activate protein kinase C (PKC), respectively (Fig. 49-5). The responses of gastric parietal cells to simulation of muscarinic acetylcholine receptor are apparently similar, because in isolated rabbit gastric glands, carbachol stimulated the release of Ca2+ from intracellular pools and Ca2+ influx across the plasma membrane (223). Carbachol also induced inhibitory κB (IκB) kinase in isolated canine gastric parietal cells, via intracellular Ca2+- and PKC-dependent pathways (224). Detailed reviews of muscarinic acetylcholine receptor signaling (222) and regulation (225) in other systems have been published.

Calcium-Sensing Receptors Ligand Binding. Although the distribution of calcium-sensing receptors is widespread, only one class has been described in mammals (226). Calcium-sensing receptors are abundant in the parathyroid glands and kidney, where they regulate parathyroid hormone secretion and calcium reabsorption from the renal tubules, respectively (226). Calcium-sensing receptors are also present in the gastric mucosa (227–229). Functional studies suggest that the affinity of the calciumsensing receptor for Ca2+ is approximately 2 mM (226,230), but the precise details of the Ca2+-binding site have not yet been defined. Agonists and antagonists of the calcium-sensing receptor have been developed (231). Agonists such as R-568 were initially developed for the treatment of hyperparathyroidism (231). A phase I trial of the effect of R-568 on gastrin secretion in healthy subjects reported increased serum gastrin in only one of six individuals (232). There have been no reports of the gastric effects of the antagonists such as NSP 2143 that were developed originally as a treatment for osteoporosis (231). Structure. The calcium-sensing receptor belongs to the seven-transmembrane domain receptor family. The nucleotide sequence encoding the calcium-sensing receptor, which was first cloned from a bovine parathyroid cDNA library (233), revealed the presence of a large 600-amino-acid N-terminal

M3 receptor

H2 receptor

Adenyl cyclase

Phospholipase C

PIP2

IP3 + DAG

Ca2+ mobilization

ATP

Protein kinase C

Cyclic AMP

Protein kinase A

FIG. 49-5. Signaling mechanisms for the M3 and H2 receptors that control acid secretion. The muscarinic acetylcholine M3 receptor and the histamine H2 receptor are taken as examples. Binding of acetylcholine to the M3 receptor activates phospholipase C, which hydrolyzes phosphatidyliositol4,5-bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG) (222). These breakdown products, in turn, mobilize Ca2+ from intracellular stores and activate protein kinase C, respectively. Binding of histamine to the H2 receptor activates both phospholipase C and adenyl cyclase (349). The latter enzyme converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (AMP), which, in turn, activates protein kinase A.

1234 / CHAPTER 49 extracellular domain (226). Sequences of the human, bovine, rat, and mouse receptors are now available. The solubilized receptor is a homodimer of 220,000-Da molecular mass (234), with disulfide bonds between extracellular domains (235). Mutations in the gene encoding the calcium-sensing receptor result in alterations in serum calcium concentrations (236). Both loss- and gain-of-function mutations in the gene have been described, but their effect on gastric acid secretion has not been reported. The availability of mice lacking the calcium-sensing receptor (237) should allow more detailed investigation of the gastric biology of this receptor. Gastric Expression. Cultured human antral G cells have been shown to express the calcium-sensing receptor (227). Receptor mRNA was detected by RT-PCR, and receptor protein by immunohistochemistry (227). Immunohistochemistry also shows calcium-sensing receptors in mucous, chief, and parietal cells in rat gastric glands (229), and in the case of parietal cells, this result has been confirmed by functional studies by fluorescence microscopy (238). Signaling. Multiple intracellular signaling pathways are affected by the calcium-sensing receptor (226,239). Activation of phospholipase A2, C, and D and inhibition of adenyl cyclase have all been demonstrated in bovine parathyroid cells. In cultured antral G cells, the observation that Ca2+-stimulated gastrin release was blocked by a phospholipase C inhibitor, but not by inhibition of adenyl cyclase, suggested that phospholipase C activation was more important in this cell type (230). These pathways have not been investigated in parietal cells. Gastrin/Cholecystokinin Receptors Ligand Binding. Two distinct receptors for amidated peptides of the gastrin/CCK family have been described (240,241). The CCK-1 receptor is found in most of the alimentary tract and discrete parts of the brain. The CCK-2 receptor is widely distributed in the brain and is also found in the stomach. Receptors binding nonamidated, progastrinderived peptides also have been recognized (242,243). Previously, it had been generally assumed that only amidated forms of gastrin were biologically active, but there is now abundant evidence that gastrin17gly and other nonamidated gastrins are able to stimulate proliferation of the normal colonic mucosa and gastrointestinal cell lines and to enhance gastrin-stimulated acid secretion (244). The CCK-2 receptor can be readily distinguished from the related CCK-1 receptor on the basis of agonist-binding profiles or with selective antagonists. The CCK-2 receptor binds CCK8 and gastrin17 with similar nanomolar affinity and does not require ligand sulfation for binding, whereas the CCK-1 receptor binds sulfated CCK8 with a maximum affinity of 20 pM, but has greater than 1000-fold lower affinity for unsulfated CCK8 or for gastrin17. Several structurally unrelated antagonists that possess both nanomolar affinities and high selectivities for either CCK-1 or CCK-2 receptors are now available. The gastrin17gly receptor does not recognize amidated gastrin17 or antagonists selective for

CCK-1 or CCK-2 receptors (242,243,245). The observation that binding of ferric ions is essential for the biological activity of gastrin17gly (246), but not of amidated gastrin17 (247), may assist in the cloning of the gastrin17gly receptor. Structure. The cDNA encoding the rat CCK-1 (241) and canine CCK-2 (240) receptors were first cloned in 1992. The sequences of the human, guinea pig, and rat CCK-1 receptors, as well as the human, cow, dog, Mastomys natalensis (an African rodent), rabbit, and rat CCK-2 receptors, are now known. The CCK-1 and CCK-2 receptors both belong to the family of receptors with seven-transmembrane segments; in humans, 50% of the amino acids are identical. Comparison of all known sequences in the family shows that the identities are clustered in the transmembrane helices and in the first and second intracellular loops. The binding properties of the cloned CCK-1 and CCK-2 receptors expressed in COS cells are generally similar between species, with the exception of the canine CCK-2 receptor (248). The structures of the receptors for gastrin17gly have not yet been determined (242,243,245). The CCK-1 and CCK-2 receptors were identified as membrane glycoproteins by covalent cross-linking of iodinated peptides and by glycosidase treatment of the resultant complexes. The CCK-1 receptor on rat pancreatic acinar membranes appeared on SDS-polyacrylamide gel electrophoresis as a diffuse band of Mr 85 to 95,000 (249), with a protein core of Mr 42,000 (250). The CCK-2 receptor on partially purified canine parietal cells also migrated as a diffuse band, with an average Mr of 74,000 (251). Homodimerization of both CCK-1 and CCK-2 receptors has been observed (252). Naturally occurring mutants lacking the first extracellular domain of the CCK-1 (253) and CCK-2 receptors (254) have been described. The affinities of the truncated receptors for both agonists and antagonists were reduced approximately 10-fold. A C-terminally truncated inactive variant form of the human CCK-1 receptor also has been reported (255). Variant forms of the human CCK-2 receptor with deletions (256) and insertions (257) in the third intracellular loop caused by alternative splicing also have been detected. The variant receptor with the insertion has been reported to increase constitutively both intracellular Ca2+ concentrations (257) and Src tyrosine kinase activity (258). Expression. There is now abundant evidence that gastric ECL cells express CCK-2 receptors. For example, a panel of CCK-2 receptor antagonists blocked gastrin-stimulated release of pancreastatin or histamine from enriched rat gastric ECL cells (259,260). CCK-2 receptor mRNA and protein have been detected in human gastric ECL cells by PCR in situ and by immunohistochemistry and confocal microscopy, respectively (261,262). These observations have been confirmed in rats by binding of fluorescein-labeled CCK8 to ECL cells in vascularly perfused stomachs (263) and by PCR analysis of single ECL cells isolated by laser-assisted microdissection (264). Gastrin appears to be essential for correct ECL cell development, because the numbers of ECL cells containing histamine were significantly reduced in the gastric mucosa of CCK-2 receptor–deficient mice

REGULATION OF GASTRIC ACID SECRETION / 1235 (265–267). Conversely, hypergastrinemia results in ECL cell hyperplasia in rats (268,269) and humans (270), and treatment of rats with a CCK-2 receptor antagonist blocked ECL cell development (46). Data also indicate that gastric D cells express CCK-1 receptors (262). CCK-1 receptor was detected at the mRNA level by PCR in situ and at the protein level by immunohistochemistry and confocal microscopy (262). These observations are consistent with previous reports of CCK-dependent release of somatostatin from canine fundic D cells (271,272), of an increase in CCK-stimulated acid secretion in CCK-1 receptor–deficient rats after treatment with a CCK-1 receptor antagonist (273), and of an increase in somatostatin mRNA and inhibition of gastric acid secretion in gastrin/CCK double-knockout mice after treatment with CCK (274). The controversy that has raged over the presence of gastrin receptors on parietal cells may be approaching resolution (37). Functional receptors for gastrin were demonstrated on enriched populations of canine (209), rabbit (275–277), and rat (278) parietal cells. In human gastric mucosa, CCK-2 receptors were detected in parietal cells at the mRNA level by PCR in situ and at the protein level by immunohistochemistry and confocal microscopy (261,262). Perhaps the most convincing evidence has been the demonstration by PCR that 25% of single rat parietal cells isolated by laserassisted microdissection contain CCK-2 receptor mRNA (264). However, the possibility remains that functional CCK-2 receptors may not be expressed in rat parietal cells, because no binding of fluorescein-labeled CCK8 was detected in the midglandular region of the corpus of vascularly perfused rat stomachs (263). The observation that induction of acid secretion by gastrin, but not carbachol, was completely abolished in histamine H2 receptor–deficient mice (57) is consistent with the latter conclusion. In contrast, an increase in gastric pH was observed when histamine H2 receptor–deficient mice were treated with a CCK-2 receptor antagonist (279). Signaling Mechanisms. Secretion. CCK-2 receptors control secretion from parietal cells via increases in intracellular Ca2+ (280) and inositol triphosphate (281,282; for early references, see the review by Chew and coworkers [280]). The gastrin-dependent effects in parietal cells are blocked by pertussis toxin and are unaffected by cholera toxin, and hence are likely to be mediated by interaction of the CCK-2 receptor with a Gi-like protein (283). Activation of similar signaling pathways downstream of the CCK-2 receptor in ECL cells results in histamine release (284) via inositol triphosphate–dependent release of Ca2+ from intracellular stores (285). Proliferation. Gastrin stimulates proliferation via the CCK-2 receptor in many cell types. In ECL cells from the African rodent Mastomys natalensis, stimulation of the CCK-2 receptor resulted in increased Ras-GTP, tyrosine phosphorylation and activation of MAPK, and increased proliferation (284). In small-cell lung carcinoma cell lines that express the CCK-2 receptor, addition of the appropriate ligand has been shown to result in mobilization of intracellular Ca2+ and ultimately mitogenesis, and both

effects were blocked by a CCK-2 receptor–selective antagonist (286,287). The mechanisms involved have been studied in detail in fibroblasts transfected with the CCK-2 receptor. In this system, gastrin-stimulated release of inositol triphosphate leads to increases in intracellular Ca2+ and mitogenesis (288,289). Raf kinase and MAPK were activated by pathways independent of Gi-like proteins or PKC (290). Tyrosine phosphorylation of several proteins including focal adhesion kinase and paxillin (288), induction of the early response genes c-fos and c-myc (288), and increased expression of cyclooxygenase-2 (291) and of several cyclins (292) also were observed. The mechanisms by which the CCK-2 receptor stimulates proliferation also have been studied extensively in the rat pancreatic carcinoma cell line AR4-2J. Gastrin activated MAPK by both phosphatidylinositol 3-kinase–dependent and –independent pathways (293,294) and MAPK activation were essential for both gastrin-dependent stimulation of proliferation (295) and inhibition of apoptosis (296). Gastrin also reduced apoptosis via a calcium-dependent, PKCindependent phosphorylation of the kinase Akt (297). Dominant-negative mutants of the GTP-binding proteins Ras, Rac, Rho, and Cdc42 were used to demonstrate that Ras was required for the activation of both MAPK and Akt, whereas Rho and Cdc42 were required for the activation of Akt only (296). Hence, mitogenic signaling by the CCK-2 receptor in ECL cells, fibroblasts, and AR4-2J cells apparently involves the MAPK cascade. In contrast, less is known about the mitogenic signaling pathways downstream from receptors for gastrin17gly. In the mouse gastric cell line, immortomouse gastric epithelium (IMGE) gastrin17gly stimulated proliferation via activation of phosphatidylinositol 3-kinase and MAPK (298). Transcriptional activation via stimulation of c-jun kinase also has been reported in response to gastrin17gly in the rat pancreatic carcinoma cell line AR4-2J cells (290) and in the human colorectal carcinoma cell lines LoVo and HT29 (299). The mechanism by which gastrin17gly induced expression of the gene encoding the α-subunit of the gastric H+,K+-ATPase in canine parietal cells has not been defined (300). Gastrin-Releasing Peptide Receptors Ligand Binding. In mammals, the family of receptors for gastrin-releasing peptide (GRP) is limited to three members: the GRP receptor (also called BB-1), the neuromedin B (NMB) receptor (BB-2), and BRS-3 (BB-3) (301). The GRP receptor binds bombesin with 60-fold higher affinity than the NMB receptor, whereas the NMB receptor binds NMB with 60-fold higher affinity than the GRP receptor (302). Although the natural ligand of BRS-3 has not yet been identified, and BRS-3 does not recognize either bombesin or NMB, a selective BRS-3 agonist has been developed (302). However, despite the description of numerous GRP receptor– selective (303) and some NMB receptor–selective antagonists (304), attempts to develop BRS-3–selective antagonists have not yet been successful.

1236 / CHAPTER 49 Structure. All three classes of GRP receptors belong to the seven-transmembrane domain receptor family. The first family member to be cloned was the mouse GRP receptor, the cDNA for which was isolated from Swiss 3T3 fibroblasts (305). The cDNAs encoding the NMB receptor and BRS-3 were subsequently isolated from a human small-cell lung carcinoma line (306) and from guinea pig uterus (307), respectively. The homology between the three human receptors is approximately 50%. The sequences of the human, mouse, rat, and chicken GRP receptor, the human, mouse, and rat NMB receptor, and the human, rhesus monkey, sheep, mouse, rat, guinea pig, and chicken BRS-3 receptors are now known. Although the human gene encoding the GRP receptor is interrupted by two introns, there is as yet no evidence for the existence of splice variants (308). Gastric Expression. Bombesin stimulates gastrin release from cultured human antral G cells, and stimulation is blocked by a bombesin antagonist (309). A GRP antagonist also inhibits gastrin release in response to intravenous GRP in vivo (310). The presence of mRNA for both GRP receptor and BRS-3, but not NMB receptor, in cell populations that contained 20% to 40% G cells was demonstrated by RTPCR (311). Because BRS-3 does not respond to bombesin at physiological doses, and because more than 85% of the bombesin-responsive cells contained immunoreactive gastrin, it is likely that antral G cells express GRP receptor. However, direct identification of the cell types that express the two receptors is still lacking. The functional role of the three receptors could also be investigated in GRP receptor–deficient (312), NMB receptor–deficient (313), or BRS-3–deficient mice (314). Bombesin was recognized as a stimulant of somatostatin secretion by the rat stomach more than 20 years ago (315). Similar effects also have been observed in mouse stomach (316). In rats, bombesin stimulates the release of somatostatin from cultured fundic D cells (317), but the receptor involved has not been defined, and no such stimulation is observed in cultured human D cells (318). Signaling. Signaling pathways downstream from the GRP receptor have been investigated extensively in cultured human antral G cells. Studies with selective inhibitors indicated that bombesin-stimulated gastrin release required activation of phospholipase C (319), a subsequent increase in inositol triphosphate (311), and release of Ca2+ from intracellular stores (319). Bombesin-stimulated gastrin release was also dependent on influx of extracellular Ca2+ via L-type Ca2+ channels (311,320). Although PKCµ was not required for bombesin-stimulated gastrin release, PKCγ was involved in the desensitization of the bombesin response (321). Histamine Receptors Ligand Binding. The known histamine receptors can be divided into four classes (192). Although the affinity of H1 and H2 receptors for histamine is in the micromolar range (322), both H3 and H4 receptors bind histamine with nanomolar affinity (192). A large number of selective

agonists have been developed for H1 (e.g., substituted 2-phenyl histamines) and H2 receptors (amthamine) (322). An even greater number of selective antagonists is available for H1 (mepyramine) and H2 (cimetidine) receptors (322). The H3 and H4 receptors share similar affinities for several agonists and antagonists. For example, the H3 antagonist thioperamide, which does not bind to H1 and H2 receptors, has similar potency at the H4 receptor (192). (R)-α-methyl-histamine and N-methyl-histamine both act as agonists at H3 and H4 receptors, but their potency is at least 40 times greater at the H3 receptor (192). Interestingly, the bacterium H. pylori produces an enzyme capable of generating N-methyl-histamine from histamine (190). Selective H4 receptor agonists (323) and antagonists (324) have been reported. Structure. All four classes of histamine receptors belong to the seven-transmembrane domain receptor family. The H1 and H2 receptor cDNA were both cloned in 1991 from bovine adrenal medulla (325) and canine gastric parietal cell (326) cDNA libraries, respectively. Some years elapsed before the cloning of the H3 receptor from a human thalamus cDNA library (327) and the H4 receptor from human leucocyte cDNA (328). Interestingly, the four receptors are not closely related in sequence, with the percentage identity ranging from 20% (H2/H3) to 43% (H3/H4) (192,329). Species comparisons indicate significant similarities in H2 receptor sequences, with 83% to 86% identity among human, rat, guinea pig, and dog (330). In the case of H3 receptor sequences, the similarity is even greater, with 92% identity among human, chimpanzee, orangutan, rat, mouse, guinea pig, and dog (331); in contrast, H4 receptor sequences are more divergent, with the rat, mouse, and guinea pig sequences 69%, 68%, and 65% identical to the human H4 receptor, respectively (332). The molecular masses of the H1 (333), H2 (333), and H3 (334) receptors were determined in cross-linking experiments to be 56, 59, and 70 kDa, respectively (see Table 49-1). Several splice variants of the rat H3 receptor have been described (331). The variants differ in the sequence of the third intracellular loop and in the presence of an extra eight C-terminal amino acids (331). The biological relevance of these variants is open to question, because there has been a continuing debate over whether the variants have different affinities for agonists and antagonists (summarized in Hancock and colleagues [331]). In any case, no splice variants have been detected in humans (335). In the case of the H1 and H2 receptors, splice variants are not expected because both genes lack introns (325,326). Gastric Expression. H2 receptors have been detected on gastric parietal cells by a variety of techniques. In situ hybridization with antisense riboprobes (336), autoradiography with 125 I-aminopotentidine (336), and confocal microscopy (337) all confirm earlier functional studies (338). In H2 receptor null mice, stomachs were enlarged because of hyperplasia of parietal, ECL, and mucous neck cells, and there was marked hypergastrinemia (279). In contrast, it is still unclear whether parietal cells express functional H3 receptors. Although H3 agonists inhibited gastric acid secretion in isolated rabbit fundic glands (339),

REGULATION OF GASTRIC ACID SECRETION / 1237 the stimulatory effect of an agonist for H3 receptors (N-methyl-histamine) in isolated rabbit parietal cells was mediated solely by the H2 receptor (195). Many functional studies have suggested that gastric ECL cells express H3 receptors (see review by Coruzzi and colleagues [340]). For example, in dogs, peripheral administration of the H3 receptor agonist (R)-α-methyl-histamine reduced the stimulation of acid secretion in response to ECL cell activation by pentagastrin (341). Despite the abundance of functional studies, we are not aware of any direct demonstration of H3 receptors at the mRNA or protein level in gastric ECL cells. Given the similarity in agonist affinities and antagonist profiles between the H3 and H4 receptors, there is an urgent need to define the class of histamine receptors on ECL cells at the structural level and through functional studies with selective H4 receptor agonists (323) and antagonists (324), or through investigations with H3 receptor–deficient mice (342,343). There also has been some controversy over the presence of H3 receptors on gastric D cells (344). Superfusion of human, dog, and rat antral segments (345), of rat fundic mucosal segments (346), or of isolated mouse stomach (347) with the H3 agonist (R)-α-methyl-histamine decreased somatostatin and increased histamine secretion, whereas the H3 antagonist thioperamide had the opposite effect. In contrast, the H3 agonist had no effect on somatostatin release from cultured rabbit fundic D cells (348). Signaling. H2 receptor stimulation leads to activation of both adenyl cyclase and phospholipase C (see Fig. 49-5) in membranes isolated from purified canine parietal cells (349). The phospholipase C–catalyzed increase in inositol triphosphate concentrations is presumably responsible for the increase in intracellular Ca2+ observed in single cultured rabbit parietal cells (350). In contrast, stimulation of the H3 receptor results in inhibition of N- and P-type Ca2+ channels (351) and of forskolin-stimulated adenyl cyclase activity (327). More detailed reviews of H2 (322) and H3 (331) receptor signaling in other cell types are available in the literature. Pituitary Adenylyl Cyclase–Activating Polypeptide Receptors Ligand Binding. Two classes of receptors have been described for peptides of the PACAP/VIP family (352). The PAC1 receptor has a much higher affinity for PACAP than for VIP, whereas the more closely related VPAC1 and VPAC2 receptors have similar nanomolar affinities for PACAP and VIP. Removal of the first five residues of PACAP1-38 reduced binding affinity for the PAC1 receptor 10-fold, but completely abolished the ability of the peptide to stimulate adenyl cyclase activity (353). A naturally occurring 61-residue peptide called maxadilan isolated from the salivary gland of the sand fly Lutzomia lingipalpis has been shown to act as a PAC1 agonist, even though it shows no significant sequence homology with PACAP (354). Surprisingly, deletion of maxadilan residues 25 through 41 generates a specific antagonist of the PAC1 receptor (355).

Structure. The PAC1, VPAC1, and VPAC2 receptors all belong to the secretin receptor subbranch of the seventransmembrane domain receptor family. The PAC1 receptor cDNA was first cloned from a rat pancreatic acinar cell line (356), and sequences of the human, bovine, rat, mouse, goldfish, and frog receptors are now available (352). Crosslinking experiments showed molecular masses ranging from 60 (357) to 65 kDa (358), presumably caused by species differences in glycosylation (see Table 49-1). Several splice variants of the human PAC1 receptor have been described. Although PACAP had similar affinity for four splice variants with different sequences in the third intracellular loop and stimulated adenyl cyclase and phospholipase C with similar potency, the variants differed in their maximal response to PACAP when total inositol phosphate was measured (359). Variants differing in their N-terminal extracellular domains had significantly different affinities for PACAP38, PACAP27, and VIP (360). Gastric Expression. Gastric ECL cells express PAC1 receptor cDNA, but not VPAC1 or VPAC2 receptor cDNA (144). All 4 splice variants in the third intracellular loop were detected by RT-PCR, but only a single protein was detected by Western blotting (361), and its size of 48 kDa was considerably smaller than that reported by other workers (see Table 49-1). In contrast, gastric D cells appear to express VPAC receptors, because both PACAP and VIP increase intracellular Ca2+ and stimulate somatostatin release with similar efficacy (74). The inhibitory effects of peripheral PACAP may be largely through stimulation of somatostatin release from D cells, because the effects were not observed in SSTR2 knockout mice or in wild-type mice after treatment with the SSTR2 receptor antagonist PRL-2903 (362). Parietal cells do not appear to express PAC1 or VPAC receptors because the PACAP-induced increase in intracellular Ca2+ in superfused gastric glands was completely blocked by the histamine antagonist ranitidine (74). Signaling. Treatment of ECL cells with PACAP resulted in increased intracellular Ca2+, which, in turn, stimulated histamine release (144). The increase in Ca2+ was dependent on increased entry of extracellular Ca2+ via L-type and receptor-operated channels (143). The observation that depletion of intracellular Ca2+ stores with thapsigargin did not affect secretion suggested that mobilization of intracellular Ca2+ stores was not involved (143). Somatostatin Receptors Ligand Binding. In mammals, five classes of receptors have been described for peptides of the somatostatin/ cortistatin family (363). The receptors arise from five separate genes on different chromosomes, with the SST2A and SST2B receptors derived from the same gene by alternative splicing. The gene duplications that caused the somatostatin receptor family are likely to have occurred at least 450 million years ago, because homologues of all five genes have been recognized in fish genomes. All five receptors have similar affinities for somatostatin 14.

1238 / CHAPTER 49 Many selective somatostatin agonists and antagonists have been developed (363). For example, octreotide, which targets SST2, SST3, and SST5 receptors, has widespread clinical applications as a somatostatin analogue because of its greater stability in vivo. The antagonist PRL2970 and its analogs, which bind to SST2, SST3, and SST5 receptors with similar affinity (364), have been particularly useful in investigations of the role of the SST2 receptor in the gastrointestinal tract (316). Structure. The SST receptors all belong to the seventransmembrane domain receptor family. Partial amino acid sequences of the SST2 receptor were obtained from GH4C1 pituitary cells (365), and SST2 receptor nucleotide sequences were first cloned from a human genomic library (366) and from a rat brain cDNA library (367). Sequences of the human, bovine, rat, mouse, dog, pig, and puffer fish receptors are now available. As mentioned earlier, two splice variants of the SST2 receptor have been described (368). The variants differ in the length of their intracellular C termini. The sequence of part of the unique C-terminal region of the longer SST2a variant was confirmed at the protein level (365). Western blotting showed a single protein of 72,000 Da molecular mass (368). Gastric Expression. A general survey suggested that mRNAs encoding all five somatostatin receptor subtypes were expressed in epithelial cells of rat gastric mucosa (369). Receptor autoradiography showed that SST1 receptors were the predominant subtype in human gastric mucosa (370). A study of isolated rat gastric ECL cells detected expression of SST2 receptor cDNA, and treatment with a SST2-selective agonist reduced intracellular Ca2+ (371). Whereas the ECL cells expressed the SST2A splice variant, only the SST2B splice variant was found in rat parietal cells (372). The occurrence of SST2 receptors on both ECL and parietal cells has been independently confirmed in mice in which the lacZ reporter gene was inserted into the SST2 gene (63). In anaethetized SST2 receptor null mice, gastric pH values (3.8 ± 0.3) were much lower than in anaethetized wild-type animals (7.1 ± 0.1), and basal acid output was 10-fold greater over a 2-hour period (373). A more recent study with preferential agonists in wild-type mice confirmed that the inhibitory effects of somatostatin were mediated solely by the SST2 receptor (374). Signaling. Activation of the SST2 receptor modulates multiple signaling pathways (363). Somatostatin treatment reduces adenyl cyclase (375), inhibits voltage-dependent Ca2+ channels (376), and activates an alternative MAPK pathway (377). Occupation of the SST2 receptor results in its phosphorylation by Src. Direct interaction between the phosphorylated SST2 receptor and the Src homology-2 (SH2) domain-containing tyrosine phosphatases SHP-1 and -2 is necessary for phosphatase activation (378). In many cell types, treatment with somatostatin results in SST2 down-regulation via receptor internalization (363). For example, in transfected pituitary cells, internalization follows agonist-induced phosphorylation of the third intracellular loop and the C-terminal tail of the SST2A receptor (379). Internalization rates in transfected HEK 293 cells are not

affected by SST2A homodimerization, but can be reduced by heterodimerization with the SST3 receptor (380). Such processes have not yet been examined in detail in gastric cells. Conclusion. The most important receptors controlling acid secretion are the muscarinic acetylcholine M3 receptor, the gastrin CCK-2 receptor, the histamine H2 receptor, the PACAP PAC1 receptor, and the somatostatin SST2 receptor. All five receptors are members of the seven-transmembrane receptor family, and amino acid sequence comparisons show close homology for all except the PAC1 receptor (Fig. 49-6). The cellular localization of the receptors within the gastric mucosa is summarized in Table 49-2. Integrated Control of Gastric Acid Secretion Phases of Gastric Acid Secretion There are four recognized phases of gastric acid secretion: basal phase, cephalic phase, gastric phase, and intestinal phase. However, it needs to be remembered that the regulatory mechanisms are not discrete, but rather are a continuum with the acid secretory response at any instant representing the integrated effect of stimulatory and inhibitory influences. The different inputs regulating gastric acid secretion are summarized in Figure 49-1 and are discussed in the following section. Basal Acid Secretion Most humans secrete acid in the basal state with a mean around 3 nmol/hr and an upper limit of normal of 10 nmol/hr in male and 5 nmol/hr in female individuals. Basal acid secretion is lowest in the morning and highest in the afternoon (381,382). Peak acid output determined after intravenous administration of a maximally effective dose of pentagastrin is about 10-fold greater than basal output (383). Cephalic Phase Gastric acid secretion increases before food even reaches the stomach. The thought, sight, smell, and taste of food increase gastric acid secretion and together account for a third to a half of the total response (384). Pavlov demonstrated nearly 100 years ago that the vagus nerve was involved (385). The mechanism involves central stimulation of the vagus nerve, and the acetylcholine released from the nerve endings may act directly on the parietal cell, as well as indirectly via stimulation of the release of histamine from the ECL cell and gastrin from the antrum (386) and via PACAP-mediated histamine release from the ECL cell (144,387). The signal is transmitted from the cerebral cortex and hypothalamus to the dorsal motor nucleus of the vagus (DMNV) in the medulla oblongata. Stimulatory efferent preganglionic fibers of the vagus originating from the DMNV innervate the stomach (388). These axons terminate in the stomach wall where postganglionic neurons directly innervate the parietal, ECL, G, and D cells. Stimulation of

REGULATION OF GASTRIC ACID SECRETION / 1239 PAC-1

Somatostatin-2 GRP CCK-2 Histamine-H2 Acetylcholine-M3

0.1

Ca-Sensing

FIG. 49-6. Phylogenetic tree for the receptors that control gastric acid secretion. The amino acid sequences of the muscarinic acetylcholine M3 receptor, the calcium-sensing receptor, the gastrin cholecystokinin 2 (CCK-2) receptor, the gastrin-releasing peptide (GRP) receptor, the histamine H2 receptor, the pituitary adenylyl cyclase–activating polypeptide (PACAP) PAC1 receptor, and the somatostatin 2 receptor were aligned with the program T-Coffee (518). The resultant phylogenetic tree was constructed with the program Treeview (519).

the DMNV increases gastric acid secretion, whereas ablation eliminates the central control of acid secretion (389,390). Sensory input from the stomach is integrated in the nucleus tractus solitarius (NTS) and mediated through the DMNV (391). The NTS also responds to hypoglycemia, resulting in an increase in acid secretion mediated through the DMNV (392). Beginning in the mid 1970s, a series of studies began to unravel the relative contributions of either direct vagal activation of the parietal cell or intermediate release of gastrin and histamine. Proximal vagotomy virtually abolished the acid response to sham feeding in humans (393) and decreased the acid response to 2-deoxyglucose (a vagal

stimulant) in dogs (394). One of the mediators on the parietal cell is acetylcholine because atropine attenuated the gastric acid response to sham feeding in humans (395) and abolished the acid response to sham feeding, insulin, or deoxyglucose in dogs (396). The increase in gastrin in the cephalic phase is only modest (384,393). The relatively small increase is probably the result of the activation of an inhibitory vagal cholinergic pathway perhaps involving somatostatin (397), because atropine or proximal vagotomy potentiates gastrin released during modified sham feeding (393,395). Until recently it was believed that the increase in gastrin, although small, was essential for a normal acid response to cephalic stimulation

TABLE 49-2. Receptor subtypes on D, enterochromaffin-like, and parietal cells Receptor Acetylcholine Ca2+-sensing Gastrin GRP Histamine PACAP Somatostatin

Known subtypes

G cell

M1, M2, M3, M4, M5 CAS CCK-1, CCK-2 GRP, NMB, BRS-3 H1, H2, H3, H4 PAC1, VPAC1, VPAC2 SSTR1, SSTR2a, SSTR2b, SSTR3, SSTR4, SSTR5

M3a

D cell

ECL cell

Parietal cell

M3

b

CAS

c

c

CCK-1

CCK-2

M3 CAS CCK-2

GRP

d

c

c

c

H3a VPAC1 or 2

H3 (or H4)d PAC1 SSTR2a

H2

c c

b

c

b

SSTR2b

Receptors positively identified in a given cell type are indicated. aBoth positive and negative reports have been published, sometimes because of species differences. bAbsence of the known subtypes of that class of receptors from a given cell type. cAbsence of data. dThe peptide has a biological effect, but the receptor subtype has not been defined. BRS-3, bombesin receptor subtype 3; CAS, calcium-sensing; CCK, cholecystokinin; ECL, enterochromaffin-like; GRP, gastrinreleasing peptide; NMB, neuromedin B; PACAP, pituitary adenylyl cyclase–activating polypeptide; PAC1, type 1 PACAP receptor; SSTR2, type 2 somatostatin receptor; VPAC1, vasoactive intestinal peptide receptor 1.

1240 / CHAPTER 49 because administration of an anti-gastrin monoclonal antibody abolished the acid response to sham feeding (398) and antral mucosectomy reduced the acid response by 75% (396). The vagal, noncholinergic transmitter for gastrin release was thought to be GRP because GRP neurons are located in the fundus and antrum (399), and GRP infusion to humans and other species stimulates both gastrin and acid secretion (399,400). However, Hildebrand and colleagues (310) demonstrated that a GRP antagonist blocked acid secretion after sham feeding, although the increase in gastrin was unchanged. This observation suggested that endogenous GRP may be a physiologic regulator of gastric acid secretion independent of gastrin release. Indeed, this surprising finding is consistent with cephalic release of gastrin being determined not by a stimulant, but by inhibition of the release of an inhibitor such as somatostatin (397,401,402). The conundrum that H2 antagonists blocked about half of vagally induced acid secretion (403) although acetylcholine was only a poor activator of ECL cells (128) has been resolved with the finding that PACAP is a potent stimulant of histamine release from the ECL cell (144,387). PACAP, a member of the glucagon family, is present in gastric postganglionic nerves and is released by vagal activation (387,404). PACAP stimulates histamine release from ECL cells through the PAC1 receptor and somatostatin from D cells through the VPAC receptor (144). Thus, the acid secretory outcome of vagal activation and release of PACAP can be variable depending on the balance between stimulatory and inhibitory effects (144,387). With the finding of dual localization of peptides in gut and brain, it is not surprising that many peptides in the central nervous system have modulating roles on gastric secretions (405). As shown in Table 49-3, there is a host of central stimulants and inhibitors of gastric acid secretion. Thyrotropin-releasing hormone (TRH) was the first brain peptide shown to have an effect on gastric acid secretion (406). Subsequent localization and functional studies suggested that TRH in the dorsal vagal complex rather than in the hypothalamus activated the central vagal cholinergic input into the stomach (407). A number of peptides have opposing central and peripheral actions. For example, central TABLE 49-3. Peptides that affect gastric acid secretion when administered into the central nervous system Stimulants

Inhibitors

Thyrotropin-releasing hormone (TRH) Somatostatin Neuropeptide Y (NPY) Galanin Peptide YY (PYY) Orexin

Corticotropin-releasing factor (CRF) β-Endorphin Bombesin Neurotensin Calcitonin Calcitonin gene–related peptide (CGRP) Interleukin-1 (IL-1)

Modified from Geoghegan JG, Pappas TN. Central peptidergic control of gastric acid secretion. Gut 1997;40:164–166, by permission.

administration of bombesin, NPY, or galanin stimulates acid secretion, whereas peripheral administration has the opposite effect (405). Somatostatin is a potent inhibitor of gastric acid secretion when given peripherally (408), but can be a central stimulant (409). NPY is of particular interest because it is a potent stimulant of feeding (410), and central administration increases gastric and pancreatic secretions in a manner that mimics the response occurring in preparation for a meal (411). Interestingly, another central stimulant of feeding, orexin, also increases pancreatic and gastric secretion via a vagally dependent mechanism (412,413). The central effect of ghrelin is not resolved with one study in conscious rats reporting a decrease (414), whereas in urethane-anesthetized animals, an increase was demonstrated (415). Peripheral administration of ghrelin to anesthetized rats stimulated gastric acid secretion via vagally dependent mechanisms (416). However, subsequent studies in conscious rats showed that ghrelin had no effect on gastric acid secretion (414,417). An outstanding issue for many of the above peptides is whether the effects are physiologic or pharmacologic. Gastric Phase The gastric phase accounts for about 50% of the total acid response to a meal and is the sum of a physical component caused by distension of the stomach and a chemical component from the effect of absorbed luminal nutrients. As determined by intragastric titration, gastric acid secretion increases rapidly after a meal to the same output achieved after a maximally effective dose of pentagastrin or histamine (Fig. 49-7A) (418). As shown in Figure 49-7B, the gastric pH actually increases because of the buffering capacity of food and because the pH of most foodstuffs is greater than the gastric pH. The increased pH serves to sustain the increased gastric acid secretion until the food is emptied or buffering capacity is exceeded. Distension. Distension activates stretch receptors in the body and antrum, which, in turn, initiate a long vagovagal reflex arc. The components of this reflex are vagal afferents with their nuclei in the nodose ganglion and vagal efferents with their nuclei in dorsal motor nuclei of the vagus nerve. The magnitude and mechanisms of action are species dependent, with humans having a smaller, less gastrindependent response than dogs (419,420). In humans, the increase in gastrin is small and does not account for the total acid response to distension (419). However, in dogs, distension-induced acid secretion is abolished after administration of an anti-gastrin monoclonal antibody (420), whereas in rats, the same antibody produced a 60% reduction (421). The acid response to distension is reduced by vagotomy, histamine (H2), or muscarinic receptor blockade (421,422). Luminal Nutrients. The major stimulants are peptic digests of proteins, ethanol, coffee, and calcium. Proteins are poor stimulants of acid secretion, but the breakdown products (amino acids and amines) stimulate acid secretion predominantly through the release of gastrin (423). Phenylalanine and tryptophan are the more potent amino acids (424).

REGULATION OF GASTRIC ACID SECRETION / 1241 A

PAO

35 30 25

Acid 20 secretion 15 (mmol/hr) 10 5

B

0

60 120 180 Time (min) after meal

240

0

60 120 180 Time (min) after meal

240

5 4 3 pH

2 1

FIG. 49-7. Acid secretion and intragastric pH after eating a steak meal (mean ± standard error). Acid secretion (A) was measured by in vivo intragastric titration to a pH of 5.5 in 6 subjects. Intragastric pH (B) was allowed to seek its natural level on another day in 10 subjects. Peak acid output (PAO) is also indicated. (Reproduced from Feldman M. Gastric secretion. In: Feldman M, Sleisenger MH, Fordtran JS, Friedman LS, eds. Sleisenger & Fordtran’s gastrointestinal and liver disease: pathophysiology, diagnosis, management. 7th ed. Philadelphia: WB Saunders, 2002;797–809.)

Gastrin is the principal mediator of the postprandial increases in gastric acid secretion in dog (420), rat (421), and human (381,425). In humans, the response to food can be reproduced by an intravenous gastrin infusion; in animals, the response can be blocked by anti-gastrin antibodies (420,421,425). Studies using H2 antagonists suggest that the effect is not through a direct effect on the parietal cell, but indirectly by gastrin-induced release of histamine (421). A number of groups have used vascularly perfused stomachs or superfused segments of antral and fundic mucosa from rats and mice to define the local paracrine and neural reflexes regulating acid secretion (426–429). Mechanical distension or chemical stimulation in the antrum results in cholinergic and GRPergic stimulation of gastrin secretion and cholinergic inhibition of somatostatin release. In the fundus, there is a direct cholinergic stimulation of acid secretion, a cholinergic inhibition of somatostatin release, and a gastrin-mediated increase in acid secretion. The latter effect is by a direct effect on the parietal cell and via gastrinstimulated release of histamine from ECL cells. The inhibition

of somatostatin release serves to accentuate the stimulatory effects (427–430). Although these potential mechanisms also have been demonstrated individually in vivo, it has been difficult to assign quantitative importance to the different inputs. These inconsistencies relate to species differences and to comparisons between in vivo and in vitro responses. An attempt to model mathematically the food-stimulated regulation of gastric acid secretion based on experimentally verified regulated processes has been reported (431). These modeling studies confirm the importance of the central and neural inputs and of food, as well as the key contributions of gastrin and histamine, but they emphasize that an intact negative feedback loop involving somatostatin is critical for the regulation of gastric acid secretion (431). The major inhibitory regulator of gastric acid secretion is an increase in intragastric acidity, and as noted earlier, the mediator is predominantly somatostatin. Luminal acidification reduces acid and gastrin secretion in response to nutrients, sham feeding, and antral distention (432,433). The threshold is a pH of 3.0, and at pH 1.0, acid output is abolished. Both in vitro and in vivo studies suggest that somatostatin acting by paracrine and endocrine pathways inhibits parietal cell function both directly and by reduction of gastrin secretion (10,430,434,435). Patients with duodenal ulcers have a reduced ability to inhibit the gastric phase of gastric acid secretion and are less sensitive to the inhibitory effects of a low intraluminal pH (433,436). Intestinal Phase The major inputs on acid secretion in the intestinal phase are inhibitory, and are initiated by nutrients in the intestinal lumen. However, peptone perfusion of the upper intestine of man increases duodenal gastrin secretion and gastric acid secretion (437). Studies in patients after gastrectomy indicate that the duodenum is the source of gastrin and that gastrin34 is preferentially released (438). The observation that entry of nutrients into the intestine results in a stimulation of gastric acid secretion and potentiates the effect of gastrin (439) prompted an intense search for the “intestinal phase hormone” or enterooxyntin. However, the initial report of a bioactive ileal polypeptide termed porcine intestinal polypeptide or gastrotropin has not been confirmed, and the chemical nature of the enterooxyntin is unknown (440–442). Infusion of fat, acid, or hyperosmolar glucose solutions into the upper small intestine inhibits acid secretion by the release from the intestine of a circulating inhibitory factor. This factor has been termed enterogastrone, but it is likely that the response is elicited by a number of inhibitory factors. As summarized by Lloyd and Debas (443), there are at least three requirements for a substance to be considered an enterogastrone: release in response to the presence of fat in the intestine, inhibition of gastric acid secretion after administration of the substance in physiologic amounts, and reversal of the inhibitory effect of intestinal fat by blockade of the factor’s receptor. However, these requirements do not sufficiently take into account the interactions between

1242 / CHAPTER 49 the various putative enterogastrones so that infusion or blockade of one particular factor may be a too stringent criterion. The first chemically characterized enterogastrones included gastric inhibitory polypeptide (GIP), CCK, and secretin. Claims also have been made for neurotensin, somatostatin, PYY, and glucagon-like peptide-1 and -2 (GLP-1, GLP-2). The evidence for each of these substances is considered here in turn. Intestinal nutrients, predominantly fat and not acid, increase circulating CCK, and infusions of CCK inhibit gastric acid secretion (444,445). However, the dose of CCK required to obtain an effect was often pharmacologic, and thus the role of CCK as an enterogastrone was thought to be minor. More recent studies using specific CCK-1 receptor antagonists showed that blockade of this receptor reversed intestinal fat–induced acid inhibition of meal-stimulated gastric acid secretion in dogs (446,447), and to a lesser extent in rats (448). In humans, CCK-1 receptor blockade resulted in a marked accentuation of the postprandial increase in gastric acid secretion and converted CCK-8 into a potent acid secretagogue (449,450). The inhibitory effect of CCK involves the release of gastric somatostatin via activation of CCK-1 receptors (272,449,450). The somatostatin, in turn, acts on the parietal cells. This dual effect of CCK stimulating both somatostatin and gastrin secretion explains why CCK is equipotent to gastrin in vitro, whereas in vivo CCK infusions

(acting through the CCK-2 receptor) can result in small increases or decreases in gastric acid secretion (445) (Fig. 49-8). Indeed, mice lacking both gastrin and CCK have a net increase in gastric acidity presumably through removal of CCK-mediated somatostatin release (274,451). In contrast, transgenic mice with gastrin deficiency alone are hypochlorhydric (48). After the isolation of GIP from the small intestine, and the observations that GIP was released by a meal and that GIP infusion inhibited acid secretion, it was believed that GIP was the long sought after enterogastrone (452). However, early preparations were contaminated with CCK making interpretation of these results difficult. Nevertheless, the observation that antibodies to GIP potentiated peptonestimulated acid secretion in dogs supported a role for GIP as an enterogastrone (453). Peptide YY is localized in the distal gut (454) and released by a meal (72). Intravenous administration of PYY inhibits meal-stimulated gastric acid secretion (455). However, PYY immunoneutralization did not reduce the inhibition of gastric acid secretion by intestinal perfusion with fat (447), suggesting that PYY does not have a physiologic role as an enterogastrone. There is substantial evidence for secretin being an enterogastrone in a number of species including rat (456), dog (457), and human (458,459). Duodenal acidification and to

CCK1 Antral mucosa

-

G cell

D cell SST

Gastrin

CCK

CCK2

CCK2

Parietal cell

ECL cell

CCK1 D cell

Histamine

SST

Oxyntic mucosa

FIG. 49-8. Interactions among gastrin, cholecystokinin (CCK), histamine, and somatostatin in the control of parietal cell function. Gastrin stimulates acid secretion primarily through release of histamine via activation of CCK2 receptors on enterochromaffin-like (ECL) cells. CCK counterbalances gastrin action through release of somatostatin (SST) from antral or fundic D cells, which inhibit histamine release from ECL cells, as well as gastrin release from G cells. Despite its nanomolar affinity for CCK2 receptors on parietal and ECL cells representing the positive effector pathway, the net effect of CCK on acid secretion is inhibitory. (Reproduced from Schmidt and Schmitz [451], by permission.)

REGULATION OF GASTRIC ACID SECRETION / 1243 a lesser extent fat stimulate secretin release (460). Infusion of secretin inhibits, whereas anti-secretin antibody potentiates, meal- and pentagastrin-stimulated acid secretion (456,457,459,461). Potential mechanisms include activation of vagal afferent fibers and release of somatostatin and PGE2 (461–463). The tridecapeptide neurotensin is another putative enterogastrone because it is located in the ileum and is released by a fatty meal. In addition, infusion of neurotensin inhibits acid secretion, and immunization with an anti-neurotensin antibody blocks fat-induced inhibition of acid secretion (464,465). The inhibitory effect of neurotensin is associated with an increase in somatostatin, but whether this is causative has not been determined (466). A number of proglucagon-derived peptides are produced in the L cells of the small intestine, and their release by a meal inhibits gastric acid secretion (467). Infusion of GLP-1 (proglucagon 78-107) or GLP-2 (proglucagon 126-150) inhibits gastric acid secretion via vagally dependent mechanisms (468,469). Oxyntomodulin (proglucagon 33-69) is coreleased with the other proglucagon-derived peptides during a meal and inhibits gastric acid secretion, although the doses used are probably supraphysiologic (470). As with a number of other enterogastrones, the inhibitory effect of the proglucagon-derived peptides is associated with the release of somatostatin (471,472). Major Regulators of Gastric Acid Secretion Histamine The cell biology of the histamine-containing ECL cell and the regulation of ECL cell activity and growth are considered in the Enterochromaffin-like Cell section earlier in this chapter. This section considers the functional role of histamine in the regulation of gastric acid secretion. Although mast cells contain the majority of gastric histamine, the regulated source of histamine involved in the control of gastric acid secretion is the ECL cell (473). The observation that H2 antagonists block gastrin-, acetylcholine-, as well as histamine-stimulated acid secretion confirmed the notion that histamine is a key regulator of acid secretion (474). The long-standing controversy over whether histamine is the final common mediator of secretagogue-stimulated acid secretion or whether histamine acts to potentiate the effect of other secretagogues on the parietal cell appears to be resolved in that both actions occur, although to different extents. Thus, gastrin primarily stimulates histamine release from the ECL cell through the CCK-2 receptor, which activates the parietal cell via the H2 receptor (20,386). A secondary mechanism is by a direct action of gastrin on the parietal cell (475). This conclusion is supported by studies with H2 receptor– and HDC-deficient mice. Gastric pH was increased in H2 receptor–deficient mice (59) and further increased after administration of muscarinic M3 and gastrin antagonists (279). Another group reported a normal basal pH level in H2 receptor–deficient mice, although the effect of gastrin was abolished (57). HDC-deficient mice have an

increased gastric pH, whereas mast cell–deficient mice have a normal gastric pH, confirming that the ECL cell is the source of bioactive histamine (125,476). Activation of H3 receptors reduces gastric acid secretion in cats and dogs (477,478), although in rats, no effect or an increase in gastric acid secretion has been reported (340,347). The mechanism is predominantly by histamine activation of H3 receptors located on ECL cells leading to a reduction in histamine synthesis and release (340). Thus, the effect of histamine appears to be a balance between activation of the stimulatory H2 receptor and the inhibitory H3 receptor (340). A further influence is that H3 activation decreases, whereas H2 receptor activation stimulates, somatostatin secretion (347,479). Overall, H3 receptors probably play only a minor modulating role in the control of gastric acid secretion (19), and the contribution in humans has not been determined. Acetylcholine The vagus nerve is an important regulator of gastric acid secretion. For instance, the cephalic phase of gastric acid secretion is totally dependent on an intact vagus nerve, and until the advent of H2 antagonists and proton pump inhibitors, truncal or highly selective vagotomy was a frequent treatment for peptic ulcer disease (480). The vagus nerve does not innervate the parietal, ECL, gastrin-containing, or somatostatin-containing cells directly, but rather synapses with ganglion cells of the enteric nervous system. These postganglionic fibers can be cholinergic (releasing acetylcholine) or peptidergic (containing peptides such as GRP, VIP, and PACAP). Often a single fiber can contain a variety of neurotransmitters (see Chapter 21). Acetylcholine binds to the M3 receptor on the parietal cell to increase acid secretion, but is a poor stimulant of histamine from fundic ECL cells (62,76,128). Cholinergic stimuli still increase acid secretion in H2 receptor– or HDC-deficient mice, demonstrating a histamine-independent effect (57,125). The major mediator of vagal release of histamine is PACAP (144). This conclusion is consistent with the finding that H2 receptor antagonists can partially inhibit central stimulation of acid secretion (403). The released histamine is delivered to the parietal cell in a paracrine manner and activates the parietal cell via a H2 receptor where it potentiates the direct action of acetylcholine. In the antrum, acetylcholine stimulates the secretion of gastrin from the G cell into the circulation. Gastrin then functions in an endocrine fashion via a CCK-2 receptor to stimulate the parietal cell, either directly or indirectly by the release of histamine from the fundic ECL cell (128). The cholinergic stimulation of gastrin is potentiated by the simultaneous inhibition of somatostatin (428). An M3 receptor–deficient mouse has been reported, and as expected, these mice have a lower acid secretion (214). Acid secretion in response to all secretagogues (gastrin, histamine, carbachol) was reduced to about half the secretion in wild-type mice. Plasma and antral gastrin levels were markedly increased probably as a result of the increased intragastric pH level, although, surprisingly, there was no

1244 / CHAPTER 49 oxyntic mucosal hypertrophy, which suggests that the M3 receptor may be important for the trophic effect of gastrin (214). Thus, the M3 receptor–deficient mice establish that M3 receptors play a key role in basal and stimulated acid secretion. Gastrin The detailed biology of gastrin is discussed in Chapter 4. This section presents features relevant to the role of gastrin in the regulation of gastric acid secretion. Gastrin is a classical gut peptide hormone, which was identified originally as a stimulant of gastric acid secretion. It is produced principally by the G cells of the gastric antrum and to a variable extent by the upper small intestine, with much lower amounts in the colon and pancreas (481,482). The term gastrin refers to the 17- or 34-amino-acid amidated end product of gastrin processing (483). The related hormone CCK, which is responsible for pancreatic enzyme secretion, has the same C-terminal tetrapeptide amide as gastrin (445). Like many other peptide hormones, the initial translation product of the gastrin gene is a large precursor molecule, preprogastrin (101 amino acids), which is converted to progastrin (80 amino acids) by cleavage of the N-terminal signal peptide (Fig. 49-9). Progastrin is processed further within secretory vesicles by endopeptidases and carboxypeptidases to yield glycine-extended gastrins. The C terminus of glycine-extended gastrin34 is then amidated by peptidyl α-amidating monooxygenase, and further proteolytic cleavage results in mature amidated gastrin17 (482). In healthy humans, progastrin and the glycine-extended gastrins comprise less than 10% of circulating gastrins (484). The C-terminal flanking peptide of progastrin is present in the antrum in approximately equimolar concentrations to gastrin amide (485,486).

The major stored forms of amidated gastrin are tyrosinesulfated and nonsulfated gastrin 17, in approximately equal concentrations. Less than 10% of antral gastrin amide is gastrin34, although duodenal gastrin is 50% gastrin34. The clearance rate of gastrin34 is about fivefold slower than gastrin17; therefore, although equal concentrations stimulate acid secretion to a similar extent, the effect of gastrin34 is more sustained (487). Between meals circulating gastrin34 predominates, probably as a result of its slower clearance and preferential release of gastrin34 from the duodenum. Meal-stimulated gastrin is mainly gastrin17. Gastrin acting through the CCK-2 receptor is the major hormonal regulator of gastric acid secretion. The gastrinacid feedback loop, whereby increased gastric acidity inhibits gastrin secretion and decreased acidity stimulates gastrin release, is central to the regulation of gastric acid secretion (481). Gastrin has a dual stimulatory effect on parietal cell function. The direct effect is via gastrin receptors on the parietal cell (475), although probably the more important pathway involves stimulation by gastrin of the synthesis of HDC and of histamine release from fundic ECL cells (117,474,488). Histamine, in turn, activates the parietal cell (474). Somatostatin is another important component because it inhibits both gastrin and gastric acid secretion. The interactions of gastrin and other regulators of gastric acidity have been discussed earlier in this chapter, as has the role of gastrin in the cephalic, gastric, and intestinal phases of gastric acid secretion. In summary, based on immunoneutralization studies in animals and gastrin infusions into humans, gastrin is the major hormonal mediator of the gastric phase of acid secretion (420,421,425,436). Gastrin also contributes to the cephalic and intestinal phases to a variable extent depending on the species, with gastrin being more important in the dog than in the rat (481). The relative

Preprogastrin -Arg-Arg 37

Progastrin

-Lys-Lys54

-Arg-Arg -

-Gly-Arg-Arg- CTFP

72 Signal peptidase -Lys-Lys-

80

-Gly-Arg-Arg-

Prohormone convertase Glycine-extended gastrin17

-Gly Transamidation

Gastrin17

-amide

FIG. 49-9. Processing of human progastrin. Conversion of preprogastrin (101 amino acids) to the mature biologically active species involves removal of the N-terminal signal sequence to form progastrin (80 amino acids). Progastrin is converted to Gly-extended intermediates by trypsin and carboxypeptidase B–like enzymes. Gastrin-gly is probably an end product, but an amidating enzyme then converts most of the gastrin-gly to form gastrin-amide (17 and 34 amino acids). CTFP, C terminal flanking peptide.

REGULATION OF GASTRIC ACID SECRETION / 1245 contribution of gastrin to the nongastric phase in humans awaits further studies with gastrin antagonists. As detailed in Chapter 51, studies with knockout mouse models (gastrin, CCK-2 receptor) and transgenic mice (gastrin overexpressing) have provided additional insight into the role of gastrin (47,48,55,265,266). As expected, CCK-2 receptor– and gastrin-deficient mice have decreased acid secretion, decreased parietal cells, and a reduction in functional activity of the histamine-containing ECL cells (47,48,265,266). Acute infusion of gastrin, cholinergic stimuli, or histamine had no effect, but partial restoration of acid secretion in gastrin-deficient mice was achieved by high-dose infusion of amidated gastrin17 for 6 days (48). Transgenic mice overexpressing amidated gastrin initially had increased parietal and ECL cell numbers and increased gastric acid secretion, but as the mice aged, gastric acid secretion decreased as a result of gastric atrophy and loss of parietal cells, eventually resulting in gastric cancer (55,489). Data suggest that precursors of gastrin-amide such as gastrin-gly also contribute to the regulation of gastric acidity, at least in the longer term (490,491). Gastrin-gly alone has no acute effect on gastric acidity (492,493), although pharmacologic doses infused for 2 hours potentiate the stimulatory effect of gastrin-amide on acid secretion in rats (493). Gastrin-gly stimulated histamine release, but at 1% of the potency of gastrin-amide (494). Inhibition of gastrin amidation for 3 days increased plasma and tissue gastrin-gly and enhanced gastric secretion (490). This enhancement has been attributed to a direct stimulatory effect by gastrin-gly on the H+,K+-ATPase (300), although the observation has not been confirmed (495). In gastrin-deficient mice administered gastrin, acid secretion increases from low levels, and then gradually decreases over a period of 2 weeks, whereas in animals receiving gastrin plus gastrin-gly, secretion was maintained over many weeks (495). The potentiation appeared to result from a direct effect on the extent and duration of parietal cell activation (495). In a subsequent study, the same group demonstrated that mice overexpressing gastrin-gly had a normal acid secretion, whereas double transgenic mice overexpressing both gastrin and gastrin-gly had an increased acid secretion compared with transgenic mice overexpressing only gastrin (491). Taken together, it appears that gastrin-gly increases gastric acidity by maintaining the integrity and activation state of the parietal cell by an unknown mechanism. This effect may be relevant to the sustained hyperchlorhydria seen in patients with H. pylori infection or Zollinger–Ellison syndrome because these patients tend to have increased ratios of nonamidated gastrins in the circulation (484). The effect of progastrin on acid secretion has not been reported, whereas the C-terminal flanking peptide of progastrin (progastrin 75-80 in humans, progastrin 75-83 in most other species), which is present in high concentrations in the antra of a number of species (for review, see Paterson and colleagues [486]), did not alter gastric acid secretion after short-term (5-minute) infusions alone or together with gastrin-amide (485). The C-terminal flanking peptide stimulated histamine release, but at 1% of

the potency of gastrin (494). The receptors mediating the effects of gastrin-gly or other nonamidated forms of gastrin have not yet been characterized, but are distinct from the CCK-2 and CCK-1 receptors that interact with amidated gastrin and CCK (482). Somatostatin Although first isolated from ovine hypothalamus, the largest store of somatostatin is in the gastrointestinal tract (496). The biology of somatostatin is reviewed in Chapter 4 and in the Somatostatin Receptors section earlier in this chapter. This section presents aspects relevant to the role of somatostatin in gastric acid secretion. Somatostatin is the major peptide inhibitor of gastric acid secretion with three distinct cellular targets: antral G cells, fundic ECL cells, and parietal cells. Although all five members of the somatostatin receptor family (SSTR1-5) are present in the gastric mucosa (369), SSTR2 is the receptor subtype predominantly involved in the regulation of gastric acid secretion and inhibition of gastrin and histamine release (371,497–499). The observation of somatostatin receptors on parietal, ECL, and gastrin cells confirms that somatostatin can act either directly on the parietal cell or by inhibition of intermediaries (371,496,500). The physiologic effect of somatostatin therefore arises both from direct inhibition of the parietal cell and indirectly by inhibition of gastrin and histamine release. Somatostatin is present in D cells of both the fundus and the antrum. Antral D cells are open-type cells with apical processes in contact with the lumen and are sensitive to changes in gastric acidity, peptides, and neuronal mediators, whereas fundic D cells are closed-type cells and respond to peptide and neuronal mediators, but not luminal contents (479,501,502). Experiments using anti-somatostatin monoclonal antibodies suggest that in rats somatostatin is the mediator of gastric acid inhibition induced by intestinal fat (464). However, similar studies in the dog did not confirm a role for somatostatin (503). The somatostatin antibody increased basal acid secretion in wild-type mice, whereas SSTR2-deficient mice had an increased basal acid secretion that was reversed by administration of an anti-gastrin antibody (373). However, these studies were performed in urethane-anesthetized mice, and because urethane stimulates gastric somatostatin secretion, the role of somatostatin in basal acid secretion is unresolved. Indeed, in conscious, SSTR2-deficient mice or somatostatin-immunoneutralized sheep, gastric acidity is normal (373,504). Somatostatin has a more important role in the modulation of stimulated, rather than basal, acid secretion. The observation that infusions of somatostatin that matched the postprandial level inhibited gastric secretion support the concept of a hormonal role in humans (408). HCl is a potent stimulant of somatostatin in animals in vitro and in vivo (426,428,501). In humans HCl alone has little effect on somatostatin but potentiates the meal-stimulated somatostatin release indicating that gastric acid participates in the postprandial increase in plasma somatostatin (434).

1246 / CHAPTER 49 The increase in somatostatin as a result of HCl infusion inhibits both the parietal cell and the G cell, and thus attenuates further increases in gastric acid secretion. There have been conflicting reports on the effects of vagal or cholinergic activation on gastric somatostatin release. These have been partially resolved by studies which distinguish between fundic and antral stores of somatostatin. Nevertheless, species differences and variations between in vitro and in vivo models using intact animals and isolated organ perfusion preclude a unified picture. Where tested, cholinergic stimulation inhibits fundic somatostatin secretion and may either stimulate or inhibit antral somatostatin secretion (426,428,501,505). The net effect in vivo is a decrease in circulating somatostatin because the rate of secretion of somatostatin from the fundus is higher than from the antrum (505). As mentioned earlier in the Intestinal Phase section that discusses enterogastrones, there is a long list of regulatory peptides that stimulate somatostatin secretion including gastrin, CCK, calcitonin gene–related peptide, peptide YY, PACAP, GRP, secretin, and proglucagon-derived peptides (reviewed by Chiba and Yamada [496]). Because most of these peptides are inhibitors of gastric acid secretion, somatostatin may be the common inhibitory mediator for both hormonal (e.g., secretin) and neural (e.g., PACAP) effectors.

DISORDERS OF GASTRIC ACID SECRETION The stomach is not essential for life provided vitamin B12 is supplied. Only a brief overview of the causes and consequences of overproduction and underproduction of gastric acid is given here. These areas have been reviewed elsewhere (12,418,506).

response to GRP infusion (400,510,511). The loss of inhibition is postulated to be the result of a lower somatostatin concentration, a reduction in the inhibitory effect of CCK, and a reduction in the inhibition of gastrin and acid secretion normally mediated by antral distension (512–515). Normal subjects infected with H. pylori and infected patients with duodenal ulcer have similar exaggerated gastrin responses, but the increase in gastric acid secretion is much greater in the patients with duodenal ulcer (400). The effect of H. pylori on gastric acid secretion is dependent on the pattern of gastritis caused by the infection. Thus, antral-predominant gastritis leads to acid hypersecretion and duodenal ulcer, whereas an atrophic pangastritis leads to reduced acid secretion (509). Small-Bowel Resection About 50% of patients who have undergone massive small-bowel resection experience development of hypergastrinemia and increased gastric acid secretion, especially in the early postoperative phase (516,517). This is generally thought to be the result of removal of intestinal factors (enterogastrones) that inhibit gastrin and gastric acid secretion. Increased Histamine Secretion Patients with foregut carcinoid tumors and overproduction of histamine and patients with systemic mastocytosis with an overproduction of histamine-producing mast cells have gastric acid hypersecretion (418).

Decreased Gastric Acid Secretion Increased Gastric Acid Secretion Zollinger–Ellison Syndrome Patients with Zollinger–Ellison syndrome have an increased circulating gastrin (generally >200 pmol/L) as a result of a gastrin-secreting tumor usually located in the pancreas (507). Because the gastrin is not regulated by the acid feedback loop, basal acid secretion is increased (>15 mEq/hr with a mean of 42 mEq/hr) (508). Stimulants of acid secretion are less effective, thus although maximal acid output is increased (mean, 63 mEq/hr), the basal acid output/maximal acid output ratio is high (508). Duodenal Ulcer Disease The majority of patients with peptic ulcer disease have increased gastric acid secretion, and most of these patients are infected with H. pylori (506). A number of disorders of gastric acid secretion regulation have been reported in subjects with H. pylori (509). These include hypergastrinemia and lack of inhibition at low pH levels, and an increased gastrin and gastric acid response, but decreased somatostatin

The potential consequences of low gastric acid secretion are protein and lipid malabsorption due to suboptimal activation of pepsins and lipase, iron-deficiency anemia, increased risk for enteric infections, and hypergastrinemia with the potential to induce ECL cell carcinomas (418). Gastritis Chronic atrophic gastritis is associated with hypochlorhydria, or in some cases achlorhydria. Gastrin level is substantially increased (often >500 pmol/L) because of loss of the gastrin-gastric acid feedback loop. The severe chronic atrophic gastritis is caused by autoimmune (type A) gastritis that destroys parietal and chief cells but spares the antrum. The resultant absence of intrinsic factor leads to pernicious anemia because of failure of cobalamin absorption in the terminal ileum (12). Acute infection with H. pylori produces a decrease in gastric acid secretion, and this decline is maintained in the chronic state if the infection remains in the corpus and body (509). Patients with body-predominant gastritis and a reduction in gastric acid output have an increased risk for development of gastric cancer (489,515).

REGULATION OF GASTRIC ACID SECRETION / 1247 Miscellaneous Reduced acid secretion is observed in some patients with gastric ulcer or gastric cancer generally as a result of the atrophic gastritis. Truncal or highly selective vagotomy reduces gastric acid secretion and was a common treatment for peptic ulcer disease (418).

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Gastroduodenal Mucosal Defense Marshall H. Montrose, Yasutada Akiba, Koji Takeuchi, and Jonathan D. Kaunitz Juxtamucosal Environment and Preepithelial Defenses, 1260 Gastroduodenal Mucous Layer, 1260 pH in the Mucous Gel Layer, 1261 Gastroduodenal Epithelial Layer, 1264 Regulation of Permeability, 1264 pH Regulation, 1265 Unique Protective Role of HCO3−, 1268 Subepithelial Defense: Gastroduodenal Mucosal Blood Flow, Neural Sensors and Effectors, and Chemical Mediators, 1273 General Concepts, 1273 Prostaglandins, 1274 Organ-Specific Mechanisms, 1274

Injury and Restitution, 1279 Daily Challenges to Epithelial Integrity, 1279 Imposed Mucosal Damage, 1279 Cell Exfoliation and the Mucoid Cap, 1280 Epithelial Restitution, 1280 Animal Models of Gastroduodenal Injury, 1281 Summary and Conclusions, 1282 Acknowledgments, 1282 References, 1282

The field of gastroduodenal mucosal defense encompasses a broad array of topics, ranging from clinical trials of ulcer prevention and healing to molecular interactions between transport proteins and signaling molecules. A comprehensive review of all of these topics, which would be hundreds of pages long and include thousands of literature citations, clearly would be unwieldy and of limited practical utility. Therefore, we have endeavored to focus on the key homeostatic

mechanisms that maintain epithelial cell viability in the face of high luminal acidity. Emphasis also is placed on mechanisms that are up-regulated or altered in response to luminal acid, because this mode of regulation implies a protective role. Again, because of the extensive number of publications addressing this topic, we focus on describing findings that bear on factors that are responsible for the gastric and duodenal epithelial resistance to impending mucosal damage induced by the luminal aggressive factors acid and pepsin. We emphasize the findings published after the late 1990s because much of the earlier work in this area has been subject to excellent reviews (e.g., see reviews by Hogan and colleagues [1], Flemström and Isenberg [2], Allen and colleagues [3], and Allen and Flemström [4]). We deemphasize mechanisms of injury and repair, particularly because restitution and repair are reviewed elsewhere in this volume (see Chapter 16). Because mucosal injury is such a commonly used end point in studies of protective mechanisms, we also describe some of the most popular injury models, with comments on their use as surrogates for clinical ulcer disease and usefulness as experimental model systems. Because the

M. H. Montrose: Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267. Y. Akiba: Brentwood Biomedical Research Institute, West Los Angeles VAMC, Los Angeles, California 90073. K. Takeuchi: Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan. J. D. Kaunitz: Department of Medicine, Division of Digestive Diseases, West Los Angeles VAMC, and UCLA School of Medicine, Los Angeles, California 90073. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1259

1260 / CHAPTER 50 stomach and the duodenum have distinct structures and defensive mechanisms, we describe each organ separately when necessary for clarity. The mucosa of the acid-exposed organs of the upper gastrointestinal (GI) tract—the esophagus, stomach, and proximal duodenum—are constantly exposed to gastric acid at concentrations that promptly necrose unprotected living cells (5). Each organ has evolved robust defense mechanisms that rely on regulated intercellular junctions, blood flow, mucus and bicarbonate secretion, specialized plasma membrane ion permeability, and cellular acid/base transport and buffering to prevent the occurrence of cellular injury. Gastroduodenal mucosal defense can be subdivided into preepithelial, epithelial, and subepithelial factors that function in the prevention of mucosal injury. This division, although arbitrary because the entire epithelium functions as a unit to prevent damage caused by noxious agents, is useful nosologically. All of these protective mechanisms are coordinated by a network of chemical messengers. Each of these contributions to mucosal defense is potentially important to human health because acid damage is a crucial aggravating factor in the formation and in the persistence of duodenal ulcers (3). Much of this chapter is predicated on the assumption that acid and pepsin are the major aggressive factors in the pathogenesis of upper GI mucosal injury. With few separate data regarding the role of pepsin, and with pepsin active only at low pH, it is difficult to address the roles of these aggressive factors separately (6–8).

JUXTAMUCOSAL ENVIRONMENT AND PREEPITHELIAL DEFENSES The gastroduodenal lumen provides unique and extreme challenges for tissue survival. The intragastric fluid has a hydrogen ion concentration that can vary a million-fold in just 30 seconds (9). Because of the reactive mixture of concentrated gastric acid and bicarbonate, duodenal luminal pCO2 can approach its theoretical limit of 75 kPa (10). The gastroduodenal lumen also contains degradative enzymes (pepsin, pancreatic proteases, peptidases, and lipases) and amphipathic bile salts that can disturb cell membranes. In one of the most remarkable examples of tissue adaptation and specialization, the gastroduodenal epithelium thrives while continuously exposed to this environment, whereas other cell types die after a 10-minute exposure to an HCl concentration greater than 1 mM (5). This section focuses on the protective mechanisms that permit gastroduodenal epithelial cells to survive these daily environmental extremes. Based on cumulative results from the past few decades, one uncontested principle is that the apical membrane of the gastroduodenal epithelium is not directly exposed to the noxious pH excursions of the bulk lumen and is protected from luminal enzyme activity. This ability to hide the epithelium from bulk luminal conditions is the result of several potential mechanisms that control the juxtamucosal environment within the submillimeter space adjacent to the tissue.

The relative importance of each mechanism is debated, but in aggregate unequivocally forms an essential first line of defense for epithelial survival.

Gastroduodenal Mucous Layer A firmly adherent mucous gel forms over the gastric surface, composed of 95% water and 5% extensively crosslinked mucin glycoproteins that are products of mucin (MUC) genes (3,11). It also contains products from acid, bicarbonate, and other secreted proteins, resulting in viscous properties and a composition that resist conventional fixation and means of study. This mucous gel layer at a minimum serves as an important physical defense against luminal constituents, but may also have a strong role in other preepithelial defenses of the stomach and duodenum (3,12,13). A hydrophobic layer facing the luminal content has been implicated in protection, presumably by retarding the diffusion of H+ toward the mucosa. The protective role of this layer has been inferred from correlative studies in which substances associated with weakened defenses such as nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt the hydrophobic barrier, whereas interventions associated with strengthening defenses, such as growth factors, increase hydrophobicity (14–17). Furthermore, hydrophobic particles such as carbon adhere to gastric mucus, further suggesting surface hydrophobic properties to the gastric mucous gel (12,18,19). These observations notwithstanding, the indirect and correlational nature of the studies, coupled with the need to measure hydrophobicity in vitro after tissue drying, have cast doubt on the relevance of these studies to mucosal protection in vivo (3). The physical structure of the mucus gel is well established. The predominant proteins are mucins, glycoproteins that are highly cross-linked. Confirming early observations (20), ultrastructural studies of the gastric surface mucous gel layer reported alternating layers of two distinct mucin classes (21). The molecular basis for this multilaminated structure is alternating layers containing predominantly MUC5AC (a mucin secreted from gastric epithelial cells in the surface and pit) and MUC6 (secreted from neck and gastric gland cells), among other protein components (22–24). The physiologic significance of the laminae is unknown, but observations suggest that it may be important to mucosal defense. Electron microscopy studies have demonstrated that Helicobacter pylori locate not only on the apical surface, but also in the mucous gel layer covering the gastric mucosa, preferentially colonizing within the MUC5AC-containing regions in the mucous gel layer (24). Moreover, there is histochemical evidence that the multilaminated structure of the surface mucous gel layer is disrupted by colonization with H. pylori, such that it is restored after eradication of H. pylori (22). Moreover, the mucous gel contains another class of protein that is important to mucosal defense, trefoil peptides. A gene family of three trefoil peptides, composed of TFF1, TFF2, and TFF3, has been identified. All of these 7- to 12-kDa

GASTRODUODENAL MUCOSAL DEFENSE / 1261 peptides share a common structure of three internal cystine bonds that yield the signature “trefoil” structure of three internal loops, and all are stored in mucous secretory cells and secreted in parallel with mucins (25). The distribution of each peptide in the normal GI tract is distinct. TFF1 is stored in the gastric pit and surface mucous cells, TFF2 is present in the gastric gland mucous cells, and TFF3 is stored in the small and large intestinal goblet cells (26–29). Secreted TFF1 and TFF2 are found in the adherent mucous gel layer; the concentration of TFF2 in rat gastric mucus has been estimated at 10 µM (27). The coordinated localization of trefoil peptides and mucins in the normal GI epithelium and in the ulcer-associated cell lineage suggests that they may organize the protection and repair of the GI mucosa (28,30). Trefoil peptides are markedly protease resistant, prolonging their survival in the gastric lumen (31). Addition of TFF2 to mucin solutions significantly increases viscosity and elasticity, in which the mucin solutions are turned into a gel-like state (32). These effects on the physical properties of mucus, however, are only one aspect of trefoil peptide actions, which include regulation of gastric acid secretion and of cellular differentiation. TFF2−/− knockout mice have modest changes in tissue morphology in unstressed animals (shorter gastric pits and gastric glands with decreased epithelial migration, no detected change in mucous cell number) with unaffected TFF1 mRNA abundance. TFF2−/− animals had increased basal net acid output (either decreased bicarbonate secretion or increased acid secretion), and a fourfold increase in number of lesions after 12-hour exposure to the nonselective cyclooxygenase (COX) inhibitor indomethacin (33). Chronic treatment with a proton-pump inhibitor (omeprazole) increased TFF2 concentration in gastric secretions, thereby promoting repair and preventing injury to the gastric mucosa in response to luminal noxious agents (34). Evidence also suggests that trefoil peptides promote epithelial restitution across damaged epithelium (see Epithelial Restitution section Perfusion

pH 7.0

later in this chapter). Thus, results suggest multiple physiologic roles for TFF2 in the promotion of mucosal healing. Given the paradigm that mucosal alterations in response to luminal acid implies a role on mucosal protection, mucus secretion rate and gel thickness are both augmented by luminal acid. We have developed a technique in which mucous gel thickness can be measured continuously in living rodents (18,35–39). Mucous gel thickness was measured noninvasively by alternately focusing between the fluorescently labeled surface epithelial cells and the mucous gel surface, as delineated by carbon particles or by fluorescent microspheres. We found, for example, that mucous gel thickness rapidly increased in response to perfused acid, but equally rapidly decreased in thickness when the acid challenge is removed. Measurement of effluent mucous glycoprotein content was consistent with increased sloughing of mucus into the perfusate when mucus was secreted rapidly, indicating that there is a dynamic relation between mucus secretion and erosion, as has been hypothesized previously (40). When the secretion rate slowed, the rapid sloughing remained, thinning the gel until a new steady state occurred. A scheme depicting our concept of how mucous gel thickness is regulated is shown in Figure 50-1. Further studies showed that the capsaicin pathway, involving acid-sensing vanilloid receptors (VR), afferent nerves, calcitonin gene-related peptide (CGRP), and nitric oxide (NO), regulates mucous gel secretion (Fig. 50-2) and that nonselective COX inhibition with indomethacin abolishes the mucous secretory response to all secretagogues, suggesting a fundamental role of prostaglandins (PGs) in duodenal mucus secretion (39).

pH in the Mucous Gel Layer This section focuses on studies of gastric surface pH where the mucosa is exposed to relatively constant concentrations pH 2.2

pH 7.0

pH 7.0

Goblet cells

Brunner's glands

PGE2

FIG. 50-1. Dynamic regulation of mucous gel thickness in response to luminal acid. In the left panel, baseline mucus secretion and the rate of sloughing into the lumen are balanced. Luminal acid creates a sudden exocytotic burst of mucus secretion from goblet cells and Brunner’s glands, which thickens the gel. The newly secreted mucus sloughs into the lumen at a greater rate, resulting in a new steady-state gel thickness. Removal of luminal acid decreases mucus secretion, decreasing gel thickness, and also initiates synthesis of new mucous granules. In contrast, PGE2 IV injection stimulates mucous secretion from Brunner’s gland followed by secretion from goblet cells. (Modified from Kaunitz and Akiba [426], by permission.)

1262 / CHAPTER 50 CNS

Sensor (VR-1)

Capsazepine

Capsaicin-treatment Blood flow Effector CGRP

NO

hCGRP8-37 L-NAME

COX PG NSAIDs

Mucus secretion

FIG. 50-2. The capsaicin pathway. A scheme for the regulation of upper gastrointestinal defense mechanisms in response to luminal acid. Inhibitors are depicted in italics. CGRP, calcitonin gene–related peptide; CNS, central nervous system; COX, cyclooxygenase; hCGRP8-37, inhibitor of human calcitonin gene–related peptide; L-NAME, NG-nitro-Larginine methyl ester; NO, nitric oxide; NSAIDs, nonsteroidal anti-inflammatory drugs; PG, prostaglandin; VR-1, vanilloid receptor 1. (Modified from Kaunitz and Akiba [426], by permission.)

of luminal acid. This is not meant to deemphasize the role of this preepithelial defense in the duodenum; the preepithelial mucous-bicarbonate layer may actually play a more important role in mucosal protection against luminal acid in the duodenum than in the stomach, because of an increased ability to dampen rapid shifts of luminal pH that are both transient and of lesser magnitude than the acid challenges faced by the gastric mucosa. Nevertheless, most research has focused on gastric surface pH. Numerous laboratories have reported a pH gradient at the gastric mucosal surface in rabbit (41), rat (42–45), human (46), frog (47,48), guinea pig (49), dog (50), and mouse (51–53). In most cases, this pH gradient has been reported as relatively alkaline directly at the tissue surface and becoming more acidic at distances farther away from the surface. This alkaline layer is caused by active bicarbonate secretion, most likely mediated by surface or pit gastric epithelial cells (42,54) (see Gastric HCO3− Secretion section later in this chapter). It is considered a major defense of the mucosa against gastric acid, but the absolute value of pH at the gastric surface is debated. Most measurements from microelectrodes have reported values near neutrality (pH 7), although results from guinea pig and frog have reported values closer to pH 4 (48,49). The use of pH-sensitive fluorescent dyes with ex vivo confocal microscopy reports a surface pH

near pH 4 in both rats and mice (42,51–53). This lack of convergence in surface pH values currently remains unexplained and represents a discrepancy of 1000-fold proton concentration. Nevertheless, all observations agree that the alkaline layer in the stomach is only a part-time defender of the epithelium, because it is not observed under all physiologically important conditions. Both microelectrode and confocal microscopy observations show that the relatively alkaline surface pH disappears when luminal pH is reduced to less than pH 2 (50,53,55–57). Thus, in the presence of ~10 mM proton, bicarbonate secretion is insufficient to neutralize stomach acid at the gastric surface. Because more than 100 mM HCl can be produced by parietal cells, this led to the common belief that the capacity of bicarbonate secretion in the stomach is only 10% of acid secretion. An microelectrode study has reported sustaining the surface pH near 7 even in the presence of luminal pH 1 (58). HCl concentration becomes the major determinant of free (active) proton concentration at extremely acidic pH. At pH less than 3 (i.e., >1mM proton), adding conventional pH buffers has negligible influence on measured proton concentrations. Simple dilution with water (or other secretions) will increase pH in this acidic realm not because of neutralization of acid/base equivalents, but because of dilution of HCl. In the absence of volume changes, large concentrations of exogenous acid or base are needed to change pH simply because such amounts are needed to ratchet up or down the prevailing HCl concentration. At less extreme luminal pH, adding pH buffer in the luminal fluid to match the resistance to pH change found at luminal pH 2 disrupted the alkaline surface layer (53). Thus, increased resistance to luminal pH changes (because of either increased pH buffers or high HCl concentration) can compromise pH control at the gastric surface by overwhelming the capacity of alkali secretion. Currently, there are three competing models to explain how gastric acid and alkali secretion impact on surface pH control. Bicarbonate secretion is stimulated (and the relatively alkaline surface layer is observed) when luminal pH decreases to fasting levels (e.g., pH 3 in fasted rodent stomach) (48,51,59). Noninvasive measurements using confocal microscopy have indicated that when luminal pH is increased to values found in the fed stomach (>pH 5), rats and mice demonstrate a dramatic change wherein the pH directly at the gastric surface was acidified by gastrin-stimulated and omeprazole-inhibitable H,K-ATPase activity (42). In this condition, the surface pH gradient was qualitatively reversed to become most acidic near the surface and more alkaline at distances further from the tissue (42,51–53). These results led to a simple model of surface pH control in which the dominant acid/base secretion contributes to hold surface pH near pH 4 (at least in rodents). This model is not universally accepted, as evidence from microelectrode studies report rat surface pH closer to pH 7 in both the presence and absence of acid secretion (45,57,60,61). These latter results support a model in which secreted acid is ejected from gastric glands under pressure (62) and propels undiluted through channels in

GASTRODUODENAL MUCOSAL DEFENSE / 1263 mucus to reach the gastric lumen (63). In these microelectrode studies of surface pH, addition of gastrin (stimulation of acid secretion) or indomethacin (inhibition of bicarbonate secretion) do not perturb surface pH value (45,58). This appears to be in direct conflict to the observations with confocal microscopy that surface pH control mediated by bicarbonate secretion is weakened by COX-1 inhibitors or COX-1 gene disruption and enhanced by exogenous addition of PGs (42,51,52). An alternative model has been proposed based on microelectrode measurements of pH within gastric gland lumens. In this model, mucins act as proton buffers that effectively transport protons from the gland to the bulk lumen at low activity (higher pH), where activated pepsin then releases acid by proteolytic cleavage of the mucin (49). In that model, it remains unclear whether proton buffering and enzymatic substrates are sufficient to produce the observed acid concentrations in the gastric lumen. The observation of a disequilibrium pH near the gastric mucosa requires a physical explanation. Luminal protons are neutralized by reaction with bicarbonate as protons diffuse toward the tissue surface, creating a juxtamucosal pH gradient where excess bicarbonate ions sustain a pH greater than the values found in the bulk lumen. Properties besides those in the mucous gel must contribute to maintenance of this extracellular microenvironment, a supposition directly supported by the observation that the steady-state surface pH gradient can extend beyond the thickness of the mucous gel layer (53,64). An unstirred layer is the simplest explanation for this observation. Such a layer always forms at the interface between an aqueous and nonaqueous compartment, restricting mixing of molecules within the unstirred layer (65). In the stomach, an unstirred layer may form at the interface between mucus and the aqueous lumen or at the interface between the epithelial surface and the luminal contents, or at both (36,64,66). Confocal microscopy confirms that access of large-molecular-weight fluorophores to the juxtamucosal space in vivo is slowed compared with access with bulk solutions (53), further supporting the role of unstirred layers in the of disequilibrium pH. A second potential mechanism is the presence of high concentrations of fixed pH buffers in the mucous gel. Nevertheless, when the surface pH buffering capacity was estimated in situ in a mouse, it was found to be negligible (2.6 mM protons/pH unit at luminal pH 3) (53). In a third mechanism, mucus may act as a diffusion barrier to protons. In vitro measurements of proton and bicarbonate diffusion have yielded diffusion coefficients that are up to 10 times slower in isolated mucus compared with saline solution (~0.5 − 2 × 10−5 cm2/sec) (36,66–68). Nevertheless, these diffusion coefficients of protons and bicarbonate in mucus are comparable with those found in saline for ions such as Na+, K+, or Cl−. Even accepting the slowest reported diffusion of protons, a third of the protons theoretically will diffuse 80 µm or greater from a starting point within 10 seconds. This distance is greater than the average mucus thickness in both rats and mice (12,42,64). There are also some concerns about diffusion measurements performed in vitro because nondestructive removal of the

tightly adherent mucus is extremely difficult (58), mucus structure and function is sensitive to environmental conditions (69,70), and the contribution of artifactual unstirred layers can be imposed by the measurement apparatus. Using twophoton microscopy to uncage a 800 molecular weight fluorophore adjacent to the gastric surface, so that its diffusion could be monitored directly ex vivo, investigators found no evidence of restricted diffusion in mucus (53) (Fig. 50-3). Thus, evidence suggests that surface pH is set by the integrated action of an unstirred layer and regulated alkali (and presumably acid) secretion, not by limiting diffusion of ions or small-molecular-weight buffers in the adherent mucous gel. Peristalsis and mixing in the stomach are predicted to lessen the unstirred layer thickness during digestion, but the extent of such an effect remains unknown. Currently available results are most relevant for the fasted stomach, with less aggressive mixing, a lower luminal pH, and alkali secretion dominating to keep luminal pH and surface pH from acidifying further.

FIG. 50-3. Representative uncaging experiment. The stomach of an omeprazole-treated (60 mg/kg intraperitoneally) and anesthetized mouse was exteriorized, and the gastric mucosa was placed on the chamber on the confocal microscope stage. The nonperfused chamber contained a strongly buffered saline solution with caged fluorescein (pH 5). A two-photon microscope was used to uncage the fluorescein in a defined region near the tissue surface, and fluorescence intensity was imaged every 0.3 second thereafter. Images are fluorescence and reflectance images overlaid. The tissue is on the right side (white), and the solution containing the caged fluorescein is on the left side (green). (See Color Plate 24.) (Modified from Baumgartner and Montrose [53], by permission.)

1264 / CHAPTER 50 GASTRODUODENAL EPITHELIAL LAYER Regulation of Permeability Apical Membranes The apical plasma membranes of the gastroduodenal cells are presumably highly adapted to resist acid. Exposure of the apical surfaces of gastric surface cells increases transmembrane potential difference, presumably by inhibition of function of epithelial sodium channels (ENaC). Few investigators have studied the plasma membrane properties of gastroduodenal epithelial cells in response to acidification of the outer surface of the apical membrane, but results have been remarkably similar. In the toad Necturus maculosus, apical acidification increased apical transmembrane resistance, an effect preceded by a transient decrease of intracellular pH (pHi) and abolished by substitution of apical cations by the organic cation N-methyl-glucamine, supporting the role of ENaC in the regulation of resistance (71), which is a finding replicated by another group (72). These findings add an additional layer of epithelial defense regulated by luminal acid, and they also are consistent with exposure of the gastric surface epithelial cells to luminal acid. Intercellular Junctions The epithelial lining of the upper GI tract, consisting of epithelial cells of many types (surface mucous cells, surface epithelial cells, parietal cells, chief cells, goblet cells, endocrine cells, and undifferentiated cells) forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Permeability across the epithelium is, in part, determined by the rate-limiting barrier of the paracellular pathway—the most apical intercellular junction referred to as the tight junction (TJ). The intricate regulation of TJ multiprotein complex is important both for the maintenance of mucosal integrity and the restitution of epithelial continuity (73). The stomach and duodenum have, as discussed earlier, a distinct structure that is an important component of overall mucosal barrier function. Part of the structure are the intercellular junctions, which in epithelia are generally termed tight and adherens junctions, as described in Chapter 61. In the early part of the twentieth century, the relative impermeability of the gastric epithelium to acid was thought to contribute to protection against acidinduced injury (74). The primary observation was that the stomach was able to maintain high [H+] for extended periods, implying that the rate of acid “back-diffusion” from lumen to plasma is quite low (75–79). Transmural electrical resistance (TEER) was also proposed as an alternate measure of mucosal permeability (80). An increase of back-diffusion rate and a decrease of transmucosal resistance by compounds associated with gastric injury (81), or by inhibition of acid secretion, reinforced the impression that nevertheless epithelial H+ permeability plays an important role in mucosal defense (82–84). The concept of TEER as a surrogate for

impending injury was further developed in the mid-1980s with the observation that transmural potential difference suddenly decreased under anoxic and related conditions (85,86). Until the 1970s, it was assumed that junctional permeability was more or less constant until gross mucosal damage occurred, at which time permeability irreversibly increased, giving rise to the term barrier breaker. A better understanding of this concept originated in the laboratory of Andrew Soll, who developed a system of isolated canine chief cell monolayers in primary culture as a surrogate system for the measurement of gastric transepithelial permeability. A striking initial finding was the increase of TEER associated with apical acidification (87), implicating the role of junctional permeability in mucosal defense. Using a far less technically demanding system, another group showed that acidification of the apical surface of gastric-derived rat gastric mucous (RGM-1) cells promptly increased TEER and decreased mannitol permeability, confirming the earlier work (88). Furthermore, they showed that the nonselective COX inhibitor indomethacin, but not the selective COX inhibitor NS-398, abolished the permeability decrease. In subsequent publications, epidermal growth factor and related growth factors were found to decrease permeability, which is consistent with their gastroprotective role (89,90). More recently, advances in the understanding of junctional structure and regulation have served as the basis for further advances in the regulation of gastroduodenal epithelial barrier function. Because filamentous or F-actin plays an important role in maintaining mucosal integrity, the F-actin inhibitor cytochalasin D was used to increase transepithelial resistance and mannitol permeability in bullfrog gastric mucosa (91). In another study, E-cadherin, a member of the cadherin family of homotypic cell–cell adhesion molecules that is expressed in the gastroduodenum (92), was up-regulated by apical acid exposure in cultured RGM-1 gastric cells, but not in intestinal epithelial cell-6 (IEC-6) intestinal epithelial cells. Like the chief cell monolayers and Necturus antrum, RGM-1 cells maintained (and perhaps even decreased) transepithelial dextran permeability when exposed to apical acid, unlike IEC-6 cells. Acid-related E-cadherin expression was related to an acid-induced “spike” of [Ca2+]i (92). E-cadherin was further implicated in the regulation of intestinal permeability by a calcium- and polyamine-dependent mechanism in another study (93). These important studies provide an initial framework for the understanding of junctional regulation in the presence of luminal acid. Another tight junctional molecule, occludin, is a component of the strands between cells. One group studied the phenotype of occludin null (−/−) mice. Although electron microscopy showed a remarkable preservation of the other strand protein claudin, the gastric epithelium of null mice was chronically inflamed and became hyperplastic (94). The duodenal mucosa has only about 5% of the total epithelial electrical resistance of the stomach, yet mucosal permeability appears also to play a prominent role in the resistance from acid injury. The transmucosal paracellular permeability of the small intestine can be measured relatively

GASTRODUODENAL MUCOSAL DEFENSE / 1265 easily with the use small organic molecules such as sugar alcohols, polyethylene glycol, and ethylene diamine tetraacetic acid (EDTA). Paracellular permeability correlates with diseases or interventions associated with mucosal injury (95). Because mucosal permeability is a good measure of mucosal integrity and is regulated by luminal acid and by other factors, the concept of duodenal junctional regulation was introduced. Many studies have originated in the laboratory of Olaf Nylander (96,97), who used small organic molecules and polymers to determine the permeability characteristics of rat duodenum. As opposed to data obtained from the stomach, luminal acid exposure increased absorption of EDTA. Subsequent studies demonstrated that the nonselective COX inhibitor indomethacin enhanced the HCl-induced permeability increase (98), implying that endogenously produced PGs played a role in this phenomenon. This permeability response to acid perfusion was enhanced by administration of the NO inhibitor N-nitro-L-arginine methyl ester, implying a role for endogenously produced NO in the genesis of this response. Subsequent work from the same group revealed that administration of the tachykinin neurokinin A, a promotility peptide, also reversibly increased duodenal permeability (99), although infusion of the peptide vasoactive intestinal peptide (VIP) substantially decreased permeability (100). pH Regulation General Principles One of the assumptions that has been made by most investigators of upper GI mucosal defense is that luminal acid places severe stress on the mucosal cells. This assumption is based on the observation that acid is required for the development of mucosal ulceration, regardless of cause (101). If acid is a final common pathway for injury, a logical assumption is that mucosal defense mechanisms serve to protect the mucosa from excessive acidification. Although there is no absolute proof in an in situ mucosal preparation that irreversible cellular acidification inevitably precedes cellular necrosis, much evidence has accumulated that strongly supports this hypothesis. Some of the earliest work addressed the overall mucosal interstitial pH (intramural pH), measured by Sb electrodes. In rabbit fundic pouches, induction of hemorrhagic shock in the presence of luminal acid was associated with profound mucosal acidification and ulceration (83), with lesser effects observed in chambered rat stomach. Acidification of amphibian antral surface cells was enhanced by compounds that are associated with experimental ulceration (102) and is associated with loss of transmembrane potential difference (103). Damage to cultured gastric RGM cells or rat duodenal villous cells in vivo correlated with decrements of intracellular pH (pHi) during acid exposure (8,104,105). Furthermore, marked cytosolic acidification is associated with necrosis and apoptosis of diverse cell types (5,106). The sequential destruction of gastroduodenal epithelial cells in response to injurious interventions has been well

documented (107,108). Regardless of insult, either luminal due to the application of ethanol or bile acids or the serosal application of acetic acid, mucosal damage proceeds in a sequence starting with swelling or “ballooning” of the villous tips, followed by progressive necrosis of the epithelial cells, leading to villous destruction. The exact mechanism by which the epithelial cells necrose is poorly understood. Because the presence of luminal acid is a characteristic prerequisite for mucosal injury, most investigators have assumed that cellular acidification precedes injury. Surprisingly, few direct data support this hypothesis, mostly because of the difficulty of measuring pHi in injured mucosa. Therefore, we have postulated that active pHi regulation is an important epithelial defense mechanism. Measurement and Regulation of Gastroduodenal Intracellular pH Measurement of gastroduodenal pHi was first reported in 1969 in a preparation of isolated dog parietal cells using the 5,5-dimethoxyazolidine-2,3-dione (DMOE) method (109). Cellular pH was measured in isolated oxyntic cells (110) and in amphibian gastric mucosa using similar methodology. pHi was higher in histamine-treated mucosae than in mucosae treated with an antisecretory compound (82). By the late 1980s, several laboratories were measuring pHi with microelectrodes in amphibian mucosa or with fluorescent dyes in other species. Acidification of the luminal surface of amphibian gastric mucosa did not reduce pHi if the perfusate pH was greater than 2 (111). A rapid cellular acidification was observed in response to perfusate of pH 2, followed by a return to new steady-state pHi of ~7.0 (103). A modest decrease of pH of the solution bathing the serosal surface, however, profoundly reduced pHi (103,111). Small organic acids, such as acetylsalicylic and acetic acids, altered transmembrane potential, with acetylsalicylic acid hyperpolarizing and acetic acid depolarizing transmembrane potential without altering pHi of Necturus gastric surface epithelial cells (112). Further studies demonstrated that high luminal pCO2 also acidified the epithelial cells, with the presence of serosal HCO3−/CO2 required to maintain pH at the basal pHi of 7.1 to 7.2; serosal application of the stilbene 4-acetamido4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) also decreased pHi (113). Serosal Na+ was necessary for maintenance of pHi and also for recovery from acidic pHi (103). Qualitatively similar results were obtained from the study of Necturus duodenum (114). Application of “barrier breaking” compounds such as taurocholate, ethanol, and aspirin in the presence of luminal pH 3 also profoundly acidified the epithelial cells (102). These studies also were performed in preparation of isolated gastric glands with the newly developed trapped, fluorescent ratio dye BCECF (2′7′-bis-2carboxyethyl-5-(and-6)-carboxyfluorescein), where sodiumproton exchange activity was localized to the basolateral membrane (115). The first use of digital imaging technology to the study of pHi in the gastroduodenum was published in 1987 (116), a technological advance enabling the measurement

1266 / CHAPTER 50 in individual cells. In isolated rabbit parietal cells, sodiumdependent, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)–sensitive, amiloride-insensitive pHi recovery from acidification was described, consistent with Na+-HCO3− cotransport, which is similar to findings obtained with frog oxyntic cells (117,118), although the existence of Na+-HCO3− cotransport in the basolateral membranes of rabbit parietal cells was disputed (119). Studies of gastric pHi regulation up to the mid-1980s are well summarized in an excellent review by Machen and Paradiso (120). Further studies indicated a role for H,K-ATPase in pHi regulation of frog oxynticopeptic cells (121). In 1991, the first measurement of pHi of gastric surface cells in living animals was accomplished, using the trapped fluorescent dye 5,6 carboxyfluorescein (122). The dye was loaded using a miniperfusion chamber placed over the gastric corpus mucosa of anesthetized rats. With the technique, basal pHi was 7.1 to 7.2 and variably decreased with the perfusate pH less than 3 (35,122–125). In rat gastric corpus in vivo, perfusion of amiloride had no effect on pHi, whereas the potent, membrane-permeant amiloride analogue 5-(N,N-hexamethylene)-amiloride

exposed by perfusion or by close arterial perfusion reduced pHi, suggesting that only the basolateral Na+-H+ exchanger (NHE) controlled pHi (123), which is in agreement with the Necturus data (126). Duodenal pHi was first measured in 1976 in rat duodenum using glass microelectrodes, where basal pHi was ~ 7.0 (127). In 1992, Kivilaakso and coworkers (114) measured pHi in Necturus duodenal epithelial cells with glass microelectrodes, finding a basal pHi of 7.05 and acidification with exposure to luminal solution of pH 2.7. Removal of serosal Na+ or HCO3−/CO2 additionally acidified the cells (114). In 1999, we measured pHi in rat duodenal epithelial cells of living rats using BCECF, in a modification of the technique used in stomach (128). Using this technique, our group demonstrated that preservation of pHi during exposure to luminally perfused acid was dependent on HCO3− uptake into the cells and was enhanced by dysfunction of the apical membrane cystic fibrosis transmembrane regulator (CFTR) (Fig. 50-4) (104,105). Confocal laser-scan microscopy also was used to measure pHi of epithelial cells in intact rat duodenum in vitro (129). A baseline pHi of 7.3 was found, with

A

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C

D

FIG. 50-4. Fluorescent images of BCECF (2′7′-bis-2-carboxyethyl-5-(and-6)-carboxyfluorescein)loaded mouse duodenal epithelial cells. (A–C) Confocal sections were taken through the mucosa to reveal the localization of the dye. (A) Confocal transverse section at the level of the villous core depicting green BCECF fluorescence in the epithelial cells surrounding the interior structures. (B) A section close to the surface of the epithelial cells showing the polygonal morphology of the BCECFloaded epithelial cells in cross section. (C) Confocal reflectance image of the same villus depicted in A and B. (D) BCECF-loaded villi, as visualized by conventional fluorescent microscopy, before acid exposure. (See Color Plate 25.) (Modified from Hirokawa and colleagues [104], by permission.)

GASTRODUODENAL MUCOSAL DEFENSE / 1267 acidification in response to perfusion of a low pH solution. Using perfused mini-Ussing chambers, or spheroids, pHi also has been measured in biopsies of human duodenal mucosa (130,131) and in duodenal epithelial cells isolated from rats (129). Mechanism of Duodenal Epithelial Acidification Duodenal epithelial cellular acidification has been observed in response to perfusion of the luminal surface with physiologic concentrations of H+. Although H+ is a permeant ionic species, its overall permeability through the lipid bilayer is low and would not account for the rapid cellular acidification observed. Mechanisms for facilitated H+ entry may include sodium-proton exchange via an apical membrane exchanger such as NHE2 or NHE3 that would be

“reversed” from its usual Na+ absorptive function. Currently, the role of apical membrane NHE isoforms on pHi is controversial (132,133). Another possibility is that H+ traverses the apical membrane as CO2, which, in the presence of carbonic anhydrase and an anion exchanger, can transport acid equivalents. This physiologic phenomenon, first described in 1942 by Jacobs and Stewart (134) for red cells, is likely to be present in the gastroduodenum, where high luminal pCO2, abundant carbonic anhydrase, and apical anion exchangers are all present. According to this mechanism, a luminal H+ combines with luminal HCO3− to yield CO2 and H2O. The CO2, a permeant gas, traverses the plasma membrane, where it is hydrated to carbonic acid, which dissociates into H+ and HCO3− . The HCO3− exits through the plasma membrane via anion exchange, completing the cycle (Fig. 50-5).

H+

HCO−3 CO2

NPPB

10

1

Apical

NHE3

SLC

CFTR

CO2 + H2O MTZ

2

CA

3

DMA

Basolateral

7

NHE1

cAMP

NBC

EP Na+

Na+

5

pHi

HCO−3

pHi

4

H+

9

HCO−3

H+

Acid sensors

6 PG production

8

DIDS

HCO−3

Indo

FIG. 50-5. Possible mechanisms for H+- and CO2-induced duodenal bicarbonate secretion. Based on currently available data, this hypothetical scheme depicts a stepwise activation of the HCO3− secretory pathway after exposure to high luminal pCO2 in rat duodenum: (1) H+ conversion to CO2 by extracellular carbonic anhydrase, and CO2 diffusion into the cell; (2) CO2 conversion to H+ and HCO3− by carbonic anhydrase (CA), inhibitable with methazolamide (MTZ); (3) intracellular acidification by H+; (4) H+ extrusion via Na+-H+ exchanger (NHE)-1, inhibitable with dimethyl amiloride (DMA); (5) submucosal acid sensor activation by H+; (6) prostaglandin (PG) production, inhibitable with indomethacin (Indo); (7) adenosine-3′,5′-cyclic monophosphate (cAMP) increase via PG receptor activation, presumably the EP3 receptor (EP); (8) sodium-bicarbonate cotransporter (NBC) activation and HCO3− loading, inhibitable with DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid); (9) intracellular alkalinization; and (10) augmented HCO3− secretion via cystic fibrosis transmembrane regulator (CFTR), inhibitable with NPPB (5-nitro-2-[3-phenylpropylamino] benzoic acid). Apical NHE-3 is unlikely to be involved, and the involvement of an apical anion exchanger (SLC) is still uncertain. Black arrows indicate CO2 and ion movements; dashed arrows indicate activation; T indicates inhibition; intracellular shading depicts pHi: acidic, (dark gray) neutral (light gray), and alkaline (white). SLC, solute carrier. Modified from Furukawa and colleagues (233).

1268 / CHAPTER 50 The “Cystic Fibrosis Paradox” Unique Protective Role of HCO3− General Principles Bicarbonate has been used clinically for centuries as a remedy for peptic ulceration. In 1948, a group studying experimental ulceration in dogs discovered that peptic ulceration was prevented by intravenous administration of HCO3− (135), a result later confirmed by another group (136). Subsequent studies have addressed the role of systemic HCO3− in upper GI mucosal injury. We have already discussed that HCO3−/CO2 in the serosal solution is necessary for maintenance of pHi in gastric antrum. Further studies indicated that systemic acidosis induced by hemorrhagic shock was associated with severe gastric injury, an effect abrogated by correction of acidosis with systemic HCO3−, but not with Tris buffer (137), leading some authors to conclude that HCO3− is uniquely important to the maintenance of gastroduodenal mucosal integrity (138). The protective mechanism of HCO3− was not well understood until the 1980s, when a series of observations provided support to a novel hypothesis, which we term the pH gradient hypothesis. Measurement of a preepithelial pH gradient (see pH in the Mucous Gel Layer section earlier in this chapter) combined with studies correlating the rate of HCO3− secretion and mucosal injury susceptibility (58,139–141) supported the hypothesis that mucosal HCO3− secretion neutralized luminal H+ in the preepithelial mucus, completely neutralizing luminal acid before its reaching the epithelial cells. This hypothesis is further supported by correlational studies, such as the observation that H. pylori infection complicated by duodenal ulcers is associated with diminished bicarbonate secretion, and eradication of Helicobacter infection restores duodenal bicarbonate secretory capacity (140). Furthermore, a strong correlation between bicarbonate secretion and mucosal injury susceptibility has been found in experimental animal models (142–144). In light of these data, bicarbonate secretion has become the most accepted mechanism by which the duodenal epithelium is protected from acid (1,2,4). Using in vivo microscopy, Synnerstad and colleagues (57) demonstrated that systemic bicarbonate reduced slCr-EDTA clearance in rat mucosa in vivo, consistent with mucosal protection. The “pH gradient hypothesis,” however, is not supported by all available data. For example, HCO3− secretion is always measured in the presence of a luminally oriented HCO3− gradient, which is probably not the case physiologically, because duodenal luminal pCO2 greatly exceeds that of plasma (10,145). Furthermore, impairment of duodenal mucosal HCO3− secretion by anion channel inhibitors or CFTR mutation decreases rather than increases mucosal injury susceptibility, mirroring the relative dearth of duodenal ulceration in patients with cystic fibrosis (CF) (104,105,146). At this point, the relative importance of duodenal cellular versus secreted HCO3− is unknown, although the absolute importance of systemic HCO3− in gastroduodenal mucosal protection is unquestioned.

CF is an inherited disease caused by homozygosity of alleles bearing CFTR mutations. Impaired duodenal and pancreatic HCO3− secretion characterizes patients afflicted with this disease, which, combined with normal or supernormal gastric acid secretion, produces abnormally high acidity in the upper GI tract (147–150). Consequences of this acidity are erosive esophageal disease, pulmonary acid reflux, and inactivation of secreted pancreatic enzymes (151–153). Despite these marked acid-related upper GI abnormalities, these patients are remarkably resistant to peptic duodenal ulceration, a phenomenon yet unexplained (146,154,155). The term CF Paradox is used to describe this apparently paradoxical protection from duodenal injury. We have reexamined the role of bicarbonate secretion in overall duodenal defense from acid, and in doing so have formulated a novel hypothesis with regard to the role of bicarbonate transport. To test these possibilities, we developed a technique for the measurement of pHi in the duodenum of anesthetized rats (104,128,156). With this system, we could perfuse solutions of varying pH through a chamber placed over the exposed duodenal mucosa, thereby simulating changes in luminal pH. We exposed the mucosa to a brief pulse of acid, which promptly decreased epithelial pHi. With removal of the acid challenge, pHi was increased to supernormal values, which indicated that cellular buffering power has increased, not decreased, during acid challenge. Furthermore, a second acid challenge acidified pHi less than the first, further confirming that acid exposure was followed by increased cellular buffering power. This somewhat surprising finding was confirmed by comparison with prior studies conducted in a variety of systems in which acid pulses were followed by pHi overshoot, indicative of increased buffering power in cells containing an active base-loading mechanism such as sodium-bicarbonate cotransport (157). Further studies indicated that this buffering power increase was inhibited by the stilbene anion transport inhibitor DIDS, which also inhibited this adaptive effect. Our studies thus were consistent with bicarbonate uptake being induced by luminal acid exposure by a DIDS-inhibitable mechanism, which is most likely a sodium-bicarbonate cotransporter (NBC), presumably located on the basolateral, blood-facing cellular pole. This finding was expected insofar as primary isolated duodenal epithelial cells recovered from acid exposure by a mechanism consistent with the activity of an NBC (158,159). In further studies of duodenal alkaline secretion, we found that titratable alkalinity of the duodenal perfusate increased substantially during luminal acid perfusion, although total CO2 content decreased somewhat. The best explanation for these data was that although acid back-diffusion increased markedly during luminal acid perfusion, bicarbonate secretion was unchanged, suggesting that HCO3− secretion did not increase during acid stress. The implications of these data, combined with our measurements of pHi, support our hypothesis that increased cellular buffering, and not bicarbonate secretion, is an important duodenal defense mechanism from acid. In this proposed mechanism,

GASTRODUODENAL MUCOSAL DEFENSE / 1269 bicarbonate secretion occurs to remove excess alkali from the cell when excess intracellular bicarbonate is no longer needed after acid challenge (Fig. 50-6). We further tested our hypothesis with the anion transport inhibitor DIDS to inhibit basolateral HCO3− uptake into the enterocyte and the anion channel inhibitor 5-nitro-2(3-phenylpropylamino) benzoic acid (NPPB) to inhibit HCO3− exit. Both inhibitors abolished bicarbonate secretion, as has been published previously (160,161). DIDS decreased pHi of the duodenal cells, whereas NPPB increased pHi. These effects on pHi were consistent with inhibition of base uptake and exit from the cell, respectively. The susceptibility of the epithelial cells to acid injury was enhanced by DIDS, but decreased by NPPB. We thus were able to uncouple bicarbonate secretion from mucosal protection, because NPPB inhibited bicarbonate secretion while reducing injury susceptibility. These data added further evidence that pHi regulation, as well as secreted bicarbonate, appears to be of importance for duodenal mucosal protection. Furthermore, we measured epithelial pHi and injury in mice transgenic for the most common severe CFTR mutation. In response to acid perfusion, the reduction of pHi was lower and injury was less in mice homozygous for the CFTR mutation (104). These data all suggest that impairment of HCO3− exit from the duodenal villous cell can protect against acidinduced injury, despite diminished HCO3− secretion.

Luminal pH 7.0 CFTR

2.0

2.0

CFTR

CFTR

CFTR

HCO−3

HCO−3 pHi

pHi

pHi

pHi

NBC

NBC

NBC

7.0

NBC

HCO−3

FIG. 50-6. Sequential response of duodenal epithelial cells to luminal acid. In the left panel, steady-state intracellular pH (pHi) ~7.1 when no acid is present. In the succeeding panels to the right, luminal acid rapidly acidifies the epithelial cells. Low pHi decreases cystic fibrosis transmembrane regulator (CFTR) conductance and intracellular [HCO3−], suppressing HCO3− secretion. Low pHi also increases the activity of the basolateral sodium-bicarbonate cotransporter (pNBC1), which, in turn, increases cellular bicarbonate concentration. When luminal pH returns to neutrality, acid diffuses out of the cell. The excess intracellular alkali increases pHi over baseline (overshoot), which activates CFTR and down-regulated adenoma (DRA), which then increases bicarbonate secretion. In the disease cystic fibrosis, a dysfunctional CFTR and DRA limit apical HCO3− exit, which increases pHi. (Modified from Kaunitz and Akiba [426], by permission.)

Mechanism of Gastroduodenal HCO3− Secretion This chapter has already described some of the molecular components of the duodenal epithelial HCO3− secretory pathway, and this section describes these components in more detail. The pancreas and duodenal epithelial cells can secrete HCO3− at high concentrations. The mechanism of this HCO3− secretion remains enigmatic, although much progress has been made regarding the identity of many of the key transport proteins and their regulation. Chapters 55, 73, 74, and 75 present detailed information regarding acid/base transporters and pancreatic secretion. Comprehensive reviews of gastroduodenal HCO3− secretion (2) and pancreatic HCO3− secretion (162) also have been published. We accordingly concentrate on the most recent data in this regard, and try not to recapitulate in too much detail the data addressing the mechanism of pancreatic HCO3− secretion, although there appear to be many similarities between pancreatic and duodenal epithelial anion secretion, such as similarly arrayed polarized acid/base transporters and the existence of high luminal CO2/HCO3− concentrations (2,10,163). Gastric HCO3− Secretion The stomach secretes HCO3−, a function that is of uncertain significance given the overwhelming simultaneous secretion of H+. Nevertheless, gastric mucosal HCO3− secretion has been implicated in the formation of a protective preepithelial alkaline layer, as described earlier (see the pH in the Mucous Gel Layer section). Measurement of gastric alkaline secretion has been inhibited by the presence of massive H+ secretion, necessitating the use of potent antisecretory compounds or measurement methods unaffected by the presence of acid secretion. Before the advent of the use of proton pump inhibitors, the use of antisecretory compounds to “unmask” underlying HCO3− secretion presented problems because HCO3− and H+ secretion may be coregulated. Perhaps the most successful means of measuring bulk HCO3− secretion is with the use of inline pH and CO2 electrodes, in which [HCO3−] is calculated using the Henderson–Hasselbalch equation (164,165). On a micro scale, a group in Bari, Italy, developed an electrophysiologic method to measure the pH of the lumen and epithelial cell pHi of individual gastric glands isolated from the frog Rana esculenta (166). Most reviews of HCO3− secretion have focused on duodenal secretion, although gastric HCO3− secretion differs from duodenal secretion in some ways. The locus of the HCO3− secreting cells has not been firmly identified, although there are data to support alkaline secretion by the oxynticopeptic cells of Rana esculenta (166,167). Suitable candidate anion exchangers have been localized to gastric surface cells in the case of anion exchanger (AE) or solute carrier (SLC) (AE2, AE4; SLC4A2, SLC4A4) (168–170) with putative anion transporter (PAT1; SLC26A6) also localized to the gastric epithelium (171). Nevertheless, isolated gastric surface cells appear to lack HCO3− secretory capability (172). The lack of CFTR expression in the gastric epithelium (173) further

1270 / CHAPTER 50 differentiates the mechanism of gastric HCO3− secretion from that of duodenum. Regulation of gastric HCO3− secretion has been studied much less intensively than has duodenal HCO3− secretion. Most of the studies are well summarized in excellent reviews (cf. Flemström [54]). We have studied the pH of the gastric preepithelial layer, which is highly strongly related to HCO3− secretion (see pH in the Mucous Gel Layer section earlier in this chapter). Gastric HCO3− secretion is EP1 dependent (174,175) (see Gastric Protection by Prostaglandins section later in this chapter). Duodenal HCO3− Secretion General Principles Duodenal HCO3− secretion is acknowledged to be an important, acid-augmented mucosal protective mechanism. The locus of secretion is thought to arise from the epithelial cells, which is the subject of the following paragraphs. Nevertheless, the Brunner’s glands that underlie the proximal epithelium also have secretory functions (176). Although their ability to secrete epidermal growth factor and mucus is unquestioned, Brunner’s glands might also secrete HCO3−. There are no direct data supporting HCO3− secretion from these structures, however, and abundant data to support epithelial HCO3− secretion in distal mammalian duodenum, which does not contain Brunner’s glands (177), or in frog duodenum, which is devoid of Brunner’s glands (178). Nevertheless, Brunner’s glands contain HCO3− transportrelated proteins such as carbonic anhydrase (CA) (179,180), and CFTR (173,181), suggesting that they may serve some HCO3− secretory function. Mechanism of Duodenal HCO3− Secretion The mechanism by which bicarbonate is secreted from the duodenal epithelial cell is controversial. Secretion is generally regarded as “basal” in the absence of prosecretory compounds or luminal acid and “stimulated” when secretion is augmented by an intervention. This distinction, although useful, can be arbitrary because the basal neural tone, for example, can not always be assessed, and can be altered by the type of anesthetic or fasting (182). Nevertheless, in vitro measurements suggest that basal secretion is electroneutral, and whereas secretion stimulated by adenosine-3′,5′-cyclic monophosphate (cAMP), for example, basal secretion is electrogenic, implying the recruitment of alternate secretory pathways by the secretagogue. As attractive as this hypothesis is, especially in light of the role of CFTR in cAMPstimulated HCO3− secretion, in vivo data have suggested that both basal and stimulated HCO3− secretion are active (183–188). Microfluorospectrophotometry has confirmed at least three classes of plasma membrane proteins attributed with acid/base transport in duodenal enterocytes. One is an amiloride-sensitive NHE that extrudes cellular acid, which

exists as NHE1 in the basolateral membrane and as NHE2 and NHE3 on the apical surface of duodenal mucosal epithelial cells (189,190). NHE1 is accepted as a “housekeeping” transporter because it is ubiquitously present on almost all cell types and is believed to remove excess acid equivalents formed from cellular metabolic processes (191). NHE2 and NHE3 are thought to help facilitate electroneutral intestinal NaCl absorption (190,192,193), although their function in the highly acidic proximal duodenum may differ from that in the more neutral jejunum and ileum. Proabsorptive transporters, such as NHE2 and NHE3, are reciprocally regulated with prosecretory transporters such as CFTR in kidney (194), which is consistent with the observation that the cellular messenger cAMP up-regulates CFTR function while down-regulating NHE3 function (195). Inhibition of apical membrane NHE2 and especially NHE3 augments human duodenal bicarbonate secretion (189). Selective apical NHE3 inhibition in rats significantly increased DBS in addition to decreasing luminal H+ entry. Interestingly, this inducible increase in duodenal bicarbonate secretion (DBS) can be reversed by concurrent selective inhibition of CFTR, indicating an underlying reciprocal CFTR-NHE interaction in which inhibition of NHE3 can up-regulate CFTR function and increase duodenal bicarbonate secretion (132). There is biochemical and functional evidence for regulatory interaction between CFTR and NHE3 (196,197), adding evidence for the suggestion that CFTR activity influences overall transcellular HCO3− transport by regulating the activity of proteins involved with HCO3− transport. Duodenal enterocytes import HCO3− from the blood plasma at the basolateral membrane by a variant of Na+:HCO3− cotransporter (NBC1), in response to decreased pHi (105,159,183,198). Ambient CO2 is also a source of intracellular HCO3− through the action of cellular CA (160, 183–185). CA is highly expressed in the cytoplasm, apical, and basolateral membranes of duodenal enterocytes (199). Of the soluble CA isoforms, CA II is abundant in the cytoplasm of epithelial cells, with soluble CA XIII being less abundant, whereas membrane-bound CA IX is expressed at the enterocyte basolateral membranes (179,200–202). The relative physiologic functions of the cytoplasmic and membrane-bound CA isoforms in the duodenal epithelium are currently unknown. Duodenal enterocytes then export HCO3− by Cl−/HCO3− exchange, as well as via an apical anion conductive pathway. The unquestioned role of CFTR in duodenal HCO3− transport is supported by numerous studies indicating that basal and acid-, cAMP and PG-stimulated HCO3− secretion is severely abrogated by CFTR absence or dysfunctional mutation (104,131,203–208). CFTR is a cAMP-regulated anion channel that can transport HCO3−, as well as Cl− (209). Although electrophysiologic studies revealed a CFTR PHCO3−:PCl− selectivity of ~0.25 (210–213), recent studies have shown that CFTR can function in an alternative conformation in which relative HCO3− selectivity is higher. Reddy and Quinton found that glutamate and ATP regulated CFTR anion selectivity in human sweat ducts (214). Further studies have indicated that CFTR relative

GASTRODUODENAL MUCOSAL DEFENSE / 1271 anion permeability is regulated by extracellular Cl− in CFTR transformed cell lines (215) and Xenopus oocytes (216). However, it is unclear whether CFTR serves directly as a bicarbonate channel, as a Cl− channel that either indirectly preserves transmembrane potential difference (217), or as a Cl− recycler that maintains transmembrane ionic gradients (218). Furthermore, HCO3− secretion in response to heatstable toxin (STA) in individuals with mutant CFTR may indicate the presence of alternative HCO3− channels (131). Moreover, CFTR is a key regulator of other cellular proteins (219), and thus may additionally up-regulate putative apical anion transporters in the duodenum (220,221). The Cl− dependence of duodenal HCO3− secretion has been controversial, with some laboratories finding no effect, whereas others found the luminal Cl− substitution inhibited but did not stimulate basal secretion, whereas others found that Cl− substitution inhibited basal secretion (160,178,209, 222–224). Nevertheless, a family of apical anion exchangers, which include down-regulated adenoma (DRA; SLCA26A3), pendrin (PDS or SLC26A4), PAT1 or SLC26A6, and AE4 or SLC4A4, have been immunolocalized to the brush-border membrane of duodenal epithelial cells and have been implicated in duodenal HCO3− secretion (171,220,225). Compelling data support coregulation of CFTR and SLC26A anion exchangers, probably through interactions involving the PSD-25/disc-large/zonula occludens-1 (PDZ) domains (220,225–229). Currently, the molecular mechanism for basal and stimulated duodenal HCO3− secretion remains controversial. Regulation of Duodenal HCO3− Secretion The most accepted physiologic stimulus is exposure to luminal acid, which produces a robust and long-lasting secretory response (230,231). Luminal acid exposure increases the generation of several mediators that augment mucosal secretion. Nevertheless, the epithelial cells might additionally be directly involved in the response to acidification because luminal acidification or increase of luminal pCO2, both of which augment HCO3− secretion, also decrease epithelial cell pHi (105,128,156,232,233). The local mechanism is not well understood, although intracellular acidification up-regulates cellular HCO3− uptake by reducing effective [HCO3−]i and possibly by up-regulating NBC1 (198,234). Furthermore, active HCO3− uptake increases [HCO3−]i upregulating soluble adenylate cyclase, which can then upregulate CFTR (235) (see Fig. 50-5). We endeavor here to focus on studies published during the early 2000s that have addressed the role of newly discovered isoforms of COX and NO and PG receptor subtypes. Regulation of duodenal HCO3− secretion has been reviewed extensively in the literature (1,2,4). Role of Prostaglandin and Nitric Oxide Prostaglandin E2 and EP Receptor Subtypes. Duodenal HCO3− secretion is stimulated by exogenously administered PGs, especially the E type. The receptor activated by PGE2 has been subdivided into four receptor subtypes, prostaglandin

type E (EP) EP1-4. We investigated, using various EP agonists and EP receptor knockout mice, the EP receptor subtype involved in the stimulatory action of PGE2 on duodenal HCO3− secretion (174,175). Duodenal HCO3− secretion is dose dependently increased by PGE2 or sulprostone (EP1/EP3 agonist), ONO-NT012 (EP3 agonist), or ONO-AE1-329 (EP4 agonist), but not by 17-phenyl PGE2 (EP1 agonist) or butaprost (EP2 agonist). The HCO3− stimulatory action of PGE2 was blocked by ONO-AE3-208, but not by the selective EP1 antagonist ONO-8711. We also found that PGE2 stimulated duodenal HCO3− secretion in both wild-type and EP1 receptor knockout mice, but not in the animals lacking EP3 receptors (175). Duodenal PGE2-stimulated HCO3− secretion thus is likely to involve the activation of EP3/EP4 receptors. From the data, it appears that activation of both receptor subtypes is required for a full secretory response, supported by the observation that marked potentiation of the HCO3− secretory response by coadministration of sulprostone (EP1/EP3 agonist) and ONO-AE1-329 (EP4 agonist) at subsecretory doses. Duodenal HCO3− secretion occurs in response to increased intracellular cAMP levels, as well as by the receptorindependent adenylate cyclase activator forskolin (222,231, 236,237). Ca2+ also functions as an intracellular mediator in HCO3− secretion, since this secretion is stimulated by A-23187 and inhibited by the removal of Ca2+ from the serosal solution (222). Indeed, the HCO3− stimulatory action of sulprostone (EP1/EP3 agonist) in the rat duodenum was significantly mitigated by verapamil and potentiated by pretreatment with isobutylmethyl xanthine (IBMX), an inhibitor of phosphodiesterase, suggesting that PGE2-stimulated HCO3− secretion is mediated by both Ca2+ and cAMP (174). EP receptor subtypes are coupled with different signal transduction systems; activation of EP1 receptors increases intracellular [Ca2+] independent of phosphoinositol (PI) turnover, whereas activation of EP2 and EP4 receptors increases intracellular cAMP (238). The EP3 receptor has four splice variants that are coupled to different signaling pathways (239). EP3A receptor is linked to activation of Gi protein, whereas EP3B and EP3C are coupled with activation of Gs protein, augmenting adenylate cyclase activity. Activation of EP3D increases [Ca2+]i by stimulating PI turnover. We confirmed that duodenal HCO3− response to ONO-AE1-329 was significantly augmented by pretreatment with IBMX, but not affected by verapamil, confirming the mediation by cAMP of the action of EP4 agonist. Role of Nitric Oxide. The influence of NO on duodenal HCO3− secretion has been controversial. We previously reported that the inhibition of NO production by NGmonomethyl-L-arginine methyl ester (L-NAME) increased duodenal HCO3− secretion in anesthetized rats (240,241), possibly by a vagally dependent mechanism (242). Guanylin, an endogenous activator of guanylate cyclase C, increased HCO3− secretion in the rat duodenum via cyclic guanosine monophosphate (cGMP) production (224). Because NO increases cGMP by stimulating soluble guanylate cyclase (243), it is likely that NO stimulates duodenal HCO3− secretion

1272 / CHAPTER 50 via cGMP. Indeed, duodenal HCO3− secretion was increased by the NO donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5nitro-3-hexenamide (NOR3), as well as by dibutyryl cGMP (dbcGMP), in the isolated bullfrog duodenum (244). Moreover, the NOR3-induced HCO3− secretory response was significantly antagonized by prior addition of methylene blue, an inhibitor of soluble guanylate cyclase, suggesting that this effect is mediated intracellularly by guanylate cyclase/cGMP. The HCO3− stimulatory action of NOR3 was similarly observed in the rat duodenum (245), supporting the contention that NO and cGMP participate in the regulation of duodenal HCO3− secretion. This secretory response is inhibited by the nonselective COX inhibitor indomethacin, implying mediation by endogenous PGs. NO or NO donors stimulate PG production; the NO donor S-nitroso-N-acetylpenicillamine stimulates PGE2 production in rat gastric epithelial cells independent of cGMP (246). We also observed that both NOR3 and dbcGMP increased duodenal mucosal PGE2 content and its release into the serosal solution. Because the HCO3− stimulatory action of NOR3 or dbcGMP in the duodenum was almost totally attenuated by indomethacin (244), endogenous or exogenous cGMP facilitates duodenal HCO3− secretion through COX activation. The mechanism of the HCO3− stimulatory action of cGMP appears to be different from that of guanylin or Escherichia coli heat-stable enterotoxin (STA). The latter two substances are thought to stimulate membrane-bound guanylate cyclase C, activating CFTR through cGMP-dependent kinase. The HCO3− stimulatory effect of guanylin or STA are resistant to indomethacin treatment, excluding the involvement of endogenous PGs in the mechanism (224). Acid-Induced Duodenal HCO3− Secretion Prostaglandin E2 and EP Receptor Subtypes. Mucosal acidification increased HCO3− secretion and PGE2 generation in the rat or mouse duodenum, responses that were significantly attenuated by indomethacin (231,245,247). These results confirm that PGE2 is a major mediator of duodenal HCO3− secretion in response to luminal acid. The acid-induced HCO3− response was totally absent in the mice with EP3 receptor gene disruption, although the PGE2 generation was increased in response to acid (231). The selective EP4 antagonist ONO-AE3-208 almost totally attenuated duodenal HCO3− secretion after mucosal acidification, supporting our hypothesis that the HCO3− stimulatory action of PGE2 is mediated by the combined activation of EP3 and EP4 receptors. Prostacyclin/IP Receptors. Duodenal mucosal acidification stimulates PG biosynthesis, with increased mucosal content of 6-keto PGF1a, as well as PGE2 (248). Prostacyclin (PGI2) or TY-10957, a stable PGI2 derivative, stimulates duodenal HCO3− secretion (249,250). Thus, PGI2 may also be involved in duodenal HCO3− response induced by acidification, in addition to PGE2. We have examined, using prostaglandin type I (IP) receptor knockout mice, the possible involvement of PGI2 and its IP receptors in this secretion and found that the presence of IP receptors is not essential for the regulatory process of acid-induced duodenal HCO3−

secretion. Then, a question arises as to why endogenous PGI2, despite its generation after acidification, does not contribute to acid-induced HCO3−secretion. Because PGI2 is generated in the vascular endothelium with a short biological half-life (251), this prostanoid might be easily degraded before reaching the epithelium, whereas PGE2 is mainly produced by the secreting surface epithelial cells. Role of Nitric Oxide and Its Interaction with Prostaglandins. Acid-induced HCO3− secretion in the rat duodenum was potently inhibited by pretreatment with L-NAME, as well as indomethacin (245). NG-nitro-L-arginine (L-NNA) inhibited acid-induced HCO3− secretion in conscious dogs (252). Similar findings were reported by Holm and colleagues (253), who showed in anesthetized pigs that luminal acidification augmented the HCO3− output in parallel with the increase of NO metabolites in the lumen, and that these responses were markedly inhibited by the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA). We also observed that luminal acidification significantly increased duodenal HCO3− secretion with concomitant luminal release of NO metabolites (NO2−/NO3−), with these responses abolished by L-NAME but not by indomethacin (245). Locally released NO thus augments duodenal HCO3− secretion in response to acid, without accompanying hemodynamic influences. PGE2 generation after mucosal acidification was blocked not only by indomethacin, but also by L-NAME in an L-arginine–sensitive manner (245,254). A secretory dose of NOR3 increased duodenal mucosal PGE2 generation, an effect suppressed by indomethacin (244,245). These results strongly suggest an interactive role for NO and PGs in the mechanism of acid-induced duodenal HCO3− response. The mechanism of acid-induced NO release is incompletely understood. Most epithelial cells, as well as submucosal cell types, can produce NO. Acid-induced HCO3− secretion is mediated via an axonal reflex pathway, in addition to endogenous PGs and NO (Fig 50-6) (236), a process attenuated by functional ablation of capsaicin-sensitive sensory neurons (255). Other studies also showed that gastric hyperemic response induced by acid back-diffusion is mediated by NO released by stimulation of sensory neurons (256). Mucosal acidification likely increases NO release through stimulation of capsaicin-sensitive sensory neurons. Several studies showed that the afferent side of the neuronal reflex pathway is influenced by PGs, probably by facilitating the neuronal excitation in response to acid, suggesting an interaction of PG and NO (255,257,258). These pathways likely are similar to those that mediate other protective response such as hyperemia and mucus release. Role of Cyclooxygenase and Nitric Oxide Synthase Isozymes. COX and NOS are key enzymes in the biosynthesis of PGs and NO, respectively. COX exists in two forms; COX-1 is a constitutive enzyme, whereas COX-2 message and protein are normally undetectable but can be induced by proinflammatory or mitogenic compounds (259,260), or even mucosal acid exposure in the stomach (261). Likewise, NOS also exists in two forms, the neuronal NOS (nNOS) is

GASTRODUODENAL MUCOSAL DEFENSE / 1273 constitutively expressed, whereas inducible NOS (iNOS) is induced by cytokines such as lipopolysaccharide (LPS) and tumor necrosis factor-α (243,262). Because the duodenal mucosa is continuously challenged by acid and food constituents, COX-2 and iNOS activity may also contribute to maintaining the HCO3− response and mucosal integrity against acid. SC-560, a selective COX-1 inhibitor, inhibited PGE2 biosynthesis and HCO3− secretion in responses to mucosal acidification (254), whereas the selective COX-2 inhibitor rofecoxib had no effect, nor was the expression of COX-2 mRNA up-regulated, indicating that enhanced PGE2 production after mucosal acidification is mediated by endogenous PGs produced by COX-1. Acid-induced duodenal HCO3− secretion was inhibited by L-NAME, in an L-arginine– dependent manner, but not by aminoguanidine at doses that selectively block iNOS but not cNOS (254). Holm and colleagues (263) reported that epithelial iNOS expression is up-regulated on acid exposure in parallel with an augmented HCO3− secretory response in the rat duodenum, a response prevented by the highly selective iNOS inhibitor, L-N61-iminoethyl-lysine. This discrepancy notwithstanding, the data confirm a role for endogenous NO in the duodenal HCO3− response to acid and may suggest that cNOS, but not iNOS, is the isoform involved in this response, at least during the acute phase of duodenal HCO3− secretion after mucosal acidification. Indeed, iNOS mRNA was not up-regulated in the duodenal mucosa after exposure to 10 mM HCl for 10 minutes, although mRNA for nNOS was constitutively expressed, but not affected, by mucosal acid challenge (254). Intracellular Mediators. All of the intracellular mediators such as cAMP, cGMP, and [Ca2+i] that regulate intestinal Cl− secretion also augment duodenal HCO3− secretion. Recently, melatonin, which is secreted by the pineal gland, but is abundantly expressed in the duodenum, was found to up-regulate HCO3− secretion (264,265). In a follow-up article, the authors found that short fasting, a common procedure preceding HCO3− measurement, diminished the secretory response to orexin A and bethanechol, without affecting the response to VIP or melatonin (182). Numerous studies have addressed the role of mediators such as PGs and NO in the regulation of duodenal HCO3−. A variety of substances including PGE2, VIP, theophylline, and adenylate cyclase–activating peptide stimulate HCO3− secretion in vivo and in vitro (174,222,236,266,267). These agents increase intracellular levels of cAMP by stimulating the adenylate cyclase or by phosphodiesterase inhibition, further supporting the role of cAMP as a mediator of duodenal HCO3− secretion. These effects are dose dependent, with high cAMP-producing effects that may be supraphysiologic (268). Furthermore, studies have demonstrated that guanylin, an endogenous activator of guanylate cyclase C, increases HCO3− secretion in the rat duodenum via cGMP, and that NOR3, the NO donor, stimulates duodenal HCO3− secretion in several species partly mediated by endogenous PGs (224,244,245). Moreover, NO donors stimulate PG

production in several organs and several types of cells including the GI epithelial cells (246,269,270).

SUBEPITHELIAL DEFENSE: GASTRODUODENAL MUCOSAL BLOOD FLOW, NEURAL SENSORS AND EFFECTORS, AND CHEMICAL MEDIATORS General Concepts Much of the regulation of preepithelial and epithelial defense mechanisms resides in the submucosa, which contains the acid sensors, neural pathways, and neuromediators that coordinate these mechanisms and responses to luminal acid. Furthermore, the submucosa contains the microcirculation, which is another acid-responsive, protective mechanism. These pathways have been discussed in detail elsewhere in this textbook (see Chapters 21, 24, 26, 27, and 31). This section endeavors to describe subepithelial defense mechanisms as they specifically apply to gastroduodenum. Mucosal blood flow is an important component of the gastroduodenal barrier function. It is under the elaborate control of the central and enteric nervous systems, autocrine/paracrine regulation of hormones and growth factors, and local mucosal production of eicosanoids. In the stomach, the presence of luminal acid increases delivery of vascular bicarbonate into the overlying mucous layer by the mucosal microcirculation, thereby neutralizing H+ ions invading from the lumen (57). This protective hyperemia is mediated by stimulation of capsaicin-sensitive extrinsic sensory nerve fibers, release of CGRP, and subsequent formation of NO, termed the capsaicin pathway (271) (see Fig. 50-2). The microcirculation in the submucosa of the organs of the upper GI tract plays an important role in the defense from injury caused by luminal acid. The overall role of mucosal blood flow, apart from its customary and accepted role as supplier of oxygen and remover of CO2 and cellular waste products, is somewhat controversial and thus far unproven. Certainly, the high-energy requirements of gastric parietal cells dictate constant circulation-delivered energy supply, but the function goes well beyond these considerations. Gastric hyperemia can be induced by luminal exposure to concentrated acid, but only in the presence of mucosal injury that decreases the high intrinsic impermeability of the gastric epithelium (256). In the stomach, for example, interventions that attenuate the hyperemic response to acid perfusion increase mucosal injury susceptibility (272–274). We have previously found that gastric hyperemia also can be induced with luminal acid in the presence of exogenous pentagastrin, simulating the postprandial gastric acid response (125). Interruption of this hyperemic response enhances mucosal injury susceptibility (275), underscoring and confirming the important role played by the gastric microcirculation in mucosal defense. The doctrine formulated by Silen and colleagues (276), which suggested that the gastric microcirculation delivered bicarbonate and

1274 / CHAPTER 50 carried away excess acid equivalents, continues to be the paradigm that serves as a useful framework for the interpretation of the data described in the following sections. Other elements of the submucosa that play an important defensive role include acid sensors, neural effector pathways, and chemical mediators. Acid sensing and regulation of blood flow are described in separate chapters, and thus are not described in detail here. This section focuses on chemical messengers originating from and receptors and enzymes located in the submucosa that have known or suspected protective functions. These mediators and receptors include the PGs, PG receptors, COX, protease-activated receptors (PARs), NO, and NOS.

Prostaglandins PGs have long been known to protect the gastroduodenal mucosa from injury, a contention supported by the clinical observations that inhibition of PG synthesis increases mucosal injury susceptibility, and that exogenous PGs can heal and prevent ulcers. PGs produced from arachidonic acid by two COX isoforms are present throughout the GI tract and bring about a wide variety of actions in the gut, including control of acid secretion, bicarbonate secretion, mucus production, mucosal blood flow, and maintenance of mucosal integrity (277,278). Exogenously administered PGs protect the GI mucosa against injury caused by stress, necrotizing agents, and NSAIDs. The pioneering study of Robert (279) was the first to demonstrate that exogenous PGs, especially PGE2, protect the stomach against necrotizing agents, a phenomenon termed gastric cytoprotection. Pharmacologic studies have classified four PGE2 receptors into four specific G protein–coupled subtypes, EP1-4 (238,280), as discussed earlier (see the Prostaglandin E2 and EP Receptor Subtypes section). The distribution of these receptors may explain the multiple effects of PGE2. Mice lacking prostanoid receptors (280–282) have been used to examine the role of specific PG receptors in mucosal protection (231,280,283). We have performed a series of experiments to determine the EP receptor subtypes mediating the GI protection afforded by PGE2, using rats and EP receptor knockout mice (174,283–286). We have also used prostanoids, subtype-specific EP receptor agonists and an antagonist, as tools to characterize the EP receptor subtypes involved in GI protection.

Organ-Specific Mechanisms Stomach Gastric Protection by Prostaglandins Numerous studies have tested the effects of PGs and subtypes on animal models of gastric injury. Gastric lesions induced by necrotizing agents such as ethanol or NSAIDs are considered the most suitable for examining the protective

action of PGE2 in the stomach (283–285). Oral administration of HCl/ethanol produced multiple bandlike lesions in the glandular mucosa, preventable by PGE2 pretreatment. This action of PGE2 was mimicked by prostanoids, such as 17-phenyl PGE2 or sulprostone specific to the EP1 receptor, and was significantly attenuated by ONO-AE-829, the selective EP1 antagonist (283). Neither butaprost, ONONT-012, nor 11-deoxy PGE1 had any effect on the gastric ulcerogenic response to HCl/ethanol. These results suggest that the protective action of PGE2 against HCl/ethanol is mediated by activation of the EP1 receptors. When the stomach is preexposed to a mild irritant such as taurocholate, the resistance of the mucosa to subsequently applied necrotizing agents increases, a phenomenon called “adaptive cytoprotection” (287). Because this effect disappears in the presence of indomethacin, a COX inhibitor, it is assumed that it is mediated through an enhanced production of endogenous PGs. Indeed, 20 mM taurocholate given orally increased the PGE2 content in the stomach and prevented the formation of gastric lesions induced by a subsequent challenge with HCl/ethanol (284), an effect antagonized by ONO-AE-829, the EP1 antagonist, suggesting that the adaptive gastric cytoprotection is mediated mainly by endogenous PGE2 through EP1 receptors. Oral administration of HCl/ethanol produced similar bandlike lesions in the stomachs of wild-type mice and those lacking EP1 or EP3 receptors. The development of these lesions was prevented by prior administration of PGE2 in both wild-type and EP3 receptor knockout mice, but not in the animals lacking EP1 receptors (283). Likewise, taurocholate acted as a mild irritant in the mouse stomach to increase production of PGE2, which resulted in prevention of HCl/ethanol-induced damage. This effect of taurocholate was significantly mitigated by pretreatment with indomethacin, as well as ONO-AE-829, the EP1 antagonist. Moreover, the protective action of taurocholate was observed in EP3 receptor knockout mice, but totally disappeared in EP1 receptor knockout animals (258,284). These results strongly suggest that EP1 receptors are essential for the cytoprotective action of PGE2, either generated endogenously or administered exogenously, in the stomach against necrotizing agents (Table 50-1). Capsaicin selectively stimulates capsaicin-sensitive afferent neurons through interaction with the vanilloid receptor VR-1 (TRPV1; see Acid Sensing and Blood Flow section) (288,289), an action abolished by chemical deafferentation and significantly attenuated by the antagonist of CGRP, as well as NOS inhibitors, consistent with the gastroprotective action of capsaicin being mediated by CGRP and NO associated with capsaicin-sensitive afferent neurons. Interestingly, the protective action of capsaicin was also significantly mitigated in the presence of indomethacin, suggesting an involvement of endogenous PGs, similar to the case of adaptive cytoprotection induced by a mild irritant (290,291). However, this effect of capsaicin was not affected by the selective EP1 antagonist ONO-AE-829, in contrast with that of taurocholate as a mild irritant (258). Neither stimulation of sensory neurons by capsaicin nor sensory deafferentation

GASTRODUODENAL MUCOSAL DEFENSE / 1275 TABLE 50-1. EP receptor subtypes in the alimentary tract Function

Action

EP receptor subtype

References

Acid-pepsin secretion Pepsin secretion Acid secretion

Increase Decrease

EP1 receptor EP3 receptor

283, 293

Bicarbonate secretion Stomach Duodenum

Increase Increase

EP1 receptor EP3/EP4 receptors

174 174, 231, 373

Mucus secretion Stomach Small intestine

Increase Increase

EP4 receptor EP3/EP4 receptors

294 286

Gastric mucosal blood flow Normal stomach Damaged stomach

Increase Increase

EP2/EP4 receptors EP1 receptors

283, 295 310, 311

Motility (circular smooth muscle) Stomach Intestine

Decrease Decrease

EP1 receptor EP4 receptor

283, 285 286, 374

affected mucosal PGE2 levels in the stomach. These results suggest that although endogenous PGs are involved in the gastric protection induced by both mild irritants and capsaicin, the mode of action appears to be different in these two cases (258,284). Although stimulation of afferent neurons by capsaicin does not increase the generation of PG in the stomach, its gastroprotective action is partly dependent on endogenous PGs. We found that the protective action of capsaicin was significantly restored even in the presence of indomethacin by prior administration of butaprost, the EP2 agonist, but not EP3 or EP4 agonist. Because capsaicininduced gastric protection was not affected by the EP1 antagonist, it is unlikely that EP1 receptors are involved in the facilitation by endogenous PGs of this action. Indeed, capsaicin exhibited gastric protection even in mice lacking EP1 and EP3 receptors, confirming that the protective action of capsaicin is not related to the activation of EP1 and EP3 receptors. Nevertheless, we found that capsaicin did not provide gastric cytoprotection against HCl/ethanol in IP receptor knockout animals (258). These findings in knockout mice suggest that IP receptors also are involved in the protective action of capsaicin in the stomach, in addition to EP2 receptors. Currently, the exact mechanism by which endogenous PGs contribute to the protective action of capsaicin remains unknown. Boku and colleagues (257) reported a lack of release of CGRP in response to mild injury in the stomach of IP receptor knockout mice. Thus, it is assumed that endogenous PGI2 plays a supportive role in the mechanism of capsaicin-induced gastric cytoprotection, probably by sensitizing capsaicin-sensitive afferent neurons. NSAIDs such as indomethacin produce gastric damage presumably by depletion of endogenous PGs by inhibiting COX activity. Gastric lesions induced by indomethacin were effectively and dose dependently prevented by supplementation of exogenous PGE2 (285,292). This effect of PGE2 was mimicked by sulprostone and 17-phenyl PGE2, both having

a potent affinity to EP1 receptors, and significantly attenuated by the EP1 antagonist ONO-AE-829, the result being similar to the protective action against HCl/ethanol (285). Neither butaprost, ONO-NT-012, nor 11-deoxy PGE1 afforded significant protection against indomethacin-induced gastric lesions. Moreover, indomethacin-induced gastric damage was similar in wild-type and in EP1 or EP3 knockout mice, although the protective action of exogenous PGE2 was observed in wild-type and EP3 receptor knockout mice, but not in mice lacking EP1 receptors. These data support the hypothesis that PGE2 prevents indomethacin-induced gastric lesions through the activation of EP1 receptors. Endogenous PGs regulate gastric acid and mucus/bicarbonate secretion and mucosal blood flow and motility that may contribute to gastric cytoprotection. According to previous studies (176,233,293–295), PGE2 inhibits acid secretion through its effects on EP3 receptors and increases mucus and bicarbonate secretion through EP4 and EP1 receptors, respectively (see Table 50-1). We also found that PGE2 has an acid stimulatory effect mediated by histamine released from enterochromaffin-like (ECL) cells through EP4 receptors. Furthermore, the acid inhibitory action of PGE2 is mediated by EP3 receptors in two ways, directly inhibiting acid secretion at the parietal cells and indirectly through inhibition of histamine release at ECL cells. In a preliminary study, we observed that gastric mucosal blood flow (GMBF) was increased by EP2 and EP4 agonists, but not EP1 agonists (283). Of interest, prostanoids exhibiting a preference for only EP1 receptors affected gastric motility and provided mucosal protection against gastric lesions induced by HCl/ethanol or indomethacin (283,285). These effects were antagonized by the EP1 antagonist ONO-AE-892, suggesting that the effect of PGE2 on motility is paralleled by a reduction in gastric mucosal damage, paralleling prior studies (290,292,296–298). Mersereau and Hinchey (296) were the first to show the importance of stomach hypermotility in

1276 / CHAPTER 50 the genesis of gastric lesions in response to NSAIDs. Indomethacin at an ulcerogenic dose enhances gastric motility and induces microcirculatory disturbances caused by abnormal mucosal compression of the gastric wall (298,299). Because neither butaprost, ONO-NT-012, nor 11-deoxy PGE1 provided any gastric protection against HCl/ethanol or indomethacin, despite increasing GMBF, it is unlikely that the gastric cytoprotection afforded by PGE2 is functionally associated with an increase of GMBF (283). Certainly, because inhibition of gastric motility may lead to attenuation of microvascular disturbances caused by stomach contraction, it is possible that prostanoids through EP1 receptors help to maintain mucosal blood flow during exposure to noxious agents. Neutrophils have been implicated in the NSAID-associated damage (300). PGE2 inhibits neutrophil functions, including chemotaxis. We confirmed that PGE2 inhibited neutrophil migration in vitro (285). The same inhibitory action was shown by both butaprost and 11-deoxy PGE1, but not by 17phenyl PGE2, sulprostone, or ONO-NT-012, clearly indicating that the antineutrophil chemotaxis action of PGE2 is mediated by activation of EP2 and EP4 receptors. Thus, it is assumed that the inhibition of neutrophil migration by itself is not sufficient to reduce the overall expression of gastric lesions in response to indomethacin. Because the increase in myeloperoxidase activity, as well as lesion formation induced by indomethacin was prevented when the enhanced gastric motility response was inhibited by atropine (298), it is likely that the neutrophil infiltration is secondarily associated with gastric hypermotility after indomethacin administration. Indeed, Melarange and colleagues (301) and other investigators showed that NSAID-induced gastric injury is neutrophil independent in the neutropenic rat, in contrast with prior findings (302). The mechanism by which PGE2 inhibits gastric motility through EP1 receptors is unknown. Milenov and Golenhofen (303) report that PGE2 relaxed the circular muscle but contracted the longitudinal muscle of the canine stomach. Narumiya and his group (304,305) report the localization of mRNA of the EP receptors along the GI tract. They showed that strong signals for EP1 transcripts occurred in the smooth muscle cells in the muscularis mucosa throughout the tract. Because EP1 receptors are coupled to phosphatidyl inositol turnover (280), it is assumed that contraction of longitudinal smooth muscle by PGE2 is associated with an increase of cytosolic calcium. Contraction of circular smooth muscle produces mucosal folds, which have been implicated in the pathogenesis of several ulcer models including indomethacininduced gastric lesions (296,298,299,306). Currently, the mechanism by which PGE2 relaxes circular smooth muscle through activation of EP1 receptors is unknown. Endogenous PGE2 also plays a role in the gastric hyperemic and protective responses after barrier disruption in the stomach as induced by bile acids. We reported that the COX-1 isozyme is involved in gastric functional responses, such as an increase of GMBF and a decrease in acid secretion, observed acutely after barrier disruption in the stomach (306–309). These functional alterations after barrier disruption

are adaptive responses of the stomach and play an important role in protecting the mucosa against acid injury by disposing of H+ and maintaining a microclimate for cellular restitution. This hyperemic response in the damaged stomach is attenuated by the EP1 antagonist ONO-8711 and disappears in EP1 receptor knockout mice, strongly suggesting mediation by the activation of EP1 receptors (310). We also showed no role for prostacyclin IP receptor in this phenomenon (311). Along the same line, another group found that capsaicin protection, which is CGRP dependent, was abrogated in homozygous IP knockout mice, leading the investigators to conclude that PGI2 modulates CGRP release (312). One infrequently considered action of PGs is potentiation of injury. Indeed, PGs likely augment gastric injury caused by the administration of exogenous histamine. Although PGE2 is usually gastroprotective, it strongly augmented gastric lesions in histamine-treated rats (313). These effects were prevented by an EP1 antagonist, were associated with increased microvascular permeability, and were not caused by acid secretion per se. Finally, one group studies the mechanism of gastroprotection at the cellular level using isolated guinea pig mucosal cells in primary culture. Ethanol decreased viability, increased the release of mitochondrial cytochromes, and increased apoptotic markers and apoptosis, all effects abrogated by PGE2. Further study indicated that the PGE2-mediated protection was mediated by EP2 and EP4 receptors and was associated with cAMP generation and protein kinase A activation (314). In summary, these studies demonstrate that PG-mediated gastroprotection involves mechanisms other than the augmentation of mucusbicarbonate secretion and mucosal blood flow, but also affects paracellular permeability, neural signaling pathways, and cellular signal transduction. Other Mediators Another signaling system has been invoked in gastroduodenal protection: the PARs (315). These receptors, which are expressed abundantly throughout the entire GI tract and connected secretory organs, are expressed mostly on smooth muscle cells, but also on the GI mucosa. As their name implies, PARs are activated by proteases, which unmask a cryptic N-terminal sequence that then serves as a tethered ligand to activate the receptor. The gastric mucosa has two of these receptors, PAR-1 and PAR-2, which are both associated with gastroprotection. Activation of PAR-2 appears to mimic the mucosal response to luminal acid with an increase of mucus secretion and mucosal blood flow accompanied by inhibition of acid secretion. This response, similar to the acid response, is dependent on the presence of capsaicin-sensitive nerves and CGRP. PAR-1, in contrast, also increases mucosal blood flow through the generation of endogenous PGs (Fig. 50-7). Several publications have highlighted the gastroprotective mechanisms of PAR activation. One group used the specific oligopeptide TFLLR to activate gastric PAR-1, finding that the exogenous peptide decreased gastric injury susceptibility

GASTRODUODENAL MUCOSAL DEFENSE / 1277 Trypsin Tryptase Factors Vila, Xa Unknown proteases Thrombin

PAR-2

• Mucosal protection (via activation of sensory neurons) • Mucus secretion ↑ • Mucus blood flow ↑ • Acid secretion ↓ • Pepsinogen secretion ↑

PAR-1

FIG. 50-7. Modulation of gastric mucosal functions by protease-activated receptor 2 (PAR-2) and PAR-1. (Modified from Kawabata [315], by permission.)

• Mucosal protection (via prostanoids) • Mucosal blood flow ↑ • Acid secretion ↑ • Pepsinogen secretion ↑

in a standard rat injury model, an effect abolished by the nonselective COX inhibitor indomethacin and the selective COX-1 inhibitor SC-560, but not by the selective COX inhibitor NS-398 (316). Although the PAR-1 agonist increased GMBF, the increase was not suppressed by indomethacin, although indomethacin abolished the PAR-1 agonist–mediated protection, suggesting a minor role of increased GMBF for the protective effect of PAR-1. Another group of receptors that may play a role in gastroduodenal defense are the peroxisome proliferator-activated receptors γ (PPAR-γ), not to be confused with PAR. PPAR-γ is part of a superfamily of nuclear receptors that play key roles in cellular differentiation and adipose tissue metabolism. PPAR-γ, which is highly expressed in gastric epithelial cells, is thought to play important roles in apoptosis and differentiation. Interestingly, NSAIDs, in addition to their COX-inhibitory activity, can act as ligands for PPAR-γ. In an interesting publication, one group found that indomethacin increases expression of the aforementioned gastric trefoil peptide TFF2 in cultured gastric epithelial cells (317). This induction was associated with augmented PPAR activity, suggesting that PPAR-γ might be involved in indomethacin-induced, augmented TFF2 expression. The effect of angiotensin II type 1 (AT1) antagonism was examined in a stress model of gastric injury. A remarkable reduction of gastric lesions produced in response to cold restraint stress was observed in the presence of an AT1 antagonist. The mechanism by which AT1 receptors may mediate mucosal injury in response to stress may include vasoconstrictor effects, suppression of proinflammatory cytokine production, and preservation of the pituitary-adrenal stress response (318). In another study, four different benzodiazepines were used in an in vitro, perfused model of gastric injury. The data were most consistent with a protective effect mediated by the γ-aminobutyric acid receptor A (GABAA) (319).

Duodenum Acid Sensing and Blood Flow The proximal duodenum is exposed to cyclical and rapid variations of luminal pH. Unlike other acid-exposed organs such as the stomach or esophagus, the duodenum has high transepithelial permeability to water and solutes, necessitating the presence of nonstructural defense mechanisms such as mucus and bicarbonate secretion and submucosal blood flow. We have shown previously that a brief exposure to intense luminal acidity, corresponding to physiologic acid stress, enhances all measured duodenal defense mechanisms, including mucus secretion/gel thickness increase, increased cellular bicarbonate concentration, and increased mucosal blood flow (Table 50-2). The mucosal sensor underlying these rapid changes, however, remains unknown, although there are good data that indicate that the acid sensor is a component of the well-known afferent branch of the enteric nervous system, with actual acid sensing transduced by a newly discovered acid-sensitive receptor. Studies using VR-1 antibodies have shown that there is intense staining of VR-1–immunoreactive nerves in the duodenal epithelium, including the lamina propria mucosa up to the villous tips and down to the pericryptal regions, the submucosal layer and intrinsic nerves (myenteric plexus) (271). These VR-1–positive nerves highly colocalize with CGRP. Furthermore, VR-1–positive neurons are present not only in the dorsal root ganglion (splanchnic afferent center), but also in the nodose ganglion (vagal afferent center) and in the myenteric plexus (intrinsic afferent center); and vagotomy, not sympathetectomy, abolishes the acid-induced hyperemic response in duodenum, suggesting that VR signaling projects to vagal afferents and intrinsic afferents, but unlikely to splanchnic afferents (270). These histologic and surgical studies confirm the physiologic observations reported earlier and have helped to confirm our supposition regarding the

1278 / CHAPTER 50 TABLE 50-2. Gastroduodenal protective mechanisms modulated by luminal acid Mechanism

Organ/Cell

References

↑ Mucus secretion ↑ Mucus gel thickness Alkalinization of preepithelial mucous gel

Rat gastric mucosa in vivo Rat duodenal mucosa in vivo Mouse stomach Rat stomach Duodenum Gastric surface cells Rat duodenal epithelial cells Chief cell monolayers Cultured rat gastric mucous (RGM)-1 cells Cultured RGM-1 cells Rat stomach Rat duodenum Rat stomach Rat duodenum Mouse duodenum Human duodenum Rat stomach Rat duodenum Rat dorsal root ganglia

126 39, 271 42, 51–53 57, 58, 375 2, 254, 255, 376, 377 71, 72 105, 156 87, 89, 90 88

↑ HCO3− secretion ↑ Apical membrane resistance ↑ Cellular buffering power ↑ Transmucosal resistance ↓ Paracellular permeability ↑ E-cadherin expression ↑ Paracellular permeability ↑ Submucosal prostaglandin and nitric oxide generation ↑ Mucosal blood flow ↑ Vanilloid receptor VR-1 (TRPV1) expression

nature of the acid-sensing protective up-regulation of duodenal defense mechanisms, differently from the gastric defenses in which the splanchnic afferents contribute to the acidinduced hyperemia (320). Other candidates for the upper GI acid sensor have been identified, including the acid-sensing ion channel (321,322). Submucosal capsaicin-sensitive afferent nerves have been strongly implicated in the mediation of gastroduodenal protection (320). These nerves are involved with the protective mucosal response to luminal acid, including increases of mucus and bicarbonate secretion and mucosal blood flow, although the actual mechanism transducing luminal acid into neural signals has not been fully defined. The cloning of the vanilloid receptor, or VR-1, which senses acid, coupled with the finding that VR-1 is present in the gastric mucosa (323), suggested the testable hypothesis that VR-1 may, in part, transduce the luminal acid signal. We found that VR-1 was strongly expressed in the gastric cell line RGM-1, and that VR-1 activation by capsaicin, the potent vanilloid resiniferatoxin, acid, and ethanol protected the cells from injury (324) in the absence of neural signaling. In another study, the physiologic stress induced in rats by water immersion increased gastric PGE2 synthesis and CGRP concentrations. The VR-1 antagonist capsazepine, selective denervation of capsaicin-sensitive nerves, and a CGRP antagonist greatly increased stress-related gastric injury, underscoring the role of this pathway in mucosal protection (325). Our studies revealed some novel observations about the nature of duodenal blood flow and its regulation. For example, inhibition of sodium-proton exchange with the potent amiloride analog dimethylamiloride inhibited the hyperemic acid response (128). Interestingly, acidification of the cytoplasm by alternate means, such as with an ammonium chloride prepulse or valinomycin, increased blood flow, also inhibitable by dimethylamiloride (128). These studies

92 378 96–98 247, 306 245, 379 231 248 126, 289, 378, 380, 381 271, 379, 382 322

suggested that acid must pass through the epithelial cell and exit via by the basolateral NHE isoform NHE1 before eliciting a hyperemic response. In further studies, we examined the sensing mechanisms underling the hyperemic response. Capsazepine, a VR-1 antagonist, abolished the hyperemic response to acid, confirming the involvement of VRs in the acid response. Further studies also confirmed that the hyperemic response was mediated by the capsaicin pathway, which includes afferent sensory nerves and release of the neuropeptide CGRP and the vasodilatory gas NO, but was not inhibited by indomethacin, a nonselective COX inhibitor (326). These studies provided data supporting our proposed mechanism of duodenal acid–induced hyperemia, including acid diffusion into the epithelial cell, basolateral extrusion via NHE1, activation of VRs on afferent nerves, CGRP release, with activation of endothelial NO synthesis and production of vasodilatory NO. Prostaglandin-Mediated Duodenal Protection As mentioned earlier, HCO3− secretion plays an important role in protection of the duodenal mucosa against luminal acid (247,327). Indeed, perfusion of the proximal duodenum with 20 mM HCl for 4 hours caused only a few hemorrhagic lesions in wild-type mice. Gene disruption of EP1 receptors did not affect the duodenal ulcerogenic response to acid perfusion because the lesion score was not significantly different compared with that of wild-type mice. In EP3 receptor knockout mice, however, acid perfusion for 4 hours provoked widespread lesions with a score approximately sixfold greater than that observed in wild-type littermates (231). Decreased HCO3− secretion in EP3 receptor knockout mice may progressively diminish the mucosal defensive response to acid and may increase mucosal susceptibility to acid injury. Thus, the presence of EP3 receptors is essential for maintaining

GASTRODUODENAL MUCOSAL DEFENSE / 1279 duodenal HCO3− secretion and mucosal integrity against luminal acid.

lesions could be the nucleating event in the creation of larger damage and erosion. This remains a fertile area for discovery.

INJURY AND RESTITUTION

Imposed Mucosal Damage

Chapter 16 is dedicated to a discussion of mucosal repair and restitution; therefore, the following discussion focuses only on how these processes interface with the need for and mechanisms of mucosal defense. In brief, it is useful to think of the tissue response to injury as being a balancing act between defense mechanisms and ongoing damage and aggressive factors. Repair of a breach in the mucosal barrier can only occur when conditions tip in favor of defense mechanisms.

Most of our knowledge about mucosal repair comes from conditions when damage is much more extensive than those envisioned to occur physiologically, and is in response to exogenous insult. As described in more detail in Chapter 16, one of the more remarkable and consistent findings in response to imposed gastric damage is that epithelial cell exfoliation occurs promptly, followed by rapid restitution of the epithelial layer. This captured the interest of early physiologists because large portions of the gastric epithelium could be temporarily lost without endangering the body. It has been noted since the work of Claude Bernard in the 1850s (336) and was amplified by the systematic work of Hollander (337) in the early twentieth century, examining the damaging effects of eugenol, the gastric irritant in clove oil. In 1949, Hollander and Goldfisher (337) defined the three phases of repair in response to surface epithelial damage as: (1) rapid reepithelialization (restitution) of the epithelial layer within an hour with flat cells; (2) transition of flat cells to columnar morphology; and (3) reformation of pits in areas where they were vanquished by earlier damage. The repair was morphologically complete in 2 days. Building on observations from William Silen’s group, the later work of Eric Lacy and colleagues (338–340) brought widespread recognition that superficial damage elicited a distinct category of response. In particular, preservation of viable pit cells and the stem cell niche in the isthmus was essential for rapid restitution of the epithelium. It was the pit cells that spread and migrated onto the surface to restore epithelial continuity (339,341). If damage included the proliferative/stem cells, it invariably produced hemorrhagic lesions and required extensive epithelial remodeling that required weeks to fully repair (338). The most prominent clinical instigators of ulceration are H. pylori and drugs (NSAIDs), but despite a burgeoning and burdensome literature on their effects on gastroduodenal function, it remains difficult to assign a firm pathogenic sequence as to how they cause extensive damage of the stomach lining. The difficulty has been in following the early events when these effectors have weakened stomach (or duodenal bulb) defenses, but not yet caused widespread lesions. For instance, the presence of NSAIDs or H. pylori may compromise repair of focal epithelial lesions such as those created by physiologic cell renewal, and these sustained microscopic lesions may be the initiating events that imperil the tissue and lead to larger erosions. Conversely, it has been conventional to ask how H. pylori influences gastric damage at different stages of infection, but it may be equally important to question how the occurrence of minor epithelial disruptions affects H. pylori colonization. H. pylori expresses chemotactic receptors that attract bacteria toward bicarbonate, urea, and arginine (342,343). Bicarbonate chemotaxis is

Daily Challenges to Epithelial Integrity As detailed in the previous section, an intact epithelial layer is required to sustain a barrier to caustic luminal contents, with evidence suggesting the key epithelial elements are TJs and a relatively proton-impermeable apical membrane (328). Surprisingly, there has been little recognition for the consequences of the physiologic renewal of the epithelial layer, which requires rapid turnover of epithelial cells. Cells within the gastric gland isthmus contain stem cells that proliferate asymmetrically and generate new pit cells that migrate outward to repopulate the surface epithelium every 3 days in the mouse (329). Consequently, a third of the surface epithelial cells must be replaced daily. In the 1980s, electron and light microscopy suggested two mechanisms for surface epithelial cell renewal. Either cells undergo necrosis and are shed, or they enter apoptosis and are engulfed by neighboring (epithelial or nonepithelial) cells (329). Necrotic cells are more frequently observed in the gastric epithelium compared with apoptotic cells (330–333). In both cases, massive degranulation of surface mucous cells accompanies cell degeneration and has been suggested as a means to provide a local gel coating to protect against the luminal environment as cells are removed from the epithelial layer (331,332,334,335). Because of the difficulty in exploring events that require dynamic study of individual cells in a living tissue, it has remained largely unexplored how (and if) cell shedding occurs without compromising the epithelial barrier. Similarly, little is known about how foodstuff produces microscopic gastric epithelial “nicks” during aggressive physical processing. Given the physical stresses of antral peristalsis and retropropulsion of gastric juices, limited cell damage must occur. Although never investigated, this injury will be difficult to detect because damaged areas will be randomly scattered and rapidly repaired. These physiologic stresses to the epithelium may have importance beyond their transient disturbance of mucosal defense. When the ability to repair a modestly disrupted epithelium is impaired, creation of such microscopic epithelial

1280 / CHAPTER 50 independent of urease activity, but elimination of chemoattractant receptors blocks bacterial colonization of the stomach (344,345).

Cell Exfoliation and the Mucoid Cap The rapid sequence of events after gastric damage has been most firmly established in the case of intact stomach transiently exposed to absolute ethanol or hypertonic NaCl. In both of these models, superficial epithelial damage is pervasive. Virtually the entire surface epithelium detaches within minutes and is shed into the lumen (340,341). The basis for cell exfoliation may be either direct damage to all the shed cells or bystander effects that drag healthy cells into the lumen attached to other cells that have been damaged. Regardless of cause, a mucosa denuded of epithelium is predicted to be a remarkably fragile structure to expose to the caustic luminal environment. In this situation, acid and protease back-diffusion is predicted to rapidly further erode the mucosa, although this does not appear to occur for reasons that remain unclear. The shed cells are held suspended directly over the tissue surface by virtue of being embedded in a “mucoid cap” composed of mucus, cell debris, fibrin, and other undefined constituents that form a coating over the surface (335,338, 341,346,347). All features of the mucoid cap have been defined based on conditions of extreme superficial gastric damage, when virtually all the surface epithelium is denuded into the lumen. This raises concerns about whether the mucoid cap is even a relevant structure when less pervasive damage occurs at the gastric surface. However, the process of creating an enhanced mucoid gel layer directly over an area with compromised cells is also supported for the opposite extreme when only one or a few cells are shed as part of the physiologic renewal of the epithelial layer (see the following section). Thus, results are consistent with smaller mucoid caps being physiologically relevant. In pervasive damage models, the mucoid cap is a compartment of high pH at the gastric surface, believed to make a safe haven for epithelial repair by trapping bicarbonate in the mucoid region (348,349). After superficial damage, is the altered surface pH control best interpreted as a measure of disrupted integrity of epithelial barrier or as site of dysregulation of pH control? Both interpretations are valid, with the key question being to identify the source(s) of alkali that appears in the stomach at the site of a damaged epithelium. Some believe that a flood of HCO3—rich extracellular fluid and blood plasma constituents flow across the epithelial breach (350), a contention supported by the presence of fibrin and albumin in the mucoid cap that overlies the region of damage in these pervasive damage models (335,341). Nevertheless, luminal addition of NSAIDs blocks the alkaline pH observed in the mucoid cap (349). The latter observation could suggest that local PG-regulated bicarbonate secretion is a more likely source of alkali, or alternatively might be explained by decreases in mucosal blood flow

caused by NSAIDs (351,352). Blood is the ultimate source of bicarbonate ions that reach the lumen, whether they are transported by epithelia or leak across a hole in the epithelial layer (353). The mucoid cap also is believed to provide an impermeable barrier to block flow of acid into the body and simultaneously block the flow of extracellular fluid into the gastric lumen. The accessibility of luminal constituents through the mucoid cap has never been appraised, although the concept of relative impermeability has been suggested by observing diffusion of HCl into isolated mucoid cap material being 100-fold slower than into conventional gastric mucus (354). Furthermore, physical removal of the mucoid cap or addition of a mucolytic agent (N-acetyl cysteine) increased egress of both hemoglobin and albumin into the lumen of the damaged stomach (346,355). There are a number of questions about whether the mucoid cap seen with focal epithelial damage also presents a permeability barrier to protect the epithelium, how rapidly the epithelium is defended from luminal acid after damage is imposed, and how bicarbonate permeation into the cap can be allowed when other substances must be impermeant.

Epithelial Restitution The paradigm of epithelial restitution was established by Hudspeth (356) in 1975, when he used a glass micropipette to pluck a single cell from an isolated sheet of gallbladder epithelium, then observed the neighboring cells filling the gap and simultaneously restoring normal transmucosal resistance within 30 minutes, Conceptually, restitution requires cells to transiently detach from neighboring cells and extracellular matrix, migrate across vacant parts of the basement membrane, and ultimately reattach cell-cell and cellsubstrate adhesions. In gastroduodenal epithelia, restitution does not require cell replication (25,357). When cells are lost from the gastric surface, they are replenished from viable cells remaining in the gastric pits. The speed of gastric restitution is remarkable. Even after denuding 95% of the rat surface epithelium in vivo by transient exposure to absolute ethanol, the migration of epithelial cells covers all but 15% within 15 minutes after removal of the irritant (341). In bullfrog stomach studied in vitro, restitution also occurs in response to hypertonic solutions, but takes 4 to 6 hours (340,353). The rapidity of the process in vivo is assumed to be physiologically important to limit the time that the epithelial barrier is breached, but has restricted study of in vivo restitution to models with extreme gastric damage, so that after imposing damage, the denuded epithelium can be found rapidly and restitution monitored quickly. A group of peptides and proteins that strongly stimulate epithelial cell migration have been identified and termed motogens. Motogens can stimulate random cell movement (chemokinesis) or directional movement along a concentration gradient of the motogen (chemotaxis). Two classes of motogens are known. One includes a wide variety of cytokines

GASTRODUODENAL MUCOSAL DEFENSE / 1281 (interleukins, interferon-γ) and epidermal, transforming, and fibroblast growth factors (EGF, TGF-α, and FGF, respectively) that bind to their membrane receptors and indirectly activate TGF-β1. TGF-β1 is localized to surface mucous cells and parietal cells (358). Many of these proteins also stimulate cellular proliferation, but part of their activity is direct stimulation of migration distinct from proliferative effects. In addition to the TGF-β–dependent pathway, trefoil peptides (introduced earlier in the pH in the Mucous Gel Layer section) are also motogens, which act via pathways independent of TGF-β, and have no mitogenic effects (357). Animals with genetic disruption in trefoil peptide genes have confirmed a role of TFF1 and TFF2 in gastric epithelial protection, but confirm additional facets in their physiologic effects beyond regulation of cell migration. TFF1 knockout mice have dysplastic gastric pits with a notable lack of mucins (glands seem approximately normal) and invariably form adenomas of the pylorus and antrum by the time mice reach 5 months of age. TFF1 knockout does not affect TFF3 expression, but does lead to complete loss of TFF2 in approximately two thirds of TFF1−/− animals (359). TFF1 knockout mice have not yet been examined in any experimental gastric damage model. As described earlier, TFF2 knockout mice have altered gastric morphology and basal function, but most notably, a striking sensitivity to NSAID damage compared with normal animals (33). Overall, results suggest that trefoil peptides have a major role in repair of epithelial damage or

sustaining epithelial cell viability, or both. Many of the observed consequences of trefoil gene disruption may be caused by the shared ability among trefoils to reduce proliferation and to keep cells from undergoing apoptosis (31,33, 360,361). In this case, the variable phenotypes among trefoil knockout mice may be caused by different cellular targets being protected by each trefoil peptide. This would help to reconcile the diverse phenotypes in the knockout mice with the observations that gastric damage increases expression of all three TFF isoforms at the margin of injury (362–364), and that exogenous addition of either TFF2 or TFF3 had similar protective effects on gastric repair (365).

ANIMAL MODELS OF GASTRODUODENAL INJURY We would like to conclude the chapter with a description of some of the commonly used rodent models of gastroduodenal injury. These models can be categorized into several classes, depending on the intended mode of action. Importantly, these model systems have been used to examine the effects of signaling pathways and other epithelial and submucosal factors, as well as an empirical readout of relative gastroprotective activity of a given compound. Experimental models of gastric damage generally separate into two categories. There are numerous extreme measures that rapidly produce abundant or deep lesions, or both, in

TABLE 50-3. Commonly used models of gastroduodenal injury Type of injury model

Example

Mechanisms identified or utility

References

Necrotizing agents

Ethanol, bile acids, supraphysiologic concentrations of acid, hypertonic saline Hemorrhagic shock followed by reperfusion

Prostaglandin-dependent mucosal defense independent of acid secretion or luminal acid

108, 275, 380, 383–390

Systemic acidosis, reactive O2 metabolites, mast-cell degranulation, and neutrophil adhesion Prostaglandin and COX-dependent mechanisms Gastric acid hypersecretion Central mechanisms, increased vagal output, microcirculatory disturbance Acid hypersecretion, deficient duodenal HCO3– secretory response; somatostatin depletion; altered redox state Quantitation of ulcer healing rate

83, 391–395

Direct cytotoxicity, apoptosis, wound healing Luminal acid-responsive and preinjury homeostatic mechanisms

419–421

Ischemia/reperfusion

NSAIDs

Indomethacin administration

Pyloric ligation Stress

“Shay ulcer” Water immersion restraint

Cysteamine

Cysteamine gavage

Acetic acid

Acetic acid application to serosa RGM-1 cells injury assay

Cultured epithelial cells Surrogate injury markers

Transepithelial permeability to small organic molecules; transepithelial potential difference; epithelial cell pHi; nuclear staining with propidium iodide

247, 275, 396, 397 380, 398–409 410–416 368, 370, 372

417, 418

97, 128, 156, 422–426

COX, cyclooxygenase; NSAIDs, nonsteroidal anti-inflammatory drugs; pHi, intracellular pH; RGM, rat gastric mucous.

1282 / CHAPTER 50 some cases selectively destroying the entire surface epithelium (e.g., absolute ethanol, boiling water, liquid nitrogen cold probe touched to the outside of intact stomach, 1–4 M NaCl). Alternatively, there are less extreme measures that produce scattered lesions of varying magnitude and location over a time course of days to months (e.g., Helicobacter, NSAIDs, physical stresses). In the former case, damage often is extreme and extensive, with the dominant concern being that damage is beyond anything encountered in physiology or in pathobiology (366). Because most clinically relevant gastric ulceration is not caused by an initial insult that deeply damages the tissue, this also encouraged use of superficial damage models. Despite these limitations, the extreme models provide some of the only reliable data on the rapidity of cell and tissue response to damage, because the damage initiation can be timed precisely Alternatively, the slow and less extreme models yield a more pathophysiologic level of damage, but suffer from a weaker power of analysis because of lesion creation at unknown times. In these models, it is difficult or impossible to follow events that occur in the first few hours (or days) after cells are damaged, which leads to confusion about the ability of a treatment to limit damage versus promote healing. Nevertheless, several of these model systems have been studied in some detail. For example, the cysteamine model of duodenal ulceration in rats was first described by Selye and Szabo in1973 (367). Subsequent articles have described the mechanism of this injury, including hypergastrinemia with increased gastric acid secretion, diminished duodenal HCO3− secretion in response to mucosal acid perfusion, depletion of somatostatin, and most recently, alterations of mucosal redox status and induction of heatinducible factor 1 (368–372). Other categories include necrotizing agents, NSAIDs, ischemia/reperfusion, topical acetic acid, pyloric ligation, physiologic and psychologic stress, cultured epithelial cells, and surrogate markers of injury (Table 50-3).

SUMMARY AND CONCLUSIONS The turn of this century has brought substantial advances to the understanding of how the gastroduodenal mucosa is protected from injury. Technical breakthroughs, such as the advent of digital imaging, live-animal microscopy, cultured epithelial cell monolayers, electrophysiology, the availability of selective receptor inhibitors and agonists, transgenic mice, Doppler flowmetry, the cloning of key ion transporters, and ion-selective fluorophores, alone and in combination, have enabled investigators to study in unprecedented detail, and in the absence of frank injury, the homeostatic and acidregulated protective mechanisms that join forces to protect the mucosa. An understanding of these mechanisms bears on basic interactions of environmentally exposed epithelial surfaces, enhancing the mechanistic understanding of disease processes.

ACKNOWLEDGMENTS This work was supported by Veterans Affairs Merit Review funding, grants from the National Institutes of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (RO1 54221, J.D.K.; RO1 DK54940, M.H.M.), and the Kyoto Pharmaceutical University “21st Century COE” Program and “Open Research” Program from the Ministry of Education, Science and Culture of Japan (K.T.).

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CHAPTER

51

Genetically Engineered Mouse Models of Gastric Physiology Linda C. Samuelson

Genetic Engineering in the Mouse, 1293 Transgenic Mice, 1294 Gene-Targeted Mice, 1295 Overview of Gastric Acid Secretion, 1296 Stimulation of Acid Secretion from the Parietal Cell, 1296 Inhibition of Acid Secretion, 1297 Cellular Organization of the Gastric Epithelium, 1298 Mice with Acid Regulatory Mutations, 1298 Gastrin Pathway Mutants, 1299 Histamine Pathway Mutants, 1301 Acetylcholine Pathway Mutants, 1303 Somatostatin Pathway Mutants, 1303 Parietal Cell Channel and Transporter Mutants, 1304 Apical Membrane Channels and Transporters, 1305

Basolateral Membrane Transporters, 1306 Other Gastric Mutants, 1306 Transgene Expression in the Gastric Mucosa, 1306 General Transgene Promoters, 1306 Parietal Cell Transgenics, 1307 Enterochromaffin-Like and Neuroendocrine Cell Transgenics, 1307 Gastrin Promoter and G-Cell Transgenics, 1308 Adenosine Deaminase Promoter and Forestomach Transgenics, 1308 Conclusion, 1308 References, 1309

GENETIC ENGINEERING IN THE MOUSE

and inhibitory pathways regulating acid secretion. Studies of genetically engineered mouse models also have provided insight into the consequences of altered gastric physiology on the cellular composition of the gastric mucosa. Transgenic technology bridges the fields of molecular genetics and integrative physiology. Genetically engineered mice contain foreign DNA sequences as a permanent component of their genomes integrated into a chromosome and transmitted to progeny in a Mendelian fashion. Thus, the effect of a transgene or engineered gene mutation on gastric physiology can be evaluated in vivo in intact animals, as well as in vitro in cells isolated from genetically modified strains. There are two distinct approaches to generate genetically engineered mice depending on whether DNA constructs are introduced into fertilized eggs (transgenic mice) or into embryonic stem (ES) cells (gene-targeted mice). Although transgenics can be generated in a number of mammalian species in addition to the mouse, gene-targeting technology

The mouse has emerged as a dominant experimental model system to investigate in vivo physiology because of the capability to generate transgenic and gene-targeted models. Genetically engineered mouse models have provided a new opportunity to investigate the importance and function of specific molecules and pathways involved in the development and function of the stomach. In addition, analysis of loss-of-function and overexpression models has allowed a better understanding of the complex stimulatory

L. C. Samuelson: Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1293

1294 / CHAPTER 51 currently is limited to the mouse. The large array of powerful tools for genome engineering, coupled with mouse genome information and the long history of mouse genetics, has allowed the mouse to emerge as an important model for analysis of gastric physiology.

Transgenic Mice

Fertilized egg Promoter/enhancer

polyA

Microinject transgene construct

The earliest developed transgenic approach involves the stable transfer of recombinant DNA molecules into the mouse germ line by microinjection into fertilized eggs. Development of transgenic mice using this procedure was first reported by several laboratories in the early 1980s (1–5). A transgene construct is microinjected into the pronucleus of zygotes, the eggs are implanted into pseudopregnant female mice, and approximately 20% of the resulting pups have the transgene integrated into their genomes (Fig. 51-1). The site of integration is random and typically contains multiple copies of the transgene in a single array. Integration commonly occurs before the first zygotic cleavage event; thus, every cell in the transgenic founder mouse, including the germ line, contains the same transgene insert. The transgene array is inherited as a Mendelian trait, and transgenic lines can be established by breeding the founder mice that arise from the microinjected eggs. Alternatively, founders can be evaluated directly. There is often inherent variability among the founders and the resulting transgenic lines because of effects of the variable integration sites on transgene expression. Occasionally (5–10%), the transgene causes a secondary gene mutation at the site of the insertion that is unrelated to the transgene phenotype (6). For this reason, and because transgene arrays can be unstable when carried on both chromosome copies, transgenic lines tend to be maintained as hemizygotes by breeding to wild-type mice in each generation. Several transgene promoter/enhancers have been used to drive expression in the gastric mucosa, including both general and cell-specific promoters. For example, the general cytomegalovirus (CMV) promoter, which is broadly expressed in numerous cell types in the mouse, has been used to alter parietal cell function (7). In addition, several cell-specific promoters, such as the parietal cell–restricted H+,K+-ATPase β-subunit promoter (8), have been used to target transgene expression to specific gastric cells. Transgene expression is overlaid on the normal complement of gene expression in the mouse; thus, only dominant transgene phenotypes can be evaluated with this approach. The transgene construct must contain a complete transcription unit, including sufficient promoter/enhancer sequences to direct expression to the cell type of interest, the cDNA or gene of interest with translational start and stop sequences, and 3′ termination polyadenylation site. Although intronless transgenes have been used in numerous studies, in some cases, introns have been determined to improve transgene expression, and thus should be included in the construct (9). After transfer into the mouse genome, transgene expression is dependent on a number of factors. The promoter/enhancer

Implant into pseudopregnant female

Pups from injected eggs

Transgenic founder

FIG. 51-1. Generation of transgenic mice. The transgene construct is microinjected into the pronucleus of a fertilized mouse egg. Generally, the male pronucleus is injected because of its larger size. The transgene construct is a complete transcription unit, including promoter/enhancer sequences to direct transgene expression to specific cells in the mouse, sequences encoding the gene of interest (filled box), and polyadenylation (polyA) site. After microinjection, the construct can integrate randomly into a chromosome, and a proportion (~20%) of the pups born after implantation into pseudopregnant females contain the transgene as a permanent component of their genomes. The pups are screened by analysis of genomic DNA to identify those containing the transgene. These transgenic founder mice can be studied directly, or they can be bred to establish transgenic lines. Random insertion of the transgene into different chromosomal locations can cause some variability in expression among the founders. For this reason, studies should include more than one transgenic line to check for insertion site–specific effects.

sequences in the construct are primarily responsible for regulating cell specificity and developmental timing of transgene expression, although interactions with other regulatory sequences near the integration site can affect the promoter activity. Thus, significant variation in expression from line to line can arise because of the differing integration sites for

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1295 each line. For this reason, it is usually recommended to include more than one independently generated transgenic line in the analysis to check for line-specific effects. Because gene expression is complex and the sequence elements regulating transcription are not easily recognized and, in some instances, are quite distant from the gene, genomic segments directing proper transgene expression must be determined empirically by testing in transgenic studies. In general, for characterizing a new promoter/enhancer fragment for transgenic expression, it is desirable to use large gene segments to improve the likelihood of including essential regulatory elements in the construct. Tissue and cell specificity must be analyzed critically in a number of independent transgenic lines to assess the specificity and efficiency of expression. The large genomic inserts in bacterial artificial chromosome (BAC) clones (100–200 kb) commonly result in transgenics with expression patterns that closely mimic the endogenous gene encompassed in the BAC. Although BAC transgenics have not yet been broadly used for analysis of gastric physiology, the development of BAC recombineering techniques facilitates the manipulation of these large clones for production of transgenes (10), and BAC transgenics should be useful for future exploration of gastric biology because they could allow expression in cell types for which effective transgene promoter/enhancers have not yet been characterized.

Gene-Targeted Mice A second technique for producing genetically engineered mouse models has been developed that takes advantage of the pluripotency of cultured mouse ES cells (11,12). This approach combines homologous recombination techniques with the ability of ES cells to contribute to the mouse germ line, to create mouse strains with specific gene modifications (Fig. 51-2). ES cells are derived from the inner cell mass of a preimplantation mouse embryo that forms the embryo proper; thus, these cells have the potential to differentiate into all cell types in the body. ES cells can be maintained in culture indefinitely in an undifferentiated state. Engineering specific genetic alterations into the ES cell genome is achieved through homologous exchange between the DNA of a targeting construct containing a gene mutation and the endogenous gene being targeted. Homologous recombination is inherently inefficient in mammalian cells, but with selection and genetic screening of numerous clones, ES cell clones with the desired targeted gene mutation can be identified. After microinjection of correctly targeted ES cells into a blastocyst, a chimeric mouse, composed of a mixture of host cells and ES-derived cells, is generated. The pluripotent ES cells can contribute to all of the tissues of the developing chimera, including the germ line. Transmission of the targeted gene mutation to progeny takes place when a germ-line chimera is bred. This establishes a gene-targeted strain containing the specific gene mutation initially engineered into the ES cells in culture. The first reports of germ-line transmission of a targeted allele were published in 1989 (13,14).

Targeting construct DNA

ES cell culture Select for cells that integrate DNA Screen for targeted clones

Microinject targeted cells

ICM

Blastocyst Implant into pseudopregnant female

Pups born from injected blastocysts

Germline chimeric mouse

FIG. 51-2. Generation of gene-targeted mice. A targeting construct is introduced into cultured embryonic stem (ES) cells, and those cells that integrate the DNA into their genomes are selected using an antibiotic resistance marker. Genetic screening of numerous colonies by Southern blotting or polymerase chain reaction identifies cell clones that integrated the targeting construct via homologous exchange to replace the endogenous gene with the mutant allele. Correctly targeted cells are microinjected into blastocysts where they combine with the host inner cell mass (ICM) and develop into chimeric mice after implantation into pseudo-pregnant females. Chimeras can be identified by their patchy coat color resulting from the mixture of cells derived from the host blastocyst and the targeted ES cells, which normally have differing coat color genes. The efficiency of chimera production varies among targeted ES clones because of loss of differentiation potential during time in culture. Germ line–competent ES cells contribute to the developing germ line of the chimera; thus, breeding the chimera results in transmission of the targeted gene mutation to progeny to form a targeted strain.

1296 / CHAPTER 51 Since then, hundreds of genetically engineered mice have been generated, including several with gastric phenotypes. A broad spectrum of gene mutations can be generated, ranging from point mutations altering a single amino acid in a protein to large deletions that remove the gene of interest (15). To date, targeted mice with mutations in genes altering gastric physiology have been largely null mutations. Because the mutant gene replaces the endogenous allele, recessive phenotypes can be analyzed by generating homozygoustargeted mice. This contrasts with transgenic models, which can be used only to analyze dominant phenotypes because the endogenous genes are left intact. Table 51-1 summarizes some of the key differences between transgenic and genetargeted mice. These differences stem primarily from the ability to manipulate ES cells in culture, and thereby create mice from clones that have undergone rare targeting events to replace the endogenous gene with an engineered mutation. The majority of ES cell lines used for gene targeting are derived from strain 129/Sv mice, because these cells are generally more germ line–efficient than ES lines derived from other mouse strains. However, the use of 129 ES lines is complicated by the extensive genetic variability observed in 129 substrains that arose over time from outcrossing 129 mice to other strains (16). In 1999, the names of the substrains of 129 mice were changed to reflect their relations to each other. Four different groups were recognized, including 129P (parental) substrains, 129S (steel) substrains, 129T (teratoma susceptible) substrains, and 129X substrains, which were contaminated early in their history and differ significantly from the other three 129 substrains. The genetic variability impacts on homologous recombination frequency, which is improved with a perfect DNA match (17). Thus, to maximize targeting efficiency for a particular experiment, it is desirable to match the ES cell substrain with that of the cloned mouse genomic DNA used for the targeting construct. Consideration of genetic background effects and appropriate controls are also complicated by 129 substrain variability. In general, gene-targeted mice used for the study of gastric physiology have been on a mixed genetic background, which results from the common practice of breeding the strain 129-derived chimera to C57BL/6 mice to establish the targeted mouse strain. The resultant mutant mice are on a mixed 129 and C57BL/6 strain background, which can vary in subsequent generations depending on the breeding strategy. It is not known how much genetic background influences various aspects of gastric physiology. Although strain-specific differences in gastric physiology have not been

studied systematically, certain strains are more susceptible to Helicobacter-induced inflammatory changes in the stomach (18), and differences in parietal cell lifespan have been reported to vary among strains (19). Thus, it is recommended that gene-targeted and transgenic lines be backcrossed to place the engineered locus on a pure genetic background to minimize physiologic variation. Because homologous recombination places the targeted gene sequences in the normal location in the chromosome with the associated genetic regulatory sequences, the normal pattern of expression from the engineered locus is maintained. This feature has been useful for characterization of patterns of gene expression or for marking cells by targeting a reporter gene such as bacterial β-galactosidase (lacZ) into the endogenous gene locus (20,21). Expression of the easily detected reporter can be examined to determine the spatial and temporal patterns of endogenous expression.

OVERVIEW OF GASTRIC ACID SECRETION Gastric physiology has been a topic of study for many decades. To describe the various transgenic and gene-targeted mouse models with alterations in gastric acid secretion, this section provides a brief overview of the mechanisms of acid secretion. (See Chapters 48 and 49 for an in-depth examination of the parietal cell and regulation of acid secretion, respectively, to achieve a richer understanding of the complex physiology of gastric acid secretion.) This chapter focuses on genetically engineered mouse models with alterations in gastric acid secretion and their contribution to our understanding of in vivo acid secretion. Before discussing the features of these mice, a summary of the current view of the components and regulatory pathways for gastric acid secretion is presented.

Stimulation of Acid Secretion from the Parietal Cell Gastric acid secretion is proportional to the amount and type of food ingested. A complex network of stimulatory and inhibitory signals regulates this process (Fig. 51-3). Endocrine, neural, and paracrine factors work in concert to regulate the secretion of acid from parietal cells, which is one of the most abundant cell types in the corpus of the stomach. The hormone gastrin, paracrine factor histamine, and neurotransmitter acetylcholine are the major acid secretogogues (22).

TABLE 51-1. Comparison of conventional transgenic and embryonic stem cell–derived genetically engineered mouse models

Transgene phenotype Insertion site Transgene copy number Reporter expression Other events

Transgenic

Gene-targeted

Dominant Random Variable (1–1000) Influenced by promoter and insertion site Insertional mutation (5–10%)

Recessive or dominant Targeted 1 per haploid genome Parallels endogenous gene Possible effect on nearby gene

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1297 Acid

H/K-ATPase

Lumen

Ca2+ M3

Parietal cAMP

Mucosa Ca2+ CCK2

H2

Acetylcholine

Histamine

Gastrin

ECL CCK2 ENS

FIG. 51-3. Stimulation of gastric acid secretion in parietal cells. Acid secretion is stimulated by the neurotransmitter acetylcholine, the paracrine factor histamine, and the hormone gastrin. Acetylcholine binds to muscarinic 3 (M3) receptors on the parietal cell to stimulate calcium signaling; histamine binding to H2 receptors primarily signals through increased cyclic adenosine monophosphate (cAMP); gastrin binds to cholecystokinin-2 (CCK2) receptors to evoke an increase in intracellular calcium. Parietal cell stimulation normally involves all three inducers proportional to the ingested meal. On stimulation, the H+,K+-ATPase pumps move from intracellular tubulovesicular membranes to the apical (canalicular) membrane, with subsequent acid secretion into the gastric lumen. Histamine release from enterochromaffinlike (ECL) cells occurs in response to both gastrin and neuronal stimuli from the enteric nervous system (ENS).

Gastrin is secreted from endocrine cells in the distal stomach in response to a meal. Circulating gastrin stimulates parietal cells directly by binding to gastrin/cholecystokinin-2 (CCK2) receptors. However, gastrin can also activate the parietal cell indirectly by stimulating histamine release from enterochromaffin-like (ECL) cells. Histamine stimulates parietal cells after binding to histamine 2 (H2) receptors. The importance of direct versus indirect stimulation of the parietal cell by gastrin is not fully understood, although the potent acid-blocking effect of H2 receptor antagonists suggests that gastrin primarily acts via the indirect histamine-dependent pathway (23). The third major stimulatory pathway involves activation by the neurotransmitter acetylcholine, which binds to muscarinic 3 (M3) receptors on the parietal cell. Neural stimulation can also activate ECL cell histamine release. Some evidence suggests that the neuropeptide pituitary adenylate cyclase–activating polypeptide (PACAP) binding to type I PACAP receptors (PAC1) on the ECL cell may be the mechanism of neural-stimulated histamine release (24–26), although this has not yet been fully defined.

In parietal cells, CCK2 and M3 receptors are thought to couple to Gq, which, on stimulation, activates phospholipase C to increase inositol triphosphate and the release of intracellular calcium (27–30). The H2 receptor in these cells couples primarily to Gs, which activates adenylate cyclase and evokes an increase in cyclic adenosine monophosphate (cAMP) (31,32), although it has been shown in some species, including the mouse, to also couple to calcium (28,33). There is in vitro evidence that increased levels of both cAMP and calcium are required for stimulated acid secretion (34,35). Because histamine release from ECL cells is stimulated by both gastrin and neural input, cAMP and calcium signaling pathways are normally activated in concert to stimulate acid secretion from the parietal cell in vivo during the complex regulation seen on ingestion of a meal (see Fig. 51-3). On agonist stimulation, parietal cells undergo distinct morphologic changes that are associated with acid secretion. Unstimulated parietal cells contain abundant intracellular membrane compartments known as tubulovesicles, which sequester H+,K+-ATPase pumps inside the cell. On stimulation, the tubulovesicles fuse with the apical (canalicular) membrane of the parietal cell, exposing H+,K+-ATPase pumps to the lumen, and thus enabling acid secretion to take place (36,37). Cessation of acid secretion involves reestablishment of the intracellular tubulovesicles and internalization of H+,K+-ATPase (36,37). Analysis of mice engineered to express a mutant form of the H+,K+-ATPase β-subunit (H/Kβ) that cannot be internalized demonstrated that sequestering the proton pump in tubulovesicles is not required for regulated acid secretion (38).

Inhibition of Acid Secretion Somatostatin has been shown to be an important physiologic regulator of acid (39) secretion that acts as a paracrine inhibitor at several different levels. Secreted from a specific endocrine cell population termed D cells, somatostatin in the corpus has been shown to inhibit ECL cell histamine release (40,41), and also to directly inhibit parietal cell acid secretion (42,43). Molecular and pharmacologic studies suggest that the somatostatin subtype 2 (sst2) receptor is the predominant receptor regulating parietal cell acid secretion (43–48) and ECL cell histamine release (41,49). D cells also are found in the more distal, antral region of the stomach near gastrinsecreting cells (G cells) where somatostatin release has been associated with paracrine inhibition of both gastrin secretion and synthesis (50,51). Thus, somatostatin is thought to inhibit acid secretion by directly inhibiting the parietal cells, as well as indirectly by suppressing histamine release from ECL cells and gastrin release from G cells. Somatostatin secretion can be stimulated by the intestinal hormone CCK binding to CCK1 receptors on the D cell (39,52,53). This is one mechanism whereby acid secretion is reduced during the intestinal phase of gastric physiology. The actions of CCK and gastrin in the stomach suggest that the CCK1 receptor is inhibitory, whereas the CCK2 receptor

1298 / CHAPTER 51 is stimulatory for acid secretion. Under normal conditions, CCK does not compete with gastrin to activate CCK2 receptors because the concentration of circulating gastrin is more than 10 times greater than circulating CCK concentration (54).

Cellular Organization of the Gastric Epithelium Parietal cells, together with the other gastric epithelial cells, are organized into gastric glands, the functional units of the gastric acid secretory system. In the acid-secreting corpus, the glands contain four characteristic regions: (1) the pit region at the top of the gland, which consists primarily of surface mucous cells; (2) the isthmus, which contains stem cells and immature progenitor cells; (3) the neck region, which contains a variety of cell types, including mucous neck cells; and (4) the base, which contains chief and endocrine cells (Fig. 51-4). Parietal cells are located in all four regions of the gastric gland, although they predominate in the lower pit, isthmus, and neck regions. The close juxtaposition of the

Mucous cells

Stem cells

Parietal cells

Pit

Isthmus

Neck

various gastric cell types facilitates paracrine regulation of cell development and function, such as ECL cell histamine release stimulating nearby parietal cells. The epithelium is constantly renewing, and progenitor stem cells that reside in the isthmus give rise to all of the different epithelial cell types in the gastric gland (55). The stem cells divide, differentiate, and then migrate toward the pit or base region of the gland. Through a series of careful microscopic studies coupled with 3H-thymidine labeling, Karam and Leblond (55–61) described the proportions and distinct turnover rates of the various cell types in the mouse gastric gland. The surface mucous cells mature and turn over in approximately 4 days (58). The rapid rate is probably necessary because of the harsh conditions at the luminal surface. Parietal cells have a lifespan of 54 days (61), whereas chief cells live longer with a turnover rate of approximately 6 months (57). The cell types in the gastric gland can be distinguished by their morphology and expression of specific markers (Table 51-2). Parietal cells can be readily identified by their large size, centrally located nuclei, numerous mitochondria and tubulovesicles, and expression of H+,K+-ATPase α- and β-subunits. The large size and structural complexity has been used to purify viable mouse parietal cells by flow cytometry (33). The surface mucous cells can be identified by their position in the pit region, staining characteristics, and expression of trefoil factor 1 (TFF1). In contrast, the mucous neck cells are small and difficult to distinguish unless stained for TFF2 or with GSII lectin. Endocrine cells are also small and difficult to identify under normal light microscopy; however, they are readily stained with antibodies to chromogranin A (CgA). Because ECL cells are the predominant endocrine cell type, CgA staining is frequently used to assess ECL cells in various mouse models. Other more specific markers for ECL cells include vesicular monoamine transporter 2 and histidine decarboxylase (HDC). Lectins also have been used to stain for specific gastric cell types (see Table 51-2) (62).

MICE WITH ACID REGULATORY MUTATIONS Chief cells

Base

FIG. 51-4. Cellular composition of the gastric glands in the mouse corpus. Stem cells in the isthmus region divide and differentiate to generate the various epithelial cell types in the gastric glands. Surface mucous cells migrate upward and populate the pit region of the stomach. The acid-secreting parietal cells migrate upward toward the lumen, as well as downward toward the base of the gland, and can be found in all four regions of the gland: pit, isthmus, neck, and base. The neck region contains parietal cells, mucous neck cells, enterochromaffin-like (ECL) cells, and other endocrine cell types. The predominant cell type in the base is the pepsinogen-secreting chief cell. Mutations altering acid secretion frequently also result in cellular changes to the gastric glands.

Several transgenic and gene-targeted mouse models with altered gastric physiology have been generated. Because acid secretion is not a vital function, these mutants are commonly viable and fertile, allowing for the careful examination of changes in gastric physiology as a consequence of specific gene mutations. These genetically engineered mouse models have provided an opportunity to reevaluate the importance and function of specific molecules and pathways for the in vivo regulation of acid secretion and for the growth and development of the gastric mucosa. Mutants in the three major acid stimulatory pathways have been described, as well as mutants in the inhibitory somatostatin pathway. Table 51-3 lists the various genetically engineered mouse models with alterations in gastric acid, including mice with regulatory mutations, and mice with mutations in critical

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1299 TABLE 51-2. Prevalence, marker expression, and lectin staining of cell types in the mouse corpus Cell type

Proportiona

Parietal cells

13%

Surface mucous cells

19%

Mucous neck cells Chief cells

7% 35%

Endocrine cells ECL cells

7%

D cells A cells

Markers

Lectin stainingb

H/K-ATPase α-subunit H/K-ATPase β-subunit Trefoil factor 1 Muc5AC Trefoil factor 2 Pepsinogen Intrinsic factor Chromogranin A Histamine Histidine decarboxylase Vesicular monoamine transporter 2 Somatostatin Ghrelin

DBA UEAI GSII

aThe proportions of mature cell types in the gastric glands were determined by Karam and Leblond (60). The enterochromaffin-like (ECL) cells represent the majority of the chromogranin A–positive endocrine cells. Immature cells make up the remainder of the cells in the gland. bDBA, Dolichos biflorus; GSII, Griffonia simplifolica II; UEAI, Ulex europaeus I.

parietal cell channels and transporters. In addition to gross acid phenotype, Table 51-3 summarizes gastric morphology, because alterations in acid secretion are commonly associated with cellular changes in the mucosa. Gastrin Pathway Mutants Gastrin Loss of Function Mice with null mutations in the genes encoding the hormone gastrin (54,63) and its receptor (64,65) have been generated by gene targeting in ES cells (see Table 51-3). These gene-targeted mouse strains have been used to investigate the in vivo importance of gastrin in the acid secretory system. Because of the extensive body of research showing that gastrin is a physiologic stimulator of acid secretion (66), it was expected that the loss of gastrin signaling would result in decreased acid. However, the impairment in acid secretion in these mutants was greater than anticipated. Gastrin mutants had marked reductions in both basal and induced acid secretion. Analysis of acid secretion in gastrin-deficient mutants was performed by perfusion in anesthetized mice with and without stimulation with acid secretogogues (54). In the CCK2 receptor–deficient mutants, acid secretion was measured with the pyloric ligation method (64). Measurement of resting gastric pH was performed in both ligand- and receptor-deficient mice (63,65). In each case, basal gastric acid levels were significantly reduced in the gastrin pathway mutants. Stimulated acid secretion was also severely impaired in gastrin-deficient mice, because acute induction with histamine, carbachol, or gastrin did not increase acid (54). This finding was surprising, because previous experimental models in which gastrin-induced acid secretion was acutely blocked with a gastrin immunoneutralizing antibody or CCK2 receptor antagonists were still responsive to other agonists (67,68). The lack of agonist-stimulated acid secretion in gastrin pathway mice is unique and suggests a fundamental

requirement of gastrin for acid secretion and/or for the development of the acid secretory system. Gastrin replacement by continuous perfusion using osmotic minipumps was able to partially restore acid secretion in gastrin-deficient mice (54). Perfusion with a combination of gastrin 17 and glycine-extended gastrin 17 enhanced repair of the acid secretory system even further, with the data suggesting that these two forms of gastrin work synergistically in the parietal cell to up-regulate acid secretion and to prevent cell degradation (69). Thus, the cellular components of the acid secretory system in this mutant are capable of secreting acid once gastrin is provided. The lack of an acid secretory response to acute gastrin treatment suggests that the gastrin-induced repair takes some time to occur. Increased cellular proliferation is associated with the repair (69). However, activation of acid secretion with 2 days of gastrin treatment suggests that the repair primarily involves alteration of preexisting cells in the mucosa of the mutants rather than development of new cells. Histologic examination of the major cell types in the gastric glands of the gastrin pathway mutants showed that gastrin is not required for development of the acid-secreting parietal cells or the other major cell lineages in the corpus (54,63–65). There was a general thinning of the gastric mucosa in CCK2 receptor–deficient mice (64), which is consistent with the known trophic effect of gastrin on parietal and ECL cells (66,70). There are conflicting reports whether there are less parietal cells in gastrin-deficient mutants, with some studies suggesting modest reductions (54,65), whereas another reporting increased numbers (33). The discrepancy might be because of the effects of the progressive gastric inflammation in these mice that results from bacterial overgrowth in the hypochlorhydric stomach (71). Although the acid defect in gastrin pathway mutants is not caused by a loss of parietal cells, there were significant functional changes to both parietal and ECL cells in these mice. There is growing evidence that the parietal cells of

1300 / CHAPTER 51 TABLE 51-3. Genetically engineered mouse strains with altered gastric acid secretion Acid

Gastrina

Gastric mucosal cell changesb

Method

References

Gastrin KO

Reduced

Absent

Thin mucosa: altered parietal and ECL cells

Targeting

54, 63

CCK2 receptor KO

Reduced

High

Targeting

64, 65

Gastrin/CCK KO

Alteredc

Absent

Targeted

72

Gastrin overexpression

Increased

High

Thin mucosa: less parietal and ECL cells Less, but more active, parietal cells Hyperplasia: increased parietal and ECL cells; older mice: atrophy with loss of parietal cells

Transgenic

79–81

Histamine H2 receptor KO

Alteredd

High

Targeting

84, 85, 88

HDC KO

Reduced

High

Targeting

86, 87, 90

H2/CCK2 receptor KO

Reduced

High

Targeting

88

Acetylcholine M3 receptor KO M5 receptor KO

Reduced Impaired

High ND

Normal Normal

Targeting Targeting

95 96

Somatostatin Receptor 2 KO Somatostatin KO

Increased Increased

Normal High

ND Normal

Targeting Targeting

98 99, 141

Parietal cell acid secretory machinery Absent H+,K+-ATPase α KO H+,K+-ATPase β KO Absent

High High

Targeting Targeting

103 82, 105

H/Kβ/gastrin KO

Absent

Absent

Targeting

105

H+,K+-ATPase β mutant KCNQ1 KO

Increased

ND

Abnormal parietal cells; dysplasia Abnormal parietal cells; hyperplasia; loss of chief cells; increased immature cells Abnormal parietal cells; loss of chief cells Hyperplasia

Transgenic

7

Absent or Low

High

Abnormal parietal cells; hyperplasia

110, 111

NHE2 (Na+-H+) KO

Increased Increasedg

Targeting

115

AE2 (Cl−/HCO3−) KO

Absent

Increasedg

Progressive loss of parietal and chief cells Few parietal and chief cells; surface mucous cell hyperplasia Surface mucous cell hyperplasia; less and abnormal parietal cells

114

NHE4 (Na+-H+) KO

Progressive lossf Reduced

Targeting/ Inducede Targeting

Targeting

116

Miscellaneous TFF2 KO

Increased

Low

Targeting

119

Foxl1 KO

Low

ND

Targeting

120–122

Mouse model Gastrin

aCirculating

Hyperplasia: increased parietal and ECL cells Hyperplasia: increased parietal and ECL cells Thin mucosa: reduced parietal and ECL cells

Thin mucosa, decreased mucous neck cells; decreased proliferation Hypertrophy; unresponsive parietal cells; surface mucous cell hyperplasia

plasma gastrin levels. text for a description of acid secretory and mucosal cell changes. cGastrin/CCK double-mutant mice had near-normal secretory responses to histamine, but they did not respond to acute gastrin or carbachol treatment. dH receptor–deficient mice are characterized by low/normal basal acid and lack of responsiveness to histamine and gastrin. 2 eGene mutation caused by an x-ray–induced balanced translocation. fProgressive parietal cell death results in progressive loss of acid secretion. gCirculating gastrin was not measured; gastrin messenger RNA abundance was increased. CCK2, cholecystokinin-2; ECL, enterochromaffin-like; HDC, histidine decarboxylase; KO, knockout; ND, not determined. bSee

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1301 gastrin-deficient mice are immature; thus, repair may involve the terminal maturation of the cells. Immaturity is suggested by reduced expression of the parietal cell marker H+,K+-ATPase (54,72), and by smaller parietal cell size (33). Because parietal cells grow in size as they mature, a smaller size would be consistent with immature cells (73). The gastrin repair characteristics suggest that the existing cellular defects can be fairly rapidly repaired because increased acid secretion is observed as early as 2 days after gastrin replacement (69). Although gastrin does not affect parietal cell lifespan, there is an effect on migration along the gland axis. Gastrindeficient mice have a greater proportion of parietal cells migrating upward toward the lumen than wild-type mice (19). Although loss of gastrin signaling does not have a large affect on ECL cell number, there is a significant effect on the positioning of the ECL cells in the gastric gland, with clustering toward the base of the gland in gastrin-deficient mice (54,72,74). Differentiated markers of ECL function are also reduced, including gastric histamine content, as well as expression of CgA and the histamine biosynthetic enzyme HDC (54,63,74). Ultrastructural analysis of CCK2 receptor– deficient and gastrin-CCK double-mutant mice showed that ECL cells lack histamine secretory vesicles (72,74). Together, these studies suggest that ECL cell histamine synthesis and storage is dependent on gastrin. Therefore, reductions in histamine and gastrin stimulation of the parietal cell are characteristic of gastrin pathway mutants. It is unclear what aspects of the impaired acid secretion are caused by loss of direct gastrin stimulation of the parietal cell versus loss of histamine stimulation of the parietal cell. The lack of response to acute histamine stimulation in gastrin-deficient mice (75) suggests that either the acid secretory machinery is not fully functional in these mice or that histamine signaling alone is not sufficient to induce acid secretion in vivo. The defect in basal and induced acid secretion was likely caused by the loss of gastrin stimulation of both parietal and ECL cells. Gastrin-Cholecystokinin Double-Mutant Mice Gastrin and CCK make up one family of peptide hormones. Both bind with nearly equal affinity to the CCK2 receptor, whereas CCK is specific for the CCK1 receptor (76). Circulating gastrin is more than 10-fold greater than circulating CCK (54); thus, CCK2-stimulated responses are largely gastrin specific. Circulating CCK is not increased in gastrin-deficient mice (54,72), suggesting that CCK is not up-regulated to compensate for loss of gastrin. To assess this point further, gastric physiology in gastrin-CCK doublemutant mice was compared with gastrin-deficient mice. Surprisingly, instead of further reducing acid secretion as a result of loss of residual CCK stimulation of CCK2 receptors, acid secretion was significantly increased in the double mutant mice (72). This occurred despite a more severe ECL cell defect in the double mutant mice, with reduced HDC activity and significantly less secretory vesicles than gastrin-deficient mice (72). The ECL cell phenotype was

similar to CCK2 receptor–deficient mice (74). In contrast with the ECL cell impairment, parietal cells were hyperactive in the double-mutant mice. Although parietal cell numbers were slightly reduced, the proportion of actively secreting parietal cells in freely fed mice was greater than in wild-type mice (72). The acid secretory response to pyloric ligation was similar to wild-type, and histamine response was normal in double-mutant mice, although there was no response to carbachol (72). The increased acid secretion in the doublemutant mice contrasts with low acid in both gastrin-deficient and CCK2 receptor–deficient mutants. The data suggest that net acid output is determined by balancing stimulatory gastrin signaling at CCK2 receptors and inhibitory CCK signaling at CCK1 receptors (77). CCK-deficient mice do not have any obvious stomach defects; however, acid secretion has not been analyzed (78). Gastrin Overexpression It is useful to compare the phenotypes of gastrin overexpression with gastrin loss of function to further investigate in vivo gastrin function. The INS-GAS transgenic mouse exhibits a twofold increase in circulating amidated gastrin resulting from the expression of a human gastrin transgene in pancreatic β cells through use of a 0.4-kb rat insulin I promoter (79). At 4 months of age, basal acid was increased approximately threefold in this mutant, which is consistent with the idea that gastrin is a key inducer of the acid secretory system (80). There was also an increase in the number of parietal cells observed at this age. In general, mouse models with increased circulating gastrin exhibit gastric mucosal cell hyperplasia with increased parietal and ECL cells when examined at young ages (see Table 51-3). Interestingly, the increased acid secretion seen in the INSGAS transgenics was lost as the mice aged, because gastric atrophy developed resulting in the loss of parietal cells and the development of mucous cell hyperplasia (80). Similar gastric histopathology was observed in a second gastrin transgenic mouse model that exhibited a sixfold increase of amidated gastrin (81). The changes that occurred with aging in these transgenics underscore the importance of examining mice at various ages for analysis of gastric physiology. Indeed, age-related changes in acid secretion and mucosal cell histology have been detected in other studies for both wild-type and mutant mice (80–82). Thus, to fully understand the effect of an engineered mutation on gastric physiology, acid secretion and mucosal cell changes should be evaluated in mice of various ages.

Histamine Pathway Mutants Histamine Regulation of Gastric Acid The important contribution of histamine to the regulation of gastric acid secretion has been demonstrated by the effectiveness of H2 receptor antagonists to block acid (23,83).

1302 / CHAPTER 51 Moreover, histamine receptor antagonists have been shown to block responses to gastrin and carbachol, suggesting that histamine is the most significant inducer of acid secretion (83). Because histamine is such a potent inducer of acid and also may provide a necessary parietal cell cAMP signal, it was reasonable to predict that both basal and induced acid secretion would be impaired in histamine pathway mouse mutants. Thus, it was surprising that histamine pathway disruption by targeted mutagenesis showed a less severe acid secretory impairment than in gastrin pathway mutants (see Table 51-3). Two different histamine pathway genes have been mutated in mouse strains, including the H2 receptor (84,85) and the histamine biosynthetic enzyme HDC (86). These mutants share many features, including near-normal basal acid content, induction of acid secretion after carbachol administration, and loss of gastrin responsiveness. Note that histamine responses are enhanced in the HDC mutant, and, as expected, are abolished in H2 receptor mutants (84–87). Hypergastrinemia was also observed in these mice (84–86), which is a common observation for mouse mutants with low acid secretion (see Table 51-3). Circulating gastrin is regulated by acid content in the stomach by a classic feedback mechanism in which gastrin synthesis and secretion are increased when acid is low. The observation of increased circulating gastrin in these mouse mutants suggests that gastrin secretion is increased to compensate for the loss in histamine signaling to maintain acidity in the stomach lumen. This is supported by the enhanced effect of the gastrin receptor antagonist YM022 to block acid secretion in H2 receptor–deficient mice (85,88), as well as the markedly reduced acid secretion observed in H2 receptor-CCK2 receptor double-mutant mice (88). The lack of an acid secretory response to gastrin administration in the histamine pathway mutant mice might suggest that gastrin signaling is already at maximum levels in these mice. However, increased acid secretion in response to gastrin stimulation has been observed in overexpression transgenics with similar levels of hypergastrinemia (81). Thus, it is unlikely that the lack of gastrin responsiveness in the histamine pathway mutants is caused by maximal stimulation or desensitization of gastrin stimulation of the parietal cells. However, it is also possible that parietal cells in the mutants have altered agonist responses. Acetylcholine signaling also appears to be important for maintaining low basal pH in histamine pathway mutants, as demonstrated by decreased acid secretion after antagonist treatment (85,86,88). In contrast with gastrin, histamine pathway mutants have been shown in some studies to be responsive to carbachol (84–86). The observation that histamine is not required for basal or carbachol-induced acid secretion suggests that other pathways may also increase parietal cell cAMP in vivo. The H2 receptor and HDC mutants would be expected to similarly disrupt parietal cell histamine signaling to the parietal cell, which is thought to be H2 receptor–mediated. Indeed, these two mutants had many similarities, as indicated earlier. However, notably, these two mutants also showed

important differences. Measurement of basal acid secretion demonstrated a significant reduction in the HDC-deficient mouse compared with wild-type control animals (86,87). In contrast, H2 receptor–deficient mice had apparently normal basal acid secretion (84,85). Non–H2 receptor–mediated effects could contribute to the difference in basal acid. Loss of HDC would affect histamine production throughout the body, and all histamine signaling would be disrupted, including H1, H2, and H3 receptor–mediated effects; in contrast, only H2 receptor–mediated processes would be blocked in the H2 receptor mutant mouse. There is some evidence that regulation of acid secretion may include H3 receptor–mediated processes. For example, in a study using isolated mouse stomachs, the H3 receptor antagonist thioperamide was shown to increase somatostatin, decrease histamine, and decrease acid in a dose-dependent manner (89). Thus, it is possible that somatostatin may be up-regulated in the HDC mutant through this pathway, resulting in reduced acid secretion. There has been significant variation in the reported gastric phenotype of HDC-deficient mice. For example, two studies noted near-normal basal acid secretion (86,87), whereas a third study measured markedly increased resting pH (90). Carbachol responses were also variable, with one study reporting significant, although low, responses (86) and another study reporting no carbachol response (87). The basis for the variability is unclear. However, there are two confounding issues that may explain some of the differing results. First, it has been reported that HDC-deficient mice may be able to use histamine from dietary sources (91). Thus, diet could affect histamine content in mutants housed at different locations to cause variability in gastric physiology. Second, there were differences in methodology of study that might explain some variability. Gastric acid output was measured in some studies under urethane anesthesia (86,87). Because urethane is known to stimulate somatostatin release (92,93), acid secretion measured under these conditions is not comparable with studies that do not use this anesthetic. Comparison of Histamine Pathway and Gastrin Pathway Mutants Comparison of the phenotype of gastrin pathway with histamine pathway mutant mice provides insight into the importance of direct versus indirect gastrin stimulation of the parietal cell, which has been a point of some debate. Mice with gastrin pathway mutations would be expected to lose both direct and indirect stimulation, whereas histamine pathway mutants would maintain direct gastrin effects on the parietal cell (see Fig. 51-3). Indeed, the data show that the gastrin pathway mutants had a more severe phenotype. Both basal and induced acid secretion were severely impaired in gastrin-deficient mice (54), whereas histamine pathway mutants had near-normal basal secretion (84,86). Thus, gastrin must at least partially stimulate the gastric acid secretory system via a histamine-independent pathway, suggesting some component of direct stimulation of the parietal cell. Indeed, parietal cells are known to have CCK2

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1303 receptors, and isolated parietal cells can respond to gastrin in vitro (76). In contrast, it has been determined that H2 receptor antagonists are effective in vivo acid blockers, even after stimulation with gastrin (83). Thus, it is evident that in vivo acid secretion involves both gastrin and histamine stimulation of the parietal cell. Analysis of H2 receptor-CCK2 receptor double-mutant mice also provides evidence to support independent roles for both gastrin and histamine stimulation of the acid secretory system. Double-mutant mice exhibited severely impaired acid secretion with near-neutral basal pH (88). The impairment is more severe than the defects in either of the single mutants, suggesting that aspects of both the gastrin and histamine pathways can operate independent of the other (88). Mucosal Hypertrophy in Histamine Pathway Mutants Another phenotype observed in the histamine pathway mutants is mucosal hypertrophy. In the H2 receptor–deficient mouse, stomach wet weight was significantly increased, with increased numbers of parietal and ECL cells (84,85,88). In addition, there was an increase in cellular proliferation, as measured by 5-bromo-2′-deoxyuridine incorporation or proliferating cell nuclear antigen staining (84,85,88). The hypertrophy is apparent in the H2 receptor mutant at 10 weeks of age and leads to grossly enlarged mucosal folds. Hypertrophy was not originally noted in the HDC-deficient mouse (86). However, subsequent studies reported hypertrophy associated with increased parietal and ECL cells as early as 3 months of age (90). Hypergastrinemia is the likely explanation for the hypertrophy, because treatment with the gastrin receptor antagonist YM022 resulted in reduced mucosal thickness and decreased parietal and ECL cells in HDC-deficient mice (90). The importance of gastrin for the mucosal hypertrophy is also suggested by the marked atrophy and decreased parietal and ECL cells observed in H2 receptorCCK2 receptor double-mutant mice (88). The data suggest that high circulating gastrin in the histamine pathway mutants is acting as a growth factor to increase proliferation and parietal and ECL cell numbers. Aberrant gastric glands develop with the emergence of dilated glands and cystic structures by 12 months of age (88). Similar to gastrin-deficient mice, H2 receptor–deficient mice have smaller parietal cells (84).

Acetylcholine Pathway Mutants Acetylcholine, the third major inducer of acid secretion, is thought to stimulate parietal cells directly via M3 receptors, as well as indirectly by stimulating histamine release from ECL cells (see Fig. 51-3). The role that the various muscarinic receptor subtypes play in gastric acid secretion has been difficult to address because of the lack of agonists and antagonists with a high degree of receptor subtype selectivity. Gene-targeted mouse models are useful to discern the importance of the various muscarinic receptor subtypes for a number of physiologic processes (94). M3 receptor–deficient

mice were analyzed to investigate the role of acetylcholine in the regulation of acid secretion (95,96) (see Table 51-3). These mice have reduced basal gastric acid secretion and increased resting intragastric pH, which is consistent with the important role that neuronal stimulation has on acid secretion, and also with M3 receptors mediating acetylcholine signaling in parietal cells. The mutant mice responded to carbachol, gastrin, and histamine induction; however, the responses are significantly less than those in wild-type mice (95). Consistent with the impaired acid secretion is the observation of decreased numbers of activated parietal cells (95). Hypergastrinemia was also observed in the mutant, suggesting that feedback mechanisms were operating to compensate for lower parietal cell stimulation by increasing circulating gastrin (95). Surprisingly, despite the hypergastrinemia, there was no mucosal hypertrophy detected in M3 receptor–deficient mutant mice, even when examined in mice up to 2 years of age (95). This finding is in contrast with the general observation of gastric hypertrophy in other hypergastrinemic mutants (with the exception of gastrin receptor–deficient mice; see Table 51-3) and suggests that gastrin stimulation of mucosal cell growth requires M3 receptors. The mechanism for this effect is not apparent. Targeted mutation of M5 receptors also induced an acid phenotype, with lower carbachol responses detected in mutant mice (96). Examination of expression of the various subtypes by reverse transcriptase polymerase chain reaction demonstrated that the M5 receptor was detected in the stomach, but not in the oxyntic or antral mucosa, suggesting localization in the enteric nervous system (96). In contrast, M3 receptor messenger RNA (mRNA) was detected in both oxyntic and antral mucosa. Both M3 and M5 receptor mutants had blunted histamine release in response to carbachol, suggesting that indirect activation of the parietal cell is reduced (96). A critical role for histamine in mediating carbachol responses was demonstrated by effective blocking of carbachol-induced acid secretion with the H2 receptor antagonist famotidine. M5 receptor–deficient mice had normal mucosal histology; gastrin levels were not measured (96).

Somatostatin Pathway Mutants Somatostatin is thought to be a paracrine regulator of acid secretion that operates at several levels, including inhibition of the parietal cell, inhibition of the ECL cell, and inhibition of gastrin release from G cells. Somatostatin regulation is complicated, with five different receptor subtypes. Molecular and pharmacologic studies suggested that the sst2 receptor is the predominant receptor regulating acid secretion (41,43–49). The production of an sst2 receptor–deficient mouse model by Zheng and colleagues (97) enabled Martinez and coworkers (98) to investigate the consequences of loss of all of the isoforms of this receptor for gastric acid secretion. Analysis of acid secretion in the mutant supported the conclusion that somatostatin, acting through the sst2 receptor, is a physiologic inhibitor of gastric acid secretion (see Table 51-3).

1304 / CHAPTER 51 normal in somatostatin-deficient mice, although this has not been studied in detail (99). It will be important to complete the analysis of gastric physiology and morphology in sst2 receptor– and somatostatin-deficient mouse (101,102) strains to better understand the mechanisms of somatostatin inhibition of acid secretion and to assess cellular alterations in the gastric mucosa.

Measurement of resting intragastric pH showed no differences between sst2 receptor–deficient and wild-type control mice, suggesting that this receptor does not mediate tonic inhibition of acid secretion by somatostatin. However, a significant phenotype was uncovered when studies were performed with urethane anesthesia, which has the interesting property of stimulating gastric somatostatin release (92,93). Acid secretion in anesthetized wild-type mice was approximately 10-fold less than in sst2 receptor–deficient mice (98). This result suggested that the sst2 receptor normally mediates inhibition of acid secretion when somatostatin levels are increased. Interestingly, there were no changes in circulating gastrin levels, suggesting that somatostatin inhibition of gastrin release does not occur through an sst2 receptor–mediated pathway. There was, however, hypergastrinemia in somatostatin-deficient mice, confirming that somatostatin inhibits gastrin secretion through a different receptor subtype (99). Further study of sst2 receptor–deficient mice showed that the CCK analog bombesin inhibits gastric acid secretion via a somatostatin-dependent pathway (100). Gastric morphology was not examined in sst2 receptor– deficient mice; thus, it is unknown whether the cellular composition of the mucosa is altered in concert with the changes in acid secretion. Gastric morphology looks relatively

PARIETAL CELL CHANNEL AND TRANSPORTER MUTANTS Genetic engineering in the mouse also has allowed the investigation of the roles of several ion transporters and channels expressed in parietal cells for gastric acid secretion. Analysis of loss-of-function mutations created by gene targeting has identified several critical channels and transporters (see Table 51-3). In general, these mutants have a more severe impairment in gastric acid secretion than the regulatory mutants described earlier, which results in achlorhydria, hypergastrinemia, and gastric mucosal hypertrophy. A model for ion flow in the parietal cell based in part on current gene-targeting studies is presented in Figure 51-5. High concentrations of HCl and KCl are secreted across the

Apical secretion:~150 mM HCl ~20 mM KCl Cl− K+

KCNQ1

H+ H/K-ATPase K+ CO2+H2O

CA II

H+ + HCO3−

Cl− HCO3−

2K+ Na/K-ATPase

AE2

Na+ 3Na+ NHE4 H+

FIG. 51-5. Parietal cell channels and transporters involved in acid secretion as determined from gene-targeted mouse models. Hydrogen (H+) ions are generated in the cell by carbonic anhydrase II (CA II). The H+,K+-ATPase pump, composed of α- and β-subunits, is responsible for transport of H+ ions across the apical membrane into the lumen. Gene disruption of either subunit results in achlorhydria. Several K+ channels are reported to be expressed in parietal cells. Targeted disruption of the KCNQ1 channel results in a severe impairment in acid secretion, suggesting that this channel plays a critical role in acid secretion. Whether other K+ channels also contribute to acid secretion remains to be determined. Loss of the chloride-bicarbonate exchanger AE2 from the basolateral membrane results in achlorhydria, demonstrating its important function to transport HCO3− out of the cell to counter the apical flow of H+, and transport of Cl− into the cell for apical secretion. There are several Na+-H+ ion exchangers (NHE) on the basolateral membrane of parietal cells. NHE4 loss-offunction mutants are achlorhydric, suggesting that this is the critical exchanger tied to acid secretion. Na+,K+-ATPase is thought to be required for K+ entry and for generation of the membrane potential to activate apical channels.

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1305 apical membrane in activated parietal cells. Carbonic anhydrase II (CA II) is thought to generate H+ for transport out of the cell. The H+,K+-ATPase in the canalicular membrane is responsible for movement of H+ across the apical membrane in exchange for K+. This activity is dependent on activation of potassium channels in the apical membrane to provide K+ for function of H+,K+-ATPase. There is also a Cl− channel in the apical membrane, which has not been tested by analysis of mutant mice. A number of basolateral exchangers are also required for gastric acid secretion. HCO3− exit across the basolateral membrane via the AE2 (Cl−-HCO3−) exchanger matches H+ flow across the apical membrane to support HCl secretion. Coupled activity of the NHE4 (Na+H+) and AE2 (Cl−-HCO3−) exchangers load the cell with NaCl and provide Na+ to drive Na+,K+-ATPase, which is required for generation of a membrane potential to activate apical secretion.

Apical Membrane Channels and Transporters Proton Pump Mutants Gene-targeting experiments have generated mice with mutations in the α- and β-subunits of the H+,K+-ATPase pump. Analysis of these mutants has demonstrated that, as expected, both subunits of the proton pump are essential for normal acid secretion (82,103,104). The acid secretory phenotypes of the two mutants are similar, with achlorhydria detected in both α- and β-subunit–deficient mice (see Table 51-3). In addition, plasma gastrin levels were markedly increased to compensate for the low acid. Major alterations in parietal cell morphology were also noted in both the αand β-subunit–deficient mouse models, including dilated canaliculi, short microvilli, and loss of tubulovesicular membranes. However, differences were noted between the two H+,K+-ATPase subunit mutants in the cellular makeup of the gastric glands. In particular, whereas the H+,K+ATPase β-subunit–deficient mouse showed significant decreases in chief cells (82), there was no difference in chief cell numbers detected in the α-subunit–deficient mouse, although there was a dramatic reduction in expression of the chief cell marker pepsinogen (103). This suggested that the β-subunit of H+,K+-ATPase is required for the normal maintenance and distribution of several cell types within the gastric glands, whereas the α-subunit appears to be important for the normal morphology of only parietal cells. H+,K+ATPase β-subunit–deficient mice exhibited extensive mucosal hyperplasia (82), which was not seen in the αsubunit mutant, although they developed dysplastic glands with cystic structures as they aged (103). In the H+,K+-ATPase β-subunit mutant, it was unknown whether the abnormal parietal cell morphology and hyperplasia were caused by increased gastrin levels, loss of acid, or some other factor. To address which aspects of the phenotype of the H+,K+-ATPase β-subunit–deficient strain might be caused by high gastrin levels, these mice were crossed

with gastrin-deficient mice (105). The H+,K+-ATPase β-subunit/gastrin-deficient double-mutant mice showed abnormal parietal cell morphology, with loss of tubulovesicular membranes, suggesting that this aspect of the phenotype of H+,K+-ATPase β-subunit mutant mice was not caused by high gastrin. However, the hyperplasia and increased numbers of immature cells observed in H+,K+ATPase β-subunit mice were not apparent in double-mutant mice, suggesting that these aspects of the original mutant mouse were caused by the increased levels of gastrin. Hypergastrinemia also was not responsible for the lack of chief cells because this feature remained in the doublemutant mouse (105). Potassium Channel Mutant The H+,K+-ATPase functions as an electroneutral pump exchanging H+ for K+ across the apical membrane. The identification of the apical potassium channel associated with the proton pump has been elusive and somewhat controversial. A few different K+ channels have been reported to be expressed in parietal cells (106). The voltage-gated KCNQ1 channel is abundant and apparently colocalizes with H+,K+ATPase (107–109). A knockout mouse model was created to test the importance of KCNQ1 in normal physiology (see Table 51-3). These mice have significantly impaired acid secretion, which is consistent with this channel playing a critical role in the parietal cell (110). The gastric lumen was near neutral pH (6–7) in the mutant. In addition, as has been observed in other mouse mutants with low acid secretion, these mice are hypergastrinemic. Also similar to other hypergastrinemic mouse mutants, KCNQ1-deficient mice had mucosal hypertrophy. By 3 months of age, stomach weight was threefold greater in the mutant, with increased cellular proliferation (110). Parietal cells were vacuolated, and there was an expansion of mucous neck cells and decreased chief cells in the mutant. Similar results were reported for a mouse strain that carries a balanced chromosomal translocation that mutates the KCNQ1 gene (111). Hyperplasia was detected as early as 8 days after birth in these mice (111). More than 100 KCNQ1 mutations have been found in families associated with cardiac arrhythmia and deafness. Gastric phenotypes have not been reported in these families. However, the marked phenotype in the mouse mutant suggests that mutation of KCNQ1 in humans may be associated with gastric dysfunction. The impaired acid secretion in KCNQ1 mutant mice suggests that it is a likely physiologic K+ channel associated with H+,K+-ATPase function. This is supported by studies showing the effects of selective inhibitors of this K+ channel on acid secretion in isolated rat gastric glands (109). However, there are other K+ channels in parietal cells that also could be involved in this process. Of particular interest is Kir2.1, because of its localization in secretory membranes of rabbit parietal cells, and because it is activated by protein kinase A and acid (112). Another candidate is Kir4.1, which has been localized to the apical membrane of rat parietal cells (113).

1306 / CHAPTER 51 Basolateral Membrane Transporters Other mouse models have been engineered with disruptions in key basolateral transporters required for acid secretion, including Na+-H+ exchanger isoform 2 (NHE2)- and NHE4-deficient strains (114,115), and a Cl−-HCO3− anion exchanger (AE2)–deficient mutant (116) (see Table 51-3). NHE2-deficient mice were shown to have a progressive loss of acid secretion caused by the necrotic death of parietal cells (114). Young mice (18–19 days) have normal gastric pH levels. However, as the mice age, gastric pH levels increase to slightly alkaline values consistent with the extensive loss of parietal cells. The observation of normal gastric pH levels in young mice suggests that NHE2 is not required for acid secretion, but is essential for viability of the parietal cell. It has been suggested that this exchanger is involved in pH regulation (117). In contrast, NHE4-deficient mice show a consistent loss of acid secretion at all ages, suggesting that this exchanger is required for gastric acid secretion (115). The NHE4-deficient mutant had few parietal cells and chief cells and correspondingly reduced concentrations of mRNA for the H+,K+-ATPase subunits and the chief cell markers pepsinogen and intrinsic factor (115). Apoptosis was increased and necrotic parietal cells were identified with highly vacuolated cytoplasm. Because there were few parietal cells in the mutant, a definitive role for NHE4 in acid secretion was not proved. Instead, it may be necessary for parietal cell development or survival. The few parietal cells in the mutant were smaller and exhibited limited development of canalicular membrane and an absence of tubulovesicles (115). NHE4-deficient mice have increased gastrin mRNA, suggesting compensatory up-regulation in response to low acid secretion (115). Targeted mutation of the AE2 Cl–-HCO3– exchanger resulted in achlorhydria, with alkaline intraluminal pH (116). These mice are emaciated, toothless, and growth retarded, with most dying around the time of weaning (116,118). Parietal cells were reduced in number, with small size, poorly developed canalicular membranes, and absence of tubulovesicles (116). The achlorhydria and lack of development of a mature secretory membrane in parietal cells suggest that AE2 is a central component of basolateral ion transport required for acid secretion.

OTHER GASTRIC MUTANTS Two additional mouse mutants have been described with alterations in gastric acid secretion (see Table 51-3). In contrast with the abundance of mouse mutants with reduced acid, TFF2-deficient mice were shown to have increased acid secretion (119). In the corpus, TFF2 is expressed primarily in mucous neck cells (see Table 51-2). TFF2-deficient mice were viable and fertile without gross gastrointestinal abnormalities. However, examination of the stomach showed decreases in mucosal thickness, proliferation rate, and mucous neck cells. Parietal cell numbers were normal, but

activated parietal cells were increased, and both basal and bethanechol-induced gastric acid were increased approximately twofold (119). As might be expected with high gastric acid, circulating gastrin is low to compensate for the low luminal pH. The mechanism of TFF2 regulation of acid secretion currently is unknown. Targeted disruption of the mesenchymal transcription factor Foxl1 resulted in mice with low acid secretion (120–122). The gastric glands in this mutant were hyperplastic and disorganized; however, the number of parietal cells detected by Dolichos biflorus (DBA) staining was not significantly altered (121,122). Parietal cell function was depressed, however, with low H+,K+-ATPase expression, no response to bethanechol, and a severely blunted response to histamine. Consistent with reduced fusion of tubulovesicular membranes at the apical surface, expression of synaptosomal associated protein 25 (SNAP-25) was reduced in the Foxl1 mutant (121). The mechanism for the parietal cell defect is not fully understood because Foxl1 expression is limited to the gastric mesenchyme.

TRANSGENE EXPRESSION IN THE GASTRIC MUCOSA Several transgene promoters have been used to drive cellspecific expression in the gastric mucosa, including parietal cells, neuroendocrine cells, G cells, and the squamous epithelium of the forestomach (Table 51-4). In the future it will be advantageous to define promoters to direct transgene expression to additional cell types in the gastric mucosa, including mucous pit and neck cells, chief cells, D cells, and ECL cells. The use of recombineered BAC transgenes may be a useful approach to define new cell-specific promoters in the stomach (10,123). The ability to manipulate the physiology of gastric cells through transgene expression is a useful tool to understand the complex interactions that occur in the gastric mucosa to regulate acid secretion.

General Transgene Promoters An elegant transgenic mouse model was described by Courtois-Coutry and colleagues (7) in which parietal cells were shown to constitutively secrete acid. In this study, a transgene was constructed that contained the H+,K+-ATPase β-subunit with a mutation in the internalization signal (Y20A), resulting in the inability of H+,K+-ATPase to be resequestered from the apical membrane, leading to high acid secretion. The CMV promoter was used to drive expression of the H+,K+-ATPase β mutant transgene. Although this is a strong constitutive promoter with expression in a number of tissues, functional changes were limited to cells that express H+,K+-ATPase, including parietal cells in the stomach (7) and renal tubule epithelial cells (124). The H+,K+ATPase β transgenic mice were shown to hypersecrete acid, and over the course of several months they developed gastric

GENETICALLY ENGINEERED MOUSE MODELS OF GASTRIC PHYSIOLOGY / 1307 TABLE 51-4. Cell-specific expression of transgene promoters in the stomach Promoter

Cell-specific expression

H/Kβ

Parietal cell

Chromogranin A Neurogenin 3 Gastrin

Neuroendocrine cells Endocrine precursors G cell

Adenosine deaminase (ADA)

Forestomach squamous epithelium

aGenomic

DNA fragmenta

Species

References

−1035 bp to +24 bp −13.5 kb to −29 bp −4.8 kb to +42 bp −81 kb to +102 kb BACb −450 bp to +550 (rat exon 1) 4-kb human exons 2 and 3 −6.4 kb to +90

Mouse Mouse Mouse Mouse Chimeric

8, 73, 126, 127 128 129 123 79, 135

Mouse

136

−4.4 kb to −3.3 kb and −750 bp to +90

Mouse

137

fragments used to construct the transgenes are indicated. Numbers refer to the start of transcription (+1). 3 bacterial artificial chromosome (BAC) #RPCI-23-121F10.

bNeurogenin

ulcers and a hypertrophic gastropathy resembling Ménétrièr’s disease (7). Gastrin levels were not measured in this study, but the increased acid would be expected to result in reduced circulating gastrin. To test the importance of internalization on regulated acid secretion, mice were generated that only expressed the H/Kβ mutant by crossing gene-targeted mice that had a H/Kβ null mutation (82) to transgenic mice expressing the Y20A mutation (7). Thus, the double-mutant mice only expressed the internalization-deficient form of H/Kβ. Parietal cells in these mice did not contain tubulovesicles, and the H+,K+ATPase pump resided exclusively on the apical membrane. Nevertheless, histamine-stimulated acid secretion was similar to wild-type mice, demonstrating that control of acid secretion can occur independent of internalization of the proton pump (38). Another study used the CMV promoter to express the regenerating gene product (Reg) protein in transgenic mice (125). The stomach exhibited increased proliferation, with increased mucosal thickness and expansion of parietal and chief cells (125). Whether this effect is caused by expression within the stomach or by a secondary effect of the expected widespread expression of this transgene throughout the body was not addressed.

Parietal Cell Transgenics The best-characterized transgene promoter for expression in the gastric mucosa is the mouse H+,K+-ATPase β-subunit promoter. This promoter has been used in a number of different experiments with transgene expression directed to parietal cells of transgenic mice (8,73,126–128). Most studies have used a ~1-kb mouse H+,K+-ATPase β-subunit promoter fragment containing sequences extending from −1053 to +24 bp (see Table 51-4). Transcription from this promoter was specific for the parietal cell lineage, because transgene expression was shown to be absent in both precursors, and differentiated members of the mucous pit and chief cell lineages.

Moreover, transgene expression was observed in the majority of parietal cells, as detected by coexpression with H+,K+ATPase. In one study, the H+,K+-ATPase β transgene was expressed in more than 95% of the parietal cells in three of four transgenic lines that were generated (8), which demonstrates the effectiveness of this promoter for parietal cell expression. Although the transgenic mouse experiments that used this promoter previously focused on developmental questions, it will be a powerful promoter for future experiments in which parietal cell physiology can be manipulated.

Enterochromaffin-Like and Neuroendocrine Cell Transgenics CgA is involved in processing or sorting of proteins in secretory granules in neuroendocrine cells. Neuroendocrine expression of a luciferase reporter gene has been achieved in transgenic mice using the mouse CgA promoter (129). In the stomach, the transgene was predominantly expressed in ECL cells, which is the most abundant neuroendocrine cell type in the mouse corpus. Expression in some D and G cells also was observed. In addition to this pattern of expression in the stomach, the transgene was expressed in other tissues known to express endogenous CgA, including intestine, adrenal gland, pancreas, and brain. In general, transgene expression was selective for the neuroendocrine system and was similar to endogenous CgA expression. In gastric ECL cells, CgA secretion and gene expression is regulated by gastrin (130–134), and accordingly, the CgA transgene promoter also was shown to be regulated by gastrin (129). With omeprazole treatment, which blocks H+,K+-ATPase and normally induces hypergastrinemia, there was a fourfold increase in transgene expression. Although the CgA promoter fragment defined in this study will be a powerful tool for manipulating neuroendocrine cells in future studies of gastric physiology, it would also be desirable to define ECL cell– and D cell–specific transgene promoters to independently manipulate these cell types.

1308 / CHAPTER 51 Another transgene that targets gastric endocrine cells is the mouse neurogenin 3 BAC clone used by Leiter and colleagues (123). Lineage tracking experiments allowed this group to conclude that the neurogenin 3 transcription factor is expressed in progenitor cells that differentiate into endocrine cells in different regions of the gastrointestinal tract. Expression varies along the gut axis, with extensive expression in intestinal and pancreatic endocrine cells and only subpopulations of endocrine cells in the stomach. Only a small number of endocrine cells in the acid-secreting portion of the stomach are derived from neurogenin 3–expressing precursors (123).

Gastrin Promoter and G-Cell Transgenics Transgene expression in G cells was achieved by creating a rat/human gastrin gene chimera (rGAS-hGAS) (79). A 1-kb fragment containing the rat gastrin promoter and first exon was fused to a 4-kb fragment containing human gastrin exons 2 and 3 (see Table 51-4). In the stomach, this transgene was expressed specifically in the G cells in the antrum. The pattern of transgene expression was similar to endogenous gastrin gene expression, with the exception of higher transgene expression observed in the duodenum. Two other gastrin transgenes were examined at the same time, including rGAS-hGH, which contained the 1-kb rat promoter fused to a human growth hormone reporter, and hGAS-hGAS, which contained a 2-kb human gastrin promoter linked to the 4-kb human gastrin segment (79). Neither of these transgenes was expressed in the stomach, demonstrating that both the rat and human gastrin segments are required for G cell– specific expression. A derivative of the chimeric transgene was also used in a study by Zhukova and colleagues (135) in which a human insulin gene was inserted into the rat/human clone (Gas-Ins). Similar to the results of Wang and colleagues (79), gastric antral–specific transgene expression in G cells also was detected in this study, although in contrast with the previous study, no expression was detected in duodenum. This chimeric sequence will be useful for directing further transgenes into gastric G cells. However, better definition of the specific rat and human gastrin sequences required for proper expression would make this a more useful system.

Adenosine Deaminase Promoter and Forestomach Transgenics An adenosine deaminase (ADA) promoter element has been described to direct high-level transgene expression to the stratified squamous epithelium in the forestomach of the mouse (136,137) (see Table 51-4). ADA is a purine catabolic enzyme that is expressed ubiquitously, but the level of expression varies markedly among different tissues. In mice, the greatest levels of ADA occur in the gastrointestinal tract, including the squamous epithelium that lines the tongue, esophagus, and forestomach, where the enzyme can account

for as much as 20% of soluble protein (138). Initially, transgenic mouse studies with the ADA promoter showed high expression with a 6.4-kb promoter fragment (136). This was followed up with smaller constructs, leading to the identification of a 1.1-kb upstream flanking sequence that is required for high-level expression when paired with the natural promoter. Although this promoter was used for directing expression of a reporter gene, it would be useful in future studies to direct high-level expression of substances for secretion into the lumen of the stomach to test their importance for regulation of gastric physiology. Another promoter that has been used to express transgenes in the gastric epithelium is the mouse cytokeratin 19 promoter (139). A 2.1-kb transgene promoter has been reported to drive expression in the squamous epithelium of the forestomach, as well as in proliferating cells in the gastric glands (139,140). Expression in other epithelial tissues in the gastrointestinal system also was observed (139). There is some variability in the reported expression pattern of this promoter in the stomach; thus, further definition of cell specificity would be helpful.

CONCLUSION Integrative genomics, the study of gene function in genetically engineered animal models, takes advantage of the genome sequencing effort and the powerful technologies to manipulate the mouse genome. Genes for many of the key regulators of the stomach have been identified and cloned, and many knockout models have been produced that have allowed further investigation of the roles of these regulatory factors in the function of the gastric acid secretory system. Although the data obtained from many genetically engineered mouse models have supported the findings from more classical pharmacologic and physiologic experiments, the acid secretory phenotypes of other models were unexpected, often resulting in the need to reexamine the roles of certain regulators. In particular, the finding that basal acid secretion was normal in H2 receptor–deficient mice was unexpected given that H2 receptor antagonists are extremely effective blockers of acid secretion in mouse and human. This finding alone suggested that there might be another histamine pathway in parietal cells, that cAMP signaling is not necessary, or that gastrin and/or acetylcholine alone are sufficient for normal acid secretion. It is clear that combining pharmacologic and physiologic experiments with the generation of new genetically engineered mouse mutants will be required to fully understand the complex regulation of the gastric acid secretory system. In the future, promoters that have been defined for cell-specific transgene expression in the gastric mucosa can be used to create transgenics with altered gastric secretory function. For example, manipulation of the cell signaling pathways in parietal cells using the mouse H+,K+-ATPase β promoter would be useful to help define the critical components for activation of acid secretion.

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Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion. J Clin Invest 1998;101:1243–1253. Gawenis LR, Greeb JM, Prasad V, Grisham C, Sanford LP, Doetschman T, Andringa A, Miller ML, Shull GE. Impaired gastric acid secretion in mice with a targeted disruption of the NHE4 Na+/H+ exchanger. J Biol Chem 2005;280:12781–12789. Gawenis LR, Ledoussal C, Judd LM, Prasad V, Alper SL, Stuart-Tilley A, Woo AL, Grisham C, Sanford LP, Doetschman T, Miller ML, Shull GE. Mice with a targeted disruption of the AE2 Cl− /HCO3− exchanger are achlorhydric. J Biol Chem 2004;279: 30531–30539. Rossmann H, Sonnentag T, Heinzmann A, Seidler B, Bachmann O, Vieillard-Baron D, Gregor M, Seidler U. Differential expression and regulation of Na(+)/H(+) exchanger isoforms in rabbit parietal and mucous cells. Am J Physiol Gastrointest Liver Physiol 2001;281: G447–G458. Medina JF, Recalde S, Prieto J, Lecanda J, Saez E, Funk CD, Vecino P, van Roon MA, Ottenhoff R, Bosma PJ, Bakker CT, Elferink RP. Anion exchanger 2 is essential for spermiogenesis in mice. Proc Natl Acad Sci U S A 2003;100:15847–15852. Farrell JJ, Taupin D, Koh TJ, Chen D, Zhao CM, Podolsky DK, Wang TC. TFF2/SP-deficient mice show decreased gastric proliferation, increased acid secretion, and increased susceptibility to NSAID injury. J Clin Invest 2002;109:193–204. Kaestner KH, Silberg DG, Traber PG, Schutz G. The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation. Genes Dev 1997;11:1583–1595. Kato Y, Fukamachi H, Takano-Maruyama M, Aoe T, Murahashi Y, Horie S, Suzuki Y, Saito Y, Koseki H, Ohno H. Reduction of SNAP25 in acid secretion defect of Foxl1−/− gastric parietal cells. Biochem Biophys Res Commun 2004;320:766–772. Fukamachi H, Fukuda K, Suzuki M, Furumoto T, Ichinose M, Shimizu S, Tsuchiya S, Horie S, Suzuki Y, Saito Y, Watanabe K, Taniguchi M, Koseki H. Mesenchymal transcription factor Fkh6 is essential for the development and differentiation of parietal cells. Biochem Biophys Res Commun 2001;280:1069–1076. Schonhoff SE, Giel-Moloney M, Leiter AB. Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types. Dev Biol 2004;270:443–454. Wang T, Courtois-Coutry N, Giebisch G, Caplan MJ. A tyrosine-based signal regulates H-K-ATPase-mediated potassium reabsorption in the kidney. Am J Physiol 1998;275:F818–F826. Miyaoka Y, Kadowaki Y, Ishihara S, Ose T, Fukuhara H, Kazumori H, Takasawa S, Okamoto H, Chiba T, Kinoshita Y. Transgenic overexpression of Reg protein caused gastric cell proliferation and differentiation along parietal cell and chief cell lineages. Oncogene 2004;23:3572–3579. Li Q, Karam SM, Gordon JI. Simian virus 40 T antigen-induced amplification of pre-parietal cells in transgenic mice. Effects on other gastric epithelial cell lineages and evidence for a p53-independent apoptotic mechanism that operates in a committed progenitor. J Biol Chem 1995;270:15777–15788. Li Q, Karam SM, Gordon JI. Diphtheria toxin-mediated ablation of parietal cells in the stomach of transgenic mice. J Biol Chem 1996; 271:3671–3676. Canfield V, West AB, Goldenring JR, Levenson R. Genetic ablation of parietal cells in transgenic mice: a new model for analyzing cell lineage relationships in the gastric mucosa. Proc Natl Acad Sci U S A 1996;93:2431–2435. Hocker M, Raychowdhury R, Plath T, Wu H, O’Connor DT, Wiedenmann B, Rosewicz S, Wang TC. Sp1 and CREB mediate gastrin-dependent regulation of chromogranin A promoter activity in gastric carcinoma cells. J Biol Chem 1998;273:34000–34007. Watkinson A, Dockray GJ. Functional control of chromogranin A and B concentrations in the body of the rat stomach. Regul Pept 1992;40: 51–61. Dimaline R, Evans D, Forster ER, Sandvik AK, Dockray GJ. Control of gastric corpus chromogranin A messenger RNA abundance in the rat. Am J Physiol 1993;264:G583–G588. Syversen U, Mignon M, Bonfils S, Kristensen A, Waldum HL. Chromogranin A and pancreastatin-like immunoreactivity in serum of gastrinoma patients. Acta Oncol 1993;32:161–165. Borch K, Stridsberg M, Burman P, Rehfeld JF. Basal chromogranin A and gastrin concentrations in circulation correlate to endocrine cell

1312 / CHAPTER 51 134.

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proliferation in type-A gastritis. Scand J Gastroenterol 1997;32: 198–202. Hocker M, Cramer T, O’Connor DT, Rosewicz S, Wiedenmann B, Wang TC. Neuroendocrine-specific and gastrin-dependent expression of a chromogranin A-luciferase fusion gene in transgenic mice. Gastroenterology 2001;121:43–55. Zhukova E, Afshar A, Ko J, Popper P, Pham T, Sternini C, Walsh JH. Expression of the human insulin gene in the gastric G cells of transgenic mice. Transgenic Res 2001;10:329–341. Winston JH, Hanten GR, Overbeek PA, Kellems RE. 5′ flanking sequences of the murine adenosine deaminase gene direct expression of a reporter gene to specific prenatal and postnatal tissues in transgenic mice. J Biol Chem 1992;267:13472–13479. Xu PA, Winston JH, Datta SK, Kellems RE. Regulation of forestomach-specific expression of the murine adenosine deaminase gene. J Biol Chem 1999;274:10316–10323.

138. Chinsky JM, Ramamurthy V, Fanslow WC, Ingolia DE, Blackburn MR, Shaffer KT, Higley HR, Trentin JJ, Rudolph FB, Knudsen TB, et al. Developmental expression of adenosine deaminase in the upper alimentary tract of mice. Differentiation 1990;42:172–183. 139. Brembeck FH, Moffett J, Wang TC, Rustgi AK. The keratin 19 promoter is potent for cell-specific targeting of genes in transgenic mice. Gastroenterology 2001;120:1720–1728. 140. Oshima H, Oshima M, Inaba K, Taketo MM. Hyperplastic gastric tumors induced by activated macrophages in COX-2/mPGES-1 transgenic mice. EMBO J 2004;23:1669–1678. 141. Zavros Y, Eaton KA, Kang W, Rathinavelu S, Katukuri V, Kao JY, Samuelson LC, Merchant JL. Chronic gastritis in the hypochlorhydric gastrin-deficient mouse progresses to adenocarcinoma. Oncogene 2005; 24:2354–2366.

CHAPTER

52

Structure–Function Relations in the Pancreatic Acinar Cell Fred S. Gorelick and James D. Jamieson Organization of the Exocrine Pancreas, 1313 Pancreatic Development, 1314 Structural Organization, 1316 Extracellular Matrix, 1316 Intercellular Junctions, 1317 Functional Responses of the Acinar Cell: Protein Synthesis, Vectorial Transport, Modifications, and Sorting, 1319 Protein Synthesis, 1321 Vectorial Movement of Proteins, 1321 Protein Modifications, 1322 Sorting and Concentration of Nascent Proteins, 1323

Cell Signaling, 1326 Cell-Surface Receptors, 1326 G Protein–Coupled Receptors, 1327 Calcium Signaling, 1327 Secretion, 1330 Exocytosis, 1331 Apical Membrane Expansion and Retrieval, 1332 Secretory Protein Extrusion, Solubilization, and Delivery into the Duct System, 1333 Acknowledgments, 1334 References, 1334

The exocrine pancreas has two major physiologic functions: it supplies the enzymes and enzyme precursors (zymogens) that are needed for digesting lipids, carbohydrates, and proteins, and it secretes a bicarbonate-rich fluid that neutralizes acidic gastric secretions, thus providing the correct pH for duodenal digestion by pancreatic enzymes. The exocrine pancreas is composed of two major cell types, acinar cells and duct cells (Fig. 52-1). The acinar cell is responsible for the synthesis and storage of digestive enzymes, whereas duct cells secrete chloride and bicarbonate. This chapter focuses on the function of the acinar cell. In addition to its central role in digestion, the acinar cell has been a key model for scientific studies. Thus, many of the steps responsible for regulating protein synthesis and export and cell signaling were described using the pancreatic acinar cell (Fig. 52-2).

ORGANIZATION OF THE EXOCRINE PANCREAS

F. S. Gorelick: Department of Medicine, VA Healthcare CT, and Yale University School of Medicine, West Haven, Connecticut 06516. J. D. Jamieson: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

The pancreas is a retroperitoneal organ that is composed of exocrine and endocrine glands. About 85% of the gland is exocrine and about 10% is extracellular matrix (ECM). Because the duct cells and blood vessels comprise about 4% of the volume of the gland, the endocrine pancreas represents only less than 2% of pancreatic mass. The glandular portion of the pancreas may decrease in amount in diseases such as chronic pancreatitis or pancreatic cancer. The fraction of endocrine tissue may vary depending on various pathologic conditions such as diabetes. The fundamental structural units in the exocrine pancreas are macroscopically visible lobules. Each lobule is drained by interlobular ducts that combine to form larger interlobular ducts. These ducts gradually enlarge and coalesce to form the main pancreatic duct that delivers secretions into the duodenum. The fundamental secretory unit of the exocrine pancreas is the acinus, a collection of ~20 to 200 acinar cells, adjacent centroacinar cells, and proximal small ducts (Figs. 52-2 and 52-3A). The collective function of the cells in the acinus is to mix exocytosed secretory proteins from individual acini

1313

1314 / CHAPTER 52

A

B

FIG. 52-1. Major cell types of the exocrine pancreas. (A) Acinar cell electron-dense zymogen granules in its apical region (arrowheads). (B) Duct cell with numerous mitochondria (arrowheads).

with water and electrolytes to form a mixture that can flow into the distal ductular system. In contrast to other glands such as the salivary glands, the exocrine pancreatic duct system does not always end in blind-ended lumens surrounded by acinar cells (Fig. 52-3A). Thus, wax-cast and microscopic reconstruction studies have shown that the most proximal ducts are often branched and interconnect with groups of acinar cells, frequently forming a collar around the duct, as well as the typical blind-ended gland structure (see arrowhead in Fig. 52-3B) (1).

PANCREATIC DEVELOPMENT The exocrine pancreas arises from two anlagen of the primitive foregut: a dorsal portion coming from the dorsum

A

of the duodenum, which forms a portion of the head and uncinate process and all of the body and tail, and a ventral portion derived from the primitive bile duct, which forms the remainder of the head and uncinate process (Fig. 52-4). Generally, the ducts draining the dorsal and ventral pancreas fuse at about 6 weeks gestation in humans, with the ventral duct providing the main conduit of drainage into the duodenum. After birth, the different origins of the pancreas are reflected by the occasional persistence of separate dorsal and ventral ducts (known as pancreas divisum) and a greater concentration of some hormones in the ventral or dorsal pancreatic bud. The arterial supply arises from branches of the splenic artery, which form arcades with the pancreatic branches of the gastroduodenal and superior mesenteric arteries. The autonomic innervation is both parasympathetic

B

FIG. 52-2. Organization of the pancreatic acinus. (A) Groups of acinar cells identified by electron-dense zymogen granules at their apical pole (arrow) empty their contents into the lumen, which connects to a small ducts lined by cuboidal cells (arrowhead). (B) Sketch shows gland organization; note that acinar groups sometimes surround the duct and do not form a terminal gland.

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1315

A

B

FIG. 52-3. Demonstration of small duct structure in the pancreas. (A) Retrograde injection of India ink into pancreatic ducts outlines the termination of small ducts in the acinar lumen (al). (B) Wax cast of pancreatic ducts also demonstrated that although most small ducts are terminal structures, some such as one near the top of the field form loops (arrowhead). (Modified from Ashizawa and colleagues [1], by permission.)

and sympathetic through splenic subdivisions of the celiac plexus. As is the case for all endodermal glandular derivatives of the primitive gut endoderm, epithelial evagination is accompanied by preservation of transepithelial integrity despite massive movements of epithelial sheets and rapid cell division as the epithelium expands, invades the adjacent mesenchyme, and differentiates (2). Pancreatic growth and differentiation is regulated by the sequential activation of specific genes. Critical for regulating the onset of pancreatogenesis is activation of the Pdx1 gene. A complex and not fully described series of signals from the mesenchyme, adjacent tissues, and the epithelium orchestrate the subsequent development of the endocrine and exocrine pancreas. The programs that mediate

Formation

development of the dorsal and ventral pancreas are not identical. For example, in the earliest stages, the dorsal bud requires signals from the notochord and later from the aorta, whereas ventral bud development requires signals that arise from the nearby developing liver and heart. Release of fibroblast growth factor by the developing heart and its interactions with sonic hedgehog are required to drive differentiation of a portion of the ventral bud to form liver and not pancreas (3,4). Downstream of Pdx1 in pancreatic development is activation of NOTCH1. Final differentiation of the endocrine and endocrine pancreas requires the sequential activation of distinct genes for each lineage. For example, Ptf1α followed by Mist1 activation are required for exocrine differentiation. Similarly, pancreatic innervation and vascularization requires

Rotation

Fusion

Common bile duct

Ventral pancreas

Ventral pancreatic duct

Dorsal pancreas

Dorsal duct 5 weeks

6-7 weeks

8 weeks

FIG. 52-4. Pancreatic development involves the fusion of two pancreatic anlages to form the adult pancreas. (Used with the permission of the American Gastroenterological Association Bethesda, MD.)

1316 / CHAPTER 52 the sequential activation of distinct genes (2). The importance of some of these developmental pathways, especially those that drive cell proliferation and transformation, is underscored by their potential roles in pancreatic tumorigenesis (5,6). Despite the complex tubular-acinar structure in the mature gland, beginning with the cuboidal epithelial cells lining the opening of the main pancreatic duct at the sphincter of Oddi and continuing to the termination of the smallest ductal/acinar structures, the epithelium maintains its continuity, ensuring that the ductal or acinar luminal space maintains its physical separation from the interstitial space (see later). Notably, intercalated duct cells mark the end of the acinar cell lumen and the beginning of the duct system. Examination of Fig. 52-5 shows that the intercalated duct cells are connected to each other and to adjacent acinar cells via junctional complexes typical of all epithelia.

STRUCTURAL ORGANIZATION Extracellular Matrix Typical of all epithelia, acinar units and ducts of the pancreas are embedded in an ECM. All cells found in the ECM synthesize ECM molecules; ECM precursor or stem cells are derived from embryonic mesenchyme. Thus, the resident cells of the ECM, with the exception of transients such as phagocytic and immune cells, are capable of forming all of the cellular and molecular elements of the ECM. The ECM has a number of functions. First, it helps to organize and support the epithelium. Collagen and elastin fibrils provide tensile strength and resiliency, respectively, for the gland proper, whereas reticular fibrils of collagen type III form a meshwork that supports the parenchyma as it does in all glandular structures. The proteoglycans in which the structural elements of the ECM of the gland are embedded serve as a reservoir for water and electrolytes. The ECM binds cytokines and growth factors, and thus makes them available when needed. The ECM also acts as a physical

FIG. 52-5. Tight junctions (arrows) interconnect acinar cells (recognized by their zymogen granule content) and small duct cells (DC).

barrier for infectious agents and is the site where phagocytic and immune cells play their roles during infections and inflammatory processes. A number of nonepithelial cell types including macrophages, stellate (Ito) cells, mast cells, and cells of the T- and B-lymphocytic line are embedded in the ECM. Finally, formation of the majority of the ECM appears to be under the control of the stellate cell (7,8). In the resting state, the prominent vitamin A–filled vesicles in their cytoplasm can identify these cells (Fig. 52-6). After stimulation, the vitamin A–laden vesicles disappear, and these cells begin to proliferate and produce collagen. Such stimulation can occur during development, tissue repair, or regeneration. However, some pathologic conditions such as chronic pancreatitis and pancreatic cancers are associated with persistent stellate cell activation and excess pancreatic fibrosis. There is some evidence that excessive fibrosis may play a role in mediating pancreatic growth and the development of neoplasia. Integrating the ECM with the epithelial cells of the pancreas is the basement membrane (Fig. 52-7). The basement membrane directly underlies the basal plasma membrane of the epithelium and is produced by the epithelial cells. It is composed of a cage of type IV collagen in which is embedded a number of basement membrane matrix components including negatively charged heparin sulfate proteoglycans and linker molecules including laminin, nidogen, and perlecan. The linker molecule laminin has an important role in integrating the structure and function of the overlying epithelium with the ECM. Laminin is a multivalent molecule with binding sites for integrins on the basal plasma membrane of epithelial cells, proteoglycans, ECM collagen, and the surfaces of ECM cells such as fibroblasts, adipocytes, and capillary endothelial cells. Because of these multivalent functions and the molecules to which it binds, laminin ensures that the epithelium and the ECM are effectively a structural continuum.

FIG. 52-6. Pancreatic stellate cells are filled with highly refractile droplets that store vitamin A. These disappear from the cell when it is activated and transformed into the collagenproducing phenotype. (Courtesy of Minoti Apte.)

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1317

FIG. 52-7. Transmission electron microscopy demonstrating the basal regions of adjacent acinar cells (arrowheads) with their associated basement membranes and intervening collagen fibrils (arrow) in the extracellular space.

In fact, it has become evident that there is extensive functional interaction between epithelial cells and the ECM and that integrins play a central role in this. Integrins comprise a large family of transmembrane proteins that are located in the basal plasma membranes of virtually all epithelial cells (9). In this location, they serve as receptors for various extracellular ligands, notably laminin. Although traditionally they have been viewed as mechanical links between the overlying epithelium and the basement membrane at focal contacts, and hence to the ECM, it is now clear that their function is much broader. By virtue of the fact that the cytoplasmic portion of integrins is linked indirectly to the actin cytoskeleton and to organized signal arrays within the cell, integrins are able to transmit information from the extracellular environment into the cell. This information takes a variety of forms that are now being understood, including activation of signaling pathways for growth and differentiation and activation of transcription. Thus, signals from outside the cell arising in the external environment/ECM can affect cell function in the short term, such as potentially changing the patterns of transcription of secretory proteins, or they may have long-term effects such as progression to neoplasia. An interesting corollary to the fact that integrins link the ECM to the actin cytoskeleton and signaling arrays is the suggestion that changes in ECM shape and rigidity can be transmitted into the cell and activate signaling pathways, providing for additional ways in which the ECM can influence transcription and translation (9).

providing an impermeant barrier to the intermixing of ions and other molecules between luminal contents and the interstitial space (10). As shown in Figure 52-8, transmission electron microscopy demonstrates that tight junctions are discrete electron-dense structures within closely opposed regions of plasma membrane. Filamentous bands on the cytoplasmic side of tight junctions represent regions of attachment of cytoplasmic actin filaments. Freeze-fracture preparations that reveal the distribution of proteins within membranes show that tight junctions are composed of interdigitating bands that form a contiguous collar around the apical region of the cell (Fig. 52-9). Our understanding of the structure and function of tight junctions has been improved by the discovery that a family of tetra-spanning membrane proteins, the claudins (from the Latin claudere meaning “to close”), forms the basis for the permeability properties of tight junctions. Homophilic and heterophilic claudin complexes form strong adhesive structures between epithelial cells. Claudins are expressed in an epithelial-specific fashion; for example, claudin 16 is highly expressed in epithelial cells of the proximal tubules of the kidney, but little is found in other tissues. In the pancreas, claudin 2 is found only in junctions of the duct epithelia, claudin 5 is limited to junctions between acinar cells, and claudins 3 and 4 are in both locations (11). Compelling evidence now exists that claudins form the structural basis of tight junctions (12,13). It is also clear that tight junctions form the structural and functional basis for the paracellular pathway, now recognized as a highly selective barrier that both restricts the paracellular movement of large and small molecules and facilitates passage of certain ions between the lumen and interstitial space. Ion selectivity of the paracellular pathway is determined by the charges of amino acids on the extracellular domain of tight junction claudins (14). Although mutations of claudins in the duct or acinar epithelium of the pancreas have not been reported, claudin mutations are the cause of several human diseases,

Intercellular Junctions All cells in an epithelium, including the duct and acinar cells of the pancreas, are attached to each other by junctional complexes. These are functionally classified into three groups: (1) sealing or tight junctions; (2) adhering junctions; and (3) communicating junctions. These junctional structures are distinguished by their distribution, morphologic appearance, protein composition, and function. Tight Junctions Sealing or tight junctions are always found closest to the lumen of glands and ducts and are classically described as

FIG. 52-8. Transmission electron microscopy demonstrating junctional complexes between two adjacent cells including a tight junction (arrow), adherens zonule (arrowhead), and a desmosome just below. Note the actin filaments in the cytoplasm associated with the tight and adhering zonules; intermediate filaments are associated with desmosomes. L, lumen.

1318 / CHAPTER 52

FIG. 52-9. Freeze-fracture image of apical region of pancreatic acinar cells showing the interconnected fibrils (arrow) that form the tight junctions. Above the tight junction region, microvilli project into the acinar lumen (arrowheads).

such as familial hypomagnesemia caused by mutations in claudin 16 (also known as paracellin-1) in distal tubules of the kidney (15). The central role of tight junctions is to provide the structural and functional basis for the paracellular pathway by restricting the bidirectional movement of molecules (Fig. 52-10). Another important role of the long sealing strands generated by claudins in tight junctions is to prevent the intermixing by diffusion of membrane proteins and lipids between the apical and basolateral domains of epithelial cells. Other major proteins associated with tight junctions include the transmembrane protein occludin and multiple cytoplasmic proteins including ZO1 and ZO2.

ZO1 and ZO2 and possibly other proteins such as cingulin indirectly link claudins and possibly occludens to the actin cytoskeleton. Because of this association, it has been suggested that actin microfilaments, containing myosin II, may be involved in modulation of the paracellular pathway in response to signals. A variety of signaling molecules including cytokines can up-regulate or down-regulate paracellular permeability. These act by affecting the levels of junctional proteins or by affecting their phosphorylation. For example, transforming growth factor-α increases permeability, whereas epidermal growth factor decreases permeability. The tight junction and other components of junctional complexes that form an impermeant barrier (see Fig. 52-10) become disordered early in the course of experimental pancreatitis (16). This is associated with an increase in paracellular permeability. It is likely that this increase in paracellular permeability allows duct contents to escape into the intracellular space. This phenomenon could contribute to the decrease in pancreatic secretions and increase in serum levels of pancreatic enzymes observed early in the course of acute pancreatitis. Adhering Zonules and Desmosomes

FIG. 52-10. Transmission electron microscopy demonstrating junctional region of adjacent duct cells after an intravenous injection of lanthanum. Note that the electron-dense marker moves up between the lateral membranes (arrowhead), but is stopped from entering the duct lumen (DL) in the region of the tight junction (arrow). (Reproduced from Fallon and colleagues [16], by permission.)

All cells in an epithelium are mechanically connected to each other via two types of junctions, both of which use homotypic interactions between cadherins expressed and localized in neighboring cells. These are adhering zonules that form beltlike arrays of adhering junctions immediately beneath tight junctions in all epithelia and desmosomes, which form punctuate adhering zones between cells of epithelia subject to mechanical shear. The cytoplasmic portion of cadherins in adhering zonules is associated with anchoring proteins that, in turn, interact with cytoplasmic actin filaments. It is believed that the contraction of actin filaments in the adhering zonule belt results in a purse string constriction of the apices of several epithelial cells, thereby causing the epithelium to evaginate as occurs during gland morphogenesis. Desmosomes, in contrast, have their cytoplasmic anchor

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1319 proteins associated with intermediate filaments. Because intermediate filaments do not possess plus and minus ends, as do actin and microtubules, the filaments associated with desmosomes do not have contractile function and serve primarily to resist mechanical shear among cells in an epithelium. All epithelial cells are also associated with their underlying basement membrane via hemidesmosomes, which have cytoplasmic cytoskeletal attachments that are also coupled to intermediate filaments, but have transmembrane linker proteins that are members of the integrin family of transmembrane proteins. As discussed earlier, integrins interact with ligands in the basement membrane. Gap Junctions Epithelial cells are also functionally connected to each other through gap junctions that allow direct interactions between the cytoplasms of adjacent acinar cells (Figs. 52-11 and 52-12). A distinct protein family known as the connexins assembles into hexamers to form small transmembrane pores between cells, known as connexons. Large, rounded groupings of connexons forming gap junctions are found in the lateral membranes of adjacent cells. The connexon pores demonstrate size and ionic selectivity and do not allow molecules with molecular weights greater than 1000 to pass. Their main physiologic functions are to coordinate signaling between cells by allowing the movement of signaling molecules such as calcium, inositol-1,4,5-trisphosphate (IP3), and cyclic adenosine monophosphate (cAMP) through the junction. Thus, hormonal signals impinging on one or a few cells in an acinus are coupled to synchronize the exocytosis of zymogen granules. The channels also close rapidly in response to intracellular acidification or high levels of cytosolic calcium, preventing spread of damage between cells in a wounded epithelium. The importance of acinar cell gap junctions has been examined using chemical inhibitors and mutant mice that are deficient in specific connexins. Several generalizations can be drawn from these studies. First, pancreatic acinar cells are coupled by gap junctions containing connexins 26 and 32 (17,18). In mice lacking connexin 32 (see Fig. 52-12), the number of gap junctions is greatly reduced, and intercellular communication of dye is inhibited (19). Interestingly, knockout

mice exhibited increased enzyme secretion, whereas electrical coupling between acinar cells was maintained (17). These findings suggest that gap junctions might down-modulate pancreatic secretion. Furthermore, electrical coupling might be mediated through connexin 26. Notably, the connexin 32−/− mice exhibited a much more severe form of cerulein (a cholecystokinin [CCK] analogue) hyperstimulation pancreatitis than wild-type control mice. These studies suggest that the overall effect of the gap junctions formed by connexin 32 in pancreatic acinar cells is to dampen both basal secretion and the cellular response to hyperstimulation.

FUNCTIONAL RESPONSES OF THE ACINAR CELL: PROTEIN SYNTHESIS, VECTORIAL TRANSPORT, MODIFICATIONS, AND SORTING The exocrine pancreas has the greatest daily rates of protein synthesis of any organ in the body. Thus, 1000 to 1500 ml pancreatic exocrine secretion, with 10 to 100 g per liter of protein, reach the small intestine each day. The pancreatic acinar cell is elegantly designed to meet the requirements for both high levels of protein synthesis and the storage and release of secretory proteins. These distinctive features have made the pancreatic acinar cell a key system for biologic studies. Indeed, the intracellular itinerary of nascent secretory proteins was first described in the pancreatic acinar cell. Synthesis and intracellular movement of export proteins is an ordered and sequential process that involves specific organelles. It begins with nascent protein synthesis in the endoplasmic reticulum (ER), vesicular transport of nascent proteins to the Golgi complex, their movement to zymogen granules, and finally secretion from the storage compartment by regulated exocytosis (20). The major organelles responsible for protein synthesis and storage are shown in Figure 52-13. After synthesis, newly formed proteins undergo covalent modifications and assume their tertiary structure with the aid of chaperone proteins that facilitate and monitor correct folding, a process termed quality control. As nascent secretory proteins undergo vectorial anterograde transport, they become concentrated and are segregated away from nonsecretory proteins, such as proteins that will reside in

FIG. 52-11. Transmission electron microscopy demonstrating a linear dense region (arrowheads) of membranes from adjacent acinar cells typical of a gap junction.

1320 / CHAPTER 52

A

B

C

D

FIG. 52-12. Acinar cell gap junction structure and function. (A) Gap junction structure in shown as circumscribed regions of dense, evenly spaced particles in the plasma membrane by freeze fracture. (B) Loss of gap junction in connexin 32−/− knockout mouse. (C) Dye transfer among a group of acinar cells after injection into a single cell. (D) Lack of dye transfer in 32−/− knockout mouse. (Modified from Chanson and colleagues [17], by permission.)

FIG. 52-13. Transmission electron microscopy of the major components of the protein biosynthetic pathway included rough endoplasmic reticulum (R), vesicular carriers (V), the Golgi complex (G), and storage granules (S). (Courtesy of M. Farquhar and G. Palade.)

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1321 lysosomes, or newly synthesized membrane proteins destined for turnover of the plasma membrane. This review emphasizes mechanisms relevant to soluble secretory proteins.

Protein Synthesis An extensive rough-surfaced endoplasmic reticulum (RER) of the acinar cell is required to meet its requirements for high levels of protein synthesis (20). Although the ER is most evident at the base of the acinar cell, it extends up to the apical region of the cell (Fig. 52-14). The width of the ER cisternae ranges from 20 to 80 nm and enlarges during active protein synthesis; all cisterna appear to be contiguous. Sites of active protein synthesis are marked by regularly placed rosettes of electron-dense ~30-nm ribosomes that are attached to the cytosolic face of the ER membrane (see Fig. 52-14). These represent areas active in translation and are ribosomes associated with messenger RNA (mRNA). ER with associated ribosomes is designated RER, whereas ER devoid of ribosomes is known as smooth ER (SER). Morphologic and functional studies provide evidence that there are mechanisms for regional specialization within the ER. For example, in some regions of the ER ribosomes are attached to the cytoplasmic face at regular intervals (RER) while other regions of the ER are devoid of ribosomes. Functional specialization of the ER is demonstrated by calcium signaling. Thus, the initial increase in cytosolic calcium in the acinar cell occurs at the apex of the cell and represents release from specific regions of the ER. This particular phenomenon may be result of localization of the IP3 calcium channel to the SER at the apical-most region of the acinar cell (see later). Other ER resident proteins are localized to specific cell regions. Although the mechanisms for establishing domains within the ER remain unclear, lipid rafts composed of distinct lipids and the tethering of ER proteins to specific proteins in the cortical skeleton are likely to have roles. The ribosome is formed by large and small subunits of the ribosome macromolecular complexes of proteins and specific

ribosomal RNA; the large and small subunit can be distinguished by transmission electron microscopy. New protein synthesis takes place in a groove that lies between the two subunits and is directed by mRNA. Specific groups of soluble proteins regulate the initiation, elongation, and termination phases of protein synthesis. The first synthesized component of a secretory protein is a specific amino-terminal extension known as the signal sequence. The signal sequence is a 15- to 50-amino-acid peptide that contains a hydrophobic core. Also known as a leader sequence, the signal allows nascent proteins to enter the ER through a macromolecular multiprotein channel known as the translocon (21,22). This signal sequence binds to a signal recognition particle (SRP) that directs the attachment of the early nascent protein and mRNA to the ER by attachment to its receptor (23). After attachment, the SRP is released and the signal peptide enters the ER protein-conducting channel. The translocon also mediates the insertion of transmembrane proteins into the ER membrane and has a role in protein degradation (as discussed elsewhere in the literature). In addition to its critical role in the initial steps of protein synthesis, the ER has other specialized functions that include cell signaling, protein folding, and specific protein modifications.

Vectorial Movement of Proteins The sequential movement of nascent proteins between secretory compartments was first demonstrated using the pancreatic acinar cell and pulse-chase protocols (24). In this type of study, isolated cells or tissues were transiently incubated with tracer amounts of a radioactive amino acid, and then this was washed away and replaced with an unlabeled amino acid. Nascent proteins incorporate the pulse of radioactive amino acids and can be followed as they traverse the cell. The labeled proteins can be detected using autoradiography (Fig. 52-15) or by assaying isolated subcellular fractions for radioactive amino acids incorporated into proteins. As shown in Figure 52-15, this approach demonstrated that

FIG. 52-14. Transmission electron microscopy demonstrating dense rough endoplasmic reticulum (RER) of acinar cell under low and high magnification (inset). Note that the electron-dense ribosomes (arrowheads) align on the cytoplasmic face of the RER.

1322 / CHAPTER 52 ER -disulfide bond formation -addition N-linked CHO -folding

Golgi -modification CHO -sorting of lysosomal enzyme

A ER

0

B

Glg

15

30 45 Time (min)

ZG

60

D

Modifications of nascent proteins

C FIG. 52-15. Temporal itinerary of nascent secretory proteins in acinar cells. (A) Nascent proteins are detected over condensing vacuoles using electron microscopic autoradiography. (B) Kinetics of appearance of nascent proteins in subcellular compartments determined by autoradiography. ER, endoplasmic reticulum; Glg, golgi; ZG, zymogen granules. (C) Summary of the itinerary of most export proteins in the acinar cell. (D) Summary of major posttranslational modifications of secretory proteins in acinar cells. CHO, carbohydrate; ER, endoplasmic reticulum.

nascent proteins moved through the acinar cell in a vectorial manner as a function of time. Subsequent studies have examined the conditions for nascent proteins entering the ER, the regulation of vesicular movement between compartments, and the distinct protein modifications that occur in specific organelles. The details of protein trafficking and maturation are discussed in the following sections. Nascent secretory proteins are subject to a variety of modifications; many of these occur within specific cellular organelles (see Fig. 52-15). Protein modifications often require the activity of accessory resident proteins that are concentrated in specific organelles. The mechanisms that restrict resident proteins to specific organelles are not well understood. However, many soluble resident ER proteins contain the amino acid sequence KDEL or a similar sequence that binds the ER resident KDEL receptor. Although the KDEL receptor and KDEL proteins escape from the ER and travel to the Golgi, they are efficiently retrieved using retrograde vesicular transport back to the ER. Similarly, a KKxx cytoplasmic sequence that binds to COPI proteins enables ER membrane proteins to be returned from the Golgi complex to the ER.

Protein Modifications Cleavage of the Signal Peptide and Folding One of the first modifications of secretory proteins within the ER is the proteolytic removal of the signal peptide. The cleavage is mediated by a specific ER protease (signal peptidase) and occurs within the translocon after a large portion of the nascent protein has entered the ER cisterna. The signal peptidase belongs to a class of proteases that cleaves proteins at or within membranes (25). Removal of the signal peptide traps the nascent protein in the secretory pathway unless the protein misfolds. Protein folding is a critical ER event that requires the coordinated and iterative interactions of a number of accessory proteins. The ER resident proteins calnexin, heat shock protein (HSP70), calreticulin, and Bip participate in this process. Calnexin is a calcium-dependent lectin that anchors nascent proteins in the ER, whereas HSP70 and other accessory proteins directly fold nascent proteins (26,27). The attachment of nascent glycoproteins to calnexin during the folding process is mediated through a carbohydrate residue (28). However, the mechanism responsible for

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1323 binding of nonglycosylated proteins, such as proteins destined for export by the acinar cell, to accessory folding proteins is unknown. Proper protein folding and development of tertiary protein structure often requires covalent and noncovalent modifications within the ER. These include glycosylation, proline hydroxylation, and disulfide bond formation. The latter is regulated by protein disulfide isomerase activity. Secretory proteins are rich in disulfide bonds because they have a greater need for protection from an oxidative environment when they exit the cell, in contrast with cytosolic proteins, which reside in a reducing environment. Specialized protein modifications also occur in the Golgi complex and include modification of carbohydrate residues, sialylation, sulfation, and addition of mannose 6-phosphate residues to lysosomal enzymes. Protein Misfolding and Degradation Proteins that are not properly folded are detected by an ER quality-control system. Most misfolded membrane proteins are immediately degraded by a multiple protein assemblage that resides in the cytoplasm and is known as the proteasome. Because misfolded proteins are formed within the ER but are degraded in the cytoplasm, they must cross the ER membrane. This is accomplished by their retrograde transport through the protein channels in the ER such as the translocon. As a misfolded protein begins to emerge into the cytosol through the ER protein channel, multiple copies of a small protein known as ubiquitin are attached. The polyubiquitin is a targeting mechanism that directs the misfolded protein to the proteasome for proteolytic degradation. The proteasome is a large (>1 × 106 Da) macromolecular protein complex that has a cylindrical structure with a cap on one end to regulate protein entry. Access of ubiquitinated proteins to the core of the proteasome is ATP dependent. Multiple classes of proteases within this core degrade ubiquitinated proteins. The proteasome is present in soluble and membraneassociated forms. ER membrane–associated proteasomes appear to be located in close proximity to the translocon. Such a distribution would make degradation of misfolded proteins efficient. Misfolded membrane and soluble proteins can follow slightly different itineraries. Although misfolded membrane proteins are immediately degraded, at least some soluble proteins must transit to the Golgi complex and return to the ER for degradation by the proteasome. Whether misfolded acinar cell proteins follow this paradigm remains unclear. Endoplasmic Reticulum Stress and the Unfolded Protein Response Sometimes the cellular mechanisms for degrading misfolded proteins are overwhelmed, and unfolded proteins accumulate. The high rates of protein synthesis by the pancreatic acinar cell might make it particularly susceptible to protein misfolding. Accumulation of unfolded proteins leads to activation of a complex feedback mechanism known as the ER stress response. The first step in this response is

to block general protein synthesis while increasing the synthesis of proteins involved in ER folding and export. When this measure is not successful and the levels of unfolded proteins continue to increase, additional components of the ER stress response are activated. These can lead to apoptotic or programmed cell death. There is immunocytochemical evidence that the early phases of an ER stress response may be activated regularly in the pancreatic acinar cell. Furthermore, it is possible that the ER stress response plays a role in pancreatic diseases, including some forms of hereditary pancreatitis that cause misfolding of cationic trypsinogen, or cystic fibrosis. Hyperstimulation of the acinar cell has been observed to lead to massive accumulations of concentrated secretory proteins, termed intracisternal granules, in the lumen of the RER (Fig. 52-16). This may be the morphologic reflection of the unfolded protein stress response. Sorting and Concentration of Nascent Proteins Endoplasmic Reticulum to Golgi Secretory proteins are stored efficiently at high concentrations in zymogen granules by steps that sequentially enrich the proteins several hundred fold more than levels found in the ER lumen (29,30). They are also segregated away from other soluble proteins, most notably lysosomal enzymes, as they move to their final storage site. The first step in the concentration process takes place as nascent secretory proteins are preparing to be transported in vesicles from the ER. These vesicles emerge from distinct protrusions from the RER, known as transitional elements, that are devoid of ribosomes and are close to the Golgi complex (see Fig. 52-13). After budding, the small (~90 nm) vesicles with their nascent protein cargo move to an intermediate compartment known as the vesicular tubular complex (VTC). Proteins are then transferred from the VTC to the Golgi complex by direct fusion or by vesicles that bud from the VTC. Movement of nascent proteins from the ER is mediated by addition of the COPII coatamer protein complex to the cytoplasmic side of the ER membrane (31–33). The COPII coat proteins are required for cargo selection, vesicle budding from the ER, and forward trafficking. The coat is assembled sequentially with addition of the small GTPase, Sar1, followed by the soluble complexes of Sec23/24 and Sec13/31. Accessory proteins may function as a scaffold for assembly and regulate the GTPase activity of Sar1. The mechanisms for cargo selection are not fully understood. Some studies suggest that putative cargo may be concentrated by a molecular scaffold at the transitional elements of the ER (34). The selection of some transmembrane proteins for cargo appears to largely take place through the binding of distinct sequences on their cytoplasmic domain to Sec24. The COPII proteins are concentrated on the forming vesicles, and transmembrane proteins that bind Sec24 also are enriched. Transmembrane proteins are enriched 5- to 10-fold in the COPII vesicle compared with the nearby ER.

1324 / CHAPTER 52

FIG. 52-16. Transmission electron microscopy of pancreatic acinar cells after long-term stimulation with a cholinergic agonist, carbachol. The round, electron-dense structures represent aggregated proteins within a dilated endoplasmic reticulum (ER). These findings are typical of ER stress.

The mechanism for concentrating soluble proteins remains uncertain, but several findings demonstrate that it must be different than for transmembrane proteins. First, whereas transmembrane proteins are greatly concentrated as they emerge in COPII vesicles, soluble proteins do not become substantially concentrated over the ER lumen until they reach the VTC (Fig. 52-17) (30). Second, mechanisms described for concentrating soluble glycoproteins in other systems are relevant to a limited number of pancreatic proteins. Thus, the carbohydrate moiety on some soluble glycosylated proteins binds to the lectin domain on the luminal domain of the transmembrane protein ERGIC-58. It is possible that glycosylated pancreatic secretory protein such as GP2 and muclin are concentrated as they leave the ER by binding to ERGIC-58. However, other mechanisms must account for concentrations of most secretory proteins observed in the acinar cell. It is possible that muclin or other zymogen granule membrane proteins may participate in the concentration of export proteins. The net effect of the enrichment mechanism is to concentrate soluble proteins such as amylase, trypsinogen, and chymotrypsinogen 10- to 20-fold between the ER lumen and the Golgi complex, and even further as they move toward the zymogen granule (Fig. 52-17). Golgi Complex to Trans-Golgi Network The Golgi complex receives nascent proteins that have been carried from the ER in vesicles. Multiple membrane stacks, transport vesicles, and the trans-Golgi network (TGN) form the Golgi complex. The cis-Golgi is closest to the nucleus, whereas the trans-Golgi is the most distal. Unlike the ER, the cisternae of the Golgi stacks are not contiguous,

and their widths are not uniform (Fig. 52-18). Thus, the central area of a Golgi stack is flattened, and the lateral region is enlarged. The lateral enlargement is likely required to accommodate the vesicular traffic in this domain. Some resident Golgi proteins are concentrated within distinct domains. For example, the structural proteins GRASP 65 and GM130 are restricted to the cis-Golgi, whereas the structural protein GRASP 55 localizes to the trans-Golgi (35). The mechanism responsible for restricting Golgi resident proteins to distinct domains is not fully understood, although new families of Golgi coat proteins are believed to be involved. However, the selective distribution of Golgi enzymes likely reflects specific and sequential roles in protein processing. The Golgi is anchored in position by microtubules and other specific proteins (36). Small (~100-nm) vesicles transport proteins in an anterograde and retrograde manner between the Golgi stacks. Thus, whereas secretory proteins are being moved anterograde, ER and Golgi resident proteins undergo retrograde transport and are returned to their proper localizations the ER and Golgi. Retrograde protein transport requires COPI coat proteins; COPI proteins also mediate some antegrade traffic in the Golgi complex. The TGN is formed by tubules and vesicles that arise from the trans-Golgi stacks. However, the TGN is biochemically distinct from the Golgi complex. For example, proteins such as TGN-38 are much more concentrated in the TGN compared with the cis- and medial Golgi. Specific protein modifications take place in the Golgi complex and TGN and are catalyzed by resident proteins. Thus, terminal carbohydrate residues are modified, mannose 6-phosphate residues are attached to lysosomal enzymes, and proteins are sialylated and sulfated. The pancreatic

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1325

FIG. 52-17. Immunogold labeling of acinar cells (left) with antibodies to amylase (5 nm) and chymotrypsinogen (10 nm), demonstrating that they become progressively concentrated as they move sequentially from the endoplasmic reticulum (ER) to the trans-Golgi (G), immature secretory granules (ISG), and finally mature secretory granules (SG). (Right) The extent of concentration varies among secretory proteins. Am, amylase; Chtg, chymotrypsinogen; ProCA, procarboxypeptidase; VTC, vesicular tubular complex. (Courtesy of H. Slot and Oprins and colleagues [30].)

acinar cell generates few O-linked glycoproteins; this modification appears to take place exclusively in the Golgi complex. A gradient of decreasing pH is found in the Golgi complex and TGN. Thus, the pH within the Golgi stacks is estimated to be just less than 7, whereas within the TGN it may be as low as 6 (37). The low pH in the TGN may have a role in protein sorting. Thus, sulfation of glycoproteins and a low-pH

environment may be required for sorting digestive enzymes to the zymogen granule (38). A primary target for this pH-dependent modification appears to be a zymogen granule transmembrane protein with a luminal glycosylated domain, muclin (39). Preliminary studies suggest that muclin may bind specific secretory proteins in a pH-dependent manner. Thus, muclin may participate in sorting of secretory proteins from the Golgi and TGN to zymogen granules.

FIG. 52-18. Electron micrographs of the Golgi complex in acinar cells. (Left) Smooth-surface stacked cisternae and associated small vesicles responsible for anterograde and retrograde transport between the endoplasmic reticulum and the Golgi and between Golgi stacks. (Right) Lower magnification electron micrograph showing the trans or entry side of the Golgi (left) and the trans or exit side where forming secretory granules with electron-dense content can be seen.

1326 / CHAPTER 52 One of the best-characterized protein sorting mechanisms for lysosomal enzymes occurs in the Golgi complex. Mannose 6-phosphate receptors on the Golgi complex recognize the mannose 6-phosphate address marker on lysosomal enzymes. After binding its lysosomal enzyme cargo, mannose 6-phosphate receptors are concentrated in clathrincoated vesicles. The subsequent fusion of these vesicles with endosomes results in acidification of the vesicle and dissociation of the lysosomal enzyme from the receptor. The lysosomal enzyme then is transported by an endosomal carrier to the lysosome. The mannose 6-phosphate receptor recycles back to the Golgi complex. Trans-Golgi Network to Zymogen Granules The TGN is a major setting for protein sorting. In addition to directing lysosomal enzymes to lysosomes, budding vesicles that form the constitutive (unregulated) pathway, as well as developing zymogen granules, arise from this compartment (40). Distinct vesicular carriers with morphologically different coats, or lacking coats, are found budding from the TGN and reflect the varying cargoes that emerge from this compartment. Studies of the TGN in living cells have shown that it is a dynamic structure with tubules protruding and retracting along microtubules. The expanded terminal regions of the TGN form precursors to zymogen granules known as condensing vacuoles (CVs). These structures are larger than mature zymogen granules and their content is less electron dense (Fig. 52-19). Maturation of CVs to zymogen granules involves a reduction in the amount of membrane by budding of small vesicles. Some studies have suggested that vesicles formed from CVs comprise those found in the constitutive-like secretory pathway described elsewhere (41,42). Budding from CVs may provide a mechanism for retrieving lysosomal enzymes that have been sorted into the regulated secretory pathway (43). Thus, both the chloride-dependent and -independent mannose 6-phosphate receptors have been localized to vesicles arising from CVs (Fig. 52-20). Whether these vesicles return

FIG. 52-20. Transmission electron microscopy of Cl− mannose 6-phosphate receptor detected by immunogold labeling on frozen sections. Note that the label is concentrated on a vesicle (arrows) that is budding from an immature secretory granule (ISG). (Modified from Klumperman and colleagues [43], by permission.)

lysosomal enzymes to the Golgi complex or other cellular compartments, or direct them to the cell surface for secretion, is unknown. The mechanism for concentrating secretory proteins in the zymogen granule is unclear. Several proteins are concentrated in this zymogen granule membrane or are attached to its inner leaflet. These include syncollin, GP2, and muclin. The role of these proteins in zymogen granule formation and condensation remains unclear. However, as mentioned earlier, muclin appears to be the best candidate for concentrating the predominately basic secretory granule through an interaction with the acidic residues of muclin in the acidic environment of the TGN.

CELL SIGNALING Cell-Surface Receptors

FIG. 52-19. Transmission electron microscopy demonstrating larger and less electron-dense immature granules and smaller, denser secretory granules.

Receptors on the acinar cell surface translate extracellular signals into cellular responses. Receptors for CCK, a hormone released by the intestine in response to a meal, are localized to the basolateral plasma membrane of the acinar cell, accessible to circulating hormones released into the interstitial space, and available to transmitters liberated from nerve terminals adjacent to the basolateral plasma membrane. The most important biologic effect of receptor activation is the secretion of digestive enzymes from the acinar cell into the lumen of the acinus in response to food in the gastrointestinal tract. However, other acinar cell receptors, such as those for insulin, somatostatin, and tumor necrosis factor-α, may mediate patterns of protein synthesis, have protective functions, or cause cell injury. Receptor activation leads to generation of specific molecules that are usually soluble and known as second

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1327 messengers. Although the acinar cell plasma membrane has both G protein–coupled and tyrosine kinase receptors, only the former have a central role in regulating secretion.

G Protein–Coupled Receptors The acinar cell has two major classes of G protein– coupled receptors on its cell surface that primarily increase either intracellular calcium or cAMP. Those that generate a calcium signal include the M1 and M3 types of muscarinic receptors, the CCK1 and CCK2 receptors, and the bombesin (gastrin-releasing peptide) receptor. Receptors that induce a cAMP signal in the acinar cell include those that bind secretin, vasoactive intestinal polypeptide, and pituitary adenylate cyclase activating peptide. Although most functional studies have examined intracellular calcium and cAMP, additional cellular signals are activated by these receptors. For example, CCK and muscarinic agonists cause cGMP to increase in the acinar cell, and some receptors activate tyrosine and phosphatidylinositol 3-kinase pathways. Although this review focuses on the physiologic effects of receptor stimulation, it is important to note that supraphysiologic stimulation of acinar cell receptors causes distinct patterns of both cell signaling and cellular responses. For example, supraphysiologic stimulation of CCK or muscarinic receptors on the acinar cell results in suppression of maximal enzyme secretion and the pathologic activation of digestive zymogens in the acinar cell. Notably, the responses elicited by supraphysiologic CCK stimulation are used to generate acute pancreatitis in animal models and might be relevant to the pathogenesis of alcohol-induced pancreatitis. The reason for having receptors on the acinar cell that respond with a calcium or cAMP response is likely related to the synergism between these two signaling pathways. Thus, the secretory response to increasing both messengers is greater than to each alone. This provides a mechanism to modulate the pancreatic secretion in response to intestinal nutrients and pH. The purpose of several classes of receptors with similar signaling responses is unclear, but presumably provides redundancy should one system fail. There are notable differences in acinar cell-surface receptors and their associated messenger pathways among species. For example, it appears that acinar cells of rats, but possibly not humans, have a CCK1 receptor. Although CCK may not interact directly with the human acinar cell, this hormone does regulate the secretory response to a meal. Thus, CCK released from small intestinal I cells regulates a major component of human pancreatic secretion by stimulation of CCK1 receptors. The explanation for the phenomena is that, in humans, CCK indirectly stimulates pancreatic secretion by activating muscarinic neural mechanisms (44,45). In addition to ligand binding, factors such as receptor phosphorylation, interactions with other regulatory proteins such as β-arrestins, and internalization regulate the function of acinar cell-surface receptors. The number of many cellsurface receptors is regulated by their exocytic insertion and

endocytosis. Removal of receptors by endocytosis represents a common mechanism for terminating cell signaling by cellsurface receptors. In this process, ligand binding causes receptor aggregation and the assembly of cytoplasmic proteins to form a clathrin coat. The forming coat concentrates proteins within a patch of membrane that is destined to be an endocytic vesicle. After the cargo has been concentrated, the clathrin coat proteins deform the membrane and form a protein lattice that surrounds endocytic vesicles. These endocytic vesicles can follow several intracellular itineraries that lead to return of intact receptor to the cell surface, or receptor degradation. The vesicles can also enter a recycling endocytic compartment in which they may be temporarily stored within the cell for subsequent reinsertion into the plasma membrane. The distribution of receptors on the acinar cell surface might be related to the patterns of cell signaling. Thus, two receptor signaling mechanisms have been proposed to account for the generation of the initial apical calcium signal in the acinar cell: (1) positioning of the receptor near the apical domain, or (2) diffusion of soluble signals over a distance from the basolateral receptor to the apical pole of the cell. Using caged carbamylcholine, Ashby and coworkers (46) determined that activation of muscarinic receptors on the basal membrane must release soluble signaling molecules to initiate an apical calcium signal. Thus, localization of receptors near the apical pole of the acinar cell is not required to generate the initial apical calcium signal. Additional cell-surface receptors might have specific biological functions on the acinar cell. For example, the proteinase-activated receptors (PAR2) are found on acinar cells. When activated by trypsin, these G protein–coupled receptors stimulated an increase in cytosolic calcium and activation of mitogen-activated protein kinase (47). Furthermore, activation of the PAR2 receptor reduced injury in cellular and in vivo models of pancreatitis. Thus, it has been suggested that when pathologically activated extracellular trypsin interacts with the membrane of acinar cells, it activates protective mechanisms through the PAR2 receptor. The molecular mechanisms of this protective response remain unclear. Calcium Signaling Cytosolic Secretagogue-induced transient increases in cytosolic calcium are required to stimulate acinar cell secretion (Fig. 52-21). Pathologic increases in cytosolic calcium can be caused by supramaximal concentrations of CCK or acetylcholine analogs; in vivo, these treatments can cause acute pancreatitis. The calcium increases elicited by these agonists exhibit distinct spatial and temporal characteristics that are coupled to physiologic and pathologic responses. Refinements in probes that detect intracellular calcium, instrumentation, and microinjection techniques have modified our view of this critical response. In unstimulated conditions, cytosolic calcium is estimated to be about 50 nM. Soon after stimulation by muscarinic agonists or CCK, there

1328 / CHAPTER 52 900 Physiologic

800

Supraphysiologic 700 Calcium 600 fluorescence % of baseline 500 400

CCK

300 200 100 0

0

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FIG. 52-21. Cytosolic calcium signal in pancreatic acinar cells stimulated by either physiologic or supraphysiologic concentrations of cholecystokinin (CCK).

is a rapid increase in cytosolic calcium that is first detected at the apical pole of the acinar cell (Fig. 52-22). This wave sweeps toward the basolateral region of the cell at an estimated 10 to 30 µm/sec. Whereas previous studies suggested that the peak calcium response was about 300 nM, more recent investigations using lower affinity calcium-sensitive dyes have found levels may be an order of magnitude (5–20 µM) greater in the apical region. Thus, the initial increase in intracellular calcium is localized to the sites of zymogen granule secretion in the apical region of the acinar cell (48–50). The mechanism for both generating an apical signal and propagating the calcium wave toward the cell base appears to be coupled to the distribution of distinct calcium release channels in the membrane of the ER. IP3-sensitive calcium channels are concentrated at the apical region of the acinar cell, just below the plasma membrane (Fig. 52-23). 250

Cytosolic Ca2+ (nM)

Apical Basolateral

200

150

100 Begin ACh

50 37

39

41

43

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FIG. 52-22. Appearance of initial cytosolic calcium signal at the apical pole of the acinar cell after stimulation with acetylcholine (ACh). (Courtesy of M. Nathanson.)

The calcium-sensitive ryanodine (RYR) receptor calcium channel is distributed in the lower two thirds of the acinar cell. After receptor stimulation and activation of phospholipase C, IP3 is released from the membrane and can diffuse to apical IP3 receptors (Fig. 52-24). The IP3 receptors release calcium that diffuses toward the base of the cell to stimulate opening of the RYR. This later phase is known as calcium-induced calcium release. Calcium channels within the plasma membrane later open and contribute both to the cytosolic calcium signal and to extracellular calcium refill of calcium stores. Mitochondria may also contribute to the regulation of this calcium signal by two mechanisms (51). First, concentrations of mitochondria within specific regions of the acinar cell (Fig. 52-25) may provide high levels of ATP locally to drive energy-dependent ion transport. Second, mitochondria can rapidly take up calcium to reduce its levels in the cytosol. Several additional mechanisms help remove calcium from the cytosol and terminate the calcium signal. There is also evidence that activation of different cellsurface receptors may generate cytosolic calcium signals with distinct spatiotemporal relations. For example, activation of acinar cell muscarinic receptors causes calcium release from ER stores, whereas stimulation of CCK receptors causes release from both the ER and lysosomes (52). Thus, multiple second messengers regulate the release of calcium into the cytosol; inositol-1,4,5-phosphate or cyclic adenosine diphosphate ribose signal is required for release from the ER, whereas nicotinic acid adenine dinucleotide phosphate mediates release of calcium from acidic lysosomal organelles. Although it is expected that these distinct patterns of calcium release would result in prominent functional differences, none have yet been described. The transient increases of the cytosolic calcium signal are essential for a physiologic response, whereas prolonged

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1329 Ca2+ release into cytosol

Ca2+ removal from cytosol

1

1

2

2

3 3

FIG. 52-23. Major mechanisms regulating cytosolic calcium signaling. Calcium release into the cytosol is mediated by entry of endoplasmic reticulum (ER) stores through the inositol-1,4,5-trisphosphate channel (1) and the ryanodine receptor calcium channel (2) and from the extracellular space through plasma membrane calcium channels (3). Calcium removal from the cytosol is mediated by ATPases that pump calcium into the ER (1) and through the plasma membrane (3) and by mitochondrial uptake (2).

increases are toxic. The most important mechanisms for removing calcium from the cytosol are the calcium-activated ATPases on the ER and plasma membrane that pump calcium back into ER stores and out of the cell, respectively (see Fig. 52-23). Mitochondrial uptake and sodium-calcium exchange may also remove calcium from the cytosol. Together, they form an elegant and interrelated mechanism both for generating increases in cytosolic calcium with distinct temporal and spatial characteristics and for returning calcium to baseline levels in preparation for another round of stimulation.

FIG. 52-24. Distribution of inositol-1,4,5-trisphosphate– sensitive calcium channels (arrowhead) in the apical region of acinar cells detected by immunofluorescence. This receptor is apical of the dark regions of the acinar cell that represent zymogen granules (arrow). (Courtesy of S. Husain and W. Grant.)

Nuclear Studies have shown that calcium signals can travel into the nucleus (53). Furthermore, transcription of some genes has been shown to be calcium dependent. Studies have shown that nuclei contain deep indentations lined by ER

FIG. 52-25. Acinar cell mitochondria are localized to specific regions of the acinar cell including the base of storage granules, around the nucleus, and along the basolateral membrane. This distribution may provide high local levels of adenosine triphosphate and allow the mitochondria to participate in calcium signaling. (Modified from Johnson and colleagues [51], by permission.)

1330 / CHAPTER 52

A

B

FIG. 52-26. Confocal micrographs demonstrating nucleoplasmic reticulum detected by (A) generation of an intranuclear calcium signal and (B) intranuclear labeling of a marker of the endoplasmic reticulum. (Courtesy of M. Nathanson and Echevarria and colleagues [54].)

(Fig. 52-26) (54). Calcium is released from these structures directly into the nucleus. This provides a mechanism to generate high regional increases of calcium within the nucleus that are independent of cytosolic increases. Increases in nuclear calcium may selectively regulate gene transcription (55).

including exocrine and endocrine cells, possess a so-called constitutive secretory pathway that is proposed to be the route by which newly synthesized membrane proteins reach the plasma membrane and is the pathway for delivery of ECM and basement membrane components (56). This pathway may have evolved before those involved in regulated secretion

SECRETION A major physiologic response of the acinar cell to stimulation is release of zymogens and digestive enzymes into the acinar lumen. Studies indicate that regulated secretory cells such as the pancreatic and parotid acinar cells possess as many as four potential pathways for the secretion of proteins: constitutive, constitutive-like, minor regulated, and regulated (42). As indicated in Figure 52-27, regulated secretory granules begin to form in the TGN with progressive selection and concentration of secretory proteins to form CVs. CVs bud off from the TGN and are now termed immature secretory granules (ISGs). In the course of formation of mature secretory granules, excess membrane must be removed because the surface area of granules is considerably less than that of CV or ISGs. Removal of excess membrane is accomplished by pinching off of vesicles from CV and ISGs. The vesicles, carrying a sample of newly synthesized secretory protein, now enter secondary pathways that are termed constitutive-like and minor regulated pathways. A consequence of removal of excess membrane from the nascent secretory granules is that the secretory granule membrane is refined to its final simplified composition, which appears unique compared with the membranes of other compartments in the cell. Finally, it has long been recognized that all cells,

C R

SG CL

ISG

MR

FIG. 52-27. Pathways leading to secretion in the pancreatic acinar cell. Several major secretory pathways emerge from immature granules including storage granules (ISG) through the regulated pathway (R), constitutive-like (CL) and minor regulated (MR) compartments. A constitutive pathway probably emerges from the trans-Golgi network. The bulk of secretion takes place through the regulated pathway. (Modified from Huang and colleagues [42], by permission.)

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1331 to take care of fundamental cell housekeeping functions. Vesicles that comprise the constitutive pathway likely arise from the TGN, but their precise origins remain unclear (41). Furthermore, there is some evidence that the constitutive pathway may be formed by tubules that transiently extend to the plasma membrane from the TGN (40). Constitutive pathway vesicles likely traffic to both the apical and basolateral domains of polarized epithelial cells, though this remains speculative because of the absence of markers for the constitutive pathway. Current evidence suggests that the constitutive-like and minor regulated pathways are responsible for delivering secretory proteins by exocytosis at a low rate to accommodate digestion in the duodenum between meals (basal or resting secretion). Secretory proteins that enter either of these two pathways consist primarily of newly synthesized proteins. The minor regulated pathway appears to deliver secretory proteins directly to the acinar cell surface, where they are released by exocytosis. The minor regulated pathway is more responsive to exocytosis with secretagogue stimulation than are mature secretory granules. The constitutive-like pathway differs from the minor regulated pathway in that it is unresponsive to secretagogues and follows an indirect route to the cell surface that may channel through the endosomal compartment. Constitutive-like and minor regulated secretion are quantitatively much smaller than regulated secretion. For example, only about 1% to 2% of total enzyme content might be released per hour by the constitutive-like pathway and only 2% to 4% by the minor regulated pathway. Regulated secretion arises from a storage pool that excludes newly synthesized secretory proteins and is built up slowly during the interdigestive phase. It is able to release 15% to 30% of the secretory protein content of the gland by classical regulated exocytosis and is maximally stimulated after ingestion of a meal, when release of massive amounts of digestive enzymes and zymogens (considering zymogens as a category of digestive enzyme) are required at rates greater than can be attained by protein synthesis alone. Zymogen granule exocytosis does not appear to contribute to resting secretion. The purpose of these distinct mechanisms of secretion is unknown, but they likely serve both to ensure that some digestive enzymes will be present in the small intestine at all times and to provide a secretory response that is proportional to luminal nutrients. The minor regulated pathway could serve to increase enzyme secretion in response to smaller quantities of food than presented by a full meal. Finally, a novel role for the constitutive and minor regulated compartments suggested by Castle and colleagues (57) is that they might deliver the target membrane soluble N-ethylmaleimide–sensitive factor attachment protein receptors (t-SNAREs) necessary for subsequent zymogen granule fusion with the apical membrane.

Exocytosis The release of zymogen granule content into the lumen of the acinus requires fusion of the vesicular membrane with the

apical plasma membrane, as shown in Figure 52-28. Three key steps are recognized in this process (Fig. 52-29). First, the zymogen granule must move from its site of formation in the trans-Golgi to the apical region of the cell. This event likely requires active involvement of contractile elements, particularly actin and associated motor proteins, in movement of the zymogen granule to its apical plasma membrane target. Notably, in the resting interphase between rounds of exocytosis, an apical actin terminal web presumably negatively regulates resting secretion in that an actin mesh is always found between zymogen granules and the apical plasma membrane (58–60). Once in the apical region of the cell, the membrane of the zymogen granule must recognize and become tethered to the apical plasma membrane with which it will fuse. Atomic force microscopy has suggested that specific structures found on the apical membrane might represent the sites of zymogen granule docking and exocytosis (Fig. 52-30). This also implies that the actin meshwork beneath the apical membrane must be dissociated for close membrane apposition to occur. The SNARE hypothesis for membrane recognition and fusion, which appears to be a generalized mechanism for all cells examined, is particularly relevant for the pancreatic acinar cell where specific interactions between the zymogen granule membrane and the apical plasma membrane are needed to ensure that exocytosis of digestive enzymes and proenzymes occurs into the acinar lumen. Endobrevin/vesicleassociated membrane protein isoform 8 (VAMP-8), appears to function as the key vesicle SNARE (v-SNARE) for zymogen secretion, whereas VAMP-3 does not appear to have an important role. An as yet unidentified t-SNARE and synaptosomal-associated protein of 25 kDa (SNAP-25) presumably exist on the apical plasma membrane of the acinar cell.

FIG. 52-28. Electron micrograph of apical region of an acinar cell showing a secretory granule closely apposed to the apical plasma membrane (left), and an image of zymogen granule membrane that has been incorporated into the apical plasmalemma after exocytosis (right), allowing for continuity of the granule content with the acinar lumen. (Courtesy of George E. Palade.)

1332 / CHAPTER 52 Lumen

FIG. 52-29. Events leading to regulated exocytosis and compensatory membrane retrieval in acinar cells. Actin-coated secretory granules move through the subapical actin network followed by docking and fusion of granule membrane with the apical plasma membrane. Dissociation of Rab3D, a small guanosine triphosphate–binding protein, may regulate these steps. After membrane fusion and release of secretory proteins into the acinar lumen, excess membrane is removed from the cell surface and internalized using a clathrin-requiring process. (Reproduced from Valentijn and colleagues [62,64], by permission.)

Nonetheless, specific interaction of the zymogen granule with the apical plasma membrane is critical, because if zymogen granules were to promiscuously fuse with the basolateral plasma membrane, enzymes and proenzymes from the granule contents could enter the interstitial space and cause injury if activated. After interaction and tethering of the zymogen granule with the apical plasma membrane t-SNARE/SNAP-25, a prefusion complex is formed. Four α helices, two from SNAP-25 and one each from the granules v-SNARE and the apical t-SNARE, form a tight coiled coil that pulls the granule and apical membranes together in a step that requires ATP. As a

result of close apposition, the cytoplasmic faces of the granule and apical membrane fuse, exposing the hydrophobic cores of the two membranes (61). This prefusion pore continues to widen until fusion of the two membranes is complete and the zymogen granule contents are free to diffuse into the centroacinar lumen.

Apical Membrane Expansion and Retrieval As a consequence of fusion of the zymogen granule with the apical plasma membrane, two problems arise. First, if all

FIG. 52-30. Proposed mechanism for partial fusion of secretory granules with the apical plasma membrane and release of secretory proteins through a transient pore. (Modified from Schneider SW, Sritharan KC, Geibel JP, Oberleithner H, Jena BP. Surface dynamics in living acinar cells imaged by atomic force microscopy: identification of plasma membrane structures involved in exocytosis. Proc Natl Acad Sci U S A 1997;94:316–321, by permission.)

STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1333

FIG. 52-31. Transmission electron microscopy of images showing sequence of formation of clathrincoated pits, invagination, and pinching off of endosomal vesicles during compensatory membrane retrieval after exocytosis (62,64).

of the granule membranes were to be inserted simultaneously into the apical plasma membrane (~900 µm2), the apical plasma membrane (~30 µm2) would increase in area ~30-fold. Clearly, this does not happen. One observes a transient increase in the surface area of the apical plasma membrane, but as exocytosis continues, even at maximal physiologic rates, the area of the luminal space returns to baseline. It is now clear that, as is the case for all regulated secretory systems (including the nerve terminal where events are best understood at the molecular level), co-terminus compensatory membrane retrieval accounts for maintenance of the luminal surface area. Evidence indicates that, in the acinar cell, patches of membrane adjacent to sites of zymogen granule membrane fusion evaginate from the apical plasma membrane and become coated with clathrin (Fig. 52-31). Pinching off and retrieval of the clathrin-coated vesicles appears to involve homologues of the cytoplasmic proteins that are required for synaptic vesicle retrieval from the nerve terminal after neurotransmitter release. The actin terminal web clearly is involved in membrane retrieval (60). Thus, if the actin cytoskeleton is depolymerized experimentally, membrane retrieval does not keep pace with continued exocytosis and the area of the apical membrane expands massively, accompanied by retention of pooled secretory proteins in an expanded acinar lumen. The fate of the internalized membrane is not clearly understood, but in chronically stimulated acinar cells, enlargement of the membrane surface area of the Golgi complex is observed. What is unclear, however, is the composition of the membrane retrieved from the apical plasma membrane. One would presume that it would consist of excess membrane contributed by fused zymogen granules, but this does not appear to be the case. In any event, the net result is maintenance of the area of the apical plasma membrane and retention or reconstitution of the specificity of its protein and lipid composition, because subsequent rounds of regulated exocytosis can take place even in the absence of continuing protein synthesis.

This is followed by solubilization of the relatively concentrated zymogen granule content, which is required for normal flow of enzymes and proenzymes through the duct system and into the small intestine. Solubilization is accomplished by two major mechanisms: (1) secretion of fluid and electrolytes from acinar, centroacinar, and duct epithelial cells; and (2) the mixing of these contents by cilia located on the apical surfaces of centroacinar and duct cells. Centroacinar cells, as seen in Figure 52-5, have the hallmarks of fluid and electrolyte secreting cells, being endowed with numerous mitochondria and abundant smooth ER. Interestingly, zymogen granule content is solubilized efficiently at pH levels greater than ~7.5. Because centroacinar and duct cells secrete a bicarbonaterich fluid with a pH of ~8, the relatively alkaline fluid secreted into the acinar lumen at the same time as exocytosis is initiated presumably plays an important role in solubilizing zymogen granule content as soon as continuity is established between the acinar lumen and the fusing zymogen granule. Centroacinar and small duct cells also possess a single flagellum (Fig. 52-32). We have observed these flagella to beat rapidly in the centroacinar lumen of isolated pancreatic acini. In addition to mixing of content, they have been postulated to act as flow sensors (63).

Secretory Protein Extrusion, Solubilization, and Delivery into the Duct System The efficient entry of zymogen granule content into the duct system requires contraction of the actin cytoskeleton (62).

FIG. 52-32. Electron micrograph of longitudinal cross section of flagella (arrowheads) in duct lumen.

1334 / CHAPTER 52 ACKNOWLEDGMENTS The authors thank Sohail Husain for his helpful comments. Support for investigative activities was provided by the National Institutes of Health (grant DK 52401, F.S.G.) and a Veterans Administration Merit Award (F.S.G.).

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STRUCTURE–FUNCTION RELATIONS IN THE PANCREATIC ACINAR CELL / 1335 48. Leite MF, Burgstahler AD, Nathanson MH. Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini. Gastroenterology 2002;122:415–427. 49. Gerasimenko OV, Gerasimenko JV, Belan PV, Petersen OH. Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca from single isolated pancreatic zymogen granules. Cell 1996;84:473–480. 50. Yule DI, Essington TE, Williams JA. Pilocarpine and carbachol exhibit markedly different patterns of Ca2? signaling in rat pancreatic acinar cells. Am J Physiol 1993;264:G786–G791. 51. Johnson PR, Dolman NJ, Pope M, Vaillant C, Petersen OH, Tepikin AV, Erdemli G. Non-uniform distribution of mitochondria in pancreatic acinar cells. Cell Tissue Res 2003;313:37–45. 52. Yamasaki M, Masgrau R, Morgan AJ, Churchill GC, Patel S, Ashcroft SJ, Galione A. Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells. J Biol Chem 2004;279:7234–7240. 53. Leite MF, Thrower EC, Echevarria W, Koulen P, Hirata K, Bennett AM, Ehrlich BE, Nathanson MH. Nuclear and cytosolic calcium are regulated independently. Proc Natl Acad Sci U S A 2003;100:2975–2980. 54. Echevarria W, Leite MF, Guerra MT, Zipfel WR, Nathanson MH. Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum. Nat Cell Biol 2003;5:440–446. 55. Hardingham GE, Chawla S, Johnson CM, Bading H. Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression. Nature 1997;385:260–265. 56. Arvan P, Castle JD. Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas. J Cell Biol 1987;104:243–252.

57. Castle AM, Huang AY, Castle JD. The minor regulated pathway, a rapid component of salivary secretion, may provide docking/fusion sites for granule exocytosis at the apical surface of acinar cells. J Cell Sci 2002;115:2963–2973. 58. Muallem S, Kwiatkowska K, Xu X, Yin HL. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell Biol 1995;128:589–598. 59. Williams J. Effects of cytochalasin B on pancreatic acinar cell structure and secretion. Cell Tissue Res 1977;179:453–466. 60. Valentijn JA, Valentijn K, Pastore LM, Jamieson JD. Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D. Proc Natl Acad Sci U S A 2000;97:1091–1095. 61. Bonifacino JS, Glick BS. The mechanisms of vesicle budding and fusion. Cell 2004;116:153–166. 62. Valentijn KM, Gumkowski FD, Jamieson JD. The subapical actin cytoskeleton regulates secretion and membrane retrieval in pancreatic acinar cells. J Cell Sci 1999;112:81–96. 63. Bertelli E, Regoli M. A morphological study of the primary cilia in the rat pancreatic ductal system: ultrastructural features and variability. Acta Anat (Basel) 1994;151:194–197. 64. Valentijn K, Valentijn JA, Jamieson JD. Role of actin in regulated exocytosis and compensatory membrane retrieval: insights from an old acquaintance. Biochem Biophys Res Commun 1999;266:652–661.

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53

Stimulus-Secretion Coupling in Pancreatic Acinar Cells John A. Williams and David I. Yule Receptors, 1338 Transmembrane Signaling, 1338 Heterotrimeric G Proteins, 1338 Membrane Effectors, 1339 Intracellular Messengers, 1339 Inositol 1,4,5-Trisphosphate, 1339 Intracellular Ca2+, 1341 1,2 Diacylglycerol, 1350 Role of Cyclic Nucleotides in Pancreatic Acinar Secretion, 1351 Other Phenomena Related to Intracellular Messengers, 1352 Interaction of Intracellular Messengers, 1353 Intracellular Messenger–Induced Secretion, 1353 Action of Intracellular Messengers, 1354

Cyclic Nucleotide–Activated Kinases, 1354 Protein Kinase C, 1355 Ca2+/Calmodulin-Activated Kinases, 1355 Tyrosine Kinases, 1356 Protein Phosphatases, 1356 Secretagogue Changes in Cellular Protein Phosphorylation, 1357 Mechanisms of Exocytosis, 1358 Visualization of Exocytosis, 1358 Rab Proteins, 1359 SNARE Proteins, 1360 Cytoskeleton, 1361 Role of Ion Channels in Exocytosis, 1361 Endocytosis, 1362 References, 1362

The major function of pancreatic acinar cells is to synthesize and secrete a variety of digestive enzymes. Secretion is regulated by the gut hormones cholecystokinin (CCK) and secretin and by the neurotransmitters acetylcholine (ACh), vasoactive intestinal polypeptide (VIP), and gastrin-releasing peptide (GRP). Other peptides can act in vivo to stimulate or inhibit secretion, but their physiologic relevance is less clear. In addition, secretion can be modulated at the acinar cell level by other regulatory molecules including insulin, insulinlike growth factor, and somatostatin. All of these agents

interact initially with receptors on the plasma membrane of acinar cells. The steps after receptor occupancy that lead to secretion of either proteins or ions are generally referred to as stimulus-secretion coupling, a term coined in the pioneering work of Douglas (1). Although the term originally applied to the role of Ca2+ in mediating secretion of chromaffin and other cells, it is now generally used in the broader sense. It is operationally useful to divide stimulus-secretion coupling into consideration of transmembrane signaling, intracellular messengers, effectors, and exocytosis. Intracellular messengers include ions, cyclic nucleotides, phospholipids, and gaseous messengers. Effectors are the molecules activated by the intracellular messengers, which often are involved in specific phosphorylation events. The final steps of exocytosis in which zymogen granule fuses with the luminal plasma membrane are now being understood molecularly in the context of regulated membrane fusion. Finally, it has become clear that secretagogues trigger other regulatory

J. A. Williams: Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109. D. I. Yule: Departments of Pharmacology and Physiology, University of Rochester Medical School, Rochester, New York 14642. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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1338 / CHAPTER 53 events and pathways, as well as those described here, to mediate digestive enzyme synthesis and other acinar cell functions. These pathways have been reviewed elsewhere (2,3).

RECEPTORS The receptors for the major pancreatic secretagogues are all G protein–coupled membrane proteins with seventransmembrane domains (4). These have been characterized by their biologic response, ligand binding, and through molecular cloning and expression of native and mutated receptors. Because of their physiologic importance and their use in studying stimulus-secretion coupling, the form and characteristics of CCK and muscarinic ACh receptors are reviewed briefly. Two distinct CCK receptors have been characterized and denoted as A and B, or more recently as types 1 and 2 (5,6). CCKA receptors are highly specific for CCK, which contains a sulfated tyrosine seven amino acids from the C terminus, whereas CCKB receptors bind CCK and gastrin equally and do not require a sulfated tyrosine. Most pancreatic studies have focused on CCKA receptors because they are the predominant form on rodent acinar cells, gall bladder smooth muscle, and vagal afferent nerves. Human acinar cells do not have either form of CCK receptor (7), and the action of CCK is mediated by activation of afferent nerve endings (8). CCKA receptors wherever evaluated are able to exist in both high- and low-affinity states, and different biological actions have been assigned to a particular affinity state (9). This assignment has been greatly facilitated by the use of the CCK analog JMV-180, which acts as an agonist at high-affinity CCKA receptors and an antagonist of the lowaffinity state. There also has been considerable progress in understanding the structural basis of how CCK peptides interact with the receptor, as reviewed in the literature (10). The muscarinic cholinergic receptor on pancreatic acini of both the rodent and human pancreas has been generally characterized as a M3 isoform (7), although some evidence exists for an M1-like receptor (11). Studies of gene-deleted mice show the presence of both M1 and M3 receptors in salivary glands (12). As with the CCK receptor, muscarinic receptors can exist in high- and low-affinity receptor states, and high concentrations of cholinergic agonists induce reduced amylase release from isolated acini, similar to results with CCK. By contrast, bombesin acts on GRP receptors that show only one affinity state and induce amylase secretion without inhibition by high concentrations. In general, for all G protein–coupled receptors, the transmembrane domains may form a pocket for binding small molecules, whereas the external amino terminus and external loops may also be important in the interaction with peptide molecules. The third cytoplasmic loop is believed to be the primary site interacting with heterotrimeric G proteins, whereas the cytoplasmic carboxyl terminal may be involved with receptor regulation such as desensitization and downregulation in response to both phosphorylation and interaction with other proteins.

TRANSMEMBRANE SIGNALING Heterotrimeric G Proteins The pancreatic secretagogue receptors convey information by interacting with heterotrimeric G proteins or G proteins. These G proteins are made up of α-, β-, and γ-subunits with the latter two normally existing as a βγ complex. There are at least 16 α-, 5 β-, and 12 γ-subunits in the human and mouse genome, and the significance of this heterogeneity is not fully understood. The four major classes or families are Gq, Gs, Gi/o, and G12/13 (13). The G proteins responsible for secretion have long been assumed to belong to the Gq family, which includes αq, α11, α14, and α15. Of these, αq, α11, and α14 have been identified in acinar cells (14,15). Microinjection of a neutralizing antibody to a common region of these three G proteins blocked Ca2+ signaling induced by CCK or carbamylcholine (CCh) (15). Ca2+ signaling was normal in response to CCK, CCh, and bombesin in acini prepared from mice in which each of the three α-subunits was deleted genetically and in a α11/α14 double knockout mouse (14). This finding was interpreted as indicating that the three receptors can couple through multiple α-subunits of the Gq family. Gs is almost certainly responsible for coupling the receptors for secretin and VIP to adenylate cyclase (AC) and can be activated selectively by cholera toxin. Gi members can be inactivated by pertussis toxin and mediate the inhibition of AC brought about by somatostatin acting on somatostatin receptor 2. G12 and G13 can be activated by CCK and mediate the activation of Rho (16). All of these G proteins are primarily located in the plasma membrane, but are also present on intracellular membranes (17). All α- and γ-subunits bear posttranslational lipid modifications to mediate their attachment to membranes. The generally accepted model for heterotrimeric G-protein activation is based on the α-subunit possession of a guanine nucleotide-binding site, which in the resting state is occupied by guanosine diphosphate (GDP). After the receptor binds its ligand it interacts with the G protein to catalyze the release of GDP followed by the binding of guanosine triphosphate (GTP). The α-subunit then dissociates from the βγ complex and both can activate their effectors. Evidence suggests that activation can also involve a conformational change without full dissociation (18). The system amplifies because the lifetime of the GTP-α subunit complex is much longer than that of the hormone-receptor complex. Eventually, GTP is cleaved by intrinsic GTPase activity, or at a faster rate when mediated by a GTPase-activating protein (GAP) such as the RGS (Regulators of G-protein Signaling) proteins (19,20). RGS4, as well as RGS2, is a potent inhibitor of Ca2+ signaling in pancreatic acini transduced by different Gq-coupled receptors (21). RGS4 and RGS1 were more potent in inhibiting CCh than CCK, whereas RGS2 was equipotent. The crystal structures for the inactive (GDP-bound), active (GTPbound), and RGS-α complexes have been solved and provide a framework for understanding how heterotrimeric G proteins work as molecular switches (22).

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1339 Membrane Effectors

INTRACELLULAR MESSENGERS

The major membrane effector activated by Gq family members in response to CCK, CCh, and bombesin is a phosphoinositide-specific phospholipase C (PLC) that cleaves phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) to form inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). As discussed later in this chapter, IP3 binds to an intracellular receptor that functions as a gated Ca2+ channel, and thereby initiates the release of sequestered Ca2+, whereas DAG activates protein kinase C (PKC). There are at least 12 isoforms of PLC that are categorized into 5 families of enzymes denoted β1-4, γ1-2, δ1-4, ε, and ς , with ς being sperm specific (23). All types act on both phosphatidylinositol (PI) and its polyphosphorylated derivatives phosphatidylinositol4-phosphate (PI[4]P) and PI(4,5)P2, but at physiologic calcium levels, the polyphosphoinositides are the preferred substrate. Four PLCβ isoforms exist and are the main form activated by G protein–coupled receptors, whereas PLCγ isoforms are activated by growth factor receptors with tyrosine kinase activity. The β1 form has been shown to be activated by purified αq (24). Western blotting has shown the presence of PLCβ1, β3, γ1, and δ1 on rat pancreatic acinar membranes (25). Interestingly, anti–PLCβ1 antibody inhibited activation of PLC activated by CCK and bombesin much more than it did stimulation by CCh, whereas anti–PLCβ3 had a stronger effect on CCh stimulation. These results imply distinct pathways or structural complexes of receptor/G protein/PLC. Epidermal growth factor was shown by similar techniques to activate PLCγ1 (26). AC is the membrane effector synthesizing cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) and is found in all mammalian tissues. Molecular cloning has identified nine genes coding for membraneassociated AC and one soluble form present in the testis (27). These proteins contain two six-transmembrane domain regions separated by a large cytoplasmic loop with a second large cytoplasmic domain at the C terminus. There are two forms stimulated by Ca2+ and calmodulin (AC1 and AC3), but they are not present in the pancreas. The diterpene forskolin activates all membrane-bound isomers except AC9. Most tissues possess more than one form, but the type present in acinar cells is unclear. The structural interactions between αs and αi with AC are becoming known. The activities of some cyclases can also be regulated by posttranslational modifications including phosphorylation and nitrosylation. Secretin and VIP activate AC in pancreatic acini through Gs; CCK at high concentrations can also activate pancreatic AC. Other membrane effectors that may be activated by α or βγ G-protein subunits in pancreatic acinar cells include phosphatidyl choline–specific PLC and PLD, phospholipase A2 (PLA2) (28), the Na+-H+ exchanger, and various ion channels. It is unclear, however, whether these are regulated by G proteins directly or by intracellular messengers. There is direct evidence for an action of a βγ complex to regulate the IP3 receptor (29).

Inositol 1,4,5-Trisphosphate Binding of all major pancreatic acinar cell secretagogues results in the stimulation by Gαq/11 of PLCβ activity, the hydrolysis of PI(4,5)P2, and the subsequent formation of IP3 and DAG. Both molecules are established as primary intracellular messengers in the exocrine pancreas. The development of our current understanding of this pathway has been reviewed extensively elsewhere, and thus is covered only briefly in this chapter (30,31). Interestingly, however, this knowledge is in large part due to experimental observations made in exocrine cells, particularly those from the pancreas. For example, in the 1950s, Hokin and Hokin (32) first suggested the involvement of phosphoinositides in signal transduction after demonstrating that cholinergic stimulation increased 32P incorporation into polyphosphoinositides extracted from pigeon pancreas. This phenomenon was later demonstrated to be common to a variety of agonists in many cell types and became known as the “PI” or phosphoinositide effect. Although Michell (33) had noted a correlation between agonists that elicited the PI effect and those that resulted in Ca2+ mobilization, two studies in 1983 definitively established the connection. Initially, Berridge (34) demonstrated that agonists actually hydrolyze polyphosphoinositides and not phosphatidylinositol, resulting in the formation of inositol trisphosphate. Streb and colleagues (35) then showed that IP3 added to a suspension of permeabilized rat pancreatic acinar cells resulted in Ca2+ release from a nonmitochondrial intracellular store. Importantly, these authors demonstrated that Ca2+ release was specific for the IP3 isomer in that other inositol phosphates lacked activity (35). After stimulation with agonists, IP3 is formed rapidly. In pancreatic acini prelabeled with [3H]-inositol, stimulation with maximal concentrations of CCK, muscarinic agonist, or bombesin results in the formation of [3H]-IP3, which can be measured as early as 5 seconds and accumulates linearly with time (36). High-pressure liquid chromatography (HPLC) analysis of the [3H] products found multiple isomers of inositol trisphosphate, including inositol-1,3,4-trisphosphate in addition to IP3. These studies indicated that levels of IP3 peaked twofold to fivefold greater than basal levels within 5 seconds, and then gradually decreased to levels that were still increased about 10 minutes after exposure to agonist. In contrast, the time course of inositol-1,3,4-trisphosphate generation lagged about 15 to 40 seconds after the peak of IP3 levels and continued to increase during the exposure to agonist (37–39). A competitive binding assay for IP3 also has been developed that can be used to measure the actual mass of IP3. This convenient assay is based on binding of [3H]-IP3 to membranes prepared from tissues that are rich in IP3 receptors such as cerebellum. Studies using this assay, have shown in rat pancreatic acini that maximal concentrations of agonists increase IP3 20- to 100-fold from a basal level of 1 to 5 pmol/mg protein to 60 to 100 pmol/mg protein (Fig. 53-1).

1340 / CHAPTER 53

1,4,5–IP3 (pmol/mg protein)

150

100 CCK 10 nM CCK 30 pM Basal

50

0

0

15

30

60

300

900

Time (sec)

FIG. 53-1. Time course of cholecystokinin (CCK)-stimulated inositol 1,4,5-trisphosphate (1,4,5-IP3) formation in rat pancreatic acini. 1,4,5-IP3 mass was measured using a competitive binding assay. Stimulation with a maximal concentration of CCK (10 nM) resulted in a rapid increase in 1,4,5-IP3. The level of 1,4,5-IP3 peaked in 5 seconds and subsequently decreased to a lower, but still increased, level. (Reproduced from Matozaki and Williams [40], by permission.)

The peak occurs within 5 seconds and subsequently decays to smaller, but still increased, levels over time. Maximal concentrations of CCK, bombesin, and CCh have all been reported to increase IP3 (40–42). It has, however, been difficult to detect changes in PI metabolism at physiologic concentrations of agonist, although Ca2+ changes and enzyme secretion can be readily demonstrated (40). Although this is a phenomenon reported in other cell types, it is formally possible that Ca2+ changes stimulated by physiologic concentrations of agonists in pancreatic acinar cells results from the formation of alternative second messengers. Nevertheless, the observation that these same Ca2+ changes stimulated by low concentrations of agonist are inhibited either by antagonizing Gαq, inhibiting PLC, or blocking the IP3 receptor (IP3R) (15,43–45) suggests that this failure to detect IP3 is probably a result of lack of sensitivity of current assay procedures performed in suspensions of cells. A further assay that shows promise for assaying IP3 in single pancreatic acinar cells and perhaps at physiologically relevant concentrations of agonist is through the imaging of the translocation of the pleckstrin homology (PH) domain of PLCδ fused to green fluorescent protein variants (46,47). This PH domain has a high affinity for PIP2 and IP3, and under basal conditions, is primarily associated with the plasma membrane through binding to PIP2. On agonist stimulation, the decrease in PIP2, together with its relatively high affinity for IP3, facilitates translocation of the tagged PH domain to the cytoplasm. In mouse pancreatic acinar cells expressing this construct, it has been shown that stimulation with ACh results in translocation of the probe to the cytoplasm, in a manner consistent with IP3 production. In contrast, photolysis

of caged Ca2+ did not result in translocation (48). Although some early work showed that many forms of PLC are sensitive to increasing Ca2+, the failure of Ca2+ released by photolysis of caged Ca2+ to increase IP3 is consistent with the majority of previous reports showing that PI hydrolysis in pancreatic acinar cells is insensitive to maneuvers that increase cytosolic Ca2+ (40,49,50). The early HPLC data are consistent with a major pathway for IP3 metabolism, which in IP3 is primarily metabolized via IP3 3-kinases, resulting in the phosphorylation of IP3 to (1,3,4,5-inositol tetrakisphosphate) (51). This kinase gene family consists of three members designated ITPK (inositol trisphosphate kinase) A, B, and C (52). Reports suggest that the B and particularly C isoforms are abundantly expressed in pancreas (53,54). Interestingly, although the A and B isoforms of this enzyme are potently activated by Ca2+/calmodulin, the activity of the C isoform is unaffected by increasing Ca2+ level (54). The impact of coexpression of multiple isoforms of this enzyme on inositol phosphate metabolism and Ca2+ signaling in pancreas remains to be assessed. 1,3,4,5-IP4 can be subsequently dephosphorylated by a 5 monophosphatase to yield inositol-1,3,4-trisphosphate. The routes of metabolism of inositol phosphates are ever expanding (see reviews by Abel and colleagues [55] and Shears [56]), and pathways are present that can further phosphorylate inositol-1,3,4-trisphosphate to yield higher phosphates such as 1,3,4,6-IP4, IP5, and IP6, or dephosphorylate inositol-1,3,4-trisphosphate to yield 1-IP or 3-IP. A second alternative pathway for metabolism of IP3 uses a number of phosphatase enzymes to sequentially dephosphorylate IP3 to 1,4-IP2 and 4-IP (55,56). The route of metabolism that

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1341 predominates may depend on the strength of stimulus because the ITPK enzymes appear to have a greater Km but lower Vmax than the phosphatases. Although no physiologic role for the various inositol phosphates produced after IP3 formation has been demonstrated conclusively, 1,3,4,5-IP4 is formed rapidly in pancreatic acini (39). Based on the energy requirement for its synthesis and the characteristic short half-life of 1,3,4,5-IP4, it has been suggested that this molecule also functions as a second messenger. This idea is strengthened by the discovery of specific binding proteins for 1,3,4,5-IP4 such as GAP1IP4BP, a Ras GAP that specifically binds IP4 through its PH domain (57). It has been suggested that IP4, in part functioning through GAP1IP4BP, may play a role in gating Ca2+ entry or potentiation of the Ca2+ releasing effects of IP3 (58). However, no definitive evidence for such a role has been reported in pancreatic acinar cells.

Intracellular Ca2+ Temporal Properties of Ca2+ Signals The use of Ca2+-sensitive fluorescent dyes and reporter constructs, together with sophisticated digital imaging techniques at the single-cell and subcellular levels, has resulted in the rapid expansion of our understanding of the temporal and spatial complexity of intracellular Ca2+ signals in pancreatic acinar cells. These techniques have largely superseded other methods. Therefore, for the purposes of this chapter, early evidence indicating that PI-linked pancreatic secretagogues elicited changes in Ca2+ obtained using 45Ca2+ flux measurement, Ca2+-selective microelectrodes, Ca2+-sensitive photoproteins, and fluorescent Ca2+ measurements in bulk suspension is not discussed in detail. Response to Maximal Agonist Concentrations Initial experiments monitoring [Ca2+]i by microfluorometry of single cells using the dye Fura-2 indicated that maximal concentrations of the secretagogues CCK, ACh, or bombesin evoked a similar Ca2+ signal. On addition of either agonist, [Ca2+]i increased rapidly 5- to 12-fold from a basal value of ~75 to 150 nM Ca2+ to reach a peak of approximately 1 µM within seconds. This peak then declined over 2 to 5 minutes to reach a new plateau level around 100 nM greater than basal value, which is maintained as long as the agonist is present (42,59–61) (Fig. 53-2A). The initial peak was shown to be the result of Ca2+ release from intracellular stores, because the early transient response was essentially unaffected by removal of extracellular Ca2+, whereas the later plateau phase was absent (60–63). Removal of extracellular Ca2+ during the plateau phase resulted in the rapid attenuation of the signal, thus indicating an absolute dependence on extracellular Ca2+ for this maintained phase and implying the presence of a mechanism for Ca2+ influx from the extracellular milieu (see Fig. 53-2B). This influx is not blocked

by antagonists of voltage-gated Ca2+ channels, but is attenuated by lanthanides (62,63). Ca2+ influx is also sensitive to changes in extracellular pH, being enhanced by alkaline and inhibited in acidic conditions (62,63). Functionally, Ca2+ influx can be initiated by substantial depletion of intracellular Ca2+ pools, the so called store-operated or capacitative Ca2+ entry (SOCE) pathway (64). This pathway can be readily demonstrated by inhibition of endoplasmic reticulum (ER) Ca2+ pumps with the plant sesquiterpene lactone thapsigargin, which results in Ca2+ influx independent of receptor activation and PI hydrolysis (63–66). Because marked depletion of ER Ca2+ occurs only on stimulation with maximal concentrations of secretagogues (67), it is not clear whether this pathway is relevant except under pathologic conditions. Indeed, it has been suggested that activation of this pathway is responsible for the inappropriate intracellular activation of trypsin that occurs in models of acute pancreatitis (68,69). The molecular identity of the SOCE channel, together with the physiologic activation of the pathway, remains to be elucidated. Many mechanisms have been proposed to provide the link between store depletion and Ca2+ entry. These include the production of a soluble messenger after depletion of the ER, conformational coupling between the IP3R and the Ca2+ influx channel, and a secretion-type model whereby channels are inserted in the plasma membrane after store depletion (70,71). In support of the later idea, it has been reported that stabilization of the actin cytoskeleton terminal web, which presumably would interfere with trafficking of channels to the plasma membrane, inhibits Ca2+ influx (72). In addition, toxins that specifically cleave synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein isoform 2 (VAMP-2), proteins involved in exocytosis, attenuate Ca2+ influx in pancreatic acinar cells (73). Regarding the identity of the SOCE channel, it has been suggested that a member of the transient receptor potential (TRP) family may be a good candidate (74). Although it is possible that a TRP protein is a constituent of this pathway, no definitive evidence currently is available in support of this contention in pancreatic acinar cells. Response to Physiologic Agonist Concentrations In contrast with the situation with maximal concentrations of agonists, single-cell microfluorometry showed that lower physiologic concentrations of agonist resulted in the generation of Ca2+ oscillations (59–61,75,76). These oscillations are characterized by repetitive, regular cycles of increased and subsequently decreasing Ca2+ levels. In single acinar cells or small acini, physiologic concentrations of secretagogues (1–50 pM CCK; 50–300 nM ACh) induce after a latency of 30 seconds to 2 minutes fairly regular Ca2+ oscillations at a frequency of between 1 and 6 cycles per minute (Fig. 53-3). The maximal global [Ca2+]i reached during the release phase is generally between 200 nM and 1 µM. At least in mouse pancreatic acinar cells, the global temporal profile stimulated by ACh differs from that

1342 / CHAPTER 53 10 µM CCh

600

[Ca2+]i (nM)

400

200 100 sec

A Ca2+ −free 10 µM CCh

2.5 mM Ca2+

0.2 ∆ ratio units

B

100 s

FIG. 53-2. Intracellular [Ca2+] ([Ca2+]i) changes evoked by maximal concentrations of secretagogues in single cells. Stimulation with maximal concentrations of secretagogues results in a characteristic “peak and plateau”–type Ca2+ signal. (A) In a single fura-2–loaded rat pancreatic acinar cell, stimulation with 10 µM CCh resulted in a sharp increase in [Ca2+]i, which subsequently declined to a new plateau level that was maintained throughout the period of secretagogue application. (B) In the absence of extracellular Ca2+, the initial peak can be initiated, but is only transient, indicating that the initial phase of the response is a result of Ca2+ release from intracellular stores. Readmission of extracellular Ca2+ restores the “plateau” phase of the response, indicating that Ca2+ influx is required to maintain this portion of the response. CCh, carbamylcholine.

250 nM CCh

100 nM

100 nM

5 pM CCK

50 s

A

50 s

B FIG. 53-3. Physiologic concentrations of agonists evoke Ca2+ oscillations. In single mouse pancreatic acinar cells, low concentrations of secretagogue (50–400 nM acetylcholine [ACh] or 1–50 pM cholecystokinin [CCK]) evoked repetitive Ca2+ transients termed Ca2+ oscillations. In small clusters of mouse acinar cells, muscarinic receptor and CCK stimulation resulted in distinct global temporal patterns of Ca2+ signal. (A) Carbamylcholine (CCh) stimulation results in sinusoidal oscillations superimposed on an increased baseline, whereas in B, CCK stimulation results in much less frequent, broader transients that originated and returned to basal intracellular [Ca2+] levels between transients. (Reproduced from Straub and colleagues [79], by permission.)

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1343 stimulated by CCK and bombesin (77–80) (see Fig. 53-3). Oscillations stimulated by peptide secretagogues tend to be characterized by slow, relatively long-lived transients originating and returning to basal levels between Ca2+ spikes, whereas ACh-induced oscillations are characterized by faster short-lasting transients originating from an elevated plateau. Oscillations can be mimicked by agents that activate G proteins, such as GTPγS, sodium fluoride, or mastoparan, and by introduction of IP3 into the cell, for example, via dialysis from a whole-cell patch-clamp pipette (78,81,82). These data indicate strongly the involvement of the PI signaling cascade in the generation of Ca2+ oscillations. Oscillations are primarily the result of cycles of intracellular Ca2+ release and ATP-dependent reuptake, because the oscillations can be initiated in the absence of extracellular Ca2+ and are inhibited by agents that deplete ATP or inhibit the Ca2+ pump on the ER (61,75,83). Although Ca2+ release during each cycle only minimally depletes the intracellular Ca2+ store (67) and reuptake is efficient (84), extracellular Ca2+ is required to sustain the oscillations and ultimately to maintain the level of Ca2+ in the intracellular store (61,77). Currently, it is unclear whether Ca2+ influx stimulated at physiologic concentrations is mediated through a similar mechanism to that observed with maximal concentrations of agonists. An electrophysiologic study of mouse pancreatic acinar cells failed to detect SOCE currents after stimulation with physiologically relevant concentrations of agonists. In contrast, under these conditions, it was reported that a channel activated by arachidonic acid was predominately responsible for Ca2+ influx (85). Oscillating levels of IP3 are not necessary per se for oscillatory behavior, because nonmetabolizable IP3 is capable of initiating Ca2+ oscillations in mouse pancreatic acinar cells (81). These data indicate that the mechanism underlying Ca2+ oscillations is most likely the result of an inherent property of the Ca2+ release mechanism. Nevertheless, these data do not preclude the possibility that on agonist stimulation the [IP3] itself fluctuates and contributes to the kinetics of Ca2+ release. One mechanism whereby oscillating IP3 could occur is through periodic activation cycles of RGS proteins. RGS proteins stimulate the GTPase activity of Gα-subunits, thereby terminating the stimulus for activation of effectors such as PLCβ. In rat pancreatic acinar cells, infusion of RGS proteins via the patch-pipette results in dampening of Ca2+ signals (21,44,86,87). Interestingly, the common catalytic core of RGS proteins, the so-called RGS box, is much less effective than infusion of full-length RGS proteins (86). In addition, specific RGS proteins appear to affect the Ca2+ signals generated by different secretagogues differentially (21). These observations may indicate that individual RGS proteins are associated in a signaling complex with specific secretagogue receptors and other signaling proteins. This interaction may impact the kinetics of IP3 production and contribute to the agonist-specific characteristics of secretagogue-stimulated Ca2+ signals in pancreatic acinar cells (45).

Spatial Properties of Ca2+ Signals Single-cell microfluorometry has provided a wealth of data regarding the general temporal properties of Ca2+ signals in pancreatic acinar cells. However, by the nature of the measurements, the experiments represent the global [Ca2+]i as a mean value integrated from throughout the cell. Digital imaging techniques, however, allow the monitoring of [Ca2+]i at the subcellular level and in multiple cells of a coupled acinus. Probes are available with a choice of spectral characteristics and affinities for Ca2+ and with the ability to be targeted to various cellular compartments. The combination of the flexibility of the probes plus the imaginative use of digital imaging techniques has showed that the Ca2+ signal displays remarkable spatial intricacy that appears to be fundamental for the appropriate activation of downstream effectors. An early study by Kasai and Augustine (88) using digital imaging of small acinar clusters demonstrated a profound spatial heterogeneity in the Ca2+ signal after stimulation with a maximal concentration of ACh (Fig. 53-4A). Stimulation resulted in the initiation of Ca2+ release in the apical region of the cell immediately below the luminal plasma membrane and the subsequent spread of the signal as a wave toward the basal aspects of the cell (88). This Ca2+ wave has generally been reported to travel across the cell at a speed of between 5 and 45 µm/sec, which is consistent with the speed observed in other cell types (88–93). These data were somewhat counterintuitive because contemporary studies had demonstrated that secretagogues receptors were expressed on the basolateral face of the cell (94), and it was known that the ER, the presumed site of Ca2+ release, was present throughout the cell. A study using line-scanning confocal microscopy later confirmed that a similar pattern of apical to basal Ca2+ wave was initiated by high concentrations of CCK (89). Using similar techniques at physiologic concentrations of agonists, investigators have also shown Ca2+ signals to initiate in the apical region of acinar cells (95,96). This initial site of Ca2+ release has been termed the trigger zone (95). Ca2+ release invariably occurs at this specialized site even under conditions where stimulation of agonist is restricted to the basal region by focal application of agonist. This has been most elegantly demonstrated by focal flash photolysis of caged carbachol contained in a whole-cell patch-clamp pipette isolated in the base of the cell (97). Notably, however, the apical portion of the acinus, immediately proximal to the tight junctions, may express a relatively high number of secretagogue receptors because this area has been reported to be most sensitive to focal agonist stimulation (98,99). At threshold concentrations of ACh, repetitive, short-lasting Ca2+ transients are initiated that strikingly are contained to the apical third of the cell and do not propagate to the basal region (95,96) (see Fig. 53-4B). These spikes, although short lived, have been shown using low-affinity Ca2+ indicators to be of large amplitude in the order of 1 to 4 µM Ca2+ (100). At intermediate concentrations of ACh, apically initiated global Ca2+ transients dominate, often superimposed on a slight global

1344 / CHAPTER 53 FIG. 53-4. Spatial characteristics of Ca2+ signals in pancreatic acini. Digital imaging of Ca2+ indicators shows spatial homogeneity in agonist-stimulated Ca2+ signals. (A) Stimulation of a triplet of mouse pancreatic acinar cells (a) with a maximal concentration of acetylcholine (ACh) results in the initiation of the Ca2+ signal in the extreme apical portion of the acinar cells (b). The signal subsequently spread toward the basal aspects of each cell. The pseudo-color scale indicates the levels of intracellular [Ca2+] ([Ca2+]i). (B) A single pancreatic acinar cell is stimulated with a threshold concentration of ACh. Ca2+ signals are again initiated in the apical portion of the cell, but remain in the apical third of the cell without spreading to the basal aspects of the cell (Ab–Ah). The kinetic recorded from an apical region of interest (yellow trace in B) and from the basal region (blue trace) demonstrates that [Ca2+]i increases are observed only in the apical pole of the acinar cell under these conditions. (C) Changes in Ca2+ after photolytic liberation of inositol 1,4,5-trisphosphate (IP3) from a caged precursor induced into a single mouse acinar cell via a whole-cell patch-clamp pipette. After global increase of IP3, Ca2+ changes initially occurred at the apical pole of the acinar cell and spread to the basal pole in a similar fashion to secretagogue stimulation. The kinetic shows the Ca2+ changes in the apical (blue trace) versus basal pole (red trace) of the cell, together with the activation of a chloride conductance as measured by whole-cell patch clamp. (See Color Plate 26.) (A: Reproduced from Kasai and Augustine [88], by permission; B: Reproduced from Kasai and colleagues [95], by permission; C: Reproduced from Giovannucci and colleagues [111], by permission.)

A

B

500 msec

10 % ∆ F/F0

100 pA 2 sec

C

flash

increase of [Ca2+]i. The frequency of these transients corresponds to the frequency of oscillations noted in microfluorometry studies. Low concentrations of CCK predominately result in apically initiated global Ca2+ signals that are of longer duration (77,78). In studies of whole-cell patchclamped acinar cells, where the Ca2+ buffering of the cell is set by dialysis from the patch pipette, the broad CCKinduced transients have been reported to be preceded by

short-lasting, apically localized transients (101). Studies indicate that the precise site of initiation of each transient by specific agonists is similar, but possibly not identical (102). The site of initiation of each Ca2+ transient in the trigger zone is nevertheless tightly coupled functionally to both the exocytosis of zymogen granules and the activation of Cl− channels required for the process of fluid secretion from the pancreatic acinar cells (100,103).

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1345 Cell–Cell Communication Individual cells in the pancreatic acinus are extensively coupled by the expression of gap junctional proteins (104). These channels effectively allow the passage of small molecules up to a molecular mass of 1 to 2 kDa between cells, and furthermore provide electrical coupling of large numbers of cells in the acinus. Although stimulation of small acini with maximal concentrations of agonist results in the Ca2+-dependent closure of these junctions (105,106), at physiologic levels of CCK and bombesin where global Ca2+ transients predominate, the junctions remain open and Ca2+ signals appear to spread as waves between cells (106,107). In contrast, brief, apically confined transients initiated by threshold concentrations of ACh have been reported not to propagate between coupled cells (106). Each cycle of CCKinduced intercellular Ca2+ signaling has been reported to be initiated by a “pacemaker cell” (106). This pacemaker presumably represents the individual cell within the acinus that is most sensitive to agonist. The propagation of a Ca2+ wave between adjacent cells obviously requires relatively long-range messengers. It appears that IP3 and small amounts of Ca2+ are capable of diffusing between coupled cells to act in concert in this manner providing a signal to synchronize the intercellular Ca2+ wave. The primary evidence for this contention is that a Ca2+ signal can be observed in neighboring cells when IP3 is injected into an unstimulated individual cell. In addition, whereas Ca2+ injected into a resting cell fails to measurably increase [Ca2+]i in adjacent cells, microinjection of Ca2+ into cells previously stimulated with threshold concentrations of CCK leads to a measurable increase in [Ca2+]i in neighboring cells (106). These data are consistent with Ca2+ acting to facilitate further Ca2+ release from intracellular stores, which is described in detail in the remainder of this chapter. The physiologic function of propagating Ca2+ waves in pancreatic acinar cells is not currently firmly established. A reasonable proposal, however, is that gap-junctional communication represents a mechanism to increase the responsiveness of an acinus to threshold concentrations of agonist. In this scenario, the acinus is rendered as sensitive to secretagogue stimulation as the pacemaker cell. In support of this idea, isolated single cells are much less sensitive to secretagogue stimulation than isolated acini (108), and in addition, experimental maneuvers that increase gap-junctional permeability lead to increased secretagogue-induced amylase secretion (107).

Cellular Mechanisms Underlying the Spatiotemporal Ca2+ Signaling Inositol 1,4,5-Trisphosphate–Induced Ca2+ Release Initial analysis of secretagogue-elicited changes in the content of intracellular organelles demonstrated that Ca2+ was lost from the ER without significant changes in other subcellular compartments such as mitochondria or zymogen granules (109). Subsequently, IP3-induced Ca2+ release was

shown to occur from rough ER vesicles, but not from mitochondria or vesicles prepared from plasma membrane (110). More recent data show that the initial highly localized Ca2+ release can be mimicked by global, uniform application of IP3 either through the patch pipette or via flash photolysis from a caged precursor (91,96,111,112) (see Fig. 53-4C). To rationalize these early data with the current spatial information regarding the initiation of Ca2+ release, we suggest that the trigger zone must represent a specialized region of ER that is highly sensitive to IP3. Studies have shown that this exquisite sensitivity to IP3 is in all probability a result of the abundant expression of IP3Rs in the extreme apical region of pancreatic acini (83,113,114). IP3R were first isolated and cloned from cerebellum, and they subsequently have been shown to represent a family of three proteins named the type 1 IP3R (IP3R-1), type 2 IP3R (IP3R-2), and type 3 IP3R (IP3R-3), which are all related to the ryanodine receptor (RyR) Ca2+ release channel (115–117). Initially, it was reported that IP3R-3 was expressed in the apical pole (114). Later studies, however, showed that all three subtypes had essentially identical expression; all IP3Rs were excluded from areas containing zymogen granules and were apparent immediately below the apical and lateral plasma membrane (83,113) (Fig. 53-5). This localization is essentially identical to the “terminal web” of actin-based cytoskeleton in this region. By this technique no other significant localization of IP3R was noted except for moderate expression on perinuclear structures (83,113). The later distribution is consistent with a report of IP3-induced Ca2+ release from isolated nuclei prepared from mouse pancreatic acinar cells (118). A study where IP3 was released from a caged precursor in various localized regions of mouse acinar cells has also functionally confirmed that the apical region of the cell is more sensitive to IP3 than the basal area of the cell (112). These data were later confirmed by a study imaging permeabilized pancreatic acini (119). Quantitative Western analysis and polymerase chain reaction (PCR) have indicated that there is approximately equal expression of IP3R-3 and IP3R-2 in pancreatic acinar cells making up ~90% of the total complement of IP3R (120,121). The functional IP3R is formed cotranslationally by the tetrameric association of four individual receptor subunits (122,123). In pancreatic acinar cells, there is evidence that the channel can form a heterotetramer because multiple types of IP3R can be detected in immunoprecipitates of specific individual receptor types (124). Each subunit has a binding site for IP3 toward the N terminus, which is formed by a cluster of positively charged amino acids thought to coordinate the negatively charged phosphate groups of IP3 (125). The Kd for binding of IP3 to pancreatic membranes has been reported to be between 1 and 7 nM, a value similar to that reported for other peripheral tissues such as liver (111,126). The C terminus of each subunit is postulated to span intracellular membranes six times, and a single cationselective pore is formed from this region of the protein in the tetrameric receptor. When purified or expressed in a

1346 / CHAPTER 53

A

C

B

D FIG. 53-5. Localization of inositol 1,4,5-trisphosphate receptor (IP3R) in pancreatic slices. The localization of IP3R-1 (A), IP3R-2 (B), and IP3R-3 (C) was determined with specific antibodies to individual IP3R types and visualization by confocal microscopy. IP3R of all types predominately localized to the extreme apical pole of acinar cells, immediately below the luminal plasma membrane (arrows in A–D; compare localization of zymogen granules visualized by staining for amylase in D). IP3R-1 and IP3R-3 also localized to perinuclear structures (arrowheads in A–C). (Reproduced from Yule and colleagues [113], by permission.)

heterologous system and then reconstituted in planar lipid bilayers, the protein can be demonstrated to function as an IP3-gated cation channel with many of the characteristics of the release channel (127). That the outer nuclear membrane is continuous with the ER has been exploited in patch-clamp experiments to study IP3R channel activity in isolated nuclei from Xenopus leavis oocytes and Cos cells (128,129). These experiments have provided insight into the activity and regulation of the channel in a native membrane. Whereas the IP3 binding pocket and channel pore are highly conserved between IP3R family members, the intervening sequence between the binding region and pore is more divergent and consists of the so-called regulatory and coupling or modulatory domain. This region consisting of ~1600 amino acids is thought to be important in modulating the Ca2+ release properties of the IP3R. Indeed, Ca2+ release through the IP3R is markedly influenced by many factors, most importantly by Ca2+ itself (130,131). The majority of studies have indicated that all forms of the IP3R are biphasically regulated by Ca2+. [Ca2+] in the range of 0.5 to 1 µM increases the steady-state open probability of the channel, whereas at higher concentrations, the activity decreases (130, 131). This property of the IP3R is thought to be fundamentally

important in the generation of the different spatial and temporal pattern of Ca2+ signals observed in cells (132). In pancreatic acinar cells, IP3-induced Ca2+ release has been shown to be inhibited when Ca2+ is increased and enhanced when Ca2+ is buffered with chelators (133,134). The [Ca2+]i, together with the range of action of Ca2+, can be manipulated by dialyzing cells with buffers exhibiting differing on-rates for Ca2+ binding. In pancreatic acinar cells, restriction of the range of action of Ca2+ using the slow on-rate buffer EGTA resulted in spatially restricted IP3-induced spikes and the attenuation of global waves consistent with EGTA, inhibiting the positive effect of Ca2+ to facilitate Ca2+ release between spatially separated release sites. In contrast, the fast on-rate buffer BAPTA resulted in larger monotonic Ca2+ release, which was interpreted to reflect the loss of local Ca2+ inhibition of IP3R (135). IP3R activity is also influenced through interaction with numerous factors such as proteins, adenine nucleotides, and phosphorylation, in particular by cyclic nucleotide–dependent kinases (see reviews by Patel and colleagues [122] and Patterson and colleagues [136]). The IP3R-1 represents one of the major substrates for phosphorylation by protein kinase A (PKA) in brain, and thus represents a potentially important locus for cross talk between the cAMP and Ca2+

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1347 signaling systems (137). In pancreatic acinar cells, PKA activation results in phosphorylation of IP3R-3 (79,80). Functionally, phosphorylation of IP3R in pancreatic acinar cells correlates with IP3-induced Ca2+ release, which is decreased for the magnitude and kinetics of Ca2+ release (79,138). The IP3R-3 and the regulatory subunit of PKA can be coimmunoprecipitated, and this colocalization appears to be important in functionally targeting PKA to the IP3R, because a peptide that disrupts binding of PKA to scaffolding proteins negates the effect of activating PKA on Ca2+ release (79,138). Physiologically relevant concentrations of CCK, but not ACh, also result in PKA-dependent phosphorylation of IP3R (79). This observation is consistent with earlier reports that CCK stimulation leads to an increase in cAMP and PKA activation. CCK-induced phosphorylation of IP3R may contribute to the specific characteristics of CCK-induced Ca2+ signals because maneuvers that interfere with PKA activation disrupt the pattern of CCK-induced, but not ACh-mediated, Ca2+ signaling. Conversely, increasing cAMP converts ACh-induced Ca2+ signaling characteristics into signals that resemble CCK stimulation (138,139). CCK-stimulated signaling is not affected by increasing cAMP or by stimulation with VIP, presumably because PKA activation and phosphorylation of IP3R has already occurred (75,138). CCK and bombesin stimulation result in Ca2+ signals with similar characteristics, and this may be related to that bombesin stimulation also results in phosphorylation of IP3R in mouse pancreatic acinar cells (42,79). In vitro, IP3R can be phosphorylated by PKC, Ca2+/calmodulindependent kinase II, and tyrosine kinases of the src family (122,136). Although no direct evidence has been reported regarding phosphorylation of IP3R by these pathways in pancreatic acinar cells, activation of PKC has been shown to inhibit Ca2+ release in permeabilized pancreatic acinar cells and to attenuate Ca2+ oscillations stimulated by secretagogues or by direct G-protein activation, without an effect on PI hydrolysis (82,140). Thus, the possibility exists that the IP3R is a substrate for other kinases in pancreatic acinar cells. Infusion of G-protein βγ-subunits into pancreatic acinar cells also has been shown to induce Ca2+ release. An initial report attributed this to the activation of PLCβ2 by βγ and the subsequent production of IP3 (141). A later report, however, demonstrated that the Gβγ-induced Ca2+ release was independent of IP3 production and the result of a direct interaction of Gβγ with IP3R (29). This association was confirmed by coimmunoprecipitation and was shown to increase the open probability of IP3R in a manner independent of IP3. This interaction of IP3R and βγ may be important for the action of Gαi-linked agonists in pancreatic acinar cells. Ryanodine Receptor–Induced Ca2+ Release Evidence exists for Ca2+ release initiated through activation of RyRs in pancreatic acinar cells. This family of channels, best studied as the Ca2+ release channel in skeletal and cardiac muscle, is classified as belonging to the same gene superfamily as IP3Rs. Indeed, RyRs are modulated by similar

regulators and share some sequence homology with the IP3Rs, especially in the putative Ca2+-conducting pore region of the C terminus. However, whereas IP3Rs have an absolute requirement for IP3 with Ca2+ as an important coagonist for gating, RyRs only require Ca2+ to open through a process termed calcium-induced calcium release (CICR). The functional expression of RyR in pancreatic acinar cells has been indicated by a number of studies; for example, microinjection of Ca2+ in the presence of the IP3R antagonist heparin results in Ca2+ release in mouse pancreatic acinar cells (95). In addition, treatment of pancreatic acinar cells with high concentrations of ryanodine known to block RyRs dampens secretagogue-induced Ca2+ signals (89,91), and one report has shown that low concentrations of ryanodine, which permanently opens the RyR in a subconductance state, results in Ca2+ release (142). In muscle cells, caffeine activates RyRs and results in emptying of sarcoplasmic reticulum Ca2+ stores. In contrast, the majority of reports from pancreatic acinar cells indicate that caffeine does not elicit Ca2+ release and actually inhibits secretagogue-induced Ca2+ signaling through an action to inhibit PLC and IP3R-mediated Ca2+ release (142,143). This later effect of caffeine, together with presumably much lower numbers of RyRs in pancreatic acinar cells, probably explains the absence of caffeine-induced Ca2+ release. The physical presence of RyRs has, however, been difficult to demonstrate in pancreatic acinar cells, with conflicting positive and negative reports of expression. For example, in one study, RyRs could be detected using Western analysis in salivary gland acinar cells, but not in pancreatic acinar cells (83). In contrast, an initial study reported the expression using PCR analysis of RyR type 2 (RyR2), but not RyR type 1 or 3 (RyR1 and RyR3, respectively), in mRNA extracted from rat pancreatic acini (144). However, a later report using single-cell PCR and Western analysis demonstrated that all three types of RyRs were expressed in pancreatic acini (145). In all probability, this inconsistency is related to the relatively low expression of RyRs in pancreas compared with muscle cells. Although IP3Rs have a well-defined localization in pancreatic acinar cells, several studies have reported that RyRs have a more diffuse distribution. Immunohistochemistry and studies with fluorescently labeled ryanodine have indicated that RyRs are distributed throughout acinar cells with perhaps the greatest concentration in the basal aspect of the cell (91,144). As a result of this localization to areas of the cell with low levels of IP3R, it has been suggested that activation of RyRs plays an important role in the propagation of Ca2+ signals from the initial release of Ca2+ in the trigger zone to the basal aspects of the cell. In support of this contention, high concentrations of ryanodine have been shown to slow or spatially limit the spread of Ca2+ waves in mouse pancreatic acinar cells (89,91) (Fig. 53-6). In a number of cell types, there is evidence that in addition to activation by Ca2+, the activity of RyRs is modulated by cyclic adenosine diphosphate ribose (cADPr). cADPr was first suggested to be a Ca2+-releasing second messenger based

1348 / CHAPTER 53 I

II 0.2% ∆F/F0 15 min

I

3s II

∆F/F0

A

100 µM ryanodine 10 µM CCh I

II

III

IV

V 0.2% ∆F/F0

3 min

6 min

I

6 min

3 min

3s

IV

B FIG. 53-6. Contribution of ryanodine receptor (RyR) to global Ca2+ signals in pancreatic acinar cells. Global Ca2+ signals were initiated by photolysis of caged inositol 1,4,5-trisphosphate (IP3). (A) Images and kinetic traces show that global Ca2+ signals can be initiated multiple times after exposure to IP3. However, as shown in B, exposure to a high concentration of ryanodine, known to inhibit RyR, leads to a slowing in the progression of the Ca2+ wave and to the restriction of the signal to the apical pole of the cell after stimulation with IP3 (compare absence of ryanodine in stimulation I with that obtained after exposure to ryanodine for 15 minutes in stimulation IV). CCh, carbamylcholine. (See Color Plate 27.) (Reproduced from Straub and colleagues [91], by permission.)

on experiments performed in sea urchin eggs, where it was shown to release Ca2+ and function as a messenger during fertilization (146). Subsequently, it has been shown to release Ca2+ and satisfy some of the criteria for a second messenger in mammalian systems including lymphocytes, pancreatic β cells, and cardiac myocytes (147–149). Ca2+ release after cADPr exposure is inhibited by blocking concentrations of ryanodine and appears to reduce the threshold for CICR through RyRs (150). However, opinion is divided whether it functions through a direct effect on the RyRs, or indirectly through interaction with an accessory protein such as FKBP 12.6 or calmodulin (151,152). In pancreatic acinar cells, the intracellular application of cADPr results in Ca2+ release. This observation has been reported in both rat and mouse acini with either whole-cell pipette dialysis of cADPr, liberation of cADPr from a caged precursor by two-photon flash photolysis, or direct application

to permeabilized acini (119,153–156). In addition cADPr has been reported to release Ca2+ from a rat pancreatic microsomal preparation (157). The spatial localization of cADPrinduced Ca2+ release remains to be resolved conclusively. In an initial report, cADPr introduced by dialysis from a patchclamp pipette into mouse pancreatic acinar cells resulted in Ca2+ release from the apical pole of the cell (153). This Ca2+ release was blocked by ryanodine and, interestingly, by the IP3R antagonist heparin. These data were interpreted as indicating that Ca2+ release mediated by cADPr was dependent on both IP3Rs and RyRs, presumably because highly localized Ca2+ release initially through RyRs sensitized neighboring IP3Rs to basal levels of IP3 (153). Consistent with this view, it was shown that low concentrations of ryanodine also resulted in Ca2+ release from the apical pole in a manner apparently dependent on functional IP3Rs (142). In contrast, in a study more consistent with the localization of the majority

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1349 of RyRs, selective local uncaging of cADPr in different regions of an acinar cell by two-photon photolysis reported that the basal aspect of the cell was more sensitive to cADPr (154). These data are consistent with a report that, in permeabilized acini, the apical pole exhibited a higher affinity for IP3, but cADPr released Ca2+ exclusively from the basal aspects of the cell (119). Important questions to address that are required to establish cADPr as a bona fide Ca2+-releasing second messenger in pancreatic acinar cells are to demonstrate that the molecule is produced after secretagogue stimulation, and that it is necessary for Ca2+ release. To these ends it has been reported that CCK and ACh, but not bombesin, stimulate the activity of cytosolic ADP-ribosyl cyclase activity, resulting in the production of cADPr (158). One such enzyme that possesses this activity is CD38. Ca2+-signaling events in acinar cells prepared from CD38 null mice are dampened and appear reminiscent of RyR blockade (159). In addition, 8-NH2cADPr, a structural analog of cADPr that antagonizes the effect of cADPr, has been reported to block CCK and bombesin, but not ACh or IP3-induced Ca2+ signals, in mouse pancreatic acinar cells (160,161). This later result, although not internally consistent with the ability of ACh to induce the formation of cADPr, has been suggested to indicate that CCK stimulation preferentially couples to the generation of cADPr, and thus may account for some of the distinct characteristics of reported CCK-induced Ca2+ signals. Nicotinic Acid Dinucleotide Phosphate–Induced Ca2+ Release An additional putative messenger that potently induces Ca2+ release in pancreatic acinar cells is nicotinic acid adenine dinucleotide phosphate (NAADP). Once again, the activity of this agent was first reported to play a role in invertebrate fertilization. In contrast with IP3 and cADPr, the receptor for NAADP has yet to be identified. Although it has been reported that NAADP-induced Ca2+ release from nuclei isolated from mouse pancreatic acini is dependent on RyR, (118) the majority of evidence suggests that the receptor is likely to represent a novel Ca2+ release channel, because although the activity of IP3Rs and RyRs exhibits a bell-shaped dependence on Ca2+, the putative NAADP receptor does not support CICR (162). Although this property of NAADP is not well suited to play a role in the propagation of Ca2+ waves, it has been suggested that NAADP is required to initiate Ca2+ signals, and this initial Ca2+ increase subsequently recruits IP3Rs and RyRs. This idea is supported by the observation that NAADP introduced via the patch pipette into mouse pancreatic acinar cells results in Ca2+ release in the apical pole but that this release is absolutely dependent on both IP3Rs and RyRs (155). These data suggest that Ca2+ release through NAADP is quantitatively small, but ideally localized to sensitize IP3Rs and RyRs. Although IP3Rs and RyRs reside in the ER, it has been suggested that NAADP primarily acts on a distinct store, probably an acidic lysosome– related organelle (163). This idea is primarily based on the

observation that NAADP-induced Ca2+ signaling, but not cADPr or IP3R-induced Ca2+ release, is inhibited by experimental maneuvers that either inhibit vacuolar type H+-ATPase or result in osmotic disruption of lysosomes (163). The concentration–response relation for NAADP-induced Ca2+ release also displays unique properties. NAADP-induced Ca2+ release is biphasic; nanomolar levels induce Ca2+ release, whereas micromolar concentrations fail to release Ca2+, but do render the mechanism refractory to subsequent stimulation (164). Provocatively, exposure of mouse pancreatic acinar cells to inactivating concentrations of NAADP also renders cells refractory to stimulation with threshold concentrations of CCK, but not ACh or bombesin, and disruption of the NAADP releasable store selectively disrupts CCK-induced Ca2+ signals (164,165). These data suggest that, under these conditions, CCK and NAADP use a common mechanism to induce Ca2+ release. Importantly, physiologically relevant concentrations of CCK can also be shown to result in the production of NAADP, whereas ACh stimulation does not result in measurable accumulation (166). Thus, accumulating evidence suggests that CCK-induced production of NAADP may contribute to the distinct characteristics of CCK-induced Ca2+ signals in mouse pancreatic acinar cells. Ca2+ Clearance After a [Ca2+]i increase, mechanisms must be present to reduce [Ca2+]i rapidly and efficiently during the declining phase of Ca2+ oscillations, and ultimately to terminate Ca2+ signals after secretagogue removal. In addition, the resting [Ca2+]i of 50 to 200 nM must be maintained in the event of a large concentration gradient from the cell exterior, a leak from the ER, and the negative intracellular potential, all of which would tend to drive Ca2+ into the cytoplasm. Homeostasis is accomplished by a variety of pumps and transporters that have specific distribution on both the plasma membrane and the membranes of various organelles. Tepikin and colleagues (167,168), using a technique where the extracellular [Ca2+] is monitored by an indicator in a small volume of extracellular fluid, demonstrated that Ca2+ is extruded across the plasma membrane after agonist stimulation. This in all probability occurs by Ca2+ pumps of the plasma membrane Ca2+-ATPase gene family (PMCA). Ca2+ATPase activity is present in plasma membrane preparations isolated from pancreatic acinar cells, and immunoblotting demonstrates the expression of PMCA family members (83,169,170). On supramaximal stimulation with CCK or ACh, the amount of Ca2+ lost from the cell approximates the entire agonist-releasable pool (171). Ca2+ extrusion also occurs during more physiologic stimulation with CCK, and the activity follows the [Ca2+]i (167). PMCA is also important for maintaining resting [Ca2+]i because its activity can be demonstrated at basal [Ca2+]i. Indeed, the rate of pumping has been shown to have a steep dependence on [Ca2+]i (Hill coefficient of ~3) and is effectively saturated at [Ca2+] greater than 400 nM (172). The activity of the PMCA can also be increased by agonist stimulation in a manner independent of

1350 / CHAPTER 53 Ca2+, and this may reflect modulation of the activity of the pumps by phosphorylation or association with regulatory proteins (173). PMCA is not homogeneously distributed over the entire plasma membrane and appears to be most abundant in the luminal and lateral plasma membrane (83). The localization of PMCA correlates with the site of most apparent Ca2+ pumping activity because Ca2+ extrusion occurs preferentially from the apical aspects of mouse pancreatic acinar cells when monitored using an indicator with limited diffusional mobility (174). Ca2+ pumps are also expressed on ER membranes (175,176). Indeed, the ER in pancreatic acinar cells has been shown to function effectively as a single continuous Ca2+ store (67,177). This has been best illustrated by demonstrating that ER Ca2+ pump activity exclusively at the basal pole can recharge the ER after maximal agonist stimulation such that Ca2+ signals can be initiated at the apical pole (177). ER Ca2+-ATPases belong to the sacroplasmic and ER Ca2+ATPase gene family (SERCA). Both SERCA 2A and SERCA 2B have been reported to be expressed in pancreatic acinar cells, and specific subcellular distributions of specific SERCA pumps have been reported (83). SERCA 2A appears to have distribution similar to IP3Rs exclusively in the apical pole, whereas SERCA 2B predominately resides in the basal aspects of the cell. All SERCA pumps are inhibited by thapsigargin, and treatment results in Ca2+ leak from the ER store. In pancreatic acinar cells, thapsigargin treatment results in a uniform increase in [Ca2+]i and abolishes the characteristic secretagogue-stimulated, apically initiated Ca2+ wave even before the ER is fully depleted (83). The later observation indicates that the microenvironment created by SERCA pumps is crucial for the initiation of Ca2+ signals in the apical pole during Ca2+ oscillations. This could occur as a function of the SERCA pump controlling either the cytosolic [Ca2+] or local luminal ER [Ca2+] in the apical pole, because both SERCA pumps and IP3Rs are markedly influenced by the [Ca2+] on both faces of the ER membrane (84,178). A further Ca2+ uptake mechanism that plays a significant role in spatially shaping Ca2+ signals in pancreatic acinar cells is through Ca2+ sequestration into mitochondria. Mitochondrial Ca2+ uptake occurs via a Ca2+ uniporter as a function of the large electrical potential across the inner mitochondrial membrane (179). Because the Ca2+ uniporter is a relatively low-affinity, high-capacity transporter, it was generally believed that mitochondrial Ca2+ uptake was relevant only under pathologic conditions when the [Ca2+] was significantly increased for prolonged periods. However, with the advent of mitochondrially targeted indicators, it has become clear that mitochondria function during normal physiologic Ca2+ signaling in many cell types (180). Moreover, it is now thought that mitochondrial Ca2+ uptake is important not only for shaping cytosolic Ca2+ signals, but also for stimulating the production of ATP, because key enzymes in the Krebs cycle are Ca2+ dependent (181). It appears that the privileged localization of mitochondria close to Ca2+ release sites, where Ca2+ is presumably high, allows the Ca2+ uniporter to function effectively under these

conditions. In pancreatic acinar cells, mitochondria are indeed in close proximity to Ca2+ release sites because stimulation with physiologic concentrations of both CCK and ACh, which increase global [Ca2+]i only to submicromolar levels, lead to mitochondrial Ca2+ uptake (182–184). Furthermore, mitochondrial Ca2+ uptake in pancreatic acinar cells is coupled to the conversion of NAD to NADH, and thus presumably to metabolism (183). In common with other Ca2+ clearance mechanisms in acini, energized mitochondria also have specific subcellular localization. The majority of studies have reported that mitochondria are concentrated in a perigranular “belt” (91,185) (Fig. 53-7A), together with additional further subpopulations surrounding the nucleus and immediately below the basolateral plasma membrane (182). Strikingly, it appears that these perigranular mitochondria play a role in limiting the spread of Ca2+ signals on stimulation with threshold concentrations of agonist because disruption of mitochondrial Ca2+ uptake facilitates the spread of normally spatially confined Ca2+ transients (91,185) (see Fig. 53-7B).

1,2 Diacylglycerol A second messenger role is also well established for DAG, the endogenous activator of PKC (30,186). Stimulation with maximal concentrations of PLC-coupled agonists leads to a rapid increase in DAG. In contrast with the transient IP3 formation, in the continued presence of agonist, DAG levels continue to increase over many minutes (40,42,187). Using a mass assay for DAG, one can detect an initial peak after 5-second stimulation with a maximal dose of agonist that is concomitant with the peak in IP3 (40). This initial peak is of similar size to IP3; moreover, the two show identical concentration dependence on CCK. At lower concentrations of agonist (3–50 pM CCK), in which PI turnover is not readily detectable, this early peak is absent. Only the later increase is apparent. Moreover, the partial agonist analog of CCK, JMV180, which fails to measurably increase IP3, also does not induce an early peak, but rather only the later phase of DAG production (40). The later increase in DAG does not correlate well with PI(4,5)P2 hydrolysis, whether for magnitude, time course, or dependence on agonist concentration. DAG formed from the hydrolysis of other membrane phospholipids, such as phosphatidylcholine, is important in a variety of cell types (188). In pancreatic acini prelabeled with [3H]choline, the dose-dependent release of [3H]choline metabolites, predominantly phosphorylcholine, can be detected on stimulation with CCK, CCh, or bombesin (40), indicating that this may be an important source of DAG in pancreatic acinar cells. In support of this view, a further study has demonstrated a decrease in [14C]phosphatidylcholine on stimulation with carbachol in cells prelabeled with [14C]glycerol (189). Phosphatidylcholine hydrolysis may reflect direct G-protein control, but as it has been demonstrated that phorbol ester and Ca2+ ionophore can also cause phosphatidylcholine hydrolysis in acinar cells (40), it is more likely to be controlled secondarily by Ca2+ and DAG originating from initial PI(4,5)P2 hydrolysis.

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1351

A 0.5 µM FCCP

I

II

0.1% ∆F/F0 15 s

3 min

∆F/F0

B FIG. 53-7. Functional consequences of mitochondrial distribution in pancreatic acini. (A) Localization of active mitochondria was visualized by confocal microscopy in living mouse pancreatic acini loaded with mitotracker red. Mitotracker red fluorescence merged with a phase image of a small mouse acini. The predominant localization of mitochondria is to a perigranular belt surrounding the zymogen granules. (B) Photolysis of threshold concentrations of inositol 1,4,5-trisphosphate (IP3) resulted in apically limited Ca2+ signals, as shown in the images and kinetic traces in region I (red trace indicates apical region of interest [ROI]; black trace indicates basal ROI). In the same cell, after disruption of the mitochondrial membrane potential, and thus mitochondrial Ca2+ uptake by FCCP, an identical exposure to IP3 (region II) resulted in a global Ca2+ signal. These data suggest that functional mitochondria are required to constrain Ca2+ signals in the apical portion of the acinar cell. (See Color Plate 28.) (Reproduced from Straub and colleagues [91], by permission.)

Role of Cyclic Nucleotides in Pancreatic Acinar Secretion Together with calcium, cAMP can play a central role in regulating pancreatic acinar secretory activity (190). This view is based on the following lines of evidence: (1) several hormones or neurotransmitters that stimulate protein or fluid secretion, or both, from acini likewise augment cellular cAMP levels; (2) synthetic analogs of cAMP, such as dibutyryl-cAMP and 8-bromo-cAMP, stimulate protein and electrolyte secretion from the acinar epithelium; and (3) agents that stimulate AC or inhibit cyclic nucleotide phosphodiesterase activity either evoke acinar secretory activity

themselves or potentiate the stimulation of secretion elicited by other agonists that increase cAMP. The magnitude of the effect of increasing cAMP on enzyme secretion is species dependent. Guinea pig acini show a strong secretory response to cAMP and have been the primary model used to evaluate the effects of different regulatory peptides that act through this intracellular messenger. Rat and mouse acini show a small response to cAMP, but one that also potentiates the response to calcium mobilizing agents, as shown in Figure 53-8 (191). By contrast, acini from cat, dog, and human show almost no response (192). Polypeptides in the secretin/VIP/glucagon family appear to augment acinar secretory activity via modulation of

1352 / CHAPTER 53 20

15 Plus CCK

10

Amylase release (% total/30 min)

5

0

VIP alone

0

10−11

A 50

10−10 10−9 VIP (M)

10−8

10−7

CCK + VIP

40

30

20

CCK alone

10

0

B

0

3

10 30 CCK8 (pM)

100

300

FIG. 53-8. Interaction of cholecystokinin octapeptide (CCK8) and vasoactive intestinal peptide (VIP) on amylase release from isolated mouse pancreatic acini. (Reproduced from Burnham and colleagues [191], by permission.)

cAMP levels. Of these, secretin and VIP have received the most attention. In suspensions of dispersed acini prepared from guinea pig pancreas, secretin and VIP both elicit maximal increases in cAMP levels ranging from 8- to 30-fold (193). In the presence of millimolar concentrations of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-lmethylxanthine (IBMX), a larger increase in cAMP (320-fold) was observed (194). More recently, other endogenous regulatory molecules have been shown to increase acinar cAMP. The brain-gut peptide histidine isoleucine (PHI), hypothalamic growth hormone–releasing factor (GHRF), and pituitary adenylate cyclase–activating polypeptide (PACAP-38), all of which are structurally homologous to VIP, increase cAMP in guinea pig acini, most likely by interacting with VIP receptors (4). Similarly, a variety of peptides isolated from Gila monster venom increase cAMP by interacting with VIP receptors. Other peptides increase cAMP in acinar cells via distinct receptors, including calcitonin gene–related peptide and glucagon-like peptide-1. Somatostatin, which in vivo inhibits amylase release, inhibits the increase in cAMP induced by secretin and VIP in guinea pig (195) and rat (196) pancreatic acini. This effect is believed to be mediated by a Gi protein, because its effect is blocked by pertussis

toxin. The significance of this inhibition to the action of somatostatin in vivo, however, is unclear; somatostatin has not been shown to inhibit amylase release by pancreatic acini in vitro. The observed increase of cAMP levels by secretin and VIP results from activation of AC in acinar cell plasma membranes; this conclusion is supported by both biochemical and cytochemical studies. Hormone-responsive AC activity has been demonstrated in pancreatic particulate fractions and purified plasma membrane fractions (196,197). Both secretin and VIP stimulate cAMP formation, with the former peptide being both substantially more potent and slightly more effective. CCK at high concentrations also can activate pancreatic AC, but this is not believed to be related to its physiologic action. AC has been localized cytochemically in fragments of rat pancreas (198). In these electron microscopic studies, precipitates indicative of AC activity were uniformly localized along the basolateral plasma membranes of acinar cells. The intensity of precipitation was markedly increased by adding secretin to the incubation medium. Compounds that either stimulate AC or inhibit cyclic nucleotide phosphodiesterase activity also affect pancreatic cAMP metabolism. Cholera toxin, which activates AC by covalent modification of its stimulatory guanine nucleotide– binding regulatory protein, increased cellular cAMP in pancreatic acini (199). Forskolin, a diterpene isolated from Coleus roots that is a potent activator of AC and acts directly on the catalytic subunit of the enzyme complex, elicited a 30-fold increase of cAMP in dispersed rat pancreatic acinar cells (200). Phosphodiesterase inhibitors such as IBMX, theophylline, and Ro 20-174 alone have little effect on acinar cAMP levels, although they markedly potentiate the stimulatory effects of secretin and VIP (193,201).

Other Phenomena Related to Intracellular Messengers Another possible intracellular messenger involved in stimulus-secretion coupling is cyclic guanosine monophosphate (cGMP). Intracellular levels of cGMP are increased by ACh, CCK, and calcium (190). However, increasing intracellular cGMP levels by adding permeant derivatives such as dibutyryl cGMP or 8Br-cGMP or by activating endogenous guanylate cyclase with nitrosourea or sodium nitroprusside has little or no effect on Ca2+ fluxes or amylase release. The increase in cGMP produced by soluble guanylate cyclase was originally thought to be mediated by Ca2+, but later studies suggest it is by nitric oxide (NO) (202). cGMP has been suggested to mediate Ca2+ influx induced by depletion of Ca2+ stores (203), but this effect has not been seen by others (204). NO could affect secretion and other acinar cell functions by effects on cGMP or by cGMP-independent actions. Pancreatic acini contain nitric oxide synthase (NOS) activity measured either by conversion of arginine to citrulline or by accumulation of nitrite and nitrate products of NO breakdown (205). NOS inhibitors such as NG-nitro-L-arginine and NG-monomethyl-L-arginine block NO production and inhibit

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1353 pancreatic secretion in vivo, but their effects on amylase release from isolated acini are controversial, with two reports of inhibition (205,206) and six reports showing no effect on CCK or carbachol-stimulated secretion (207,208). NOS is now known to be made up of three isoforms: endothelial NOS (eNOS) and neuronal NOS (nNOS), which have constitutive activity regulated by calcium, and inducible NOS (iNOS), which is not calcium activated, but rather is inducible by certain cytokines. Most NOS in pancreas is eNOS present in endothelial cells or nNOS present in neurons. Both Western blotting and immunohistochemistry suggest that eNOS is the form associated with acinar cells (207). Genetic deletion of eNOS in mice reduces stimulated pancreatic secretion in vivo, similar to a number of studies with NOS inhibitors, but has no effect on secretion by isolated mouse acini (209). Thus, the weight of current evidence does not support a role for NO in acinar stimulussecretion coupling, and the function of NO and its downstream mediator cGMP is unclear. Pancreatic secretagogues (CCK, carbachol) increase the level of free arachidonic acid in acinar cells either through the sequential action of a PLC and diglyceride lipase or by activation of a cytoplasmic PLA2. The effects of arachidonic acid on amylase release have been modest and conflicting, as reviewed previously (190). More recently, Tsunoda and Owyang (210) suggested that PLA2 activation was involved in signaling by high-affinity CCK receptors in that these authors reported that a PLA2 inhibitor ONO-RS-082 blocked Ca2+ oscillations induced by the high-affinity CCK agonist JMV-180 and reduced amylase secretion stimulated by JMV-180. They later reported that a PLA2-activating peptide, PLAP, would induce Ca2+ spiking, production of arachidonic acid metabolites, and amylase release (211). PLAP has structural similarity to heterotrimeric G-protein β-subunit, and its action was partially blocked by antibody to Gβ. Using various inhibitors, Tsunoda and Owyang (212) suggested that this action of high-affinity CCK receptor was mediated by a β-subunit of a Gq protein. Such a model receives support from the finding that Gβγ can induce Ca2+-mediated chloride oscillations (29). Unfortunately, little subsequent work has been done in this area, and it remains a model of unclear significance. Arachidonic acid is metabolized in acinar cells by both cyclooxygenase and lipoxygenase pathways, resulting in the synthesis of prostaglandins and leukotrienes. Both cyclooxygenase and lipoxygenase inhibitors have been shown to block arachidonic acid metabolism, but in general they fail to affect enzyme secretion induced by secretagogues (190). Currently, there is little convincing evidence for prostaglandins or leukotrienes as intracellular messengers in acinar cell stimulus-secretion coupling. A number of newer signaling pathways have been demonstrated in acinar cells that are activated by pancreatic secretagogues (3). These include the mitogen-activated protein kinase (MAPK) cascades leading to extracellular signal– regulated kinases (ERKs), c-Jun N-terminal kinase (JNKs), p38 MAPK, and the phosphatidylinositol 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR)/p70S6K signaling

pathway. These pathways primarily regulate growth, protein synthesis, and gene expression and are not believed to be directly related to stimulus-secretion coupling. PI3K has been suggested to be involved in a mode of exocytosis induced by supramaximal concentrations of ACh based on such secretion being inhibited by the PI3K inhibitor Wortmanin (213). Similarly, PI3K inhibitors and PI3Kγ gene detection reduced the release of intracellular Ca2+ by supramaximal CCK (214). However, conventional amylase secretion induced by physiologic CCK or cerulein was not affected by Wortmanin, another PI3K inhibitor LY294002, or the mTOR inhibitor rapamycin (215,216). It is also unclear whether activation of PI3K serves as a signaling pathway or whether the products of PI3K are simply necessary for vesicular fusion that might mediate insertion of a calcium channel into the plasma membrane.

Interaction of Intracellular Messengers It has been known for some time that when a Ca2+-mediated secretagogue is combined with a cAMP-mediated secretagogue, the protein secretory response is greater than the additive response to the two individual secretagogues (217). This was observed initially for guinea pig pancreas, where both classes of agonists are potent secretagogues, and later for mouse and rat pancreas (191), where cAMP-mediated secretagogues alone have little effect (see Fig. 53-8). This result was also observed when receptors were bypassed by the use of the ionophore A23187 to increase [Ca2+], and this was combined with a cAMP derivative, forskolin, or cholera toxin. Neither class of secretagogue alters the mediating events induced by the other, thus the potentiation of enzyme secretion must occur at a later step in cell activation. However, this potentiation does not apply to all cellular responses. Stimulation of Na+ transport, when acted on by Ca2+- and cAMP-mediated secretagogues, is additive (218), and the increase in glucose transport induced by Ca2+-mediated agents is not altered by adding VIP (191).

INTRACELLULAR MESSENGER–INDUCED SECRETION The ability of lipophilic messenger derivatives, such as dibutyryl cAMP, to mimic the action of certain hormones provided early evidence for the importance of intracellular messengers. In the case of secretagogues such as CCK and carbachol, the discovery of Ca2+ ionophores allowed artificial increase of intracellular Ca2+, which can drive amylase release. More recently, thapsigargin has been shown to inhibit the microsomal Ca2+-ATPase, and thereby release sequestered Ca2+ into the cytoplasm. The increase in DAG has been mimicked with water-soluble DAG derivatives or, more commonly, by using phorbol esters to directly activate PKC as a substitute for DAG. The combination of Ca2+ ionophore such as 12-0-tetradecanoylphorbol 13-acetate (TPA) or thapsigargin together with an active phorbol ester

1354 / CHAPTER 53 induces amylase secretion comparable with CCK or carbachol (Fig. 53-9). The problem with many of these approaches is that they are qualitative at best in mimicking intracellular increase of messengers and require molecules to be lipophilic to ensure cellular penetration. An alternative approach is to permeabilize the cell membrane, while leaving intracellular organelles and the exocytotic machinery intact. This allows equilibration of intracellular and extracellular milieu such that ionic conditions can be set and hydrophilic molecules introduced into the cell. Depending on the size of the induced pores, cytoplasmic proteins will be retained or will leak from the cell. Permeabilization techniques were initially used to demonstrate Ca2+-induced secretion. Knight and Koh (219) used electropermeabilization whereby cytosolic proteins remain within the cell; they reported a 50% effective concentration for Ca2+ of 2 µM in stimulating amylase release. Digitonin and saponin, which make large holes with cytoplasmic leakage, allowed Ca2+-stimulated release, but an EC50 for Ca2+ of 20 to 100 µM for amylase secretion was observed (220), which is clearly higher than observed in intact acinar cells. The bacterial toxins streptolysin O and staphylococcal α toxin have been used for controlled permeabilization of cells. SLO produces large pores, allowing proteins up to 400 kDa to enter and exit, whereas α toxin produces small pores, permitting molecules up to 2 to 4 kDa to penetrate. Using these toxins, Ca2+ stimulated amylase secretion, with an EC50 of 0.50 µM in mouse acini (221) and 1.4 µM in rat acini (222,223). Permeabilization experiments have showed that Ca2+-induced secretion is dependent on ATP and enhanced by the addition of TPA, GTPγS, and cAMP

ACTION OF INTRACELLULAR MESSENGERS The intracellular messengers active in pancreatic acinar cells have been identified and characterized. Less is known, however, of the mechanisms by which they act to induce release of zymogen granule content by exocytosis, as well as to affect fluid secretion, protein synthesis, and gene expression. Although other mechanisms may exist, all the intracellular messengers activate protein kinases and phosphatases, and thereby regulate the state of protein phosphorylation. Furthermore, considerable data exist indicating that changes in the phosphorylation of regulatory proteins are involved in the mediation of the action of hormones and neurotransmitters in a variety of tissues and physiologic responses. It therefore appears likely that protein kinases and phosphatases are involved in the overall process of secretion, although their specific targets and whether a phosphorylation event is an actual trigger for secretion is poorly understood.

30

25 Amylase release (% of total)

(219–222,224–226). A later study evaluating the time course of secretion showed there was an initial ATP-independent component that requires previous ATP in the cell to “prime” a small subset of releasable granules (227). Although GTPγS could be acting in part by activating PLC, several lines of evidence suggest a distinct GTP-binding protein is involved in the terminal steps of exocytosis. First, the combined effect of GTPγS and TPA is greater than that of TPA alone (221). Second, GDPβS, an inhibitor of G proteins, reduces Ca2+stimulated release (222). Third, both low-molecular-weight and heterotrimeric G proteins have been identified on the secretory granule membrane (228–232). In addition to allowing ionic control, permeabilization allows the entry and exit of peptides and proteins. It was observed in early studies that SLO-permeabilized cells rapidly lost their ability to secrete (221). This was shown to be caused by leakage of cytosolic protein in that it was not observed with α toxin and could be reversed by adding brain cytosolic protein (223). One of the proteins that can produce this action is CRHSP-28, a Ca2+-regulated phosphoprotein previously localized to the apical pole of acinar cells (233). Subsequently, CRHSP-28 was shown to bind to Annexin VI on zymogen granules (234). Permeabilization also allows entry of peptides designed to mimic effector domains or block interactions. SLO also allows entry of antibodies. Such studies have been used to support a role for Rab3, Rab4, Gαq (230,235,236), and a tyrosine phosphatase (237) in control of amylase release.

20

15

10

5

0 Control

CCK

TG

TPA

TG + TPA

FIG. 53-9. Synergism of amylase release from rat pancreatic acini induced by the sacroplasmic and endoplasmic reticulum Ca2+-ATPase gene family (SERCA) inhibitor thapsigargin (TG; 3 µM) and the phorbol ester TPA (1 µM). Together, the two are able to reproduce the effect of a maximal concentration of cholecystokinin (CCK; 100 pM).

Cyclic Nucleotide–Activated Kinases Most of the known effects of cAMP are mediated by cAMP-activated protein kinase, also known as PKA (238). Binding of cAMP to the regulatory subunit of the kinase results in dissociation and activation of the catalytic subunit. PKA activity has been observed in pancreatic acini from

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1355 a variety of species, and distinct cAMP- and cGMP-activated kinases each with appropriate cyclic nucleotide binding have been described (239). Two isoforms of PKA (types I and II) have been described that differ in their regulatory subunits (RI and RII), and both are present in pancreas. Immunocytochemical studies have shown RI and RII immunoreactivity in zymogen granules and in the cisternae of rough ER (240). PKA can also be targeted to specific organelles or molecules through interaction with A-kinase anchoring proteins (AKAPs). Currently, physiologic substrates of PKA in acinar cells are largely unknown. In situ activation of PKA in response to hormones can be estimated by homogenizing cells and measuring kinase activity of the free catalytic subunit without further addition of cAMP (which when added yields total kinase activity). Such studies have shown that secretin and VIP activate this kinase with a reasonably good correlation to the increase in cAMP levels (239,241). CCK, but not carbachol, can activate PKA, which is consistent with that high concentrations of CCK can increase cAMP (242). This effect appears to be produced by the low-affinity CCK receptor. PKA can also be activated directly without an increase in cAMP by the Smad3/4 complex induced by transforming growth factor-β (243). Moreover, PKA-independent actions of cAMP exist. One target is a cAMP-regulated guanine nucleotide exchange factor (GEF) now referred to as Epac. Epac2 is known to promote insulin secretion from pancreatic β cells most likely to be interacting with the Rab3 effector protein Rim (244). Epac also has guanine nucleotide exchange activity toward the small G protein Rap1, which is present on zymogen granules (unpublished data). Both PKA- and non–PKAmediated actions are most likely to be related to the secretion or potentiation of secretion induced by VIP and secretin. The action of CCK through cAMP and PKA to phosphorylate the IP3R also helps shape Ca2+ signaling (79,80).

or RACKs (247). Different PKCs have their own RACKs, thus allowing isoform specificity. In pancreatic acini PKC, activity can be activated by several receptors including those for CCK, ACh, and bombesin. Phorbol ester by virtue of its DAG-like structure can also stimulate amylase secretion and potentiate secretion stimulated by calcium (see Fig. 53-9). Relatively specific general inhibitors of PKC such as bisindoylmaleimide or down-regulation of PKC by overnight incubation with phorbol ester, which should affect classical and novel PKCs, totally inhibit amylase secretion release induced by phorbol ester and inhibit secretion stimulated by CCK or carbachol by 40% to 50%. Although the molecular targets of PKC action are largely unknown, PKC activity has been shown to be present and to phosphorylate proteins in subcellular fractions containing zymogen granules (248,249). One target of PKC is the myristoylated alanine-rich C-kinase substrate (MARCKS) protein (250). Pancreatic acini contain four specific PKC isoforms: α, δ, ε, and ξ (251–253). Based on stimulation of translocation and kinase assay of immunoprecipitated forms, CCK and carbachol activate the first three and probably all four. There are inhibitors specific for classical PKCs only, and Rotterlin has some specificity for PKCδ. Using these inhibitors and dominant-negative constructs introduced by adenovirus, CCK has also been shown to increase the tyrosyl phosphorylation of PKCδ, which is required for its activation, but not translocation (254), and PKCδ appeared to be the PKC isoform responsible for amylase release (253). In similar studies using cell-permeant peptides to block the interaction with a specific RACK, PKCδ and ε were shown to mediate nuclear factor-κB activation by CCK (255) and PKCε to block ERK activation (253). Thus, different PKC isoforms appear to mediate different functions in acinar cells with PKCδ-mediating effects on secretion.

Ca2+/Calmodulin-Activated Kinases Protein Kinase C PKC is a group of phospholipid-dependent serine/ threonine kinases implicated in a variety of biological functions including proliferation, differentiation, and secretion (245,246). The PKC family is divided into three subgroups by their structure and activation requirements: conventional PKCs (α, βI, βII, and γ), which are DAG and Ca2+ dependent; novel PKCs (δ, ε, η, and θ), which require DAG but are Ca2+ independent; and atypical PKCs (ξ, λ, υ, and µ), which are independent of both DAG and Ca2+. There are four constant (C) domains in PKC: the C1 domain contains the binding site for DAG, the C2 domain contains the Ca2+binding site, the C3 domain contains the ATP-binding site, and the C4 domain contains the substrate recognition site. Classical PKCs contain all four C domains, whereas the other two groups lack C2 or both C1 and C2. All PKC isoforms are primarily located in the cytoplasm at rest and on activation transfer to a membrane environment where they interact with docking proteins termed receptors for activated C kinases,

Although it is possible for Ca2+ to bind directly to an enzyme, most actions of Ca2+ are initiated by binding to proteins without enzymatic activity. With the exception of troponin in skeletal muscle, the most abundant Ca2+-binding protein is calmodulin. Calmodulin is a ubiquitous 19-kDa acidic protein that binds 4 moles Ca2+ per mole protein. The binding of Ca2+ occurs first to two sites on the carboxylterminal half of the molecule, followed by binding with lower affinity to two sites in the amino-terminal half of the molecule. Calmodulin is responsible for activating several enzymes in the pancreas, including multiple protein kinases, a protein phosphatase, a cyclic nucleotide phosphodiesterase, and NOS. Other pancreatic calmodulin-binding proteins also have been identified by a gel-overlay binding technique (256). In contrast with a single ubiquitous cAMP-activated kinase, a number of distinct Ca2+-activated calmodulin-dependent (CaM) kinases exist that differ in their substrate specificities. Some, such as myosin light chain kinase (MLCK) and phosphorylase kinase, have distinct substrate proteins,

1356 / CHAPTER 53 whereas CaM kinase II (CaMKII) can phosphorylate a large number of proteins, and is therefore sometimes referred to as a multifunctional protein kinase (257). Five of these kinases have been identified or purified from pancreas, or both, including MLCK (258), CaMKI (259), CaMKII (260,261), CaMKIII (259), and CaMKIV (204). CaMKI was originally isolated as a result of its ability to phosphorylate the synaptic vesicle protein synapsin 1. Using two different antibodies, investigators have identified CaMKI in rat pancreas, but immunohistochemistry showed that it was predominantly expressed in islets of Langerhans with little or no staining in acinar cells (262). CaMKII has been studied extensively in brain and other tissues. It is encoded by four genes, α, β, γ, and δ, each of which also exhibits alternative splicing, resulting in highly homologous proteins of 50 to 60 kDa containing an N-terminal catalytic domain, a conserved central regulatory domain that contains both the calmodulin-binding domain and an autoinhibitory domain, and a C-terminal subunit association domain. The holoenzyme is an oligomer of 12 subunits arranged as 2 stacked hexameric rings (263). In the absence of bound Ca2+/calmodulin, the kinase is maintained in an inactive configuration as a result of the interaction of the autoinhibitory domain with the catalytic domain. On activation by Ca2+/calmodulin binding, the kinase undergoes an autophosphorylation on threonine 286 (α isoform), rendering the kinase active even after calmodulin dissociates. This prolonged activation of individual subunits allows the enzyme to decode the frequency of Ca2+ oscillations and participate in synaptic plasticity in the brain. Pancreatic CaMKII was purified as a single 50-kDa subunit similar in size to the brain α-subunit (260). Although there is little information regarding the specific isoform present in acini, most nonneural tissues contain predominately δ and γ. CaMKII appears present in acinar cells based on Western blotting of acini (204) and immunocytochemical localization to the apical region of acinar cells (264). Moreover, CCK, CCh, and bombesin (but not JMV-180) stimulation rapidly induced the appearance of Ca2+/calmodulin-insensitive activity as measured with autocamtide 2 as a specific substrate (265). However, neither the importance of CaMKII in secretion nor the nature of its specific substrates is known. Proposed inhibitors of CaMKII including KN-62 and KN-93 have little effect on amylase secretion. CaMKIII is known to be relatively specific for its substrate elongation factor 2 (eEF2), a 100-kDa protein involved in ribosomal translation of RNA. It is a monomeric protein of 140 kDa and can itself be regulated by a variety of kinases. The regulation of eEF2 phosphorylation by CCK in vitro has been reported (266). CaMKIV is a more recently discovered monomeric kinase of 40 kDa that has high sequence homology with the catalytic and regulatory domains of CaMKII and broad substrate specificity, although somewhat different from CaMKII. It has been shown to be present in rat pancreatic acini and to be activated by CCK and CCH (204). Because there are no specific inhibitors of CaMKIV, its importance in acinar secretion currently is unknown.

MLCK from pancreas, as well as other tissues, specifically, phosphorylates myosin light chain. Pancreatic MLCK is a 138-kDa protein and requires both Ca2+ and calmodulin (258). Its activity is believed to be responsible for the increased myosin light chain phosphorylation seen in acini stimulated with CCK (267).

Tyrosine Kinases CCK is known to stimulate tyrosine phosphorylation of acinar cell proteins, and this was suggested to mediate hormone-induced amylase release in that the tyrosine kinase inhibitors genistein and staurosporine inhibited amylase secretion (268). However, genistein and tyrophostins were shown at the high concentrations used to have multiple effects on acinar Ca2+ signaling including inhibition of phosphoinositide hydrolysis (269,270). More recent studies have evaluated the tyrosine phosphorylation of Shc, insulin receptor substrate-1, ERKs, JNKs, and p38 MAPK, all of which are involved in a variety of pathways related to growth or gene expression (3). Another group of tyrosine-phosphorylated proteins are part of the focal adhesion complex. Garcia and colleagues (271) identified the kinase p125 focal adhesion kinase (FAK) and its substrate paxillin as being tyrosyl phosphorylated in response to CCK. This phosphorylation was shown to be induced by physiologic levels of CCK and to require an intact actin cytoskeleton (271). More recently, Pyk2, a tyrosine kinase related to p125FAK but activated by Ca2+, also was shown to be tyrosine phosphorylated and activated by CCK (254). Activation of Src or a family member Yes or Lyn may also be related to the activation of FAK or PyK2 (272,273). Some of these changes are likely related to the high concentration effect of CCK to alter the actin cytoskeleton, but whether they are related to physiologic stimulussecretion coupling remains unclear. Finally, carbachol and CCK stimulation have been shown to increase the tyrosyl phosphorylation of PKCδ and its association with PyK2 in acini (274,275).

Protein Phosphatases Regulation of cellular phosphoproteins also involves protein phosphatases as the state of phosphorylation represents a balance between kinase and phosphatase activity. Four different classes of serine/threonine-specific protein phosphatases have been identified in eukaryotic cells (276): types 1, 2A, 2B, and 2C. Protein phosphatase type 1 (PP1) was originally defined by its ability to dephosphorylate the β-subunit of phosphorylase kinase and by being regulated by two small heat-stable proteins termed inhibitor-1 and inhibitor-2. PP2, which dephosphorylates the α-subunit of phosphorylase kinase, was subdivided into three distinct enzymes, PP2A, PP2B, and PP2C, in that PP2B was dependent on Ca2+, PP2C was dependent of Mg2+, and PP2A was independent of divalent cations. Phosphatase inhibitors have

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1357 been discovered that also aid in classifying enzyme activity. The marine toxin okadaic acid is a potent inhibitor of PP2A, a modest inhibitor of PP1, and a weak inhibitor of PP2B. Calyculin A is a strong inhibitor of both types 1 and 2A, whereas cyclosporine A and FK506 are potent and selective inhibitors of PP2B, the Ca2+/calmodulin-activated phosphatase also known as calcineurin. Rat pancreas was shown to contain both types 1 and 2 phosphatase activity based on dephosphorylation of phosphorylase kinase (277). Later these were shown to be predominantly types 1 and 2A (278). Ca2+-activated phosphatase was purified from pancreatic cytosol and was shown to consist of two subunits and to be activated by calmodulin (277). Although phosphatase activity is present in cytosol, much of the specific actions of phosphatases in various cell types are through targeting via specific subunits or anchoring proteins. Protein phosphatase inhibitors also have been shown to affect acinar cell secretion. Both okadaic acid and calyculin A inhibit amylase secretion at relatively high concentrations (279–281). Because they inhibit Ca2+-stimulated secretion in permeabilized acini, their action may be on a postreceptor step in stimulus-secretion coupling (279,282). Whereas the calyculin effects are most likely on a type 1 phosphatase, the okadaic acid effects were assigned to type 2A or 2B by different investigators. Because of the multiple roles of phosphatases, these effects cannot be considered specific, and okadaic acid also caused fragmentation of the Golgi disrupting intracellular protein transport (281). Inhibitors of the type 2B phosphatase, cyclosporine A, and FK506 also have been reported to partially inhibit secretion in dispersed acini (283,284). However, the concentrations of CsA used may have affected the mitochondrial permeability pore transition, and in our hands, the more specific FK506 did not affect amylase secretion, although it did potently block dephosphorylation of the calcineurin substrate CRHSP-24 (285). Other studies have also suggested that the tyrosine phosphatase inhibitor vanadate can also affect secretion, although one study showed increased amylase release (286) and another showed decreased amylase release induced by CCK (287).

Secretagogue Changes in Cellular Protein Phosphorylation Pancreatic secretagogues alter the phosphorylation of a number of identified and unidentified proteins as shown by two-dimensional gel electrophoresis of protein from 32 P-labeled acini (250,284,288). Evaluation of 500 phosphoprotein spots yielded 27 secretagogue-regulated proteins with some increasing and some decreasing with different time courses (250). Two heat-stable serine-phosphorylated proteins regulated by Ca2+ have been purified and characterized molecularly as novel proteins termed CRHSP-24 and CRHSP-28 (289,290). CRHSP-24, which is dephosphorylated, is the major calcineurin substrate in acini, whereas CRHSP-28, which has increased phosphorylation, is localized in the

secretory pole and may play a role in secretion as discussed later. A number of proteins have been identified using phospho-specific antibodies that play roles in signal transduction cascades and protein synthesis. Some of these are affected by secretin, VIP, and cAMP, as well as by CCh, CCK, and phorbol ester. Part of the problem in relating phosphorylation events to secretion is that so many cell functions are regulated by phosphorylation. Future progress is more likely to come from first identifying specific proteins related to secretion, and then considering phosphorylation as a potential regulatory mechanism. Figure 53-10 summarizes the role of intracellular messengers and effectors in pancreatic enzyme secretion in simplified form. This model is largely based on research on rat, mouse, and guinea pig pancreatic acini. Current knowledge is that the components are present in all species except for the CCK receptors, which are not present in human acini (7).

Secretin

VIP

Bombesin CCK

Ach

PI-P2 AC

Gs

Gq PLC

IP + 3 DAG

ATP Ca2+

cAMP Ca2+

CAM

PK-A

PP

PK

PK-C

Altered phosphorylation of structural and regulatory proteins

FIG. 53-10. Schematic diagram of stimulus-secretion coupling of pancreatic acinar cell protein secretion. The binding of hormones and neurotransmitters to their membrane-spanning receptors (shown here only as distinct conceptual units) leads to the G protein–coupled formation of intracellular messengers. Receptors for cholecystokinin (CCK), acetylcholine (ACh), and bombesin are coupled by Gq to the activation of a PLCβ, which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3), which, in turn, leads to release of Ca2+ from intracellular stores, accompanied by stimulation of Ca2+ influx. The stimulation of influx may be related to the state of refilling of the pool. The simultaneous formation of diacylglycerol (DAG) by phospholipase C (PLC) activates protein kinase C (PKC). Receptors for vasoactive intestinal polypeptide (VIP) and secretin are coupled by Gs to the stimulation of adenylate cyclase (AC). The formation of the intracellular messengers cyclic adenosine monophosphate (cAMP), Ca2+, and DAG influence either directly or indirectly the activity of kinases and phosphatases. ATP, adenosine triphosphate; CAM, calmodulin; PK, protein kinase; PP, protein phosphatase.

1358 / CHAPTER 53 The cAMP-PKA pathway stimulated by VIP and secretin is much more important in the guinea pig.

have been reviewed extensively (292). We focus here on what is known concerning zymogen granule exocytosis.

MECHANISMS OF EXOCYTOSIS

Visualization of Exocytosis

The final steps in stimulus-secretion coupling involve fusion of the zymogen granule with the apical plasma membrane, release of granule content proteins into the acinar lumen, and retrieval of the granule membrane (Fig. 53-11). Although control of this process involves intracellular regulatory signals such as Ca2+, cyclic nucleotides, and protein kinases, successful secretion into the acinar lumina also involves transport and targeting of both zymogen granules and apical membrane proteins. This process involves the cytoskeleton including both microtubules and actin-based components. The major proteins involved with exocytosis share basic mechanisms with other vesicular transport systems that are conserved from yeast to neurons (291,292). Two major families of proteins are the soluble N-ethylmaleimide– sensitive factor attachment protein receptors (SNAREs) and small G proteins of the Rab family. Initially, the physical contact between the donor membrane (zymogen granule) and the “acceptor” membrane (apical plasma membrane) is variably termed tethering or docking. In most fusion reactions, this is orchestrated by Rab proteins, which recruit other effector molecules. After membrane attachment, fusion is initiated by the action of the SNARE proteins, which bridge the fusing membranes and quite likely provide the energy necessary for fusion. These processes and molecules, which are involved in intracellular transport, exocytosis, and endocytosis,

The concept of exocytosis was developed in part based on images of emptying fused granules observed by the electron microscope (293). Such images from pancreatic tissue are relatively uncommon, and they usually have been ascribed to a short lifetime that was not capturable by the required fixation. Approaches to quantitate changes in membrane surface area of living cells by capacitance or fluorescent lipid tracers such as FM1-43 have been only modestly successful and made more difficult by the simultaneous occurrence of exocytosis and endocytosis (294,295). Real-time video recording with differential interference contrast and comparison of adjacent frames has been used to visualize the rapid disappearance of granules (296,297). These studies showed rapid early events at the apical membrane indicating that granules fuse while undergoing only short movements, and that fusion events appeared independent. In a more recent approach, Nemoto and colleagues (298) used two-photon confocal microscopy of a fluorescent aqueous marker such as sulforhodamine-B or Texas Red-dextran to visualize exocytotic images. These tracers penetrate into the lumina and rapidly enter the granule after fusion, where they can be visualized (Fig. 53-12). Fused zymogen granules maintained their appearance for a mean lifetime of 220 seconds and could serve as targets for sequential fusion of other granules. It was observed that granule-granule fusion only follows an

Tethering

Rab Rab

1

2

3

t-SNARE

vSNARE Docking

Motor proteins

Budding Microtubule

Actin filaments

4

Trans-Golgi network

Fusion

Actin coated granules

SNARE complex Plasma membrane

FIG. 53-11. Model of the steps in exocytosis of zymogen granules including movement to the luminal membrane, passage through the terminal web of actin filaments, tethering, docking, and fusion. Note for simplicity that not all of the soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex proteins are shown.

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1359 Dextran

Phalloidin

Overlay

Control

A

10 µm

CCK

B

5 µm

FIG. 53-12. Visualization of exocytosis and actin coating of zymogen granules by two-photon confocal microscopy. Acini were incubated with cascade blue dextran (left, red) to label the lumen and Alexa 488-phalloidin (middle, green) to label filamentous actin; an overlaid image is shown on the right. (A) Unstimulated; luminal areas are visualized as red wider lines surrounded by apical membranes coated thickly with F-actin. (B) Stimulated with 100 pM cholecystokinin (CCK) for 60 sec and show selective actin coating of granules undergoing exocytosis. Images were generated from stacks of 10 XY images at an internal of 1 µm. (See Color Plate 29.) (Courtesy of T. Nemoto, T. Kojima and H. Kasai, National Institute of Physiological Sciences and PRESTO, Japan Science and Technology Agency; bottom panels reproduced from Nemoto and colleagues [301], by permission.)

initial exocytotic event, suggesting that apical membrane proteins must diffuse into the fused granule membrane to confer fusion competency. However, a similar latency averaging 9 seconds between subsequent events and the initial event argues against this. Subsequent studies have shown that after exocytosis, the membrane lipids do not mix over hundreds of seconds (299), but that a SNARE protein, syntaxin-2, does diffuse into the granule membrane over a few minutes (300). This approach to visualizing exocytosis has been extended using a fixable luminal tracer that allows simultaneous immunocytochemistry and has been used to show that granules undergoing exocytosis became rapidly coated with actin, which was shown to be necessary for compound exocytosis (301). The fixation technique also allows similar events to be visualized using standard onephoton confocal microscopy (302). Finally, the atomic force microscopy also has been used to observe depressions on the surface of the apical region of isolated single acinar cells, which correlated with amylase release, although their exact nature was unclear (303).

Rab Proteins Rab proteins are small monomeric G proteins of the Ras superfamily. There are more than 60 Rab proteins in the mammalian genome; all share a common structure for binding and hydrolyzing GTP and possess a C terminus that is

modified by the addition of hydrophobic geranylgeranyl groups, which allow them to attach to membranes (304,305). They have unique regions that convey targeting specificity and the ability to interact with effector molecules. All small G proteins are molecular switches that are “on” when liganded with GTP. They are activated by GEFs, which may also play a role in targeting the G protein, and are turned “off ” by a Rab GAP. Inactive GDP liganded G protein is primarily present in the cytosol bound to a GDI (guanine dissociation inhibitor) protein. Thus, Rab proteins cycle between GTP and GDP liganded forms and between a membrane-attached and a cytosolic state. A number of Rab proteins have been recognized or reported on pancreatic zymogen granules, including Rabs 3D, 4, 5, 11, and 27B (228,229,236,306–308). Some of these may be involved in granule formation or endocytosis after fusion. Most attention has focused on Rab3D because Rab3 proteins are involved with exocytosis in a variety of cell types. There are four Rab3 isoforms (A, B, C, and D), with Rab3A being the predominant form on synaptic vesicles. Rab3D appears to be the primary form on large secretory granules such as in the acinar cells of pancreas, parotid, and lacrimal glands (229,232). Although 3D appears to be the only Rab3 on acinar cell zymogen granules, other Rab3 isoforms are present in pancreatic AR42J cells (309). Rab3D plays a positive role in acinar cell secretion, with overexpression in transgenic mice increasing (310) and acute expression of a dominant-negative form decreasing secretion (311).

1360 / CHAPTER 53 However, secretion was reported as normal in a Rab3D genetargeted mouse, although granules were larger (312). Almost all the Rab3D on zymogen granules exist in the GTPliganded state, although this is reduced by overexpression of dominant negatives (313). Little is known concerning Rab3 GEFs or effector proteins in acini. Suggested possible mechanisms of action of Rab3D include induction of fusion by the effector domain (314), controlling the number of granules docked at release sites (315), or regulating actin polymerization around the zymogen granule (316). The closest relatives of Rab3 within the Rab family are Rab27A and 27B. They also have been identified as playing a role in exocytosis in multiple secretory cells. Rab27B has been identified on zymogen granules, and acute overexpression of constitutively active 27B enhances, whereas a dominant-negative inhibits, secretion (228). These results are similar to studies of other secretory cells including islet β cells, pituitary cells, and parotid (317). Rab27A plays a role in melanosome transport that involves a linker protein Slac2a or melanophillin and an unconventional myosin, myosin Va (318). A number of homologous linker proteins have been identified as Slps (synaptotagmin-like proteins) or Slacs (Slp homologue lacking C2 domains). One termed granulophillin plays a role in islet secretion, but is not present in acini (319). Several species appear to be associated with zymogen granules (Chen X, Williams JA, and Andrews PC, unpublished data). The nonconventional myosin Vc also has been identified in acini (320). Another distinct Rab effector, Noc2, has been reported to interact with both Rab3 isoforms and Rab27A in β cells (321). Interestingly, Noc2-deficient mice show impaired insulin secretion, but also an accumulation of acinar cell zymogen granules and impaired in vitro amylase secretion (322). Further work clearly is important to establish the role of Rab3 and Rab27 in acinar cell secretion, and whether they are redundant or regulate different processes. Less is known of the other Rab proteins reported on zymogen granules including Rab11 (308), Rab4 (236), and Rab5 (307). Rab11 is thought to function in membrane recycling and is known to interact with myosin Vb through the linker protein Rab11-FIP2 (323).

SNARE Proteins SNARE proteins were originally identified as membraneattached receptors for soluble N-ethylmaleimide–sensitive factor (NSF) attachment proteins (SNAPs) (291). There are more than 35 mammalian SNARE proteins that share a homologous sequence, the SNARE motif. The best characterized are the synaptic SNARE complex made up of synaptobrevin/VAMP on the vesicle and syntaxin 1 and SNAP-25 on the plasma membrane. SNARE proteins associate into a core complex of four parallel helical bundles, with SNAP-25 providing two and the other two proteins each providing one bundle. Assembly of the core complex proceeds in a zipperlike fashion that provides energy for fusion. Afterward, the complex is disassembled by the ATPase NSF in concert with

SNAPs as cofactors. Assembly of SNARE complexes needs to be regulated, and this is carried out by SNARE modulatory proteins such as Munc18 and Tomosyn, which bind syntaxin, preventing its participation in assembly, and Munc13, which participates in vesicle priming. Other regulators involved with synaptic vesicle fusion include the Ca2+ sensor synaptotagmin and small soluble proteins termed complexins. Important tools in defining the role of SNARE proteins have been the selective proteases tetanus toxin and botulinum toxins, which cleave SNARE proteins, preventing their assembly. Pancreatic acinar cells contain SNARE proteins, although the exact complex mediating exocytosis is not fully understood. VAMP-1 (synaptobrevin), VAMP-3 (cellulobrevin), and VAMP-8 (endobrevin) have been identified on zymogen granules (324–326). Pancreatic VAMP-2 and cellulobrevin were shown to be sensitive to tetanus toxin, but the toxin had only a modest inhibitory effect on amylase secretion (324). VAMP-8, which is not toxin sensitive, was shown to be the major isoform mediating exocytosis through the use of VAMP-8 knockout mice, which showed an almost complete block in pancreatic secretion (326). Acinar cells contain multiple forms of syntaxin including 2 through 4 (327). By immunocytochemistry, syntaxin 2 was localized to the apical plasma membrane, syntaxin 4 was most abundant on the basolateral membrane, and syntaxin 3 was most abundant on the zymogen granules. A study using botulinum neurotoxin C was interpreted as indicating that syntaxin 2 mediates fusion of zymogen granules with the plasma membrane, whereas syntaxin 3 might mediate zymogen granule/zymogen granule fusion (328). However, VAMP-8 was shown to exist in a complex with syntaxin 4 (326). The third SNARE protein SNAP-25 is restricted to neurons and is not present in acinar cells. However, two broadly expressed isoforms, SNAP-23 (329) and SNAP-29 (X. Chen and J. A. Williams, unpublished data), are present. SNAP-23 was predominantly localized on the basolateral plasma membrane (329), but also appeared in an immunoprecipitated complex with VAMP-8 and syntaxin 4 (326). Because syntaxin 4 is primarily on the basolateral membrane, SNAP-23 has been suggested to mediate basolateral fusion (330). However, a truncated mutant of SNAP-23 introduced by an adenovirus was shown to inhibit stimulated amylase release (331). Thus, it appears highly likely that a SNARE complex is involved in zymogen granule exocytosis. Although the exact makeup is not fully understood, the best evidence is for VAMP-8, SNAP-23, and either syntaxin 2 or 4. Other SNARE-associated proteins and potential interacting proteins also have been identified. Munc18c was identified in acinar cells, localized to the basolateral membrane, and shown to dissociate in response to high concentrations of CCK or PKC (330). This was suggested to mediate basolateral secretion. Another more novel protein, syncollin, was originally identified in pancreas as a syntaxin-binding protein (332). It was then shown to be located on the inner surface of the granule membrane, whereas syncollin knockout mice showed relatively normal secretion (333); both observations suggest it might not be important for secretion. However, a more

STIMULUS-SECRETION COUPLING IN PANCREATIC ACINAR CELLS / 1361 recent study of the knockout mice showed a reduction in both amylase release and exocytotic images (334). Other proteins reported on the zymogen granule and possibly playing a role in secretion include cysteine string protein (335) and the heterotrimeric G-protein subunits αq/11 (230) and Gαo and Gαs (231).

Cytoskeleton The acinar cell cytoskeleton clearly plays a role in the mechanism of secretion both indirectly by maintaining the required cellular architecture and compartmentation and directly by providing motile force to move granules and to regulate the process of exocytosis. As for other cells, the acinar cell cytoskeleton is made up of three components: microtubules, intermediate filaments, and microfilaments. Currently, there is little evidence for an active role of intermediate filaments. Microtubules are thought to play a role in directing vesicular traffic such as zymogen granules toward the apical pole and endocytosed membrane vesicles toward the cell center. In this regard, microtubules running toward the apical area have their minus end at the apical end (336). Early studies of the effect of disruption of microtubules showed that secretion of stored amylase was not inhibited, although secretion of newly synthesized protein was inhibited (337,338). The main class of microtubule-based motor proteins in acinar cells is the kinesins, of which most move toward the plus end of the microtubule, but a few move in the minus direction. Kinesins have been reported to be localized to zymogen granules, and microinjection of a kinesin antibody inhibited zymogen granule movement (339). Addition of exogenous kinesin was reported to enhance whereas a monoclonal antibody against kinesin inhibited amylase secretion from permeabilized acini, but the effect was directed at the cAMP-stimulated component secretion (340). CCK, secretin, and phorbol ester all enhanced the phosphorylation of kinesin heavy chain (339). The molecular identity of kinesin molecules and their receptors on granules remains to be established. The actin-based cytoskeleton in acinar cells is dominated by the terminal web made up of bands of filaments running across the cell just under the apical plasma membrane. This structure includes filamentous actin, which can be readily labeled with fluorescent tagged phalloidin, but also contains conventional myosin (II), tropomyosin, and α actinin (341). Actin (presumably G actin) has been shown by electron microscopic immunocytochemistry to be associated with zymogen granules (342). Myosin I (343) and myosin Vc (320) also have been localized in the apical pole associated with granules. A reduction in apical filamentous actin has been reported in response to physiologic CCK accompanied by cell shape changes (344), although the effect is not as prominent as the widely reported decrease in response to supramaximal CCK stimulation. These authors reported that apical myosin II remained relatively unchanged, but that phosphorylation of myosin light chain was enhanced.

The role of the actin cytoskeleton also has been studied using a number of chemical agents affecting actin polymerization. Cytochalasins B and D, which disrupt microfilaments, partially inhibit secretion, but have large effects on acinar structure with luminal expansion (345,346). Latrunculin A, a cell-permeant actin monomer-sequestering agent, inhibits amylase secretion without greatly affecting cell structure (344,347). Latrunculin may, however, be acting by blocking polymerization because much of the filamentous actin remains intact. In a study in permeabilized acini, introduction of B-thymosin a actin monomer binding protein was shown to enhance amylase release even without an increase in Ca2+ (348). This was interpreted to be in support of the hypothesis that the apical terminal web is a barrier to granules reaching release sites. However, high concentrations of monomer binding proteins or phalloidin, which promotes actin polymerization, blocked secretion, indicating a positive role for actin in exocytosis (348). Subsequently, a high concentration of jasplakinolide, a cell-permeant actin polymerizer, also was shown to inhibit secretion (316). These data could be interpreted as indicating that local dynamic changes in actin polymerization are required for secretion. Such reorganization has been reported in that zymogen granules undergoing exocytosis have been reported to become coated with filamentous actin (301,302,316). However, this coating may occur after fusion and promote exit of granule content into the lumen (301,302). Evidence also exists for secretagogue effects on actinmodulating proteins. CCK is known to activate MLCK, leading to myosin phosphorylation (267,344). CCK also activates p38 MAPK, which leads through MAPKAP kinase 2 to phosphorylation of heat shock protein 27, a known regulator of actin polymerization (349). In addition, CCK and muscarinic receptor signaling can activate the small G proteins Rho and Rac in acini (347). Moreover, introduction of dominant-negative Rho and Rac into acini via adenoviral vectors inhibits amylase release (347). Rho and Rac are known to regulate a number of effectors influencing both microtubules and the actin cytoskeleton, thus future studies of this area in acini should be fruitful.

Role of Ion Channels in Exocytosis The chemiosmotic theory of exocytosis proposed that anion conductances in the secretory granule membrane play a crucial role in Ca2+-dependent secretion. In this model, an H+-ATPase pumps protons into granules setting up a pH gradient that would pull anions in through a regulated conductance causing osmotic swelling that would overcome repulsive forces and drive fusion. However, zymogen granules are only mildly acidic, and evidence for ATPase in the membrane is lacking. There is, however, good functional evidence for both anion and K+ channels in the zymogen granule membrane, and various proposals of how these might participate in fusion or in flushing out granule contents after fusion exist (350). Two types of anion channels have

1362 / CHAPTER 53 been identified, a C1C-2 chloride channel (351) and a CLCA1 calcium-activated anion channel permeable to HCO3+ (352). A K+ conductance is also present in zymogen granules based on swelling studies and pharmacologically is most like KCNQ1 (350). Current evidence suggests that granule swelling takes place after fusion and that any ion flux promotes discharge of the granule content and promotes granule membrane retrieval.

Endocytosis For secretion to occur normally and continuously, the zymogen granule membrane must be retrieved and recycled by the process of endocytosis. Expansion of the luminal membrane can occur with secretion, but is followed by a return to normal dimensions with an increase in the size of the Golgi and the appearance of abundant clathrin-coated vesicles in the apical portion of the cell (see Chapter 52). In various secretory cells, membrane retrieval occurs as a rapid pinching of the secretory vesicle (kiss and run), or after fusion of the granule membrane as a patch in the secretory membrane (kiss and collapse). In acinar cells, there is no evidence for retrieval of intact granule membrane, and endocytosis from both the inserted intact zymogen granule membrane and the apical membrane has been reported (299,353). Apical endocytosis has been shown to require an alkaline pH (354) and to be associated with cleavage of GPI-anchored proteins especially GP-2 (355). Actin is well known to be required for endocytosis and may be associated with a Src family tyrosine kinase, which is required in acini for endocytosis (356). Rab 4, which is associated with apical actin in acinar cells, is also known to be involved with early endocytosis and has been proposed to do so in acinar membrane retrieval (357).

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54

Cell Physiology of Pancreatic Ducts Barry E. Argent, Michael A. Gray, Martin C. Steward, and R. Maynard Case Patterns of Pancreatic Electrolyte Secretion, 1371 Structural Basis of Secretion, 1372 Advances in Studying Duct Cell Physiology, 1375 Mechanisms of Ductal Electrolyte Secretion, 1378 Basolateral Transport Mechanisms, 1379 Apical Transport Mechanisms, 1381 Carbonic Anhydrase, 1384

Paracellular Cation Transport, 1384 An Updated Model for Ductal Electrolyte Secretion, 1384 Regulation of Ductal Secretion, 1385 Stimulatory Pathways, 1386 Inhibitory Pathways, 1389 Concluding Remarks, 1391 References, 1391

Pancreatic juice is the product of two distinct types of secretory processes that may be referred to as protein (enzyme) secretion and electrolyte secretion. Enzymes, after their synthesis, intracellular trafficking, and storage, are secreted by exocytosis from acinar cells, as described in detail in Chapters 52 and 53. Electrolyte secretion is achieved by the coordinated vectorial transport of selected ions across an epithelium, accompanied by water in isotonic proportions. In the pancreas, the most significant of these ions is bicarbonate (HCO3−), and as described in detail in the following section, it is now generally accepted that the secretion of HCO3−-rich fluid occurs largely (perhaps exclusively) in pancreatic ducts. In humans, these two processes result in the daily secretion of about 2.5 L of HCO3−-rich fluid containing 6 to 20 g enzymes. Although the role of the enzymes in digestion is clear, the functions of pancreatic electrolyte secretion are less precise, but include acting as a vehicle for transporting the enzymes to the duodenum where the HCO3− helps to neutralize gastric acid, and thus increase duodenal pH. Pancreatic HCO3− may also aid disaggregation of secreted enzymes after their exocytosis (1). Because of this, altered

HCO3− transport may contribute to the pathology of pancreatitis, as it is known to do in cystic fibrosis (CF).

B. E. Argent and M. A. Gray: Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom. M. C. Steward and R. M. Case: Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

PATTERNS OF PANCREATIC ELECTROLYTE SECRETION This chapter focuses on analyzing the transport mechanisms by which pancreatic duct cells secrete HCO3− (and hence fluid) and their regulation. As background to these analyses, it is important to recognize that both transport and regulatory mechanisms differ substantially between species. This is important for two reasons. First, data obtained in one species, especially those involving models of pancreatic disease, cannot be assumed to be relevant to humans (and hence human disease). Second, transport or regulatory mechanisms, or both, that are dominant in one species may be present to a much lesser extent in humans (and therefore unrecognized), where they may account for otherwise inexplicable symptoms or be amenable to therapeutic intervention. As a result of these differences, the volume and composition of pancreatic juice secreted in response to a variety of stimuli can vary markedly between species. These differences, which we have previously described in detail (2,3), are summarized in Table 54-1 and are illustrated for the guinea pig and rat in Figure 54-1. References to these differences are made throughout the main sections of this chapter, and the following is a summary of the major differences and their relevance: 1. In all species, secretin evokes a HCO3−-rich secretion. However, its volume varies and is especially small in

1371

1372 / CHAPTER 54 TABLE 54-1. Species-dependent patterns of pancreatic electrolyte secretion Species (key references)

Stimulus

Human (235), dog (236,237), and cat (150,238)

Spontaneous + Secretin + CCK + Vagus Spontaneous + Secretin + CCK + Vagus Spontaneous + Secretin + CCK + Vagus (carbachol) Spontaneous + Secretin + CCK + Vagus Spontaneous + Secretin + CCK + Vagus Spontaneous + Secretin + CCK + Vagus (carbachol)

Rat (57,239)

Rabbit (240)

Pig (241,242)

Guinea pig (211)

Hamster (243)

Volume

Maximum [HCO3−] (mM)

0(+) +++++ + + + ++ +++ ++ ++ +++ ++ +++ 0(+) +++++ ++ ++++ + +++++ +++ +++ + ++++ + ++

— 145 60 a

25 70 30 a

60 130 110 120 a

160 35 150 95 150 140 120 60 140 40 80

This table gives an idea of the response to stimuli given alone: potentiation often occurs when stimuli are given together. Most data were obtained from studies on anesthetized animals: quantitative differences may occur in conscious animals, especially in the rat, in which secretion is increased in conscious animals. aUnknown. CCK, cholecystokinin. Modified from Case RM, Argent BE (5) by permission.

the rat (about fivefold less than the cat per gram of whole pancreas). 2. There is a reciprocal relation between HCO3− and Cl− concentrations: as secretory rate increases, so does HCO3− concentration, with a corresponding reduction in Cl− concentration. At maximal flow rates, HCO3− concentration peaks around 140 mM in most species except the rat, where the maximum is about 70 mM. 3. The response to cholecystokinin (CCK) is very variable. In dog, cat, and human, CCK on its own evokes little or no fluid secretion. In the guinea pig, it evokes a copious, HCO3−-rich secretion, most probably from the ducts. In the rat, it evokes a Cl− rich secretion from acini. 4. The response to vagal stimulation also varies. In those species (guinea pig, pig) in which the vagus nerve contains many VIPergic neurons, stimulation evokes a brisk, HCO3−-rich secretion (presumably from the ducts). In other species, the effect of vagal (cholinergic) stimulation is less clear. 5. Although there are few reliable data concerning secretion in the mouse pancreas, our observations on isolated ducts suggest that the mouse is similar to the rat (4). In conclusion, the patterns of pancreatic electrolyte secretion observed in laboratory species that are now most commonly studied differ from those in species that were

studied previously (dog, cat) and that are closer to the pattern seen in humans. These differences are most marked in the rat (and probably also the mouse) for reasons that are becoming increasingly clear. This is obviously important in interpreting physiologic studies on pancreatic ducts, many of which have used rat or mouse tissue. It is also probably important (but usually ignored) when devising models of pancreatic disease, most of which involve the use of rats or mice. In addition to these classical stimulatory mechanisms involving secretin, CCK, and the vagus nerve, many other candidate stimulatory and inhibitory mechanisms undoubtedly influence duct cell secretion. We have previously documented these at length (5), and the more significant mechanisms are considered later in this chapter.

STRUCTURAL BASIS OF SECRETION The pancreas develops from two endodermal outgrowths of the primitive gut, the dorsal and ventral pancreatic buds, which fuse and give rise to the branched, ductal mass characteristic of an exocrine gland. In this process of development, duct cells give rise to acinar and islet cells. After pancreatic morphogenesis is complete, duct cells retain a limited capacity to form new duct cells and to differentiate into islet cells, but not into acinar cells (6,7). Scanning electron micrographs of

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1373 Na+

Na+ Concentration (mmol/l)

120

Cl−

Cl−

HCO−3

80

Cl− 40

HCO−3

HCO−3 K+

0 0

K+

K+

4

A

0 4 0 Secretory rate (µl/g/min)

B

4

8

C

160

Concentration (mmol/l)

Na+

Na+

Na+ Na+ + K+− Cl−

Na+ + K+− Cl−

120

80

40 Cl−

Cl− K+

0 0

20

40

K+

60 0 Secretory rate (µl/min)

D

20

40

E

FIG. 54-1. Volume and ionic composition of pancreatic juice. Electrolyte composition of pancreatic secretion in anesthetized rat stimulated by secretin (A), cerulein (B), or a combination of secretin and cerulein (C). These curves are derived from data in Sewell and Young (57). Electrolyte composition of pancreatic secretion in anesthetized guinea pig stimulated with secretin (D) or cholecystokinin octapeptide (CCK-8) (E). The broken line represents residual anions (Na+ + K+ − Cl−) and is almost all bicarbonate. (A–C: Reproduced from Case and Argent [50], by permission; D, E: Modified from Padfield and colleagues [211], by permission.)

the ductal system in the rat pancreas are shown in Figure 54-2. Egerbacher and Bock (8) have reviewed the structure of the ductal tree. The terminal branches of this tree are called intercalated ducts. Cells comprising the distal end of each intercalated duct line an acinar lumen, where they are known as centroacinar cells. There is some evidence that intercalated ducts can link several acini in series (9,10). Intercalated ducts connect with intralobular ducts that run within lobules of pancreatic tissue. These, in turn, empty into interlobular ducts (running between lobules), which join the main pancreatic duct. In rat, mouse, and hamster, interlobular ducts empty into a common bile/pancreatic duct (11). In all species studied, unmyelinated nerves travel in the lamina propria of all ducts. Nerve endings are found near the basal surface of the ductal epithelium, from which they are almost always

separated by a basal lamina (12). These fibers are largely cholinergic or adrenergic (12,13), although VIPergic fibers also are common. A variety of other peptidergic neurons also have been observed using immunocytologic techniques (see Table 2 in Case and Argent [5]). For cell number, centroacinar and ductal epithelial cells comprise about 11% of total pancreatic cells in the rat (14), of which 80% are intralobular. However, because these cells are small, they account for an even smaller proportion of gland volume—4% in guinea pig (15) and probably even less in the rat (6). Assuming that ductal epithelial cells are solely responsible for HCO3− and fluid secretion, depending on species, they can secrete their own volume in about 2 minutes. Despite this impressive performance, they look rather unimpressive in structural studies in a large variety of species (there are several reviews in the literature [2,8], including

1374 / CHAPTER 54

a

b FIG. 54-2. Scanning electron micrographs showing the ductal system of the rat pancreas. (a) Small proximal ducts (arrows) have a characteristic smooth basolateral surface and branch repeatedly without changing diameter. Arrowheads indicate junctions of the proximal ducts with the acini (A). (b) Larger distal ducts (D) have an uneven basal surface and collect secretions from the small proximal ducts. Scale bars = 20 µm. (Reproduced from Takahashi-Iwanaga and colleagues [18], by permission.)

human [12,16], rat [10,13,17,18], and cat [8]) (Fig. 54-3). Indeed, misled by this simple structure, Ferraz de Carvalho (19) wrongly concluded as recently as 1980: “Considering ultrastructural features as a whole, we cannot ascribe to the epithelial cells of the small ducts [of mink] a significant role in the phenomena of secretion and absorption.” Thus, centroacinar cells and the epithelial cells of intercalated and intralobular ducts (sometimes called principal cells) have a similar rather simple structure. They are rather flattened cells containing a sparse endoplasmic reticulum/Golgi complex (as would be expected), but they are also not especially endowed with mitochondria, and intracellular and intercellular canaliculi are absent (see Fig. 54-3). Proceeding down the ductal tree into the interlobular ducts, principal cells become more columnar and are joined by other specialized cells capable of secreting mucus and a variety of peptides (8,12,13,20). A reexamination of the ductal tree in

the rat also has demonstrated the existence in interlobular ducts of secretory canaliculi opening into the duct lumen, as well as lateral processes for cellular interdigitation (18) (see Fig. 54-3). Although principal duct cells look superficially similar throughout the ductal tree, there is now clear evidence of functional differences in duct categories, both within and between species. Thus, for example, in the human pancreas, the key membrane transport proteins involved in HCO3− and water secretion appear to be expressed most highly in the intercalated ducts (21–25). However, in the rat, immunolocalization data (25–28) and the ultrastructural features just described (18) suggest that the interlobular ducts may be more important as sites of secretion in this species. Complexity is further enhanced by the finding that distal sections of the ductal tree may also be sites of HCO3− reabsorption or “salvage” (29).

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1375

a

b FIG. 54-3. Fine structure of rat pancreatic ducts as shown by transmission electron microscopy. (a) Proximal duct. Note that few organelles are present in the cytoplasm of the cells and the lateral plasma membranes are smooth (arrows). A indicates acinar cell. (b) A distal duct running in a pancreatic lobule. Note the folding of the lateral plasma membrane (arrows) and that the cells contain numerous mitochondria (M). Arrowheads in the lumen indicate secretory canaliculi containing numerous microvilli. Occasionally, lateral processes of a duct cell (P) distort the basal region of a neighboring cell. Scale bar = 2 µm. (Reproduced from Takahashi-Iwanaga and colleagues [18], by permission.)

ADVANCES IN STUDYING DUCT CELL PHYSIOLOGY Until the mid 1980s, essentially all studies of pancreatic HCO3− secretion were performed on intact animals, usually dog, cat, or rabbit, or on whole-gland preparations isolated from the same species, as we have reviewed previously (3). However, in 1986, our first physiologic study on ducts isolated from rat pancreas heralded a new era in the analysis of pancreatic duct cell secretion (30). It led to the publication in 1988 of four innovative studies using this preparation. 1. Membrane potential measurements in microperfused ducts revealed the presence of a secretin-stimulated

Cl− conductance and a Cl−-HCO3− exchanger in the apical membrane (31). 2. Similar studies identified a K+ conductance and an Na+ -H+ exchanger (NHE) in the basolateral membrane (32). 3. Patch-clamp studies defined the properties of the apical Cl− channel (subsequently identified as cystic fibrosis transmembrane conductance regulator [CFTR]) (33). 4. Intracellular pH (pHi) measurements confirmed the presence of NHE and Cl−-HCO3− exchangers (34). Together with previous work on isolated glands, these studies led to the model of rat pancreatic HCO3− secretion that appeared in the previous edition of this book (35) and that is reproduced in this chapter as Figure 54-7. In the

1376 / CHAPTER 54

A HCO−3 /CO2

9.0

Forskolin

8.5

Luminal pH

8.0

7.5

7.0

6.5 0

5

B

10 Time (min)

15

20

FIG. 54-4. Measurement of HCO3− secretion by isolated pancreatic ducts. (A) Interlobular duct isolated by collagenase digestion and microdissection from guinea pig pancreas. The ends of the duct have sealed during overnight tissue culture. Glass pipettes are used to hold the duct and fill it with a weakly buffered solution containing the pH-sensitive fluoroprobe 2′7′-bis(2-carboxyethyl)5(6)-carboxyfluorescein (BCECF)-dextran. (B) Changes in luminal pH in a duct filled with a nominally Cl−-free solution. The rapid increase in luminal pH after stimulation with 1 µM forskolin results from Cl−-independent HCO3− secretion to the lumen. (Reproduced from Ishiguro and colleagues [39], by permission.)

previous edition, we state that “it is rather difficult to envisage how the secretory mechanism shown [in that Figure] could generate juice HCO3− concentrations of 140-150 mM which are typical of [other species]…Either these species use a completely different mechanism to secrete HCO3−, or their duct cells have additional transport processes that can raise luminal HCO3− and lower luminal Cl−.” This latter conclusion was not far from the mark. However, as discussed in this chapter, it is the rat that has additional transport processes that reduce luminal HCO3−, rather than the other way round. Faced with this problem (i.e., how to generate an HCO3− concentration of 140 mM), we decided to begin studies on the guinea pig pancreas. At the same time, we and others adopted new experimental techniques that have led to significant progress in solving the problem. Particularly valuable have been intracellular fluorescent indicators for pHi, Na+ (36),

and Cl− (37), which, combined with simultaneous microperfusion of the duct lumen (38), have helped in the identification and localization of transporters to the apical and basolateral membranes. By injecting fluorescent probes for pH and Cl− into the lumen of isolated ducts (39), we also have been able to measure net HCO3− secretion directly (Fig. 54-4). In our initial studies on isolated ducts, analysis of secretory rate was restricted to measuring the volume of fluid accumulated in the duct after 1 hour (30). Refinements using fluorescent imaging (40) and video microscopy (4) have allowed us to record secretion rate continuously (Fig. 54-5), whereas concurrent fluorometric measurements of fluid secretion and luminal pH (40) have allowed changes in HCO3− secretion to be followed. Much progress also has been made in the identification, localization, and characterization of membrane transport proteins. Identification has been achieved by cloning and

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1377

200 µm

A

B

29947

36031

C

D

Forskolin

Relative luminal volume

1.35 1.3 1.25 1.2 1.15 1.1 1.05 1 HCO−3

Hepes

E

0

5

10

15 Time (min)

20

25

30

FIG. 54-5. Measurement of fluid secretion by isolated pancreatic ducts. (A, B) Bright-field images of a sealed interlobular duct segment from guinea pig pancreas before and after stimulation with forskolin. (C, D) The increase in luminal volume is calculated from the increase in luminal area as determined by image analysis. (E) Changes in relative luminal volume (normalized to initial volume) showing the HCO3− dependence of fluid secretion in guinea pig pancreatic ducts. (Reproduced from Fernández-Salazar and colleagues [4], by permission.)

reverse transcriptase-polymerase chain reaction (RT-PCR), even from isolated ducts (26). Immunohistochemical localization has been achieved using isoform-specific antibodies and has been fruitful in identifying and localizing ductal transporters, especially in less accessible tissue such as human pancreas (Fig. 54-6). Characterization of individual transporters and channels has progressed through the use of heterologous expression systems (e.g., Xenopus oocytes and HEK293 cells), and these also have been used to study interactions between coexpressed transport proteins (41). Cell lines derived from human ductal adenocarcinomas offer the possibility of Ussing chamber experiments (42,43) and the chance to evaluate the effects of genetic manipulation.

However, most are probably derived from the larger ducts of the human pancreas (44) and may therefore have a different phenotype from cells in the proximal part of the ductal tree, which, as discussed earlier, are thought to be the major site of HCO3− secretion in human pancreas. Notwithstanding these phenotypic differences, primary cultures from main duct epithelium may also be helpful. Bovine duct cells produce confluent monolayers that respond to secretin (45,46), whereas canine duct cells have been grown successfully without transformation in long-term culture on filters (47,48). Immortalized pancreatic cell lines also have been obtained from both normal and CF mice using the ImmortoMouse transgene (49).

1378 / CHAPTER 54

B

E

C

A

D

F

FIG. 54-6. Immunohistochemical localization of transporters in human pancreas. (A) Peroxidase reaction product (brown) showing the localization of aquaporin 1 (AQP1) water channels at the luminal and basolateral membranes of centroacinar cells, intercalated ducts, and intralobular ducts. (B–D) Immunofluorescence double labeling showing AQP1 (B, green) and cystic fibrosis transmembrane conductance regulator (C, red) and their colocalization (D, yellow) at the apical membrane of intercalated ducts. (E) Localization of pancreatic sodium-bicarbonate cotransporter (pNBC1; green) in intercalated ducts (red: actin; blue: nuclei). (F) pNBC1 expression at the basolateral membrane (arrowheads) of a small interlobular duct (L=duct lumen). (See Color Plate 30.) (A–D: Modified from Burghardt and colleagues [21], by permission; E: Reproduced from Satoh and colleagues [25], by permission; F: Reproduced from Marino and colleagues [23], by permission.)

MECHANISMS OF DUCTAL ELECTROLYTE SECRETION The particular challenge in understanding secretion in the pancreatic duct is to establish how the ductal epithelial cells in some species secrete HCO3− ions at concentrations as high as 140 mM. Early studies on whole-gland preparations of the pancreas (see reviews by Case and Argent [2,50], Kuijpers and De Pont [51], Schulz [52], and Novak [53]) showed that most of the HCO3− in pancreatic juice was derived from plasma, rather than from cellular metabolism. Furthermore, because HCO3− secretion occurred against an electrochemical gradient, it had to be an active process. Its dependence on Na+, K+, and Cl− and its sensitivity to ouabain and other transport inhibitors suggested important roles for Na+,K+-ATPase, Na+-H+, and Cl−-HCO3− exchangers and carbonic anhydrase (CA), and it also was shown that secretin-evoked secretion was mediated by an increase in intracellular cyclic adenosine monophosphate (cAMP).

Electrophysiologic studies performed in the late 1980s on duct segments isolated from rat pancreas (31,33) demonstrated a crucial role for a cAMP-activated Cl− conductance in the apical membrane, subsequently identified as CFTR (54). These and other electrophysiologic studies (32), together with early fluorometric measurements of intracellular pH (34), led to the model for ductal HCO3− secretion shown in Figure 54-7. In this model for HCO3− secretion by the rat duct cell, CO2 enters the cell by simple diffusion through the basolateral membrane and is hydrated by CA. Intracellular HCO3− is generated by the extrusion of protons back across the basolateral membrane via NHE. The HCO3− ions are then secreted across the apical membrane by Cl−-HCO3− exchange at a rate that depends on the availability of luminal Cl−. This, in turn, is regulated by efflux of Cl− via the cAMP-activated Cl− channel, which acts as the main control point for regulation by secretin. The resulting transfer of negative charge across the apical membrane to the lumen is balanced by a net efflux

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1379 Tight junction Apical membrane

Basolateral membrane CO2

CO2 H+

H2O CA

HCO−3

Na+

Cl− 3Na+

2K+

~

extrusion of H+, as described earlier, or alternatively by basolateral uptake of HCO3− itself. During the 1990s, it became apparent that H+ extrusion may be achieved by both NHE and H+-ATPase activity. More importantly, it appears that HCO3− uptake also occurs by Na+-HCO3− cotransport. It also should be remembered that significant amounts of Cl− are secreted by the exocrine pancreas, much of which may well derive from the ductal epithelium. This too requires a basolateral uptake mechanism, the identity of which has been neglected until quite recently. Na+-H+ Exchangers

Cl− K+

Na+ LUMEN

FIG. 54-7. Original model for electrolyte secretion by pancreatic duct epithelium. HCO3− is secreted across the apical membrane in exchange for Cl−, which is supplied to the duct lumen by an apical membrane Cl− conductance. Intracellular HCO3− is generated from CO2 through the action of carbonic anhydrase (CA), and H+ is extruded across the basolateral membrane by Na+-H+ exchange. Figure is based on electrophysiologic studies of interlobular ducts isolated from rat pancreas (31–33).

The existence of a basolateral NHE in pancreatic duct cells is supported by measurements of pHi, which show an Na+dependent, amiloride-sensitive recovery from intracellular acidification, usually induced experimentally by application of a short pulse of NH4+ ions, in the absence of extracellular HCO3− (34,36,38,59,60). Identification of the exchanger as NHE1, a ubiquitous member of the SLC9 gene family (61), follows from pharmacologic studies using the isoformspecific inhibitor HOE694 (29) and from immunohistochemistry using isoform-specific antibodies (29,62). NHE1 is widely associated with a housekeeping role in pHi regulation and undoubtedly plays this role in the regulation of duct cell pHi, but its contribution to HCO3− secretion varies significantly among species and between the unstimulated and secretin-evoked conditions. Inhibition of the exchanger with amiloride has no effect on secretin-evoked secretion in rabbit (63) or pig ducts (64,65) and relatively little effect in rat (66), cat (67), and guinea pig (36,39). These findings have led to the search for other basolateral mechanisms that could contribute to the supply of intracellular HCO3−.

of K+ ions through basolateral K+ channels. Cl– also creates a transepithelial potential difference that drives the paracellular flow of cations, predominantly Na+, through the tight junctions to the lumen. Intracellular K+ is replenished, and the inward gradient for Na+ is maintained, by the continuous activity of the basolateral Na+,K+-ATPase. This basic scheme continues to hold well, and our computational modeling studies (55,56) have shown that it accounts well for many of the characteristics of secretion by the pancreatic ducts of the rat, a species in which maximal secreted HCO3− concentrations are about 70 mM (57). However, as mentioned in the previous section, the model cannot adequately account for the greater concentrations of HCO3− (≥140 mM) secreted by the pancreatic ducts of species such as the guinea pig, cat, dog, and human (5). Significant progress has been made in establishing the molecular identities of the transporters involved in secretion and the interactions that occur between them. But, as we have explained in detail elsewhere (58), the identity of the apical exit pathway for HCO3− ions in species that secrete 140 mM HCO3− remains uncertain.

In the pig and, to a lesser extent, other species, a basolateral H+-ATPase also contributes to proton extrusion (68). Experimentally, the activity of a proton pump is observed as a Na+- and HCO3−-independent recovery of pHi after intracellular acidification (69). Its sensitivity to bafilomycin A1 and its association with tubulovesicles that become inserted into the basolateral membrane after stimulation with secretin (70,71) suggest that it is a vesicular-type H+-ATPase or V-ATPase (72). Although this type of pump activity can be demonstrated in the ductal epithelia of several species after stimulation (38,59), it is not necessarily active in the range of pHi values that exist during normal secretion. Inhibition of the H+-ATPase undoubtedly causes a significant reduction in secretion in the pig (73), but it has relatively little effect in other species (39,40).

Basolateral Transport Mechanisms

Na+-HCO3− Cotransporters

HCO3−

In principle, uptake across the basolateral membrane could be achieved either by hydration of intracellular CO2 and

H+-ATPase

Duct cells in several species use a basolateral Na+-HCO3− cotransporter to maintain intracellular pH (36,38,59,60,69). This is seen as an Na+- and HCO3−-dependent recovery of pHi

1380 / CHAPTER 54 after acid loading, which is insensitive to amiloride analogues, but inhibited by stilbene derivatives such as H2DIDS (dihydro-4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). The Na+-HCO3− cotransporter (NBC1) expressed in the pancreas (74,75) is now known to be a splice variant of the NBC1 originally cloned from the kidney (76). It is a member of the SLC4 family of HCO3− transporters (77). Immunohistochemistry shows that the “pancreatic” pNBC1 variant is expressed at the basolateral membranes of intercalated, intralobular, and interlobular ducts in the pancreas (23,25, 75,78) (see Fig. 54-6E and F), and it is also present in many other tissues (77). The cotransporter carries two HCO3− ions into the duct cell with every Na+ ion (79), and its electrogenicity probably helps to maintain the negative membrane potential required to drive HCO3− and Cl− ions across the apical membrane (80). Separate and combined application of amiloride and H2DIDS during secretin-evoked secretion indicate that, in guinea pig duct cells at least, this transporter is responsible for as much as 75% of the basolateral HCO3− uptake, whereas the NHE contributes significantly less to bicarbonate loading (36,40). However, as already indicated, the relative contributions of the three HCO3− uptake pathways described here may vary significantly among species (58,81). Anion Exchangers Also present at the basolateral membrane of pancreatic duct cells is a Cl−-HCO3− exchanger. Its activity is detected as a DIDS-sensitive increase in pHi, caused by HCO3− entry, when Cl− ions are removed from the fluid bathing the basolateral membrane (38,60). The transporter is probably AE2 (82,83), which mediates a neutral 1:1 exchange of anions and, like pNBC1, is a member of the SLC4 gene family (77). If active during secretin stimulation, the exchanger would allow HCO3− ions taken up across the basolateral membrane to leak back out of the cell, thus dissipating the supply of HCO3− ions for secretion. In doing so, however, it would bring Cl− ions into the cell, thereby enhancing the driving force for apical Cl− secretion, as appears to be the case when rat duct cells are stimulated with bombesin (84). Not surprisingly, in species such as the guinea pig, which secrete high concentrations of HCO3−, secretin stimulation appears to inhibit the basolateral anion exchanger (37). Cation-Chloride Cotransporters Whereas ductal secretion in the guinea pig is totally dependent on HCO3− (40), ducts of other species, including the rat and mouse, can secrete a Cl− rich fluid in the absence of HCO3− (4). This process is blocked by bumetanide and is therefore likely to be driven by a member of the SLC12 cation-chloride cotransporter superfamily (85), most probably a Na+-K+-2Cl− cotransporter (NKCC1) located at the basolateral membrane. Although bumetanide has little effect on secretion by the pig pancreas (64), our studies with bumetanide suggest that NKCC1 contributes about 40% of the fluid secreted by rat and mouse ducts (4), perhaps

explaining why these species do not secrete particularly high HCO3− concentrations. K+ Channels As in other secretory epithelia, basolateral K+ channels play a vital role in maintaining the membrane potential of the ductal cells during secretion. The basolateral efflux of K+ ions helps to balance the depolarizing effect of Cl− and HCO3− efflux across the apical membrane, as well providing a sink for the continuous uptake of K+ by the basolateral Na+,K+ATPase. Despite their obvious importance in secretion, little is known about the molecular identity or the functional properties of pancreatic K+ channels. In unstimulated rat duct cells, the basolateral K+ conductance is pH dependent and more sensitive to block by Ba2+ than tetraethylammonium (TEA) (32,86). Our data suggest that this conductance may be attributable to an 82 pS channel that has been detected in single-channel patch-clamp studies (87). Stimulation with secretin leads to a marked increase in basolateral K+ conductance, although the hyperpolarizing effect of this is usually masked by the depolarizing effect of the accompanying increase in apical Cl− conductance (88,89). We have shown that this secretin-induced increase in basolateral K+ conductance is largely due to the activation of maxi-K+ channels by protein kinase A (PKA) (90). These channels are also Ca2+ sensitive and voltage dependent, with a linear current-voltage (I-V) relation and a conductance of about 200 pS. They are blocked by TEA, as well as by Ba2+ (87), both of which completely inhibit secretin-evoked fluid secretion in isolated rat ducts (84). In slow whole-cell patch recordings from rat duct cells, the endogenous resting K+ conductance was also shown to be inhibited by purinergic agents (91,92), suggesting that purinoreceptor stimulation could oppose secretion (see Inhibitory Pathways). Studies on other species are lacking, although a number of groups have identified Ca2+-activated basolateral K+ conductances in a variety of cultured pancreatic cell lines (43,93–95). In cultured dog pancreatic duct cells (94) and the human pancreatic duct cell lines CAPAN-1 (43) and CFPAC-1 (93), pharmacologic evidence suggests that the underlying channels are the charybdotoxin-sensitive maxi-K+ channels, whereas in the human pancreatic duct adenocarcinoma cell line (HPAF) cells, the conductance was more consistent with the presence of small- and intermediateconductance, Ca2+-activated K+ channels (95). RT-PCR indicated the expression of KCNN4 in these cells, which is consistent with the pharmacologic data (95). Na+,K+-ATPase With the possible exception of species such as the pig, in which an H+-ATPase also makes a significant contribution, the primary driving force for electrolyte secretion in pancreatic duct cells derives from the basolaterally located Na+,K+ATPase. Various histochemical techniques show strong basolateral labeling for this protein in the intercalated ducts

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1381 in the cat (96) and dog pancreas (97) and the interlobular ducts in the rat (98). The function of the pump is clearly to maintain the inward Na+ gradient that drives the basolateral uptake of HCO3− and Cl− by NHE1, pNBC1, and NKCC1. Its electrogenicity may also make a small contribution to the maintenance of the membrane potential (32). Water Channels Because a large fraction of the fluid output of the exocrine pancreas is believed to derive from the ductal epithelium, which represents just a small fraction of the total cellular mass of the organ, it would not be surprising to find aquaporin (AQP) water channels expressed in these cells to enhance the transepithelial water permeability, and thereby facilitate the production of a near-isotonic secretion. Although AQP8 was first identified in the pancreas and liver (99), this isoform appears to be confined to the apical membrane of pancreatic acinar cells (100). Only recently has it become clear that the isoform expressed in the duct cells is the fairly ubiquitous AQP1. This is present in both the basolateral and apical membranes of the interlobular ducts of the rat (26,27), but is more strongly expressed in the intercalated and intralobular ducts of the human pancreas (21) (see Fig. 54-6A). These distribution patterns match closely those of CFTR (see Fig. 54-6B to D), suggesting that the principal site of fluid secretion in the ductal tree may differ among species.

Apical Transport Mechanisms Two alternative exit pathways have been proposed for HCO3− secretion across the apical membrane of pancreatic duct cells: exchange with luminal Cl− via a Cl−-HCO3− exchanger, and diffusion through an anion channel. There now appears to be good evidence that both mechanisms exist, although their relative contributions may differ among species and possibly among different segments of the ductal tree or at different stages in the secretory response (58). Crucial evidence that HCO3− could be secreted in the absence of luminal Cl− came from our micropuncture experiments on secretin-stimulated guinea pig ducts (39,40) (see Fig. 54-4). Anion Exchangers The observed dependence of HCO3− secretion on Cl− led to the idea that secretion might involve a Cl−-HCO3− exchanger (84,101,102). Its localization at the apical membrane was deduced first from electrophysiologic data (31), and subsequently confirmed by pHi measurements in microperfused ducts, which showed a DIDS-sensitive alkalinization after substitution of luminal Cl− (38,60). The likely involvement of the apical exchanger in HCO3− secretion was supported by observation of a marked increase in activity after stimulation with secretin or forskolin (41,103), and a reduction in

activity after exposure to substance P (SP) (104), which inhibits fluid secretion (105,106). The increase in activity of the apical anion exchanger during secretion appears to be strongly dependent on interactions with CFTR (107,108), and interestingly, the exchanger is successfully stimulated by a number of CFTR mutants that lack Cl− channel activity (41). This appears to correlate with a good retention of pancreatic function in patients carrying those mutations (109). It appears likely that the apical exchanger is not one of the AE group of SLC4 anion exchangers, but is instead a member of the SLC26 family of sulfate transporters (110). This is a large and diverse family of exchangers with broad anion selectivity and variable stoichiometries. Prime candidates for the exchanger in the pancreatic duct are SLC26A3 (down-regulated in adenoma [DRA]) (111), which is expressed apically in the larger ducts of the mouse pancreas (83), and SLC26A6 (putative anion transporter-1 [PAT1]), which is found in the interlobular ducts of human pancreas and in pancreatic cell lines (83,112,113). SLC26A6 expressed in oocytes supports DIDS-sensitive Cl−-HCO3− exchange (114,115) and interacts with CFTR both directly (116,117) and through PDZ-binding domains, linking them both to scaffolding proteins (107,118). As mentioned at the beginning of this chapter, it has always been difficult to envisage how a Cl−-HCO3− exchanger would be able to secrete HCO3− ions into a luminal fluid containing 140 mM HCO3− (55,56). Under such conditions, a neutral 1:1 exchanger would be expected to reverse and mediate HCO3− reabsorption rather than secretion. The discovery that SLC26A3 and SLC26A6 are electrogenic (119,120) raised the possibility that this would help to account for the secretion of 140 mM HCO3− (116). However, our calculations suggest that even with the more favorable 1Cl−:2HCO3− stoichiometry proposed for SLC26A6, the exchanger would be operating close to equilibrium (i.e., very slowly) during the secretion of 140 mM HCO3− (58), and there is some experimental evidence to support this (60). Taken together with the observation that guinea pig ducts will secrete HCO3− into a luminal fluid nominally free of Cl− (39,40), we have to conclude that neither of the SLC26 transporters provides the main route for HCO3− efflux across the apical membrane during maximal secretion. Cystic Fibrosis Transmembrane Conductance Regulator The existence of an apical, cAMP-stimulated Cl− conductance was first demonstrated in interlobular ducts isolated from rat pancreas (31,33). Our patch-clamp studies showed small-conductance Cl− channels in this membrane with the I-V relation, anion selectivity, and pharmacology characteristic of CFTR (33,54,121). Similar channels have since been reported in a number of other species and cell lines (47,122–126), and immunohistochemistry has confirmed the expression of CFTR protein at the apical membrane of intercalated duct cells in human pancreas (22,24) (see Fig. 54-6C and D) and intralobular and interlobular ducts in rat pancreas (28).

1382 / CHAPTER 54 The profound pancreatic dysfunction associated with CF undoubtedly indicates a central role for CFTR in pancreatic secretion. However, it has become unclear whether the role of CFTR is primarily to provide a source of luminal Cl− to support Cl−-HCO3− exchange, as originally proposed (31,33), or whether its main role is to activate the apical anion exchanger (41,107), as described earlier. The observation that HCO3− secretion can occur in the nominal absence of Cl− (39,40) and the conclusion (see earlier) that no known anion exchanger could secrete 140 mM HCO3− have led to the evaluation of a third possibility, namely, CFTR itself acts as a HCO3− conductance. Measurements of intracellular Cl− and HCO3− concentrations and membrane potential in guinea pig duct cells indicate that the electrochemical gradient for HCO3− would favor diffusive efflux through an apical channel even during maximal secretion into 140 mM luminal HCO3− (80). In contrast, the permeability of the CFTR channel to HCO3− has always been considered too low relative to Cl− (124,127–129) to make this a realistic possibility. Some work, however, suggests that the Cl−-HCO3− selectivity of CFTR may be regulated by various intracellular and extracellular factors (130). In particular, low extracellular (luminal) Cl− concentrations may shift the selectivity of CFTR toward HCO3− (131), as well as limit the activity of CFTR, through a novel negative feedback mechanism (132). The effect of changing luminal [Cl−] on CFTR activity is illustrated in Figure 54-8. This latter effect will help to limit apical membrane depolarization caused by the large apical membrane anion conductance and, together with the shift in CFTR selectivity, will favor HCO3− efflux (secretion) through CFTR.

Furthermore, because the ducts of species that secrete high HCO3− concentrations may lack basolateral uptake mechanisms for Cl−, the competition between Cl− and HCO3− for efflux through CFTR may shift strongly in favor of HCO3− once intracellular and luminal Cl− concentrations have fallen to the low values that are observed during maximal secretion (4,133). Ca2+-Activated Cl− Channels Our whole-cell current recordings from mouse duct cells have showed a Ca2+-activated Cl− conductance in the apical membrane markedly larger than that caused by CFTR (Fig. 54-9) (123,134,135). The presence of these Ca2+-activated Cl− channels (CaCCs) in the duct cell could explain why CF null mice show rather little pancreatic pathology (136). In contrast, in rat and guinea pig pancreatic duct cells, CaCC current density is much lower than CFTR current density (see Fig. 54-9). We have shown that CaCCs are also expressed in human duct cells at relatively a high density (see Fig. 54-9) (137), making them a potential therapeutic target in patients with CF (135). Activated over a physiologic range of intracellular Ca2+ concentrations (10 nM to 1 µM), CaCCs exhibit slight outward rectification when maximally activated and, unlike CFTR, are more permeable to I− than Cl−. Generally, these channels are blocked by extracellular DIDS and niflumic acid, although clear species differences do exist (135). Single-channel data are scarce, but Ho and colleagues (138), using the cultured human pancreatic cell line CFPAC (which was originally derived from a patient with CF), found that the underlying channels had a small unitary conductance

High [Cl]

Low [Cl]

1 pA 10 s

0.5 pA 1s

A

B

FIG. 54-8. External Cl− modulates cystic fibrosis transmembrane conductance regulator (CFTR) gating. Cell-free, outside-out patch-clamp data obtained from baby hamster kidney (BHK) cells stably transfected with CFTR at a membrane potential of −50 mV. Solid lines to the left of the traces represent the current level when all channels are closed. (Top left) Trace shows single-channel records obtained in the presence of 155.5 mM external Cl−. (Top right) Trace shows current records after reducing external Cl− to 35.5 mM in the same experiment. Expanded sections at higher time base also are shown to illustrate the marked change in CFTR channel activity. From analysis of open-state probability, the shift from high to low external [Cl−] reduced channel activity by ~70%. (Modified from Wright and colleagues [132], by permission.)

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1383 600

Mouse

Human G. Pig Current density (pA/pF)

Rat

CaCC

CFTR

−600

FIG. 54-9. Mean current density of cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+-activated Cl− channel (CaCC) in pancreatic duct cells from different species. The maximal stimulated current density (pA/pF) was measured at Erev ±60 mV using fast whole-cell current recordings. Whole-cell currents were obtained from I/V plots made over the voltage range ±100 mV and normalized to cell capacitance. CFTR data were taken from the following references: rat (54), mouse (123,134), and guinea pig (124); CaCC data were taken from the following sources: mouse (123,134) and human (137). CaCC data for rat and guinea pig are unpublished observations (M. A. Gray and B. E. Argent). Note that we currently do not have current density values for CFTR in human pancreatic duct cells.

of about 1 pS. In excised membrane patches, channel activity was stimulated by increasing bath (cytosolic) calcium and by purified calmodulin-dependent protein kinase II (139). Interestingly, the latter stimulation was selectively inhibited by the inositol metabolite, Ins(3,4,5,6)P4 (138,139). Inhibition of short-circuit current (Isc) by purinergic receptor–generated IP4 was previously shown to occur in basolaterally permeabilized monolayers of CFPAC cells (140), suggesting that this inositol phosphate is a physiologically relevant regulator of Ca2+-dependent anion secretion via inhibition of CaCCs. Although there is no clear consensus on the molecular identity of CaCCs, observations that Ca2+-mobilizing agonists such as acetylcholine (ACh) (141), luminal adenosine triphosphate (ATP) (142), and luminal Ca2+ (143) stimulate ductal secretion suggests that they have a significant physiologic role. Limited data indicate that they have a lower permeability to HCO3− than Cl− (135,144), but there is some evidence that they are capable of supporting HCO3− secretion in the CFPAC-1 cell line (145). K+ Channels Studies on intact rat ducts have found little evidence for any significant resting apical membrane K+ conductance (86). However, our molecular, biochemical, and electrical measurements on HPAF cells have provided clear evidence for the presence of an apical K+ conductance with properties that are consistent with TWIK-related acid sensitive K+ channel 2 (TASK-2) (95). The conductance was inhibited by clofilium, but not chromanol 293B, which are both inhibitors of cAMP-activated K+ channels. Interestingly, addition of clofilium reduced basal transepithelial Isc when

applied to the mucosal (luminal), but not the serosal (basolateral), side of the cell monolayer (95). Because TASK-2 channels are activated by extracellular alkalinization over a range that is observed in pancreatic juice (146), they could play an important role in anion secretion by offsetting the apical membrane depolarization caused by anion efflux through CFTR. Na+-H+ Exchangers Perhaps surprisingly, NHE activity has been observed at the apical membrane of main duct cells in both rat (38), bovine (147), and human pancreas (147a). Its role there is probably to effectively retrieve luminal HCO3− when secretion slows down or ceases altogether. The reabsorption of HCO3− would help to account for the relatively acidic and Cl−-rich juice that is collected from the main duct after a period of stasis (1,148–150), and perhaps serves to minimize the risk that pancreatic enzymes become activated within the ductal tree. Although both NHE2 and NHE3 have been detected by immunohistochemistry in mouse main ducts, knockout mouse data suggest that only NHE3 is active in normal animals (29). Increase of intracellular cAMP by stimulation with forskolin significantly inhibits the exchanger, and this, similar to the stimulation of the apical anion exchanger described earlier, may occur through a direct physical interaction with CFTR (151). Na+-HCO3− Cotransporters A second mechanism for the reabsorption of secreted HCO3− in the main duct of the mouse pancreas is probably provided

1384 / CHAPTER 54 by an electroneutral Na+-HCO3− cotransporter, NBCn1 (or NBC3) (152). This is another member of the SLC4 gene family with a clearly defined role in HCO3− retrieval in mouse salivary ducts (153). It too is inhibited by CFTR in coexpression studies, suggesting that there may be reciprocal control of HCO3− secretion and reabsorption through interactions among CFTR, SLC26 exchangers, NHE3, and NBCn1 linked by PDZ domains to scaffold proteins. Water Channels As already mentioned, the AQP1 water channel is present in both the basolateral and apical membranes of the interlobular ducts of the rat pancreas (26,27) and in the intercalated and intralobular ducts of the human pancreas (21). In the latter, however, there is also evidence for coexpression of AQP5 at the apical membrane, and its distribution maps closely onto that of CFTR in the ductal system, with most of the labeling concentrated in the smaller, proximal ducts (21). Interestingly, this is the isoform most closely associated with other fluid-secreting exocrine glands such as the salivary and lacrimal glands and the mucosal glands of the airways (154).

Carbonic Anhydrase The CA inhibitor acetazolamide has significant inhibitory effects on pancreatic secretion in many different species (63,150,155–162). This has normally been taken as evidence that much of the secreted HCO3− is derived from the hydration of intracellular CO2. Some work, however, suggests that this is not necessarily true, because some HCO3− transporters are thought to require a close physical association with CA to achieve their full activity (163,164). Strong interdependencies between anion exchangers and CAII and CAIV in expression systems have led to the concept of a HCO3− transport “metabolon” (165,166). The role of CA in these protein complexes may be to prevent the development of local pH gradients in the vicinity of the transporters, rather than simply to generate HCO3− from CO2 (167). The CA isozymes expressed in pancreatic duct cells are thought to include: (1) soluble CAII, which is present throughout the cytosol (168,169) and in close association with the apical membrane (170); (2) membrane-anchored CAIV with extracellular activity at the apical membrane (170,171); and (3) membrane-spanning CAIX and CAXII with extracellular activity at the basolateral membrane (172). Until the associations of these isozymes with the apical and basolateral transporters are known, the inhibitory effects of acetazolamide on HCO3− secretion in the pancreatic duct clearly should be interpreted with caution.

Paracellular Cation Transport Electrophysiologic studies indicate that the transepithelial electrical resistance of microperfused rat interlobular ducts

is approximately 50 Ω.cm2 (31,32,86). This places the ductal epithelium firmly in the “leaky” category; one consequence of this is that the transepithelial potential is never greater than a few millivolts. The low paracellular resistance also ensures that the apical and basolateral membrane potentials are closely coupled and never differ by more than a few millivolts. Thus, for example, the depolarization of the apical membrane as a result of secretin-stimulated Cl− and HCO3− efflux would be instantly transmitted to the basolateral membrane, where it would enhance the driving force for the electrogenic pNBC1 cotransporter. Although not proven, the low resistance of the paracellular pathway is probably caused by a large cation-selective permeability through the tight junction barrier, and this is believed to provide the transport pathway for the majority of the secreted cations, predominantly Na+ and, to a lesser extent, K+.

An Updated Model for Ductal Electrolyte Secretion The basic elements of the model shown in Figure 54-7 are now firmly established and account for many aspects of secretin-evoked ductal secretion. However, additional transporters, summarized in Figure 54-10, have now been identified and these as well as interactions between them help to explain how some species secrete greater HCO3− concentrations than others. If we consider first the poorer HCO3− secretors such as the rat and mouse, it is clear that the presence of a basolateral NKCC1 cotransporter (4), and perhaps the basolateral AE2 exchanger, enables their ducts to sustain the uptake of Cl−, as well as HCO3−. The resulting driving force for Cl− secretion and the preferential selectivity of CFTR for Cl− would therefore explain why the secreted HCO3− concentration never increases to greater than 70 mM in these species. At the apical membrane, HCO3− efflux is almost certainly achieved mainly by Cl−-HCO3− exchange, probably via an electrogenic SLC26 exchanger operating in parallel with and stimulated by the CFTR Cl− channels. Although it has been suggested that species such as the guinea pig secrete greater HCO3− concentrations as a result of sequential modification of a primary secretion as it flows through the ductal system (56,116,119), studies on isolated duct segments indicate that they are capable of generating a HCO3−-rich fluid at a single site (40). But it appears likely that this is not achieved instantly after stimulation. At first, the secretory mechanism is probably similar to that already described for the rat and mouse and is totally dependent on luminal Cl−. In the unstimulated condition (Fig. 54-11A), Cl− uptake by the basolateral AE2 ensures that there is a mixed secretion of HCO3− and Cl−. After stimulation (see Fig. 54-11B), the suppression of basolateral Cl− uptake ensures that the efflux of Cl− across the apical membrane diminishes and the luminal concentration of HCO3− steadily increases. As it does so, the apical anion exchanger approaches equilibrium, and the low luminal Cl− concentration causes CFTR to switch its selectivity in favor of HCO3− ions (131) (see Fig. 54-11C). The driving force for HCO3− is thereafter maintained by the

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1385

LUMEN

∼

V-ATPase

2HCO3− HCO3−

AE2

Cl−

AQP1

H2O

Cl−

K+

TASK2

Na+

NHE3

∼

Na+ NBCn1

AQP1 AQP5

Cl−

Cl−

HCO3− H2O

HCO3−

HCO3−

H+

K+

Low [HCO3− ] L

Unstimulated

2HCO3−

Na+ 3Na+

Cl−

A CaCC

2Cl−

Maxi-K etc.

CFTR

Cl−

Na+ K+

Na, K-ATPase 2K+

SLC26A6

Cl− HCO3−

Na+

pNBC1

Cl−

H+

Cl− HCO3−

HCO3−

Na+

NKCC1

2HCO3−

H+

NHE1

HCO3−

Na+

Cl−

Low [HCO3− ] L

Stimulated

B

Na+

Na+

FIG. 54-10. Electrolyte transporters expressed in pancreatic duct epithelium. The molecular identities of the channels and transporters proposed in the original model (see Fig. 54-7), and most of those characterized in subsequent functional studies, have now been established with reasonable certainty. Not all of the transporters shown in this diagram are expressed in all segments of the duct or in all species. In particular, the apical Na+-H+ exchanger (NHE3) and Na+-HCO3− cotransporter (NBCn1) are confined mainly to the larger interlobular and main ducts, where they are thought to be involved in the “salvage” of luminal HCO3− when secretion ceases. AQP1, aquaporin 1; CaCC, Ca2+-activated Cl− channel; CFTR, cystic fibrosis transmembrane conductance regulator; NKCC1, Na+-K+-2Cl− cotransporter; pNBC1, pancreatic sodiumbicarbonate cotransporter; SLC, solute carrier; TASK2, TWIK-related acid sensitive K+ channel 2.

basolateral pNBC1 and H+ extruders, whereas Cl− secretion declines to a low level simply through the lack of basolateral uptake.

REGULATION OF DUCTAL SECRETION In vivo studies on whole animals have shown that the physiologic control of pancreatic secretion in response to a meal is a complex process that involves the interaction of multiple hormonal and neural pathways (see reviews by Case and Argent [5] and Nathan and Liddle [173], as well as Chapter 55). Here, we focus on in vitro studies, using either isolated ducts or duct cell cultures, that have provided information about the neurotransmitter and hormonal receptors

HCO3−

2HCO3−

Cl− HCO3−

Cl− Stimulated

HCO3− High [HCO3− ] L

C FIG. 54-11. Sequential mechanism for the secretin-evoked generation of high luminal HCO3− concentrations. (A) In unstimulated ducts, a mixed secretion of Cl− and HCO3− is generated through the basolateral uptake of Cl− and HCO3− and the parallel operation of the apical anion exchanger and cystic fibrosis transmembrane conductance regulator (CFTR). (B) After stimulation, increased activity of the luminal transporters and basolateral pancreatic sodium-bicarbonate cotransporter (pNBC1), together with suppression of basolateral Cl− uptake, lead to increasing HCO3− and decreasing Cl− concentrations in the lumen. (C) The decline in intracellular and luminal Cl− favors HCO3− secretion via CFTR, whereas the apical anion exchanger is inhibited or approaches equilibrium. At this point, Cl− secretion virtually ceases, and the secreted fluid becomes rich in HCO3−. For clarity, only the principal transporters thought to be involved in HCO3− secretion are shown. (Modified from Steward and colleagues [58], by permission.)

expressed on the duct cell and their associated signaling pathways. Since the previous edition of this textbook was published in 1994 (35), the number of receptor types expressed on the duct cell has grown substantially, and two new concepts have emerged for the control of ductal HCO3− and fluid secretion. First, it has become apparent that

1386 / CHAPTER 54 receptors are expressed on the apical membrane of the duct cell, and that ductal secretion can be modulated by luminal signals. Second, there is now good evidence for inhibitory control of HCO3− secretion by a variety of receptors expressed on the duct cell (Fig. 54-12).

Stimulatory Pathways Cyclic Adenosine Monophosphate Pathway Secretin, Vasoactive Intestinal Peptide, and β-Adrenergic Agonists The peptide hormone secretin is probably the most important physiologic regulator of pancreatic ductal secretion (2,5,35,50), and we have shown that the peptide stimulates fluid and HCO3− secretion from isolated rat (30,84) and guinea pig pancreatic ducts (40,174). The dose of the hormone required for a half-maximal response was about 0.5 nM in guinea pig ducts and about 10 pM in rat ducts; these values are similar to those required for half-maximal fluid secretion from the in vitro perfused rat pancreas (see Argent and colleagues [30] for more information) and for half-maximal depolarization of the duct cell basolateral membrane potential (Vbl) (88). Secretin increases cAMP

Blood

accumulation in isolated ducts (175–177), and both the electrophysiologic (31) and fluid secretory effects of secretin can be mimicked by dibutyryl cAMP and forskolin (30,39,174). The general consensus is that cAMP activates protein kinase A, which, in turn, phosphorylates CFTR in the apical membrane of the duct cell (see Mechanisms of Ductal Electrolyte Secretion section earlier in this chapter). Some species differences emerge (probably explained by species-dependent receptor expression) when attempting to correlate the effect of agonists other than secretin on duct cell cAMP with their ability to increase fluid secretion. For instance, a high dose of vasoactive intestinal peptide (VIP; 10 nM) has only a weak effect on fluid secretion from isolated rat ducts (105) and increases cAMP in rat ductal fragments only at doses in excess of 100 nM (175). However, VIP has been reported to be as effective as secretin in increasing cAMP levels in isolated guinea pig ducts (177) and in depolarizing rat duct cells (89). β-Adrenergic receptors are usually coupled to adenylyl cyclase, and isoprenaline causes a depolarization of Vbl (178) and evokes fluid secretion from rat ducts (87). However, isoprenaline has no effect on cAMP levels in guinea pig ducts (177). The crucial role of secretin-stimulated cAMP increase in the regulation of duct cell HCO3− secretion has been recognized for many years and cannot be overstated. However, the

Duct cell

Lumen

Stimulation Secretin, VIP, β-adrenergic agonists

cAMP

cGMP ACh, trypsin, histamine, ATII

CCK, bombesin

Ca2+

Guanylin, uroguanylin ATP, Ca2+

?

Inhibition Substance P ACh + secretin

PKC

AVP, ATP, 5HT*

Ca2+ (*Na+)

FIG. 54-12. Regulation of duct cell secretion by hormones and neurotransmitters. Diagram summarizes our current understanding about the expression of hormone and neurotransmitter receptors on the basolateral and apical membranes of the pancreatic duct cell. Also shown is the effect (stimulation or inhibition) of activating these receptors on duct cell secretion. Data come from experiments on either isolated ducts or primary duct cell cultures where either secretion or short-circuit current was measured. Neurotensin receptors are also present on the basolateral membrane of the duct cell, but the effect of neurotensin on ductal secretion has not been tested. The hormones and neurotransmitters are grouped to reflect the intracellular signaling pathways that they use. Question mark indicates unknown. ACh, acetylcholine; ATII, angiotensin II; AVP, arginine vasopressin; cAMP, cyclic AMP; cGMP, cyclic GMP; CCK, cholecystokinin; 5HT, 5-hydroxytryptamine; PKC, protein kinase C; VIP, vasoactive intestinal peptide.

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1387 following sections focus on more recently discovered stimulatory and inhibitory mechanisms. Cyclic Guanosine Monophosphate Pathway Guanylin and Uroguanylin Guanylin and uroguanylin are short peptides that exhibit a structural homology to Escherichia coli heat-stable enterotoxins (STA) (see Kulaksiz and colleagues [179]). In the gut, these peptides are present in enterochromaffin and mucous cells within the epithelium and are released luminally. Once in the lumen, they stimulate the guanylate cyclase C (GC-C) receptor on intestinal epithelial cells, leading to an increase in intracellular cyclic guanosine monophosphate (cGMP). The cGMP activates cGMP-dependent protein kinase (cGKII), which, in turn, phosphorylates CFTR to increase fluid and electrolyte secretion (180). It is now apparent that guanylin and uroguanylin are also present in the centroacinar and ductal epithelial cells of the human (179,181) and rat pancreas (182). Also expressed in the same cells are the other components of this cell signaling system, GC-C, cGKII, and CFTR (179,181,182). Furthermore, activation of CFTR-like Cl− currents by guanylin and STA has been observed in CAPAN-1 cells (179). Thus, the possibility exits that guanylin and uroguanylin present within the ductal epithelial cells could, via a luminal pathway, activate HCO3− secretion. However, this hypothesis has not been tested directly by examining whether luminal guanylin and uroguanylin stimulate HCO3− and fluid secretion from isolated pancreatic ducts. Moreover, the physiologic stimulus for guanylin release from duct cells into pancreatic juice is unknown; thus, the status of guanylin/uroguanylin as either a physiologic or pathophysiologic regulator of pancreatic ductal secretion remains to be established. Calcium Pathway Acetylcholine Historically, the stimulatory effect of ACh on the intact pancreas was ascribed entirely to its effect on acinar cells (because enzyme secretion also is stimulated). However, we have shown that ACh is an effective stimulant of fluid secretion from rat (141) and guinea pig ducts (174). The response to ACh is blocked by atropine (141,174), and M2 and M3 muscarinic receptors are expressed in isolated ducts (183). In fact, the density of muscarinic receptors on the duct cell is about sevenfold greater than on acinar cells (183). Because pancreatic ducts are innervated by cholinergic neurons (13), ACh is likely to play a physiologic role in the regulation of ductal fluid transport. Simultaneous activation of ductal fluid transport and acinar enzyme secretion would be physiologically advantageous for flushing enzymes along the ductal tree toward the duodenum. ACh and its analogue carbachol both increase the intracellular Ca2+ concentration ([Ca2+]i) in duct cells (38,91,141, 184–186) and cause a marked depolarization of Vbl (91). The usual [Ca2+]i response consists of an initial peak followed by

a sustained plateau, probably reflecting release of Ca2+ ions from intracellular stores and Ca2+ influx from the extracellular space, respectively (38,91,141,184,185). A study of the spatiotemporal dynamics of these [Ca2+]i signals suggested that [Ca2+]i oscillations may occur in the duct cells (186). It has been reported that pancreatic duct cells have a relatively high permeability to Ca2+, insofar as removal of extracellular Ca2+ causes a rapid decline in [Ca2+]i (91,141,184). The nature of this Ca2+ flux pathway in the basolateral membrane of the duct cell has not been established; however, either a Ca2+ channel (184) or a Na+-Ca2+ exchanger may be involved (187,188). Importantly, ACh causes a dose-dependent increase in [Ca2+]i (91,141,184), and the dose–response curves for AChinduced fluid secretion and the increase in [Ca2+]i are similar (141). Moreover, the Ca2+ ionophore, ionomycin, also increases [Ca2+]i and mimics the effect of ACh on fluid secretion (141). Until recently it was generally assumed that an increase in [Ca2+]i would stimulate ductal secretion by activating the CaCCs expressed in the duct cell (123,134, 135,137). However, this may not be the only mechanism involved because it has been reported that an increase in [Ca2+]i can also activate luminal Cl−-HCO3− exchange in cells that express CFTR (108). Adenosine Triphosphate (Luminal) Purinergic P2 receptors (P2R) are expressed on both the apical and basolateral membranes of the duct cell (48,91,92, 189–194). P2R are divided into two subgroups, P2Y, which are G protein–coupled receptors, and P2X, which are ligandgated ion channels. The spatial distribution of P2R on the duct cell is somewhat controversial, but members of both the P2Y and P2X subgroups are expressed on the apical and basolateral membranes (48,92,190,191,194). Using isolated guinea pig ducts, we have shown that application of ATP (and UTP) to either the luminal or basolateral membranes is associated with an increase in [Ca2+]i (142). Because the effects of luminal and basolateral ATP on [Ca2+]i were additive, we concluded that there must be two separate pools of receptors expressed on each side of the duct cell (142). We also showed that luminal application of ATP (and UTP) stimulated fluid and HCO3− secretion, with the maximal response (at 1 µM ATP) being equal to about 75% of the maximum response to secretin (142). In contrast, basolateral application of ATP inhibited spontaneous secretion by 52% and secretin-evoked secretion by 41% (142). A possible explanation for these different effects of luminal and basolateral ATP has been provided by Novak and her colleagues (92,195). Using functional and molecular criteria, this group postulated the expression of P2Y2 and P2Y4 purinoreceptors on the basolateral membrane of the duct cell and P2X4 and P2X7 on the apical membrane. Stimulation of the apical P2X receptors caused a monophasic increase in [Ca2+]i that was dependent on extracellular Ca2+ and explained by opening of the P2X receptor–associated nonselective cation channel, resulting in Na+ and Ca2+ influx into the duct cell (92). An increase in

1388 / CHAPTER 54 [Ca2+]i at the apical membrane of the duct cell would be expected to open CaCCs, which would explain the observed stimulation of secretion (142). However, Novak and her colleagues (92,195) found no evidence for an increase in Cl− conductance when the apical P2X receptors were activated, indicating that the secretory response to luminal ATP must be mediated by some other mechanism. One possibility is that the increase in [Ca2+]i activates electroneutral Cl−-HCO3− exchange (108); however, neither BzATP (a P2X agonist) nor ATP itself had any effect on the activity of Cl−-HCO3− exchangers, NHEs, or NaHCO3 cotransporters in the duct cell (195). In contrast with these findings, Muallem’s group (190) has reported a more complex pattern of P2R expression in rat duct cells with the apical and basolateral membranes containing members of both the P2Y and P2X subgroups. ATP is contained within the zymogen granules in acinar cells, and thus will be released into the duct lumen when enzyme secretion is stimulated (196). A paracrine link between enzyme secretion and the stimulation of ductal fluid secretion via luminal ATP would be physiologically advantageous for washing enzymes down the ductal tree. Moreover, luminal release of ATP from acini in response to cholinergic stimulation may explain why ACh potentiates ductal HCO3− secretion evoked by secretin (196). However, whether luminal ATP would ever increase to a level that stimulated the duct cell is debatable because both acini and ducts express the ectoATPase CD39 (197), and ATP concentrations in pancreatic juice have been reported to be either low or undetectable (197,198). However, these ATP measurements were made on juice collected from the main duct and might not reflect the situation in the lumen of the smaller ducts where HCO3− is secreted. Ca2+ Ions (Luminal) Pancreatic juice contains millimolar concentrations of Ca2+, most of which is secreted together with enzymes from the acinar cells. Given the Ca2+ concentrations that exist in pancreatic juice, there is a risk that calcium carbonate stones could form in the alkaline environment of the tubular ductal system, especially during the interdigestive periods when juice flow rate is low (149). We have shown that, in part, the pancreas guards against stone formation by expressing a Ca2+-sensing receptor (CaR) on the apical membrane of the duct cell (143,199). CaR is a G protein–linked receptor that responds to millimolar concentrations of Ca2+, consistent with a role in sensing extracellular Ca2+ levels. Activation of the duct cell CaR with gadolinium (a potent ligand) causes an increase in intracellular Ca2+ concentration and a marked increase in HCO3− secretion (143,199). We proposed that the resultant dilution of the luminal contents will prevent stone formation, and therefore duct blockage (143,199). Some support for this idea comes from the observation that pancreatitis is occasionally associated with hypocalciuric hypercalcemia, an inherited disease caused by inactivating mutations in CaR (200).

Trypsin The proteinase-activated receptor-2 (PAR-2) is a G protein–coupled, Ca2+-mobilizing receptor. In the presence of trypsin, PAR-2 is cleaved within its extracellular NH2terminal domain, which exposes a tethered ligand that binds and activates the receptor. Functional studies in primary cultures of duct cells (201) and the human duct cell lines CAPAN-1 (108,202) and CFPAC-1 (108) have indicated that PAR-2 is expressed on the basolateral membrane. In these experimental models, activation of the basolateral PAR-2 initiates an intracellular Ca2+ signal that stimulates Isc (201) and Cl−/HCO3− exchange activity (108), which is consistent with a stimulatory effect on ductal secretion. In contrast, bovine main duct expresses PAR-2 on the apical membrane (203). Activation of this apical PAR-2 reduced basal HCO3− secretion, but had no effect on secretin-stimulated secretion (203). Whether the PAR-2 receptor has any role in the control of ductal HCO3− secretion under physiologic conditions is uncertain, because activated trypsin should not be present in the duct lumen or systemic circulation. However, in pancreatitis, trypsin that leaked across the basolateral membrane of the acinar cell would stimulate ductal secretion and promote clearance of toxins and debris from the duct lumen. Pancreatic hypersecretion is observed during the early phase of acute necrotizing pancreatitis (204) and is accompanied by a simultaneous decrease in protein output (205). Interestingly, activation of PAR-2 reduced the pathologic effects of cerulein-induced pancreatitis observed in the rat in vivo (202). Angiotensin II Angiotensin II (AT II) can stimulate anion secretion (Isc) in the CFPAC ductal cell line (206,207). Both basolateral and apical application of AT II were effective in increasing anion secretion, and the effect of AT II was associated with an increase in intracellular Ca2+ and cAMP concentrations (206,207). Pharmacologic evidence suggested that predominantly A1 receptors were involved in the response (206,207). More recently, similar findings have been reported for primary duct cell cultures, and in addition, it was shown that AT II activated an ion channel that had similar characteristics to those of CFTR (208). Whether AT II is important in the physiologic or pathophysiologic control of pancreatic ductal secretion remains to be determined. Histamine Histamine is quite a potent stimulant of HCO3− secretion from the dog pancreas (5), and a study on primary cultures of canine duct cells has shown that histamine has a direct effect on the ductal epithelium (209). Acting via basolateral H1 receptors, histamine increased [Ca2+]i, activating Ca2+sensitive Cl− and K+ channels and stimulating an increase in Isc in polarized duct cell monolayers (209).

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1389 Neurotensin, Cholecystokinin, and Bombesin A number of other agonists, in addition to those mentioned earlier, can affect duct cell [Ca2+]i and, in some cases, the Vbl. Neurotensin receptors are present on about 20% of rat duct cells, and this peptide has a small effect on [Ca2+]i and also depolarizes Vbl (89,91). Whether CCK increases [Ca2+]i is controversial; two groups have reported an effect in rat ducts (38,210), whereas others report either a small response (91) or no effect of this peptide (141,184). In this context, it should be mentioned that CCK has no effect on the duct cell Vbl (89), and that cerulein, an analogue of CCK, does not increase fluid secretion from isolated rat ducts (30). However, we have shown that CCK acts as a potent stimulant of HCO3− secretion from the in vivo guinea pig pancreas (211) and, via activation of CCK1 receptors, from isolated guinea pig ducts (174). We also have reported that bombesin has a clear stimulatory effect on ductal secretion in the rat (84,105) and guinea pig, which involves activation of the gastrin-releasing peptide receptor (174). The intracellular messenger system associated with the stimulatory effect of bombesin remains to be identified. We have shown that the peptide has no effect on cAMP accumulation (141), and either no effect (141) or only a small effect on [Ca2+]i has been reported (38,91). Moreover, bombesin has no effect on the Vbl of rat duct cells (89). Finally, secretin, VIP, adrenaline, and phenylephrine usually do not increase [Ca2+]i (141,184), although one report exists showing that secretin will initiate [Ca2+]i oscillations in rat duct cells (210). In summary, these data clearly show that a Ca2+ pathway exists for the activation of ductal secretion, in addition to the classical cAMP pathway used by secretin, VIP, and β-adrenergic agonists. Good evidence exists to indicate that ACh, ATP, luminal Ca2+, trypsin, AT II, and histamine use the Ca2+ pathway, although whether it is used by CCK and bombesin is controversial. It is important to understand this Ca2+ pathway because it could form a route to enhance duct cell HCO3− and fluid secretion in the inherited disease CF. Certainly, CF transgenic mice that do not express CFTR, but which have a high density of CaCCs in their duct cells, do not exhibit the pancreatic pathology characteristic of CF in humans (123,134).

Inhibitory Pathways Inhibitory pathways may be physiologically important for limiting the hydrostatic pressure that develops within the lumen of the duct (thus preventing leakage of enzymes into the parenchyma of the gland) and for switching off pancreatic secretion after a meal. Somatostatin (212,213) and peptide YY (214,215) both inhibit secretion from the intact pancreas and are generally considered to work by interfering with the neural or hormonal control of the gland, or both, although one study has reported that somatostatin decreased cAMP

production in isolated ducts (177). In contrast, SP (104,105), 5-hydroxytryptamine (5-HT) (216), arginine vasopressin (AVP) (217), and basolateral ATP (142) have all been shown to inhibit fluid or HCO3− secretion, or both, from isolated pancreatic ducts, and thus must act directly on the ductal epithelium. Substance P The neuropeptide SP has been identified in the pancreas of the dog, rat, mouse, and guinea pig (218,219) and has been shown to inhibit fluid secretion from the intact gland of the dog (220,221) and rat (222) and from the perfused rat pancreas (223). The inhibitory effect of SP in the perfused rat pancreas has been ascribed to an indirect neural mechanism because the SP-mediated inhibition of secretinstimulated fluid secretion was blocked by tetrodotoxin (223). However, we have shown that SP is a potent inhibitor of basal and secretin-stimulated secretion from isolated rat (105) and guinea pig pancreatic ducts (104,219), suggesting also a direct action on the ductal epithelium. The inhibitory effect of SP on isolated ducts is dose dependent, is reversed by spantide, a SP receptor antagonist (104,105), and is not associated with a change in the membrane potential of the duct cell (89). Importantly, SP has no effect on intracellular cAMP levels (141) and also inhibits fluid secretion in response to dibutyryl cAMP (105). From these observations, we have proposed that the inhibitory effect of SP occurs downstream from the generation of cAMP in the intracellular signaling pathway (105). Our data indicate that SP reduces ductal HCO3− secretion by inhibiting a Cl−-dependent HCO3− transport step, most probably a SLC26 family anion exchanger, on the apical membrane of the duct cell (104,219). SP interacts with tachykinin receptors, which are seventransmembrane-spanning receptors coupled to the Gq/G11 family of G proteins (224), and which can activate the Ca2+ and protein kinase C (PKC) intracellular signaling pathways. The involvement of intracellular Ca2+ in the inhibitory effect of SP is unlikely because the peptide has been reported to have either no effect on [Ca2+]i (225) or to cause only a small increase at high doses (91). However, we have shown that a variety of PKC isoforms are expressed in pancreatic ducts (219), and activation of PKC with phorbol esters inhibits pancreatic secretion both in vivo (226) and in isolated ducts (104,106,219). Importantly, we also found that phorbol esters inhibited fluid secretion from isolated ducts in response to forskolin, but had no effect on cAMP accumulation in response to this stimulant (106). Thus, like the effect of SP (105), the inhibitory effect of phorbol ester appears to occur downstream from the generation of cAMP in the intracellular signaling pathway (106). Confirmation that the effect of SP is mediated by PKC comes from our experiments in which we showed that bisindolylmaleimide (a highly selective, cell-permeable, PKC inhibitor) caused a dosedependent reversal of the inhibitory effect of SP (219).

1390 / CHAPTER 54 Inhibitory effects of PKC on Cl−-HCO3− and Cl−-OH− exchange processes have been reported in hepatocytes (227) and in the human Caco-2 intestinal cell line (228). In Caco-2 cells, the inhibitory effect of PKC activation on Cl−-OH− exchange was blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and by a specific inhibitor of the novel, Ca2+-independent PKCε (228). PI3K phosphorylates plasma membrane inositol phospholipids on the 3-carbon of the inositol ring, and the enzyme can be activated, perhaps indirectly, by phorbol esters. The 3-phosphorylated inositol lipid products have themselves been shown to activate the Ca2+independent, novel, PKC isoforms, possibly by acting as membrane-docking molecules (see Saksena and colleagues [228] for references). Activation of PKC in Madin–Darby canine kidney and Caco-2 cells has been shown to be associated with an increase in apical membrane endocytosis, which could, in theory, reduce the number of transporters in the apical membrane (229). It has been reported that PKCmediated internalization of the human amino acid transporter hCAT-1 underlies the inhibitory effect of PKC on amino acid transport in Caco-2 cells (230). Combined Doses of Secretin and Acetylcholine It is well known from in vivo studies that low doses of cholinergic agonists can potentiate the effect of secretin on pancreatic HCO3− secretion (see Case and Argent [2]). Some of this potentiation may result from luminal ATP (released from acinar cells together with enzymes after ACh stimulation) activating P2X and P2Y receptors on apical membrane of the duct cell (92,190,195). Similarly, in isolated rat ducts, we have shown that the combination of low doses of secretin and ACh (by themselves subthreshold) causes a significant increase in fluid secretion (106). Because isolated ducts are free of acinar tissue and duct cells do not release ATP after stimulation with ACh (195,196), our data suggest that the Ca2+ and cAMP signaling pathways in the duct cell can interact to potentiate secretion. In contrast, we have also shown that when isolated ducts were exposed to greater doses of either secretin or ACh (which on their own caused near-maximal fluid secretion) plus varying doses of the other stimulant, fluid secretion was markedly inhibited (106). It was not necessary to expose the isolated ducts to high doses of both stimulants to observe this inhibitory effect; for example, 0.1 µM ACh (which alone has no effect on fluid secretion) completely inhibited the response to 0.1 nM secretin (a near-maximal dose). This inhibitory effect of ACh was blocked by atropine, indicating a requirement for ACh to interact with muscarinic receptors (106). We also reported that the inhibitory effect of ACh was reversed by the PKC inhibitors staurosporine and H-7 and mimicked by phorbol esters, strongly suggesting the involvement of PKC (106). Moreover, ACh and phorbol ester also inhibited fluid secretin stimulated by forskolin, but had no effect on intracellular cAMP generation, placing the site at which PKC inhibits ductal fluid secretion downstream of cAMP in the

secretory mechanism (106). These observations are puzzling, because on its own, ACh acts as a stimulant of ductal fluid secretion, whereas when combined with secretin, it can inhibit secretion. A possible explanation is that PKC can phosphorylate a presumed target protein in the duct cell only when cAMP is increased; that is, there may be a requirement for prior phosphorylation of the target(s) of PKC by PKA before inhibition of secretion can occur. Whatever the explanation, it is clear that increase of PKC activity in the duct cell, whether by the tachykinin SP, by ACh, or by phorbol esters can inhibit ductal fluid and HCO3− secretion. The physiologic relevance of these observations is uncertain; however, they emphasize the potential for complex interactions between agonists at the level of the duct cell. 5-Hydroxytryptamine Enterochromaffin cells containing 5-HT are present throughout the ductal system of the pancreas, including the small interlobular and intralobular ducts, which are the major site of HCO3− secretion (216). Studies on isolated guinea pig ducts have shown that addition of 5-HT to the basolateral (but not luminal) side of the epithelium inhibits basal, secretin-, and ACh-stimulated fluid secretion (216). The inhibitory effect of 5-HT was concentration dependent (IC50 = 30 nM for secretin-stimulated secretion) with maximum inhibition being observed at 100 nM 5-HT (216). Pharmacologic studies identified the 5-HT3 receptor as mediating the inhibitory effect (216). The 5-HT3 receptor is a ligand-gated nonselective cation channel, and it was postulated that an increase in intracellular Na+ after receptor activation would reduce HCO3− accumulation dependent on the basolateral NBC1 and NHE (216). Consistent with this idea, exposure of duct cells to 5-HT caused a transient increase in [Ca2+]i (consistent with opening of the nonselective cation channel) and a decline in pHi (consistent with an increase in intracellular Na+ slowing NBC1 and NHE) (216). Furthermore, increasing the hydrostatic pressure in the pancreatic duct inhibited secretin-stimulated secretion in vivo, and this effect was significantly reduced by a 5-HT3 receptor antagonist (216). Taken together, these observations suggest that an increase in hydrostatic pressure in the duct lumen (mimicking ductal blockage or stricture) leads to the release of 5-HT across the basolateral membrane of the enterochromaffin cells, which then binds to 5-HT3 receptors on the duct cells to arrest secretion (216). Arginine Vasopressin AVP inhibits pancreatic secretion in conscious (231,232) and anesthetized dogs (233). Because AVP is a potent vasoconstrictor, these inhibitory effects might be explained by a reduction in blood flow, and therefore O2 supply, rather than a direct effect on the ductal epithelium. In the in vitro perfused guinea pig pancreas, a preparation in which perfusate flow (and O2 supply) can be maintained constant in the face of

CELL PHYSIOLOGY OF PANCREATIC DUCTS / 1391 vasoconstriction, AVP caused a modest 15% inhibition of secretin-stimulated fluid secretion (217). In contrast, when tested on isolated guinea pig ducts, AVP caused a 30% decrease in fluid secretion (217). Thus, AVP does have a direct, albeit modest, inhibitory effect on the duct cell. AVP had no effect on cAMP accumulation in isolated ducts, but increased Ca2+ release from intracellular stores and Ca2+ efflux across the plasma membrane (217). How these changes in Ca2+ metabolism lead to inhibition of secretion is unclear; however, if Ins(3,4,5,6)P4 was produced in response to stimulation with AVP, then this might inhibit CaCCs in the duct cell (138). Adenosine Triphosphate (Basolateral) In contrast with the stimulatory effect of luminal ATP (see earlier), we have shown that basolateral ATP inhibits fluid and HCO3− secretion from pancreatic ducts (142). Activation of the basolateral P2Y receptors caused a biphasic increase in [Ca2+]i, probably explained by activation of Ca2+ influx and subsequent Ca2+ release from intracellular stores (92). There was an associated marked reduction in basolateral K+ conductance (i.e., closure of K+ channels), which would explain the inhibition of fluid and HCO3− secretion (92). Interestingly, ATP has been reported to inhibit secretinstimulated secretion from the in vivo dog pancreas (234). The mechanism by which P2Y2 receptors close basolateral K+ channels is unclear; however, stimulation of the duct cell with ATP is associated with a decline in pHi (92), which would inhibit the pH-sensitive K+ channels expressed in this cell (86). ATP is released from the basolateral side of the acinar cell after cholinergic stimulation (196); however, whether this ATP reaches the ducts and the reason why it would be physiologically useful to inhibit ductal fluid secretion under these conditions remain unknown. In summary, these studies show that inhibition of ductal fluid and HCO3− secretion can occur at the level of the duct cell, as well as by interference with the hormonal and neural pathways that normally stimulate secretion. Certainly, activation of PKC markedly inhibits ductal secretion, and we are beginning to understand how this occurs. There are also some puzzling observations; for instance, basolateral ATP and vasopressin both increase [Ca2+]i, but inhibit secretion. In contrast, ACh and a number of other agonists also increase [Ca2+]i, but have a stimulatory effect on secretion, suggesting perhaps that Ca2+ signals must be compartmentalized in the duct cell.

CONCLUDING REMARKS In conclusion, significant advances have been made in our understanding of the cellular physiology of the duct cell since the previous edition of this textbook was published in 1994. At that time, studies on isolated ducts were still in their infancy. Nevertheless, the basic cellular model for ductal

HCO3− secretion that we proposed in 1994 (based mainly on data from rat ducts) has stood the test of time, although modifications are needed to explain the secretion of nearisotonic NaHCO3 by some species, including humans. There is general agreement about HCO3− uptake mechanisms at the basolateral membrane of the duct cell; however, the mechanism by which HCO3− is secreted across the apical membrane remains controversial. CFTR is clearly a key player (witness the effects of CF on the pancreas), but whether CFTR acts as a HCO3− channel or whether its role is to activate SLC26 anion exchangers is an area of intense interest. Recently, it also has become apparent that the ionic environment within the duct lumen, in particular the Cl− concentration, can regulate both the activity of CFTR and its relative permeability to Cl− and HCO3− ions. Understanding the molecular basis for these effects and the role they play in the mechanism of HCO3− secretion will be a focus of current and future research. It will also be important to firmly establish which SLC26 isoforms are expressed in the duct cell and whether their expression varies along the ductal tree. Finally, secretory studies on isolated ducts have shown that the duct cell possesses a plethora of hormone and neurotransmitter receptors that, when activated, can either stimulate or inhibit secretion. That some of these receptors are expressed on the luminal membrane indicates that secretory function can be controlled by luminal and blood-borne signals, emphasizing that the physiologic control of ductal secretion in vivo is indeed complex.

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125. 126. 127. 128.

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Regulation of Pancreatic Secretion Rodger A. Liddle Patterns of Secretion, 1397 Basal Secretion, 1397 Integrated Response to Meals, 1398 Phases of the Meal Response, 1400 Cephalic Phase, 1400 Gastric Phase, 1400 Intestinal Phase, 1401 Absorbed Nutrient Phase, 1403 Neural and Hormonal Regulators, 1404 Neural Mechanisms, 1404 Hormonal Mechanisms, 1410 Feedback Regulation of Pancreatic Secretion, 1417 Inhibition of Pancreatic Secretion, 1420 Glucagon, 1420 Glucagon-Like Peptide-1, 1421

Somatostatin, 1421 Pancreatic Polypeptide, 1422 Peptide YY, 1422 Pancreastatin, 1422 Pancreatic Function Testing, 1423 Tests of Pancreatic Synthetic or Metabolic Activity, 1423 Measurements of Pancreatic Enzymes in Blood, 1424 Pancreatic Enzymes in Stool, 1424 Pancreatic Polypeptide, 1424 References, 1424

During the late 1990s and early 2000s, substantial advances have been made in our understanding of the neural and hormonal processes that are involved in the regulation of pancreatic secretion. Moreover, novel feedback mechanisms have been identified that integrate intestinal signals with pancreatic secretory responses. Although these processes are complex, they illustrate the highly regulated nature that is needed for maintaining sufficient secretion of pancreatic enzymes that are essential for nutrition.

of the meal and is associated with the ingestion, digestion, and absorption of food. Meal-induced secretion is thought to be the most important aspect of pancreatic exocrine function because lack of pancreatic enzyme secretion results in malabsorption and maldigestion of nutrients and general poor nutrition. Interestingly, however, the amount of pancreatic secretions that are present in the intestine during the basal condition may be sufficient to facilitate substantial enzymatic degradation of ingested foods and prevent malnutrition. These observations suggest that the exocrine pancreas functions at a level that is considerably greater than the minimum necessary for complete digestion of food. Despite this finding, most studies of pancreatic function have been devoted to studying stimulated, rather than basal, secretion.

PATTERNS OF SECRETION Basal secretion occurs when food has emptied from the stomach and the small intestine and is associated with fasting. Meal-induced pancreatic secretion occurs after the ingestion

Basal Secretion

R. A. Liddle: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

The rate of basal pancreatic exocrine secretion is generally expressed as a percentage of the maximal amount that the pancreas can secrete when stimulated by a secretagogue such as cholecystokinin (CCK). When expressed as a percentage of the maximal pancreatic capacity to secrete enzymes,

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1398 / CHAPTER 55 values for basal enzyme secretion range from 10% of maximal in cats to 20% in humans and 30% of maximal in rats (1). Basal secretion of bicarbonate, however, is often only 1% to 2% of the maximal secretory rate compared with administration of exogenous secretin, and there is considerably less species variation with the exception being the rat in which basal bicarbonate secretion is 25% of maximal secretion (2). Therefore, the basal secretory rate of pancreatic enzymes may be adequate to prevent malabsorption of ingested nutrients because frank malabsorption generally appears only when pancreatic enzyme secretion is reduced to 10% (3). Several different mechanisms are responsible for basal pancreatic exocrine secretion and could be caused by: (1) an automaticity of the gland; (2) regulation by low levels of gastrointestinal hormones such as CCK or secretin; or (3) release of neurotransmitters such as acetylcholine. In vitro, pancreatic acinar cells demonstrate basal enzyme release, although it is unknown what is responsible for this basal exocytosis. In experimental animals such as dogs and rats, basal secretion is primarily caused by cholinergic innervation, because atropine blocks basal secretion and CCK receptor antagonists have been shown repeatedly to have no effect on basal pancreatic secretion (4–10). The inhibitory effect of anticholinergic drugs is probably caused by blockade of the muscarinic receptor on pancreatic acinar cells that blocks the effects of acetylcholine released from postganglionic pancreatic nerves. Moreover, basal levels of CCK are not sufficiently high to stimulate pancreatic exocrine secretion. Therefore, in contrast with secretin that appears to play a role in basal pancreatic secretion, there is strong evidence that CCK is not important in regulating basal pancreatic secretion in rats (11). In humans, however, varying reports have suggested that CCK and cholinergic inputs may contribute to basal pancreatic enzyme secretion. Profound inhibition of pancreatic secretion has been seen after atropine administration in humans, and some reports indicate that CCK receptor antagonists may reduce pancreatic secretion (12–17). These findings indicate either that low levels of circulating CCK are sufficient to stimulate human pancreatic secretion, or that CCK released as a peptidergic transmitter from pancreatic neurons contributes to basal secretion. Pancreatic bicarbonate secretion is largely regulated by secretin. Basal pancreatic bicarbonate secretion correlates with plasma secretin levels; however, cholinergic inputs also affect bicarbonate release because atropine decreases both basal and secretin-stimulated bicarbonate secretion (18–20). It is likely that basal bicarbonate release is augmented by acetylcholine released locally from nerves in the pancreas. Interestingly, the patterns of pancreatic secretion appear to be more complex if one examines secretory rates over time. Even under basal conditions, over several minutes, the secretory rate of pancreatic secretion varies. There are brief increases in bicarbonate and enzyme secretion that occur every 60 to 120 minutes. These bursts of pancreatic secretory activity coincide with periods of increased motor activity of the stomach and duodenum that are associated with the migrating motor complex (MMC) (21–23,25–27).

Simultaneous with increases in pancreatic secretion, there are increases in gastric acid secretion and biliary flow into the duodenum. All of these actions are associated with increase of motilin levels in the blood. In addition, pancreatic polypeptide (PP) levels correlate well with the antral phase II motor activity and pancreatic enzyme secretion (24). These activities appear to be cholinergically mediated because atropine administration or ganglionic blockers abolish the periodic spikes in basal enzyme secretion. Administration of motilin prematurely initiates pancreatic secretion that is seen during the basal period and shortens the time between peaks of secretory activity. Conversely, immunoneutralization of motilin with specific antiserum abolishes the cyclic pattern of pancreatic secretion (25). Administration of PP inhibits basal pancreatic secretion, and immunoneutralization with PP antiserum augments the peak in pancreatic secretion that is seen in the basal period (26). These findings are consistent with the overall belief that the MMC functions as a housekeeper to eliminate chyme, debris, and other secretions during the interdigestive period and to keep microbial populations in check. Although it is reduced, the periodic basal pattern of pancreatic secretion persists despite duodenectomy or autotransplantation of the pancreas (27,28), indicating that independent of any obvious hormonal or neural influences, the pancreas is able to generate an endogenous periodicity that is most likely caused by activation of postganglionic cholinergic neural activity. These findings are consistent with electrophysiologic observations documenting spontaneous ganglionic neural activity (29). A circadian rhythm to pancreatic secretion has been described in rats in which there is a peak in secretion during the dark phase of the day/night cycle (30).

Integrated Response to Meals The major function of the exocrine pancreas is to facilitate the efficient digestion of food and absorption of micronutrients. The function of pancreatic enzymes is to break down macronutrients such as proteins to small peptides and amino acids, triglycerides to fatty acids and monoglycerides, and carbohydrates to sugars that can be absorbed easily from the small intestine. Pancreatic bicarbonate secretion is important for neutralizing gastric acid and creating an intraluminal environment with a pH that is hospitable to the action of enzymes especially in fat digestion (31). Unfortunately, little is known about the actual pancreatic secretory process involved in the normal digestion of meals in humans. This problem is due in large part to difficulties in sampling intestinal secretions with and without food in the lumen without altering the normal physiologic function of the pancreas, biliary system, and intestine that together constitute an integrated response to a meal. Attempting to measure pancreatic juice free of biliary secretions requires cannulation of the pancreatic duct or diversion of pancreatic juice flow from the intestine. Diversion of pancreatic juice from the intestine disturbs the normal milieu by removing one or more factors

REGULATION OF PANCREATIC SECRETION / 1399 that are either involved in neutralizing gastric acid or necessary for maintaining the proper intestinal environment in which intestinal releasing factors may function (see later). When pancreatic juice is diverted from the intestine, pancreatic bicarbonate is no longer present and the gastric acid entering the duodenum may avoid neutralization. Gastric acid is also a potent stimulus of secretin release, which, in turn, stimulates pancreatic fluid and bicarbonate secretion. Interestingly, in dogs, the flow of pancreatic juice rich in bicarbonate increases after the diversion of pancreatic juice from the intestine. Although this fluid is rich in bicarbonate, the amount of enzymes in the juice does not increase (32). This may mean that the intragastric digestion of proteins to small peptides and amino acids and triglycerides to fatty acids is sufficient to stimulate pancreatic secretion when these nutrients reach the intestine. In contrast, in rats, diverting pancreatic juice stimulates pancreatic enzyme secretion to near-maximal amounts similar to that produced by exogenous CCK. Quantifying pancreatic secretion associated with meals in humans is extremely difficult; therefore, there is little quantitative information available. Sampling from the duodenum is fraught with hazards. Concentration measurements are notoriously unreliable because the volume of duodenal contents may vary. Moreover, duodenal bicarbonate is the product not only of pancreatic secretion, but also biliary and intestinal sources. There are also important species differences because bicarbonate secretion in the pig is much less relative to biliary bicarbonate production, whereas pancreatic bicarbonate is the major source for neutralization of acid chyme in dogs (33). Maximum bicarbonate concentration in humans may be as high as 150 mM, which is approximately twice as great as that in the rat. Measurement of trypsin is also difficult because it requires enzymatic activation of trypsinogen, and the active form binds avidly to food, making its quantification complex. The physical nature of food is an important factor in the regulation of meal-stimulated pancreatic responses. When comparing a solid form of a meal with the same food homogenized to a liquid, the total pancreatic trypsin output was the same; however, the secretory response was prolonged (34), which is consistent with solid food emptying from the stomach at a slower rate than liquids. When liquid meals were delivered through a gastric tube to healthy individuals several times per day, the pancreatic secretory response was sixfold greater than basal levels over a 24-hour period (35). The maximal pancreatic enzyme response to fat occurs at low rates of fat delivery to the intestine. When the fat content of the meal was increased, there was no further stimulation of pancreatic enzyme secretion; however, when the protein content was increased, there was nearly a twofold increase in meal-stimulated pancreatic enzyme secretion (36,37). The effects of food on gastric emptying rates may also affect pancreatic secretory responses. The duration of pancreatic secretion correlated with the time required for the stomach to empty (35). As long as food was emptying from the stomach, pancreatic secretion was maintained at a

high level; when the upper small intestine was free of food, pancreatic secretion declined. Importantly, delivery of food further down the intestine has an inhibitory effect on pancreatic secretion. A detailed study examined the temporal interrelations among gastric emptying, plasma CCK, PP and peptide YY (PYY) levels, and pancreatic and biliary secretion (38). Increasing the rates of protein and fat delivery to the duodenum produced an initial increase in pancreatic secretion and plasma CCK levels that peaked, but after 4 hours declined despite food continuing to empty from the stomach to the duodenum. This finding was surprising because it was thought that nutrients were the dominant factor affecting both CCK and pancreatic exocrine secretion. The later appearance of PP and PYY, which are inhibitory to both CCK release and pancreatic secretion, may explain the late postprandial decline. This study illustrates that the postprandial pancreatic secretory response is complex and is the product of integrated stimulatory and inhibitory factors. Glucose alone causes a vigorous pancreatic secretory response, although it is not a potent stimulus of CCK release (39), suggesting that a neural mechanism may be involved (40). It is possible that these results could be caused by distention of the stomach because balloon distention causes a similar pancreatic secretory response (41,42). Overall, meal-stimulated pancreatic secretion is 50% to 60% of the maximal secretory capacity of the organ (43–45). Pancreatic bicarbonate secretion has been best studied in dogs. After a single meal there are distinct peaks of pancreatic secretion (46). The first peak occurs within the 3 hours after eating and contains fluid that is high in protein, but low in bicarbonate. This peak constitutes 25% of the maximal volume output. The second peak occurs 10 to 12 hours after eating and contains fluid that is high in bicarbonate, but low in protein. Pancreatic secretory rates returned to basal after approximately 16 hours (46). The differences in bicarbonate output are probably related to the acid nature of gastric contents. It is likely that free acid concentrations are low early after eating; therefore, little acid chyme reaches the duodenum to stimulate bicarbonate production. However, late in the course when food has emptied from the stomach, gastric contents are unbuffered and substantial gastric acid enters the duodenum and stimulates a bicarbonate-rich pancreatic juice. This concept is supported by the observation that the second peak of pancreatic secretion is blocked by cimetidine (47). Meal-stimulated bicarbonate and enzyme responses are consistently found to be less than maximal rates of secretion regardless of the species studied including dogs, rats, and humans. But although the reasons for this are not entirely clear, they may be related to slow rates of gastric emptying. With delayed delivery of nutrients to the duodenum there may be submaximal pancreatic stimuli such as the gastrointestinal hormones CCK and secretin. There also may be simultaneous or subsequent release of inhibitors of pancreatic secretion as food travels farther down the intestine. Furthermore, as absorption of nutrients occurs, they are no longer present in the lumen of the intestine to stimulate

1400 / CHAPTER 55 pancreatic responses. All of these possibilities may contribute to postprandial pancreatic secretion that is less than that induced by CCK alone.

PHASES OF THE MEAL RESPONSE There are four major physiologic processes used to describe pancreatic secretion. These include cephalic, gastric, intestinal, and absorbed nutrient phases, which describe the sites at which signals to the pancreas originate. Each of the phases involves both secretory and inhibitory inputs, although the overall effect is overwhelmingly stimulatory. The secretory processes involve multiple levels of regulation including neurohormonal and hormonal–hormonal interactions. Although many of the steps that stimulate pancreatic secretion appear to be redundant, the system ensures that adequate enzymes are available for digestion. In general, after a meal, there is a temporal relation in which the cephalic phase contributes to pancreatic secretion before initiation of the gastric and intestinal phases, respectively. The absorbed nutrient phase includes inputs from each of the other three phases and involves the effects of nutrients absorbed into the blood to affect pancreatic secretion. Although it is important to understand the contribution of each of these phases to pancreatic secretion, it is more important to recognize that with normal feeding there is considerable overlap. Therefore, the integrated response to a meal results from the combination of all phases for physiologic regulation of pancreatic secretion.

Cephalic Phase The cephalic phase of secretion results from inputs including the sight, smell, taste, and act of eating food. Although these processes are commonly thought of as stimulating pancreatic secretion, they may also generate inhibitory signals when eating is associated with unpleasant features such as unattractive, malodorous, or bad-tasting food. The cephalic phase of secretion has been produced in humans by presenting them with food that they see, smell, and taste, but do not swallow (a process that is known as modified sham feeding). In animals, food can be diverted from the esophagus by a surgically prepared esophageal or gastric fistula, and sham feeding can occur by allowing these animals to eat and swallow while preventing food from entering the stomach. In both dogs and humans, sham feeding stimulates low volumes of pancreatic secretions that are rich in enzymes, but low in bicarbonate. The total pancreatic secretory response to sham feeding is approximately 25% to 50% of maximal (13,14,48). Secretion of the islet hormone, PP, increases with sham feeding and has been used as a marker of vagal innervation of the pancreas (49). In humans, the duration of pancreatic response to modified sham feeding is brief, lasting approximately 60 minutes, and ceases at the conclusion of sham feeding (50). If swallowing is included

in sham feeding, the pancreatic secretory and PP responses are much greater (51). In dogs, the pancreatic enzyme response to sham feeding lasts more than 4 hours (52). There is substantial experimental data to support the concept that cephalic stimulation of pancreatic secretion is mediated by the vagus nerve. First, cholinergic agonists produce a pancreatic secretory response similar to that of cephalic stimulation (53). Second, the vagus nerve is the major source of cholinergic neurotransmitters to the pancreas. Third, electrical nerve stimulation of the vagus nerve or administration of 2-deoxyglucose (2-DG), which causes hypoglycemia (and initiates a vagal response), stimulated pancreatic juice flow similar to that of sham feeding (54). Finally, vagotomy blocked these responses. In anesthetized rats, pancreatic fluid and protein output after electrical nerve stimulation or 2-DG was partially blocked by atropine (55). Thus, although the vagus nerve carries fibers that bear peptidergic transmitters, as well as acetylcholine, these data indicate that acetylcholine is the dominant neurotransmitter. The role of peptidergic efferent fibers in sham feeding is largely unknown. Sham feeding is also a major stimulus of gastric secretion that may contribute to stimulation of pancreatic secretion through the release of secretin (13). Interestingly, it has been shown that mental stress produced by intense problem solving can also stimulate pancreatic enzyme secretion in humans (56). In dogs, both sham feeding and electrical vagal nerve stimulation caused the release of CCK (57,58). However, modified sham feeding does not stimulate CCK release in humans (14). The regions of the dorsal and ventral anterior hypothalamus, including the medial hypothalamus, dorsomedial and ventromedial nuclei, and mammillary bodies, appear to generate signals for pancreatic secretion (59). Determining the neurotransmitters and peptides that are involved in regulating these processes has been approached by examining effects of substances administered into the central nervous system. In rats, central administration of β-endorphin, calcitonin gene–related peptide (CGRP), and corticotropin-releasing factor inhibit pancreatic secretion (60–62). In contrast, thyrotropin-releasing hormone stimulates pancreatic secretion through the vagus nerve and involves both muscarinic and vasoactive intestinal peptide (VIP) receptors (62).

Gastric Phase The gastric phase of pancreatic secretion has been difficult to study in unanesthetized intact animals and humans. Other than testing the effects of gastric distention by installation of inert substances or balloon dilation of the stomach, it has been problematic to examine the effects of foods or other nutrients on pancreatic secretion because of the chemical properties of the nutrients themselves that stimulate neural reflexes and cause the release of hormones. Gastrin is the best studied gastrointestinal hormone and is a major regulator of gastric acid secretion. Although early

REGULATION OF PANCREATIC SECRETION / 1401 reports suggested that gastrin was also a potent stimulus to pancreatic secretion, these conclusions have been shown to be incorrect. Plasma gastrin levels that are required for stimulation of pancreatic secretion are considerably greater than those that occur after a meal (63). The gastric phase is responsible for about 10% of the pancreatic secretory response to a meal. This phase of pancreatic secretion is mediated primarily by gastropancreatic reflexes. Balloon distention of the stomach stimulates pancreatic secretion that is rich in pancreatic enzymes (41,64,65). Installation of alkaline fluid into the gastric antrum stimulates pancreatic secretion and gastrin release. In contrast, installation of acid fluid into the stomach causes a similar increase in pancreatic secretion without increasing serum gastrin levels. Each of the responses to distention is blocked by atropine or truncal vagotomy (64,66). Therefore, it appears that gastric contributions to pancreatic secretion are mediated by vagovagal cholinergic reflexes that originate in the stomach and terminate in the pancreas. Separate gastropancreatic reflexes have been identified originating in both the oxyntic and antral regions of the stomach (67,68). The antropancreatic reflex can be blocked by hexamethonium or atropine and is independent of gastrin because it is not blocked by inhibiting gastrin release via antral acidification (68,69). The stomach is also important in preparing food for delivery to the intestine, where nutrients can stimulate the intestinal phase of pancreatic secretion. By the action of pepsin and gastric lipases, proteins are digested to peptides and triglycerides to fatty acids and monoglycerides, respectively. More extensive degradation of protein by enzymes other than pepsin does not further increase the pancreatic stimulatory activity of pepsin digests, suggesting that gastric digestion of protein is sufficient to produce protein products that initiate the intestinal phase of pancreatic secretion. Acid-suppressive therapy has been reported to impair assimilation of ingested protein, and peptic digestion of proteins is important for the ability of some proteins to stimulate pancreatic secretion (70,71). In clinical situations, release of pancreatic enzymes is reduced in humans who have had gastric operations that alter gastric digestion or emptying (43,72). Of course, gastricemptying rates are critical for the delivery of nutrients to the intestine that are involved in the stimulation of neural reflexes and hormones that regulate pancreatic secretion.

Intestinal Phase The intestinal phase of pancreatic secretion begins when food and chyme empty from the stomach into the intestine. Under normal conditions, the pancreas is already primed by cephalic and gastric influences that have increased blood flow to the pancreas and initiated secretion. The intestinal phase is easier to study than the other phases because solutions can be instilled directly into the intestinal lumen. Nevertheless, the interactions of various food components such as fats, proteins, carbohydrates, and their breakdown products with neural and hormonal factors are complex. In the intestine,

pancreatic secretions serve two major purposes. First, pancreatic bicarbonate neutralizes gastric acid delivered to the duodenum. Second, pancreatic enzymes break down proteins, fats, and carbohydrates to their constituent components that can then be absorbed. The intestinal phase of pancreatic secretion can contribute as much as 70% to the postprandial secretory response. Role of Gastric Acid Most studies investigating the role of acid on pancreatic bicarbonate secretion have been performed by instilling acid solutions into various regions of the small intestine. Instillation of HCl into the duodenum is a potent stimulus of pancreatic bicarbonate secretion. However, gastric acid that is delivered to the intestine after a meal is strongly buffered by food, primarily proteins. The pH of the duodenum is 2.0 to 3.0 in the first few centimeters; however, there is a steep gradient and the pH increases to 5.0 to 6.0 in the midduodenum (73,74). This increase in the pH in the more distal portion of the duodenum is due to pancreatic bicarbonate secretion stimulated in large part by the release of secretin from the intestinal mucosa. Although instilling acid solutions into the duodenum is not the same as delivery of gastric acid normally produced by the stomach, there is substantial experimental evidence that gastric acid–induced release of secretin after a meal stimulates pancreatic secretion. It has been shown that the pancreatic bicarbonate response to a meal is twofold greater in dogs in which the pancreatic juice has been diverted from the intestine, indicating that pancreatic juice in the intestine is necessary to neutralize the intestinal contents and that the lack of this neutralization results in greater bicarbonate secretion (32,75). In addition, administration of the histamine H2 receptor blocker cimetidine, which blocks gastric acid production, has been found to substantially reduce pancreatic bicarbonate response to a meal (76). Interestingly, the pancreatic bicarbonate response to a liquid meal is related to the amount of free, unbuffered H+ entering the duodenum rather than the total amount of buffered acid (77). Finally, in dogs, maintaining the pH of a liquid gastric meal to greater than 4.5 resulted in little pancreatic bicarbonate secretion; however, secretion increased substantially as the pH values were reduced. Therefore, it appears that there is a pH threshold of 4.5 in the intestine that is important for stimulation of pancreatic secretion (78). Moreover, it can be concluded that gastric acid provides the postprandial stimulus for pancreatic bicarbonate secretion. Further studies indicated that the bicarbonate response is proportional to the load of acid entering the intestine. In addition, a pH-dependent mechanism exists for stimulation by acid (79,80). The pancreatic response to acid is also dependent on the length of small intestine that is exposed to a pH less than 4.5. The best studies have been conducted in dogs where perfusion of a 10-cm segment of duodenum with HCl increased pancreatic bicarbonate secretion up to 10% of the response produced when the entire small intestine was perfused.

1402 / CHAPTER 55 Perfusion of a 45-cm segment of duodenum and jejunum caused a response that was 75% of that of the entire small intestine (81). Studies in other species have been less rigorous in design, making it difficult to formulate strong conclusions about the precise role of acid regulating the intestinal phase of pancreatic secretion. Contributions of Proteins, Peptides, and Amino Acids to Pancreatic Secretion Most studies examining the ability of protein and amino acids to stimulate pancreatic secretion have been performed in dogs, rats, and humans (82). In dogs, intact proteins such as casein, albumin, and gelatin did not stimulate pancreatic secretion (83). However, enzymatic degradation converted these proteins into small peptides and amino acids that then were effective stimulants of pancreatic enzyme secretion (84). Amino acids and peptides are only weak stimulants of pancreatic fluid and bicarbonate secretion. Of all the amino acids, only certain amino acids are effective stimulants of pancreatic enzyme secretion (84–86). The aromatic amino acids phenylalanine and tryptophan appear to be the most potent in dogs and humans. Moreover, only L-amino acids can stimulate pancreatic secretion, which is consistent with the overall metabolic importance of these stereoisomers (87). Studies conducted in anesthetized animals have been fraught with difficulty in interpretation because anesthesia may interfere with neural or hormonal responses to nutrient stimulation of the intestine and pancreas. Although under experimental conditions amino acids can stimulate pancreatic secretion, overall, peptides may be the more physiologically relevant secretagogues because small peptides are much more abundant than amino acids in the lumen of the intestine after a meal (88). Dipeptides and tripeptides containing phenylalanine and tryptophan are effective stimulants of pancreatic secretion, as are longer peptides generated by pepsin digestion of proteins (83,89). The amount of pancreatic secretion produced by intraluminal administration of amino acids or peptides is much less than that produced by maximal doses of exogenously administered CCK (83,90,91). This finding indicates that either: (1) intraluminal amino acids or peptides are incapable of stimulating maximal release of hormones or neural signals that stimulate secretion, or (2) inhibitors of secretion are also produced together with the stimulatory signals. The mechanisms by which intestinal factors stimulate pancreatic secretion are incompletely understood; however, release of CCK into the circulation and the stimulation of cholinergic reflexes are thought to be most important (92). Little is known about the precise cellular and molecular processes by which amino acids or peptides interact with cells of the intestinal mucosa to initiate these responses. For example, it has been proposed that there are specific receptors or transporters on enterocytes that bind amino acids or peptides and generate intracellular signals stimulating hormone release or a neural reflex. Studies on isolated intestinal cells studied in vitro indicate that proteins and peptides

have little effect on CCK release and other factors are probably involved (93) (see later). The pancreatic response to intraluminal infusion of amino acids is concentration dependent (85). It appears as though amino acid concentrations of 8 mM are necessary to stimulate pancreatic secretion. Furthermore, as was described for acid stimulation of bicarbonate secretion, the length of intestine that is exposed to amino acids is also important for assessing pancreatic responses. In dogs, perfusion of the first 10 cm of duodenum with phenylalanine did not stimulate pancreatic secretion. However, perfusion of 45 cm of small intestine caused a significant increase in pancreatic enzyme output, but this was still less than what was seen with perfusion of the entire small intestine (85). Thus, the pancreatic response to amino acids is dependent on the entire load of nutrients, not just the concentration of nutrients. It has been suggested that some amino acids such as tryptophan may stimulate pancreatic secretion at concentrations as low as 3 mM (94). In humans only the proximal small intestine is involved in the stimulatory actions of pancreatic secretion. Amino acids stimulated pancreatic secretion only when perfused into the proximal 180 cm of small intestine, and no response occurred when amino acids were perfused at 230 cm from the pylorus, which would be the approximate level of the ileum (95). Therefore, the intestinal mechanisms that stimulate pancreatic secretion appear to be confined to the duodenum and jejunum. Addition of acid to amino acid (87,96) or peptide (80) preparations potentiates the pancreatic bicarbonate response. However, pancreatic enzyme secretion does not increase to greater than that produced by peptides or amino acids in the absence of acid, and there is no potentiation between amino acids or peptides with oleate or monoolein on pancreatic enzyme output (91,97). The pancreatic response to dietary protein differs in the rat. Intestinal or intragastric administration of certain proteins such as casein or soy protein potently stimulates pancreatic enzyme secretion (98). However, hydrolyzed casein does not stimulate pancreatic secretion, and amino acids are much less effective than in other species. The effects of proteins on pancreatic secretion appear to be mediated by the release of CCK (99). Contributions of Dietary Fat to Pancreatic Secretion Intact triglycerides when administered into the intestine do not stimulate pancreatic secretion in dogs (100); however, fatty acids of more than eight carbons stimulate both enzyme and bicarbonate release in dogs, rats, and humans (90,100–102). It also appears that fatty acids are only effective stimulants of pancreatic secretion when in a micellar form. After lipase digestion of fatty acids in humans, monoglycerides stimulate the pancreas, but glycerol does not (91,100). Not all fatty acids are equal in their ability to stimulate pancreatic secretion. In humans, fatty acids of 8, 12, and 18 carbon atoms are effective stimulants, and the order of potency for stimulating enzyme output is C18>C12>C8 (102). The explanation for differences in potency is not entirely clear, but it does not appear to be caused by rates of

REGULATION OF PANCREATIC SECRETION / 1403 fatty acid absorption (103). In a model of CCK cells in vitro, fatty acids of medium-chain length were shown to stimulate CCK release, raising the possibility that the effects of fatty acids in the intestine are mediated through release of CCK (104,105). However, oleate did not stimulate CCK release from isolated rat intestinal cells containing CCK without producing nonspecific effects on LDH release (a sign of cell toxicity) (93). Delivery of fatty acids in micellar form to rat intestinal cells in vitro was not performed. In dogs, intestinal perfusion with sodium oleate caused a greater increase in pancreatic enzyme secretion than perfusion with phenylalanine, with pancreatic secretory outputs reaching levels that were 70% of the maximal secretion seen with high doses of exogenous CCK (90,100). Similarly, in humans, intestinal perfusion of 10 mM monoolein produced pancreatic secretion approximating that caused by a maximal dose of exogenous CCK (91). Intestinal fatty acids also produced a robust pancreatic bicarbonate response that was as high as 70% of the maximal response to exogenous secretin in dogs (90). In contrast with the effects seen on enzyme secretion, the order of fatty acid chain length on bicarbonate secretion is the reverse, with shorter chain fatty acids (e.g., C8) being more potent than longer chain fatty acids (e.g., C18). Consequently, monoolein is a weak stimulant of bicarbonate secretion. Fatty acid stimulation of bicarbonate secretion occurs at a neutral or alkaline pH, and fatty acids do not interact with acid to potentiate bicarbonate secretion (97,103). By virtue of their ability to inhibit gastric acid secretion and gastric emptying and to stimulate pancreatic bicarbonate output, dietary fats and fatty acids can influence the pH of the proximal small intestine. Role of Bile Acids in Regulating Pancreatic Secretion Under normal conditions, bile acids are secreted into the intestinal lumen where they form micelles with fatty acids, triglycerides, and phospholipids. Consequently, free bile acids are in low concentrations in the intestine. It is possible that bile acids interact with the intestinal mucosa to elicit some response. Alternatively, bile acids may solubilize triglycerides and their digestion products that could affect pancreatic secretion. However, the overall importance of bile acids in regulating pancreatic secretion is not well understood. Little or no effect was seen when bile acids were injected into the intestine of dogs or humans, and only with a large bolus were bile acids shown to stimulate pancreatic secretion (91,100, 106). In contrast, bile and taurocholic acid were shown to inhibit the pancreatic response to digestion products of protein and fat (91). How these studies relate to the normal physiologic condition in which bile acids continually drain into the intestine from the liver or greater amounts delivered via gallbladder contraction is unclear. In humans, intraduodenal infusion of bile and the bile salt sodium-taurodeoxycholate stimulated secretin release; therefore, it is possible that the effect of bile on pancreatic bicarbonate and fluid secretion may be caused by release of secretin (107). The effects of bile on pancreatic enzyme secretion were inhibited by atropine, indicating a cholinergic mechanism is involved (52).

Bile does not modify the pancreatic response to exogenously administered CCK or secretin (91,108). In rats, diversion of bile from the intestine stimulates pancreatic secretion (109,110). This may be caused by destruction of intraluminal pancreatic enzymes in the absence of bile that leads to increased levels of endogenously produced releasing factors that exert a positive effect on pancreatic secretion through the release of CCK. This phenomenon of feedback regulation is demonstrated easily in the rat and differs somewhat in other species (see the following discussion). Other Factors Distention of the intestine with a balloon in anesthetized dogs did not affect pancreatic secretion (111); however, an inhibitory effect has been seen in conscious animals (112–114). It was reported that distention-induced inhibition of pancreatic secretion could be blocked by topical applications of lidocaine or intravenous atropine, indicating that neural pathways were involved. α- and β-adrenergic blockers did not have an effect. None of the studies related balloon distention to forces that may occur physiologically with a meal. With duodenal infusion of saline in humans, increasing volumes (0.2, 0.8, and 3.2 ml/min) produced gated increases in pancreatic enzyme and bicarbonate secretion (115). Maximal secretion was approximately 20% of that observed with exogenous CCK. Osmolality may also influence pancreatic secretory responses (116). Infusions of mannitol at concentrations up to 520 mOsm/kg were shown to stimulate pancreatic secretion to levels approximately 20% of maximum (115). However, greater levels of osmolality may actually inhibit pancreatic secretion. Calcium and magnesium salts perfused into the intestine of dogs or humans stimulated pancreatic secretion to levels similar to those of maximal doses of CCK (117,118). Specifically, CaCl2, MgSO4, and MgCl2 stimulated pancreatic secretion when perfused into the intestine or administered intravenously (119). It is possible that calcium and magnesium stimulate endogenous neural or hormonal pathways in the intestine. It is also conceivable that absorbed calcium and magnesium stimulate the pancreas directly (120). This is not surprising because extracellular calcium is necessary for enzyme secretion from pancreatic acinar cells in vitro. Perfusion of isotonic glucose into the intestine has little effect on pancreatic secretion in dogs, rats, or humans (84, 101,121).

Absorbed Nutrient Phase The absorbed nutrient phase refers to the concept that nutrients once absorbed from the intestine may directly stimulate pancreatic secretion. Such an action would represent a direct effect of nutrients on the pancreas or an indirect effect through the ability of nutrients to stimulate the release of hormones or neurotransmitters that may influence pancreatic exocrine secretion.

1404 / CHAPTER 55 Amino Acids The effects of intravenous infusion of amino acids have been controversial. Infusion of a mixture of amino acids has been shown to stimulate pancreatic enzyme secretion in rabbits, dogs, and humans (122–125). Interestingly, a mixture of 17 amino acids produced a modest increase in pancreatic secretion, whereas greater doses of phenylalanine or tryptophan produced stronger responses (123). However, a number of other investigators were unable to show any increase in pancreatic secretion after the intravenous administration of amino acids (121,126–128). More frequently, intravenous infusion of amino acids has been reported to actually inhibit pancreatic enzyme secretion in response to either intraduodenal amino acids or exogenous CCK (81,95,129,130). Fats Studies testing the effects of fatty acid infusions on pancreatic secretion have been limited. Intravenous infusion of the fatty acid decanoate had no effect on pancreatic secretion in dogs (100). Longer chain fatty acids could not be used because of their hemolytic effects. Two groups of investigators reported that intravenous infusions of emulsified triglycerides stimulated pancreatic enzyme secretion in dogs and humans (123,131). However, several other groups were unable to reproduce these findings (125,127,132). None of the studies correlated lipid levels in plasma after intravenous infusion with those that occur normally after a meal. The effects of other intravenous fatty acids or triglyceride preparations have not been tested. Other Nutrients A single report indicated that intraarterial administration of glucose stimulated pancreatic secretion of amylase in rats (133). In contrast, however, other investigators reported that infusion of glucose to produce hyperglycemia actually inhibited pancreatic enzyme and bicarbonate secretion (125). Large amounts of calcium infused intravenously have been shown to stimulate pancreatic enzyme secretion in dogs, cats, and humans, but not rats (120,134–137). Overall, the evidence that nutrients absorbed after a meal may have significant effects on pancreatic exocrine secretion are weak. However, the studies described earlier are provocative and illustrate the need to correlate levels of amino, lipids, and glucose that occur after meals with those achieved after intravenous infusion to accurately assess the effects on pancreatic secretion.

NEURAL AND HORMONAL REGULATORS For many decades it was thought that the vagus nerve and the hormones CCK and secretin were the only regulators of pancreatic secretion. However, it is now clear that multiple neural pathways involving a number of different

neurotransmitters and neuropeptides, as well as a variety of hormones, can influence pancreatic secretion in either a stimulatory or inhibitory manner. These pathways are discussed in the following section.

Neural Mechanisms The gastrointestinal tract and the pancreas are the only peripheral organs that have a well-developed neural plexus. The intrinsic nerve plexus of the pancreas receives inputs from both the parasympathetic and sympathetic nervous systems. Parasympathetic nerves innervating the pancreas originate primarily in the dorsal vagal nucleus of the brain. Most of these fibers run through the posterior vagal nerve trunk and terminate as preganglionic vagal nerves on pancreatic ganglia. Sympathetic innervation of the pancreas is supplied by neurons with cell bodies in the celiac and superior mesenteric ganglia. These nerve cells send fibers to the nerve cell bodies in pancreatic ganglia, acinar and duct cells, blood vessels, and islets. However, the sympathetic innervation of the exocrine cells of the pancreas is less than that of blood vessels and islets (138). Pancreatic ganglia integrate neuronal inputs of both the exocrine and endocrine pancreas. These ganglia are innervated by vagal preganglionic, sympathetic postganglionic, enteric, and sensory fibers (138). Postganglionic nerves containing cholinergic, noradrenergic, peptidergic, and nitrergic transmitters surround most acinar clusters and regulate exocrine secretion (139). A major role for intrapancreatic cholinergic neurons being involved in stimulation of pancreatic secretion has evolved from numerous experimental studies. These neurons receive input from both the central nervous system through the cephalic phase of pancreatic secretion and via vagovagal reflexes that involve both the afferent and efferent limbs of the vagus nerve. Vagovagal reflexes are initiated by gastric and intestinal stimulation. These pathways converge on cholinergic intrapancreatic neurons that release acetylcholine. Acetylcholine, in turn, binds to muscarinic receptors on pancreatic acinar and duct cells to stimulate both enzyme and bicarbonate secretion. The effects of acetylcholine potentially are aided in most species by the hormones secretin, VIP, and perhaps gastrin-releasing peptide (GRP). Furthermore, VIP released from peptidergic nerves in the pancreas also regulates pancreatic secretion. Vagal Innervation Electrical stimulation of the vagus nerve trunks activates efferent vagal nerve fibers causing pancreatic secretion (140,141). In most species, with the pig being an exception, electrical stimulation of the vagus nerve causes greater release of pancreatic enzymes relative to fluid and bicarbonate. Similar to electrical stimulation, insulin-induced hypoglycemia or administration of 2-DG also potently stimulates pancreatic secretion (142–145). Approximately 50% of the

REGULATION OF PANCREATIC SECRETION / 1405 as well as higher centers. The gastric and intestinal phases of the meal response activate both chemical and stretch receptors. Together, these stimuli generate a response that is manifest by vagal efferent signaling. Interestingly, it is still unknown how the specific chemical signals such as acid, amino acids, peptides, fatty acids, monoglycerides, and divalent cations actually activate the vagus nerve. Electrical activation of sites within the brain and the vagus nerve has been recorded after gastric distention and intestinal perfusion with amino acids (151). Intriguing studies examining the timing of pancreatic responses after stimulation provided unique insights into the regulation of pancreatic secretion by vagovagal reflexes and intestinally generated hormonal signals (152) (Fig. 55-1). The latency of pancreatic secretion after intraintestinal administration of tryptophan or sodium oleate was only approximately 20 seconds. However, direct administration of CCK into the portal vein did not increase pancreatic secretion until 30 seconds. Moreover, administration of atropine or truncal vagotomy attenuated the response to the intestinal stimulants, but did not alter the response to CCK. These observations indicate that intestinal stimulants activate a non-CCK mechanism to affect pancreatic secretion, and this pathway likely involves a cholinergic reflex. The effects of atropine on pancreatic secretion have been fairly uniform throughout all species tested (7,13,153–156). Atropine decreased pancreatic enzyme secretion more than it reduced fluid or bicarbonate. Surgical vagotomy also had

maximal CCK-induced response can be produced by vagal stimulation (142). Vagotomy eliminates most of the effects of insulin or 2-DG on pancreatic secretion (144). It is believed that each of these methods to stimulate the vagus nerve causes the release of acetylcholine, which activates muscarinic receptors on the pancreas because each action can be blocked by atropine (140,146). Conversely, treatment of animals with acetylcholine or other muscarinic cholinergic agonists stimulates pancreatic enzyme secretion (53). A number of other observations have clarified the role of cholinergic innervation in the pancreas. Intrapancreatic nerves and ganglia, identified by histologic examination and electron microscopy, have been found to lie in close approximation to both pancreatic ducts and acinar cells (137). Moreover, the pancreas contains the enzymes choline acetyltransferase and acetylcholinesterase which are involved in the synthesis and inactivation, respectively, of acetylcholine (147–149). In studies using pancreas removed from experimental animals, stimulation of the vagal nerve trunks stimulated enzyme secretion in the pancreas in situ (146). Finally, using radiolabeled antagonists, specific muscarinic receptors have been identified on pancreatic acinar cells (150). All of these data provide strong support for direct cholinergic stimulation of pancreatic enzyme and bicarbonate secretion. During the cephalic phase of secretion, afferent input to the vagus nerve comes from olfactory and gustatory receptors,

3.0 LATENCY 37 sec CCK ip

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FIG. 55-1. Latency of pancreatic amylase secretion after intraportal injection of cholecystokinin (CCK) or intraduodenal oleate. Amylase response as a function of time was measured in dogs treated with either intraportal injection of CCK (0.66 U/kg) (top) or intraduodenal injection of sodium oleate (1 mmol). These findings indicate that the pancreatic amylase response to intraduodenal oleate occurs more rapidly than intraportal injection of CCK. (Reproduced from Singer and colleagues [152], by permission.)

1406 / CHAPTER 55 a selective effect on reducing enzyme release and reduced both basal and stimulated secretion (142,155,157–159). The observation that atropine further reduced basal pancreatic secretion after vagotomy suggests that intrapancreatic neurons were still active after vagotomy. Notably, however, in the conscious rat, atropine does not inhibit the pancreatic secretory response to CCK or the response to release of endogenous CCK (6,9,160). Muscarinic antagonists consistently have been shown to inhibit the pancreatic response to intestinal HCl, although the effects of vagotomy have been less reproducible (142,159,161–164). Doses of atropine as low as 5 µg/kg have been shown to inhibit the effects of HCl-induced pancreatic secretion (165). These effects are seen when low acid loads are instilled, but can be overwhelmed by high amounts of HCl (7). Application of topical anesthetics to the intestinal mucosa inhibited the effects of HCl on pancreatic enzyme, fluid, and bicarbonate secretion (166–168). Although it would be possible to interpret these results as indication that local cholinergic mechanisms mediate acidinduced release of secretin and CCK, using specific secretin radioimmunoassays, investigators have shown neither atropine nor vagotomy to reduce acid-induced secretin release (161,169). Furthermore, vagal stimulation with insulin hypoglycemia or 2-DG did not stimulate secretin secretion (19,161,170). Thus, it appears that the inhibitory effects of local anesthetics on acid-induced pancreatic secretion are caused by interference of neural receptors for HCl that normally are responsible for the afferent limb of an enteropancreatic reflex, rather than a direct effect on gastrointestinal hormone release. An explanation for the variable effects of vagotomy on secretin-induced bicarbonate secretion remains unknown. An increased response to secretin after vagotomy, as has been observed by some, may be caused by increased sensitivity of the pancreas caused by chronic lack of exposure to acid, leading to increased secretin secretion and down-regulation of the secretin receptors (169,171). Support for this concept comes from the observation that chronic exposure to secretin leads to decreases in secretin sensitivity thought to be caused by down-regulation of the secretin receptor. Anticholinergic drugs inhibit bicarbonate and enzyme secretion in response to exogenous secretin. This effect is seen with low doses of secretin, but is lost with greater doses (172). Vagal input potentiates the bicarbonate response to secretin and is blocked by atropine (172). Together, these data indicate that acetylcholine released from intrapancreatic nerves potentiates the effects of secretin on pancreatic duct cells. The inhibitory effects of atropine and vagotomy on HCl-induced pancreatic bicarbonate secretion are most likely caused by interference with initiation of intrapancreatic reflexes mediated by cholinergic neurons, rather than direct effects on secretin release. It has been shown that in isolated, perfused rat pancreas, electrical field stimulation potentiated secretin-stimulated pancreatic secretion. This effect was reduced by atropine (173) or an antiGRP serum (174). Together, atropine and anti-GRP serum completely abolished the enhanced secretin effect, suggesting

that acetylcholine and GRP released from intrapancreatic nerves potentiate secretin-stimulated pancreatic secretion. In isolated rat pancreatic ducts, secretin-stimulated fluid secretion was potentiated by acetylcholine (175). Pancreatic secretion stimulated by intestinal perfusion with amino acids, fatty acids, and peptides was inhibited by vagotomy and anticholinergic drugs (12,94,142,152,157, 158,176–178). The secretory response of the autotransplanted pancreas was only 40% to 50% of that of the intact pancreas (94). Although it has been proposed that cholinergic influences mediate the release of CCK, there is little experimental evidence to suggest that CCK release is regulated directly or indirectly by cholinergic nerves. However, topical application of anesthetic drugs to the intestine blocks the pancreatic enzyme response to intestinal perfusion with amino acids (167,179). In the autotransplanted pancreas in which the gland is entirely denervated, the response to CCK is unaltered (178). Furthermore, the enzyme secretory response of the autotransplanted pancreas in response to intestinal stimulants is unaltered by vagotomy or atropine, whereas the intact pancreas is strongly inhibited by each of these maneuvers. These findings indicate that the effects of vagotomy and atropine on the intact pancreas are not caused by inhibition of CCK release or other hormones. In support of this conclusion was the finding that measurements of blood levels of CCK were unaltered in animals treated with vagotomy after oral administration of fats (180,181). Atropine has been shown to delay the CCK response to intraduodenal administration of corn oil in humans; however, the total integrated CCK response indicated by the area under the curve was unchanged (182). The response to exogenous CCK was also shown to be unaltered by vagotomy (180). Moreover, enzyme secretion from isolated pancreatic acini in vitro was not affected by atropine (183). In the isolated pancreas in situ, there was no evidence for cholinergic influences on CCKstimulated enzyme secretion. All of these data together indicate that the inhibitory effects of vagotomy and anticholinergic drugs on pancreatic enzyme secretion in response to intestinal stimulants are mediated by enteropancreatic reflexes rather than by a direct effect on CCK secretion. Quantitatively, approximately half of the enzyme response to intestinal stimulants such as amino acids and fatty acids is mediated by neural pathways that are largely cholinergic and vagovagal. However, when the effects of different loads of intestinal stimulants are considered, it appears that vagal cholinergic reflexes are the major mediators of pancreatic secretion in response to low intestinal loads, but hormones may mediate pancreatic responses to high loads of intestinal stimulants (184,185). Anatomic evidence of enteropancreatic innervation has been provided by histochemical studies in the rat demonstrating that enteric neurons actually project to pancreatic ganglia (186,187). Binding studies using isolated pancreatic acinar cells and specific muscarinic receptor antagonists have shown that M1 and M3 receptors reside on pancreatic acinar cells. These studies have been confirmed by detection of

REGULATION OF PANCREATIC SECRETION / 1407 messenger RNA (mRNA) for each receptor subtype. M1 receptors also are involved in regulating pancreatic secretion probably through a nonacinar presynaptic mechanism (188). In summary, there is good evidence for direct cholinergic stimulation of pancreatic enzyme secretion that mediates the cephalic, gastric, and intestinal phases of pancreatic enzyme secretion in response to a meal. Moreover, cholinergic reflexes potentiate the bicarbonate response to secretin. Enteropancreatic vagovagal cholinergic reflexes are the primary mediators of pancreatic enzyme secretion in response to low loads of proteins, amino acids, fatty acids, and HCl. These data should not be taken as evidence, however, that hormonal influences, primarily of CCK, do not also play an important role in regulating meal-stimulated pancreatic secretion. CCK is the major mediator of pancreatic enzyme secretion when high loads of protein, amino acids, and fatty acids are instilled into the intestine. The interrelation between CCK and cholinergic influences regulating pancreatic secretion are discussed in the following section. Sympathetic nerves of the pancreas do not appear to mediate pancreatic secretory responses to intestinal stimuli. Vasoactive Intestinal Peptide Electrical stimulation of the vagus nerve trunks stimulates pancreatic enzyme and bicarbonate secretion (146,189). This stimulation achieves pancreatic secretory levels approximately 50% of the response seen with exogenous secretin. This effect was not caused by the release of enteric hormones because removal of the stomach, small intestine, and colon had no effect on vagally stimulated pancreatic secretion. The effect of vagal stimulation on enzyme but not bicarbonate secretion was blocked by atropine. However, hexamethonium, which blocks ganglionic transmission, prevented both enzyme and bicarbonate secretion. Pancreatic bicarbonate and enzymes were stimulated by acetylcholine, but these effects were lower than those seen with electrical stimulation (140). In addition to effects on secretion, electrical stimulation of the vagus nerve also increases pancreatic blood flow and local vasodilation (190). In the vascularly perfused pig pancreas, a pancreatic bicarbonate response to vagal stimulation was retained. These findings suggested that another transmitter besides acetylcholine was involved in the pancreatic bicarbonate response to electrical stimulation. A body of work has now implicated VIP as the transmitter most likely responsible for fluid and bicarbonate secretion and vasodilation (191). Data to support VIP as a neural peptide involved in regulating pancreatic fluid and bicarbonate secretion came from two primary observations. First, VIP-containing neurons are found within the pancreas (192). These fibers surround intrapancreatic ganglia and pancreatic ducts. Second, VIP can directly stimulate pancreatic bicarbonate secretion from the isolated vascularly perfused pancreas (191,193). Subsequent studies demonstrated that electrical stimulation of the vagus nerve after atropine treatment resulted in an increase in bicarbonate secretion and an increase in VIP in

the venous effluent from the pancreas (189). Moreover, the pattern of VIP release paralleled the secretion of fluid and bicarbonate, and the amount of VIP in pancreatic venous effluent was similar to that required to stimulate pancreatic secretion with intraarterial injection (191). Administration of somatostatin blocked the bicarbonate response and reduced VIP in the pancreatic effluent, but did not alter the pancreatic secretory response to exogenous VIP. Additional information was provided by the demonstration that the VIP and pancreatic bicarbonate responses were blocked by hexamethonium or administration of an antiserum to VIP (191). VIP was released by cholinergic agonists acting on nicotinic receptors, suggesting that VIPergic neurons are activated by preganglionic parasympathetic, cholinergic fibers (194). Although these studies provide compelling evidence to support the role of VIP in regulating pancreatic fluid and bicarbonate responses to vagal stimulation, there is limited information on intact animals or humans. In the guinea pig pancreas, depolarization of nerves causes enzyme secretion by a noncholinergic, nonadrenergic mechanism (195). VIP can reproduce these effects. In many species including pigs, guinea pigs, cats, and turkeys, VIP is a full agonist; however, in rats, dogs, and humans, VIP appears to be a weak partial agonist (196). Thus, although exogenous VIP appears to be almost as efficacious as secretin (it is only 1% as potent for stimulation of pancreatic bicarbonate secretion) in some species, it is less efficacious in rats, dogs, and humans (197–199), and vagal stimulation does not release high amounts of VIP (141,143,145,200,201). Currently, it is difficult to conclude that VIP plays a major role in regulating pancreatic fluid and bicarbonate secretion in vivo, and it is most likely that it serves as a neurotransmitter that is involved in mediating pancreatic vasodilation and increasing blood flow. Further studies indicated that multiple mediators are involved in the pancreatic secretory response to electrical stimulation of the vagus nerve (202). Gastrin-Releasing Peptide GRP immunoreactivity has been identified in nerve fibers surrounding pancreatic acini and ducts and nerve cell bodies of intrapancreatic ganglia (203). GRP strongly stimulates pancreatic enzyme, fluid, and bicarbonate secretion in humans, pigs, dogs, and rats and enzyme secretion in vitro in mice, rats, and guinea pigs (204–206). In pigs, the response to GRP is approximately 40% of the secretion produced by maximal amounts of secretin and nearly 100% of the enzyme response to CCK (204). Moreover, the GRP receptor has been identified in pancreatic cancer cells and mediates enzyme secretion. However, the effects of GRP appear to be species-specific because the GRP analog bombesin does not stimulate pancreatic secretion in the dog. For many years it was believed that the effects of GRP on the pancreas were mediated by release of CCK. Although GRP is an effective stimulant of CCK release, several studies demonstrated that the actions of GRP on the pancreas are clearly independent of CCK. Intestinal resection, which removes

1408 / CHAPTER 55 CCK cells, did not affect GRP stimulation of pancreatic secretion in rabbits (207). Similar to CCK, GRP also stimulates pancreatic growth. However, this effect persists even in the presence of CCK receptor blockade (208,209). Electrical stimulation of the vagus nerve stimulated pancreatic enzyme secretion and increased GRP in venous effluent from the pancreas (174,204). In the isolated perfused porcine pancreas, atropine inhibited pancreatic enzyme secretion, but did not alter the GRP effluent from the pancreas, although it did inhibit enzyme secretion in response to exogenous GRP or electrical nerve stimulation. Pancreatic enzyme secretion in response to vagal stimulation also was inhibited by GRP desensitization, GRP antagonists, or GRP immunoneutralization (210). GRP and its analog bombesin also have been shown to stimulate acetylcholine release (211). These findings indicate that GRP stimulates release of acetylcholine from postganglionic intrapancreatic nerves in addition to stimulating the acinar cell directly. This concept is supported by the observation that tetrodotoxin inhibited GRP-stimulated amylase release by more than 70% in rat pancreatic lobules (206). Notably, however, neural blockade did not completely inhibit GRP-stimulated secretion, and a small proportion of the actions of GRP is through direct effects on pancreatic acinar cells. Additional studies are needed to fully characterize the role of GRP within the pancreas and intrapancreatic neurons. Other Peptide Neurotransmitters Immunohistochemical staining has demonstrated a number of gastrointestinal peptides in nerves of the pancreas. In vitro studies in isolated pancreatic acini have also shown that these peptides may have stimulatory or inhibitory activities. CCK, GRP, neurotensin, peptide histidine isoleucine, and CGRP have all been found in cell bodies of intrapancreatic neurons and stimulate pancreatic secretion from acinar cells (212–219). Inhibitory peptides include substance P, enkephalin, and neuropeptide Y (NPY) (212,213,215, 220,221). NPY is released from the porcine pancreas after stimulation of the vagus nerve or splanchnic nerves innervating the pancreas (222). NPY has no effects on basal pancreatic secretion and only modest inhibitory effects on secretin- or CCK-stimulated secretion (223). However, it does increase vascular resistance in blood vessels of the pancreas, and it is believed that NPY is involved in the regulation of pancreatic blood flow. Various molecular forms of CCK have been identified in the pancreas; however, it is unclear whether there is an important role for CCK as an intrapancreatic neurotransmitter that regulates pancreatic exocrine secretion because there was no detectable CCK in the venous effluent of the isolated perfused pig pancreas after vagal stimulation (191). Adrenergic Nerves Adrenergic nerves containing the neurotransmitter norepinephrine are believed to exert inhibitory influences on pancreatic secretion. However, studies to establish these

effects have been difficult because of the many diverse effects of sympathetic nerve stimulation or nonspecific effects after administration of adrenergic agents. The cell bodies of norepinephrine-containing nerves that innervate the pancreas are found in the celiac ganglion. Fibers from these cells extend to nerves in the intrapancreatic ganglia and to blood vessels, ducts, and islets (213). It appears as though there is little noradrenergic innervation of acinar cells when applying immunohistochemical techniques. Electrical stimulation of splanchnic nerves to the pancreas inhibited pancreatic secretion (140,224). Conversely, cutting splanchnic nerves was reported to increase pancreatic secretion (225). Celiac denervation has been shown to reduce pancreatic secretion by ∼70%, whereas increasing blood flow by 350%. This finding suggests that there was disruption not only of stimulatory fibers, but also of sympathetic fibers that maintain tonic constriction of pancreatic vessels (226). Functional studies examining the effect of adrenergic transmitters on pancreatic secretion have been fraught with hazard because of the wide-ranging effects of norepinephrine on multiple processes including blood pressure, blood flow, neural reflexes, and release of hormones. Various reports describe norepinephrine stimulating, inhibiting, or having no effect on pancreatic secretion (61,213,224,227–230). In contrast, in rats and dogs, specific β receptor agonists inhibit fluid and enzyme secretion during both the basal and postprandial periods and after stimulation with secretin or CCK (230–234). Overall, the effects of α and β receptor antagonists have not been helpful in increasing our understanding of adrenergic regulation of pancreatic secretion. Administration of α receptor blockers have been shown to increase, decrease, or have no effect on basal or stimulated pancreatic secretion (235–238). Other studies have been conflicting, with reports that β-blockers may increase, decrease, or have no effect on pancreatic secretion (140,224,225,228,232,235,237,239–241). Norepinephrine is believed to be the primary transmitter released by intrapancreatic adrenergic nerves. In vivo, norepinephrine has been shown to inhibit basal pancreatic flow and that stimulated by secretin in the vascularly perfused dog pancreas (242). It was thought that part of this effect was caused by vasoconstriction and decreased blood flow. In contrast with the effects on secretin-stimulated secretion, norepinephrine augmented the stimulation of pancreatic juice and enzyme secretion caused by CCK in the perfused dog pancreas. Moreover, norepinephrine stimulated enzyme secretion from fragments of rat pancreas in vitro (243,244). This effect was blocked by β but not α receptor blockers. The β receptor agonist isoproterenol stimulated fluid and enzyme secretion in the in vitro rat pancreas (244). This effect was mediated by the β1 receptor and was accompanied by large increases in cyclic adenosine monophosphate (cAMP) without changing intracellular calcium. Taken together, the studies of adrenergic regulation of pancreatic secretion do not lead to a clear conclusion. In vitro studies are difficult to extrapolate to the in vivo situation, and in vivo studies are complicated by the widespread effects of adrenergic agonists and antagonists. Nevertheless, in humans,

REGULATION OF PANCREATIC SECRETION / 1409 a selective β2-adrenergic receptor agonist has a weak inhibitory effect on CCK-stimulated enzyme secretion (245). Although pharmacologic effects can be demonstrated in vitro, currently there is insufficient evidence to conclude that adrenergic regulation of pancreatic exocrine secretion is important beyond inhibiting pancreatic juice and bicarbonate secretion that likely occurs through direct and indirect effects on pancreatic blood flow. Dopamine Overall dopamine has a secretin-like effect on the pancreas and stimulates fluid and bicarbonate secretion with little effect on pancreatic enzyme output. The effect of dopamine is blocked by dopamine receptor antagonists, but is unaffected by α or β receptor antagonists (246–249). Pancreatic acinar cells have been shown to express receptors for dopamine (250). In dogs, these receptors are linked to activation of adenylate cyclase and stimulation of fluid and bicarbonate secretion (251). Dopamine causes little enzyme secretion from either acinar cells in vitro or the pancreas in vivo (252,253). In rats and dogs, high doses of dopamine stimulate secretion of pancreatic fluid (230,248). This effect is inhibited in rats by propranolol, but not by the dopamine receptor antagonist haloperidol (228), indicating that the effects of dopamine are mediated through the β-adrenergic receptor rather than the dopamine receptor. Dopamine has been reported to inhibit pancreatic enzyme secretion in rats (230,254). In humans, dopamine has been reported to have little effect alone, but did inhibit CCK-stimulated pancreatic enzyme secretion (58,255,256) Dopamine-containing neurons have been identified in pancreatic tissue (257). Serotonin Serotonergic nerves of the pancreas appear to originate in the intestine. Intrapancreatic nerves have been shown to take up serotonin, and pancreatic acinar cells have been shown to remove serotonin from plasma and secrete serotonin, suggesting that serotonin release occurs physiologically within the pancreas (258–262). In anesthetized rats, the 5-hydroxytryptamine subtype 2 (5-HT2) serotonin antagonist ketanserin and the 5-HT3 antagonist ondansetron each inhibited secretin-stimulated pancreatic volume and bicarbonate secretion (263), suggesting that serotonin may mediate the effects of secretin through both 5-HT2 and 5-HT3 receptor subtypes. Interestingly, both of these serotonin antagonists also have been reported to inhibit secretin release induced by duodenal acid. Nevertheless, despite these advances, the overall functional significance of serotonin in pancreatic secretion still remains largely unknown. Nitric Oxide Nitric oxide (NO) is involved in the control of a number of physiologic and pathophysiologic functions in the gastrointestinal tract. In the pancreas, NO can affect secretory activity and pancreatic blood flow (264). As a volatile gas,

it is not practical to directly measure NO in biological tissues; therefore, studies on the localization of NO have been done by examining nitric oxide synthase (NOS), the enzyme responsible for the production of NO. Pharmacologic tools have been used to study the physiologic actions of NO in animals, organ preparations, and cells. The actions of NO have been deduced by using NO donors, NOS inhibitors, agents that can inactivate NO (such as superoxide-generating compounds), or agents that stabilize NO (e.g., superoxide dismutase). NO is unusual compared with other signaling molecules that bind to receptors in the cell membrane because NO can penetrate cells to interact directly with what is considered to be its primary target, guanylate cyclase (265). In this manner, NO activates the enzyme to produce cyclic guanosine monophosphate (cGMP). Immunohistochemical studies using antisera against NOS isoforms have shown that the enzyme responsible for synthesis of NO is abundant in nerve fibers of the pancreas of all species including the mouse, rat, chicken, cat, monkey, hamster, guinea pig, and human. Neuronal NOS (nNOS) positive nerve fibers are found in intrapancreatic ganglion cells, with many also containing other neuropeptides including VIP (266,267). These nerve fibers course through interlobular and interacinar spaces of the pancreas. Nerve fibers of either intrapancreatic or extrapancreatic ganglion cells including nitrergic enteropancreatic neurons and viscerosensory fibers also contain nNOS (268). These nerve fibers are found in perivascular, periacinar, and periductal regions of the exocrine pancreas and surround and innervate pancreatic islets. Islet cells may costore NOS and somatostatin (269). A constitutive form of NOS found in endothelial cells (eNOS) has been localized to vascular endothelium of the pancreas. Some acinar cells and duct endothelial cells have been shown to have nNOS-like immunoreactivity. The nitrergic supply of the exocrine pancreas and blood vessels is similar in every species examined. Ascertaining the functional role of NO in regulating pancreatic secretion has been complicated by the wide distribution of NO-producing cells and the wide range of effects of NO on a number of different cell types and tissues (270). In vivo studies are often difficult to interpret because of the effects of NO on blood flow, neurotransmission, and release of other hormones and transmitters. In general, in vivo studies indicate that NO stimulates exocrine pancreatic secretion. In rats, basal pancreatic secretion was reduced after NOS inhibition, and these effects were reversed by high doses of L-arginine (271,272). In addition, inhibition of NO synthesis significantly reduced pancreatic secretion stimulated by high doses of cerulein, pancreatic juice diversion, or feeding a meal (271,272). In humans, the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), in a dosedependent manner, reduced pancreatic enzyme but not bicarbonate or fluid secretion stimulated by secretin and cerulein. This effect also was reversed by L-arginine (273). In conscious dogs prepared with chronic pancreatic fistulae, it was found that pancreatic protein output induced by either sham feeding, a meal, or infusion of secretin plus CCK were all inhibited by the NOS inhibitor N(G)-nitro-L-arginine

1410 / CHAPTER 55 (L-NOARG) (274). Although the study failed to show any effects of L-NOARG on basal pancreatic protein secretion, others have shown an increase in the frequency of the MMC and suppression of phase III increases in pancreatic secretion (275). This finding indicates that NO may exert tonic effects to inhibit intestinal motility and stimulate pancreatic secretion during the basal state. This concept is supported by studies in rats in which basal pancreatic secretion is attenuated by NOS inhibitors (271,272). In conscious rats, secretin-stimulated pancreatic secretion was inhibited by the NOS inhibitor N-nitro-L-arginine ester (L-NAME), an effect that was reversed by L-arginine (276). NO donors have been used to test the effects of exogenous NO on pancreatic secretion. Both glyceryl trinitrate and sodium nitroprusside increased basal pancreatic secretion in conscious dogs and anesthetized cats, but had no effect on meal-stimulated pancreatic responses (274,275). The mechanism by which NO may affect pancreatic exocrine secretion remains a matter of controversy. In rats and dogs, it has been shown in vivo that attenuation of pancreatic secretion with NOS blockade was associated with a reduction in pancreatic blood flow (271,274). Accompanying studies showed that the NOS inhibitor did not affect isolated pancreatic acini, leading to the conclusion that endogenous NO exerted its effects on pancreatic secretion through alterations in pancreatic blood flow. However, additional studies indicate that other mechanisms are probably involved. It has been shown that the effects of NOS inhibitors on pancreatic secretion do not always parallel changes in blood flow. In the isolated perfused porcine pancreas, both electrical vagal nerve stimulation and treatment with VIP stimulated pancreatic secretion and induced vascular relaxation. The NOS inhibitors L-NOARG and L-NAME reduced pancreatic secretory responses to both stimuli, but did not affect vascular relaxation (277). In anesthetized cats, the NOS inhibitor L-NMMA blocked secretin-stimulated pancreatic secretion, but did not affect vascular blood flow (278). Evidence for NO having a direct effect on pancreatic secretion comes from studies on isolated pancreatic acini and lobules. Treatment of pancreatic acini with the NOS substrate L-arginine increased nitrite and cGMP levels and amylase release. These effects were blocked by L-NMMA and L-NOARG (268). NOS inhibitors also blocked carbachol-stimulated amylase release. The effects of NO on pancreatic acinar cell secretion appear to be mediated by an increase in cGMP. Using in vitro pancreatic segments, investigators used electrical field stimulation or acetylcholine to stimulate amylase output (265). Both of these responses were reduced by treatment with 10−3 M sodium nitroprusside, an NO donor, indicating that NO affected pancreatic secretion by reducing endogenous neurotransmitter release. To confirm this possibility, investigators conducted studies in pancreatic segments preloaded with [3H]choline, a method that has been used to estimate neuronal acetylcholine release. [3H]choline efflux was increased after electrical field stimulation and was inhibited in the presence of sodium nitroprusside. Sodium nitroprusside had no effect on the pancreatic secretory response to

acetylcholine, thus supporting the concept that NO acts at a presynaptic site on intrapancreatic neurons. In summary, these findings indicate that NO may have direct stimulatory effects on pancreatic acinar cell secretion and indirect effects by increasing pancreatic blood flow or through modulation of intrapancreatic parasympathetic nerves.

Hormonal Mechanisms The observation that intestinal stimulants produced similar pancreatic responses from innervated pancreas and denervated, autotransplanted pancreas suggested that pancreatic secretion was under the exclusive regulation of circulating hormones (84). To function as a hormone that stimulates pancreatic secretion, several criteria must be met. First, if such a hormone plays a physiologic role in postprandial pancreatic secretion, it must be released into the circulation after ingestion of the meal. Second, levels of the hormone in the blood that occur after a meal should be similar to the levels that are required of an exogenously infused hormone to affect pancreatic secretion. Third, strong evidence for the existence of a stimulatory regulator of pancreatic secretion would be the demonstration that administration of a specific hormone receptor antagonist or an antiserum that blocked the hormone in question reversed the stimulatory effects on pancreatic secretion. Although we now appreciate that hormones interact with neural factors, hormonal transmitters provide a major stimulus to pancreatic secretion. Historically, secretin and CCK have been considered the major hormones regulating pancreatic secretion. Our understanding of the manner in which each of these hormones interact with the pancreas has increased substantially. Secretin Secretin is the most potent stimulant of pancreatic fluid and bicarbonate secretion (279). Produced by enteroendocrine cells of the upper small intestine, secretin is released during the intestinal phase of a meal. Acid secreted from the stomach into the duodenum is the major stimulant of secretin, although ingested fatty acids may also contribute to secretin release (280). One of the major difficulties in studying secretin physiology has been the lack of sufficiently sensitive and specific secretin radioimmunoassays. It is likely that many of the early reports on secretin levels in plasma were performed with insufficiently sensitive or specific assays. Consequently, it was difficult to measure the experimentally induced or postprandial secretin response. More recently, however, improved assays have led to the conclusion that postprandial secretin levels can be accurately measured in experimental animals and humans. Despite more robust assays, a number of studies did not find significant increases in plasma secretin levels after a meal (281–284), whereas others found statistically significant increases in the

REGULATION OF PANCREATIC SECRETION / 1411 iv Rabbit serum

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0 1.2

1.0 Normal serum Bicarbonate output (mEq/15 min.)

mean values for secretin after a meal (285). Taken together, it appears that the postprandial secretin response is small in humans, amounting to approximately a doubling of basal levels (285,286). In contrast, in dogs, secretin responses are much greater, reaching levels that are 4- to 10-fold greater than basal levels (76). Some of the differences in postprandial secretin levels may be because of the type of macronutrients that were tested (287). For example, liquid meals buffer gastric acid more completely than mixed solid meals and did not contribute as much to the secretin response to gastric acid. This may explain why the increase in secretin levels after a liquid meal was not seen for approximately 100 minutes in one study (44). It would be natural to ask if such small increases in plasma secretin levels are sufficient to affect pancreatic exocrine secretion. Multiple studies have demonstrated that the pancreas is sensitive to secretin. Significant increases in pancreatic fluid and bicarbonate secretion have been demonstrated with continuous intravenous secretin infusions as low as 1 to 2.8 pmol/kg/hr (44,288–290). Bolus injections of secretin as low as 0.125 pmol/kg stimulated fluid and bicarbonate secretion in humans (291,292). Infusion of exogenous secretin that reproduces postprandial secretin blood levels stimulated pancreatic bicarbonate secretion equivalent to 10% of the maximal pancreatic secretory response, indicating that other hormones or neurotransmitters contribute to postprandial pancreatic bicarbonate secretion in humans (288,291). Interpretation of these studies may not be straightforward because of the specificities of secretin antisera and the molecular forms of endogenous secretin that may in exist in blood. For example, many secretin antibodies do not recognize glycine-extended secretin, which has been shown to be present in blood and can stimulate pancreatic secretion (293). In dogs, intestinal perfusion with HCl stimulated three times more bicarbonate secretion than an exogenous infusion of secretin that reproduced similar blood secretin levels (294). These data indicate that secretin is responsible for some of the pancreatic bicarbonate secretion caused by intestinal acid or a meal. The contribution of secretin to bicarbonate secretion should not be minimized. Notably, the pH threshold in the intestinal lumen for stimulating secretin release and pancreatic bicarbonate are the same (295,296). Moreover, the timing for delivery of acid to the duodenum, initiation of pancreatic bicarbonate secretion, and stimulation of secretin release is also similar (287). In humans who are achlorhydric or in whom gastric acid is neutralized by sodium bicarbonate, plasma secretin levels were low (285,286). In dogs, administration of antisecretin antiserum reduced the pancreatic secretory response to luminal stimulation by both sodium oleate and exogenous secretin (297) (Fig. 55-2). The failure of exogenous secretin to reproduce the entire pancreatic bicarbonate response to intestinal acid or a meal may be because of the contributions of other neurotransmitters and hormones such as acetylcholine and CCK that act synergistically on the pancreas. This hypothesis is supported by experimental data demonstrating that intestinal perfusion with amino acids in dogs potentiated the pancreatic

0.8

0.6

0.4 Anti-secretin serum

0.2

0

−60

0

60

120

Time (minutes)

FIG. 55-2. Effects of immunoneutralization of secretin on pancreatic bicarbonate secretion in dogs. The effects of intravenous normal rabbit serum (solid line) and antisecretin serum (dotted line) on the concentration and output of pancreatic bicarbonate are shown in response to feeding a meat meal. Each value represents a mean ± standard error of nine experiments in nine dogs with pancreatic fistulae. (Reproduced from Chey and colleagues [297], by permission.)

bicarbonate response to exogenous secretin (200). In addition, in several species, electrical stimulation of the vagus nerve potentiated the effects of exogenous secretin (140,200,298). The potentiation of the secretin response by electrical stimulation of the vagus nerve in cats was not blocked by atropine or adrenergic blockers, suggesting that other neurotransmitters are involved (200). It also has been shown in dogs that basal pancreatic bicarbonate and enzyme secretion were inhibited by atropine (299). However, in animals in which the pancreas was denervated, secretin-stimulated protein and bicarbonate secretion potentiated by CCK were unaffected by atropine treatment, indicating that cholinergic elements are not involved in CCK-induced enzyme secretion. Furthermore, potentiation of CCK-induced bicarbonate output by secretin does not depend on extrinsic neural or cholinergic elements (299). CCK has been shown to potentiate the action of secretin in humans (29,49,73,290,300,301), although CCK did not account for all of the potentiation of endogenous secretin-stimulated bicarbonate secretion.

1412 / CHAPTER 55 In most species studied other than the rat, the actions of secretin on pancreatic secretion were sensitive to atropine. In humans, pancreatic bicarbonate secretion stimulated by secretin and its potentiation by CCK were inhibited by atropine (163). However, even in the rat, the effects of secretin are dependent on vagal afferent signaling. Chemical ablation of vagal afferent fibers with topical application of capsaicin reduced pancreatic secretion produced by physiologic but not pharmacologic doses of secretin (302). It is likely that acetylcholine released from intrapancreatic nerves contributed to these effects on bicarbonate secretion. Potentiation of secretin-stimulated pancreatic secretion by basal cholinergic activity may explain why even low levels of secretin that occur after a meal as measured by radioimmunoassay are sufficient to stimulate bicarbonate output (298). Insulin plays a permissive role on the action of secretin and CCK (303). Immunoneutralization of insulin with specific insulin antisera in rats substantially inhibited pancreatic secretion stimulated by either a meal or secretin plus CCK (304). These findings are likely to be physiologically relevant because acinar cells are exposed to high concentrations of insulin via a vascular portal network in which venous blood flows from islets and bathes pancreatic acini. Cholecystokinin CCK is released from specific enteroendocrine cells of the small intestine known as I cells. I cells are more abundant in the proximal small intestine and become gradually less abundant farther down the small intestine. The major dietary components that stimulate CCK release are fats and protein. In certain species, such as the rat, CCK release is controlled by intraluminal proteases, and an active feedback system participates in release of a putative intestinally secreted CCK-releasing factor. One of the major problems that have plagued the CCK field has been difficulty in developing sensitive and specific CCK assays. Several radioimmunoassays and a bioassay have been developed that have sufficient sensitivity to detect what are low basal levels of CCK in plasma (305–313). Another problem has been the appearance of various molecular forms of CCK in blood. Molecular forms ranging in size from CCK-8 to CCK-58 have been described in dogs, rats, and humans (314–316). It is important that assays detect all of the potentially biologically active molecular forms to accurately estimate relevant CCK concentrations in blood. More recently, using techniques to prevent CCK degradation in plasma, investigators found that the large form of CCK, CCK-58, was the predominant form in dogs and humans and was the only form detected in the rat (314,316,317). Interestingly, evidence is accumulating that CCK-58 may have activity distinct from shorter forms (318). All biologically active forms of CCK possess an intact carboxyl terminus. Therefore, bioassays that measure biologically active hormone or radioimmunoassays directed at the carboxyl terminus of CCK and can distinguish CCK from gastrin (which shares an identical pentapeptide carboxyl terminus) should provide similar estimates of CCK

activity. Basal CCK levels are as low as 0.5 to 1 pM and increase to 10 to 16 pM after a mixed meal (44,307, 319–321). Of the specific nutrients that stimulate CCK release, fats and protein or amino acids are the most potent (307,309–311). Although some groups have reported that HCl stimulates CCK release (310,321), this has not been found by other investigators (307,320,322). A transient increase in CCK has been observed after ingestion of glucose, but not water or saline (319). In studies in which plasma CCK levels and pancreatic exocrine secretion were measured simultaneously with the infusion of exogenous CCK, it was found that pancreatic enzyme secretion could be stimulated by low levels of CCK (44). The physiologic relevance of this finding in humans has been assessed by comparing plasma CCK levels and pancreatic secretory responses after a meal. The investigators of that study concluded that because plasma CCK levels and pancreatic enzyme secretion after a meal were similar to those that occurred after the infusion of cerulein, endogenously released CCK is a major hormonal regulator of pancreatic exocrine secretion. However, one of the difficulties in interpreting the results of the study was that an average of CCK levels was used to estimate the total CCK responses and individual CCK levels could not be reproduced identically by infusion of exogenous hormone. Further support for CCK playing an important role in normal pancreatic secretion has come from studies using specific CCK receptor antagonists. The CCK blocker proglumide administered before intestinal perfusion of either amino acids or a fat emulsion, or the infusion of exogenous CCK-8, significantly decreased pancreatic enzyme secretion (323). The specificity of this response was further confirmed by the observation that proglumide did not alter pancreatic secretion stimulated by a cholinergic agonist. The CCK antagonist loxiglumide also has been shown to inhibit pancreatic enzyme secretion stimulated by CCK plus secretin (324). In humans, blockade of CCK receptors with loxiglumide or devazepide decreased pancreatic secretion to 40% to 60% of that after a meal (16,325,326). In vitro, a number of studies have demonstrated that CCK potentiates secretin-stimulated enzyme secretion in pancreatic acini from mice, rats, and guinea pig pancreas (327–329). In humans, pancreatic bicarbonate secretion in response to secretin is potentiated by exogenous CCK (290). However, studies have not been done to determine whether potentiation of secretin-stimulated pancreatic secretion occurs with physiologic levels of CCK in vivo. It did not appear as though CCK-stimulated pancreatic enzyme secretion was potentiated by secretin in dogs or humans (44,290,300). Notably, however, potentiation of pancreatic secretion by a variety of hormones and transmitters that are thought to be important in the physiologic pancreatic secretion in other species do not appear to be operational in dogs (300). It has been demonstrated in anesthetized rats that atropine and hexamethonium almost completely blocked the pancreatic enzyme response to low but not high doses of CCK (330). Furthermore, transection of vagal afferent nerves also

REGULATION OF PANCREATIC SECRETION / 1413 blocked the effects of CCK (331). In this study, CCK did not increase pancreatic secretion to greater than that produced by bethanechol, thus refuting the proposal that direct stimulation of pancreatic acinar cells by CCK requires a basal vagal cholinergic tone. In dogs, enzyme secretion produced by low but not high doses of exogenous CCK was inhibited by atropine (177). Similar findings have been observed in humans in which CCK infusion producing plasma CCK levels similar to those that occur after a meal stimulated pancreatic enzyme secretion that could be inhibited by atropine (332,333) (Fig. 55-3). These findings suggest that CCK acts through a neuronal pathway rather than directly

A

CCK alone

Net increase in Trypsin (KU/hr)

CCK + Atropine

20

15

10

5

0

B

10 20 CCK infusion (ng/kg/hr)

40

CCK alone

70 Net increase in Lipase (KU/hr)

5

CCK + Atropine

60 50 40 30 20 10 0

5

20 10 CCK infusion (ng/kg/hr)

40

FIG. 55-3. Effect of cholecystokinin (CCK) on pancreatic enzyme secretion and inhibition with atropine in humans. CCK-8 infusion evoked a dose-dependent increase in trypsin (A) and lipase (B) output in human volunteers. Atropine administration nearly abolished CCK-8–stimulated enzyme release at lower doses of 5 and 10 ng/kg/hr, but was relatively less potent at higher doses (20 and 40 ng/kg/hr) (f < 0.01, analysis of variance for repeated measures; mean ± standard error; n = 6). (Reproduced from Soudah and colleagues [333], by permission.)

on the pancreas to stimulate exocrine secretion. Earlier reports failed to demonstrate that vagotomy reduced CCKstimulated pancreatic secretion (6,9,178); however, these may be explained by differences in the lengths of time from vagotomy to experimentation. The pancreas has a unique ability to regain function after vagotomy, and if this is not taken into consideration, differences in secretory responses may change over time, which could alter experimental interpretation (334). Capsaicin is a neurotoxin that targets small-diameter sensory neural fibers. Local application of capsaicin to the vagus nerve reduced pancreatic secretion in response to physiologic doses of CCK in a manner similar to that of vagotomy or atropine administration (331). In addition, gastroduodenal but not jejunal application of capsaicin blocked pancreatic secretion in response to physiologic doses of CCK. Together, these findings indicate that at physiologic concentrations, CCK simulates pancreatic secretion through a capsaicin-sensitive afferent vagal pathway that originates in the gastroduodenal mucosa (335) (Fig. 55-4). Several anatomic and electrophysiologic studies support a role for CCK acting on the vagus nerve. CCK receptors have been identified in the rat vagus nerve by autoradiography (336). Moreover, CCK receptors reside on the presynaptic portion of vagal afferent nerve terminals (337). In agreement with the functional studies described earlier, perivagal capsaicin treatment reduced the binding of CCK to the vagus nerve (331). In rats, CCK stimulated vagal afferent nerves, as recorded by microelectrodes implanted in the nodose ganglia (331). Additional electrophysiologic and secretion studies used in combination with the CCK analog JMV180 have characterized both high- and low-affinity vagal CCK receptors (338). JMV-180 acts as an agonist on high-affinity CCK receptors and as an antagonist on low-affinity CCK receptors. In rats, the CCK1 receptor antagonist L-364,718 blocked pancreatic secretion stimulated by JMV-180 (339). However, acute vagotomy in anesthetized rats and perivagal capsaicin in conscious rats blocked this response, indicating that JMV-180 stimulated pancreatic secretion through a vagal afferent mechanism. Interestingly, JMV-180 did not block pancreatic secretion in response to physiologic doses of CCK. To assess the site of action of endogenous CCK, investigators found JMV-180 to enhance, rather than block, the pancreatic secretory response to intraduodenal administration of casein. These findings indicate that both endogenous and exogenous CCK stimulated pancreatic secretion by acting on high-affinity CCK receptors. Substantial insight into the mechanism by which CCK affects the human pancreas has been provided by characterization of CCK receptor subtypes. CCK1 receptors have been identified on rat pancreatic acinar cells using functional, biochemical, and molecular biological methods. Radioligand binding to isolated acini, receptor cross-linking, receptor autoradiography, Northern blot analysis, and in vitro secretion studies all confirmed the existence of CCK1 receptors (335,340–344). In contrast with the rodent, it has been difficult to demonstrate CCK1 receptors in the human pancreas.

1414 / CHAPTER 55 A

Intact rat

B

Chronic vagotomy

Afferent vagal

Afferent vagal CCK (Physiologic doses)

Efferent vagal

Efferent vagal ACh Intrinsic neuron CCK (Supraphysiologic doses)

ACh CCK CCK (Physiologic (Supraphysiologic doses) doses)

FIG. 55-4. Sites of action for cholecystokinin (CCK) regulation of pancreatic secretion in rats. (A) Physiologic levels of plasma act via stimulation of the vagal afferent pathways. In contrast, supraphysiologic plasma CCK levels act on intrapancreatic neurons and, to a larger extent, on pancreatic acini. (B) Adaptive changes occur after chronic vagotomy involving recruitment of intraduodenal cholinergic neurons that activate a gastrin-releasing peptide neural pathway to stimulate secretion. ACh, acetylcholine. (Reproduced from Li and Owyang [334], by permission.)

Little CCK1 receptor mRNA was detectable by reverse transcriptase-polymerase chain reaction in the human pancreas (344,345). However, CCK2 receptor was expressed in human pancreas and appeared to be the predominant CCK receptor subtype (346,347). Demonstration of CCK2 receptors on acinar cells of the human pancreas has been difficult. Immunohistochemical studies using specific CCK receptor antisera found most staining on somatostatin-containing D cells of the islets of Langerhans and CCK1 receptors were identified only on insulin-secreting cells in the fetal human pancreas (348–350). There is no conclusive evidence that either CCK1 or CCK2 receptors are present on human pancreatic acinar cells. Functional studies also failed to demonstrate CCK stimulation of enzyme secretion from human pancreatic acini, although difficulties in obtaining viable tissue brought some results into question. The best studies have been done in preparations of isolated human pancreatic acini that demonstrated robust secretory responses (346,351) (Fig. 55-5). In these studies, muscarinic cholinergic agonists stimulated increases in both intracellular Ca2+ and enzyme secretion, but CCK had no effect (346). Further evidence for the lack of functional CCK receptors was provided studies in which the CCK2 receptor was artificially inserted into human acinar cells by adenoviral-mediated transfer of CCK receptor DNA (346). CCK-stimulated increases in intracellular Ca2+ were seen after but not before CCK receptor gene transfer. Together, these findings indicate that human pancreatic acinar cells do not normally express functional CCK receptors. A series of studies have shown that CCK acts in concert with serotonin to stimulate postprandial pancreatic enzyme

secretion. In rats, the CCK receptor antagonist L-364,718 reduced postprandial pancreatic enzyme secretion by 54%, whereas the combination of L-364,718 and ketanserin, a 5-HT2 antagonist, and ICS-205,930, a 5-HT3 antagonist, reduced pancreatic secretion by 94% (352). Electrophysiologic studies have demonstrated that serotonin and serotonin analogs activate 5-HT3 receptors on nerve terminals within the ferret gastric and duodenal mucosa and stimulate firing of vagal afferent fibers (353,354). Recordings from the nodose ganglia were activated by intraluminal osmotic loads or administration of carbohydrates, effects that were antagonized by the 5-HT3 antagonist granisetron (355). Even intraluminal administration of serotonin stimulated afferent vagal firing through 5-HT3 receptors and stimulated pancreatic protein output. These effects were blocked by the 5-HT3 antagonist ondansetron, acute vagotomy, administration of methscopolamine, or perivagal application of capsaicin (356). Agents that deplete the mucosa of serotonin, such as p-chlorophenylalanine, eliminated neuronal activation within the nodose ganglia normally stimulated by intraluminal factors. Thus, it appears that intraluminal chemical stimuli activate 5-HT3 receptors on mucosal vagal afferent fibers. In contrast, mechanical stimulation of the mucosa stimulated both 5-HT3 and 5-HT2 receptors on vagal afferents to stimulate pancreatic secretion (352). Together, these findings indicate that 5-HT and CCK both activate vagal afferent pathways that originate in the intestinal mucosa and stimulate pancreatic secretion. The ability of CCK to potentiate the actions of 5-HT were demonstrated in studies in which infusion of subthreshold doses of CCK-8, which by themselves did not stimulate pancreatic secretion, when accompanied by

REGULATION OF PANCREATIC SECRETION / 1415 7 Carbachol

Amylase release (% total/30 min)

6 AdCCKB + CCK8

5 4 3 2

CCK8 Gastrin

1 0 0

−11

−10

−9 −8 −7 −6 Secretagogue (Log M)

−5

−4

−3

FIG. 55-5. Human pancreatic acini secrete amylase in response to carbachol, but not cholecystokinin (CCK)-8 or gastrin. Isolated human pancreatic acini, either uninfected or infected for 4 hours with an adenovirus that expresses the human CCK2 receptor (AdCCKB), were incubated with increasing concentrations of CCK-8, gastrin, or carbachol at 37°C for 30 minutes. The concentration of amylase released into the medium was measured using a colorimetric reagent and was expressed as a percentage of initial acinar amylase content. Data are the means of three separate experiments. (Reproduced from Ji and Bi [346], by permission.)

intraduodenal infusion of maltose or hyperosmotic saline (that activate 5-HT receptors), produced greater pancreatic secretion than either agent alone. Electrophysiologic studies have identified some neurons within the nodose ganglia possessing high-affinity CCK receptors that respond to intraluminal infusion of serotonin. In these studies, neurons were stimulated with 5-HT and subthreshold doses of CCK that by themselves produced no measurable electrophysiologic effect. The effects of 5-HT were blocked by the CCK receptor antagonist CR-1409 (357), demonstrating that CCK potentiated the actions of 5-HT. These potentiating actions of CCK and serotonin may explain why even small postprandial increases in plasma CCK levels are sufficient to stimulate pancreatic enzyme secretion (335). One group of investigators has found that intraduodenal administration of CCK-8 stimulated pancreatic secretion (358). This effect was blocked by muscarinic receptor antagonists or CCK antagonists, providing additional support that CCK from the intestine stimulates pancreatic secretion through vagal reflexes. It is important to emphasize that although other gastrointestinal hormones including CCK have been detected in the lumen of the intestine, the physiologic relevance of intraluminal CCK is unknown. Nevertheless, under physiologic conditions, CCK acts primarily through vagovagal and possibly short enteropancreatic reflexes to stimulate pancreatic secretion. Neurotensin It has been demonstrated in several species including humans that neurotensin stimulates pancreatic secretion (359–361). Intestinal administration of fatty acids increased plasma neurotensin levels, and the pattern of neurotensin-stimulated

pancreatic secretion is similar to that stimulated by intestinal fat (362). However, it is unclear that neurotensin is a physiologic regulator of pancreatic secretion because the amounts of neurotensin infused produced greater plasma neurotensin levels than occurred after a meal or after intestinal perfusion with fatty acids. In humans, neurotensin stimulated bicarbonate secretion, but actually decreased enzyme secretion stimulated by secretin and cerulein (363). Another group of investigators has found that neurotensin accentuated the enzyme response, but not the bicarbonate response to secretin, and the bicarbonate but not the enzyme response to cerulein (364). In rats, it was observed that neurotensin inhibited basal pancreatic secretion, but did not affect secretin- or CCK-stimulated secretion (365). The conflicting results in humans and the lack of inclusive experimental data in animals, together with the high doses of neurotensin that were required for stimulating pancreatic secretion, make it unlikely that neurotensin has a role in regulating meal-stimulated pancreatic secretion (364). Xenin Xenin is a 25-amino-acid peptide that is structurally related to neurotensin (366). It is produced by endocrine cells of the gastric mucosa and has biological actions that are similar to neurotensin. Xenin is released into the circulation after ingestion of the meal. In the gastrointestinal tract, xenin binds to the neurotensin receptor and inhibits acid secretion and pancreatic exocrine secretion. Neurotensin receptor antagonists block some of the actions of xenin, indicating that these two peptides may interact differently with this receptor. Although the biological actions of xenin in pancreatic secretion have been demonstrated by infusion of exogenous

1416 / CHAPTER 55 peptide, the physiologic role of xenin in pancreatic exocrine secretion is unknown. Histamine Although the principal effect of histamine on the gastrointestinal tract is regulation of gastric acid secretion, it also has been shown to have effects on pancreatic exocrine secretion. Histamine-immunoreactive mast cells are distributed throughout the pancreas in many species including humans, dogs, fox, sheep, pigs, cattle, and rats (367). In the human pancreas, histamine is more abundant in the head portion compared with the tail (368). Mast cells are primarily located around blood vessels in the pancreas and are less abundant in the acinar and endocrine portions of the gland. In the guinea pig pancreas, mast cells also are associated with peptidergic neurons, especially those containing substance P (367). Because of its location around blood vessels and its known vasodilatory effects, it has been postulated that histamine plays a role in mediating pancreatic blood flow, particularly in the microcirculation. Pancreatic acinar cells express both H1 and H2 histamine receptors (369,370). H1 receptors are coupled to inositol phosphate metabolism and generation of intracellular calcium as an intracellular signal. H2 receptors are coupled to Gs and, on stimulation, result in generation of cAMP (371). H3 receptors are located on presynaptic nerve terminals and inhibit the release of neurotransmitters (372). Histamine is found in pancreatic juice of dogs after stimulation with secretin or pilocarpine. In anesthetized dogs and the isolated dog pancreas, histamine stimulated pancreatic juice secretion, but appears to be less effective in the conscious dog and in rats (367,373). Histamine directly stimulated pancreatic enzyme secretion from pancreatic lobules in vitro from the rat, rabbit, and guinea pig (372). The effects of histamine were dose dependent, however, the concentrations needed to achieve maximal stimulation of enzyme secretion (∼10−3 M) were much greater than those of traditional hormonal secretagogues such as CCK or secretin (371). Histamine is a powerful stimulant of gastric acid secretion by virtue of its ability to activate H2 receptors on parietal cells. Through its actions on acid secretion and the ability of acid to stimulate secretin release, histamine may indirectly affect pancreatic fluid and bicarbonate secretion. Therefore, in vivo studies of the effects of histamine and histamine receptor antagonists on pancreatic fluid and bicarbonate secretion should be interpreted with caution. The effects of specific histamine receptor antagonists on pancreatic exocrine secretion have varied depending on the animal species, the state of anesthesia, and the type of antagonist used. In humans, cimetidine, famotidine, and ranitidine, which are commonly used H2 receptor blockers, did not produce clear inhibitory effects on either food- or secretin-stimulated pancreatic secretion (374–376). In the anesthetized guinea pig, histamine, secretin, and CCK stimulated pancreatic juice flow and enzyme secretion. However, the effects of secretin and CCK-8 were much

larger than those obtained with histamine. When histamine was infused with secretin, there was marked potentiation in both juice flow and enzyme output. When histamine was administered with CCK, there was only potentiation of CCK-stimulated fluid output (377). This finding suggests that histamine potentiates CCK-stimulated effects similar to those of secretin and may signal through a common intracellular mediator such as cAMP. In vivo, the effects of histamine on the pancreas are complex and probably reflect a combination of different effects of histamine signaling through its receptor subtypes. The histamine H3 agonist (R)- a-methylhistamine stimulated amylase secretion from isolated guinea pig pancreatic lobules (372), but had inhibitory effects on pancreatic juice and protein output stimulated by electrical field stimulation. These effects were inhibited by selective H3 blockers. The findings of the study suggested that stimulation of H3 receptors in the pancreas decreased fluid and enzyme output by inhibiting acetylcholine release from intrapancreatic nerves. Taken together, these findings indicate that histamine, which functions through paracrine actions in the pancreas, can exert secretory effects via activation of the H1 or H2 receptors on pancreatic acinar cells or inhibitory effects through actions via the H3 receptor on presynaptic neurons. It is possible that under conditions in which mast cells are more abundant in the pancreas such as pancreatitis, pancreatic exocrine secretion could be altered by histamine. However, current evidence does not support a strong role for histamine as a regulator of pancreatic secretion under normal conditions. Proteinase-Activated Receptor Signaling Proteinase-activated receptors (PARs) comprise a novel family of G protein–coupled membrane receptors that are activated by proteolytic cleavage. Four types of PAR receptors (PAR-1, -2, -3, and -4) have been identified. PAR-1, -3, and -4 are activated by thrombin, whereas PAR-2 is activated by trypsin, tryptase, and possibly other proteinases. Activation of PARs occurs by proteolytic cleavage of the amino-terminal portion of the extracellular region of the receptor, allowing a new amino terminus to bind the receptor as a tethered ligand. PAR-1, -2, and -4 also can be activated by applying synthetic peptides based on the receptor-activated sequence of each receptor (such as SLIGKV for the human PAR-2). PARs transmit signals by activating the G protein Gq/11 with subsequent activation of phospholipase C (PLC). The gastrointestinal tract is particularly abundant in PAR-1 and -2. PAR-2 (but not the other PARs) is involved in regulation of pancreatic exocrine secretion. Administration of the PAR-2–activating peptide stimulated pancreatic juice secretion in anesthetized rats (378). PAR-2 activation also stimulated amylase release from isolated pancreatic acini in vitro, as well as in conscious mice. Therefore, the effect of PAR-2 activation appears to be directly on the acinar cell. In dog pancreatic duct cells, PAR-2 agonists activated ion channels that are likely involved in pancreatic juice secretion (378,379). Although it is difficult to determine the precise

REGULATION OF PANCREATIC SECRETION / 1417 role of PAR-2 in the regulation of pancreatic exocrine secretion, the location of the receptor on acinar cells and the abundance of proteinases within the pancreas and inflammatory cells that can migrate to the pancreas raise the possibility that PAR signaling may be important under physiologic or pathophysiologic conditions.

Feedback Regulation of Pancreatic Secretion In 1972, Green and Lyman (380) discovered that installation of trypsin inhibitor into the upper small intestine of the rat stimulated pancreatic enzyme secretion. Furthermore, surgical diversion of the bile-pancreatic duct to remove mixed bile and pancreatic juice produced a potent stimulus for exocrine secretion, and this stimulation was suppressed by intraduodenal infusion of trypsin (380). These observations formed the basis for the hypothesis that a negative feedback mechanism existed by which signals for pancreatic secretion emanating from the gut are controlled by the presence or absence of proteases in the intestinal lumen. These observations have generated considerable interest in the mechanisms that regulate pancreatic secretion in experimental animals and humans. Most studies examining feedback regulation of the pancreas have been conducted in rats. Diversion of pancreatic juice in unanesthetized animals produced pancreatic responses that were similar in magnitude to those of exogenous administration of CCK. During diversion of bilepancreatic juice, there was no additional increase in pancreatic secretion with the administration of exogenous CCK, carbachol, insulin, or 2-DG (2,381). However, in anesthetized rats, exogenous CCK and 2-DG were able to strongly stimulate pancreatic secretion even in the presence of bile-pancreatic juice diversion, implying that anesthesia interfered with the generation of a signal coming from the intestine, but did not interfere with the ability of the pancreas to respond to secretagogue stimulation. The pancreatic bicarbonate response to secretin or infusion of intestinal acid was not altered with pancreatic diversion, indicating that bicarbonate secretion was not regulated by a feedback mechanism other than by neutralizing gastric acid through secretion of pancreatic bicarbonate. Diversion of bile alone stimulated pancreatic secretion; however, this effect was only partially abolished by the infusion of bile acids (109,382,383). The effect of bile diversion was thought to be related to degradation of intestinal proteases in the absence of bile, reducing protease activity, and thus initiating the feedback response. Return of as little as 10% of the bile-pancreatic juice abolished the effects of diversion on pancreatic secretion (110). This implies that approximately 90% of pancreatic juice needs to be eliminated before an increase in pancreatic secretion is observed through the feedback mechanism. Feedback regulation of pancreatic secretion may involve factors other than just intraluminal proteases. In rats prepared with gastric fistulae, the pancreatic response to

diversion was significantly reduced when gastric flow was eliminated from the intestine (384). This response may have been caused by gastric acid because intestinal perfusion with HCl in rats with an open fistula increased pancreatic secretion in the presence of pancreatic juice diversion. Therefore, gastric acid entering the duodenum may play a role in feedback regulation of pancreatic secretion. Additional studies have examined the effects of specific enzymes on bile-pancreatic duct diversion. Installation of trypsin, chymotrypsin, or elastase into the upper small intestine suppressed the pancreatic secretory response to diversion; however, amylase and lipase had no effect (380,382,385–389). The ability of these enzymes to modify feedback regulation was confined to the upper third of the intestine (386). These effects also have been shown to be caused by the proteolytic activity because inactive protease zymogens (chymotrypsinogen) did not modify the effects of pancreatic juice diversion (385). The effects also were limited to the lumen of the gut because intravenous administration of enzymes did not modify pancreatic secretion (386). Further evidence for the role of intraluminal proteases in feedback regulation comes from extensive studies testing different types of protease inhibitors. In the absence of pancreatic juice diversion, a variety of protease inhibitors have been shown to be effective stimulants of pancreatic secretion. These include soybean trypsin inhibitor, potato chymotrypsin inhibitor, aprotinin, pancreatic secretory trypsin inhibitor (PSTI), and some other synthetic trypsin inhibitors (380,387,390–393). However, like trypsin, the effects of these inhibitors on pancreatic secretion occurred only when they were instilled into the proximal third of the small intestine. Intact proteins such as casein or soy protein instilled into the stomach or duodenum were potent stimulants of pancreatic secretion (380,391,394). In contrast with other proteins such as albumin and lactalbumin, hydrolyzed proteins, small peptides, and amino acids were not effective stimulants of pancreatic secretion (380,391,394). The abilities of certain proteins to stimulate secretion correlated with their susceptibilities to serve as substrates for trypsin and perhaps other proteases (99,391). It has been hypothesized that dietary proteins could behave as transient “protease inhibitors” when instilled into the gut because they serve as substrates for these enzymes and could temporarily bind proteases within the intestinal lumen. Bile-pancreatic juice diversion not only stimulates pancreatic secretion in many species, but also promotes pancreatic growth in rats (395). Hamsters appear to be similar to rats in that pancreatic diversion increased pancreatic secretion and growth (396). Moreover, in rats and hamsters, prolonged dietary feeding of trypsin inhibitors and meals that are rich in proteins, such as casein, increased pancreatic growth, pancreatic enzyme synthesis, and even caused pancreatic adenomas. Feeding raw soybean flour to chickens stimulated pancreatic growth; however, adding trypsin inhibitor to the diets of pigs or monkeys did not have any effect on pancreatic growth (397).

1418 / CHAPTER 55 Only one group has been able to demonstrate a positive effect of pancreatic juice diversion on pancreatic secretion in the dog (398), and this has not been reproduced by others (32,142). Intraduodenal trypsin inhibitors have not been shown to stimulate pancreatic secretion in dogs. Accordingly, there are few data to suggest that intestinal feedback regulation of pancreatic secretion exists in the dog. Pancreatic juice diversion has been reported to stimulate pancreatic secretion in the cow (399). The existence of feedback regulation of pancreatic secretion in humans has been controversial because these studies have been more difficult to interpret. Studies instilling trypsin inhibitors into the duodenum have not consistently demonstrated an effect on pancreatic secretion. Specifically, the protease inhibitors aprotinin and FOY-305, which inhibit trypsin but have little effect on chymotrypsin, did not significantly stimulate enzyme secretion in humans (301,400,401). Interestingly, human trypsin may be resistant to some trypsin inhibitors such as those prepared from soybean (402,403). Therefore, the type of trypsin inhibitor used may be critical for assessing the protease-sensitive component responsible for regulating pancreatic secretion. A Bowman–Birk soybean trypsin inhibitor that inhibits chymotrypsin and elastase caused significant stimulation of pancreatic secretion when instilled into the duodenum of human volunteers (404). Together, these studies indicate that some combination of trypsin, chymotrypsin, and elastase participate in the regulation of pancreatic secretion and that inhibition of each of these may be necessary to effectively stimulate pancreatic secretion in humans. In summary, it appears as though active proteolytic enzymes in the upper small intestine are central to a feedback mechanism regulating pancreatic secretion in rats, mice, hamsters, chickens, cows, pigs, and humans. Role of Cholecystokinin in Feedback Regulation Although it had been postulated that the effects of pancreatic diversion on pancreatic secretion were mediated by CCK, it was not until the mid 1980s that assays sensitive and specific enough to actually measure blood levels of CCK became available. A specific bioassay and several radioimmunoassays have been used to estimate circulating CCK levels (305–313). Since then, studies have demonstrated that installation of trypsin inhibitor into the stomach of rats was a potent stimulus of CCK secretion, and that dietary proteins that were potent stimulants of pancreatic secretion were also effective stimulants of CCK release (305). It also was shown that dietary proteins that effectively stimulated CCK secretion were ineffective with the coadministration of exogenous trypsin (99). Moreover, diversion of bile-pancreatic juice significantly increased plasma CCK levels (405). Substantial evidence indicated that CCK mediated the effects of trypsin inhibitor feeding and bile-pancreatic juice diversion on pancreatic growth. This has been demonstrated in rodents, where not only did each of these interventions increase plasma CCK levels, but these effects were completely

blocked by CCK receptor antagonists (406,407). In rats, a dynamic relation was noted between pancreatic growth responses and CCK levels during the course of feeding a high-protein diet (408). During the first few days of feeding a high-casein diet, plasma CCK levels were high. Over the ensuing week, pancreatic size increased substantially, after which plasma CCK levels declined to basal levels. It appeared as though casein-stimulated CCK release caused pancreatic growth. It was proposed that pancreatic hypertrophy was accompanied by an increase in pancreatic enzyme output establishing a new steady-state in which sufficient enzymes were produced to reduce the effects of a high-protein diet on CCK release. These findings suggested that a dynamic interaction existed among dietary, hormonal, and pancreatic secretory and growth responses. Cholecystokinin-Releasing Factor The mechanism by which intraluminal proteases modulated pancreatic secretion had long been a subject of speculation. In 1989, it was demonstrated that stimulation of pancreatic secretion and CCK release occurring in the absence of luminal trypsin was caused by a trypsin-sensitive CCK-releasing peptide that was secreted into the intestinal lumen (409). This “CCK-releasing factor” stimulated CCK release when introduced to the intestinal lumen and was sensitive to trypsin degradation. According to the hypothesis, in the rat, the CCK-releasing factor is secreted spontaneously into the lumen of the proximal small intestine. In the presence of proteolytic enzymes, the releasing factor is degraded and inactive. However, when bile-pancreatic juice is diverted or protease inhibitors are present, the releasing factor remains intact and is able to stimulate CCK secretion. Proteins appear to stimulate pancreatic secretion by complexing with enzymes within the intestinal lumen and protecting the CCK-releasing factor from proteolytic degradation. The releasing factor is thereby preserved so that it can stimulate the CCK cell to secrete CCK. CCK, in turn, stimulates pancreatic enzyme secretion. This hypothesis is supported by the observation that intact proteins stimulated CCK secretion in vivo, but not from isolated mucosal cells in vitro (93,99). The factors regulating production and secretion of the CCK-releasing factors are largely unknown. It is controversial whether cholinergic innervation is important for regulation of CCK-releasing factor–mediated pancreatic secretion. In anesthetized rats, atropine was found to abolish the pancreatic secretory response and increases in plasma CCK levels caused by bile-pancreatic juice diversion (410,411). However, in conscious rats, atropine had no effect on pancreatic secretion stimulated by intraduodenal infusion of dietary proteins or trypsin inhibitor (412). Moreover, pancreatic secretion and CCK release were unaffected by atropine treatment in conscious rats after bile-pancreatic juice diversion (413). In a model of vascularly perfused intestinal segments, in which CCK release could be measured after intraluminal administration of food and other

REGULATION OF PANCREATIC SECRETION / 1419 stimulants, luminal nutrients were found to stimulate CCK release without activating intramural neurons (414). Somatostatin inhibited increases in plasma CCK levels accompanying bile-pancreatic juice diversion by inhibiting the release of CCK-releasing factor (415). Two groups, at the same time, purified from rat intestinal juice or porcine intestinal extracts different substances that possessed CCK-releasing activities (416,417). A 72-aminoacid protein was isolated from washings of the rat small intestine (416). By sequential purification and bioassay of CCK-releasing activity, a novel peptide was discovered, named luminal CCK-releasing factor (LCRF). When introduced into the lumen of the upper small intestine, the amino-terminal 35-amino fragment of LCRF exhibited full biological activity for stimulating pancreatic exocrine secretion. The stimulatory activity of intestinal washings was eliminated by immunoneutralization with a specific LCRF antiserum. Histochemical staining demonstrated immunoreactive LCRF in intestinal mucosal cells and enteric nerves (418). It was proposed that LCRF released into the lumen of the intestine interacts with the apical surface of CCK cells to stimulate CCK secretion. This possibility has been supported by studies demonstrating that LCRF stimulated CCK release from isolated human intestinal mucosal cells in vitro and from the CCK-containing cell line, STC-1 (419). Therefore, LCRF had a direct effect on both murine and human CCK cells. Diazepam-binding inhibitor (DBI) is an 89-amino-acid protein that is abundant in the central nervous system and peripheral nerves and serves as a natural inhibitor of diazepam binding. In 1996, DBI was purified from porcine intestinal extracts using a bioassay method for screening CCK-releasing activity (417). It was proposed that DBI was responsible for the feedback regulation of pancreatic secretion and the postprandial release of CCK. The following findings were demonstrated in rats to support this hypothesis: (1) intraduodenal administration of the 18-amino-acid fragment DBI, DBI33-50, increased pancreatic secretion and plasma CCK levels; (2) increases in luminal DBI immunoreactivities paralleled changes in plasma CCK levels during diversion of pancreatic juice; and (3) intraduodenal administration of DBI antiserum reduced pancreatic secretion and release of CCK during diversion of bile-pancreatic juice (420). DBI immunostaining and in situ hybridization studies in the rat demonstrated that it was widely distributed in the villi of the upper intestine (421). Therefore, it is conceivable that the apical surface of CCK cells would be exposed to DBI secreted into the lumen of the intestine. In vitro studies demonstrated that DBI33-50 stimulated CCK release from the murine CCK-containing cell line, STC-1, by eliciting Ca2+ oscillations through a voltage-dependent L-type Ca2+ channel (422). Monitor peptide, also known as PSTI-I, is a 61-amino-acid peptide that was originally purified from pancreatic juice (392). When instilled into the lumen of the intestine, monitor peptide stimulated pancreatic secretion by virtue of stimulating CCK release (423). Although monitor peptide possesses trypsin inhibitor activity, its ability to stimulate CCK secretion

appears to be independent of this effect because monitor peptide could directly stimulate CCK release from isolated intestinal mucosal cells in vitro (424,425). Monitor peptide is produced by pancreatic exocrine cells and is secreted into the pancreatic duct eventually reaching the duodenum; therefore, the physiologic action of monitor peptide differs substantially from that of intestinal CCK-releasing factors. Because monitor peptide is secreted in pancreatic juice, the levels in the intestine are not increased unless pancreatic secretion is stimulated. Therefore, monitor peptide does not account for the negative feedback responses caused by diversion of bile-pancreatic juice, which appears to be mediated by the intestinal CCK-releasing factor. Although the physiologic role of monitor peptide is unknown, it could either mediate protein-stimulated pancreatic secretion or potentiate CCK release and pancreatic secretion once the process of pancreatic secretion has been initiated. Therefore, monitor peptide could provide a positive feedback mechanism reinforcing meal-stimulated secretion. Experimentally, two lines of evidence suggest that monitor peptide may participate in a positive feedback loop. First, in rats fed a high-protein diet, monitor peptide gene expression in the pancreas was increased (426). Second, administration of exogenous CCK increased monitor peptide mRNA levels, suggesting that the effects of diet were mediated by CCK (427). Monitor peptide can interact directly with CCK cells. Radiolabeled monitor peptide has been shown to bind to intestinal mucosal cells (428). In vitro studies have shown that monitor peptide directly stimulated CCK secretion from isolated rat intestinal mucosal cells and did so in a dosedependent manner (424,425). Secretion required extracellular calcium because studies with the calcium chelator, ethylenediaminetetraacetic acid, and the calcium channel blocker MnCl2 each blocked monitor peptide-stimulated CCK release. These data indicate that intracellular calcium is a likely mediator of monitoring peptide-stimulated signaling. Not only do these findings indicate that monitor peptide has a direct effect on CCK cells, but also that its actions are not dependent on neural influences. A putative monitor peptide receptor has been partially characterized in dispersed intestinal mucosal cells from rat jejunum (429). Specific monitor peptide binding was demonstrated to be reversible, temperature dependent, and pH dependent, which is consistent with receptor binding. Autoradiography of an affinity cross-linked complex using 125 I-labeled monitor peptide showed a potential receptor with a molecular mass of 33 or 53 kDa in the reduced or unreduced form, respectively. These studies provide evidence that monitor peptide binds to specific cell-surface receptors on intestinal mucosal cells to stimulate CCK secretion. Because monitor peptide is present in the intestinal lumen, it is believed that these receptors reside on the apical surface of mucosal CCK cells. In this position, monitor peptide receptors would be available to bind monitor peptide and initiate a cascade of signaling events leading to CCK secretion.

1420 / CHAPTER 55 Feedback Regulation of Secretin Secretin release and pancreatic bicarbonate secretion also have been proposed to be regulated by a feedback system involving intraluminal protease activity (398). Several observations support this hypothesis. Diversion of pancreatic juice from the duodenum produced increases in blood secretin levels and pancreatic secretion in anesthetized rats (430). Effects on both secretin release and pancreatic exocrine secretion were inhibited by reinfusion of pancreatic juice. It was proposed that the effects of intraduodenal proteases on secretin release were caused by degradation of a secretin-releasing peptide. This hypothesis was based on evidence that acid-induced release of secretin was mediated by a protease-sensitive substance in duodenal juice (431). In anesthetized rats, intestinal perfusate was collected in the presence of duodenal acid infusion. This material, when reinfused into the intestine, stimulated pancreatic bicarbonate secretion. A similar material has been found in dogs that, when instilled into the duodenum of recipient rats, stimulated pancreatic bicarbonate secretion. Moreover, bicarbonate secretion was inhibited by intravenous infusion of a specific antisecretin serum (432). On further purification, the aminoterminal sequence of 31 residues of the putative secretinreleasing peptide was found to be identical to canine pancreatic phospholipase A2 (PLA2) (433). In support of the concept that PLA2 may be a functional secretin-releasing peptide, was the observation that acid infusion in the duodenum was shown to stimulate pancreatic PLA2 immunoreactivity and to increase pancreatic bicarbonate secretion. In addition, treatment of intestinal washings collected from rats after acid infusion of the duodenum with anti-PLA2 serum abolished the stimulatory effects on secretin release and pancreatic secretion in recipient rats (434). Interestingly, pancreatic PLA2 has been shown to stimulate secretin release in secretin-producing cells in vitro (435). These findings suggest that pancreatic PLA2 acts as a secretin-releasing factor. However, porcine pancreatic PLA2 was unable to stimulate secretin release in rats, raising the possibility that other factors might be involved (279).

INHIBITION OF PANCREATIC SECRETION There is a tendency to think only of stimulatory factors that regulate pancreatic exocrine secretion; however, the net effect of neural-, hormonal-, or meal-stimulated effects on the pancreas is likely to be the combined result of both stimulatory and inhibitory influences. A number of observations support the concept that pancreatic secretion is regulated by physiologic inhibitors. First, after a meal, there was a brief peak in pancreatic enzyme and bicarbonate secretion that approximated the maximal responses seen after exogenous secretin or CCK administration, but the remainder of the postprandial pancreatic secretory response was much less than the maximal response (44,46,75,436). Second, both cephalic and gastric phases of pancreatic secretion were

much less than maximal responses (13,14,65,68,437,438). Third, intestinal perfusion with amino acids produced a pancreatic response that was much less than the maximal response seen after infusion of exogenous CCK (323). Fourth, intestinal perfusion with hypertonic solutions of saline or glucose inhibited secretin-stimulated pancreatic bicarbonate secretion or the effects of secretin plus CCK on pancreatic enzyme secretion (165,439,440). Fifth, despite the stimulatory effects of fatty acids in the proximal small intestine, installation of sodium oleate into the distal small intestine or colon inhibited pancreatic secretion (441–444). In humans, pancreatic exocrine secretion stimulated endogenously was inhibited 80% by ileal perfusion with either carbohydrates or fat at concentrations that occurred physiologically in the late postprandial state (445). It is now clear that ingestion of a meal causes the release of gastrointestinal hormones that have inhibitory effects on the pancreas. In addition, it is possible that inhibitory reflexes are initiated after eating that also directly affect the pancreas or the release of stimulatory hormones such as CCK and secretin or intrapancreatic neurotransmitters such as acetylcholine or VIP. As has been discussed previously, absorbed nutrients such as glucose and amino acids may exert inhibitory effects on the pancreas, but these are also likely to be mediated by hormones. To function as an inhibitory hormone that regulates pancreatic secretion, several criteria must be met. First, if such a hormone plays a physiologic role in postprandial pancreatic secretion, it must be released into the circulation after ingestion of the meal. Second, levels of the hormone in the blood that occur after a meal should be similar to the levels that are required of an exogenously infused hormone to affect pancreatic secretion. Third, strong evidence for the existence of an inhibitory regulator of pancreatic secretion would be the demonstration that administration of a specific hormone receptor antagonist or an antiserum that blocked the hormone in question reversed the inhibitory effects on pancreatic secretion. However, there have been few studies in which reversal of a putative hormonal inhibitor of pancreatic secretion has been demonstrated by a receptor antagonist or specific antiserum. Caution must be used in interpreting these results because it is still possible that a hormone or inhibitory transmitter may act in concert with other factors. In this circumstance, the agent alone may have an inhibitory effect that is apparent only in the presence of other bioactive substances. To date, six peptides have been studied in sufficient detail to qualify as physiologic inhibitory regulators of pancreatic secretion. These include glucagon, glucagon-like peptide (GLP), somatostatin, PP, PYY, and pancreastatin.

Glucagon Ingestion of a meal stimulates large increases in plasma glucagon levels. These physiologic levels of glucagon can be reproduced in humans by infusion of exogenous glucagon at a rate of 2 µg/kg/hr in humans (446,447). Administration of

REGULATION OF PANCREATIC SECRETION / 1421 additional glucagon can inhibit pancreatic secretion stimulated by either a meal, intestinal perfusion of HCl or amino acids, or administration of exogenous CCK (448–455). Glucagon administration also has been reported to reduce secretin-stimulated bicarbonate and CCK-stimulated enzyme secretion in some studies (447,448,452,456–460). Because low doses of glucagon can inhibit pancreatic secretion in humans, it is likely that glucagon has a physiologic inhibitory effect on the exocrine pancreas. Studies of glucagon effects on pancreatic acinar cells, acini, or pancreatic lobules in vitro have been less clear. High concentrations of glucagon have been reported to inhibit basal enzyme secretion, but potentiate CCK-stimulated secretion (461–464). Therefore, the mechanism by which glucagon may inhibit the pancreas in vivo remains incompletely understood.

did not inhibit pancreatic exocrine secretion induced by direct vagal nerve stimulation. These effects occurred at plasma concentrations only slightly greater than those observed after a meal in healthy individuals and were similar to those seen in individuals with rapid gastric emptying, indicating that these were likely to be physiologic effects of GLP-1. These findings suggest that GLP-1 exerts its inhibitory effects on the pancreas through the vagus nerve, but does not alter peripheral transmission of vagal impulses or intrinsic excitatory neurons. Therefore, the site of GLP-1 action appears to be at either the level of the central nervous system or the afferent pathway to the brainstem. It is possible that the inhibitory effects of GLP-1 on pancreatic secretion stimulated by intestinal infusion of amino acids was caused by a reduction of vagal, cholinergic inputs, which were important for intestinal stimulation of pancreatic secretion.

Glucagon-Like Peptide-1

Somatostatin

Glucagon-like peptide-1 (GLP-1) is expressed in specific endocrine cells of the small intestine and, like most gastrointestinal hormones, is released into the blood in response to a meal. Although it is most widely recognized for its effects on carbohydrate metabolism, GLP-1 also inhibits gastric acid secretion and gastric motility in humans and is thought to be one of the hormones responsible for the “ileal brake” (465,466). Studies in humans have demonstrated that GLP-1 inhibited gastric acid and pancreatic secretion, as well as gastric emptying, when infused in amounts that produce plasma concentrations similar to those observed after a meal (465–467). Because gastric acid emptying into the duodenum stimulates secretin release, which can increase pancreatic fluid and bicarbonate secretion, it was important to distinguish the effects of GLP-1 on inhibition of gastric acid from its effects on pancreatic secretion. A linear relation between gastric emptying and pancreatic exocrine secretion was unchanged by GLP-1 infusion, indicating that at least part of the inhibitory effect of GLP-1 on meal-stimulated pancreatic secretion was caused by its inhibitory effect on gastric emptying (466). However, additional studies indicated that GLP-1 inhibited pancreatic secretion independent of effects on the stomach. Intravenous infusion of GLP-1 in humans inhibited pancreatic secretion stimulated by intraduodenal perfusion of amino acids (468). Interestingly, in the isolated, perfused porcine pancreas, GLP-1 did not inhibit pancreatic secretion induced by vagal nerve stimulation, indicating that GLP-1 does not directly affect the pancreas (469). Moreover, GLP-1 had no inhibitory effect on secretin- and CCK-induced pancreatic secretion (470). Similarly, in anesthetized pigs, infusions of GLP-1 did not affect secretin-stimulated pancreatic secretion or secretion induced by electrical stimulation of the vagus nerve (140). In anesthetized pigs with splanchnic nerve denervation, insulin-induced hypoglycemia strongly stimulated pancreatic bicarbonate and protein secretion, which was inhibited by GLP-1 (470). However, GLP-1

Somatostatin is a unique gastrointestinal peptide that has broad inhibitory effects on both the release of other hormones and their target tissues. Two forms of somatostatin exist, somatostatin-14 and -28. Both exhibit inhibitory activities, although their specificity for somatostatin receptor subtypes differs somewhat. Somatostatin is a prototypical paracrine transmitter that is released from somatostatin-containing D cells and acts on adjacent cells. However, somatostatin also is released into the blood after a meal, and considerable work has been devoted to characterizing the role of somatostatin levels in the circulation (471). Administration of exogenous somatostatin inhibited pancreatic bicarbonate secretion stimulated by a meal or intestinal perfusion of amino acids, sodium oleate, or HCl (472–478). Somatostatin also inhibited secretin- or secretin plus CCK-induced pancreatic bicarbonate secretion (476,479–483). These effects were seen at lower doses of secretin and occurred with both somatostatin-14 and -28. Somatostatin also inhibited CCK-stimulated enzyme secretion (473,477,480–482,484). Somatostatin receptors have been identified on pancreatic acinar cells of many species (485). In vitro, somatostatin inhibited secretin- or VIP-stimulated increases in cAMP levels (485). However, high doses of somatostatin were required to inhibit enzyme secretion in response to these and other peptides such as CCK (485–487). Somatostatin applied to the isolated perfused rat pancreas did not inhibit exocrine pancreatic secretion stimulated by cerulein, secretin, acetylcholine, or electrical vagal nerve stimulation (488). Additional studies demonstrating that somatostatin inhibited pancreatic secretion in vivo, but not in vitro, indicated that the inhibitory effects were indirect and are not through direct actions on pancreatic acinar cells (489–492). Somatostatin has been shown to inhibit VIP secretion after vagal nerve stimulation, but did not inhibit the pancreatic secretory response to exogenous VIP administration (189). Although the precise mechanisms by which somatostatin inhibits pancreatic secretion are not completely known, it is

1422 / CHAPTER 55 likely that somatostatin inhibits the release of both hormones and neurotransmitters that are involved in stimulating pancreatic secretion.

Pancreatic Polypeptide PP levels in the blood also increase after a meal. The increase was 5- to 10-fold greater than basal levels, and similar blood levels could be produced by infusion of exogenous PP at doses of approximately 100 pmol/kg/hr in humans (493). PP also is released by sham feeding, duodenal acidification, secretin, CCK, gastrin, or GRP (92,493–500). The physiologic relevance of PP in pancreatic secretion has been deduced from studies in which physiologic doses of PP have been shown to inhibit pancreatic bicarbonate and enzyme secretion in response to a meal, intestinal perfusion with amino acids, oleate, HCl, secretin, CCK, and sham feeding (499,501). Moreover, PP was shown to inhibit pancreatic secretion in response to secretin, CCK, and cholinergic agonists, but did not affect the release of either secretin or CCK (497,502). In dogs, infusion of a PP antiserum was found to increase pancreatic secretion (26). PP receptors have not been identified in pancreatic acinar cells, and PP does not appear to inhibit pancreatic enzyme secretion from pancreatic acini in vitro (503). Therefore, it appears that PP inhibits pancreatic secretion through an indirect cholinergic mechanism (504). Notably, glucagon, somatostatin, and PP are all found in pancreatic islets. Furthermore, they are all released into the endocrine-exocrine portal circulation of the pancreas (505). This is an interesting anatomic arrangement in which pancreatic acinar and duct cells are exposed to high concentrations of islet hormones. Therefore, it is possible that much greater concentrations of peptides are produced locally to affect pancreatic exocrine function than would be predicted by measuring peripheral venous levels of these peptides. It would be interesting to determine the effects of intraarterial administration of these peptides on pancreatic enzyme and bicarbonate secretion at concentrations that produce normal postprandial levels of peptide in the venous effluent drainage of the pancreas.

Peptide YY PYY (506) is a member of the PP family and is structurally similar to both PP and NPY (507). In the gastrointestinal tract, PYY is found in the ileum and colon (508,509). Like most gastrointestinal hormones, blood levels of PYY increase after a meal (510). Perfusion of the distal small intestine with fatty acids was a strong stimulus for PYY secretion in dogs (511). Infusion of exogenous PYY inhibited both meal-stimulated and secretin- and CCK-stimulated pancreatic secretion (507,511,512). Importantly, the doses of PYY that reduced pancreatic secretion produced plasma

levels that were similar to those that normally occurred after a meal. PYY also inhibited gastric acid secretion, but this effect was distinctly different from that of PP, which had no effects on acid secretion from the stomach (513). The inhibitory effects of PYY on pancreatic secretion were unlikely to be directly on the pancreas, but appeared to be dependent on vagal innervation (514). It has been demonstrated that PYY acts on the area postrema in the central nervous system to inhibit CCK-stimulated pancreatic secretion (515). The importance of PYY in pancreatic secretion in humans is unclear with the report that PYY did not inhibit secretion stimulated by secretin plus CCK-8 (516). Therefore, there may be species differences in susceptibility to the inhibitory effects of PYY on pancreatic secretion. Nevertheless, it is generally believed that PYY mediates the inhibitory effect of ileal and colonic intestinal fatty acids on pancreatic and gastric secretions that is commonly known as the “ileal break.” PYY may also be the “pancreatone” that has been extracted from the mucosa of the ileum and colon that has inhibitory effects on gastric acid secretion.

Pancreastatin Pancreastatin is a 49-amino-acid peptide that was first discovered in the porcine pancreas (517). Pancreastatin is found in many cells of the neuroendocrine system and is produced from the proteolytic cleavage of its precursor, chromogranin A (518). Pancreastatin has been localized to insulin-containing β cells, glucagon-containing α cells, and somatostatin-containing D cells of the pancreatic islet (519). Most chromogranin A originates from chromaffin tissue, and it is believed that this may be the major (and indirect) source of circulating pancreastatin. Porcine pancreastatin levels have been shown to increase 50% in response to a meal, and perfused porcine pancreas pancreastatin was released in parallel with insulin (520). There was also a vagal component to its regulation because electrical stimulation of the vagus nerve also stimulated pancreastatin release (521). In humans, pancreastatin levels in blood increased in response to intraluminal infusion of a mixed meal or after glucose ingestion (522,523). The effects of pancreastatin on pancreatic exocrine secretion have been investigated in experimental animals. Pancreastatin was shown to inhibit pancreatic secretion after a meal, CCK-8 administration, or central vagal nerve stimulation (524). The inhibitory effects of pancreastatin appeared to be mediated by the presynaptic modulation of acetylcholine release in intrapancreatic nerves (525). It is believed that pancreastatin exerts its primary physiologic effects through local paracrine actions. Because of its location and high concentration in pancreatic islets, it is thought that pancreastatin modulates pancreatic exocrine secretion through the islet-acinar portal system.

REGULATION OF PANCREATIC SECRETION / 1423 PANCREATIC FUNCTION TESTING A variety of different methods have been developed to assess the secretory function of the human exocrine pancreas, and measurements of pancreatic secretion have been used to determine abnormal function of the pancreas (526). These are commonly known as tests of pancreatic function and are usually performed to determine whether an individual has pancreatic insufficiency or chronic pancreatitis. Diseases of the pancreas leading to chronic pancreatitis are usually associated with histologic damage to the gland. Clinically, the anatomy of pancreas can be assessed using imaging studies such as ultrasound, computed tomography, magnetic resonance imaging, and cholangiopancreatography, and alterations in the parenchyma of the gland or the pancreatic duct are often used for diagnostic purposes. However, tests of pancreatic function attempt to measure changes in the secretory capacity of the pancreas. These tests rely on their ability to detect damage to the pancreas that is less obvious and often less advanced than imaging techniques. Secretions of the pancreas are approximately 1 L/day and consist of water, bicarbonate, and digestive enzymes. Tests of pancreatic function are based on measuring pancreatic bicarbonate or enzyme secretion or secondary effects caused by the lack of enzymes in the intestine. Pancreatic fluid secretion is generally not used as a direct measurement because of the variability in adequate sampling. Direct assessment of pancreatic function is accomplished by collecting and measuring pancreatic secretions or determining pancreatic secretory capacity. Because basal pancreatic secretion can vary, tests usually are done by measuring pancreatic responses to administered secretagogues such as secretin or CCK (or a CCK analogue such as cerulein). Pancreatic secretions are collected by a tube placed into the duodenum maintained under continuous aspiration to collect duodenal/pancreatic juice. To minimize contamination of gastric secretions, a tube with a port to aspirate gastric juice generally is used. In addition, it is often helpful to use a nonabsorbable perfusion marker such as polyethylene glycol, which can be infused into the stomach to measure contamination by gastric fluid or introduced into the duodenum to assess the adequacy of duodenal collections. One of the first pancreatic function tests to be developed was the Lundh test, which entailed having the subject ingest a test meal consisting of 5% protein, 6% fat, and 15% carbohydrate in a liquid form. Duodenal contents then were aspirated for 2 hours, and the pancreatic enzymes trypsin, amylase, or lipase was measured. The sensitivity of this test for assessing chronic pancreatitis and pancreatic insufficiency was 66% to 94% (527,528). It has subsequently been found, however, that using a hormonal secretagogue appears to be a more sensitive test. Administration of a supraphysiologic stimulus to the pancreas with the secretagogues secretin and/or CCK, is more reproducible than the Lundh meal test. Different investigators have used either secretin,

CCK, or a combination of the two agents, which were given by intravenous bolus injection or continuous infusion. Doses of secretin of 0.5 to 4 CU/kg body weight typically are used. An advantage of the secretin test is that pancreatic bicarbonate can be more easily measured than with ingestion of a meal. Either peak bicarbonate concentration or total bicarbonate output or volume can be measured. The sensitivity of this test for diagnostic pancreatic insufficiency has been reported to range from 67% to 94% and is generally more sensitive than imaging techniques such as endoscopic retrograde pancreatography. Some investigators have estimated that this test is only sensitive enough to detect reduced pancreatic function when 30% to 50% of the function of the gland has been lost. Currently, there are not uniform, unanimously accepted, and well-standardized values for direct pancreatic function testing. Therefore, it is incumbent on each center to standardize its own methods including establishing reference ranges, assessing assay variabilities, and determining sensitivities and specificities for healthy and diseased populations.

Tests of Pancreatic Synthetic or Metabolic Activity The pancreas is a metabolically active organ with tremendous protein secretory capacity. Radioactively labeled amino acids injected intravenously are taken up rapidly by the pancreas and synthesized into digestive enzymes. Because of this rapid incorporation into proteins, 75 Se-methionine has been used as the basis of a pancreatic function test. In this test, 75Se-methionine is given as an intravenous bolus followed by stimulation of pancreatic secretion with secretin-CCK. Duodenal contents then are aspirated to measure protein-bound radioactivity and reflect pancreatic exocrine function. The sensitivity and specificity of this test appears to be less than that of the secretinCCK stimulation test and does not obviate the need for duodenal intubation. Based on the rapid metabolic activity of the pancreas, it was proposed that a substantial amount of the circulating amino acids were consumed by the pancreas and incorporated into digestive enzymes. It was postulated that a diseased pancreas would be less able to extract amino acids from the blood compared with a healthy pancreas. Plasma levels of amino acids have been measured in healthy individuals and in patients with chronic pancreatitis before and after stimulation of the pancreas with secretin plus CCK. A decrease in plasma amino acid concentrations of less than 12% indicated pancreatic exocrine insufficiency (529). However, the sensitivity of this test was limited and did not adequately discriminate between individuals with normal pancreatic function and those with mild to moderate disease (530). Therefore, because of the lack of specificity, this test has been largely abandoned.

1424 / CHAPTER 55 Measurements of Pancreatic Enzymes in Blood Measurements of pancreatic enzymes in blood are of limited usefulness in assessing pancreatic function. Serum amylase or lipase levels have no relation to pancreatic function. In general, serum trypsinogen levels are not sensitive enough to indicate pancreatic insufficiency, and only low levels (<20 ng/ml) have been associated with chronic pancreatitis (531). However, levels this low were seen only in advanced disease and were accompanied with steatorrhea. Pancreatic Enzymes in Stool Reduced secretion of pancreatic enzymes into the intestine may be reflected in the enzyme content in stool. Trypsin activity in stool has been measured in patients with chronic pancreatitis, but has been found to be both nonspecific and insensitive as a diagnostic test. However, chymotrypsin appears to be more stable than trypsin and has been used as an assay of pancreatic function. Most studies report sensitivities for the fecal chymotrypsin assay of 72% to 90% (532,533). This test also has been used to predict pancreatic insufficiency in children with cystic fibrosis (534,535). Enthusiasm on the part of investigators has been dampened by the realization that although fecal chymotrypsin is a reliable test for advanced chronic pancreatitis, it is unreliable for early disease (536,537). More recently, pancreatic elastase-1 has been evaluated as a measure of pancreatic exocrine function. This pancreatic protease appears to undergo minimal degradation during intestinal transit and is enriched sixfold in feces compared with duodenal juice. Using an enzyme-linked immunoabsorbent assay, a good correlation between fecal elastase-1 levels and duodenal juice amylase, lipase, and trypsin levels were reported in both healthy individuals and patients with chronic pancreatitis (538,539). However, similar to fecal chymotrypsin, fecal elastase measurements are insensitive for detection of early chronic pancreatitis or mild pancreatic insufficiency (540,541). Pancreatic Polypeptide PP is produced by specific cells in pancreatic islets, and serum PP levels have been proposed as a test of chronic pancreatitis (542). Normally, circulating PP levels increase following ingestion of a meal or stimulation with CCK. It was postulated that with reduced function of the pancreas stimulated PP levels would be reduced in patients with chronic pancreatitis. However, several studies have reported that PP levels are too variable to be useful as a test of pancreatic function (543–545).

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Bile Formation and the Enterohepatic Circulation Paul A. Dawson, Benjamin L. Shneider, and Alan F. Hofmann Biosynthesis, Chemistry, and Physical Chemistry of Bile Acids, 1438 Biosynthesis of Bile Acids, 1438 Bacterial Biotransformation of Bile Acids during Enterohepatic Cycling, 1441 Chemistry and Physical Chemistry of Bile Acids, 1443 Structure–Physiochemical Relations, 1444 Enterohepatic Circulation, 1444 Bile Secretion and Hepatic Bile Acid Transport, 1446 Overview of Bile Secretion, 1446 Bile Acid–Independent Canalicular Bile Flow, 1447 Overview of Hepatic Bile Acid Transport, 1447 Na+-Dependent Bile Acid Uptake across the Sinusoidal Membrane of the Hepatocyte, 1448 Na+-Independent Bile Acid Uptake across the Sinusoidal Membrane of the Hepatocyte, 1449 Transporters Mediating Bile Acid Efflux across the Sinusoidal Membrane of the Hepatocyte, 1449

Canalicular Bile Acid Transport, 1450 Ductular Modification of Bile, 1450 Cholehepatic Shunt Pathway, 1450 Concentration of Bile in the Gallbladder, 1450 Overview of Intestinal Absorption of Bile Acids, 1451 Molecular Mechanisms for Intestinal Bile Acid Transport, 1451 Regulation of Intestinal Bile Acid Transport, 1452 Functions and Dysfunctions of Bile Acids in the Intestine, 1454 Small Intestine, 1454 Large Intestine, 1455 Therapeutic Uses of Bile Acid Agonists and Antagonists in Clinical Medicine, 1455 Bile Acid Agonists, 1455 Bile Acid Antagonists and Modifiers of Bile Acid Metabolism, 1456 References, 1456

Bile formation plays a critical role in intestinal lipid digestion and absorption, cholesterol homeostasis, and the excretion of lipid-soluble xenobiotics, endobiotics, and heavy metals. The maintenance of hepatic bile formation is essential for

normal liver function, and the production of bile is itself dependent on hepatic synthesis, transport, and canalicular secretion of bile acids, the predominant organic anions in bile. Hepatic bile acid secretion requires an intact enterohepatic circulation because most of the bile acids secreted by the hepatocyte were previously secreted into the small intestine and have undergone enterohepatic cycling. Normal hepatic and gastrointestinal physiology can be profoundly affected by disturbances in bile acid synthesis, biliary secretion, and enterohepatic cycling. Bile fulfills the requirements for a digestive secretion, because its major constituent, bile acids, facilitates lipid absorption from the intestine and also promotes sterility of small intestinal content. However, bile is also an excretory secretion because it serves as the major route of elimination

P. A. Dawson: Department of Internal Medicine, Section of Gastroenterology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157. B. L. Shneider: Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029. A. F. Hofmann: Department of Medicine, Division of Gastroenterology, University of California, San Diego, La Jolla, California 92093. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1437

1438 / CHAPTER 56 for a number of substances that are typically protein bound in plasma and cannot be eliminated by glomerular filtration. These include cholesterol, bilirubin, heavy metals, and lipophilic endobiotics or xenobiotics in conjugated or unconjugated form. Bile acids, themselves, are the major end product of cholesterol catabolism and represent a major route for cholesterol elimination from the body. The cloning and functional analysis of many of the important hepatic, biliary, and intestinal transporters has helped define the molecular mechanisms involved in bile formation and the enterohepatic circulation of bile acids. In addition, previously defined inherited clinical phenotypes such as several forms of pediatric liver disease, as well as primary bile acid malabsorption, are now known to be associated with mutations in these transporter genes, underscoring their importance. This chapter reviews the physiology of bile formation and the enterohepatic circulation of bile acids, with particular emphasis on the transporters involved. The physiologic functions of bile acids, as well as the use of bile acid agonists and antagonists in clinical medicine, also are mentioned briefly.

BIOSYNTHESIS, CHEMISTRY, AND PHYSICAL CHEMISTRY OF BILE ACIDS Biosynthesis of Bile Acids In all vertebrates examined to date, cholesterol is converted to water-soluble derivatives that are excreted in bile. As a group, these molecules are termed bile salts and would be included in the ST (sterol lipids) category in the recently proposed comprehensive classification of lipids (1). Bile salts consist mainly of sulfate conjugates of bile alcohols and of taurine (or glycine) conjugates of bile acids. In this chapter, the term bile acids is used to denote all types of bile salts including bile alcohol sulfates, as well as bile acids. Note that C24 bile acids (with a carboxyl group at C-24) are the predominant form in most mammals (2).

The rate-limiting enzyme in the classical or neutral pathway for bile acid synthesis in most vertebrates is cholesterol 7α-hydroxylase (CYP7A1), a cytochrome P450 hydroxylase that is expressed only in the hepatocyte (3). As a result of the activity of CYP7A1 and that cholesterol has a 3-hydroxy group, all bile alcohols and bile acids must have a hydroxy group at the 3 and the 7 positions. In bile alcohols, the terminal functional group on the side chain is a primary hydroxyl group (at C27) by definition. Thus, bile alcohols have one or more hydroxy groups on the side chain, and most bile alcohols have three hydroxy groups on their steroid nucleus. Consequently, bile alcohols commonly have four, five, or six hydroxy groups altogether (4). Bile alcohols are insoluble and must be sulfated on the side chain terminal hydroxy group at the C-27 position. In a variety of ancient vertebrates such as cartilaginous fish, herbivorous bony fish, and four ancient mammals (elephant, manatee, hyrax, and rhinoceros), bile alcohol sulfates are the predominant organic anion present in bile (2,5,6). In contrast with bile alcohols, the side chain of bile acids ends in a carboxyl group, by definition. In most mammals and some species of birds, bony birds, and snakes, the C8 side chain of cholesterol undergoes a modified β-oxidation to form a five-carbon isopentanoic acid side chain. Such 24-carbon bile acids are termed cholanoic acids to distinguish them from 27-carbon cholestanoic acids. The names and structures of the common mammalian bile acids are shown in Table 56-1. Bile acids are synthesized from cholesterol in the pericentral hepatocytes of the hepatic acini. In this process, the hydrophobic substrate cholesterol is converted to a watersoluble, amphipathic product through a series of sterol ring hydroxylations and side-chain oxidation steps. Bile acids formed in the hepatocyte are termed primary bile acids to distinguish them from secondary bile acids, which are formed when intestinal bacterial enzymes modify the hydroxy groups of cholic acid or chenodeoxycholic acid (CDCA) by dehydrogenation (oxidation of a hydroxy group to an oxo group), epimerization (changing an α-hydroxy group to a β-hydroxy

TABLE 56-1. Names and structures of common mammalian bile acids Common name

Position of hydroxy groups

Type

Chenodeoxycholic acid group 1. Chenodeoxycholic acid 2. Ursodeoxycholic acid 3. Lithocholic acid

3α, 7α 3α, 7β 3α

Primary Primary (or secondary) Secondary

Cholic acid group 4. Cholic acid 5. Deoxycholic acid

3α, 7α, 12α 3α, 12α

Primary Secondary

Hyocholic acid group 6. α-Hyocholic acid 7. Hyodeoxycholic acid

3α, 6α, 7α 3α, 6α

Primary Secondary

Muricholic acid group 8. α-Muricholic acid 9. β-Muricholic acid 10. ω-Muricholic acid 11. Murideoxycholic acid

3α, 3α, 3α, 3α,

Primary Secondary Secondary Secondary

6β, 7α 6β, 7β 6α, 7β 6β

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1439 group, or vice versa), or dehydroxylation. Hepatic bile acid synthesis originally was thought to involve one major pathway, the “classical” or neutral pathway (CYP7A1 pathway) that favors cholic acid biosynthesis. This paradigm was later modified by the discovery of a second pathway, the “alternative” or acidic pathway (oxysterol 7α-hydroxylase [CYP7B1] pathway) that favors CDCA biosynthesis (3). In the neutral pathway, the enzyme CYP7A1 converts cholesterol directly into 7α-hydroxycholesterol. In the alternative acidic pathway, cholesterol is first converted by C-24, C-25, or C-27 sterol hydroxylases into oxysterols, the major species being 27-hydroxycholesterol, before oxysterol 7αhydroxylation (3,7–9). The process of bile acid biosynthesis and conjugation is complex and requires the action of 17 different enzymes. Bile acid biosynthesis involves the following reactions: (1) initiation of synthesis by 7-hydroxylation of sterol precursor; (2) additional modifications to the ring structure that eliminate the ∆5,6 double bond of cholesterol and also form a 5βA/B cis ring juncture between the A and B rings of the steroid nucleus; (3) oxidation and shortening of the side chain; and (4) conjugation of the bile acid with an amino acid (3). The exact order of steps in the biosynthetic pathway remains unclear because many of the intermediates serve as substrates for more than one biosynthetic enzyme. The enzymes can be divided into two broad groups. The first group performs modifications to the sterol ring structure, whereas the second group modifies the sterol side chain (3). In the classical neutral pathway, sterol ring modifications precede side chain changes, whereas the side chain modifications occur before or during changes to the sterol ring structure in the acidic pathway. In either pathway, 27hydroxylation and oxidation are carried out in the mitochondria. These reactions then are followed by oxidation catalyzed by peroxisomal enzymes to form a 27-carbon bile acid with subsequent elimination of a 3-carbon fragment to give the 5-carbon isopentanoic acid side chain (10). Figure 56-1 summarizes the bile acid biosynthetic pathway. Of the two major biosynthetic pathways, the neutral pathway is thought to be quantitatively more important in humans (11). The evidence supporting this conclusion includes biosynthetic studies using tritiated bile acid precursors and analysis of patients with inborn errors of bile acid synthesis. In healthy adults without gallbladders, [3H]7-hydroxycholesterol, but not [3H]27-hydroxycholesterol, was efficiently converted to cholic acid and CDCA (12), underscoring the minor role of the CYP27 pathway in adults. This conclusion is also supported by the finding that bile acid production was decreased greatly in an adult patient with an inherited CYP7A1 defect in the neutral pathway (13). However, in contrast with adults, the acidic pathway may be dominant in human neonates, as is evident by the apparent lack of CYP7A1 in newborns and the finding of severe cholestatic liver disease in an infant with an inherited oxysterol 7α-hydroxylase gene (CYP7B1) defect (14,15). In the mouse, the neutral Cyp7a1 pathway is the major route for bile acid synthesis and is the predominant pathway in the neonate (7,8,16,17). Induction of the mouse oxysterol 7α-hydroxylase enzyme (Cyp7b1) in the

alternative pathway does not occur until almost 4 weeks of age (16). Of the two major pathways for bile acid biosynthesis, CYP7A1, the major enzyme of the neutral pathway, is partly controlled by a negative bile acid feedback loop, whereas CYP27A1 and CYP7B1, major enzymes of the acidic pathway, are not subject to regulation by bile acids. Feedback inhibition of CYP7A1 had been recognized for many years in that bile acid synthesis is decreased after administration of hydrophobic bile acids and increased by interruption of the enterohepatic circulation by creation of a biliary fistula, ileal resection, or administration of bile acid sequestrants (18). The molecular mechanisms responsible for this negative feedback regulation of bile acid biosynthesis have only recently been elucidated. In one mechanism, excess bile acids indirectly feed back to suppress expression of CYP7A1 by acting as ligands for the farnesoid X receptor (FXR) in the hepatocyte (19,20). The bile acid receptor FXR transcriptionally activates the gene for the orphan nuclear receptor, small heterodimer partner (SHP). SHP, in turn, acts by antagonizing yet another orphan nuclear receptor, liver receptor homolog-1 (LRH-1), that is required for expression of the CYP7A1 gene (21,22). Bile acids can also inhibit CYP7A1 gene transcription in an SHP-independent fashion (23,24). In one such pathway, FXR induces the hepatic expression of human fibroblast growth factor-19 (FGF-19) that is secreted and activates the hepatic FGF-4 receptor. Activation of the FGF-4 receptor then down-regulates CYP7A1 through the c-Jun NH2-terminal kinase (JNK) pathway (23–28). A more interesting variation on this pathway has begun to emerge from a variety of studies using mouse models where bile acids taken up by the ileal enterocytes bind to FXR and induce expression of fibroblast growth factor-15 (FGF-15), the mouse orthologue of human FGF-19. FGF-15, which is not made by the mouse liver (29), is then secreted by the ileal enterocytes and carried to the liver in the portal circulation. At the liver, FGF-15 activates the FGF-4 receptor and down-regulates CYP7A1 via the JNK pathway. These complex molecular titrations allow bile acid synthesis to be linked to both intestinal and hepatic bile acid levels. This pathway also helps to explain a puzzling experimental observation where intravenous infusion of bile acids into the bile-fistula rat was not as potent in down-regulating hepatic bile acid synthesis as intraduodenal infusion of bile acids (30). Figure 56-2 shows a model summarizing the regulation of CYP7A1. In contrast with the neutral (CYP7A1) pathway, the alternative or acidic pathway for bile acid synthesis appears to be regulated by cholesterol delivery to the mitochondria, the site of 27-hydroxylation (31–33). The process is analogous to steroid hormone biosynthesis in the adrenal gland, where steroidogenic acute regulatory protein (StAR)–mediated transfer of cholesterol to the mitochondria is rate limiting (34). Because the liver expresses several different StAR-related lipid transfer (START) domain-containing proteins (35), it is not yet clear whether StAR itself mediates cholesterol transfer to the mitochondrial inner membrane for bile acid synthesis, or whether other related START domain proteins are involved (33,36,37).

1440 / CHAPTER 56 Neutral pathway 22 23 20 17 24 25 13 16

21 12

11 1

9

2 HO

8 14 15 7

10 5

4

Acidic pathway

27 26

Cyp27

HO

6

COO−

CH2OH

HO

Cyp7A1 Cyp7B1 HO

OH COO−

O

HO

OH

OH

Cyp8B OH

O

O

OH

Cyp27

OH

HO

COO−

Cyp27 O

HO

OH

CH2OH

CH2OH

OH

OH

OH

OH

COOH COOH

COOH

HO HO

OH

Cholic acid

HO

OH

OH OH

Chenodeoxycholic acid / Muricholic acid (humans) (mice)

FIG. 56-1. The classical pathway for bile acid synthesis begins with cholesterol 7α-hydroxylase (CYP7A1), which converts cholesterol into 7-hydroxycholesterol. Bile acid intermediates produced by this pathway are substrates for the sterol 12α-hydroxylase (CYP8B1), the rate-determining step in the production of cholic acid. The level of CYP8B1 expression determines the relative amount of cholic acid produced, and this pathway mainly produces cholic acid in humans. Several alternate pathways exist for bile acid synthesis. In the acidic pathway, cholesterol is first converted into 27-hydroxycholesterol by the sterol 27-hydroxylase (CYP27). The conversion of 27-hydroxycholesterol to a bile acid intermediate is catalyzed by the oxysterol 7α-hydroxylase (CYP7B1). The acidic pathway preferentially produces chenodeoxycholic acid in humans and muricholic acid in mice.

The default bile acid is CDCA, which has hydroxy groups in the α configuration at positions C-3 and C-7. Most natural bile acids formed by the hepatocyte can be considered derivatives of CDCA. In humans, the newly synthesized (primary) bile acids are CDCA and cholic acid, a trihydroxy bile acid with hydroxy groups at the C-3, C-7, and C-12 positions. Cholic acid and CDCA are the dominant primary bile acids in most mammals. In the majority of mammalian species, primary bile acids are trihydroxy bile acids, formed by hydroxylating CDCA or an intermediate in CDCA synthesis (2,38). As noted earlier, hydroxylation occurs at C-12 in humans, at C-6 (the muricholic and hyocholic acid families) in pigs, rats and mice, and at C-1 and C-15 in Australian marsupials (2). Other sites of hydroxylation are still being identified. The reason for adding a third hydroxy group to the steroid nucleus is unclear. One explanation is that CDCA is

intrinsically hepatotoxic. Another explanation is lithocholic acid (LCA), the major bacterial biotransformation product of CDCA, is extremely hepatotoxic (39,40). After their biosynthesis, bile acids are conjugated with taurine (aminoethanesulfonic acid) or glycine via an amide bond linking the carboxyl group of the bile acid with the amino group of a taurine or glycine. Such conjugates are termed N-acyl amidates or aminoacyl amidates (38), because the term conjugated bile acids also encompasses sulfate and glucuronate conjugates. Bile acids are conjugated with taurine in most nonmammals, but in mammals, conjugation may be with taurine, glycine, or both. In humans, the majority of biliary bile acids are conjugated to glycine. Conjugation with taurine or glycine increases the hydrophilicity of the bile acid and the acidic strength of the side chain, in essence converting a weak acid (pKa ~5.0) to

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1441 inherited defects in bile acid conjugation have a clinical phenotype of malabsorption of dietary triglyceride and fat-soluble vitamins (44,45). In addition to taurine and glycine, bile acids can be conjugated via an ester linkage with sulfate, glucuronate, or N-acetyl glucosamine on the nuclear hydroxyl groups of Nacyl amidated or unconjugated bile acids. Bile acids can also be conjugated with glucuronate on the carboxyl group of the side chain. The only one of these “nonamidation pathways” that is important in health (in humans) is sulfation of LCA. The sulfated LCA N-acyl amidates are secreted into bile, but are not substrates for the ileal transport system and are rapidly lost from the body (46,47). As a result, LCA conjugates comprise less than 5% of biliary bile acids in humans (48) and other vertebrates analyzed to date. Under certain pathophysiologic conditions such as cholestasis, bile acid sulfation and additional hydroxylation are increased, and these modifications function to decrease the cytotoxicity of the circulating bile acids and to promote their renal elimination (49–51).

FIG. 56-2. Mechanisms responsible for feedback negative regulation of hepatic bile acid synthesis. In the hepatocyte, bile acids (BA) bind to and activate the nuclear receptor farnesoid X receptor (FXR). FXR binds to the promoters and induces expression of small heterodimer partner (SHP) and fibroblast growth factor-19 (FGF-19; in humans). SHP, in turn, interacts with the liver receptor homolog-1 (LRH-1) or hepatocyte nuclear factor (HNF)-4α to decrease expression of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1), respectively. FGF-19 is secreted and interacts with its cognate receptor fibroblast growth factor receptor 4 (FGFR-4) to repress expression of CYP7A1 and CYP8B1 via the c-Jun NH2-terminal kinase (JNK) pathway. In a variation on this pathway (bottom), bile acids taken up into the ileal enterocyte bind to and activate FXR. FXR then induces expression of FGF15 (mouse). The FGF15 is secreted into the portal circulation and carried to the liver, where it binds FGFR-4 and represses expression of CYP7A1 and CYP8B1 via the JNK pathway.

a strong acid (pKa ~3.9 for the glycine conjugate; pKa < 2.0 for the taurine conjugate) (41). As a result, conjugated bile acids are almost completely ionized at the pH conditions prevailing in the biliary tract and small intestine. The physiologic consequence of conjugation is to decrease the passive diffusion of bile acids across cell membranes during their transit through the biliary tree and small intestine (42). Consequently, conjugated bile acids are absorbed only if a specific membrane carrier is present. Conjugated bile acids are also more soluble than unconjugated bile acids at acidic pH and more resistant to precipitation in the presence of high concentrations of calcium than the unconjugated species (43). The net effect of conjugation is to maintain high intralumenal, micellar concentrations of bile acids in bile to solubilize cholesterol and to facilitate lipid digestion and absorption down the length of the small intestine. The importance of bile acid conjugation is nicely illustrated by the finding that

Bacterial Biotransformation of Bile Acids during Enterohepatic Cycling Most of the conjugated bile acids secreted into the small intestine are efficiently absorbed intact. However, bile acids are also metabolized during their passage through the intestine by the endogenous bacterial flora. Modification of primary bile acids by the intestinal bacterial flora is important for several reasons. First, such bacterial changes decrease the aqueous solubility of bile acids and increase their hydrophobicity, resulting in a marked reduction of the monomeric concentration of bile acids (52,53). This, in turn, reduces the flux of bile acids through the colonic epithelium. Second, the composition of the circulating pool of bile acids is influenced by the input of secondary bile acids from the colon, and secondary bile acids may have different pharmacologic and signaling activities than their primary bile acid precursors (54). Third, some secondary bile acids such as LCA are quite hepatoxic. The feeding of CDCA to rabbits and rhesus monkeys induces severe hepatotoxicity because of the accumulation of LCA in the circulating pool of bile acids (55,56). A fraction (perhaps one third) of the bile acids secreted into the small intestine is deconjugated by the bacterial flora in the distal small intestine. The unconjugated bile acids are absorbed passively or actively and returned to the liver where they are reconjugated, mixed with newly synthesized bile acids, and resecreted into bile. This process of intestinal deconjugation and hepatic reconjugation is a normal part of bile acid metabolism, but the fraction of the bile acid pool that undergoes such deconjugation-reconjugation varies widely among species (2). A small fraction of bile acids secreted into the small intestine will escape absorption and pass into the colon where deconjugation is completed. In the colon, the action of bacterial 7α-dehydroxylase converts cholic acid to deoxycholic

1442 / CHAPTER 56 acid (DCA), a dihydroxy bile acid with hydroxy groups at the C-3 and C-12 positions, and converts CDCA to LCA, a monohydroxy bile acid with a hydroxy group at the C-3 position (57) (Fig. 56-3). In a similar fashion in rodents, the 3,6,7-trihydroxy bile acids are converted to 3,6-dihydroxy bile acids, hyodeoxycholic acid and murideoxycholic acid (38). Deconjugation and dehydroxylation of the primary bile acids cholic acid and CDCA reduces their aqueous solubility such that DCA is quite insoluble at the pH of the cecum and LCA is insoluble at body temperature (53). Note that the bile acid must first be deconjugated before dehydroxylation because the first step in 7-dehydroxylation is the formation of a coenzyme A (CoA) derivative, and this derivative can form only with weak acids such as the unconjugated side chain (57). For example, bile acids such as cholylsarcosine that are resistant to deconjugation are also resistant to dehydroxylation (58). A fraction of these 7-deoxy bile acids are absorbed from the colon and returned to the liver where they are efficiently reconjugated with glycine or taurine, and may or may not be 7α-rehydroxylated. The extent of the bile acid

7α-rehydroxylation varies considerably among species and is appreciable in rats, mice, prairie dogs, and hamsters (59,60). In contrast, humans do not 7α-rehydroxylate DCA (61,62). This species variation in 7α-rehydroxylation activity is reflected in the biliary DCA concentration that varies from 0% to 10% of bile acids in rats, mice, guinea pigs, prairie dogs, and hamsters (63–66), 15% to 30% in dogs and humans (67,68), and greater than 90% in rabbits (69). The especially high proportion of DCA in rabbit bile is explained by a combination of the efficient microbial 7α-dehydroxylation of cholic acid, the efficient absorption of DCA from the colon, and the inability to 7α-rehydroxylate (69). Hepatic 7α-rehydroxylation is considered to be a detoxification mechanism, and animals with low biliary concentrations of DCA are particularly sensitive to DCA-associated toxicity. For example, DCA produces severe acute toxicity when infused into rats and hamsters (70,71), whereas dogs, rabbits, and humans are relatively resistant. Besides undergoing 7-dehydroxylation during colonic transit, the hydroxy groups on the steroid nucleus may be modified, as noted earlier, by dehydrogenation, epimerization, or

OH COOH O

Cholic

Primary (From Cholesterol)

R-C-N-CH2COO°

OH

HO

H + NH3

COOH Chenodeoxycholic OH

HO

Cholylglycine Chenodeoxycholylglycine Deoxycholylglycine Lithocholylglycine Ursodeoxycholyglycine

CH2COO° + Glycine

Sulfolithocholylglycine +SO4 Sulfolithocholyltaurine

OH COOH

NH3+(CH2)2SO2O° + Taurine

Deoxycholic HO

Cholyltaurine Chenodeoxycholyltaurine Deoxycholyltaurine Lithocholyltaurine Ursodeoxycholyltaurine

O

Secondary (From Primary Bile Acids)

COOH

R-C-N-(CH2)2SO2O° H

Lithocholic HO

COOH Ursodeoxycholic HO

FIG. 56-3. Chemical structures of the major primary and secondary bile acids. Primary bile acids are formed in the liver, deoxycholic and lithocholic secondary bile acids are formed in the large intestine by bacterial 7-dehydroxylation of their primary bile acid precursor, and ursodeoxycholic acid is formed by bacterial epimerization of the hydroxy group at the seventh carbon atom in the distal small intestine or large intestine. Bile acids are conjugated with glycine or taurine in the hepatocyte. Before 7-dehydroxylation, bile acids are deconjugated by bacterial enzymes. Sulfation of lithocholic acid conjugates prevents ileal conservation and results in rapid excretion from the body. (Modified from Hofmann AF. Biliary secretion and excretion. The hepatobiliary component of the enterohepatic circulation of bile acids. In Johnson LR, ed. Physiology of the gastrointestinal tract. 3rd ed. New York: Raven Press, 1994;1562, by permission.)

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1443 even elimination to form an unsaturated bile acid. These secondary bile acids may be absorbed and further metabolized by the hepatocyte before secretion into bile. The reactions include hepatic sulfation, hydroxylation, and glucuronidation of LCA, epimerization of the C-7 hydroxy group of CDCA to form ursodeoxycholic acid (3α, 7β dihydroxy bile acid; UDCA), and reduction of 7-oxo-lithocholate to CDCA or its 7β-epimer, UDCA. These additional modifications are important for detoxification of LCA in some species. LCA has been found to be hepatotoxic in all species currently examined because it is a potent inhibitor of canalicular secretory function (49). As noted earlier, in humans, conjugation of LCA with sulfate blocks uptake by the ileal bile acid transporter and blocks passive intestinal absorption, and the conjugated LCA is rapidly lost from the circulating pool of bile acids. In some animals, hydroxylation of LCA is an important alternative mode of detoxification. The molecular mechanisms responsible for inducing these detoxification pathways have been elucidated and involve induction of cytosolic sulfotransferases and the cytochrome P450 enzyme, CYP3A, by the orphan nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androsterone receptor) (72–74). LCA, as well as other xenobiotic inducers, bind and directly activate PXR in the hepatocyte. The activated nuclear receptor then induces expression of modification enzymes that confer resistance to LCA toxicity. In animal models, pretreatment with PXR ligands, such as the antipruritic agent rifampicin, was protective against LCA-induced liver damage (72). In contrast, mouse models lacking PXR and CAR were extremely sensitive to LCA-induced liver damage including appearance of necrotic foci and increased serum ALT levels (74). The mechanism of protection appears to be related to the CAR/PXR coordinate induction of the major hepatic bile acid/bilirubin metabolizing and detoxifying enzymes and hepatic alternative efflux systems effects that counteract cholestasis and hepatic damage (75). It is possible that similar approaches could be used to exploit the anticholestatic actions of these nuclear receptor pathways in patients, and this is a promising area of investigation (76).

Chemistry and Physical Chemistry of Bile Acids Unconjugated natural bile acids (with an isopentanoic acid side chain) have a pKa of 5.0, and their protonated form is poorly soluble. Their aqueous solubilities range from 0.03 mM for CDCA to 0.3 mM for cholic acid (77). The bile acid anions of the common natural dihydroxy- and trihydroxyunconjugated bile acids are fully soluble. Over a narrow concentration range, bile acid anions associate to form polymolecular aggregates that are probably helical in arrangement (77). Because this behavior in solution is similar to that shown by typical ionic detergents, the aggregates are termed micelles. The concentration range over which self-association occurs is termed the critical micellization concentration (CMC), even though the self-association of bile acid molecules is much more gradual than that of typical detergents

such as dodecyl sulfate. Note that the sodium salts of the monohydroxy (3α) bile acid, LCA, and of the dihydroxy bile acid, murideoxycholic acid (3α, 6β), are insoluble at body temperature. All detergents have a temperature above which they are highly soluble (and are present as a micellar solution) and below which they are poorly soluble (and present as a simple solution of monomers). This temperature may be termed the critical micellization temperature (CMT). For natural dihydroxy- and trihydroxy-conjugated bile acids, this CMT is less than the freezing point of water. For LCA and murideoxycholic acid, the CMT is greater than body temperature (43,78). The solubility of unconjugated bile acids also increases exponentially with increasing pH because the proportion of the water-soluble anion increases exponentially with alkalinization (53). When the concentration of the anion reaches the CMC, micelle formation occurs. At greater pH values, the additional water-soluble anions form more micelles. There is thus a narrow pH range over which the solubility of unconjugated bile acids increases markedly; this pH value has been termed the critical micellization pH (CmpH). For the common unconjugated bile acids, the CmpH is between pH 6 and 8 (79). Whereas all unconjugated bile acids with an aliphatic side chain have a pKa of about 5.0, conjugation with taurine decreases the pKa of a bile acid by at least 4 units. As a result, taurine conjugates of the common dihydroxy and trihydroxy bile acids are fully water soluble at a pH greater than about 3. The effect of conjugation with glycine is more complex. Glycine conjugation has two opposing effects on the solubility of a given bile acid. The most important effect is to reduce the pKa by about 1.2 units (41). Thus, a glycine-conjugated bile acid begins to ionize at considerably lower pH than its corresponding unconjugated derivative. However, conjugation with glycine also reduces the solubility of the protonated monomer (80). Overall, the effect of enhanced ionization outweighs the lower solubility of the protonated acid such that the CmpH values for the common, natural, glycineconjugated bile acids are considerably less (pH 4.5–6) than those of the corresponding unconjugated acids (pH 6.5–8). As a result, the glycine conjugates of common dihydroxy and trihydroxy bile acids are fully soluble at the pH conditions prevailing in the human small intestine during digestion, and the same is likely to be true for other mammals. For most bile acids, the effect of conjugation with glycine or taurine on the CMC of the corresponding unconjugated bile acid is relatively small (77). For glycine conjugates, the additional hydrophobicity contributed by the methylene group appears to be balanced by the hydrophilicity of the amide bond. For taurine-conjugated bile acids, the hydrophobicity of two methylene groups appears to be almost balanced by the amide bond, as well as by the additional oxygen on the sulfonic acid group (79). The CMC of a taurine conjugate is slightly less than that of its corresponding unconjugated derivative in most instances. In addition to increasing the solubility at the slightly acidic conditions present in the proximal jejunum, conjugation with glycine or taurine prevents formation of the insoluble calcium salts. Calcium salts of the

1444 / CHAPTER 56 common, natural, unconjugated dihydroxy bile acids are rather insoluble, with solubility products of the calcium salt [Ca(BA−)2] being between 1 and 10 nM (43). However, in most circumstances, at the pH values and Ca2+ activities present in body fluids, unconjugated bile acid anions never achieve a sufficiently high concentration to form an insoluble calcium salt. The calcium salts of taurine-conjugated bile acids are fully water soluble (81). The calcium salts of glycineconjugated bile acids do not form in supersaturated solutions because of striking metastability of such solutions (82).

Structure–Physiochemical Relations Simple micelles, that is, micelles composed of bile acid anions alone, do not occur in mammals. Instead, mixed micelles composed of bile acid anions and other lipids are present in bile and the small-intestinal contents. The bile in mammals contains mixed micelles that are composed of bile acid anions, phospholipid (mostly phosphatidylcholine [PC]) cholesterol, and other lipophilic molecules (2). Heavy metals, if present, may be bound as counterions to the micelles, which have a negative charge. In small-intestinal contents, the mixed micelles are composed of bile acid anions, fatty acids (partly ionized), 2-monoglycerides, PC (originating from bile and dietary sources), fat-soluble vitamins, cholesterol (from bile and dietary sources), and plant sterols (83). If lipophilic drugs are ingested or secreted in bile, they also will be present in the mixed micelles. Other dietary constituents will partition between the aqueous phase, the micellar phase, and the oil phase (consisting of triglyceride, diglyceride, and protonated fatty acid; see Chapter 66). Fatty acids when partly ionized, as well as monoglycerides, form hydrated bilayers provided they are greater than a certain transition temperature (84). PC has similar properties. Bile acids are rigid planar amphipathic molecules that adsorb to lipid bilayers and increase the radius of curvature of the outer layer. This results in a rapid conversion of the bilayers to a mixed micelle (85). This process of conversion of a bilayer to a mixed micelle occurs in the biliary canaliculus— in the process of biliary lipid secretion—as well as in the small intestinal lumen— in the process of fat digestion (83). The mixed micelles of bile and of small-intestinal content, although of differing chemical composition, have an identical molecular arrangement by small-angle neutron scattering (86). Probably, the mixed micelle is crudely spherical, but micelles must be considered flickering clusters, with a rapid exchange of bile acids between those in the mixed micelle and those present as monomers in the surrounding aqueous phase (87). In the biliary canaliculus, PC is concentrated in the outer layer of the canalicular membrane. This concentration is mediated primarily by the multidrug resistance gene 2 (MDR2; gene name ABCB3) (88,89), a member of the ATP-binding cassette (ABC) gene family that functions as a “flippase” to move PC from the inner to the outer membrane leaflet. The phospholipid forms bilayers that bulge out in hemispheres

from the canalicular membrane and can be visualized by freeze-fracture electron microscopy (90,91). These bilayers are likely also to contain cholesterol. Cholesterol transport across the canalicular membrane is mediated in large part by additional ABC transporters, the half-transporters ABCG5 and ABCG8 (92,93). In addition to the actions of ABCG5 and ABCG8, efficient cholesterol transport into bile appears to require PC transport (94,95). Bile acids solubilize the phospholipid bilayers and convert them to mixed micelles containing bile acids, PC, and cholesterol. Human bile is quite rich in cholesterol, with the cholesterol/PC ratio ranging from 0.2 in healthy individuals to 0.4 in patients with cholesterol gallstone (96). In most mammals, this ratio is much smaller and cholesterol gallstone formation does not occur (2). Bile acids, PC, and cholesterol are termed biliary lipids. Bilirubin glucuronide is not classified as a biliary lipid because it is relatively hydrophilic, it is present in bile in much smaller concentrations than the other biliary lipids, and its concentration in bile does not appear to affect gallstone formation.

ENTEROHEPATIC CIRCULATION From an anatomic point of view, the enterohepatic circulation is divided into two independent pathways, the portal and extraportal pathways (97). The extraportal enterohepatic circulation consists primarily of the lymphatic drainage from the intestine, whereby substances secreted by the intestine pass through the lacteals into the lymphatics and drain into the superior vena cava. Although important for the movement of cholesterol and phospholipid that are transported in large chylomicron particles (98), this extraportal pathway plays little role in the enterohepatic circulation of bile acids. Bile acids undergo a portal enterohepatic circulation whereby they are secreted by the liver, pass into the intestine, are absorbed from the intestinal lumen, pass into the portal circulation, and are efficiently extracted by the liver. At a fundamental level, the enterohepatic circulation of bile acids can be thought to consist of a series of storage chambers (gallbladder, small intestine), valves (sphincter of Oddi, ileocecal valve), mechanical pumps (canaliculi, biliary tract, small intestine), and chemical pumps (hepatocyte, cholangiocyte, ileocyte). Most, if not all, of the major bile acid transport proteins responsible for the enterohepatic circulation have been identified and include the Na+-dependent hepatocyte bile acid transporter (NTCP; gene name SLC10A1), the Na+-independent bile acid and organic anion transporter (OATP1B1; gene name SLC01B1), the ATP-dependent hepatocellular bile acid export pump (BSEP; gene name ABCB11), the apical sodium-dependent bile acid transporter (ASBT; gene name SLC10A2), and the organic solute transporter (OST) α-β in the ileal enterocyte (99–101) (see Fig. 56-4 and Table 56-2). The anatomic components of the enterohepatic circulation are the liver, gallbladder, biliary tract, intestine, portal venous circulation, and to a lesser extent, the colon, systemic

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1445 Hepatocyte

BA

Na

Cholangiocyte

1

Tubule BA

2 BA

4 3

Na

A

4

BA

BA

3

Na BA

Liver Synthesis (0.2-0.6 g/d)

Filtered/ Teabsorbed

Portal venous return (>95% of biliary secretion)

Biliary secretion 4 12 cycles/d

Layer 1 Kidney

Urinary excretion (<0.5 mg/d)

Enterocyte

Ileum BA

4 ILBP

NA BA

Colon

BA

Fecal excretion (0.2-0.6 g/d)

FIG. 56-4. Enterohepatic circulation of bile acids (BA) showing the individual transport proteins responsible for bile acid transport across various epithelia including hepatocytes, cholangiocytes, ileal enterocytes, and renal proximal tubule cells. Bile salts are synthesized in the liver and excreted into the bile ducts by an adenosine triphosphate (ATP)–driven transporter (bile salt export pump) (2). Most bile salts are reabsorbed in the terminal ileum by a sodium-dependent transporter (apical sodium-dependent bile acid transporter [ASBT]) (3). ASBT is also expressed on the apical surface of cholangiocytes and renal proximal tubules. An organic anion transporter(s) is expressed on the apical surface of jejunum and ileum and may mediate low levels of sodium-independent absorption of bile salts. At the basolateral surface (4), the organic solute transporter (OST) α-β and possibly also multidrug resistance protein 3 (MRP3) mediate transport of bile salts into the portal circulation. MRP3 and OSTα-OSTβ are also expressed in renal proximal tubule cells and cholangiocytes. The enterohepatic circulation of bile salts is completed at the basolateral surface of hepatocytes by a sodium-dependent transport process (sodium-dependent taurocholate transporting polypeptide [NTCP]) (1). ILBP, ileal lipid binding protein. (Modified from Shneider [186], by permission.)

circulation, and kidney. However, efficient hepatic extraction and intestinal reabsorption largely restrict bile acids to the intestinal and hepatobiliary compartments. During fasting, bile acids are sequestered and concentrated approximately 10-fold in the gallbladder, resulting in low levels of bile acids in the small intestine, portal vein, serum, and liver. During the digestion of a large meal, the gallbladder remains contracted, and bile acids secreted by the liver bypass the

gallbladder and pass directly into the duodenum. During this period, the bile acid concentration in the small intestine is approximately 5 to 10 mmol/L. After a meal, the sphincter of Oddi contracts and the gallbladder relaxes, causing a larger fraction of the secreted bile acid to enter the gallbladder for storage. Thus, the enterohepatic cycling of bile acids increases during digestion and slows between meals and during overnight fasting. This rhythm of bile acid secretion

1446 / CHAPTER 56 TABLE 56-2. The role of human transport proteins in the enterohepatic circulation of bile acids Transporter (gene symbol)

Location

Function

Hepatocyte Bile acid–dependent bile flow NTCP (SLC10A1) OATP1B1 (SLCO1B1) OATP1B3 (SLCO1B3) MRP3 (ABCC3) MRP4 (ABCC4) BSEP (ABCB11) MDR3 (ABCB4) ABCG5 ABCG8 FIC1 (ATP8B1)

Basolateral membrane Basolateral membrane Basolateral membrane Basolateral membrane Basolateral membrane Canalicular membrane Canalicular membrane Canalicular membrane Canalicular membrane Canalicular Membrane

Na+-dependent uptake of bile acids Na+-independent uptake of bile acids Na+-independent uptake of bile acids ATP-dependent export of organic anions ATP-dependent export of organic anions ATP-dependent bile acid export ATP-dependent phosphatidylcholine export ATP-dependent sterol export ATP-dependent sterol export ATP-dependent aminophospholipid transport (?)

Bile acid–independent bile flow OATP (1A1, 1B1, 1B3) MRP1 (ABCC1) MRP2 (ABCC2)

Basolateral membrane Canalicular membrane Canalicular membrane

Na+-independent uptake of organic solutes ATP-dependent export of nonbile acid organic solutes ATP-dependent export of nonbile acid solutes

Cholangiocyte ASBT (SLC10A2) OST-α/β MRP3 (ABCC3)

Apical membrane Basolateral membrane Basolateral membrane

Bile acid uptake (cholehepatic shunt?) Na+-independent export of bile acids ATP-dependent export of organic anions

Ileal enterocyte ASBT (SLC10A2) OST-α/β MRP3 (ABCC3)

Apical membrane Basolateral membrane Basolateral membrane

Na+-dependent uptake of bile acids Na+-independent export of bile acids ATP-dependent export of organic anions

Renal proximal tubule cell ASBT (SLC10A2) OST-α/β MRP3 (ABCC3)

Apical membrane Basolateral membrane Basolateral membrane

Na+-dependent uptake of bile acids Na+-independent export of bile acids ATP-dependent export of organic anions

ABC, ATP-binding cassette; ASBT, apical Na+ bile acid transporter; ATP, adenosine triphosphate; BSEP, bile acid export pump; FIC1, familial intrahepatic cholestasis type 1; MDR, multidrug resistance protein; MRP, multidrug resistance-associated protein; NTCP, Na+-taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; OST, organic solute transporter; SLC, solute carrier; ?, proposed functions.

is maintained even after cholecystectomy. When the gallbladder is absent, bile acids are stored in the proximal small intestine. After ingestion of a meal, small-intestinal contractions propel the stored bile acids to the distal ileum where they are actively reabsorbed (102). The enterohepatic circulation of bile acids is an extremely efficient process; less than 3% of the intestinal bile acids escape reabsorption to be eliminated in the feces. Thus, most of the bile acids secreted by the hepatocyte were previously secreted into the small intestine and returned to the liver in the portal circulation. In the small intestine, bile acids are absorbed predominantly by an active transport system restricted to the terminal ileum and, to a lesser extent, by passive absorption down the length of the intestine (103). Of all the conjugated anions secreted into bile, only bile acids are actively absorbed in conjugated form by the small intestine and undergo an enterohepatic circulation. This enterohepatic cycling may be defined simply as the quotient of biliary secretion divided by synthesis (or input in the case of a drug). For bile acids, this value is about 15,000/300, or 50. For bilirubin, the value is 1.

BILE SECRETION AND HEPATIC BILE ACID TRANSPORT Overview of Bile Secretion Formation of canalicular bile is an osmotic process driven by active secretion of organic solutes into the canalicular lumen, followed by passive inflow of water, electrolytes, and other solutes (99). Canalicular bile formation has been measured using metabolically inert solutes such as mannitol and erythritol that enter bile via the paracellular route and are not absorbed in the biliary ductules. Their use in quantifying canalicular bile flow is analogous to the use of inulin for measuring the glomerular filtration rate. Canalicular bile flow is traditionally divided into two components: bile acid–dependent bile flow (bile flow caused by the secretion of bile acids) and bile acid–independent flow (bile flow attributed to active secretion of nonbile acid constituents such as glutathione) (104). Solutes such as the conjugated bile acids that are actively pumped across the canalicular membrane generate bile flow and are termed primary solutes. Other primary

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1447 solutes include conjugated bilirubin, glutathione, bicarbonate, and conjugates of various metabolites and xenobiotics. Water, plasma electrolytes, calcium, glucose, amino acids, and other low-molecular-weight solutes that flow passively into the canaliculus in response to the osmotic gradient are termed secondary solutes. The choleretic activity of each primary solute is defined as the amount of bile flow induced per amount of solute secreted. (For example, in normal saline, there are 3.3 µl per µmol of ion.) For bile acid–dependent bile flow, this value is estimated for a solute such as a bile acid by plotting bile acid output as the horizontal axis against bile flow as the vertical axis (105). The slope of this line indicates the volume of bile flow induced by the mass of bile acid secreted and has been termed the apparent choleretic activity (106). By tradition, the accompanying cation is neglected, so that the true “choleretic activity” should be corrected for the accompanying cation, that is, for monovalent bile acids, divided by 2, and for bile acid sulfates, divided by 3. The apparent choleretic activity differs among conjugated bile acid species and ranges from 8 to 25 µl of bile flow induced per µmole of bile acid secreted, and is influenced by the osmotic properties of the mixed micelles in bile, as well as the permeability of the paracellular junctions to other solutes that enter canalicular bile by solvent drag (107). The large flux of bile acid molecules through the hepatocyte is the result of the accumulation of a large mass of bile acids that cycles continuously between the liver and intestine. In contrast with glucose or fatty acids, conjugated bile acid undergo little or no metabolism. Rather, they are transported vectorially through the hepatocyte and secreted actively across the canalicular membrane. No other “endobiotic” has a trans-hepatocyte flux rate as great as bile acids. Two other major biliary constituents, phospholipid and cholesterol, do not undergo vectorial transport by the hepatocyte, but originate from hepatocyte stores. Bilirubin does undergo vectorial transport, as it enters the hepatocyte in unconjugated form, is glucuronidated during hepatocyte transport, and then is actively secreted into bile. Although there is evidence of enterohepatic cycling of bilirubin under certain pathophysiologic conditions such as bile acid malabsorption (108), in health, bilirubin does not undergo an enterohepatic circulation and biliary secretion is equal to its biosynthesis rate. As a result, its rate of canalicular transport is about 1/400 that of bile acids. During digestion, the bile acid flux is about 0.3 to 4 µmol/kg/min, depending on the species, and decreases during fasting.

Bile Acid–Independent Canalicular Bile Flow Canalicular bile flow is also generated by active secretion of other nonbile acid primary solutes by the hepatocyte and biliary epithelium (107). This bile acid–independent flow is defined operationally as the extrapolated intercept with the vertical axis for the plot of bile flow versus bile acid output,

usually in an animal subjected to acute biliary drainage. In humans, most canalicular bile flow is generated by bile acid secretion, but in rodents, the majority of bile flow appears to be induced by secretion of anions other than bile acids (107,109). For example, in the rat, the bile acid–dependent and –independent bile flow has been estimated to be approximately 50 and 70 µl/kg/min, respectively (107,109); mice genetically deficient in hepatic bile acid secretion also exhibit relatively normal levels of hepatic bile flow (110). Hepatobiliary excretion of reduced glutathione and bicarbonate (HCO3−) constitute the major components of the bile acid–independent fraction of bile flow (111–113). The ATP-dependent canalicular secretion of glutathione via the multidrug resistance protein 2 (MRP2; gene name ABCC2) plays a particularly important role. In addition to being secreted at high concentrations into bile, intralumenal catabolism of glutathione by the γ-glutamyltranspeptidase, which is present, in part, on the canalicular membrane, further increases the concentration of osmotically active solutes by hydrolyzing glutathione into its three constituent amino acids. Besides the ATP-dependent secretion of organic anions into bile, hepatic and biliary ATP-independent secretion of bicarbonate via the HCO3−/Cl− anion exchanger AE2 contributes to the bile acid–independent bile flow (114). The majority of this HCO3− secretion occurs at the level of the bile ductule cells (115) in response to stimulation by a variety of hormones and neuropeptides such as secretin and vasoactive intestinal peptide (116). Hepatic bile continues to be modified during its transit through the biliary tract by the movement of water through specific channels (aquaporins), the secretion of solutes such as bicarbonate and chloride, and the absorption of solutes such as glucose, amino acids, and bile acids (116,117).

Overview of Hepatic Bile Acid Transport Approximately 97% of bile acids secreted into bile are derived from the recirculating pool. To maintain this process, liver parenchymal cells must transport bile acids efficiently from the portal blood into bile. This vectorial transhepatocellular movement of bile acids is a concentrative transport process that is driven by a distinct set of primary (ATP-dependent) and secondary (Na+ gradient–dependent) active transport systems at the sinusoidal and canalicular plasma membranes (99). These transporters are listed in Table 56-2 and shown in Figure 56-5. Bile acid flux through the liver and the number of participating hepatocytes are variable. In the fasting state, bile acids are taken up predominantly by the periportal hepatocytes (the first hepatocytes of the liver acinus), whereas during feeding, more hepatocytes in the liver acinus participate in bile acid uptake. In periportal hepatocytes, bile acid synthesis is repressed, whereas perivenous hepatocytes actively synthesize bile acids. Thus, periportal hepatocytes primarily absorb and secrete recirculating bile acids, and perivenous cells secrete predominantly newly synthesized bile acids. The transport of recirculating bile

1448 / CHAPTER 56

FIG. 56-5. Hepatocyte and cholangiocyte transporters important for bile acid secretion. At the sinusoidal membrane, the Na+-taurocholate cotransporting polypeptide (NTCP; SLC10A1) mediates the uptake of conjugated bile acids. Sodium-dependent uptake of bile acids through the NTCP is driven by an inwardly directed sodium gradient generated by Na+,K+-ATPase (not shown). The Na+-independent bile acid uptake is mediated by organic anion transporters OATP1B1 (SLC01B1) and OATP1B3 (SLC01B3). At the canalicular membrane, bile acids are secreted via the bile salt efflux pump (BSEP; ABCB11), and sulfated or glucuronidated bile acids are secreted via the multidrug resistance protein 2 (MRP2; ABCC2). The canalicular membrane also expresses adenosine triphosphate (ATP)–dependent export pumps that transport phospholipid (multidrug resistance gene 2 [MDR3]; ABCB4), cholesterol and plant sterols (ABCG5/ABCG8), and drug metabolites (MDR1; ABCB1) into bile. FIC1 (ATP8B1) is a P-type ATPase mutated in progressive familial intrahepatic cholestasis type 1. At the sinusoidal membrane, the ATP-dependent export pumps, MRP3 and MRP4, can transport organic anions and their conjugates into the space of Disse. Within the bile ducts, conjugated bile acids are absorbed by the apical sodium-dependent bile acid transporter (ASBT; SLC10A2) expressed on the apical membrane of cholangiocytes in the large bile ducts. Bile acid may then exit at the basolateral surface into the hepatic arterial circulation via an ATP-dependent carrier, MRP3 (ABCC3), or by an anion exchange mechanism, the organic solute transporter α-β (OSTα-OSTβ). BA, bile acid; GSH, glutathione; OA, organic anion. (Modified from Arresse M, Trauner M. Molecular aspects of bile formation. Trends Mol Med 2003;9:558, by permission.)

acids through periportal hepatocytes drives the majority of bile flow (118). The concentration of bile acids in the portal blood of healthy humans is 20 to 50 µmol/L. Dihydroxy bile acids are tightly bound to albumin (about 99% bound), whereas trihydroxy bile acids are only partly bound (about 70%). Uptake by the liver is typically expressed as fractional extraction or first-pass extraction and represents the percentage of bile acids removed during a single passage through the hepatic acinus. The fractional extraction of bile acids from sinusoidal blood ranges from 50% to 90%, but remains constant regardless of systemic bile acid concentrations. Because fractional bile acid extraction remains constant despite the bile acid load, bile acid uptake is sometimes termed blood flow limited. The concentration of total bile acids in the systemic circulation reflects this efficient hepatic extraction and averages 3 to 4 µmol/L in the fasting state.

Na+-Dependent Bile Acid Uptake across the Sinusoidal Membrane of the Hepatocyte Hepatocellular uptake of bile acids occurs against an unfavorable electrochemical ion gradient and results in a 5- to 10-fold concentration gradient between the plasma filtrate present in the space of Disse and the hepatocyte cytosol. The uptake of conjugated bile acids at the sinusoidal (basolateral) membrane is mediated predominantly (>80%) by a secondary active Na+-dependent transport system. The driving force for this Na+-dependent uptake is generated by the basolateral Na+,K+-ATPase that maintains the prevailing out-to-in Na+ gradient. In contrast with conjugated bile acids, Na+-dependent uptake accounts for less than half the uptake of unconjugated bile acids such as cholic acid or UDCA. The sinusoidal NTCP (gene symbol SLC10A1) encodes a 45-kDa membrane glycoprotein that shares

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1449 sequence identity with the Na+-dependent bile acid transporter present in the ileal enterocyte and satisfies all the functional criteria for hepatocytic Na+-coupled bile acid uptake. These properties include: (1) preferential high-affinity transport of conjugated bile acids; (2) kinetics for taurocholate transport similar to isolated hepatocytes; (3) electrogenic Na+-taurocholate uptake; (4) appropriate tissue-specific expression in the liver; and (5) similar ontogeny for Na+dependent bile acid uptake and NTCP in development (119). The presence of additional sinusoidal Na+-dependent bile acid transporters has been suggested; however, the support for such transport systems is equivocal (120). Although it is likely that NTCP accounts for most, if not all, hepatic Na+-dependent bile acid transport, conclusive support awaits identification of inherited NTCP defects in patients or the generation of an NTCP knockout mouse model. Polymorphisms that are specific to different ethnic groups and that interfere with bile acid transport in vitro have been reported for the human NTCP gene (121). Unfortunately, clinical data were not available for those subjects, leaving open the possibility that an isolated NTCP gene defect may be asymptomatic because the liver also expresses Na+independent bile acid transporters. A rare disorder characterized by hypercholanemia has been described; however, NTCP expression was normal in those patients and defects in other hepatic genes were found to be responsible for this disorder (44,45). Na+-Independent Bile Acid Uptake across the Sinusoidal Membrane of the Hepatocyte Unconjugated bile acids such as cholate are taken up predominantly by hepatic Na+-independent transport systems. Whereas all the properties of hepatocellular Na+-dependent bile acid transport can be accounted for by NTCP, Na+independent bile acid transport involves several different transport systems. Identification of Na+-independent hepatic transporters was originally confounded by their broad substrate specificity, and the multiplicity of substrates could not be reconciled easily with the properties of known hepatic transport systems. The list of substrates that share apparently common transport pathways included conjugated and unconjugated bile acids, Bromsulphalein, cardiac glycosides and other neutral steroids, linear and cyclic peptides, and numerous drugs. This conundrum was resolved with discovery of the organic anion transporting polypeptide (OATP) transporter gene family. Hepatocellular uptake of albumin-bound organic molecules is mediated by a limited number of multispecific transport systems (OATPs) with partially overlapping specificity, rather than many highly specific transport systems (122,123). In general, OATP substrates tend to be albumin-bound, organic solutes with greater molecular weights (>450 Da). However, members of the OATP family also transport uncharged compounds such as ouabain or aldosterone and even transport bulky organic cations such as N-(4, 4-azo-n-pentyl)-21-deoxy-ajmalinium.

The OATP genes encode 12 potential transmembrane domain proteins that share no sequence identity with the Na+-dependent bile acid transporters. The driving force for OATP-mediated organic anion uptake appears to be anion exchange, with coupling of bile acid uptake to HCO3−, or more likely, glutathione efflux (124,125). The OATP-type transporters constitute a large gene family with more than 36 members identified in human, mouse, and rat tissues. The original HUGO Gene Nomenclature Committee designation for the superfamily of OATP solute carriers was SLC21A. However, confusion related to species differences has led to the adoption of a new species-independent classification and nomenclature system, designated OATP/ SLC0 (123). The human carriers, OATP1B1 (SLC01B1 gene; original protein name OATP-C) and OATP1B3 (gene symbol SLC01B3; original protein name OATP8) exhibit partially overlapping substrate specificities and account for the majority of hepatic Na+-independent bile acid clearance. OATP2B1 (SLC02B1 gene; original protein name OATP-B) does not transport bile acids, but functions in human liver together with OATP1B1 and OATP1B3 to transport organic anions and drugs. These human OATPs transport Bromsulphalein; bilirubin glucuronides; steroid metabolites such as estradiol-17β-glucuronide and estrone-3-sulfate; arachidonic acid products such as prostaglandin E2, thromboxane B2, and leukotriene C4; and a wide variety of drugs such as pravastatin, digoxin, and fexofenadine (126). Because the majority of hepatic-conjugated bile acid uptake is sodium dependent, the major physiologic role of these broadspecificity solute carriers may be hepatic clearance of nonbile acid substrates such as endogenous amphipathic metabolites and xenobiotics.

Transporters Mediating Bile Acid Efflux across the Sinusoidal Membrane of the Hepatocyte An area of current active investigation is the cloning and characterization of transporters that mediate the efflux of bile acids and other organic solutes from the interior of the hepatocyte into the space of Disse. Much of this work has focused on members of the MRP (gene family ABCC) family of ATP-dependent transporters including MRP3 and MRP4 (127–129). The transporter MRP4 has been shown to mediate the cotransport of conjugated (N-acyl amidated) bile acids and glutathione (130). The suggestion has been made that bile acids efflux, and then are taken up again by the hepatocyte, with the result that there is continuous regurgitation and uptake as bile acids are transported down the hepatic acinus (131). These members of the MRP family, and possibly other transporters, may promote bile acid efflux in cholestasis (99,101,132), thus reducing the concentration in the hepatocyte and decreasing the likelihood of apoptosis/necrosis (76). Efflux of bile acid sulfates promotes renal excretion because sulfated bile acids are less protein bound and are not reabsorbed from the glomerular filtrate by the tubular epithelial cells.

1450 / CHAPTER 56 Canalicular Bile Acid Transport Canalicular secretion is the rate-limiting step in hepatic transport of bile acids from blood into bile. Whereas bile acid concentrations within the hepatocyte are in the micromolar range, canalicular bile acid concentrations are more than 1000-fold greater, necessitating active transport across the canalicular membrane. Functional evidence of ATP-dependent bile acid transport by the canalicular membrane has existed for more than a decade (133). However, molecular identification of the canalicular bile acid transporter proved elusive until members of the ABC family of transporters were investigated more closely. Functional expression and characterization studies showed that a novel ABC transporter closely related to the MDR1/P-glycoprotein gene is the canalicular bile acid transporter (134). This 160-kDa protein (originally named “sister of P-glycoprotein” [Spgp]) has been shown to efficiently transport conjugated bile acids and was subsequently renamed the bile salt export pump (BSEP; gene symbol ABCB11). The human BSEP gene locus on chromosome 2q24 is linked to progressive familial intrahepatic cholestasis type 2 (PFIC2), a hepatic disorder characterized by biliary bile acid concentrations less than 1% of normal and an absence of BSEP from the canalicular membrane. The role of BSEP as the major canalicular bile acid efflux pump was confirmed by the identification of mutations in the BSEP gene causing dysfunction of the gene product in patients with PFIC2 (135,136). The ability of BSEP to transport solutes in addition to bile acids is a new area of investigation, and it has been shown that the hepatic 3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitor pravastatin is a BSEP substrate (137). Based on the fairly broad substrate specificity exhibited by the related MDR1 transporter, it appears likely that other substrates will be identified. A proposed mechanism of hepatotoxicity for drugs inducing cholestasis is inhibition of BSEP (138,139). An area of active research is the development of polarized cells expressing both a bile acid uptake protein such as NTCP and the canalicular BSEP; such cells mediate vectorial transport of conjugated bile acids and may be useful for detecting inhibition of either bile acid uptake or secretion by drug candidates (140).

Ductular Modification of Bile Hepatic bile is modified further during its transit in the biliary tract via the action of ductular epithelial cells (cholangiocytes). These modifications include the carrier-mediated absorption of solutes such as glucose, amino acids, and bile acids, hydrolysis of glutathione by γ-glutamylpeptidase, and active secretion of ions such as chloride and bicarbonate (116,117). Bile may be transiently hypertonic as it enters the finest biliary ductules, but the paracellular junctions are freely permeable to water, and the inflow of water quickly renders bile isosmotic (141). Absorption of solutes is likely to occur in the smallest biliary ductules, whereas secretion is likely to occur in the

larger ductules (142,143). Secretion of chloride ion occurs via cystic fibrosis transmembrane conductance regulator (CFTR) and non-CFTR pathways (144,145), and bicarbonate may be secreted directly, or more likely, be generated by chloride-bicarbonate exchange (116). The secretion of chloride is promoted by hormones such as secretin and VIP and inhibited by somatostatin. Water enters bile in response to ion secretion. It may enter via aquaporin pores in the apical membranes of cholangiocytes or via the paracellular junctions (146). Calcium enters bile paracellularly, and the Ca2+ activity of ductular bile is similar to that of plasma. The PC in bile is thought not to be absorbed in the biliary ductules, but cholesterol absorption may occur.

Cholehepatic Shunt Pathway The cholehepatic shunt pathway originally was proposed to explain the bicarbonate-rich choleresis after administration of unconjugated C24 dihydroxy bile acids such as norUDCA (a C23 bile acid with a C4 side chain) or UDCA when given at high rates to the isolated perfused liver. In that cycle, unconjugated dihydroxy bile acids are secreted into bile, passively absorbed in protonated form by cholangiocytes, returned to the hepatocyte via the periductular capillary plexus, and resecreted into bile, thereby generating a bicarbonate anion (147). Hypercholeresis can also be induced by certain drugs that are lipophilic, weak organic acids such as the nonsteroidal anti-inflammatory drug sulindac (148). However, the physiologic significance of the cholehepatic shunt pathway for hepatic bile secretion in the healthy animal was uncertain because bile contains few unconjugated bile acids and the conjugated bile acids would be ionized and unable to diffuse passively across the biliary epithelium. The subsequent discovery that the biliary epithelium expresses the ASBT (SLC10A2 gene) (149–151), as well as a mechanism for basolateral export of bile acids (152), provided a molecular basis for the cholehepatic shunt pathway. The presence of a cholehepatic shunt pathway implies that the flux of bile acids through the hepatocyte could be greater than the load presented to the liver or recovered in bile (153). However, because bile acids are ultimately secreted from the biliary tree, the quantitative significance of this pathway under normal physiologic conditions is uncertain. Under cholestatic and pathophysiologic conditions, cholehepatic shunting may become an important escape route for bile acids in the biliary tract (127). Moreover, bile acid uptake into cholangiocytes may be important for activating cellular signaling pathways important in the regulation of secretory and proliferative events in the biliary tree (151).

CONCENTRATION OF BILE IN THE GALLBLADDER During the fasting state and between meals, hepatic bile enters the gallbladder. Entry occurs because hepatic bile is secreted with a pressure of about 20 cm water; that is, in turn,

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1451 derived from canalicular contractions and osmotic secretion of canalicular and ductular bile (154). This pressure, when the sphincter of Oddi is closed, is sufficient to fill the gallbladder, where the musculature relaxes secondary to neurohormonal stimuli, among them somatostatin. During bile storage in the gallbladder, bile is concentrated by the removal of electrolytes and water. Concentration is mediated by the gallbladder epithelium (155). On the apical membrane, a double ion exchange system (Na+-H+; Cl−-HCO3−) moves sodium and chloride ions into the cell (156), whereas hydrogen ions and bicarbonate ions are exchanged into the lumen where they combine to form water and CO2 (157,158). This remarkable process is indicated by the acidification of bile during storage. At the basolateral domain, sodium and chloride ions are effluxed, generating an osmotic force for water and filterable electrolytes to flow through the paracellular junctions. Ca2+ ion concentrations increase in bile because of Donnan equilibrium effects (82,159). The bile acid anions are too large to enter the paracellular junctions. There is modest absorption of phospholipid and cholesterol, such that bile becomes less saturated in cholesterol with prolonged storage (160). In healthy individuals, but not in subjects with gallstones, there is little change in the conjugated bilirubin species (monoglucuronides and diglucuronides) during storage, but it is possible that there is some slight hydrolysis of the ester bonds of these molecules, either occurring spontaneously or by an enzyme-catalyzed mechanism (161). Unconjugated bilirubin can be absorbed passively by the gallbladder epithelium (162), be solubilized by the mixed micelles in bile (163), or precipitate from solution as a calcium salt. Calcium bilirubinate crystals, if present together with cholesterol crystals, may be detected by ultrasonic examination of the gallbladder, and the mass of associated crystals is termed sludge (164). Alternatively, the crystals may be expelled into the small intestine with gallbladder contraction. Human bile, but the bile of no other animal, often is saturated or supersaturated with cholesterol (2). Cholesterolsupersaturated bile appears to suppress gallbladder emptying. Together with nucleating factors, this sets the stage for cholesterol gallstone formation, a common malady of caucasian individuals, especially if they are obese.

OVERVIEW OF INTESTINAL ABSORPTION OF BILE ACIDS Bile acids enter the small intestine together with other biliary constituents and facilitate absorption of dietary lipids and fat-soluble vitamins. Conjugated bile acids are absorbed from the intestine by multiple mechanisms. Some passive absorption probably occurs throughout the small intestine through the occasional paracellular junction that is sufficiently large to permit passage of the conjugated bile acid anion (165). In the duodenum, which is transiently acidic because of bursts of gastric emptying, there can be passive absorption of protonated glycine dihydroxy conjugates. In addition,

there may be low levels of carrier-mediated transport by as yet unidentified transporters throughout the entire small intestine (166). Numerous observations indicate, however, that the terminal ileum is the major site of bile acid reabsorption. For example, intestinal perfusion studies in humans have shown that ileal absorption is greater than jejunal absorption, and that profound bile acid malabsorption occurs after ileal resection (167). Studies in rodents using in situ perfused intestinal segments to measure bile acid absorption have also demonstrated that ileal bile acid transport is a highcapacity system sufficient to account for the biliary output of bile acids. The consensus from this work was that the ileal active transport system is the major route for conjugated bile acid uptake (168). A fraction (10–50% depending on the bile acid species) of the bile acids returning in the portal circulation escapes hepatic extraction and spills into the systemic circulation. The binding of bile acids to plasma proteins decreases their glomerular filtration and minimizes urinary excretion. In healthy humans, the kidney filters approximately 100 µmol bile acids each day. Remarkably, only 1 to 2 µmol are excreted in the urine because of highly efficient tubular reabsorption (169). Even in patients with cholestatic liver disease, in whom plasma bile acid concentrations are increased, the 24-hour urinary excretion of nonsulfated bile acids is significantly less than the quantity that undergoes glomerular filtration (170). Subsequent studies have shown that bile acids in the glomerular filtrate are actively reabsorbed from the renal tubules, and this process contributes to the increase in bile acid concentrations found in peripheral blood of patients with obstructive liver disease (171). Bile acid conservation by the renal proximal tubule is mediated by the same transporter (SLC10A2 gene) that is present in the ileal enterocyte (46,172). Thus, renal and ileal vectorial transport both function to conserve the bile acid pool. Molecular Mechanisms for Intestinal Bile Acid Transport Ileal Na+-Dependent Bile Acid Uptake Bile acids are actively transported across the ileal brushborder membrane by the well-characterized ileal ASBT (46, 173). The relation between the Na+-coupled bile acid transport mediated by the hepatocyte, cholangiocyte, ileal enterocyte, and renal tubule cell was resolved with the cloning of the bile acid cotransporters from those tissues (46,149,174). The liver (NTCP; SLC10A1) and ileal (ASBT; SLC10A2) Na+-bile acid cotransporters are distinct gene products that share considerable amino acid identity and structural similarity (175). In contrast, the ileal, renal, and cholangiocyte carriers are all products of the same gene. The inwardly directed Na+ gradient maintained by the basolateral Na+, K+-ATPase, as well as the negative intracellular potential, provides the driving force for ASBT-mediated bile acid uptake.

1452 / CHAPTER 56 The ASBT transports all major species of bile acids, but favors trihydroxy over dihydroxy bile acids and conjugated over unconjugated species (46). The properties of ASBT satisfied all the functional criteria for ileal active bile acid uptake. These properties include: (1) a strict sodium dependence for bile acid transport (46); (2) narrow substrate specificity encompassing only conjugated and unconjugated bile acids with negligible uptake of other organic anions (47); (3) small intestinal expression limited to the terminal ileum; (4) similar ontogeny for ileal sodium-dependent taurocholate uptake and ASBT expression (176); and (5) elimination of the enterohepatic cycling of bile acids by targeted deletion of the ASBT gene in mice (177). Finally, inherited mutations in the human ASBT gene cause primary bile acid malabsorption, an intestinal disorder associated with interruption of the enterohepatic circulation of bile acids and fat malabsorption (173). This final finding clearly demonstrates that most intestinal bile acid absorption in humans is mediated by the ASBT. Little is known about the intracellular transport of bile acids in the ileal enterocyte. The ileal lipid binding protein (ILBP, also called the ileal bile acid binding protein; gene symbol FABP6) is a member of the fatty acid binding protein family and is a major ileal enterocyte cytosolic protein (178,179). ILBP was thought to be involved in the transcellular transport of bile acids; however, a mouse model lacking appreciable ILBP expression exhibited no impairment of intestinal bile acid absorption (180). These results indicate that ILBP is not essential for ileal bile acid transport. It is likely that ILBP functions to bind bile acids when their intracellular concentration is markedly increased, thereby protecting the ileal enterocyte from bile acid cytotoxicity. Ileal Basolateral Bile Acid Transport Whereas ASBT mediates bile acid uptake across the apical brush-border membrane, less is known about the proteins responsible for bile acid export across the basolateral membrane. Several possible mechanisms for basolateral bile acid transport had been proposed, including a dedicated transporter, multiple transporters with broad substrate specificity that includes bile acids, or a combination of both systems. Among these were an alternatively spliced form of the rat Asbt (t-Asbt) (181) and the multidrug resistance-associated protein 3 (Mrp3; gene name Abcc3) (128,182). However, it is unlikely that the t-Asbt plays an important role in basolateral bile acid transport because there is little or no ileal t-Asbt expression in humans, mice, and hamsters. Although Mrp3 was an attractive candidate for the ileal basolateral bile acid transporter based on its tissue expression and bile acid transport properties (128,183), studies in Mrp3 null mice indicate that Mrp3 also plays little role in intestinal bile acid absorption (184,185). Considering the high flux of bile acids across the ileal enterocyte (186), it is likely that a dedicated basolateral bile acid transporter is required. The Ostα-Ostβ has been identified

as a potential ileal basolateral bile acid transporter based in part on its hypothesized regulation by bile acids (187,188). Ostα-Ostβ is a heteromeric solute transporter that requires the coexpression of two distinct gene products, a 340-amino-acid, 7-potential transmembrane domain protein (Ostα) and a 128-amino-acid single-transmembrane ancillary polypeptide (Ostβ) (187–189). Although the functions of the individual subunits have not yet been elucidated, it has been shown that coexpression of both Ostα and Ostβ is essential for delivery of the individual proteins to the plasma membrane (187). Ostα and Ostβ mRNA expression closely parallel one another in mouse tissues, and they are expressed at particularly high levels in terminal ileum. Although it is likely that Ostα-Ostβ accounts for a significant portion of the ileal basolateral bile acid export, conclusive support awaits identification of inherited OST gene defects in patients or the generation of an Ost knockout mouse model. Regulation of Intestinal Bile Acid Transport Regulation of Gene Expression ASBT is expressed in tissues that serve to facilitate the enterohepatic circulation of bile acids. It is found on the apical membrane of ileal enterocytes, proximal renal convoluted tubule cells, large cholangiocytes, and gallbladder epithelial cells (151,190–193). The molecular mechanisms controlling the tissue-specific expression of ASBT are unknown. ASBT activity and expression is predominantly found in villous but not crypt enterocytes, as would be expected from its role in intestinal bile acid reclamation (176,194). Expression is tightly restricted to the terminal ileum in mice, hamster, rats, and humans (195–199). The mechanism restricting ASBT expression along the longitudinal axis of the intestine is not well understood, although it appears to correlate with the expression of c-jun (200). After intestinal resection or transposition, compensatory responses in ASBT expression occur only in regions of the small intestine that natively express ASBT (199,201,202). In addition to its restricted tissue expression, the ASBT expression is regulated during development by its substrates, by hormones, and by cytokines. Table 56-3 shows the transcription factors known to be involved in regulating ASBT expression (203,204). Developmental Regulation Intestinal bile acid transport undergoes complex regulation during normal development. Descriptive analyses of this ontogeny have been performed in dogs, humans, rats, rabbits, mice, chickens, and guinea pigs (205–211).The predominant focus of study has been on apical bile acid transport, whereas data on basolateral transport are scant. The precise time of onset of ileal transport activity in the fetal period has not been determined. In rats and mice, ASBT activity in the ileum is present just before birth and is subsequently repressed in the early postnatal period (196,212). Marked induction

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1453 TABLE 56-3. Transcriptional regulation of the apical sodium-dependent bile acid transporter Transcription factor

Species

Activity

Function

Reference

HNF-1α c-Jun

Mouse Rat

Activation Activation

203 200

c-Fos FXR-SHP

Rat Mouse Rabbit Human Mouse Human Human Human Rat

Repression Repression

Basal activity Basal activity Ontogeny Mediates repression by inflammatory cytokines Negative feedback regulation by bile acids

Activation Activation Activation Activation Activation

Basal activity Mediates activation Mediates activation Mediates activation Mediates activation

LRH-1 RAR GR PPARα VDR

by by by by

retinoic acid glucocorticoids ciprofibrates vitamin D3

245 229 230 231 229 231 240 204

FXR, farnesoid X receptor; GR, glucocorticoid receptor; HNF, hepatic nuclear factor; LRH-1, liver receptor homolog-1; PPAR, peroxisomal proliferator-activated receptor; SHP, small heterodimer partner; RAR, retinoic acid receptor; VDR, vitamin D receptor.

of apical bile acid transport at the time of weaning has been carefully described in dogs, guinea pigs, rats, rabbits, and chickens (205–210). In rat and mouse ileum, this process is associated with an up-regulation of ASBT mRNA and protein (176). Unfortunately, the mechanisms underlying the developmental stage-specific expression of ASBT are poorly understood. Corticosteroids, thyroid hormone, and bile acid feeding can precociously induce up-regulation of intestinal bile acid transport before weaning (213–217). A potential clue to the mechanism underlying the ontogenic regulation of ASBT is the finding that the expression of the same gene product in the kidney is unchanged during this developmental period (172). In the rat, both transcriptional and posttranscriptional mechanisms appear to be involved in the regulation of ASBT expression. Measurement of steady-state ASBT mRNA levels by Northern blotting and ASBT transcriptional analyses by nuclear run-on assays have been performed in developing rat intestines and kidneys (172,176). On postnatal day 7, there is 10-fold more ASBT mRNA in the intestine than in the kidney, despite identical transcription rates. Between postnatal days 7 and 28, there is a greater than 200-fold increase in ASBT steady-state mRNA levels, but only a 10-fold increase in transcription. These two findings strongly suggest that both ASBT transcription and mRNA stability change during normal intestinal development. Preliminary findings suggest that activator protein-1 (AP-1) and the FXR may be relevant transcription factors involved in ontogeny of ASBT (200,218). Regulation by Bile Acids The regulation of ASBT expression by bile acids has been an area of controversy. Conflicting observations have been published, reporting that ASBT or ileal bile acid transport, or both, is up-regulated, down-regulated, or unregulated bile acids (219–222). Differences in experimental models, methods for measuring ASBT protein or activity, and species may account for some of the discrepancies that have been

observed (223). Studies in humans using a bile acid analogue, 75 Se-homocholic acid-taurine, suggest that negative feedback regulation of intestinal bile acid transport is operational in humans (224). Analysis of bile acid responsiveness is a fundamentally complex issue because it represents a systems biology question. Attempts to alter bile acid homeostasis, in vivo, are always accompanied by a plethora of compensatory responses in the enterohepatic circulation including changes in hepatic synthesis and excretion of bile acids. As such, it may be difficult to predict the consequences for flux of bile acids across the ileum in response to simple interventions such as bile acid or sequestrant feeding. Cloning of the Asbt gene has permitted analyses of bile acid responsiveness to focus on changes in mRNA or protein expression and have avoided some of the problems associated with trying to assess actual transport activity. Because the Asbt gene is expressed in a variety of tissues, the regulatory responses that are observed may be tissue specific. Negative-feedback regulation of ileal Asbt by bile acid was observed in mice and guinea pigs fed either taurocholate or cholestyramine (225). In contrast, cholic acid feeding in rats led to up-regulation of ileal Asbt mRNA levels (221). In a more recent study bile duct ligation, bile acid feeding, and sequestrant feeding in rats led to no changes in ileal Asbt expression (222) at the mRNA and protein levels (226). Differential changes in cholangiocyte versus ileal Asbt expression have been described in rats fed either taurocholate or cholestyramine (227). Finally, multiple clinical studies indicate that bile acid feeding induces only modest increases in bile acid secretion or in the bile acid pool size, strongly suggesting that there is some kind of negative feedback regulation of ileal transport in humans. Overall, it has been difficult to attain a clear picture of the bile acid responsiveness of Asbt, although the ileal data have favored negative feedback regulation in most species. Advances in our understanding of the molecular mechanisms responsible for bile acid signaling have helped to resolve many of these controversies. Acting through nuclear

1454 / CHAPTER 56 receptors, bile acids have been shown to either positively or negatively regulate the transcription of a wide variety of genes involved in lipid metabolism (228). Positive feedback regulation is mediated by the direct effect of a complex of bile acids and the FXR. Notable examples include transcriptional activation of the ILBP and the hepatic canalicular BSEP. Negative feedback regulation is more complex, as described earlier for the CYP7A1. Analysis of the mouse ASBT promoter indicated the presence of an LRH-1 element that is down-regulated by bile acids acting through the FXR-SHP pathway (229). Remarkably, the rat ASBT promoter lacks a functional LRH-1 element, explaining the lack of bile acid down-regulation in that species. The rabbit ASBT is regulated by bile acids in a similar fashion to the mouse (230). Human ASBT regulation can be modeled in Caco-2 cells because they express the human ASBT gene. Bile acid treatment of Caco-2 cells causes down-regulation of ASBT expression (231). Negative feedback regulation of human ASBT is mediated by the FXR-SHP cascade, although in humans, SHP antagonizes the activation of ASBT by the retinoic acid receptor and not LRH-1 (231). These in vitro studies of Caco-2 cells correlate well with investigations of humans and raise the possibility that regulatory responses in the ileum may be counterproductive in the setting of cholestasis (232). In fact, anomalous bile acid responsiveness of ASBT may underlie part of the pathophysiology of Byler’s disease, which is characterized by diminished FXR activity and increased ASBT expression (233). Regulation by Intestinal Inflammation and Corticosteroid Effects Bile acid malabsorption in inflammatory bowel disease, especially in the setting of ileitis, has been well characterized (234–237). Bile acid synthesis is increased several fold in response to the observed bile acid malabsorption. In patients with Crohn’s disease, the postprandial increase in serum bile acids, an indirect marker of ileal bile acid reabsorption, is diminished (238,239) and ASBT protein expression is reduced (240). Similarly, 75SeHCAT retention is reduced in patients with ileitis (241,242). Several animal models of ileitis have been developed, which have facilitated the analysis of this phenomenon. Eimeria magna infection of rabbits leads to ileitis, which is associated with reduced sodium-dependent bile acid transport of bile acids and reduced expression of Asbt protein (243). Similarly, 2,4,6trinitrobenzenesulfonic acid or indomethacin treatment lead to intestinal inflammation associated with reduced Asbt expression (244,245). The molecular mechanisms underlying inflammatorymediated repression of Asbt are linked to the effects of the transcription factor c-fos (245). The Asbt promoter contains AP-1 binding elements (200). There are two distinct ciselements near the transcription initiation start site for Asbt. The upstream AP-1 element binds a c-jun homodimer that is involved in transactivation of the Asbt gene (200).

In contrast, the downstream AP-1 element binds a c-jun/cfos heterodimer, and occupancy of this element is associated with transcriptional repression of Asbt (245). In the setting of intestinal inflammation, c-fos is phosphorylated and translocates into the nucleus. Where it binds leading to the downstream AP-1 element to transcriptional repression of ASBT expression and commensurate down-regulation of its activity. Corticosteroids have had a long-standing use in the treatment of inflammatory bowel disease. Exogenous corticosteroids can induce precocious expression of Asbt and also up-regulate Asbt expression in mature animals (246). Budesonide treatment of healthy volunteers induced a 34% increase in ASBT protein expression in ileal biopsies (240). This effect is mediated through the glucocorticoid receptor binding to specific glucocorticoid response elements in the human ASBT promoter (240). Thus, the beneficial effects of corticosteroids in inflammatory bowel disease may involve not only an effect on inflammatory cytokines, but also a direct effect on ASBT expression.

FUNCTIONS AND DYSFUNCTIONS OF BILE ACIDS IN THE INTESTINE Small Intestine Bile acids form mixed micelles with dietary lipids (and/or their hydrolysis products) and fat-soluble vitamins, enhancing their diffusion through the unstirred aqueous layer surrounding the intestinal epithelium (83,247,248). Whether there are other physiologic advantages to the presence of a micellar phase in the small intestine remains to be clarified. Several lines of evidence suggest that the presence of a micellar phase (and possibly the presence of solubilized fatty acids) acts to prevent bacterial proliferation in the small intestine. Bacterial overgrowth occurs in biliary fistula or bile duct ligated animals, as well as in animals with experimental cirrhosis that have decreased bile acid secretion (249). In cirrhotic rats with bacterial overgrowth, feeding of conjugated bile acids abolishes bacterial overgrowth, decreases bacterial translocation to intestinal lymph nodes, and decreases endotoxemia (250). This effect of bile acids could be caused by their bacteriostatic effects, or could be an indirect effect mediated by bile acids stimulating increased microbial defenses via interacting with enterocyte FXR, or both might occur. There are multiple disturbances of the enterohepatic circulation of bile acids that occur in patients. These include impaired bile acid biosynthesis and conjugation, impaired canalicular transport, and obstruction of the biliary tract. The enterohepatic circulation may be interrupted by creation of a biliary fistula or ileal dysfunction, and its efficiency decreased by administration of bile acid sequestrants (251). When there is ileal dysfunction or resection causing bile acid malabsorption, the compensatory increase in hepatocyte bile acid biosynthesis may be sufficient to virtually compensate for

BILE FORMATION AND THE ENTEROHEPATIC CIRCULATION / 1455 the bile acid malabsorption (167). However, the increased flux of bile acids into the colon may induce water and electrolyte secretion and is manifest clinically as diarrhea (251, 252). If the ileal dysfunction is sufficiently great, the compensatory increase in synthesis, about 3 to 4 g/day, is insufficient to restore normal bile acid secretion, about 12 to 15 g/day (102). Under these circumstances, bile acid concentration in the proximal small intestine decreases, and malabsorption of water-insoluble fatty acids and fat-soluble vitamins occurs (253,254). Bacterial proliferation in the proximal small intestine may lead to greatly increased bile acid deconjugation (255). Dietary oxalate is usually precipitated by Ca2+ ions in the small intestinal lumen. When the concentration of bile acids is decreased to submicellar concentrations in the small intestine, as occurs in patients with severe ileal dysfunction or resection, dietary fatty acids act as a sink for Ca2+ ions, greatly decreasing the intraluminal activity of Ca2+ ions (256). As a result, oxalate passes into solution. Some of this oxalate is absorbed in the colon, leading to hyperoxaluria and an increased risk for renal stone formation (257,258).

Large Intestine In health, the concentration of bile acids in the aqueous phase of large intestinal contents is less than 1 mM because deconjugation-dehydroxylation results in the formation of unconjugated 7-deoxy monohydroxy and dihydroxy bile acids. These bile acids are far less soluble than their conjugated dihydroxy and trihydroxy precursors. In addition, the unconjugated secondary bile acids are membrane permeable and are, in part, absorbed from the colon (259). Binding to food residues also occurs (260,261). Because of these low aqueous concentrations, it may well be that bile acids have no direct function in the colon, and their removal from solution is beneficial to the organism because it permits bacterial proliferation. Such bacterial proliferation promotes the formation of short-chain fatty acids, which are an essential nutritional source in herbivorous animals that have hindgut fermentation (262,263). When bile acid malabsorption occurs, bile acids may be at greater concentrations in the colon, where they promote secretion from the colonic epithelium by a direct effect (252), and also indirectly by activating mast cells (264,265). Bile acids also promote propulsive motility. It appears likely that not all bile acids have these pharmacologic effects. Currently, only CDCA and DCA have been shown to induce secretion and promote colonic emptying (266,267). If the intralumenal bile acid concentration is sufficiently high, bile acids may damage the paracellular junctions, permitting bile acids to be absorbed by the paracellular route and enter the cell via the basolateral domain (268,269). Whether the low bile acid concentrations present in health in the colonic lumen influence the normal process of colonic desiccation and colonic motility is unknown.

THERAPEUTIC USES OF BILE ACID AGONISTS AND ANTAGONISTS IN CLINICAL MEDICINE Bile Acid Agonists Despite their immense physiologic importance, bile acid agonists and antagonists are still relatively unimportant in clinical therapeutics. The most important use of bile acids is replacement therapy in inborn errors of bile acid biosynthesis (15). In certain defects of A- and B-ring modification, bile acid feeding suppresses the formation of cytotoxic bile acid precursors and saves the patient’s life, obviating the need for liver transplantation (271). Bile acids also retard disease progression in cerebrotendinous xanthomatosis, a disease that is caused by a deficiency in sterol 27hydroxylase (272). A second use of oral bile acids (CDCA and UDCA) is to desaturate bile in cholesterol (273). The two bile acids probably decrease biliary cholesterol secretion by two different mechanisms: CDCA inhibits HMG-CoA reductase, whereas UDCA up-regulates bile acid synthesis and decreases cholesterol intestinal absorption (274,275). Such therapy can prevent the formation of cholesterol gallstones and induce the gradual dissolution of cholesterol gallstones. Attempted gallstone dissolution with oral bile acids has fallen into disfavor because of the success of laparoscopic cholecystectomy, which is curative (276). Medical dissolution of gallstones requires months of therapy and may not always dissolve gallstones (if a noncholesterol surface is present on the surface of the gallstone); also, gallstones may recur (277). Nonetheless, it may be useful for preventing gallstones in obese subjects undergoing dramatic weight loss therapy (278) or in treatment of patients with gallstones at high risk for surgery (279). Currently, the mostly widely used form of bile acid therapy is the administration of UDCA for cholestatic liver disease, in particular, primary biliary cirrhosis (280). The UDCA dilutes out the endogenous, cytotoxic bile acids, induces hepatic detoxification mechanisms (281), and improves liver tests for aminotransferase and bilirubin levels that are markers for liver injury. In primary biliary cirrhosis, administration of UDCA decreases the rate of disease progression and probably causes a small increase in 10-year survival (282). In contrast with primary biliary cirrhosis, the therapeutic efficacy of UDCA is uncertain in primary sclerosing cholangitis (283,284), and an early trial found little benefit of UDCA for the treatment of nonalcoholic steatohepatitis (285). Cholylsarcosine, a bile acid in which cholic acid is conjugated to sarcosine (N-methylglycine), is resistant to bile acid deconjugation-dehydroxylation (58,286). It has been used to restore a micellar phase in patients with short bowel syndrome and has been shown to increase fat absorption and nutritional status in small studies (287,288). The compound may also be useful in diseases in which small intestinal propulsive motility is defective and there is bacterial overgrowth, causing excessive bacterial deconjugation (250). The drug currently remains investigational.

1456 / CHAPTER 56 Bile Acid Antagonists and Modifiers of Bile Acid Metabolism Bile acid binding agents (bile acid sequestrants) are positively charged polymers that bind bile acids by electrostatic attraction and hydrophobic bonding (289). Early sequestrants were cholestyramine and colestipol; a more recent sequestrant is colesevelam (290). Bile acid malabsorption leads to increased bile acid concentrations in the colon and an induction of electrolyte secretion (291) and propulsive colonic motility (292). Administration of bile acid sequestrants causes symptomatic relief (293), although the use of bile acid sequestrants in such conditions is “off label”; that is, FDA approval for this specific indication has not been sought. Bile acid sequestrants originally were widely used for the treatment of hypercholesterolemia (294). They continue to be used as adjuncts to statin therapy, because the combination of inhibition of CoA reductase (the pharmacodynamic action of statins) and the induction of bile acid biosynthesis causes maximal up-regulation of low-density lipoprotein (LDL) receptors and reduction of plasma LDL cholesterol levels (295). A number of pharmaceutical companies have attempted to develop ASBT inhibitors that would achieve the same effect, but these remain investigational (296–300). Although these agents have proved efficacious in reducing LDL cholesterol, one of the predicted side effects is the induction of diarrhea because of the induction of bile acid malabsorption. Such agents might be useful in cholestatic liver disease where there is inappropriate ileal conservation of bile acids (232), but the utility of this idea has not as yet been tested. In severe cholestasis, UDCA is administered routinely. One consequence of severe cholestasis is intractable pruritus (301), to which increased plasma bile acid levels appear to contribute (270). Such pruritus can be treated by dialyzing the patient’s plasma against albumin in devices that are currently in development. The cytochrome P450 inducing agent rifampicin has been shown to decrease pruritus. This agent induces 6-hydroxylation of bile acids; such 6-hydroxy bile acids may be less cytotoxic or are not retained in the enterohepatic circulation as well as CDCA and cholic acid. The clinical utility of the combination of UDCA and rifampicin is being examined, and studies have highlighted the role of the nuclear receptor signaling pathways in the actions of these drugs (76,281).

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57

Mechanisms of Hepatocyte Organic Anion Transport Allan W. Wolkoff Mechanisms of Nonbile Acid Organic Anion Uptake, 1463 Physiology, 1463 Identification of Transporters, 1464 Characteristics of Hepatocyte Organic Anion Transporting Polypeptides, 1465 Mechanisms of Bile Acid Uptake, 1468 Physiology, 1468 Identification of Transporters, 1468

Regulation of Hepatocyte Na+-Dependent Bile Acid Uptake, 1470 Organic Anion Excretion across the Bile Canaliculus, 1471 Identification of Transporters, 1471 Regulation of Hepatocyte Bile Canalicular Excretory Function, 1473 Summary, 1475 References, 1475

One of the major functions of the hepatocyte is the removal of a variety of organic anionic compounds from the blood. These compounds include various xenobiotics, as well as endogenous compounds such as bilirubin and bile acids. Many of these compounds have limited aqueous solubility and circulate bound to serum albumin. Despite being almost entirely protein bound, for the most part these organic anions are cleared rapidly from the circulation by the liver. The liver is designed to permit efficient extraction of protein-bound compounds by the hepatocyte. In comparison with other organs in which there is a tight capillary endothelium, the endothelium of the liver is fenestrated, allowing circulating proteins such as albumin to come into close proximity with hepatocytes. Although previous studies have suggested that direct interaction of albumin with the hepatocyte surface facilitates extraction of organic anions, a number of subsequent studies indicated that the organic anion is extracted from its protein carrier during the uptake process without evidence for direct protein–cell interaction. Although many of these protein-bound organic anions are lipophilic and theoretically could be taken up by hepatocytes by simple diffusion across

the lipid bilayer, studies indicate that this is unlikely. In addition, a number of proteins that are able to mediate uptake of organic anions have been identified on the hepatocyte basolateral (sinusoidal) surface. After internalization and biotransformation, many of these compounds are pumped out of the cell, across the apical (canalicular) plasma membrane, into the bile. Several proteins that are able to mediate adenosine triphosphate (ATP)–dependent excretion of these compounds have been identified on the bile canalicular plasma membrane of hepatocytes. This chapter examines mechanisms for uptake and excretion of a number of typical organic anions, including bile acids, for which the liver plays an essential role.

A. W. Wolkoff: Marion Bessin Liver Research Center and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

MECHANISMS OF NONBILE ACID ORGANIC ANION UPTAKE Physiology Influence of Albumin Binding Studies performed in the isolated perfused rat liver indicated that as much as 50% of bilirubin or sulfobromophthalein (BSP) is removed from perfusate in a single pass (1–3). Despite that these compounds are bound avidly to albumin, it is the free organic anion rather than the albumin-organic anion complex that is taken up (2,4). Subsequent investigations performed with overnight cultured rat hepatocytes showed

1463

1464 / CHAPTER 57 saturable BSP uptake that was inhibited by bilirubin (5,6). Similar to findings in the isolated perfused rat liver, ligand, but not albumin, was taken up by cultured hepatocytes; that is, albumin remained extracellular (6). The lack of specificity for albumin as a carrier was noted in studies in the isolated perfused rat liver in which there was no enhancement of bilirubin uptake when it was presented as a complex with albumin as compared with its presentation as a complex with its normally cytosolic binding protein ligandin (2) or in the absence of albumin (7). Driving Forces The driving forces for nonbile acid organic anion uptake also have been the subject of a great deal of study. Early experiments performed in isolated rat hepatocytes indicated saturable uptake of BSP without a requirement for cellular energy, as determined by lack of effect of preincubation of cells in antimycin A, rotenone, or carbonylcyanide m-chlorophenylhydrazone (8). However, subsequent studies using different metabolic inhibitors (2,4-dinitrophenol [DNP], potassium cyanide [KCN], or sodium azide/2-deoxyglucose) clearly showed that cellular energy was required for BSP uptake (5,9), and a similar difference in sensitivity of the uptake process to various metabolic inhibitors also was seen in studies of glutathione-BSP uptake by isolated rat hepatocytes (10). The physiologic significance of this differential sensitivity remains unknown. Development of a short-term cultured rat hepatocyte system facilitated studies in which the environment could be manipulated and transport characteristics and driving forces could be examined. In particular, isosmotic substitution of NaCl in the medium by sucrose resulted in an 80% reduction in uptake of BSP or bilirubin by cultured rat hepatocytes (5,6). Although this result might have signified a requirement for extracellular Na+, this was not the case. There was no significant effect on uptake of substitution of Na+ in medium by K+, Li+, or choline. In contrast, when Cl− was replaced by HCO3− or gluconate, ligand uptake was markedly reduced. Similar Cl− dependence has been described for hepatocyte uptake of bilirubin by cultured rat hepatocytes (11) and by isolated perfused rat liver (5). The mechanism for this effect remains unclear. There was no stimulation of BSP uptake by unidirectional Cl− gradients (6), although affinity of BSP for the surface of hepatocytes was approximately 10-fold greater in the presence of Cl− than in its absence. It is interesting that Cl−-dependent BSP uptake is seen in differentiated hepatocytes and is lost with time in culture and in hepatoma cell lines (12). Because this Cl−-dependent extraction of BSP from albumin appeared to be characteristic of the high-affinity hepatocyte organic anion uptake mechanism (13), it was subsequently used to develop an assay to clone the transporter using a functional expression strategy in Xenopus laevis oocytes (14) (see later). The influence of unidirectional pH gradients on BSP transport by cultured rat hepatocytes also was examined (6). These studies showed that BSP transport was stimulated by an inside-to-outside OH− gradient, consistent with OH− exchange or H+ cotransport (6).

Physiologic Modulation of Transport As noted earlier, it is possible for lipophilic molecules to cross a lipid bilayer without requiring protein facilitation. However, findings in several physiologic conditions militate against this possibility. The first finding is the process of liver regeneration after two-thirds partial hepatectomy in the rat. Within 6 hours of the regenerative stimulus, influx of bilirubin or BSP is reduced by 50%, when calculated independently of liver mass, returning to normal levels by 4 days (15). Similar studies were performed with the hypolipidemic agent nafenopin, which when administered to rats for 2 days, results in liver hypertrophy and hyperplasia that morphologically resembles regeneration (1). Notably, influx of BSP and bilirubin glucuronides was reduced by 50% in these rats, whereas bilirubin influx was normal (1). This dissociation of uptake for these subclasses of organic anions suggests that they may have partially independent uptake mechanisms. Extracellular ATP also down-regulates hepatocyte uptake of BSP (16), and this has been attributed to signal transduction mediated by a purinergic receptor that recognizes ATP−4 (16,17). Identification of Transporters Biochemical Studies The studies described earlier are consistent with the existence of an organic anion transporter(s) that is present on the basolateral (sinusoidal) plasma membrane of hepatocytes. The nature of this transporter has been the subject of investigation for many years. Initial studies described high-affinity binding of BSP to preparations of liver cell plasma membrane, and a 55-kDa protein termed BSP/bilirubin binding protein was identified (18–21). Little has been done subsequently to characterize the potential physiologic role of this protein, and it must be kept in mind that functional studies were performed at high concentrations of ligand in the absence of albumin, suggesting that low-affinity transport was examined. In addition, this protein was shown to be present on the surface of HepG2 cells (22), a cell line that lacks the high-affinity transporter and is unable to take up BSP in the presence of albumin (12). Other studies isolated a 170-kDa protein termed bilitranslocase for its ability to transport BSP and bilirubin when incorporated into liposomes (23–26). However, this protein is not hepatocyte specific (27) and is also present in HepG2 cells (28). Bilitranslocase was cloned from a rat liver expression library using a monoclonal antibody (29). The derived protein sequence is unique and has been used in a number of functional studies (30). However, analysis of the nucleotide sequence (GenBank accession number Y121780) shows that it is 93% identical to the sequence of the inverse strand of ceruloplasmin, suggesting that a cloning artifact likely occurred. A third group of studies identified a 55-kDa organic anion binding protein (oabp) after photoaffinity labeling of rat liver plasma membrane preparations by 35 S-BSP (31). However, this protein was shown to be identical to the β-subunit of F1-ATPase, an inner mitochondrial

MECHANISMS OF HEPATOCYTE ORGANIC ANION TRANSPORT / 1465 membrane protein (32). Although the protein appeared to have a form that could be identified on the plasma membrane of hepatocytes (32), its potential function in organic anion transport remains unknown. Molecular Biology Studies As discussed earlier, Cl−-dependent extraction of BSP from albumin is characteristic of the high affinity hepatocyte organic anion uptake mechanism (13). This unique transport characteristic was used to clone the transporter using a functional expression strategy in Xenopus laevis oocytes (14). A single complementary RNA (cRNA) was isolated, which, when injected into oocytes, resulted in a substantial signal of BSP uptake activity that was suppressed by Cl− substitution (33). The corresponding cDNA encodes a rat liver protein that has been named organic anion transporting polypeptide 1 (oatp1 or oatp1a1) (13,33). Computer analysis of oatp1 (oatp1a1) cDNA shows an open reading frame of 2010 nucleotides, encoding a protein with a high degree of hydrophobicity. The computer-generated model predicts 12 transmembrane domains and 4 potential N-glycosylation sites (13). However, testing and validation of this model currently have not been performed. Oatp1 (oatp1a1) expression appears to be limited to hepatocytes and epithelial cells of the choroid plexus (34,35) and the S3 segment of the proximal tubule (36). Studies using antisense knockout of oatp1 (oatp1a1) expression in Xenopus oocytes that had been injected with liver messenger RNA (mRNA) have suggested that oatp1 (oatp1a1) is responsible for a substantial fraction of organic anion transport by the liver (37), although this remains to be validated by other methods (see later discussion). Since the initial description of oatp1 (oatp1a1), more than 20 additional members of the oatp family have been described (38,39). These proteins have a relatively high degree of amino acid similarity (38), as well as overlap of transported substrates, although their tissue distributions are varied.

In particular, several members of the oatp family are expressed in hepatocytes from rats, mice, and humans (Table 57-1). These hepatocyte oatps are distributed on the basolateral plasma membrane and have overlapping substrate specificities (38). They are of similar size and have similar predicted membrane topologies and biochemical characteristics. Interestingly, multiple sequence alignment (40) shows that these oatps have 15 cysteine residues in common (Fig. 57-1) that are highly conserved over the entire oatp family. It can be hypothesized that these cysteines provide an important component of transporter function through formation of intramolecular and possibly intermolecular disulfide bonds. This is an important area that undoubtedly will be the subject of future studies. Nomenclature of the oatp family members has been confusing. In this review, proteins are indicated by a common name, followed by the suggested new name (39), which is also indicated in Table 57-1.

Characteristics of Hepatocyte Organic Anion Transporting Polypeptides Many studies have been performed using rat oatp1 (oatp1a1) as prototypical of the hepatocyte oatp family. However, where possible, studies that have been performed with other members of the oatp family are discussed in this chapter. Biochemistry Oatp1 (oatp1a1) in the liver is localized to the basolateral (sinusoidal) plasma membrane of the hepatocyte (13,36). As predicted by computer modeling, it is a hydrophobic protein and remains membrane bound even after extraction with 0.1 M Na2CO3 (36). On immunoblotting, oatp1 (oatp1a1) migrates with a molecular mass of 80 kDa. After treatment with N-glycanase, the apparent molecular mass decreases

TABLE 57-1. Members of the Oatp family that are found in liver of rat, mouse, and humans Species

Original name

Rat Rat Rat Rat Mouse Mouse Mouse Human Human

Oatp1 Oatp2 Oatp4, Lst-1 Oatp9 Oatp1 Oatp2 Oatp4, Lst-1 OATP-A OATP-C, LST-1, OATP2 OATP8 OATP-B

Human Human

Suggested new name

Old gene symbol

Suggested new gene symbol

NCBI accession number

Oatp1a1 Oatp1a4 Oatp1b2 Oatp2b1 Oatp1A1 Oatp1a4 Oatp1b2 OATP1A2 OATP1B1

Slc21a1 Slc21a5 Slc21a10 Slc21a9 Slc21a1 Slc21a5 Slc21a10 SLC21A3 SLC21A6

Slco1a1 Slco1a4 Slco1b2 Slco2b1 Slco1a1 Slco1a4 Slco1b2 SLCO1A2 SLCO1B1

NM_017111 NM_131906 NM_031650 NM_080786 NM_013797 NM_030687 NM_020495 NM_021094 NM_006446

OATP1B3 OATP2B1

SLC21A8 SLC21A9

SLCO1B3 SLCO2B1

NM_019844 NM_007256

Common names and suggested new names as proposed in Hagenbuch and Meier (39) are indicated. NCBI, National Center for Biotechnology Information.

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FIG. 57-1. Multiple sequence alignment of the organic anion transporting polypeptides (oatps) that are present in liver from rat, mouse, and humans was performed (40), and it showed that there are 15 cysteine residues in common that are highly conserved over the entire oatp family, as indicated by the asterisks. Many other regions of high homology also are shown. A functional role for these regions has not yet been elucidated.

MECHANISMS OF HEPATOCYTE ORGANIC ANION TRANSPORT / 1467 to 65 kDa, consistent with N-linked glycosylation (36,41). It has been suggested that glycosylation of oatp1 (oatp1a1) is a determinant of its trafficking to the plasma membrane, as well as possibly its transport activity (41). Function The function of oatp1 (oatp1a1) has been examined in several systems including cDNA-injected Xenopus oocytes (33,42–45), in transiently transfected HeLa cells in which oatp1 (oatp1a1) was functionally expressed using a recombinant vaccinia virus protocol (46–48), and in permanently transfected HeLa cells in which oatp1 (oatp1a1) expression was under the control of a metallothionein promoter (49,50). The best studied of these models is the oatp1 (oatp1a1)-inducible HeLa cell system in which we have found that the Km for 35 S-BSP transport is approximately 3 µM. Nontransfected cells and transfected cells in which oatp1 (oatp1a1) expression was not induced had little uptake of BSP (49). HeLa cells expressing oatp1 (oatp1a1) also exhibited Na+independent uptake of taurocholate. The Km for this process was approximately 20 µM, similar to that for taurocholate transport mediated by the Na+-dependent transporter Na+taurocholate cotransporting polypeptide (ntcp) (51). As described earlier in this chapter, earlier studies in cultured rat hepatocytes showed that BSP uptake was stimulated by an outwardly directed pH gradient, suggesting the possibility of HCO3− exchange (6). To determine whether oatp1 (oatp1a1) was a HCO3− exchanger, investigators assayed fluorescence of BCECF-loaded oatp1 (oatp1a1) stable transfectants to quantify intracellular pH as a function of transported taurocholate (50). Taurocholate rather than BSP was used in these studies because of the intense purple color of BSP at alkaline pH. These studies clearly demonstrated that oatp1 (oatp1a1) mediates organic anion-HCO3− exchange (50). Subsequent studies by Ballatori and colleagues (52,53) showed that oatp1 (oatp1a1) and oatp2 (oatp1a4) could also serve as organic anion-glutathione exchangers. Regulation of Organic Anion Transporting Polypeptide Function Extracellular Adenosine Triphosphate Extracellular ATP rapidly transduces serine phosphorylation of oatp1 (oatp1a1) via a purinergic receptor (16,17). This is accompanied within minutes by a substantial reduction in Vmax for BSP transport, whereas oatp1 (oatp1a1) localization on the plasma membrane is unaffected (17), suggesting that its transport activity is down-regulated rapidly by this serine phosphorylation event (16,17). This effect on transport is reversed rapidly by removal of extracellular ATP (16). Whether extracellular ATP is an important mediator of oatp function in vivo is unknown, but these studies suggest that the phosphorylation state of the transporter must be an important consideration when assessing alterations of its functional expression in various pathobiologic states.

Ontogenic Regulation There is also strong ontogenic regulation of oatp1 (oatp1a1) with little expression of oatp1 (oatp1a1) protein or mRNA for the first 3 weeks of life (35). Similar developmental patterns have been described for the other liver-expressed oatps in the rat with the exception of oatp4 (oatp1b2) (54,55). Expression of this protein was significant even at embryonic day 16. However, through embryonic development and early postpartum, it had an intracellular distribution and did not have the typical adult basolateral plasma membrane distribution until 29 days after birth (54). Sex Hormones and Pregnancy Hormonal regulation of expression of the oatps also has been examined. Although ethinyl estradiol administration reduces hepatic expression of rat oatp1 (oatp1a1) (56), oatp2 (oatp1a4), and oatp4 (oatp1b2) (57), this may be a consequence of the ensuing cholestasis (58–60). In adult rats, hepatic expression of oatp1 (oatp1a1) does not appear to be regulated by sex steroid hormones (61), in contrast with renal tubular expression of this transporter (46). In liver extracts from rats 20 to 21 days pregnant, expression of oatp1 (oatp1a1) also was unchanged from control, whereas expression of oatp2 (oatp1a4) was reduced by approximately 50% (62). However, this difference in oatp2 (oatp1a4) expression was not apparent when examined in basolateral plasma membrane preparations of livers from rats 19 days pregnant (63). There was no change in mRNA for oatp1 (oatp1a1) or oatp2 (oatp1a4) in livers from pregnant rats (63). In regenerating liver, after two-thirds partial hepatectomy, protein expression of oatp1 (oatp1a1) is reduced by approximately 60%, returning to normal within 1 week. Reduction in oatp2 (oatp1a4) expression is slower, reaching a nadir of 50% of normal levels by 4 days, and returning to normal by 2 weeks (64). Transcription Factors and Nuclear Hormone Receptors A role for specific transcription factors and nuclear hormone receptors in regulating expression of some of the liver oatps has been presented (65–69). In particular, murine oatp1 (oatp1a1) expression is down-regulated in hepatocyte nuclear factor 1α (HNF-1α) and HNF-4α knockout mice (67,68). Murine oatp2 (oatp1a4) expression is down-regulated in pregnane X receptor (PXR) knockout mice (70), and rat oatp2 (oatp1a4) expression also was shown to be PXR responsive in a reporter gene system (71). The genes encoding human OATP-C (OATP1B1), human OATP8 (OATP1B3), and murine oatp4 (oatp1b2) were shown to have promoters that were responsive to HNF-1α coexpression (72). In farnesoid X receptor (FXR) knockout mice, oatp1 (oatp1a1) expression was increased with cholate feeding (65). Interestingly, microsomal enzyme inducers such as phenobarbital increase expression of oatp2 (oatp1a4), but not oatp1 (oatp1a1) or oatp4 (oatp1b2) (73,74). These regulatory pathways are relatively slow as they modulate biosynthesis of

1468 / CHAPTER 57 these transporters, rather than modulating their activity directly such as would occur with most posttranslational regulatory pathways. Perhaps of even greater physiologic importance than regulation of oatps by nuclear hormone receptors is that they transport ligands that can activate these receptors (75). Regulation of oatp activity may thus have far-reaching consequences on expression and activity of a host of other cellular proteins and processes. Transported Substrates Multispecificity of Uptake The oatps mediate transport of a diverse group of compounds that have been catalogued in several reviews (38,39,76). The diversity of transported substrates is typified by oatp1 (oatp1a1), which can mediate transport of organic anions such as BSP and taurocholate (49,50,76), peptide drugs including the thrombin inhibitor CRC 220 (44) and the angiotensin-converting enzyme inhibitor enalapril (77), various steroid hormones including estradiol 17β-glucuronide (43,47), and even several cationic compounds (78). There is no clear common structural similarity of compounds that are substrates for any of the oatps. Bilirubin There has been a great deal of interest in the mechanism by which bilirubin is taken up by hepatocytes. Under normal physiologic conditions, the hepatocyte extracts this compound from the circulation rapidly, and this process has carriermediated kinetics (5,79–81). Although it has been presumed that bilirubin would be a substrate for one or more of the oatps, this remains controversial and unproven. In studies of human oatp expression after microinjection of RNA into Xenopus oocytes, it was suggested that OATP8 (OATP1B3) and OATP2 (OATP1B1) could mediate bilirubin uptake (82). Another study suggested that OATP2 (OATP1B1), but not OATP8 (OATP1B3), mediated bilirubin uptake in HEK293 cells that had been stably transfected with these transporters (83). These results in OATP2 (OATP1B1)-transfected HEK293 cells could not be reproduced in another study (11). In addition, in this latter study, an assay for temperature-dependent bilirubin uptake was established in short-term cultured rat hepatocytes (11). Using this assay, there was no bilirubin uptake in HeLa cells stably transfected with OATP2 (OATP1B1), although uptake of another organic anion (BSP) was robust (11). Although that bilirubin can pass rapidly through lipid bilayers has led some investigators to question the necessity of postulating the existence of a carrier (84,85), the kinetic characteristics of this process that is seen in various physiologic systems suggest that carrier mediation is likely. Identification of the uptake mechanism requires further study. Consideration must also be given to that although there have been many studies of oatp-mediated transport that have been conducted in cell culture and Xenopus oocyte expression systems, whether these data can be extrapolated to the in vivo situation is unclear.

Physiologic Function There is little information on the role of any of the oatps in vivo. However, several groups have shown that a number of naturally occurring polymorphisms of OATP2 (OATP1B1) in humans are associated with reduced clearance of pravastatin (86–88). Although the suggestion has been made that these polymorphisms are also associated with increased levels of serum bilirubin (89), these data are based on small numbers of patients showing small differences in bilirubin levels and are not compelling. Interestingly, there are no reports in which any of the oatps have been knocked out, although strategies for preparing specific antisense oligonucleotides have been reported but not used in a complex physiologic system (90).

MECHANISMS OF BILE ACID UPTAKE Physiology Bile acids circulate in the plasma bound to albumin (91), although they also bind to serum lipoproteins such as lowdensity lipoprotein (92). As for the nonbile acid organic anions described earlier, despite low free concentrations in the circulation, bile acids are extracted rapidly by the hepatocyte from their circulating carriers, and studies in perfused liver have shown single-pass extraction fractions as high as 80% (93). Schwarz and colleagues (94) were the first to recognize that taurocholate uptake by isolated rat hepatocytes was reduced by approximately 75% on substitution of extracellular Na+ by sucrose or K+. This has been confirmed many times by other investigators working with isolated or cultured hepatocytes (95), as well as with isolated perfused rat liver (93,96) and rat liver plasma membrane vesicles (97–101), using a variety of conjugated bile acids (95). The uptake process appears to have a Na+/bile acid stoichiometry of 2:1, indicating that it is electrogenic (102,103). Identification of Transporters Biochemical Studies Similar to the case for nonbile acid organic anions, the kinetics and characteristics of bile acid transport by hepatocytes are indicative of a carrier-mediated uptake mechanism. In an attempt to identify a hepatocyte bile acid transporter, initial studies used photoaffinity technology to covalently attach radiolabeled bile acid analogs to putative hepatocyte basolateral plasma membrane transporters. Two candidate transporters of 54 and 48 kDa were identified using this approach (104,105). Additional studies performed in rat liver basolateral plasma membranes using radiation inactivation analysis showed a minimal functional mass of 170 kDa for the Na+-dependent taurocholate transporter (106,107). However, none of these studies actually identified a Na+-dependent bile acid transporter that could be studied using biochemical techniques.

MECHANISMS OF HEPATOCYTE ORGANIC ANION TRANSPORT / 1469 Molecular Biology Studies It was not until the development of expression cloning techniques that an Na+-dependent bile acid transporter, termed ntcp, was identified (108,109). The ntcp cDNA that was cloned in Xenopus oocytes mediates Na+-dependent uptake of a number of bile acids (76,110–114). It encodes a protein with a predicted molecular mass of 39 kDa that migrates at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a result of glycosylation (76,115). On deglycosylation, the protein migrates at approximately 33 kDa (76,115). In rats, expression of ntcp is limited to hepatocytes, where it resides on the sinusoidal (basolateral) plasma membrane (116), as well as in an intracellular vesicular pool that cycles to and from the plasma membrane depending on its phosphorylation state (see later). Physiologic Role of Na+-Taurocholate Cotransporting Polypeptide and the Possibility of Other Na+-Dependent Bile Acid Transporters A physiologic role for ntcp in bile acid transport has been asserted (117), but remains unproved. The evidence in support of this suggestion is incomplete and is limited to finding similar Kms for bile acid transport in ntcp-transfected cells as compared with rat hepatocytes and parallel decreases of ntcp expression and Na+-dependent taurocholate transport in liver regeneration and in hepatocytes in culture (117). Obviously, function of other bile acid transporters could be altered under these conditions as well, and a causal relation between these perturbations and ntcp expression has not been established. The most cogent evidence that ntcp plays a major role in bile acid transport was the finding of a 95% reduction in taurocholate uptake in Xenopus laevis oocytes that had been microinjected with rat liver cRNA in the presence of nucleotides that were antisense to ntcp (37,117). Although clear-cut when these experiments were performed, it is now evident with the establishment of more complete databases that the antisense oligonucleotide that was used to inhibit ntcp translation (TAACCCATCAGAAAGCCAGA) was not specific for ntcp and could potentially inhibit synthesis of other rat liver, as well as Xenopus proteins (118). Preparation of an ntcp knockout mouse may be more conclusive regarding elucidation of the physiologic significance of this transporter, but this has not yet been reported. Although spontaneously occurring disorders in which other members of the Na+-dependent solute transport family have been mutated have been described (119–127), there have been no inheritable disorders attributable to ntcp dysfunction, although several naturally occurring polymorphisms that affect ntcp expression in vitro have been described (55). Studies in mice in which the ferrochelatase gene has been knocked out to provide a model of erythropoietic protoporphyria showed highly increased plasma bile acid levels. Ntcp and oatp1 (oatp1a1) are absent from liver plasma membrane of these mice (128). However, more than 90% of a tracer dose of 3 H-taurocholate was found in liver and bile of these mice

within 30 minutes of injection (128), indicating that bile acids could still be cleared from serum even in the absence of ntcp. Dissociation of ntcp expression and bile acid transport function also was observed in rats 7 days after bile duct ligation in which hepatic ntcp was virtually undetectable, whereas initial Na+-dependent uptake of 3H-taurocholate by hepatocytes isolated from these rats remained at 30% of control levels (129). These studies suggest the potential existence of other hepatocyte basolateral plasma membrane Na+-dependent bile acid transporters. One proposed candidate is microsomal epoxide hydrolase (mEH) (130). This 49-kDa protein was shown to reconstitute Na+-dependent taurocholate uptake in proteoliposomes (131). Although mEH expression was thought to be limited to the endoplasmic reticulum (ER), evidence has been presented that it is inserted into the ER membrane in two topologic orientations, one of which can be targeted to the plasma membrane (132). Although one study in which mEH expression and function was examined in a Syrian hamster kidney fibroblast cell line and in Xenopus laevis oocytes did not show expression of Na+-dependent taurocholate transport (133), another study performed in Madin– Darby canine kidney cells did (130). An mEH knockout mouse was reported some time ago (134). However, there have been no reports regarding serum bile acid levels, ntcp expression, or hepatocyte bile acid transport kinetics in these mice. Consequently, the potential role of mEH in bile acid transport remains to be elucidated, and the potential for additional hepatocyte bile acid transporters needs to be considered. The rationale for such experiments is exemplified by studies showing that treatment of mice with the peroxisome proliferator-activated receptor α agonist ciprofibrate substantially reduced hepatic levels of oatp1 (oatp1a1) and ntcp, but had little effect on serum bile acid levels (135). Interestingly, treatment with this drug is known to increase hepatic mEH expression (136). Substrates of the Na+-Dependent Bile Acid Transport System Specificity of the Na+-dependent bile acid uptake system was examined in isolated rat hepatocytes by studying the ability of 100 µM concentrations of test ligands to inhibit uptake of 5 µM 14C-taurocholate (137). These studies showed no influence of hydroxyl group orientation (α or β) on ability to inhibit taurocholate uptake. In contrast, the smaller the number of hydroxyl groups on the steroid ring, the more potent the inhibition, whereas substitution of hydroxyl groups by keto groups resulted in reduced inhibition of taurocholate uptake. Side chain length was also a factor in inhibitory potency, with increased inhibition of taurocholate uptake with side chains up to 11 Å and reduced inhibition with longer side chains. Because inhibition of taurocholate uptake by these compounds was competitive, the assumption is that they represent substrates for the hepatocyte Na+-dependent bile acid uptake system, although this may not necessarily be the case. Further studies examined inhibition of uptake of

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C-taurocholate into isolated rat hepatocytes by a series of trihydroxy bile acids with differing side chain length and charge (138). These studies found that long side chains (>8 Å) with no or negative charge were potent inhibitors of taurocholate uptake. Positive charge on the side chain or shortening of the side chain reduced inhibitory potency. More limited studies of the substrate specificity of ntcp have been performed. In general, for rat ntcp, there was modest relation of ntcp-mediated transport to substrate structure (76). Conjugated bile acids were transported better than unconjugated bile acids, and there was little influence on uptake of the configuration of the hydroxyl groups (α or β) on the steroid ring (76). Interestingly, rat ntcp mediated high-affinity uptake of BSP, a nonbile acid organic anion (76). Substrate specificities also were examined for rabbit ntcp and the rabbit Na+-dependent ileal bile acid transporter (114). A substrate activity model was devised for the ileal transporter (113), but a similar model does not yet exist for rabbit ntcp. Notably, compared with the rat and human ntcp counterparts, rabbit ntcp is only 75% and 82% identical, respectively. This might explain differences in substrate specificities that were reported for rat and rabbit ntcps (76,114). Regulation of Hepatocyte Na+-Dependent Bile Acid Uptake Phylogeny Na+-dependent bile acid uptake by the liver is not seen in phylogenetically old organisms such as the skate (139), although they are able to take up bile acids by Na+independent means (139). Na+-independent bile acid transport was expressed in skate liver mRNA-injected Xenopus laevis oocytes (140), and subsequently a skate homolog belonging to the oatp family was cloned (141). Other studies identified another novel protein (organic solute transporter [OST]) that could transport a number of organic solutes including taurocholate (142). Although initial studies suggested that OST was expressed in mammalian and skate liver, subsequent studies indicate that, in mammals, there is little liver expression, but abundant expression in the intestine (143).

approximately 49-kDa size seen in adults (115). This difference in apparent size was caused by differences in glycosylation (115). This dissociation of total liver ntcp expression from Na+-dependent bile acid transport is unexplained, but could be due in part to the presence of other Na+-dependent bile acid transporters with different developmental patterns. Although differences in subcellular targeting could explain these differences, plasma membrane localization of the transporter had an adult appearance by 5 days after birth (54). Liver Regeneration As is the case for nonbile acid organic anions described earlier, Na+-dependent uptake of taurocholic acid is markedly reduced 24 hours after two-thirds partial hepatectomy in the rat (146). Serum bile acid levels are substantially increased for at least 1 week compared with sham-operated control animals (64). Expression of ntcp protein is reduced by more than 90% at 24 hours of partial hepatectomy (146), and normalizes by approximately 1 week (64). Cyclic Adenosine Monophosphate Studies in rat hepatocytes showed that maximal Na+dependent taurocholic acid transport rate was increased within minutes by approximately 50% after pretreatment with cyclic adenosine monophosphate (cAMP) (147,148). This increase in taurocholate transport was associated with translocation of ntcp from an intracellular pool to the plasma membrane (149). This phenomenon was explained by the finding that ntcp exists within the hepatocyte in a phosphorylated form, and that it undergoes dephosphorylation in response to cAMP, subsequently translocating to the plasma membrane (150–152). This cAMP-dependent dephosphorylation event appears to be mediated by protein phosphatase 2B (PP2B) (153). The mechanisms that mediate ntcp trafficking through the cell are complex (148), and in addition to the factors noted earlier, use a phosphatidylinositol-3-kinase dependent pathway that may interact with actin filaments, as well as microtubules (152,154–156). Sex Hormones and Pregnancy

Ontogeny Bile acid transport also has been studied during development. The Vmax for initial Na+-dependent uptake of taurocholate by basolateral liver plasma membrane vesicles prepared from day 22 fetal, day 1, and day 14 neonates was 23%, 36%, and 47% of that seen in basolateral liver plasma membrane vesicles prepared from adults (144,145). Interestingly, ntcp mRNA levels in rat liver are less than 20% of adult levels before birth, increasing to 35% of adult levels by the first day after birth and achieving adult levels by 1 week of age (115). Ntcp protein was approximately 8% of adult levels just before birth, increasing to 82% of adult levels at 1 day after birth. However, the size of the protein was approximately 39 kDa until 4 weeks of age when it migrated at the

Na+-dependent uptake of taurocholate is greater in rat hepatocytes obtained from male versus female rats (61,157). This correlates with the finding that ntcp is reduced in basolateral plasma membrane fractions prepared from female compared with male rats (61,158). Notably, lipid fluidity was also decreased in plasma membrane fractions prepared from female compared with male rats (61). Interestingly, oatp1 (oatp1a1) levels were unchanged (61,63,158), which is distinct from findings in rat kidney (46). Further studies showed that this sexually dimorphic expression of ntcp was regulated by a number of hormones, including estrogens (159). Hormonal regulation of bile acid uptake was seen by reduction in uptake of taurocholate in hepatocytes isolated from pregnant compared with nonpregnant rats (158,160,161). Two days

MECHANISMS OF HEPATOCYTE ORGANIC ANION TRANSPORT / 1471 after birth, Na+-dependent uptake of taurocholate is increased to levels greater than those seen in nonpregnant female rats in both hepatocytes (161) and basolateral plasma membrane vesicles (162,163) prepared from female rats. In large part, this overshoot in Na+-dependent taurocholate uptake is regulated by prolactin (161–163). Whether ntcp expression correlates with altered transport of taurocholate in pregnancy remains unclear, because in one study using basolateral plasma membrane vesicles from pregnant rats, there was no change in ntcp content (63), whereas in another study, ntcp content in plasma membrane fractions prepared from pregnant rat liver was reduced by 60% (62). Whether these differences were caused by differences in membrane preparations and subcellular distribution of ntcp remains to be elucidated. Cholestasis Although a number of reviews suggest down-regulation of hepatocyte bile acid uptake in cholestasis (66,69), this appears to be another model system in which expression of ntcp does not correlate well with the physiologic situation. It is clear from a number of studies that after common bile duct ligation in the rat, ntcp protein and mRNA expression are profoundly reduced (60,129,164,165). However, a number of studies suggest that bile acid uptake after common bile duct ligation was relatively well maintained. In one study, rats were subjected to 5 days of common bile duct obstruction after which obstruction was removed (166). Serum bile acids, which had been highly increased, returned to normal within 60 to 90 minutes of relief of obstruction. This was accompanied by prompt excretion of bile acids into bile. Plasma clearance of a tracer dose of taurocholate injected at the time of relief of biliary obstruction was near normal. This normalization of transport function is faster than return of ntcp levels to normal would be expected to occur. In another study (129), hepatocytes were isolated from rats in which the common bile duct had been ligated for 7 days. Ntcp protein levels were reduced by 90%, whereas the Vmax for Na+-dependent taurocholate uptake was reduced by only 70%. When basolateral plasma membrane vesicles were prepared from livers of rats in which the common bile duct was ligated for 50 hours, Na+-dependent taurocholate uptake remained normal (167). Transport studies also were performed in isolated perfused livers from rats 24 and 72 hours after common bile duct ligation (168). Quantitation of bile acid uptake after addition of 3H-taurodeoxycholate to the perfusate (16 nmol/min/g) showed little, if any, difference in uptake between control and bile duct ligated livers. Perfusion with a larger dose of this bile acid (4000 nmol/min/g) resulted in an approximately 35% reduction in maximal uptake by livers that had undergone common bile duct obstruction for either 24 or 72 hours. In the aggregate, these studies show at most a modest reduction of hepatocyte bile acid uptake after mechanical cholestasis in the face of a profound reduction in ntcp protein and mRNA. These data imply the existence of another Na+dependent bile acid transporter in addition to ntcp, as has been suggested (129).

ORGANIC ANION EXCRETION ACROSS THE BILE CANALICULUS For the most part, organic anion uptake across the sinusoidal plasma membrane of hepatocytes represents a facilitative process in which extracellular and intracellular ligand is highly protein bound, resulting in low concentrations of free ligand. In contrast, organic anion excretion across the bile canalicular plasma membrane of the hepatocyte is an uphill process in which transport is against a large concentration gradient. Consequently, the transport mechanisms of the bile canaliculus that have been elucidated require ATP (117,118). Unlike the case for other polarized cells, the apical (bile canalicular) domain of the hepatocyte is inaccessible and difficult to study. As described later, elucidation of excretory mechanisms for bile acids and nonbile acid organic anions was greatly facilitated by study of patients with inheritable disorders of bile acid and bilirubin diglucuronide excretion using the tools of molecular genetics (118,169). Identification of Transporters Studies in Purified Bile Canalicular Plasma Membrane Vesicles This field was stimulated greatly by the ability to quantify transport of radiolabeled ligands into purified preparations of inside-out rat liver bile canalicular plasma membrane vesicles (170,171). Several studies demonstrated ATP-dependent uptake of taurocholate (172–175). Affinity labeling of these membranes with a photolabile bile salt derivative identified a 110-kDa glycoprotein (173). ATP-dependent taurocholate transport was reconstituted in proteoliposomes containing this protein (173,176), and it was presented as the ATP-dependent bile acid transporter (173,176,177). Purification and sequencing of this 110-kDa protein identified it as a known rat bile canalicular ecto-ATPase (178,179). Transfection of COS cells with the corresponding cDNA was reported as conferring ATP-dependent efflux of taurocholate (180). However, subsequent studies found that ATP-dependent bile acid transport was present in a subpopulation of canalicular membrane vesicles that had little ecto-ATPase activity (181). Similar findings were reported in liver cell lines that retained ATPdependent bile acid excretion, but had little ecto-ATPase expression (182). Molecular Biological Identification of the Bile Canalicular Bile Acid Excretory Pump More recently, it was shown that the 160-kDa bile canalicular protein originally named sister of P-glycoprotein (spgp) conferred ATP-dependent taurocholate transport in cRNAinjected Xenopus laevis oocytes, as well as in membrane vesicles purified from Sf9 cells that had been transfected with a plasmid-encoding spgp (183). This protein has now been renamed bile salt export pump (bsep) and the gene has been designated as Abcb11. That bsep is physiologically important

1472 / CHAPTER 57 has been demonstrated by finding that it is mutated in patients with the inheritable disorder progressive familial intrahepatic cholestasis type 2 (PFIC2). This disorder is associated with high levels of bile acids in serum and low levels in bile (184). Targeted inactivation of this gene in mice resulted in a much less severe phenotype (185). Although secretion of cholic acid was greatly reduced (6% of wild type) in mutant mice, total bile salt excretion was somewhat conserved (30% of wild type). This secretion was accounted for by increased excretion of tetrahydroxylated bile acids, which were not detected, in wild-type mice. These results suggest that, at least in the mouse, there may be alternative bile canalicular transporters that can mediate excretion of hydrophilic bile acids. From these most recent findings, a physiologic role for ecto-ATPase as an ATP-dependent transporter of taurocholate appears unlikely. The substrate specificity of bsep has not been examined in great detail, although it has been shown to mediate transport of the bile acids taurocholate, glycocholate, taurochenodeoxycholate, glycochenodeoxycholate, and tauroursodeoxycholate (186). In addition, a number of nonbile acid organic anionic compounds have been shown to be competitive inhibitors of bsep-mediated taurocholate transport (186,187). These compounds include rifampicin, cyclosporin, and glibenclamide. Whether these compounds are themselves substrates for bsep has not been established directly, although it has been suggested that some instances of drug-induced cholestasis may result from direct inhibition of bsep transport activity (186,187). Identification of Other Proteins that Influence Bile Canalicular Excretion of Bile Acids Study of patients with two additional phenotypically similar forms of PFIC has yielded exciting insights into additional mechanisms of bile canalicular transport function (169). PFIC1 has been found to be caused by mutation of the gene encoding a P-type ATPase, Fic1, that functions as an aminophospholipid translocator (188). It is present in greatest concentration in the intestine, but is also present in the liver (169), where it has been localized to the bile canalicular membrane (189). The relation of this transport protein to abnormal bile acid excretion remains to be elucidated (190). PFIC3 has been found to be caused by mutation of the gene encoding MDR3 (191), a bile canalicular membrane flippase that is thought to move phospholipids from the inner leaflet of the canalicular membrane to the outer leaflet, where it can form micelles with cholesterol and bile acids (192). Affected patients have low levels of phospholipids in bile (193). Interestingly, knockout mice in which expression of mdr2, the mouse homologue of human MDR3, is disrupted have a similar type of liver disease that coincides with inability of the liver to secrete phospholipids into the bile (194). Canalicular bile acid excretion is normal in mdr2/MDR3 deficiency, and it is believed that the fact that bile acid monomers are not associated with phospholipid in micelles may result in direct toxicity to cholangiocytes and hepatocytes (169).

Identification of Bile Canalicular Proteins that Mediate Excretion of Nonbile Acid Organic Anions Nonbile acid organic anions are also excreted across the bile canaliculus. The mechanism by which bilirubin glucuronides are excreted has been of long-standing interest, because when this process is disrupted in disease, patients experience jaundice and excrete these bilirubin conjugates into urine (195,196). Existence of an ATP-dependent canalicular export pump for bilirubin glucuronides was first seen in studies of bilirubin glucuronide transport by rat liver canalicular membrane vehicles (197). Notable is that this transport was deficient in vesicles prepared from TR(−) rat livers (197–199). These rats represent an animal model of the Dubin–Johnson syndrome (200), an inheritable disorder characterized by defective excretion of bilirubin glucuronides, but normal excretion of bile acids (200,201). Subsequent studies showed that an isoform of the multidrug resistance protein (MRP) was absent from the canalicular membrane of the TR(−) rats (202) and patients with the Dubin–Johnson syndrome (203,204). This bile canalicular MRP has been termed MRP2 (205). This 190-kDa protein is expressed predominantly in hepatocytes, and its amino acid sequence is 49% identical to that of MRP1, a protein with broad distribution (205). Notably, although mdr2 expression is absent in TR(−) rats, in vivo excretion of endogenous bilirubin conjugates is reduced by only 40% (200). Infusion of a bilirubin load in vivo or in isolated perfused liver showed a 98% reduction in conjugated bilirubin excretion into bile (206). These results suggest existence of other, as yet unidentified, canalicular transporters that have a low capacity for transport of bilirubin conjugates. Unexpectedly, mice in which expression of the actin scaffolding protein, radixin, had been knocked out were found to have markedly reduced expression of mrp2 at the canalicular plasma membrane, although total cell content of the protein was reduced by only 40% (207). Presumably, radixin serves as a chaperone that directly or indirectly facilitates the trafficking of mrp2 to the apical plasma membrane of hepatocytes (207). This trafficking requires the C terminus 15 amino acids of mrp2 (207,208) and is unaffected by removing the C terminus 3 amino acids that would be essential for interaction with a PDZ domain–containing protein (208,209). Mrp2 may also represent an alternative transporter for some bile acids, because it can mediate excretion of 3-glucuronide and 3-sulfate dianionic bile acids and ester sulfate conjugates of lithocholic acid (210). Taurocholate is not a substrate for mrp2, but it is transported by mrp3, a protein that is related to mrp2, but is localized to the hepatocyte sinusoidal (basolateral) plasma membrane (211). Expression of mrp3 on the basolateral membrane is highly increased in TR(−) rats (212, 213), patients with the Dubin–Johnson syndrome (214), and in cholestasis (215–218). This protein, in contrast with mrp2, can also mediate transport of taurocholate (211), and it has been suggested that it may serve as a compensatory mechanism for solute elimination from the hepatocyte when canalicular secretion is blocked (213). Other members of the mrp family also have been shown to increase in the liver in

MECHANISMS OF HEPATOCYTE ORGANIC ANION TRANSPORT / 1473 cholestasis (218,219). Their physiologic roles in health and disease remain to be elucidated. Regulation of Hepatocyte Bile Canalicular Excretory Function Phylogeny and Ontogeny Interestingly, hepatic excretion of organic anions has been highly conserved during vertebrate evolution, and homologues of mrp2 (220,221) and bsep (222,223) have been described in the liver of the skate Raja erinacei, a 200-million-year-old vertebrate. During liver regeneration in the rat after two-thirds partial hepatectomy, there is no change in protein expression of these transporters (64,224), although ontogenic studies in the rat demonstrated that adult levels were not attained until at least 4 weeks of age (225). mRNA encoding these proteins were near adult levels early in life and correlated poorly with actual protein expression (54,225). However, another study reported levels of both of these proteins near adult levels by 1 week of age, which is more in line with mRNA levels (226). The reasons for these differences are unclear. Corresponding roughly to these studies is the finding that biliary excretory function is reduced in the days after birth (227–229), although there is little information regarding the time course of maturation of this process.

endotoxin showed marked reduction of bsep and mrp2 protein levels in membrane fractions within 15 hours (237). Immunofluorescence examination showed a “fuzzy” pattern along the canalicular membrane consistent with the possibility of redistribution of these transporters to a subapical vesicular compartment (237). After 3 days of mechanical obstruction of the common bile duct in rats, bsep protein in purified plasma membrane fractions was reduced by approximately 40%, whereas mrp2 was reduced by approximately 80% (164). Similar findings were obtained for cholestasis resulting from treatment with ethinylestradiol or endotoxin (164). Although subcellular distribution of bsep, as assessed by immunofluorescence, appeared to be normal in all three models of cholestasis (164), relocalization of transporters to a vesicular compartment adjacent to the bile canalicular membrane could not be ruled out by these studies. Such intracellular relocalization resulting in functional inactivation could account for the substantial reduction in bile salt output in rats in which bile duct integrity was restored by insertion of a catheter 14 days after common bile duct ligation (164). Clear demonstration of intracellular redistribution of mrp2 was described in rat models of intrahepatic and obstructive cholestasis (238). These studies emphasize the importance of assessing both protein content and subcellular distribution as a means to better understand cellular function. Transporter Trafficking

Pregnancy When expressed per gram of liver tissue, maximal biliary excretion of bile acids and other organic anions is reduced in liver from pregnant rats (230–232). Because the liver is larger in pregnancy (231,232), when expressed per whole liver, there is little difference in maximal excretion of these compounds. When mrp2 was quantified in liver homogenates prepared from pregnant rats, it was found to be decreased by approximately 50% when equal amounts of total liver homogenate protein were compared (62). Quantification of bsep in a mixed liver plasma membrane preparation showed no difference between pregnant and nonpregnant rats, whereas mrp2 was again found to be reduced by approximately 50% (63). After birth, bsep concentration in liver was found to increase by as much as 70%, whereas there was little change in mrp2 concentration (63). In isolated perfused livers obtained from postpartum rats, maximal secretory capacity for taurocholate increased by as much as 30% (63). These effects on bsep and mrp2 protein expression could be reproduced by treating ovariectomized rats with prolactin for 1 week (63). Cholestasis Cholestasis can be defined as a syndrome resulting from impaired excretion of bile acids and impaired bile formation (59,233). Cholestasis may result from altered expression of specific transporters, as detailed earlier, or from mechanical or metabolic causes (58,69,233–236). Studies performed in cholestasis resulting from treatment of rats with

Mechanisms for trafficking of bile canalicular membrane proteins between intracellular and surface locations have been the subject of several studies (239,240). Initial experiments indicated that infusion of dibutyryl cAMP or taurocholic acid into an isolated perfused rat liver resulted in increased bile secretion (241,242). Later studies reported that treatment with these agents results in recruitment of transporters from intracellular stores to the canalicular membrane, and that this process requires intact microtubules and is regulated by phosphatidylinositol 3-kinase (PI3K) (243,244). The effects of cAMP and taurocholate on transporter recruitment to the canalicular plasma membrane appear to be independent, suggesting the existence of two intracellular pools of canalicular transporters (240,245). Whether these compartments are related to the subapical compartment and apical endocytic vesicle compartments that have been described in the hepatic WIF-B cell line (246,247) remains to be determined. Studies in which yellow fluorescent protein (YFP) tagged bsep was expressed in the polarized WIF-B9 liver cell line described abundant intracellular expression of this protein in tubulovesicular structures that also contained Rab11, a marker of recycling endosomes (248). Exchange of YFP-bsep between the canalicular plasma membrane and these intracellular structures was inhibited by colchicine, a microtubule disruptor (248). However, unlike findings in intact liver, there was no effect of cAMP, taurocholate, or PI3K inhibitors on this trafficking. The mechanistic basis for vesicular trafficking of canalicular transporters is not well understood. As noted earlier, the scaffolding protein radixin is required for bile

1474 / CHAPTER 57 in repression of the gene encoding Cyp7A1, a rate-limiting bile acid biosynthetic enzyme, thereby reducing bile acid synthesis (258,259). Expression of the gene encoding Cyp8B1, another bile acid biosynthetic enzyme, is also repressed because of interaction of SHP with LRH-1 and HNF-4α (262,263). SHP has also been described as suppressing the promoter for rat ntcp (264). In these ways, FXR is the lynchpin in an exquisitely sensitive system that coordinates bile acid synthesis and excretion (69,254). This is emphasized by the finding of severe hepatotoxicity after cholic acid feeding to FXR knockout mice (65). Interestingly, although steadystate mRNA levels for bsep are reduced in these mice (265), steady-state levels of mRNA for mrp2 and ntcp are normal (65,265). Currently, there are no reports regarding transporter protein levels or trafficking in these mice. In addition to FXR, the nuclear receptor PXR (266,267) also regulates transcription of several transporter genes (69,254,268). Its ligands include bile acids and organic anions such as rifampicin (70,75,269,270). It has been described as increasing mRNA levels of rat, mouse, and human MRP2 (258, 271), mouse oatp2 (oatp1a4) (271), mouse bsep (271), mouse mrp3 (271), and rat oatp2(oatp1a4) (71). Differences in the amino acid sequence of PXR have been related to speciesspecific responses to particular ligands (272). Interestingly, although there is no evidence that human OATP2 (OATP1B1)

canalicular localization of mrp2 (207). Whether this is related to the finding that apical targeting of mrp2 requires the presence of its carboxy terminus 15 amino acids (208) is unknown. HAX-1, a 34-kDa protein that can link proteins to cortactin (249), has been found to associate with bsep and mdr2 (250) and appears to be required for their internalization, but not delivery to the apical plasma membrane (250). The mechanism by which cortactin, HAX-1, and canalicular transporters interact remains to be elucidated. Transcriptional Regulation Recognition in 1999 (251–253) that bile acids were endogenous ligands for FXR resulted in new insights regarding regulatory mechanisms for transporter expression (69,254–257). Both unconjugated and glycine- and taurineconjugated bile acids can serve as ligands for FXR (251,253). Mrp2 (258) and bsep (259) are included in the list of target genes with transcription that is up-regulated by bile acid– bound FXR. FXR-response elements have been shown to reside in the genes encoding these transporters (258,259). Transcription of the orphan nuclear receptor known as small heterodimer partner (SHP) is also increased by FXR activation (260,261), and interaction of SHP with the orphan nuclear receptor liver receptor homolog-1 (LRH-1) results

C 2

Na+ ATP

B OA−

OA−

ATP

D

OA−

OA−

A HCO3− GSH

OA−

ATP

E

FIG. 57-2. Schematic summary of hepatocyte transport mechanisms for organic anions. Bile acids and nonbile acid organic anions can be taken up by Na+-independent (A) and -dependent (B) transporters. The group of transporters represented by A includes the organic anion transporting polypeptides (oatps), which can take up bile acids and other organic anions in exchange for negatively charged HCO3− or glutathione (GSH). B represents the Na+-dependent transporters that include the Na+-taurocholate cotransporting polypeptide (ntcp), as well as the plasma membrane form of microsomal epoxide hydrolase (mEH). D and E represent the canalicular plasma membrane adenosine triphosphate (ATP)–dependent pumps that include the bile salt excretory pump (bsep) and the multidrug resistance protein 2 (mrp2). A number of nonbile acid organic anionic compounds have been shown to be competitive inhibitors of bsep-mediated taurocholate transport. Whether these compounds are themselves substrates for bsep has not yet been established directly, although it has been suggested that some instances of drug-induced cholestasis may result from direct inhibition of bsep activity. In cholestasis, other members of the mrp family (e.g., mrp3) have increased expression on the basolateral (sinusoidal) plasma membrane (C) where they can pump potentially toxic bile acids and other organic anions out of the cell and back into the circulation.

MECHANISMS OF HEPATOCYTE ORGANIC ANION TRANSPORT / 1475 is regulated by PXR, this transporter appears to play an important role in facilitating entry into the cell of PXR ligands such as rifampicin (75). Several allelic variations of the OATP2 (OATP1B1) gene that are found in European and African Americans have markedly reduced the ability to mediate uptake of rifampicin (75). Whether PXR-mediated transporter function is altered in these individuals remains to be determined. Interestingly, FXR and PXR up-regulation may be protective against hepatocyte toxicity resulting from cholestasis (267,273). Because these nuclear hormone receptors mediate many complex events within the cell, elucidation of specific genes that ameliorate toxic events will be of great potential importance in designing new classes of pharmaceuticals (267,274).

SUMMARY Figure 57-2 summarizes the major pathways that the liver uses to take up and excrete various organic anions. The uptake pathways have a great deal of substrate overlap, and for the most part, their function in vivo requires further investigation. Function of the canalicular excretory pumps has been clarified from molecular genetic study of several naturally occurring mutations (e.g., Dubin–Johnson syndrome, progressive familial cholestasis type 2). In cholestasis, basolateral (sinusoidal) plasma membrane expression of several members of the mrp family (e.g., mrp3) is substantially increased, presumably permitting potentially toxic bile acids and other organic anions to be pumped out of the cell and back into the circulation. Future investigation into structure–function relations, as well as factors required for cell-surface expression and activity of these transporters, will be essential for ultimately understanding their function in health and disease.

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CHAPTER

58

Mechanisms of Hepatocyte Detoxification Karen F. Murray, Donald J. Messner, and Kris V. Kowdley Overview, 1483 Liver Anatomy and Function, 1483 Metabolism and Excretion, 1484 Metabolism of Toxins, 1484 Elimination of Toxins: Phase III Transport—Adenosine Triphosphate–Binding Cassette Transporter Proteins, 1490 Influence of Liver Health on Metabolism and Elimination, 1492 Protection from Damage, 1492

Hepatocyte Protective Mechanisms: Oxidant Neutralization, 1492 Hepatocyte Protective Mechanisms: Metal Sequestration, 1497 Hepatocyte Protective Mechanisms: Clearance and Regeneration, 1499 Summary, 1501 Acknowledgments, 1501 References, 1501

OVERVIEW

liver is the “lobule” model. In this model, a hepatic lobule is centered around the hepatic vein with the portal areas organized around the points of a pentagon, as shown in Figure 58-1. The liver has a dual blood supply, namely, from the hepatic artery and the portal vein, which subdivide into the terminal hepatic arterioles and portal venules. The end arterioles of the hepatic arteries also drain into the terminal portal venules. From these structures, blood leaves discrete vascular structures and freely comes into contact with hepatocytes in the sinusoids, where metabolic and synthetic activity take place. Blood subsequently drains from the sinusoids into the central veins and flows out of the liver via the hepatic veins. The hepatic lobule has been divided into three zones (zones of Rapoport) based on the difference in oxygen tension within the hepatic lobule; the oxygen tension of the blood entering the sinusoid is richest around the portal area (zone 1 of Rapoport), lowest in the region surrounding the central vein (zone 3 of Rapoport), and intermediate between zones 1 and 3 (zone 2 of Rapoport). The centrilobular region of the lobule is the most susceptible to toxic, hypoxic, and ischemic injury. One commonly cited example is hepatotoxicity of acetaminophen, which is discussed in this chapter. The portal “triad” also contains bile ducts. The bile ducts branch into smaller bile ductules and terminate in the biliary canaliculi between hepatocytes; bile drains across the biliary canalicular membrane from the hepatocytes into bile ductules and subsequently into bile ducts. The bile ducts serve a

Liver Anatomy and Function The cell types within the liver consist of hepatocytes, biliary epithelial cells, and sinusoidal-lining cells (Kupffer and endothelial cells), stellate cells (formerly known as Ito cells), and cells involved in the immune response. Hepatocytes are the predominant cells in the liver. These cells perform a wide range of metabolic activities. Hepatocytes are responsible for the synthesis of glucose (gluconeogenesis), albumin and other plasma proteins, cholesterol and bile acids, the metabolism of drugs and toxins, and are crucial to the oxidation of fatty acids. The microscopic organization of the cells within the liver is organized around the vascular supply to this organ. A widely accepted concept for the microscopic architecture of the

K. F. Murray: Children’s Hospital and Regional Medical Center, University of Washington School of Medicine, Seattle, Washington 98105. D. J. Messner: Bastyr University, Kenmore, Washington 98028. K. V. Kowdley: University of Washington, Seattle, Washington 98195. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1483

1484 / CHAPTER 58 This chapter examines detoxification by hepatocytes from two broad perspectives: (1) the mechanisms whereby drugs and toxins are metabolized by the liver, resulting in reduction of the potential of toxicity and facilitation of excretion; and (2) the mechanisms that protect the liver from autotoxicity during the process of drug and toxin metabolism, via antioxidant mechanisms and regeneration.

METABOLISM AND EXCRETION Metabolism of Toxins General Principles

Normal lobular pattern P–Portal triad; C–Central vein

FIG. 58-1. The lobular structure of the liver parenchyma. C, central vein; P, portal triad. (See Color Plate 31.)

number of different functions, including the excretion of toxins or drugs that are water soluble, secretion of bile acids, and as a central pathway for the elimination of drugs and excretion of toxins and heavy metals. An important mechanism whereby toxins can be rendered polar for subsequent elimination via bile is to be conjugated in the liver via glucuronidation, thus rendering these compounds water soluble and capable of being excreted in bile, and subsequently via the feces. Biliary epithelial cells can also further facilitate excretion of drugs and toxins by modifying the bile secreted by hepatocytes via addition of bicarbonate, water, and other compounds. Many other organic anions and cations are excreted in bile, including drugs and toxins. Normal biliary tract function is essential for maintenance of copper (Cu) homeostasis, because regulation of body Cu stores is predominantly via biliary Cu excretion. Cu toxicosis because of failure to transport this metal into the biliary canaliculus is the mechanism of liver damage in Wilson’s disease, a genetic disease caused by a loss of function mutation in the ATP7B gene. Sinusoids are unique vascular structures within the liver that contain fenestrations and are of much more variable size than capillaries. They are lined by Kupffer and endothelial cells. Kupffer cells are liver-specific macrophages that phagocytose toxins, microorganisms, and aged red blood cells. Kupffer cells also serve as a storage site for iron (Fe) salvaged from dead erythrocytes. Endothelial cells in the liver are believed to be the source of vasoactive hormones, for instance, endothelin and nitric oxide. Stellate cells, which can undergo transformation into fibroblasts after stimulation by a variety of cytokines or other mediators, appear to be important in mechanisms of hepatic fibrogenesis and fibrolysis. Once activated, stellate cells are believed to likely play an important role in both the production and degradation of collagen and extracellular matrix.

The liver plays a central role in the metabolism of drugs and toxins because of its central location between the portal (or splanchnic) circulation and the systemic circulation. Thus, the liver is the first parenchymal site of entry for orally ingested drugs and toxins bound to plasma proteins such as albumin after absorption in the gastrointestinal tract. The liver has a dual blood supply via the hepatic artery and portal vein, and thus has a high volume of blood flow, allowing for a high rate of extraction of many drugs and toxins from the circulation. In addition, a large number of enzymes in the liver are capable of transforming drugs or toxins into compounds with increased or decreased pharmacologic activity, or increased toxic potential, via oxidation/reduction reaction hydrolysis (phase I metabolism). Hepatic enzymes may also facilitate the excretion of drugs in the urine or bile by conjugating the products of phase I metabolism with other compounds, thereby rendering the phase I product more polar (phase II metabolism). Finally, products of phase II metabolism are transported into the bile ducts via phase III metabolism, which is regulated by a number of transporters localized primarily to the biliary canaliculus. Phase I Metabolism P450 Enzymatic Transformation Among the phase I biotransforming enzymatic systems, the “cytochrome P450” system is by far the most versatile and productive. With the largest number of substrates, the system is one of the most important for hepatic detoxification. The “cytochrome P450” enzymes are a large family of heme-thiolate enzymatic proteins located on the endoplasmic reticulum of hepatocytes. The term cytochrome P450 is derived from the original observation that these enzymes, isolated from hepatocyte endoplasmic reticulum microsomes, are chemically similar to mitochondrial cytochromes and are reddish in pigmentation, reflecting the heme that is part of their molecular structure. The “450” designation reflects the finding that these microsomes have peak light absorption of 450-nm wavelength light when treated chemically with a reducing agent and bound with carbon monoxide (1).

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1485 The basic reaction catalyzed by cytochrome P450s is written as follows, where S is the substrate: NADPH + H+ + O2 + SH → NADP+ + H2O + SOH The reaction is by definition a monooxygenation because of the incorporation of only one of the two oxygen atoms into the substrate. The reactions catalyzed by P450 proteins are, however, numerous and include aromatic and sidechain hydroxylation; N-, O-, and S-dealkylation; N-oxidation; N-hydroxylation; sulfoxidation; deamination; dehalogenation; and desulfuration. The resulting substrate metabolites are usually stable; however, it is at this step that many toxic metabolites are generated during hepatic drug metabolism. One of the most important groups of substrates for the cytochrome P450 system is xenobiotics, or those exogenous compounds that are “foreign to life.” This group includes toxic compounds that the body is exposed to such as therapeutic drugs, food additives, and chemicals that are used in the workplace or are industrial by-products that result in environmental contamination. Once in the body, the lipophilic quality of these compounds allows their passage across epithelial cell membranes, and ultimately into the bloodstream.

Without removal they would accumulate in cells over time and cause cellular dysfunction. As they pass through the liver via the sinusoids, however, they either passively diffuse or are actively transported into hepatocytes where they encounter, by diffusion, particular P450s able to metabolize them (2). Some compounds are successfully metabolized by only one P450, and then undergo phase II metabolism before excretion. Others, however, require metabolism by more than one P450, and hence undergo more than one phase I reaction, before undergoing phase II metabolism and excretion (Fig. 58-2). The combination of phase I and II metabolic reactions results in the transformation of a lipophilic molecule, which is difficult to excrete without reabsorption, to a polar hydrophilic molecule. This water-soluble molecule can be more readily excreted by the kidneys or via the bile into the intestine with minimal chance of enterohepatic cycling. It is now known that individual P450s represent the products of unique genes, and that the large interindividual differences in the activity of these proteins are due in part to gene mutations. The amino acid sequence of the cytochrome P450 proteins allows them to be classified into families and subfamilies based on homology. A given family of cytochrome

Canaliculus A+

B+

ATP

ATP

Phase II

CYP3A4

A

Phase II

ER

Phase I

CYP3A4

CYP2D6

B

B

Phase I

A

B

A Protein

ABC

B+

Phase III

A+

ABC

Phase III

B Sinusoid Protein

FIG. 58-2. Enzymatic transformation of toxic compounds in hepatocytes through phase I and II metabolism and cellular excretion by the adenosine triphosphate (ATP)–binding cassette (ABC) transporter proteins. ER, endoplasmic reticulum.

1486 / CHAPTER 58 P450 proteins share 40% sequence homology and are designated by an Arabic numeral. The most important proteins in drug metabolism fall into the gene families CYP1, CYP2, or CYP3. Within a family, those proteins that share 55% or more amino acid homology are further classified as a subfamily and are thus identified by a capital letter that follows the family designation, such as 2A, 2B, or 2C. A final number is added to individually identify a specific protein within a given subfamily, for instance, 2B1, 2B2, or 2B3. The CYP designation is the nomenclature common to all of the cytochrome P450 genes and proteins (1,2). In the metabolism and detoxification of therapeutic drugs, seven P450s are most important. It is now thought that CYP3A4 is responsible for metabolizing 50% of drugs (3), whereas CYP2D6 metabolizes 20%, CYP2C9 and CYP2C19 together another 15%, and CYP1A2, CYP2A6, CYP2B6 and others the remaining 15% (1,2). Although most substances that undergo phase I P450 metabolism subsequently undergo phase II conjugation before elimination from the body, the rate-limiting step is usually the P450 metabolism. Furthermore, the activity of the P450 proteins is genetically variable from individual to individual (4) and can be altered by nongenetic factors. The genetic variability is exemplified by the functional activity

of CYP2D6, the cytochrome responsible for the metabolism of 20% of medications, including those used to treat depression, psychosis, and certain cardiac arrhythmias. Individuals with absolutely no enzyme activity are termed poor metabolizers, and are more susceptible to drug toxicity from certain medications. Inheritance of this enzyme defect is autosomally recessive, requiring the acquisition of two mutant gene alleles, a finding in 5% to 10% of white people of European descent, but only 1% to 2% in those of Southeast Asian ancestry (4–7). In comparison, 20% of Asians and 5% of whites are “poor metabolizers” with respect to CYP2C19 activity, and hence can develop excessive levels of drugs such as omeprazole, which may actually enhance the efficacy of the drug in certain circumstances (6,8,9). Inheritance of this defect is similarly autosomally recessive. Although diminished catalytic activity has been associated with other cytochrome P450 enzymes because of allelic mutations, there is only one example of increased activity from a genetic cause. Gene duplication of CYP2D6 results in exceedingly rapid clearance of some CYP2D6 substrates, which could decrease the efficacy of medications metabolized through this pathway (Table 58-1). It is now known that 2% of white people are “ultrarapid” metabolizers from this defect (10).

TABLE 58-1. The major hepatic cytochrome P450 enzymes: their drug substrates, inducers, and inhibitors P450

Drug substrates

Inducers

Inhibitors

CYP1A2

Caffeine Clozapine Estradiol Theophylline Halothane Nicotine Rosiglitazone Taxol Diclofenac Ibuprofen Tolbutamide Warfarin Omeprazole

Omeprazole Tobacco smoke

Fluvoxamine Furafylline

—

Methoxazalen

Phenytoin Rifampin Rifampin Seeibarbital

—

—

Fluvoxamine Ketoconazole Fluvoxamine Quinidine

CYP2A6 CYP2C8 CYP2C9

CYP2C19 CYP2D6

CYP2E1 CYP3A4

Codeine Chlorpromazine Desipramine Dextromethorphan Encainide Haloperidol Metoprolol Acetaminophen Halothane Cyclosporin Estradiol Indinavir Lovastatin Midazolam Nifedipine Quinidine Docetaxel

Sulfafenazole

—

Ethanol Isoniazid Carbamazepine Phenobarbital Phenytoin Rifampin St. John’s wort Troglitazone

Disulfiram Delavirdine Erythromycin Grapefruit juice Ketoconazole Ritonavir Troleandomycin

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1487 Multiple unrelated nongenetic factors can also alter cytochrome P450 activity, including other medications, nutritional changes, and disease (11,12). The most notorious of these factors involve drug–drug interactions (see Table 58-1). Because many of the cytochrome P450 enzymes can bind different drug molecules, competition among multiple drugs for the same enzyme will decrease the P450 activity against any one drug. An example of this would be the decreased clearance of cyclosporin A in a patient given erythromycin, an effect caused by inhibition of CYP3A4 (13). Increased activity of P450s also can occur. In most examples of cytochrome P450 induction (see Table 58-1), the transcription of the P450 gene is increased, resulting in a greater concentration of the P450 in hepatocytes, and hence higher activity of the enzyme (14). Although not all of the P450s are inducible, for those that are inducible, the inducing molecule binds to a cytosolic receptor, and then translocates to the nucleus where it binds to the regulatory sites of the cytochrome gene. Using cyclosporin A metabolism as an example, when a patient is simultaneously given phenobarbital, cyclosporin A levels may decrease to a subtherapeutic level because of the induction of CYP3A4 by the phenobarbital. Flavin Monooxygenase Like the cytochrome P450 enzymes, oxidized flavin adenine dinucleotide (FAD)–containing monooxygenases (FMOs) localize to endoplasmic reticulum microsomes and require NADPH and O2. In addition, many of the reactions catalyzed by FMOs are also catalyzed by the cytochrome P450 enzymes. Such reactions include the oxidation of nucleophilic nitrogen, sulfur, and phosphorous heteroatoms of tertiary amines to N-oxides, secondary amines to hydroxylamines and nitrons, primary amines to hydroxylamines and oximes, and sulfur-containing drugs and phosphines to S- and P-oxides. In contrast with the P450 reactions, however, FMOs do not catalyze N-, S-, or O-dealkylation reactions. Examples of drugs metabolized via these reactions include amphetamine, imipramine, tamoxifen, thiols, and thiocarbamates. Cimetidine is an example of a sulfur-containing drug metabolized by FMO, which is, in contrast, an inhibitor of cytochrome P450. The five FMO (FMO1-5) enzymes are highly conserved across species, and all contain a glycine-rich region that binds 1 mole FAD near the active catalytic site, and also near a second glycine-rich region that binds NADPH. In contrast to the cytochrome P450 reactions, where oxygenation of the heteroatom is initially with one electron, the structure of FMO allows for two-electron oxygenation. Hence, the N-oxygenation by cytochrome P450 results in N-dealkylation, whereas it results in N-oxide formation when catalyzed by FMO. Overall, the reactions detoxify xenobiotics by reacting them with a peracid or peroxide. The FAD is reduced to FADH2 by NADPH, after which the oxidized cofactor NADP+ remains bound to the enzyme. FADH2 then binds oxygen, producing peroxide. In interaction with a substrate, the flavin peroxide is then transferred, and the FAD is restored via dehydration, releasing NADP+.

In animals and humans, the five FMO proteins are expressed in the liver, kidney, and lung to variable degrees. In humans, FMO3 is by far the most highly expressed in the liver, with the others expressed only with low activity. Interestingly, FMO3 is also the flavin monooxygenase that is responsible for the conversion of (S)-nicotine to the trans isomer of (S)nicotine N-1′-oxide, which is excreted in the urine of smokers and those using a nicotine patch. Hence, the urinary excretion of trans-(S)-nicotine N-1′-oxide can be used to probe the activity of FMO3 in vivo in humans. Similarly, the metabolism of cimetidine to cimetidine-S-oxide can be detected in the urine and is reflective of appropriate enzyme activity. The regulation of the FMOs is distinct from that of the P450 enzymes. Many compounds that induce P450 actually inhibit FMO. Indole-3-carbinol, which is found in Brussels sprouts, induces many of the P450 enzymes, but directly inhibits FMO3 (15). Alcohol Dehydrogenase Among the myriad of other enzymes involved in phase I oxidation, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are of greatest clinical importance. ADH is a zinc (Zn)-containing, cytosolic enzyme that oxidizes ethanol to acetaldehyde. ADH concentrations are greatest in the liver; however, it is also present in the kidney, lung, and gastric mucosa. The molecule is a dimeric protein consisting of two 40-kDa subunits, of which there are 6 different subunits and multiple allelic variants. The resultant ADH molecules are grouped into four major molecular classes. The class I isoenzymes are responsible for the oxidation of ethanol and other small aliphatic alcohols. The class II enzyme, in contrast, preferentially oxidizes larger aliphatic and aromatic alcohols, with minimal to no role in the oxidation of ethanol. Class III ADH substrates include the long-chain aliphatic and aromatic alcohols, and this class of ADH is largely responsible for the detoxification of formaldehyde (class III ADH is actually the same enzyme as formaldehyde dehydrogenase). Class IV ADH is the only ADH not found in the adult human liver. It is, however, present in the mucosa of the upper intestinal tract, especially the mouth, esophagus, and stomach, and is most active in oxidizing retinol, a vitamin important for cell growth and differentiation. Its localization in the upper intestinal tract, where it could convert ethanol to acetaldehyde (a potential carcinogen), and its role in retinol metabolism may implicate it in the pathogenesis of upper intestinal cancers associated with chronic alcohol ingestion. The different isoenzymes of the class I ADH have variable affinity and capacity for oxidizing ethanol to acetaldehyde. For instance, one variant, atypical ADH, is responsible for unusually rapid conversion of ethanol to acetaldehyde in 90% of the Pacific Rim Asian population, but is present in less than 20% of white people and less than 10% of African Americans, and does not occur in Native Americans or Asian Indians (16). These differences provide a biochemical explanation for the sometimes extreme ethnic differences in ethanol toxicity relative to consumption. With excessive or

1488 / CHAPTER 58 toxic ethanol ingestion, hepatic metabolism by ADH becomes saturated; however, it is the threshold at which saturation occurs that determines the point of intoxication. Biochemically, this is clear when considering that the isoenzyme activity differences for oxidizing ethanol range from a Km of 50 µM to 4 mM, where a blood ethanol level of 0.1% corresponds to 22 mM. Aldehyde Dehydrogenase ALDH oxidizes aldehydes to carboxylic acid (acetate). Twelve ALDH genes have been identified in humans, producing cytosolic, mitochondrial, and microsomal enzymes in multiple different tissue types. ALDH2 is a mitochondrial enzyme found in the liver and mucosa of the upper intestinal tract, among other tissues, and is primarily responsible for the oxidation of simple aldehydes such as acetaldehyde, which, as stated earlier, is a potential carcinogen. Approximately 50% of Pacific Rim Asians, as well as Taiwanese and Vietnamese, are deficient in ALDH2 activity. This same population has a high incidence of atypical ADH activity. In combination with the rapid metabolism of ethanol to acetaldehyde in the setting of atypical ADH, there is a rapid accumulation of acetaldehyde resulting in catecholamine release, dilation of facial blood vessels, and flushing. Acetaldehyde accumulation also causes nausea, which is likely protective against alcoholism in these individuals. Disulfiram (Antabuse) is useful clinically because it inhibits ALDH, resulting in acetaldehyde accumulation and nausea with alcohol ingestion. Other Phase I Enzymatic Reactions Hydrolysis and reduction are other reactions that contribute to phase I metabolism of toxins and drugs. Metals and compounds containing azo- or nitro- groups, aldehyde, ketone, disulfide, sulfoxide, quinone, alkene, or N-oxide moieties are reduced either enzymatically or nonenzymatically. Whereas most of the azo- and nitro- reduction is catalyzed by the intestinal microflora, the reduction of aldehydes to primary alcohols, or ketones to secondary alcohols and quinones, is catalyzed by the family of carbonyl reductases found in liver and other tissues. In the liver, carbonyl reductase activities are located both in the cytosol and in microsomes where they have different stereoselectivity. Many of the other reduction reactions occur to a minor degree in the liver, but one worthy of mention is the reduction of pyrimidines by dihydropyrimidine dehydrogenase in the hepatic cytosol. Dihydropyrimidine dehydrogenase catalyzes the reduction of pyrimidines such as 5-fluorouracil. The importance of this enzyme was exemplified by a tragic drug combination incident in Japan in 1993. Fifteen patients on Tegafur, a prodrug that is converted to 5-fluorouracil in the liver, were also given Sorivudine, an antiviral drug for the treatment of herpes zoster that was new at the time. Sorivudine is converted to an inactive intermediate metabolite by gut flora, which, in turn, is converted by dihydropyrimidine dehydrogenase to a metabolite that binds covalently to the enzyme, hence inactivating it. In these

patients taking 5-fluorouracil, this irreversible inactivation of dihydropyrimidine dehydrogenase resulted in the toxic buildup of 5-fluorouracil, and subsequent death for many of these patients (17,18). Phase I hydrolysis is catalyzed by multiple enzymes located in the serum and numerous tissues, including the liver. Two notable groups of enzymes are the carboxylesterases and epoxide hydrolases. The carboxylesterases are 60-kDa glycoproteins that are predominantly associated with the endoplasmic reticulum when in the liver. They have a central role in the generation of active metabolites from ester and amide prodrugs that are effective as cancer chemotherapeutic agents. They are also active in the metabolism of many endogenous esterified compounds such as palmityl-coenzyme A, retinyl ester, and platelet-activating factor. Epoxide hydrolase catalyzes the trans-addition of water to alkene epoxides and arene oxides, both products of cytochrome P450 metabolism. Arene oxides and alkene epoxides are highly reactive with cellular macromolecules such as DNA and proteins, and are consequently potently toxic and frequently mutagenic. Although carboxylesterases and epoxide hydrolases share no sequence homology, they both have a catalytic triad with similar topologic structure, and are hence categorized with the α/β-hydrolase fold enzymes. Phase II Metabolism Phase II metabolism results in a covalent linkage between an activated group on the parent compound or its phase I metabolite and glucuronic acid, glutathione, sulfate, acetate, or amino acids. The resultant highly polar molecule is relatively less reactive and is more expeditiously excreted in bile (see Fig. 58-2). Occurring primarily in the cytosol, these reactions are catalyzed by appropriate transferase enzymes. Glucuronidation The most important conjugation reaction, glucuronidation, is catalyzed by uridine diphosphate glucuronosyltransferases (UGTs) that are responsible for roughly 40% of the phase II conjugation of drugs. These enzymes catalyze the transfer of glucuronic acid (from uridine diphosphate glucuronic acid) to aromatic and aliphatic alcohols, carboxylic acids, amines, and free sulfhydryl groups of both endogenous and exogenous compounds, to form O-, N-, and S-glucuronides, respectively. Glucuronidation also plays an important role in the elimination of endogenous compounds including bilirubin, steroids, and fat-soluble vitamins. Although most phase II reactions take place in the cytosol, the activity of UGTs is located on microsomes of the endoplasmic reticulum. Given the dominant role that these conjugating enzymes play in the phase II metabolism of drugs and endogenous compounds, this unique localization facilitates their access to the phase I product substrates formed at the same site. Also found in the intestinal epithelium, kidney, and skin, the 15 identified UGTs are classified into 2 different families

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1489 based on more than 50% amino acid identity. Family 2 has only three subfamilies based on additional amino acid homology, whereas the members of Family 1 are further divided into individual isoforms. The multiple Family 1 isoform proteins are all encoded by a single complex gene, but alternative splicing of 12 promoters and exon 1 with exons 2 through 5 produces multiple different proteins. Although the variant isoforms have characteristic substrate specificities, there is a large degree of overlap resulting in the ability of multiple isoforms to catalyze the same glucuronidation reaction (5). Examples of diseases resulting from deficiency of UGT include Crigler–Najjar type I (complete absence of hepatic bilirubin-UGT activity) and type II (deficiency of hepatic bilirubin-UGT activity) and Gilbert syndrome (reduced hepatic bilirubin-UGT activity). The absence of bilirubinUGT results in the inability to convert bilirubin into a watersoluble form for excretion, resulting in toxic unconjugated hyperbilirubinemia. Tissue deposition of bilirubin then occurs across the blood–brain barrier and in other organs, resulting in functional impairment of the central nervous system, thyroid, and kidneys. In type II Crigler–Najjar, there is a less complete deficiency of bilirubin-UGT, resulting in less dramatic increases of unconjugated bilirubin. In addition, in contrast with Crigler–Najjar type I, patients with type II respond to phenobarbital therapy with improved glucuronidation, and hence reduced unconjugated hyperbilirubinemia. Presumably, this beneficial response to phenobarbital is because of the trophic effects of this drug on the endoplasmic reticulum, and hence increased bilirubin-UGT activity (19). The reduced bilirubin-UGT activity in Gilbert syndrome is relative mild compared with these other conditions, but still results in intermittent increases in unconjugated bilirubin in conditions of fasting or illness. Sulfation The next most common conjugation reaction does take place in the cytosol because of the action of sulfotransferases (STs). Accounting for approximately 20% of the phase II metabolic reactions, STs catalyze the transfer of inorganic sulfur from activated 3′-phosphoadenosine5′-phosphosulfate to the hydroxyl group of phenols and aliphatic alcohols. Consequently, aliphatic alcohols and other primary metabolites or parent drugs with hydroxyl groups are frequently both glucuronidated and sulfated. One prominent example is the metabolism of acetaminophen. After therapeutic doses of this medication, approximately 50% of the dose is excreted as the phenolic O-glucuronide and 30% as the O-sulfate. Animal models of UGT deficiency, in which there is significantly more susceptibility to acetaminophen hepatotoxicity, illustrate the importance of these conjugation reactions (20,21). In humans, increased susceptibility to acetaminophen-induced hepatotoxicity may occur in Gilbert syndrome with its relative decrease in bilirubin-UGT activity (22,23). Generally, however, glucuronidation and sulfation are able to compensate for each other because there is only one possible example of

glucuronidation or sulfation inhibition resulting in acetaminophen toxicity (24). Glutathione Conjugation and Acetylation N-acetyltransferases (NATs) and glutathione S-transferases (GST) together contribute approximately 25% of the phase II metabolic activity. GST is localized to many tissues, including the liver, where more than 95% of its activity is localized within the cytoplasm and less than 5% to the endoplasmic reticulum. It catalyzes the conjugation of hydrophobic, electrophilic atom-containing substrates with the tripeptide glutathione (consisting of glycine, cysteine, and glutamic acid), forming thioesters (25). The role of GST in detoxification of drugs in the liver can again be exemplified by the metabolism of acetaminophen. Although at normal doses most of the acetaminophen is metabolized via glucuronidation and sulfation, as discussed in the preceding section, a small amount is oxidized by cytochrome P450 proteins to NAPQI (N-acetyl-p-benzoquinone imine) (Fig. 58-3). NAPQI is highly reactive and covalently binds to hepatic proteins causing hepatocellular necrosis of the centrilobular cells, the primary mechanism of acetaminophen-induced hepatotoxicity (Fig. 58-4). Conjugation of NAPQI with glutathione is the main pathway for removal of NAPQI, but in the setting of acetaminophen overdose, GST becomes saturated, resulting in the toxic buildup of NAPQI and consequent hepatocellular injury. This seemingly straightforward role of GST in the detoxification of NAPQI has been challenged however; new evidence has suggested a more complicated relation. Mice with no GST activity are protected from acetaminopheninduced hepatotoxicity, despite typical biotransformation of acetaminophen to NAPQI (26). Conversely, transgenic overexpression of glutathione synthetase, with resultant increased hepatic glutathione levels, is not protective against acetaminophen-induced hepatotoxicity (27). Confusing the outlook even more are the conflicting observations that intravenous administration of glutathione peroxidase, an enzyme important in the protection against reactive oxygen species (ROS; see later), is protective against acetaminopheninduced hepatotoxicity (28), whereas the transgenic overexpression of the intracellular form of glutathione peroxidase results in more hepatotoxicity (28). These divergent observations raise questions as to the true role of glutathione conjugation in acetaminophen metabolism and protection from toxicity. A relatively minor contributor to phase II metabolism, the two NAT enzymes (NAT1 and 2) catalyze the acetylation of amines, hydrazines, and sulfonamides. Drugs metabolized by NAT therefore include isoniazid, procainamide, dapsone, hydralazine, and caffeine. Much like the genetic variability seen in the CYP protein expression, there are also variations in NAT and GST. NAT has multiple allelic variants, some of which affect its catalytic activity. These variants lead to different population frequencies with the slow-acetylator phenotype seen in 50% to 70% of American whites, African Americans, and Northern

1490 / CHAPTER 58 O C CH3

HN O

Detoxification O

C CH3

HN

CH3

N

H GS

P450

S—Protein O

O C

GSSG Protein—S—S

CH Acetaminophen

GSH Protein—SH

HN

Pr

ote

CH3

in—

O NAPQI

Sulfation glucuronidation

SH

Toxic

S—Protein O © Current Medicine

FIG. 58-3. Pathways of acetaminophen metabolism. GSH, glutathione; NAPQI, N-acetyl-p-benzoquinone imine; SH, sulfhydryls.

Europeans, but only 5% to 10% of Southeast Asians. This differential acetylation has been associated with environmental agent-induced disease such as bladder and colorectal cancer (5). Suspicion exists of a similar risk for environmental agent–induced disease related to genetic variability in the activity of GST. Methylation Thiopurine methyltransferase (TPMT) contributes only a few percent to phase II metabolism, but is an important enzyme in the metabolism of common medications used in the treatment of autoimmune conditions. TPMT is a cytosolic enzyme that methylates aromatic and heterocyclic compounds such as the thiopurine drugs azathioprine, 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG). TPMT is encoded by a single gene with allelic variants for low or high enzyme activity, resulting in differential expression, with 0.3%, 11.1%, and 88.6% of the population having low, intermediate, or high enzyme activity, respectively. Specifically, TPMT catalyzes the conversion of 6-MP to 6-methylmercaptopurine (6-MMP). With no TPMT activity, 6-MP is preferentially metabolized to thiopurines with increased risk for myelosuppression. With increased activity, however, 6-MP is preferentially metabolized to 6-MMP with enhanced risk for hepatotoxicity, and hence the proper activity of this enzyme is important for optimal hepatocyte detoxification. Evidence suggests that TPMT is inducible by mesalamine, a common concomitant drug used in the treatment of inflammatory bowel disease, with potential for altered thiopurine metabolism (29).

Elimination of Toxins: Phase III Transport— Adenosine Triphosphate–Binding Cassette Transporter Proteins After hepatocyte metabolic conversion, drugs and other potentially toxic compounds are excreted from the cell via the canalicular membrane (apical) into the bile, or across the basolateral membrane into the sinusoids to be secreted by the kidneys. Membrane transport is carried out predominantly by adenosine triphosphate (ATP)–dependent drug efflux pumps belonging to the ATP-binding cassette (ABC) family of transporter proteins (see Fig. 58-2). In addition to the excretion of potentially toxic compounds, these transport proteins are also responsible for the coupled biliary secretion of bile salts, cholesterol, phosphatidylcholine, and glutathione (30,31). The major human hepatocyte membrane transport proteins are the P-glycoproteins multidrug resistance gene 1 (MDR1), MDR3, bile salt export pump (BSEP), multidrug resistance protein 2 (MRP2), MRP1, and MRP3. Whereas MDR3 and BSEP are constitutively and specifically expressed on the canalicular membrane of the hepatocyte, MDR1 and MRP2 are also expressed in multiple other cells including those in the intestine. Because of their diverse cellular expression, MDR1 and MRP2 play a role in multidrug resistance in cancer therapy, and in the intestine, MDR1 has a reversed effect, thereby reducing serum levels of some orally absorbed medications (32,33). In contrast, MRP1 and MRP3 are expressed on the basolateral membrane of the hepatocyte, but only under certain circumstances.

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1491

V

P

A

B FIG. 58-4. Histologic consequences of acetaminophen overdose. P, portal tract; V, hepatic venule. (See Color Plate 32.)

Multidrug Resistance Gene 3 and Bile Salt Export Pump MDR3 acts as a flippase enzyme, translocating phosphatidylcholine and other phospholipids from the cytosolic leaflet to the outer leaflet of the canalicular membrane. The final excretion of phospholipids into the bile, however, is reliant on bile salt micelles (34,35). BSEP is the major canalicular export pump for bile salts in the normal liver and is the main driving force for bile secretion. Given the reliance on bile-salt micelles for the excretion of phospholipids, individuals with mutations in the BSEP gene have bile with a minimal phospholipid concentration and an abnormal bile salt profile. Termed progressive familial intrahepatic cholestasis (PFIC) type 2, patients with this condition have severe cholestasis

from birth, low γ-glutamyltransferase activity, and bile salt–induced hepatotoxicity (36,37). Individuals with mutations in the MDR3, in contrast, have bile with normal bile salt concentrations, but absent phospholipids; the free bile salts are potently cytotoxic (38,39). Characterized as PFIC 3, the clinical manifestations are similar to those seen in PFIC 2, except that γ-glutamyltransferase activity is increased and liver histology shows a more prominent pattern of ductular proliferation. Multidrug Resistance Protein 2 MRP2 is also a canalicular efflux pump, but for organic anions. It is additionally expressed in the kidney and intestine, likely contributing to its participation in multidrug

1492 / CHAPTER 58 resistance (40,41). As for other hepatic ABC proteins, the substrates of MRP2 are mostly products of phase II metabolism. Its activity also contributes to bile formation, but by transporting glutathione, a driving force in bile salt– independent flow. Regulation of MRP2 production is not entirely clear. It is known that substrates of MRP2 and some of the phase I and II enzymes can induce MRP2 expression in a dose- and time-dependent fashion, implying positive feedback on gene transcription (32,42). It is also observed, however, that gene transcription is rapidly down-regulated in the presence of endotoxins (32). Dubin–Johnson syndrome is the human clinical phenotype resultant from mutation of the gene encoding for MRP2, and hence absence of the transporter. The inability of affected individuals to excrete nonbile salt organic anions, such as bilirubin monoglucuronides and diglucuronides, results in their reflux back into the circulation (43–45). Multidrug Resistance Proteins 1 and 3 MRP1 and MRP3 are expressed on the basolateral membrane, but only under circumstances of canalicular membrane injury. (32) Specifically, MRP1 is expressed during endotoxin-mediated cholestasis (46) and liver regeneration (47), and MRP3 is increased with cholestasis and hyperbilirubinemia (48–50). In both cases, their up-regulation occurs under circumstances that down-regulate MRP2. As reverse transporters, these proteins help to reduce intracellular metabolite concentrations under circumstances of canalicular membrane transport dysfunction. Their primary substrates include glutathione S-conjugates for MRP1 and monovalent glucuronides for MRP3 (51). MRP3 may also serve an important role in organic anion export when MRP2 is down-regulated. Interestingly, patients with Dubin– Johnson syndrome have increased amounts of MRP3 in their livers (52). The potential effects of exogenous compounds on the activity of these membrane transporters must also be mentioned. Given the diversity of molecules that these transporters allow as substrates, many therapeutic medications are transported. As with the CYP proteins, medications may competitively impede the transport of other medications or endogenous substrates. In addition, inhibition can be produced by compounds acting either on the cis, or cytosolic side, or the trans, or canalicular side of the hepatocyte membrane. An example is that of in vitro inhibition of BSEP. BSEP is inhibited from the cis side by cyclosporin A, and from the trans side by estradiol-17β-glucuronide (32).

Influence of Liver Health on Metabolism and Elimination Not only are there individual variations in enzyme or transporter activity by factors that affect these individual protein functions, but metabolism by the liver is also influenced by overall organ health and host development.

Given that the liver is the main site for drug and toxin metabolism, liver disease can significantly impact these detoxification processes. In general, the severity and extent of liver damage correlates with the degree of metabolic impairment, with severe disease resulting in only 30% to 50% of the activity present in a healthy organ. Drugs that undergo significant first-pass metabolism in the liver, however, can display an increase in their bioavailability by twofold to fourfold, and when coupled with the decreased clearance, can have substantially increased pharmacologic effects and toxicity. In general, the proteins responsible for phase I metabolism are more susceptible than those of phase II (5). Age also determines variability in metabolism. Both the CYP proteins and those involved in phase II metabolism are developmentally regulated and are relatively deficient in the newborn. By 2 to 4 weeks after birth, their development increases to more mature activity levels, with the exception of UGT, which continues to increase in activity over the first two decades of life before it again declines in activity.

PROTECTION FROM DAMAGE Hepatocyte Protective Mechanisms: Oxidant Neutralization Oxygen Toxicity Oxidative metabolism of xenobiotics can produce shortlived and highly reactive intermediates capable of causing extensive damage to key biomolecules including nucleic acids, proteins, and lipids. Detoxification depends not only on metabolizing, conjugating, and excreting harmful substances, but also on protecting the hepatocyte from damage caused by these agents (Fig. 58-5). Normal metabolic reactions produce similar toxic products; the high metabolic activity of liver makes this a significant problem even in the absence of exogenous toxins. Thus, although hepatocytes are adept at neutralizing ROS, they are not always completely effective. Oxidative stress results when production of ROS exceeds the antioxidant capacity of the cell. This section describes the generation of ROS, reviews hepatocyte mechanisms for neutralizing these toxins, and discusses some of the pathophysiologic consequences of oxidative stress. Analogous generation and quenching of reactive nitrogen species (RNS) also occurs. Sources of Reactive Oxygen Species ROS include oxygen free radicals. A common reaction in aerobic organisms is the addition of one electron to molecular oxygen to form superoxide anion (O2−). In the liver, this occurs extensively in mitochondria during oxidative metabolism, as a by-product of P450 reactions in the smooth endoplasmic reticulum, and as a result of redox cycling of exogenous compounds (53). Reaction of superoxide with water gives hydroperoxyl radical (HO2*), which can be further reduced

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1493

Drug Genetic and environmental P450 influence

Redux cycling

Metabolite

Electrophile

Free radical

Superoxide anion

Covalent binding

Lipid peroxidation

Lipid peroxidation Protein-thiol oxidation

Cell injury © Current Medicine

FIG. 58-5. Mechanisms of drug-induced hepatocellular injury.

to hydrogen peroxide (H2O2). If hydrogen peroxide is not detoxified, further reduction produces hydroxyl ion (O*H), a highly reactive oxygen free radical. This key reaction is catalyzed by transition metals such as iron (Fe). The reaction of ferrous iron (II) with hydrogen peroxide to produce ferric iron (III) and hydroxyl radical was described by Fenton more than 100 years ago (54). The Fenton reaction depends on the presence of free ferrous iron (II) and is normally restricted by sequestration of catalytic iron by transferrin and ferritin, as well as rapid detoxification of hydrogen peroxide. Redox Cycling Oxidative stress is propagated and amplified by redox cycling, exemplified by redox cycling of iron. The Fenton reaction accounts for half of this process. Regeneration of Fe(II) from Fe(III) by the oxidation of superoxide anion to molecular oxygen (see Rxn 2 below) completes the process. When coupled with the Fenton reaction, highly reactive hydroxyl radicals are catalytically generated from hydrogen peroxide (the toxic precursor) and the ubiquitous superoxide anion. This cycle was first proposed in 1934 by Haber and Weiss (55). Both the Fenton and Haber–Weiss reactions are important in the liver, an organ with high oxidative metabolism of endogenous and exogenous compounds, as well as a key storage site for iron. Fenton reaction: H2O2 + Fe(II) → Fe(III) + O*H + OH− Rxn 2: O2− + Fe(III) > Fe(II) + O2 Haber–Weiss: O2− + H2O2 > O2 + O*H + OH− Superoxide anion releases catalytically active ferrous iron (II) from transport and storage proteins (such as ferritin) that bind preferentially to ferric iron (III). Consequently, reactions and processes that increase superoxide can increase the

level of bioactive free iron (56). Stimulation of Kupffer cells during an immune response has this effect in liver pathologies, including cirrhosis. Thus, although direct damage to cellular biomolecules by superoxide ion is minimal because of its reactivity with water, its ability to catalyze the redox cycling of iron makes it an important factor in oxidative stress. Analogous redox cycling occurs with many toxins, including ethanol, drugs such as adriamycin, and environmental pollutants such as carbon tetrachloride, contributing significantly to toxicity of these compounds. Most are reduced (and oxidized) by one electron transfer. Reduced intermediates react with molecular oxygen to form oxygen free radicals that cause oxidative stress, at the same time regenerating the original toxic molecule that can repeat the cycle. Hepatocytes ultimately respond by sequestering or metabolizing and excreting the toxin. Until that process is complete, however, they must be capable of detoxifying the large amounts of ROS generated by redox cycling. This occurs through direct enzymatic reduction of ROS, as well as enzymatic regeneration of small molecule reductants (57). Mechanisms of Hepatocyte Injury: Necrosis versus Apoptosis ROS damage cells by free radical transfer from an initial metabolic by-product to a variety of critical biomolecules including DNA, proteins, and lipids. Free radical addition increases the likelihood of modification through conjugation with electrophiles or covalent bond breakage (see Fig. 58-5). Physical damage, mutation, or loss of function in the short term can cause hepatocyte death, and over the longer term can contribute to the pathophysiology of liver disease. This is in addition to the cellular stress and energy consumption caused by redox cycling, which on its own can overwhelm the antioxidant capacity of hepatocytes and result in cell death. A major pathophysiologic concept that has emerged is

1494 / CHAPTER 58 that oxidative stress contributes to many diseases. Examples include cancer, cardiovascular disease, liver cirrhosis, diabetes, and others related to aging. Identifying the biochemical pathways that are activated by oxidative stress, and determining the role of those pathways in disease, is the focus of intensive research. In general terms, there are two types of cell death, necrosis and apoptosis (58). Apoptosis is an autoregulated, energydriven process that is controlled by the dying cell itself. Cell remnants can be phagocytosed by adjacent cells, but apoptosis proceeds with minimal contribution from proinflammatory immune cells (59). A typical progression might be as follows: P450-mediated oxidative metabolism of a drug or toxin, oxidative stress–induced depletion of cytoplasmic and mitochondrial glutathione, mitochondrial damage, including cytochrome c release, activation of the mitochondrial permeability transition pore, activation of caspase activity, and finally the intracellular degradation characteristic of apoptosis (60). In contrast, necrosis is not programmed and can be caused by physical or chemical damage to any of a number of cellular organelles or enzyme systems. In many cases, significant numbers of adjacent cells also become necrotic, in contrast to individual cells undergoing apoptosis. In necrosis, cellular debris and ROS are released that stimulate the immune system to initiate an inflammatory response. The involvement of infiltrating immune cells and resident Kupffer cells can then be significant in determining whether oxidative damage is quenched or potentiated. Differentiation of necrosis and apoptosis is not absolute; oxidative stress may induce damage with characteristics of both. Direct Antioxidant Enzyme Systems in the Liver The liver has a high capacity to neutralize ROS. Direct enzymatic removal of ROS is an important detoxification mechanism. This requires the coordinated responses of several antioxidant enzyme systems; individual pathways alone are insufficient (61). These include: (1) superoxide dismutase (SOD), which converts superoxide anion to hydrogen peroxide; (2) catalase and glutathione peroxidase, which convert hydrogen peroxide to water and quench the spread of lipid peroxides; (3) glutathione reductase and GST, the enzymes that regenerate reduced glutathione and conjugate it to reactive compounds; (4) epoxide hydrolase, which metabolizes reactive oxygen products; and (5) metal binding proteins such as ferritin, which chelates bioactive iron. Epoxide hydrolase and related phase I pathways have been described earlier in this chapter, and metal binding proteins are reviewed in the next section. Enzymatic mechanisms that detoxify ROS directly are described in this section, followed by a description of chemical antioxidants and the enzyme systems that maintain the cellular redox state. Superoxide Dismutase SOD initiates the detoxification of superoxide anion by converting it to hydrogen peroxide. There are three classes of SODs, with different genes, unique protein structures, and

distinct subcellular localizations (62). CuZnSOD (SOD1) is a cytosolic enzyme, MnSOD (SOD2) is mitochondrial, and extracellular SOD (SOD3) is secreted from cells. Both SOD1 and SOD3 use Cu and Zn as cofactors and have regions of structural homology. Mn-containing SOD2 is completely distinct. Expression of SOD1 is relatively constant among cell types, but can be induced by redox-active metals, H2O2, and xenobiotics. SOD1 knockout mice show increased incidence of liver tumors, likely caused by increased oxidative stress (63). SOD2 is most commonly thought of as the inducible form; its levels can increase 10-fold in response to certain drugs and cytokines. Increased mitochondrial SOD2, as well as concomitant resistance to drug-induced oxidative damage, has been proposed as a mechanism of drug resistance in some lung cancers (64). Lack of SOD2 results in increased oxidative damage in the liver (65), and overexpression protects against oxidative stress (66). In contrast, SOD3 does not play a significant role in H2O2 detoxification in hepatocytes. Catalase and Glutathione Peroxidase Conversion of hydrogen peroxide to water and molecular oxygen is an important function of catalase. This enzyme is largely restricted to peroxisomal compartments in the liver. Catalase acts as a scavenger of hydrogen peroxide and is activated at high H2O2 concentrations. It is expressed constitutively, roughly according to peroxisome content. Kupffer cells, with high levels of peroxisomes, are particularly high in catalase. In hepatocytes, glutathione peroxidase may be more important, because it has a higher affinity for hydrogen peroxide. This cytosolic enzyme uses the reducing power of glutathione to detoxify hydrogen peroxide or other peroxides and phase I electrophiles (59). As described later, redox cycling of glutathione creates a large capacity for glutathione peroxidase-mediated detoxification of H2O2 and other oxidants in hepatocytes. Chemical Antioxidants in the Liver High levels of chemical antioxidants form a main line of defense against oxidative damage. Important compounds are glutathione, thioredoxin, vitamin C, and vitamin E. Glutathione Glutathione is the major intracellular antioxidant in the liver. It is present in both reduced (glutathione) and oxidized (glutathione disulfide [GSSG]) forms at a total concentration approaching 10 mM. The precise relative amounts depend on the redox status of the cell, but in healthy cells, glutathione predominates over GSSG by as much as 100-fold (67). Glutathione is held in a reduced state by glutathione reductase and thioredoxin. Conditions of high oxidative stress lead to depletion of glutathione, which, in turn, increases the biosynthetic activity of γ-glutamylcysteine synthase to restore glutathione to normal levels. Glutathione is important as a cofactor for antioxidant enzymes, as a scavenger of ROS,

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1495 and as a reducing agent for glutaredoxin, an important molecular link to signal transduction pathways that maintain cellular redox status (68). As described earlier, phase II conjugation of glutathione to reactive electrophiles is an important detoxification mechanism catalyzed by one of many glutathione transferases found in hepatocytes (69). In the absence of additional oxidative stress, experimental depletion of glutathione from liver cytosol has only minimal effects on overall lipid peroxidation and cell survival. Challenge with exogenous free radicals after glutathione depletion elicits severe cytotoxicity, however. In humans, the concentrations and redox state of glutathione are tightly regulated, and extreme conditions are required to deplete glutathione to toxic levels. Enzymatic regulation of the glutathione redox cycle is critical for maintaining levels of glutathione and the intracellular reducing environment. Glutathione reductase reduces GSSG to form glutathione, whereas glutathione peroxidase catalyzes the reverse reaction. Oxidative stress increases GSSG and stimulates the reductase, leading to regeneration of glutathione using reducing equivalents from NADPH. Severe oxidative stress can cause the NADPH/NADP pool to turn over as often as once per minute (57), which is a significant burden on the reducing capacity of hepatocytes. Because regeneration of NADPH requires ATP, the antioxidant capacity of the liver is linked directly to its ability to generate energy (59). Thus, hepatocyte injury caused by oxidative stress depletes the capacity of the liver for mounting a sustained antioxidant response, allowing even more injury, which, in turn, leads again to less response, potentially degenerating to acute liver failure. Glutathione is compartmentalized in the cell. Most is found in the cell cytoplasm in a labile pool with a relatively short half-life. This is the pool that functions in detoxification of xenobiotics, either via antioxidant properties or phase II conjugation. Approximately 10% of cellular glutathione is found in a more stable mitochondrial pool, where it is thought to function primarily in detoxification of endogenous oxidants (57). Glutathione is imported from the cytosol, rather than being synthesized in mitochondria, but a distinct and highly active glutathione redox cycle maintains the reducing environment in this compartment. Certain conditions, including chronic ethanol exposure, inhibit glutathione import and contribute to oxidative stress in mitochondria. Depletion of mitochondrial glutathione reduces the threshold for apoptosis induced by other agents, but is not a required step in this cell death pathway (70).

oxidative stress in the pathophysiology of liver diseases including steatohepatitis and cirrhosis (73,74). Thioredoxin reductase uses NADPH to maintain levels of reduced thioredoxin in much the same way the glutathione reductase maintains glutathione. Thioredoxin levels are high in hepatocytes relative to other cell types, but are two to three orders of magnitude below glutathione (75). Thioredoxin functions together with redox factor-1 in activation of transcription factors including activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and the tumor suppressor p53, thus linking cell signaling to the cellular redox state (72,76). Thioredoxin can also sequester and detoxify heavy metals. Vitamins C and E Some vitamins may have an antioxidant role, based on their chemical properties in vitro. Two of the most active vitamins, C and E, were likely assigned roles as cellular antioxidants in part because they have no other known biochemical function in humans. In contrast, vitamin A and related compounds have numerous effects mediated by their interactions with retinoid family receptors (77). These receptor-mediated effects make putative antioxidant properties more difficult to establish. Vitamin C is water soluble and actively eliminated in urine, which may represent a pathway for iron excretion. However, its ability to chelate bioactive metals such as iron to maintain both solubility and redox activity may have deleterious effects by facilitating redox cycling and ROS generation in metabolically active tissues such as liver. This has prevented its use in iron overload diseases such as hereditary hemochromatosis (HH). Experiments in vitro, but not large-scale clinical trials, suggest mega doses of vitamin C confer cancer chemoprotective effects attributable to quenching of oxidative stress (78). The liver is important for metabolism and storage of the fat-soluble vitamins A, D, E, and K, of which vitamin E is most important as an antioxidant. Its only established cellular function is in preventing oxidative damage, particularly to membrane lipids. The most prevalent form of vitamin E, α-tocopherol, is selectively retained by the liver after absorption from the gut and packaging into very low-density lipoprotein for delivery to other tissues (59). Supplemental vitamin E can be used as an antioxidant to prevent liver damage in alcoholic liver disease, hemochromatosis, and steatohepatitis, all diseases thought to have a component involving oxidative stress. α-Tocopherol is also showing promise in cancer prevention (56).

Thioredoxin Thioredoxin is a small (12-kDa) thiol-active protein that is also important in maintaining control of intracellular redox status (71). Its effects are analogous to glutathione. Reduced thioredoxin contains a pair of active site cys-thiols that form a disulfide bond on oxidation. It is effective at scavenging hydrogen peroxide and hydroxyl radicals (72). Expression of thioredoxin is induced under conditions of oxidative stress. An increased serum level is indicative of a state of oxidative stress, and it is one factor implicating

Oxidative Stress and Disease Drug Toxicity Insufficient detoxification can damage the liver, which, in turn, causes less efficient detoxification; a degenerative cycle results. Inability to keep up with the generation of oxygen free radicals can be caused by accelerated drug metabolizing activity, particularly upon P450 induction and/or uncoupling/ futile cycling. For example, ethanol sensitizes the liver to

1496 / CHAPTER 58 acetaminophen toxicity by inducing cytochrome P450 CYP2E1, among other mechanisms. Increased oxidative metabolism and generation of ROS, together with increased GST-mediated conjugation, depletes glutathione and causes oxidative stress. Oxidative damage further impairs antioxidant capacity, continuing a degenerative cycle that results in liver-specific drug toxicity. A significant fraction of acute liver failure, particularly in older patients, occurs when the drug/toxin challenge exceeds hepatocyte capacity (59). Other examples of acute or chronic liver injury from drug-induced oxidative damage include halothane- or isoniazid-induced acute hepatitis, chlorpromazine-induced cholestasis, tamoxifen-induced steatohepatitis, methotrexate-induced fibrosis, and chemotherapy-induced (e.g., cytosine arabinoside) venoocclusive disease. Liver Disease Oxidative stress and impaired antioxidant-based protective pathways are also contributors to cause or effect, or both, in a number of liver pathologies. Viral disease including hepatitis B and C can result in liver cirrhosis, necrosis, and failure. Oxidative stress plays a major role in progression or regression of these chronic conditions, and additional toxicity from drugs or chemicals can have compounding effects (79). Oxidative stress during ischemia and reperfusion injury, shown to cause cardiovascular disease and stroke, is likely an important factor in liver transplant (80). Other conditions that create or are affected by oxidative stress include iron overload and the inflammatory response of Kupffer cells (see discussion of both later in this chapter). This brief overview helps define the scope and implications of three important points. First, oxidative stress has many causes that collectively determine redox status and hepatocyte viability. Second, diseased or damaged livers are more sensitive to additional oxidative stress caused by drugs, even at doses that would normally be safe. Finally, targeting of antioxidant pathways in the liver should have therapeutic benefit, including depletion of antioxidant capacity as an adjuvant to cancer chemotherapy (71) and antioxidant supplementation in chemoprevention. Biochemical Pathways Stimulated by Oxidative Stress or Damage Multiple biochemical signaling pathways that regulate hepatocyte proliferation, apoptosis, or both are influenced by ROS. These effects contribute to disease pathophysiology, but they also trigger stress response pathways that limit liver damage and influence repair or regeneration. The complexities of these signaling networks make it difficult to succinctly describe how oxidative stress influences cell fate, because the physiologic outcome depends on the balance of other relevant stimuli and the severity and duration of exposure to ROS. Many studies have chronicled the activation of individual pathways by ROS or ROS-generating compounds and the inhibition by antioxidants (53). Important findings include

ROS activation of the transcription factors AP-1, NF-κB, and p53, each of which can influence the expression of many genes. Oxidative stress also is known to increase gene expression by causing DNA hypomethylation, and it can alter the levels of key regulatory proteins by influencing their degradation by proteasomes (81). Two ROS-sensitive processes that illustrate some of these complexities are interhepatocyte gap junctional communication and growth factor–stimulated regeneration of hepatocytes. Gap junctions are channels that connect the cytoplasms of adjacent cells. They are composed of connexin proteins with expression that is cell type and developmentally regulated. In hepatocytes, connexin32 is the predominant form. Both connexin expression and gap junction communication are decreased by agents that induce oxidative stress and are increased by ROS scavengers (82,83). Because gap junctional shutdown restricts diffusion of oxidative metabolites out of damaged hepatocytes and conserves ATP and glutathione levels in healthy hepatocytes, redox regulation may be important in limiting acute liver damage caused by toxins. Connexin32 acts as a tumor suppressor in mice (84), and maintaining gap junctional communication may be important in preventing neoplastic disease. The links between tumor promoters, oxidative stress, and decreased gap junctional communication support this view (85). Antioxidants (e.g., vitamin E) prevent inhibition of gap junctions by liver toxicants and tumor promoters including dichlorodiphenyltrichloroethane and phenobarbital (86). Hepatocytes in adult liver are normally quiescent cells that proliferate only in response to loss of function caused by damage or removal (87). This strictly regulated proliferation reflects the need for replacing cells that are occasionally overwhelmed by the toxins they work to eliminate. There is significant overlap and interplay between pathways activated by growth factors during liver regeneration and those influenced by ROS and antioxidants such as glutathione (88). A growth factor that triggers proliferation in one setting can induce apoptosis under conditions of oxidative stress (89). Because redox status is one indicator of viability, this may serve a quality-control function in the liver by facilitating the replacement of damaged areas with healthy hepatocytes. It may also help to determine whether stem cells are recruited to this process (87). Liver regeneration is discussed in more detail at the end of this chapter. Cancer and Chemoprevention Evidence in humans and animal models suggests oxidative stress can initiate the carcinogenesis process through gene mutation, promote the survival and outgrowth of preneoplastic cells, and facilitate the progression of dysplastic nodules to metastatic disease (53,56). Initiation depends on genetic changes caused by unrepaired DNA damage. These and subsequent mutation of oncogenes or tumor suppressor genes, or both, cause cancer (90). Oxidative DNA damage is a frequent event in humans, and an important cause of gene mutation. The hydroxyl radical has been shown to produce

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1497 many of the roughly 100 forms of oxidized DNA that have been identified (91). Among the best characterized is 8-hydroxyguanosine, a mutagenic change that causes guanineto-thymine point mutations in DNA. This suggests an initiating role for hydrogen peroxide oxidation in the nucleus, a reaction that can be catalyzed by redox cycling of transition metals including iron. The high prevalence of liver cancer among hemochromatosis patients with iron overload is consistent with the idea that bioreactive iron is carcinogenic (92,93). Tumor promoters exert a differential influence on the growth, proliferation, or apoptosis of normal compared with preneoplastic cells. Many established tumor promoters also cause oxidative stress, and antioxidants can prevent experimental tumor promotion caused by agents such as hydrogen peroxide (53). This may contribute to the cancer prevention effects attributed to dietary antioxidants in liver and other tissues (94). Oxidative damage influences many biochemical pathways that regulate gene expression, proliferation, and apoptosis in cells, and it is clearly involved in the toxicities caused by many tumor-promoting agents. It is not always clear whether or how this facilitates the outgrowth of cancer cells, however; additional work is required to establish and define causal roles of oxidative stress in tumor promotion. Lack of efficacy of individual antioxidants such as vitamins C and E in large-scale cancer chemoprevention trials (78) supports the need for further investigation. Given the diversity of oxidative chemistry in cells, it is likely that a combination of antioxidants will prove significantly more beneficial than any single agent.

Hepatocyte Protective Mechanisms: Metal Sequestration

detoxification of such metals is sequestration by metal-binding proteins. The high-affinity binding to thiols and protein sulfhydryl groups that contributes to metal toxicities is also the basis for detoxification by sequestration proteins.

Mechanism of Metal Detoxification Glutathione Binding and Elimination High-affinity interaction with sulfhydryls is an important mechanism for metal detoxification. Small antioxidant peptides and sequestration proteins commonly use cysteine residues as a source of sulfhydryl-based coordination sites for Cu, Zn, Cd, and others. A notable exception is Fe, which binds poorly to sulfhydryls and is sequestered by enzymatic loading into ferritin complexes. The liver is the site of clearance of metal-binding proteins (including albumin) from blood, long-term storage of metals by sequestration proteins, and phase II–type conjugation of metals, followed by secretion to bile or back to the bloodstream for elimination. Glutathione, thioredoxin, and metallothionein (MT) are important cys-based metal-binding molecules. Binding to glutathione in the liver leads to elimination of metals in bile or transport to blood and excretion by the kidneys, similar to what occurs for drugs and other, more hydrophobic molecules (97). Importantly, although metal-glutathione binding can be of high affinity, it is noncovalent and exchangeable. This reversible binding allows glutathione to shuttle and redistribute metals between other chelators, as well as binding proteins such as MT. At the same time, increased glutathione levels in hepatocytes can transiently increase liver toxicity of some metals by facilitating their transfer from an extracellular exchangeable pool to an intracellular exchangeable pool (76).

Basis of Free Metal Toxicity

Cadmium and General Sequestration: Metallothionein

Many essential trace metals are toxic at high concentrations, and regulating the balance between adequate supply and minimal toxicity is critical. Oxidative stress is an important contributor to toxicity. Redox cycling of transition metals such as Fe, Mn, Cu, and Ni was discussed in the previous section. Depending on the chelator and the pH, each of these metals can catalyze the Fenton and Haber–Weiss reactions (95). Unless halted, this results in the catalytic generation of toxic hydroxyl radicals. Metals that are not redox active may cause oxidative stress indirectly by interfering with the sequestration or metabolism of metals that are redox active, such as Cu or Fe. They may also cause oxidative stress by blocking antioxidant defenses or by triggering an immune reaction that generates ROS (96). Metals also exert toxic effects by binding and interfering with sulfhydryl/disulfide transitions in enzymes. This can restrict enzyme conformation and activity. Toxic metals can displace endogenous metals from enzyme active sites, reducing or destroying catalytic activity. Cadmium (Cd), for example, is physically similar to Ca and can interfere with Ca-dependent regulation of enzymes involved in signaling and gene regulation. A major path for

The MTs are a family of low-molecular-weight proteins with a high cysteine content. They sequester metals including Zn, Cu, and Cd. The liver is the main organ for metal sequestration by MT. There are 10 MT genes and 4 biochemically identified isoforms that include the 2 major liver isoforms MT-1 and MT-2 (98). MT-1 contains 20 cysteine residues (of 61 total amino acids) grouped in 2 clusters that form coordination sites for up to 7 metal ions. In the absence of increased metals or other stressors, low levels of MT store Zn for use by the cell and may function as an antioxidant, based on the ability to bind hydroxyl radicals. Evidence for the role of MT in metal detoxification includes increased MT expression on challenge with toxic metals such as Cd. In addition, MT induction by high levels of Zn can protect against subsequent exposure to more highly toxic metals such as Cd. Zn-induced up-regulation of MT is an effective maintenance therapy for Cu overload as well. MT-null mice develop normally, albeit with increased sensitivity to Zn deficiency and to toxicity from Cd or other metals; overexpression of MT confers resistance to Cd toxicity (99). Tight binding to metals such as Cd and mercury (Hg) differentiates the

1498 / CHAPTER 58 detoxification function of MT from its function as a transient store of physiologically exchangeable metals such as Zn and Cu. Cd that is sequestered by MT in the liver is poorly excreted and accumulates with time (100). Iron (Fe) binds weakly and cannot induce MT; other mechanisms exist to regulate and sequester bioactive iron. Iron Sequestration: Ferritin Iron is an essential metal required for a number of key physiologic processes including oxygen metabolism. Mechanisms to ensure that the intracellular level of redox-active iron is optimized are acutely responsive to growth and metabolic needs. Because iron excretion from the body is low and essentially unregulated, iron levels are determined by the level of intake. When iron levels exceed iron needs, uptake from the gut is minimized and levels of iron regulatory proteins are adjusted (101). Under conditions of iron excess, cells, including hepatocytes, down-regulate iron import proteins such as transferrin receptors and up-regulate the iron storage protein ferritin. Hepatocytes are unique in that they also secrete hepcidin under these conditions. This hormone reduces plasma iron by inhibiting iron uptake in the small intestine, as well as iron excretion from hepatocytes and macrophages (102,103). Excess iron must be sequestered in a nontoxic form. This is accomplished primarily by binding to ferritin in hepatocytes. Ferritin is a spherelike complex composed of 24 heavy- and/or light-chain protein subunits. Its surface is permeated by ion channel-like pores that allow iron to access internal binding sites (104). Soluble Fe(II) enters the complex and is converted enzymatically to insoluble Fe(III) and deposited in the core. Up to 4000 Fe(III) atoms can be sequestered within a single ferritin complex. This process can be reversed by superoxide ion to cause release of Fe(II) from the ferritin complex. Iron release likely contributes to the pathophysiologic effects of superoxide during an inflammatory response (96). Gene knockout experiments in mice have demonstrated that ferritin is essential, and there are no known human diseases involving major defects in ferritin structure, also implying critical function. Ferritin is an efficient iron depot; iron is sequestered in ferritin at concentrations 100 trillion times the solubility limit without ferritin (105). The biosynthesis of proteins controlling iron metabolism, including ferritin, are regulated in an iron-dependent fashion. Free iron modifies the activities or levels of iron-responsive proteins (IRPs) capable of binding to iron-responsive elements (IREs) of mRNA. This regulates the translational efficiency or stability, or both, of various mRNA molecules. For ferritin, binding of iron-free IRP1 to the IRE in the ferritin mRNA decreases translational efficiency and keeps ferritin levels low when free iron is low (106). This inhibition is relieved by binding of iron to IRP1, resulting in increased translation of ferritin mRNA. Additional levels of cellular regulation are suggested by inhibitory effects of ROS (107) and serine phosphorylation (108) on levels of iron-liganded IRP1. A distinct mechanism that controls the half-life of IRP2, rather than its specific activity, is relevant for the transferrin receptor.

Iron causes oxidation and increased proteosomal degradation of IRP2 (109). Loss of IRP2 destabilizes the mRNA complexes encoding the transferrin receptor and accounts for the decreases in receptor levels caused by increased iron (110). Increased proteosomal degradation may represent a general mechanism for removing proteins damaged by oxidative stress. IRP-based mechanisms also are thought to coordinately regulate other proteins involved in iron homeostasis (101). Copper Secretion Like iron, Cu is redox active and can catalyze the generation of hydroxyl radicals. It is also an essential metal, and its metabolism is tightly regulated. Intracellular Cu binds transiently to Cu chaperones for shuttling to one of several destinations in hepatocytes (111): Cu can be incorporated biosynthetically into a number of essential enzymes, such as SOD; Cu can be delivered to the lumen of the trans-Golgi network, where it is packaged into ceruloplasmin for secretion to other cells and tissues; Cu can be bound to glutathione and eliminated to bile via the MRP2 transporter, or sequestered by MT; and finally, at high levels, it can be secreted directly. Under these conditions, the ATP7B transporter is translocated from its normal position in the trans-Golgi network to the canalicular membrane, where it pumps Cu to bile. As described in the next section, this protein is defective in Wilson’s disease (112,113). Diseases of Metal Transport Research on the pathophysiologic mechanisms of two human diseases, HH and Wilson’s disease, has provided important information on Fe and Cu metabolism and detoxification by hepatocytes. Iron Regulation and Hereditary Hemochromatosis Patients with HH and iron overload have an increased frequency of liver pathologies including fibrosis, cirrhosis, liver failure, and hepatocellular carcinoma (HCC) (113,114). The primary defect in most patients with HH is mutation of the HH (HFE) gene, which encodes a membrane protein thought ultimately to regulate the iron uptake capacity of enterocytes, possibly via influencing transferrin receptor kinetics in precursor crypt cells (115). Increased ferritin content and hemosiderin deposits in HH hepatocytes support the central role of the liver in iron sequestration and detoxification. This iron overload and the resulting clinical symptoms in homozygotic HH patients can take many years to appear, and sometimes never do, suggesting that other contributing factors are important. Thus, although phlebotomy is an effective treatment, identification of patients for which it is warranted can be difficult (113). More research is needed to fully understand the pathologic basis for this disease. For example, much remains to be learned about transferrinindependent mechanisms of iron uptake by hepatocytes.

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1499 Nontransferrin-based mechanisms for iron internalization must exist, because free iron in plasma remains low even on transferrin saturation in HH, and transferrin-null mice experience iron overload in liver and other tissues. These pathways may be particularly relevant to detoxification of iron. Copper Secretion and Wilson’s Disease Excess Cu also leads to loss of reducing equivalents in the liver and an increase in plasma lipid peroxides, together with increased frequency of cirrhosis, necrosis, and liver failure or HCC (112). Wilson’s disease, a condition characterized by impaired secretion of Cu from hepatocytes to bile, exemplifies the consequences of Cu overload. This disease is caused by mutation in the gene encoding the Cu transporter ATP7B. Without functional ATP7B, Wilson’s disease hepatocytes do not secrete Cu to bile, and they frequently do not synthesize and secrete normal levels of ceruloplasmin. Increased oxidative damage caused by increased free Cu in hepatocytes, activation of apoptotic pathways and release of free Cu to serum, and eventual Cu overload in all tissues characterize disease progression (116). These links between Fe or Cu overload and liver pathologies illustrate the toxicities of these redox-active metals and underscore the importance of metal detoxification by hepatocytes.

Hepatocyte Protective Mechanisms: Clearance and Regeneration Hepatocytes that are destroyed by toxic agents must be cleared and replaced. The processes involved are related to the severity of damage. For mild toxicities, apoptosis predominates and the response may be limited to the apoptotic hepatocytes and their close neighbors. More severe necrosis frequently involves Kupffer cells that first attack and then stimulate repair at the site of injury. A number of studies have shown that inhibition of Kupffer cell function can decrease drug-induced liver damage, whereas activation of Kupffer cells makes damage worse. Over the long term, however, Kupffer cells are required for maintaining detoxification systems in the liver. They clear dead hepatocytes and are thought to secrete cytokines that govern liver regeneration. The liver is unique among organ systems in its ability to regenerate. This property must reflect the toxicities inherent in metabolizing and clearing toxins; without it, the liver would be much less robust as a detoxifying organ. This final section on hepatocyte defense describes two physiologic mechanisms for surviving severe toxic onslaught: (1) the role of Kupffer cells in hepatocyte clearance and cytokinemediated preservation; and (2) the mechanisms involved in hepatocyte regeneration. Kupffer Cells Kupffer cells account for approximately 80% of the body population of macrophages and constitute approximately

30% of hepatic sinusoidal cells (117). Other nonparenchymal liver cell types include endothelial cells, stellate cells, oval cells, and pit cells. A major function of Kupffer cells is to remove particulate matter, foreign material, and gut-derived toxins from the portal circulation. They express multiple cell-surface phagocytic receptors that enable clearance of damaged or apoptotic cells, foreign particles, and tumor cells (118–123). They also are able to take up and detoxify the lipopolysaccharide portion of gut-derived endotoxins via scavenger receptors, macrosialin, and/or CD14 (122,124,125). Kupffer cells can be distinguished by their morphology, localization, receptor content, and primary function. Like other macrophages, Kupffer cells become activated in response to stimulation. This changes their cellular physiology from an efficient remover of particulate waste to an aggressive attacker of pathogens. Activated cells are larger than unstimulated Kupffer cells, with an increased internal membrane structure and increased ability to synthesize and secrete bioactive compounds. Phagocytic receptor content can also increase. Once activated, Kupffer cells secrete ROS (superoxide, hydrogen peroxide, nitric oxide, peroxynitrite) that stimulate fibrosis induced by neighboring stellate cells. The resulting matrix maintains liver morphology during regeneration. ROS, together with other secreted compounds (eicosanoids and proteolytic enzymes), also aid in antigen destruction (117). Cytokines secreted by activated Kupffer cells can initiate an immune response that recruits neutrophils and other cells of the immune system. Although a robust immune response is critical to defense against invading pathogens, it has no benefit in chemical detoxification, and thus is not discussed in detail here. Information on the liver as an immune organ and the immunity-related roles of Kupffer cells can be found elsewhere in the literature (126–128). Importantly, Kupffer cell distribution in resting, undamaged liver (high in periportal and central regions of the liver lobule) reflects low-level activation and recruitment to regions that are in contact with a relatively constant stream of portal endotoxins. As described earlier, necrosis results from damage to multicell structures. Unlike apoptosis, it is not regulated by the dying cell, and considerable cellular debris remains and must be removed. The clearance of aged or damaged erythrocytes by Kupffer cells has been well documented (129); an analogous process for damaged hepatocytes is thought to occur. Kupffer cell clearance of hepatocyte remnants in damaged liver allows for normal liver architecture to be restored after toxic damage. Because Kupffer cell activity can trigger further hepatocyte toxicity, as well as fibrosis and cirrhosis that impairs or prevents recovery, it is thought that low to moderate activation forms the best response to liver damage by toxins. In support of this idea, inhibition of Kupffer cell activation has been reported to decrease drug toxicity. For example, the protective effects of interleukin-10 (IL-10) in treating liver-induced drug toxicity may stem from inhibition of Kupffer cell activation (130). Conversely, activation of Kupffer cells likely contributes significantly to disease pathology in, for example, alcoholic liver disease (131).

1500 / CHAPTER 58 Therapies that limit Kupffer cell activation or the resulting cytotoxic effects may prove beneficial in drug-induced and ischemia-related liver disease. However, complete inhibition is not beneficial because of risk for infection and impaired regeneration of hepatocytes. Hepatocyte Regeneration Liver regeneration is a remarkable process that has been appreciated since at least the time of the ancient Greeks, who described it in the legend of Prometheus. It has important clinical implications. In acute acetaminophen toxicity or severe viral hepatitis, for example, treatment consists of support to keep the patient alive until dead hepatocytes are cleared and regenerated. Regeneration begins while hepatocyte remnants are being cleared by Kupffer cells. Not only are the number and mass of hepatocytes and nonparenchymal cells fully recovered, but liver architecture is largely restored (132). Even after substantial removal of diseased regions, the human liver can return to normal within weeks, while at the same time maintaining relatively normal function. Many of the critical mechanistic pieces of the hepatocyte regeneration puzzle have been identified; the current challenge is to fit the pieces together and apply this knowledge to the prevention and treatment of liver disease. Results from chemical toxicity and partial hepatectomy experiments in animals, mechanistic studies in cultured cells, and observations of human responses to disease or resection suggest several important concepts. First, proliferation of mature, differentiated hepatocytes is tightly regulated during liver regeneration. Second, many of the growth factors and cytokines responsible for this regulation are secreted by Kupffer and other nonparenchymal liver cells. Third, oval cells are stem cells found in adult liver that fuel regeneration when proliferation of existing hepatocytes cannot occur. Finally, clinical conditions characterized by extensive but imperfect liver regeneration (i.e., with cirrhosis) are linked to HCC, implicating defects in regeneration as a factor in this disease. Hepatocytes in adult liver have an estimated lifespan of roughly 1 year, and thus exist predominately in a quiescent, nondividing state (132). This situation changes dramatically upon drug-induced toxicity or partial hepatectomy. Under most conditions, hepatocyte proliferation is initiated within 12 hours and continues until liver mass is restored. In partially hepatectomized animals, hepatocytes regenerate first, followed closely by increases in Kupffer and biliary cells, and finally by endothelial cells. This process begins in cells nearest the portal vein, progresses toward the central vein, and is largely complete within 3 days. Regeneration is less coordinated after drug-induced necrosis, perhaps because the process involves cells that are themselves partially damaged. The rapid restoration of cell content is followed by a slower remodeling process in which extracellular matrix deposited by stellate cells guides the reformation of normal liver architecture.

Effects of Mitogens and Cytokines Hepatocyte regeneration is influenced by many physiologic factors and cell types. Kupffer cell activation is a critical early step; this may be initiated by phagocytosis of cell remnants from dead hepatocytes, or possibly by increased endotoxin exposure that accompanies increased portal blood flow to the damaged regions (133). Activated Kupffer cells secrete factors, including tumor necrosis factor (TNF)-α and IL-6, that act directly on hepatocytes to initiate entry into the cell cycle. ROS from Kupffer cells activate stellate cells that are a primary source of mitogenic hepatocyte growth factor (HGF) and antimitogenic transforming growth factor (TGF)-β, which are important regulators during the G1-phase of the hepatocyte cell cycle. HGF release from stellate cells can also be initiated via vascular endothelial growth factor (VEGF) binding to endothelial cells. Many growth factors and hormones from other cell types have a permissive role in hepatocyte proliferation, including insulin from the pancreas, epidermal growth factor (EGF) from the duodenum, triiodothyronine from the thyroid, and norepinephrine from the adrenal gland. Hepatocytes are well equipped to respond to external stimuli, with cell-surface receptors for many mitogens and cytokines. They are also active secretory cells and communicate through paracrine and hormonal mechanisms with the other liver cell types. Gene expression studies have identified more than 100 hepatocyte gene products that are altered during the early phases of regeneration; many are related to signal transduction and transcription factor pathways (133). Fausto (88) proposed a process of hepatocyte priming by TNF-α or IL-6, or both, that occurs early in the regeneration process. Priming is required for maximal proliferative effects of HGF, TGF-α, and EGF. The conditions needed for TNF-α to act as a priming agent, rather than as an inducer of apoptosis, are not fully established, but are influenced by antioxidant status in the hepatocyte (see Hepatocyte Protective Mechanisms: Oxidative Neutralization section earlier in this chapter). Some mitogenic factors (e.g., HGF and IL-6) also have hepatoprotective effects, based on their ability to induce antiapoptotic proteins including bcl-2 and caspase inhibitors. Signals governing the termination of regeneration are less well characterized. Under some conditions, TGF-β acts as an inhibitor of hepatocyte proliferation. TGF-β is secreted by stellate cells and may contribute to poor regeneration in areas high in fibrosis. Involvement of Oval Cells In some types of drug-induced liver toxicity, hepatocytes are too severely poisoned to proliferate. Under these conditions, hepatocyte regeneration is accomplished through the proliferation and differentiation of oval cells (87). These cells originate from adult stem cells found in the canals of Hering. Oval cell proliferation was described originally in studies on the toxicities of chemicals such as 2-acetylaminofluorene (134).

MECHANISMS OF HEPATOCYTE DETOXIFICATION / 1501 Since that time, a precursor-product relation between oval cells and hepatocytes has been demonstrated in rats (135,136). Current research is focused on identifying mitogens and cytokines that regulate proliferation and differentiation of oval cells (137). Stem cell factor and its receptor, c-kit, are both expressed in oval cell precursors and may comprise an autocrine loop governing early proliferation. In general, the effects of factors secreted by Kupffer cells and stellate cells on oval cells are similar to their effects on hepatocytes. Oncostatin M has been implicated as a cytokine required for differentiation of oval cells into hepatocytes (138). Factors that favor oval cell neoplastic transformation such as insulinlike growth factor II (IGF-II) are also of interest, because of the possibility that aberrant development of oval cells contributes to HCC (139). Importantly, recruitment of oval cells for replacement of damaged hepatocytes may be a general feature of liver defense, because proliferation of this cell type is seen in response to a variety of toxicants and in chronic liver disease (140–143).

6.

7. 8.

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10. 11. 12.

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SUMMARY

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In summary, hepatocytes are the ultimate detoxifying cells in an organ that has perfected this task, with a high concentration of nonspecific metabolizing enzymes that can handle virtually any chemical toxin, an array of mechanisms for disposing of these compounds, and robust mechanisms for self-preservation and regeneration. Continued study undoubtedly will result in therapeutic interventions that compensate for detoxification deficiencies caused by genetic and infectious disease, drug interactions and overdose, and environmental misfortune.

15.

ACKNOWLEDGMENTS

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This study was supported by National Institutes of Health grants AT000815 (D.J.M.) and DK02957 (K.V.K.).

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133. Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 2004;5:836–847. 134. Farber E. Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylamino-fluorene, and 3′-methyl-4-dimethylaminoazobenzene. Cancer Res 1956;16:142–148. 135. Evarts RP, Nagy P, Marsden E, Thorgeirsson SS. A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 1987;8:1737–1740. 136. Evarts RP, Nagy P, Nakatsukasa H, Marsden E, Thorgeirsson SS. In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res 1989;49:1541–1547. 137. Lowes KN, Croager EJ, Olynyk JK, Abraham LJ, Yeoh GC. Oval cell-mediated liver regeneration: role of cytokines and growth factors. J Gastroenterol Hepatol 2003;18:4–12. 138. Okaya A, Kitanaka J, Kitanaka N, Satake M, Kim Y, Terada K, Sugiyama T, Takemura M, Fujimoto J, Terada N, Miyajima A, Tsujimura T. Oncostatin M inhibits proliferation of rat oval cells, OC15-5, inducing differentiation into hepatocytes. Am J Pathol 2005; 166:709–719. 139. Zhang N, Siegel K, Odenthal M, Becker R, Oesch F, Dienes HP, Schirmacher P, Steinberg P. The role of insulin-like growth factor II in the malignant transformation of rat liver oval cells. Hepatology 1997;25:900–905. 140. Hsia CC, Evarts RP, Nakatsukasa H, Marsden ER, Thorgeirsson SS. Occurrence of oval-type cells in hepatitis B virus-associated human hepatocarcinogenesis. Hepatology 1992;16:1327–1333. 141. Smith PG, Yeoh GC. Chronic iron overload in rats induces oval cells in the liver. Am J Pathol 1996;149:389–398. 142. Smith PG, Tee LB, Yeoh GC. Appearance of oval cells in the liver of rats after long-term exposure to ethanol. Hepatology 1996;23:145–154. 143. Lowes KN, Brennan BA, Yeoh GC, Olynyk JK. Oval cell numbers in human chronic liver diseases are directly related to disease severity. Am J Pathol 1999;154:537–541.

CHAPTER

59

Physiology of Cholangiocytes Anatoliy I. Masyuk, Tatyana V. Masyuk, and Nicholas F. LaRusso Architecture and Phenotypic Characteristics of the Intrahepatic Biliary Ductal System (Brief Overview), 1506 Architecture, 1506 Microscopic Anatomy, 1506 Biochemical Heterogeneity, 1507 Molecular Physiology of Ductal Bile Formation, 1508 Basic Principles of Bile Formation, 1508 Cholangiocyte Transport Systems, 1508 Intracellular Signaling, 1513 Cyclic 3′,5′-Adenosine Monophosphate Signaling, 1514 Calcium Signaling, 1514 Cyclic 3′,5′-Guanosine Monophosphate Signaling, 1515

Regulation of Ductal Bile Formation, 1515 Regulation of Basal Cholangiocyte Secretion, 1516 Potentiation of Secretin-Stimulated Cholangiocyte Secretion, 1519 Inhibition of Basal and Secretin-Stimulated Cholangiocyte Secretion, 1520 Regulation by Bile-Borne Factors, 1521 Vesicular Trafficking in Mechanisms of Ductal Bile Formation, 1523 Functional Heterogeneity of Cholangiocytes, 1524 Integrated Model of Ductal Bile Formation, 1524 Concluding Remarks, 1525 Acknowledgments, 1525 References, 1525

Cholangiocytes are epithelial cells that line the lumen of bile ductules and ducts in the liver (1–7). Even though they account for only 3% to 5% of the hepatic cell population, cholangiocytes line the intrahepatic biliary ductal system with an estimated length of more than 2 km in an adult human liver (8,9). Early in the twentieth century, indirect observations suggested that cholangiocytes not only form conduits for bile produced by hepatocytes, but also play an important functional role in bile formation (9–12); however, attempts to directly define cholangiocyte functions were hindered by the lack of suitable experimental models. Our knowledge of cholangiocyte biology has advanced considerably since the early 1990s, based primarily on new experimental models such as isolated and cultured rodent and human cholangiocytes (2,13–29) and intact rodent intrahepatic bile duct units

(IBDUs) (4,30–36), which have enabled the direct study of cholangiocyte biology and have led to the conclusion that the major physiologic function of cholangiocytes is bile formation (1–7,37–52). This chapter provides an overview of what is known about cholangiocyte physiology, focusing primarily on molecular mechanisms of bile formation (i.e., cholangiocyte ion, solute, and water transport) and their regulation. Architecture and phenotypic characteristics of the intrahepatic biliary ductal system and basic principles of bile formation are described briefly in the first section of this chapter, followed by more detailed descriptions of each of the known cholangiocyte transport systems, the intracellular signaling mechanisms, and regulation of cholangiocyte secretion/absorption by a number of choleretic/cholestatic factors, including hormones, neurotransmitters, nucleotides, and bile-borne factors. An integrated model of ductal bile formation based on our current knowledge of cholangiocyte physiology is discussed at the end of the chapter. The cholangiocyte functions of pathophysiologic importance such as proliferation (1–3,5,6, 53–61), apoptosis (62–70), involvement in drug metabolism (3,6,71,72) and inflammatory processes within the liver

A. I. Masyuk, T. V. Masyuk: Department of Medicine, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. N. F. LaRusso: Departments of Medicine, Biochemistry, and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1505

1506 / CHAPTER 59 (6,72–76), interactions with the immune system (3,6,72,77,78), and microorganisms (6,72,79) have been discussed extensively elsewhere and are beyond the scope of this chapter.

ARCHITECTURE AND PHENOTYPIC CHARACTERISTICS OF THE INTRAHEPATIC BILIARY DUCTAL SYSTEM (BRIEF OVERVIEW) Architecture The intrahepatic biliary ductal system is formed by a three-dimensional network of interconnecting bile ductules and ducts of different sizes, and extends from the canals of Hering to the extrahepatic bile ducts (Fig. 59-1) (3,5,39,44, 51,80–88). The canals of Hering, lined partly by hepatocytes and partly by cholangiocytes, represent the anatomic and physiologic link between hepatocyte canaliculi and the biliary tree (80,82,89–91). More specifically, the canals are anatomic structures through which primary or canalicular bile secreted by hepatocytes enters the intrahepatic bile ducts. In humans, the intrahepatic biliary ductal system is divided according to size into ductules, small ducts (interlobular and septal) and large ducts (area, segmental, and hepatic) (see Fig. 59-1) (3,5,6,82,92,93). Bile ductules (lumen diameter

Ductules (<15 µm)

Canals of hering Interlobular ducts (15-100 µm)

< 15 µm) connect interlobular ducts (lumen diameter 15–100 µm) with the canals of Hering. Septal ducts (lumen diameter 100–300 µm) originate at the confluence of two or more interlobular ducts and drain into area ducts (lumen diameter 300–400 µm). Area ducts progress into segmental (lumen diameter 400–800 µm), and then hepatic (lumen diameter > 800 µm) ducts. In the rat, the intrahepatic biliary ductal system is divided into small bile ducts (lumen diameter < 15 µm) and large bile ducts (lumen diameter > 15 µm) (Fig. 59-2) (6,55,84,92–97). Small and large intrahepatic bile ducts, but not bile ductules, receive their blood supply from a periductal network of minute vessels, termed the peribiliary vascular plexus, which originates from hepatic artery branches and drains into the portal veins or directly into hepatic sinusoids (88,98–102). It has been suggested that the peribiliary vascular plexus plays an important role in the physiologic functions of the biliary epithelia. In particular, it may participate in the transfer of solutes between blood and bile, as well as in the supply and drainage of the substances to and from cholangiocytes (55,99,102,103).

Microscopic Anatomy In humans, small bile ducts are encircled by 4 to 5 cholangiocytes; large bile ducts may contain up to 40 cholangiocytes (3,55,80). In rats, small bile ducts are also lined by 4 to 5 cholangiocytes, whereas large bile ducts are lined by 8 to 15 cholangiocytes (Fig. 59-3A) (94,95,104). Cholangiocytes differ in size (i.e., 6–15 µm in diameter) and shape (i.e., morphologically heterogeneous), being flattened or cuboidal in small bile ducts and columnar in large bile ducts (16,80,81,89,94–96,104). Large and small cholangiocytes

Septal ducts (100-300 µm)

Area ducts (300-400 µm)

Small bile ducts (<15 µm)

Segamental ducts (400-800 µm)

Right hepatic duct (>800 µm) Left hepatic duct (>800 µm) Commom hepatic duct

FIG. 59-1. Schematic representation of the human intrahepatic biliary ductal system. The intrahepatic biliary ductal system is a three-dimensional network of interconnecting conduits of different sizes (shown in parentheses) extending from the canals of Hering, through which primary or canalicular bile enters the biliary tree, to the common hepatic duct.

Large bile ducts (>15 µm)

FIG. 59-2. A microphotograph of a portion of the rat intrahepatic biliary ductal system located close to the edge of the liver. This portion of the intrahepatic biliary tree scanned by a micro-computed tomography scanner with resolution of 6 µm contains both small and large bile ducts. The approximate size of intrahepatic bile ducts is shown in parentheses.

PHYSIOLOGY OF CHOLANGIOCYTES / 1507 A

B

Lumen

MV

TJ

APM

BPM

C

D

FIG. 59-3. (A) Transmission electron micrograph of rat liver. A large bile duct lined by 11 cholangiocytes is seen. (B) Cholangiocytes are polarized cells with apical (APM) and basolateral (BPM) plasma membrane domains. They possess well-developed microvilli (MV) and tight junctions (TJ). Scanning (C) and transmission (D) electron micrographs of cholangiocyte cilia extending from the apical surface into the ductal lumen. Inset in D shows the cross section of a primary cilium with “9+0” arrangement of its microtubules. Original magnification ×2000 (A); ×15,000 (B). Scale bars = 1 µm (C, D). (C, D: Reproduced from Masyuk and colleagues [112], by permission.)

have different nuclear-to-cytoplasmic ratios; this ratio is greater in small than in large cholangiocytes, suggesting that large cholangiocytes are more differentiated cells than small cholangiocytes (55,104). Importantly, in the rat, small cholangiocytes, which are normally mitotically dormant, may proliferate under pathologic conditions and acquire functional features of large cholangiocytes (55,59). Cholangiocytes possess discrete, specialized apical (luminal) and basolateral plasma membrane domains demarcated by tight junctions located in proximity to the apical domain between adjacent cells (16,22,105) (see Fig. 59-3B). They also possess gap junctions, that is, channels that permit direct communication between adjacent cells through the exchange of ions and small molecules (52,106). Cholangiocytes have numerous microvilli on their apical surface, which provide a fivefold increase in surface area (see Fig. 59-3B) (16,22,83,105,107,108). In addition, each cholangiocyte contains a primary cilium, a solitary, long, tubular structure projecting into the bile duct lumen (see Figs. 59-3C and D) (16,109–112). Primary cilia are expressed in most mammalian cells and are considered distinct organelles (113–117); although long known to exist, much attention has been focused on these organelles only in the last five years because of their importance in normal cell function and in disease (113–117). The primary cilium arises from the mature mother centriole of the mother/daughter pair of

centrioles and consists of a basal body and a membranebound “9+0” axoneme that contains nine outer-doublet microtubules, but lacks a central pair of microtubules and dynein arms, components typical for other types of cilia (i.e., motile cilia with “9+2” axonemes) (113–117) (see Fig. 59-3D). Primary cilia in cholangiocytes are poorly studied, and their functions are obscure. Developments have suggested (see Bile Flow section later in this chapter) that cholangiocyte cilia are likely sensory organelles involved in regulation of ductal bile formation by bile-borne regulatory factors (i.e., bile flow, osmolarity, and composition). Cholangiocytes possess an actin cytoskeleton, which is critically involved in structural and functional support of the plasma membrane establishing and maintaining cell polarity and regulating vesicle trafficking and distribution of membrane proteins (118–120). Coated pits and vesicles have been seen on both cholangiocyte apical and basolateral plasma membrane domains, suggesting that receptor-mediated endocytosis occurs in these areas (121). Cholangiocytes have sparse endoplasmic reticulum, but a prominent Golgi apparatus located near the apical membrane, small mitochondria around the nucleus, and intercellular lacunae (16,55,102–104,122). The nucleus is round to oval and often notched, and it is located basally in these cells (see Fig. 59-3B). Vesicles, lysosomes, and multivesicular bodies are present near the apical pole of cholangiocytes (44). Thus, morphologically, cholangiocytes are typical epithelial cells that provide a large surface area to the intrahepatic biliary ductal system, consistent with the occurrence of secretory and absorptive processes in this system.

Biochemical Heterogeneity Cholangiocytes are not only morphologically heterogeneous along the biliary tree, but they are also biochemically heterogeneous. Although many proteins reflecting a cholangiocyte phenotype are expressed in both small and large cholangiocytes, some proteins are expressed in small but not large cholangiocytes and vice versa. These proteins include enzymes, receptors, transporters, exchangers, and channels (55,92,94). In humans, both small and large cholangiocytes express pancreatic lipase and α-amylase, trypsin (123,124), and the Cl--HCO −3 exchanger (124,125). However, small but not large cholangiocytes express blood group antigens (i.e., A, B, H, Lewisa, Lewisb, and sialylated Lea) (126) and the antiapoptotic protein Bcl-2 (127,128). In contrast, cytochrome P4502E1 is expressed in large but not in small cholangiocytes (129,130). In the rat, both small and large cholangiocytes express endothelin (ETA and ETB) receptors and two cytokeratins, CK-7 and CK-19 (131). However, large but not small cholangiocytes express γ-glutamyltranspeptidase, alkaline phosphatase and leucine amino peptidase (132), cytochrome P4502E1 (63,64), secretin and somatostatin receptors, cystic fibrosis transmembrane conductance regulator (CFTR) and a Cl--HCO −3 exchanger (94–96), the sodium-dependent bile

1508 / CHAPTER 59 acid transporter ASBT (133), and calelectrins, proteins that regulate intracellular concentration of Ca2+ ([Ca2+]i) (134). Both small and large cholangiocytes express adenylyl cyclase (AC) isoforms, that is, membrane-bound, AC4 to AC9, and soluble AC (sAC). However, although AC4 and AC7 gene expression is relatively more abundant in small cholangiocytes, the AC8 isoform is uniquely expressed in large cholangiocytes (135). In addition, agonist-induced cyclic 3′,5′-adenosine monophosphate (cAMP) levels are greater in large cholangiocytes compared with small ones (94). In mice, a water channel, aquaporin 1 (AQP1), is expressed in both small and large cholangiocytes (136,137). Annexin V, a protein involved in membrane–membrane or membrane– cytoskeletal interactions, and in regulation of apoptosis in different cell types, is expressed in small but not large cholangiocytes (138). The functional role of annexin V in small cholangiocytes remains unknown. By microarray analysis of small and large murine cholangiocytes immortalized by the introduction of the SV40 large T-antigen gene, 230 complementary DNA (cDNA) among 4850 cDNA were differentially expressed; for example, a water channel, AQP8, caspase-9, and interleukin-2 receptor β-chain cDNA and proteins were preferentially expressed in large cholangiocytes (139). Thus, anatomically and phenotypically, the intrahepatic biliary ductal system is a complex organ, the cells of which are similar with regard to expression of a group of enzymes and marker proteins such as pancreatic lipase and α-amylase, trypsin, CK-7, CK-19, and others, but differ with regard to size, shape, and expression of a number of specific receptors and transport proteins directly involved in ductal bile formation.

MOLECULAR PHYSIOLOGY OF DUCTAL BILE FORMATION Basic Principles of Bile Formation Bile formation requires the coordinated function of both types of liver epithelial cells, that is, hepatocytes and cholangiocytes (Fig. 59-4). Chapter 56 provides a detailed description of mechanisms of bile secretion by hepatocytes. In brief, hepatocytes secrete primary or canalicular bile, an aqueous solution of organic and inorganic compounds, into a canalicular space, a minute channel between adjacent cells. Secretion of canalicular bile is an osmotic process driven predominantly by active excretion of organic solutes and ions into the bile canaliculi, followed by passive movement of water. The vectorial transport of bile salts, one of the major organic compounds of bile, and excretion of reduced glutathione and bicarbonate into the canaliculi result in “bile salt–dependent” and “bile salt–independent” canalicular bile secretion, respectively (37,41,49,52,140). Canalicular bile is subsequently delivered to the intrahepatic bile ducts, and at the level of cholangiocytes, it is extensively modified through a series of secretory and reabsorptive processes (see Fig. 59-4); thus, the final composition of bile

Hepatocyte Canalicular bile Cholangiocyte ions solutes water

Cl− HCO3− H2O

Secretion

BA Glucose

Absorption AA H2O

Ductal bile

FIG. 59-4. Schematic representation of bile formation. Hepatocytes initiate bile formation by producing canalicular bile, which is primarily composed of water, solutes, and ions. Canalicular bile is further modified by secretory and absorptive processes in cholangiocytes, resulting in production of ductal bile. Cholangiocyte secretion/extrusion of Cl−, HCO3−, and water and absorption of bile acids (BA), glucose, amino acids (AA), and water are the key transport processes in bile formation at the level of intrahepatic bile ducts. In addition, cholangiocytes transport proteins and inorganic and organic cations and anions into or from bile, completing the chemical composition of the final product.

that reaches the extrahepatic bile ducts is determined by cholangiocytes. Qualitatively, cholangiocytes modify the fluidity and alkalinity of canalicular bile by secretion of ions, primarily Cl− and HCO3−, and by reabsorption of bile salts, glucose, and amino acids. Depending on the osmotic gradients established by transported solutes and ions, water moves passively out of or into cholangiocytes; that is, it is secreted or absorbed. Net ductal bile secretion or absorption is determined by the coordinated functioning of cholangiocyte transport systems. Quantitatively, cholangiocytes, which account for only 3% to 5% of the liver cell population, are responsible for the production of approximately 40% of bile volume, depending on the species (1,4,37).

Cholangiocyte Transport Systems The formation of ductal bile by cholangiocytes is a result of transepithelial movement of ions, solute, and water with the involvement of transporters, exchangers, and channels heterogeneously expressed on the cholangiocyte apical and basolateral plasma membrane domains (Fig. 59-5). The transporting capacity of different cholangiocyte transport systems varies. Ion channels allow rapid movement of ions

PHYSIOLOGY OF CHOLANGIOCYTES / 1509 Basolateral (blood)

Apical (bile) K+

cAMP

K+

Na+

Ca2+

Cl− Cl−

Na+ K+

H+ Na+ K+ Cl− Na+ HCO3−

Cl−

HCO3−

Na+

H+

HCO3−

Cl−

Cilium

Bile acids

t-ASBT

Glucose

GLUT1

H2O

H2O

H2O

H2O MRP/ Mrp3

Organic anions

Bile acids

ASBT

Glucose

SGLT1

Amino acids MDRI/ Mdr1

Organic anions

MRP4

FIG. 59-5. Cholangiocyte transport systems. At the apical plasma membrane, cholangiocytes express cyclic 3′,5′-adenosine monophosphate (cAMP)–regulated (cystic fibrosis transmembrane conductance regulator [CFTR]) and Ca2+-regulated Cl− channels, a K+ channel (SK2), Na+-independent Cl−-HCO3− (AE2) and Na+-H+ (NHE3) exchangers, a water channel (AQP1), a Na+-dependent bile acid transporter (ASBT), a Na+-dependent glucose transporter (SGLT1), unidentified Na+-dependent and -independent amino acid transporters, and adenosine triphosphate (ATP)–dependent transmembrane efflux pumps (the multidrug resistance P-glycoproteins MDR1/Mdr1a). At the basolateral plasma membrane, cholangiocytes express a K+ channel (SK2), Na+,K+-ATPase, NHE1 exchanger, Na+-K+-Cl− cotransporter, Na+-HCO3− cotransporter (in rat), Na+-dependent Cl−-HCO3− exchanger (in humans), bile acid transporter (t-ASBT), glucose transporter (GLUT1), water channels (AQP1 and AQP4), and ATP-dependent conjugate export pumps (the multidrug resistance-associated proteins MRP3/Mrp3 and MRP4).

across the membrane at rates greater than 1010 ions per second, whereas the rate of ion transport by cotransporters/ exchangers is limited to 103 to 106 ions per second as a result of multistep processes (141).

Ion Transport (Apical) Cl − Channels Ductal bile secretion is a regulated process initiated by transport of Cl− across the cholangiocyte apical plasma membrane into the ductal lumen, which provides the driving force for fluid and electrolyte secretion. To date, four separate Cl− channels have been identified in cholangiocytes, which differ in anion permeability, sensitivity to blockers, and regulatory mechanisms. They appear to be localized, in part, to the apical domain and are capable of supporting transepithelial secretion (141,142). Cystic Fibrosis Transmembrane Conductance Regulator Cl − Channel. The CFTR is a cAMP-regulated Cl− channel expressed in human, rat, and mouse liver exclusively in cholangiocytes, providing the driving force on their apical

plasma membrane domain for Cl−-HCO3− exchange and water efflux, that is, ductal bile secretion (143–147). Under basal conditions, the Cl− permeability of the cholangiocyte apical membrane is low; after stimulation by cAMP, Cl− transport across the apical membrane increases by 20- to 40-fold (145,148). CFTR belongs to a family of adenosine triphosphate (ATP)–binding cassette (ABC) proteins with intracellular N and C termini and contains two domains spanning the plasma membrane six times each. These two transmembrane domains are separated by an intracellular regulatory domain containing phosphorylation sites for protein kinase A (PKA). At the C terminus, CFTR contains the PDZ domain, suggesting that this channel may be involved in regulation of cholangiocyte functions through interaction with other integral membrane and signaling molecules. Indeed, it has been demonstrated by patch-clamp analysis that overexpression of the protein-protein interaction domain, PDZ1, of EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50), which is normally expressed in the rat on the cholangiocyte apical membrane, in Mz-ChA1 cholangiocarcinoma cells (a model of human cholangiocytes) decreases the endogenous

1510 / CHAPTER 59 cAMP-mediated Cl− secretion, suggesting that protein–protein interactions have direct regulatory effects on CFTR Cl− transport (149). The cAMP-regulated CFTR Cl− channel has a small unitary conductance (i.e., 3–9 pS) and a linear current-voltage relation (142). Under normal conditions, cAMP-dependent PKA phosphorylates CFTR, causing channel opening and efflux of Cl ions, and/or regulates vesicular trafficking of CFTRcontaining vesicles (see Vesicular Trafficking in Mechanisms of Ductal Bile Formation section later in this chapter), which leads to an increase in the number of functional channels in the apical plasma membrane. Ca2+/Calmodulin-Dependent Protein Kinase II–Activated − Cl Channel. Functional studies demonstrate that Ca2+activated Cl− channels are expressed in human, rat, and mouse cholangiocytes and appear to complement the role of CFTR in ductal bile secretion (148,149). These channels are activated by an increase in concentration of intracellular Ca2+, which, in turn, activates Ca2+/protein kinase II. The relative contribution of Ca2+-activated Cl− channels in cholangiocyte Cl− transport in experimental models is twofold to fivefold greater than CFTR (141,142,148,150). Volume/Protein Kinase Cα–Activated Cl − Channel. The expression of a member of a family of voltage-gated Cl − channels (i.e., ClC-2) on the mRNA level has been shown in rat cholangiocytes (151). In general, ClC-2 is activated by membrane hyperpolarization and an increase in cell volume via PKCα. In cholangiocytes, this Cl− channel, the functional role of which is unknown, is activated by extracellular ATP (151). Cyclic Adenosine Monophosphate– and Ca2+-Independent − Cl Channel. A cAMP- and Ca2+-independent high Cl− conductance sensitive to pertussis toxin was reported in rat cholangiocytes (152). However, the channel providing this Cl− conductance has not yet been identified.

involved in ductal bile secretion in cooperation with CFTR and other Cl− channels providing a Cl− influx and a HCO3− efflux. Na+-H+ Exchangers (NHE2, NHE3) The electroneutral exchange of intracellular H+ for extracellular Na+, which is crucial for cellular pH and volume regulation and transepithelial Na+ transport, is performed by a family of antiporters, known generically as Na+-H+ exchangers (NHEs) and represented by 1 through 8 isoforms (NHE1-8). They are highly regulated (glyco)phosphoproteins with an N-terminal domain, which likely crosses the cell membrane 12 times and constitutes the cation exchange machinery, and a large C-terminal cytoplasmic tail, which modulates the exchanger by interacting with protein kinases and regulatory factors (165). An NHE isoform 3 (NHE3) has been identified on the rat and mouse cholangiocyte apical plasma membrane by immunofluorescence, immunoelectron microscopy, and Western blotting (166). Functional studies in IBDUs from NHE3 knockout mice and from normal rat and mice treated with the inhibitor of NHEs, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), demonstrated that NHE3 is involved in cholangiocyte fluid absorption and counterbalances fluid secretion in normal resting bile duct epithelia (166). In addition to its primary function in cholangiocyte fluid absorption, NHE3 may be a site for regulation of cholangiocyte HCO3− and fluid secretion, presumably by up-regulating CFTR via protein– protein interactions (166). The potential presence of NHE2 on the rat cholangiocyte apical plasma membrane also has been reported based on the NHEs mRNA expression and functional studies (162,167). Currently, however, no further evidence of NHE2 apical expression and function in cholangiocytes has been provided.

K+ Channels Human, rat, and mouse cholangiocytes express a Ca2+activated small conductance (SK2) K+ channel, a 63-kDa protein with six transmembrane domains activated by small increases in [Ca2+]i (144,153–160). SK2, which is an important component of stimulated Cl−, HCO3−, and water secretion, is expressed on both cholangiocyte apical and basolateral plasma membrane domains. Activation of apically located SK2 results in a small K+ conductance (about −4 µA/cm2) that may enhance the cholangiocyte Cl− secretory response to regulatory stimuli (153). Na+-Independent Cl−/HCO 3− Exchanger Na+-independent anion exchangers (AEs) represent a family of anion transporters, which mediate electroneutral and reversible exchange of Cl− and HCO3− across cell membranes. Among the four AEs identified in mammals thus far (i.e., AE1-4), only AE2 is expressed in rat and human cholangiocytes (45,46,125,161–164). AE2 is critically

Ion Transport (Basolateral) K+ Channels In contrast with apically expressed SK2 (see earlier K+ Channels section), activation of SK2 expressed on cholangiocyte basolateral plasma membrane domain results in a large K+ conductance (about −10 µA/cm2) (153). In cholangiocytes, basolaterally located K+ channels are believed to complement the functions of apical Cl− channels and are necessary for membrane hyperpolarization to maintain the electrical driving force for continued apical Cl− secretion (153). Na+-H+ Exchanger (NHE1) The first of eight NHE isoforms (i.e., NHE1) is expressed in human and rat cholangiocytes (42,161,162,168–172). The presence of NHE1 on the cholangiocyte basolateral plasma membrane was confirmed by functional studies

PHYSIOLOGY OF CHOLANGIOCYTES / 1511 (161,163). NHE1 fulfills several physiologic functions, including, but not limited to, regulation of intracellular pH (pHi), cell volume, and transepithelial HCO3− transport (45,46,167,168). Na+-HCO 3− Cotransporter (Na+-Dependent Cl−-HCO 3− Exchanger) Functional data suggest that cholangiocytes possess a specific mechanism providing the cell with HCO3− (50,161, 162,173). In rat cholangiocytes, a Na+-HCO3− cotransporter together with an NHE1 and carbonic anhydrase, an enzyme that generates HCO3− from CO2 and H2O, maintains a high concentration of intracellular HCO3− (50,161,162). In human cholangiocytes, this function is performed by the Na+dependent Cl--HCO3− exchanger together with NHE1 (50,173). Na+,K+-ATPase Na+,K+-ATPase is expressed in human and rat cholangiocytes (174–176), and as in many other epithelia, it transports Na+ and K+ against their electrochemical gradients. With each transport cycle, Na+,K+-ATPase uses the energy released by ATP hydrolysis to transport 3 ions of Na out of the cell and 2 ions of K into the cell, creating a large electrochemical gradient of ∼60 mV. This favors Na+ influx from extracellular [Na+] of ∼140 mM to intracellular [Na+] of ~14 mM, with the rate of transport of 103 to 106 ions/sec−1 (141). In cholangiocytes, the inwardly directed Na+ gradient provides the energy for coupled transport of solutes (i.e., bile acids, glucose, and amino acids). Na-K-Cl Cotransporter Functional studies suggest that rat cholangiocytes possess a basolateral, electroneutral, bumetanide-sensitive Na-K-Cl cotransporter that maintains a high concentration of intracellular Cl− (177). Under physiologic conditions in most cell types, Na-K-Cl cotransporters mediate transport of Na, K, and Cl ions into cells in an electrically neutral manner with a stoichiometry of 1Na:1K:2Cl (178). It is unknown which one of two Na-K-Cl cotransporter isoforms (i.e., NKCC1 or NKCC2) is expressed in cholangiocytes. However, because NKCC2 is thought to be present exclusively in the kidney (178), we can speculate that cholangiocytes, similar to a wide variety of secretory epithelia and nonepithelial cells, express NKCC1. In cholangiocytes, NKCC1 provides Cl− entry into cells with Na+ and K+; Cl− then is transported into the bile duct lumen via apical Cl− channels. H+-ATPase The vacuolar-type H+-ATPase involved in secretininduced H+ secretion into interstitial fluid was identified in pig cholangiocytes (179). However, no evidence was found for H+-ATPase in human (173) and rat cholangiocytes (180).

Bile Acid Transport Bile acids secreted by hepatocytes are absorbed by cholangiocytes in small amounts by different mechanisms. Unconjugated bile acids secreted by hepatocytes in an anionic form are protonated in bile and passively enter cholangiocytes across the apical plasma membrane (181). In contrast, conjugated bile acids are transported from bile into and out of cholangiocytes by several transporters expressed on the apical (i.e., Na+-dependent bile acid transporter ASBT [also known as ABAT] and an unidentified Na+-independent transporter) and basolateral (i.e., t-ASBT, multidrug resistance-associated protein 3 [MRP3]/Mrp3, MRP4, organic anion transporting polypeptide 3 [Oatp3], a bile acid carrier sensitive to 4,4′-diisothiocyanostiblene2,2′-disulfonic acid [DIDS]) plasma membrane domains (133,182–187). ASBT and t-ASBT are products of the same gene. ASBT is a 48-kDa protein identical to the bile acid transporter cloned from rat ileum and kidney (133,182). t-ASBT, a 19-kDa protein, is a truncated, alternatively spliced form of ASBT (183). ASBT is involved in uptake of taurocholic acid (TCA) across the apical plasma membrane of normal rat cholangiocytes. t-ASBT expressed in Xenopus oocytes provides effective TCA efflux. ASBT is an Na+-dependent bile acid transporter, whereas the driving force for t-ASBT remains to be identified. ASBT is an unstable and shortlived protein. In cholangiocytes, ASBT undergoes ubiquitinproteasome degradation under basal conditions, and its proteasome disposal is increased by c-Jun N-terminal kinase (JNK)–regulated serine/threonine phosphorylation (188). Considering the toxic effects associated with intracellular bile acid accumulation, it is likely that the mechanisms for bile acid efflux from cholangiocytes into the peribiliary plexus are redundant, and that multiple transporters (i.e., t-ASBT, MRP3/Mrp3, MRP4, Oatp3, and a bile acid carrier sensitive to DIDS) may be involved. The murine organic solute transporter, OST α-β, has been reported as a novel basolateral bile acid carrier responsible for bile acid efflux in the ileum and likely other ASBT-expressing tissues, including intrahepatic bile ducts (189). Glucose Transport The basal concentration of glucose in hepatic bile is low (i.e., <1.0 mM) compared with plasma (i.e., >5.0 mM) (190–192). Earlier elegant studies explored blood-bile glucose concentration differences and provided convincing evidence that glucose enters canalicular bile in equal concentration to plasma, and then is subsequently reabsorbed from bile by cholangiocytes via Na+-dependent and -independent transport mechanisms (190,191). It has become clear that cholangiocytes express a sodium-dependent glucose transporter, SGLT1, at their apical plasma membrane and a facilitative glucose transporter, GLUT1, at their basolateral plasma membrane domain (193). The Na+-independent mechanism of glucose absorption by cholangiocytes has not yet been identified.

1512 / CHAPTER 59 SGLT1 is a protein with 14 transmembrane domains and extracellularly oriented C and N termini. Domains 10 through 13 close to the C terminus form the sugar binding/ translocation domain; the Na+ binding/translocation domain is located at the N terminus (194,195). SGLT1 transports glucose via a secondary active transport mechanism: The Na+ electrochemical gradient provided by the Na+,K+-ATPase is used to transport glucose into cells against its concentration gradient (193–195). GLUT1 has 12 transmembrane domains and cytoplasmically oriented N and C termini. GLUT1 facilitates passive diffusion of glucose across the plasma membrane by an energy-independent mechanism using the glucose gradients (196,197). These two glucose transporters account for the vectorial (i.e., from bile to blood) transport of glucose, and their coordinated activities provid a plausible explanation for the low concentration of glucose in bile. Amino Acid Transport Bile contains an acidic amino acid, glutamate, and amino acids possessing a reactive sulfhydryl group (i.e., cysteine and glycine) (52). These biliary amino acids are the products of hydrolysis of the tripeptide glutathione, which is the principal driving force for “bile salt–independent” canalicular bile secretion (52,198,199). Glutathione is secreted by hepatocytes, and its degradation is catalyzed in bile by γ-glutamyltranspeptidase, an enzyme abundant on the canalicular and cholangiocyte apical plasma membrane domains (200,201). Functional studies suggest that cholangiocytes absorb biliary glutamate, cysteine, and glycine by sodium-dependent and -independent transport mechanisms different from those in hepatocytes (52,202). Absorption of these amino acids by cholangiocytes may conserve amino acids for glutathione resynthesis, and thus be an important part of the mechanisms of “bile salt–independent” canalicular bile secretion. Water Transport Because bile is a complex fluid composed of more than 98% water, an essential issue in understanding ductal bile formation is how cholangiocytes transport water. Currently, the mechanisms of water transport by intrahepatic bile ducts have not been clarified. However, the general concepts of epithelial water transport have changed dramatically in recent years, principally because of the discovery of aquaporins (AQPs), a family of water-channel proteins that, in mammals, is represented by 13 distinct AQPs (i.e., AQP0-12) (203–207). The mechanisms of AQP-mediated water transport are discussed in greater detail in Chapter 72. In brief, AQPs are small, hydrophobic proteins (26–34 kDa) consisting of 6 transmembrane domains with 5 connecting loops (A-E) and with cytoplasmically oriented amino and carboxy termini. Connecting loops B (cytoplasmic) and E (extracellular) contain NPA boxes (the signature motif asparagine-proline-alanine

conserved among family members), which form a single aqueous pore with a diameter of about 3 Å, a width sufficient to allow effective passage of water molecules in single files in either direction (203,204). In plasma membrane, AQPs are assembled as homotetramers. AQP4 forms larger oligomeric structures called intramembrane particle square arrays, or orthogonal arrays of particles, which are clusters of intramembrane particles in a systemic/geometric organization (205–207). AQPs show extremely high watertransporting capacity. For example, AQP1 transports approximately 3 × 109 water molecules per channel per second. Rat cholangiocytes express 7 of the 11 known mammalian AQPs (i.e., AQP0, AQP1, AQP4, AQP5, AQP8, AQP9, and AQP11) (205,206,208). Two of them (i.e., AQP1 and AQP4) have been well characterized by molecular, biochemical, and functional studies (209–211). AQP1 is expressed in human interlobular and terminal bile ducts (212). In the rat, in the basal state, AQP1 is principally expressed within intracellular vesicles. When cholangiocytes are stimulated by secretin, a hormone essential to regulation of ductal bile secretion (see Secretin section later in this chapter), AQP1 is translocated to the apical plasma membrane domain (210,212). In the rat, AQP1 is also present in the basolateral cholangiocyte plasma membrane, but in low amounts (210,211). In the mouse, AQP1 is present in both small and large bile ducts (35,136); however, although important, this water channel does not appear to be rate limiting for ductal bile secretion (136). Because mouse cholangiocytes also express AQP8 in substantial amounts, especially in large cholangiocytes as assessed by microarray analysis and Western blotting (139), this channel may be critical for AQP-mediated water transport in mouse biliary epithelia. AQP4 is expressed exclusively on the rat basolateral cholangiocyte plasma membrane domain (209). The high single-channel water permeability of AQP4 (approximately three times greater than that of AQP1), strongly suggests that AQP4 can balance water permeability between apical and basolateral cholangiocyte membrane domains. In contrast to AQP1, exposure of isolated rat cholangiocytes to secretin does not alter AQP4 (209). These data support the notion that AQP4 is constitutively inserted into the basolateral cholangiocyte plasma membrane domain, or alternatively, that AQP4 function involves signaling pathways other than those for secretin. Overexpression of AQP4 in mouse cholangiocytes resulted in a significant increase of AQP4mediated water transport (137). In enclosed polarized and microperfused IBDUs isolated from normal rats and mice (33–35), rapid, time-dependent water movement into or out of the lumen of IBDUs occurs when IBDUs were exposed to inward (i.e., secretory) or outward (i.e., absorptive) osmotic gradients, respectively. This effect was not inhibited by protamine, an agent that affects tight junctions, but was inhibited by HgCl2, an agent that blocks AQP-mediated water transport (34), and by specific small interfering RNA against AQP1 (213). AQP-mediated water absorption by perfused rat IBDUs in response to

PHYSIOLOGY OF CHOLANGIOCYTES / 1513 osmotic gradients generated by absorbed glucose also was observed (192). Thus, it has become clear that cholangiocytes, similar to other water-transporting cells, express AQPs and transport water in response to osmotic gradients via water channels, rather than by diffusion through the lipid bilayer or tight junctions. Transport of Proteins Cholangiocytes are involved in uptake and extrusion of proteins from and into bile. It has been shown that horseradish peroxidase can be taken up by cholangiocytes via fluid-phase endocytosis at the apical plasma membrane domain, and then may be secreted into the circulation (214). In contrast, epidermal growth factor (EGF) and polymeric IgA are transported by cholangiocytes in opposite directions. In the rat, cholangiocytes internalize EGF from the basolateral membrane by receptor-mediated endocytosis, and then transport this protein into bile by transcytosis (121). In human biliary epithelia, polymeric IgA is transported into bile by a mechanism involving the polymeric IgA receptor (103,215). Cholangiocytes transport a number of other plasma proteins into bile such as ceruloplasmin, asialoceruloplasmin, carcinoembryonic antigen, and asialocarcinoembryonic antigen (3,44,103,216–219). These proteins are transported into bile via anatomic communications existing among portal blood, lymphatic vessels, and bile ducts; the presence of fenestrae in the endothelium of the peribiliary capillaries and of vesicles in both endothelial lining cells and cholangiocytes has been interpreted as supporting the hypothesis that cholangiocytes are involved in protein translocation from blood to bile (220). Organic Anion and Cation Transport Human and rat cholangiocytes express the ATP-dependent conjugate export pumps, multidrug resistance-associated proteins MRP3/Mrp3 and MRP4 (221–224). In cholangiocytes, MRP3/Mrp3 and MRP4 might transport a wide range of organic anionic substrates to the systemic circulation including bile acids (MRP3 and MRP4) and glutathione (MRP4) absorbed from bile (187,223,225). Cholangiocytes are continuously exposed to toxic products from endogenous compounds and lipophilic drugs, many of which are organic cations excreted in canalicular bile by hepatocytes (52,140). The transmembrane organic cation gradients may favor the passive entry and accumulation of these compounds in cholangiocytes with potentially harmful effects. It has been shown that human and rat cholangiocytes express on their apical plasma membrane the multidrug resistance P-glycoprotein gene products, that is, glycosylated plasma membrane proteins, multidrug resistance 1 (MDR1) protein, and Mdr1a, respectively (176,226,227), which are known as the ATP-dependent transmembrane efflux pumps for a wide array of structurally dissimilar lipophilic compounds, including cytotoxic drugs, steroid hormones,

peptide antibiotics, calcium channel blockers, and others (228). The apical localization of MDR1 and Mdr1a in cholangiocytes (176,226,227) and Mdr1a-mediated transport of rhodamine 123, a P-glycoprotein substrate, into the lumen of rat IBDUs (227) suggest that MDR1/Mdr1a may effectively counteract accumulation of organic cations in cholangiocytes by excreting them back into bile. In addition, they potentially can excrete into bile exogenous and endogenous lipophilic compounds that enter into cholangiocytes through their basolateral plasma membrane from arterial blood (227).

Heterogeneity of Transport Systems within the Cholangiocyte Plasma Membrane As discussed earlier, transporters, exchangers, and channels are discretely distributed in the cholangiocyte apical and basolateral plasma membrane domains (see Fig. 59-5) to permit efficient regulation of vectorial bidirectional transport of ion, solute, and water across the biliary epithelium. Importantly, the lipid compositions of the cholangiocyte apical and basolateral plasma membrane domains also differ; the levels of cholesterol and phospholipids, as well as the cholesterol/phospholipid ratio, are greater in the cholangiocyte apical plasma membrane domain compared with the basolateral domain. In addition to high cholesterol, the apical plasma membrane has a high polyunsaturated fatty acid content (174,175). The increased cholesterol content gives the apical membrane rigidity that may play a role in protecting the cholangiocyte from the potentially toxic constituents in bile. Different levels of cholesterol and phospholipids in the apical and basolateral plasma membrane domains may reflect different densities of specific lipid microdomains (lipid rafts) within the membrane, which are part of the cellular machinery that in cholangiocytes may facilitate selective docking and fusion of vesicles containing functionally related proteins (i.e., channels, transporters, exchangers) necessary for ductal bile formation. Lipid rafts may also contain specific proteins that coordinate transport events on the cholangiocyte plasma membrane. One such protein, a novel epithelial scaffolding protein, Shank2, resides in the lipid rafts of the cholangiocyte apical plasma membrane, providing the cell with a PDZ domain protein known to cluster and coordinate the activities of functionally related protein complexes (229). Thus, the cholangiocyte apical and basolateral membranes differ in both protein and lipid content and composition, and this molecular diversity has functional implications, the details of which are currently being actively investigated.

INTRACELLULAR SIGNALING In cholangiocytes, the cAMP and Ca2+ signaling pathways are likely the key intracellular regulatory mechanisms involved in ductal bile formation.

1514 / CHAPTER 59 Cyclic 3′,5′-Adenosine Monophosphate Signaling The cAMP second messenger system is a prominent signaling pathway in cholangiocytes. As in other cell types, the intracellular content of cAMP results from its synthesis by ACs regulated through G protein–coupled receptors and from its hydrolyses by phosphodiesterases (230). Molecular cloning techniques have identified nine mammalian genes that encode membrane-bound ACs (AC1-9) and one gene encoding a soluble G protein–insensitive form of AC, sAC (231,232). Rat cholangiocytes express 7 ACs from 10 known in mammals, that is, 6 membrane-bound ACs (i.e., AC4-9) and sAC, suggesting the existence of multiple mechanisms of the cAMP signaling pathway in biliary epithelia (135,233). ACs can be activated or inhibited depending on the coupling of receptors to trimeric G proteins that are constituted by three subunits, α, β, and γ. There are two forms of G-protein α-subunits, the activating (i.e., αs) and the inhibitory (i.e., αi) α-subunits that determine the functional characteristics of G proteins, known as stimulatory (i.e., Gs) or inhibitory (i.e., Gi) G proteins, respectively. The αs-subunit hydrolyzes a molecule of GTP to GDP, and a complex of β- and γ-subunits anchors the G protein to the membrane. Some stimuli induce the dissociation of the αssubunit from Gs protein and binding of this subunit to ACs, resulting in an increase in cAMP levels. Other stimuli release αi-, β- and γ-subunits from the Gi protein complex, which, in turn, inhibit ACs (230). In addition, different isoforms of ACs are activated (i.e., AC8) or inhibited (i.e., AC5 and AC6) by an increase in intracellular Ca2+ (231,232), thus providing multiple mechanisms of cAMP signaling. The diverse effects of cAMP in cholangiocytes are mediated as in other cell types through the action of cAMP-dependent PKA, which modifies the activities of target proteins by phosphorylating specific serine and threonine residues (230).

Calcium Signaling Calcium signaling in cholangiocytes is initiated by two pathways: (1) activation of G protein–coupled receptors expressed on both cholangiocyte apical and basolateral plasma membrane domains; and (2) influx of extracellular Ca2+ into the cell via Ca2+ channels (Fig. 59-6). Activation of G Protein–Coupled Receptors Activation of specific G protein–coupled receptors results in production of inositol 1,4,5-trisphosphate (IP3), which diffuses into the cytoplasm to release Ca2+ from intracellular stores via its interaction with the IP3 receptors (IP3Rs) (see Fig. 59-6). IP3Rs are tetrameric IP3-gated Ca2+ channels residing within the membrane of the endoplasmic reticulum. On IP3 binding, the IP3Rs undergo large conformational changes that result in opening of the Ca2+ channels and Ca2+ release from the endoplasmic reticulum into the cytoplasm (234,235). Cholangiocytes express all three known isoforms of IP3R (i.e., types I, II, and III); however, IP3R type III is the

Cilium Receptor (P2Y) PC1

G-protein

Ca2+ PC2

TRPV4

P2X

PLC Ca2+

IP3 Ca2+ IP3R

ER

FIG. 59-6. Ca2+ signaling in cholangiocytes initiated at the apical plasma membrane domain. Activation of the G protein–coupled receptors (e.g., P2Y) results in activation of phospholipase C (PLC) and production of inositol 1,4,5trisphosphate (IP3), which via IP3 receptors (IP3Rs) induces Ca2+ release from the endoplasmic reticulum (ER) increasing intracellular concentration of Ca2+ ([Ca2+]i). [Ca2+]i may also increase as a result of entry of extracellular Ca2+ via polycystin-2 (PC2), P2X receptors, and transient receptor potential channel vanilloid subfamily 4 (TRPV4), which act as Ca2+ channels.

predominantly expressed isoform. This isoform is most concentrated near the apical plasma membrane of cholangiocytes, whereas the isoforms I and II are distributed relatively uniformly throughout the cytoplasm (236,237). Activation of any isoforms of IP3Rs leads to an increase in concentration of intracellular Ca2+; however, the patterns of [Ca2+]i signaling (i.e., Ca2+ waves and Ca2+ oscillations) depend on the type of activated IP3R isoform. It has been reported that both Ca2+ waves and Ca2+ oscillations occur in cholangiocytes, and that the type III IP3R is involved in initiation of Ca2+ waves, whereas the type I IP3R is likely involved in both the spread of Ca2+ waves and the support of Ca2+ oscillations. The role of type II IP3R in the development of [Ca2+]i signaling in cholangiocytes is unclear (236,237). Ca2+ waves induced by acetylcholine (ACh) or ATP spread in cholangiocytes from the apical to the basolateral pole. Interestingly, ACh-induced Ca2+ waves cross cholangiocytes at a speed of more than 20 microns/sec, whereas ATP-induced Ca2+ waves appear to cross cholangiocytes at a much slower speed (236). Both ACh and ATP can also induce Ca2+ oscillations in cholangiocytes. However, each of these two agonists induces distinct patterns of Ca2+ oscillations (234,238,239). ACh induces repetitive, irregular Ca2+ spikes that have a longer duration for each individual spike and a longer period between spikes compared with ATPinduced Ca2+ oscillations. Interestingly, low concentrations of ATP induce Ca2+ oscillations, whereas high concentrations induce a single transient or sustained increase in Ca2+. In contrast, ACh usually induces a single transient or sustained increase in Ca2+ rather than repetitive Ca2+ spikes, regardless

PHYSIOLOGY OF CHOLANGIOCYTES / 1515 of its concentration (239). This phenomenon may enable different stimuli to elicit distinct physiologic effects in cholangiocytes even though these stimuli act through modulation of [Ca2+]i signaling (235).

expressed in both hepatocytes and cholangiocytes. Exposing mouse cholangiocytes to hypotonicity activates TRPV4, implying that this channel might be a cellular osmosensor (N. F. LaRusso, unpublished observation).

Ca2+ Channels

Calcium-Regulated Proteins in Cholangiocytes

Until recently, the expression of Ca2+ channels on the cholangiocyte plasma membrane that allow entry of extracellular Ca2+ into the cell had not been examined. Moreover, because the direction and speed of hormone-mediated Ca2+ waves in cholangiocytes was not altered in Ca2+-free medium, it was concluded that Ca2+ channels do not contribute to the initial component of [Ca2+]i signaling (236). However, it is now evident from molecular, biochemical, and functional studies that cholangiocytes express at least three types of Ca2+ entry channels—that is, P2X receptors, polycystin-2, and transient receptor potential channel vanilloid subfamily 4 (TRPV4)—that might be involved in [Ca2+]i signaling in cholangiocytes (see Fig. 59-6).

An increase in intracellular Ca2+ initiated by extracellular stimuli may exert effects on cellular processes by interacting with various Ca2+-binding proteins, specifically with calmodulin, a 17-kDa protein that reversibly interacts with certain enzymes and other proteins, thereby altering their activities (259). The targets of the Ca2+/calmodulin complex in cholangiocytes are Ca2+/calmodulin-dependent protein kinase, cyclic nucleotide phosphodiesterase, and calcineurin, a specific phosphoprotein phosphatase (259). Ca2+ may also interact directly with Ca2+-regulated Cl− channels, K+ channels, and others that are involved in mechanisms of ductal bile formation (141,147,153). Intracellular Ca2+ is required for activation of several isoforms of protein kinase C (i.e., PKCα, PKCβ, and PKCγ) by diacylglycerol, a signaling molecule involved in regulation of cholangiocyte secretion by a number of regulatory factors (259).

P2X Receptors P2X receptors (P2X1-7) are extracellular, ATP-gated, calcium-permeable, nonselective cation channels. They have two transmembrane domains, intracellularly oriented C and N termini, and an extracellular ATP-binding site. Stimulation of P2X receptors by ATP results in an increase in [Ca2+]i (240–244). Mz-Cha-1 cells, a human cholangiocyte cell model, express four from known seven P2X receptors (i.e., P2X2, P2X3, P2X4, and P2X5) (245). Normal rat cholangiocytes express P2X2, P2X3, P2X4, and P2X7 receptors (245–247). Polycystin-2 Polycystin-2 is a 110-kDa protein containing six transmembrane-spanning domains with intracellular C and N termini. Polycystin-2 is a nonselective cation channel with multiple subconductance states and a high permeability to Ca2+. Polycystin-2 is activated by low and inhibited by high concentrations of Ca2+. Polycystin-2 has been identified in several tissues, including liver. The majority of polycystin-2 is localized in the cell to the endoplasmic reticulum where it can function as a Ca2+-permeable cation channel (248–254). However, in cholangiocytes, as in other epithelial cells containing primary cilia, polycystin-2 is expressed in apically located cilia where it colocalizes with polycystin-1 and other proteins and functions as a component of a mechanosensory complex (255) (see Bile Flow section later in this chapter). Transient Receptor Potential Channel Vanilloid Subfamily 4 (TRPV4) TRPV4, a calcium entry channel, was first described as a channel activated by hypotonicity-induced cell swelling, but it is clear now that it might integrate responses to a large variety of stimuli (256–258). TRPV4 is expressed in a broad range of tissues, including liver. In the liver, it is

Cyclic 3′,5′-Guanosine Monophosphate Signaling The involvement of cyclic 3′,5′-guanosine monophosphate (cGMP) in intracellular signaling in cholangiocytes is unclear. In a model of the isolated perfused rat liver, nitric oxide (NO) donors and cGMP, which is a product of soluble guanylate cyclase activated by NO, stimulated secretion of bile by hepatocytes, but not cholangiocytes (260–263). In a model of IBDUs isolated from normal rat, NO donors and cell-permeant cGMP analogues did not stimulate cholangiocyte HCO3− and fluid secretion (263); however, NO donors exerted a significant inhibitory effect on forskolin-stimulated fluid and HCO3− secretion by cholangiocytes via inhibition of cAMP production (75). In mouse cholangiocytes, the concentration of cGMP was increased, whereas the concentration of cAMP was decreased in response to somatostatin, a hormone that inhibits secretinstimulated cholangiocyte bicarbonate and water secretion and induces cholangiocyte water absorption (264). Thus, the true physiologic role of cGMP signaling in cholangiocytes remains obscure. Potentially, it may be involved in modulation of ductal bile formation by inhibiting cAMP-dependent cholangiocyte secretion.

REGULATION OF DUCTAL BILE FORMATION Cholangiocytes heterogeneously express numerous specific receptors (Fig. 59-7) through which ductal bile formation is regulated by hormones, regulatory peptides, neurotransmitters, and by bile-borne regulatory factors with the potential involvement of cilia.

1516 / CHAPTER 59 Basolateral (blood)

Apical (bile)

Secretin

SR

Somatostatin

sst2 P2X

VIP

?

Bombesin

?

Gastrin

CCK

Endothelin-1

ETA,B

Acetylcholine

M1,3

Phenylephrine

α/β

Dopaminergic agonists

D2

Nucleotides

GcR P2Y

Bile flow PC1

Bile osmolality TRPV4

P2Y

Nucletides P2X

FIG. 59-7. Cholangiocyte receptors. At the basolateral plasma membrane, cholangiocytes express secretin (SR), somatostatin (sst2), gastrin (CCK), endothelin (ETA,B), muscarinic (M1,3), adrenergic (α/β), dopaminergic (D2), and purinergic (P2X and P2Y) receptors. Functional studies suggest that vasoactive intestinal protein (VIP) and bombesin receptors also are expressed on this membrane domain. At the apical plasma membrane, cholangiocytes express purinergic (P2X and P2Y) and osmo- (transient receptor potential channel vanilloid subfamily 4 [TRPV4]) receptors. Cholangiocyte cilia express polycystin-1 (PC1), which functions as a mechanoreceptor. Cholangiocytes also express intracellular (cytosolic) receptors for corticosteroids (GcR). CCK, cholecystokinin.

Physiologically, ductal bile secretion is a functional response of the liver to meals. More specifically, in the postprandial state, acidic pH stimulates endocrine S cells in the duodenum and jejunum to release secretin into the portal circulation, which in the digestive phase regulates a number of physiologic functions, including secretion of bicarbonaterich ductal bile (4,5,38,39,50–52,265–268). Bicarbonate secreted by cholangiocytes determines the alkalinity, hydration, and pH of hepatic bile; minimizes the passive cholangiocyte absorption of lipophilic bile acids; and significantly contributes to the total bicarbonate requirement for digestive functions (7,51,52,265). In addition to secretin, ductal bile formation is regulated by other regulatory factors, which can be categorized into four groups: (1) regulatory factors that, similar to secretin, stimulate basal cholangiocyte secretion (i.e., vasoactive intestinal peptide [VIP], bombesin, corticosteroids, nucleotides); (2) regulatory factors that potentiate secretin-stimulated cholangiocyte secretion (i.e., neurotransmitters of cholinergic and adrenergic neurons); (3) regulatory factors that inhibit basal and secretin-stimulated cholangiocyte secretion or stimulate cholangiocyte absorption (i.e., somatostatin, gastrin, dopaminergic agonists, ET); and (4) bile-borne regulatory factors (Table 59-1). Hormones, regulatory peptides, and neurotransmitters regulate ductal bile formation through activation of specific receptors expressed on the cholangiocyte

basolateral plasma membrane domain. Nucleotides regulate ductal bile formation at both basolateral and apical poles of cholangiocytes. In this context, nucleotides belong to a group of bile-borne regulatory factors that also include, but are not limited to, bile acids, glucose, amino acids, and mechanostimuli and osmostimuli (i.e., bile flow and bile osmolarity) (see Table 59-1).

Regulation of Basal Cholangiocyte Secretion Secretin Secretin, a 27-amino-acid neuropeptide, has a major role in hormonal regulation of ductal bile formation; its choleretic effects have been known for more than 100 years (for historical background, see Kanno and colleagues [51]). Secretin stimulates bile secretion by interaction with secretin receptors expressed exclusively on cholangiocytes in the liver (268–270). Activation of secretin receptors results in an increase of intracellular cAMP levels and activation of PKA (27,94–96,164,264,268) (Fig. 59-8). PKA, in turn, activates CFTR, a cAMP-dependent Cl− channel, presumably inducing conformational changes that allow Cl− conductance, which at a single cell level increases fivefold (7,145,147,148). An increased Cl− current depolarizes the

CCKB/gastrin D2 ETA, ETB P2X, P2Y Passive transport

Gastrin Dopaminergic neurons Endothelin Nucleotides Bile acids (unconjugated) Bile acids (conjugated) Glucose Amino acids [Ca2+]i [Ca2+]i

[Ca2+]i/PKCα

Unidentified

Cl− channel/AE2 (?) Cl− channel/AE2 (?)

CFTR/AE2 Cl− channels/AE2 Cl− channels, HCO3− secretion SR, CFTR/AE2, ASBT, t-ASBT AQPs? AQPs?

CFTR/AE2

Cl− channels/AE2, Na+/HCO3− cotransporter, K+ channels Cl− channels/AE2 AE2, NHE1 CFTR/AE2

233, 255

312–318 190–192 41

131 246, 247, 306–311 181

303

299–302

289–291 264, 294–297

280–283 170 285–288

94–96, 145–147, 161–164, 166 159, 274, 275

References

α1, α1, β1, β2, adrenergic receptors; AE2, Cl−/HCO3− exchanger; AQP1, aquaporin 1; ASBT, Na+-dependent bile acid transporter; [Ca2+]i, intracellular concentration of Ca2+; cAMP, cyclic 3′,5′-adenosine monophosphate; CCK-B/gastrin, cholecystokinin B/gastrin receptor; CFTR, cystic fibrosis transmembrane conductance regulator Cl− channel; D2, dopaminergic receptor; ETA and ETB, endothelin receptors; GcR, corticosteroid receptor; M1, M3, muscarinic acetylcholine receptors; NHE1, Na+-H+ exchanger isoform 1; NHE3, Na+-H+ exchanger isoform 3; P2X and P2Y, nucleotides receptors; PC-1/2, polycystin-1/2; PKA, protein kinase A; PKC, protein kinase C; SR, secretin receptor; t-ASBT, truncated form of ASBT; SGLT1, Na+-dependent glucose transporter; sst2, somatostatin receptor subtype 2; TRPV4, osmoreceptor (transient receptor potential channel vanilloid subfamily 4); VIP, vasoactive intestinal peptide.

Bile-borne factors

Bile flow Bile osmolarity

CFTR/AE2

[Ca2+]i/PKCα, [Ca2+]i/PKCβ [Ca2+]i/PKCγ, cAMP/PKA [Ca2+]i/cAMP [Ca2+]i [Ca2+]I/PKCα

α1, α2, β1, β2 sst2

Adrenergic neurons Somatostatin

Inhibit basal and secretin-stimulated cholangiocyte secretion or stimulate cholangiocyte absorption

ASBT SGLT1 Na+-dependent and -independent transport PC-1/2 TRPV4

CFTR/AE2 SR, CFTR/AE2, AQPs

[Ca2+]i/PKC/cAMP cGMP, cAMP

Unidentified Nuclear GcR M1, M3

VIP Corticosteroids Cholinergic neurons

Potentiate secretinstimulated cholangiocyte secretion

Unidentified Gene expression [Ca2+]i /cAMP

Unidentified

CFTR/AE2/AQP1, NHE3

Bombesin

cAMP/PKA

SR

Secretin

Target

Stimulate cholangiocyte secretion

Signaling pathways

Name

Groups of factors

Receptors/Transporters

TABLE 59-1. Factors regulating ductal bile formation

PHYSIOLOGY OF CHOLANGIOCYTES / 1517

1518 / CHAPTER 59 Basolateral (blood)

Apical (bile)

Secretin

Na+

H+

nucleus

+

Na+ HCO3−

Bombesin

?

− +

PKA

cAMP

SR

Cl−

?

?

HCO3−

Cl−

+

H2O +

VIP

H+ Cl−

+

?

K+

Na+

Cilium

+

P2X

Nucleotides

Ca2+

P2Y

FIG. 59-8. Stimulation of bicarbonate-rich ductal bile secretion. Secretin induces cholangiocyte bicarbonate-rich fluid secretion via the cyclic 3′,5′-adenosine monophosphate/protein kinase A (cAMP-PKA) signaling pathway by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, resulting in extrusion of Cl ions, which, in turn, stimulate Cl−-HCO3− exchanger and subsequent secretion of HCO3−. Secreted bicarbonate ions drive passive aquaporin 1 (AQP1)-mediated water transport in response to established osmotic gradients. As a consequence of depolarization of the cell membrane induced by Cl− efflux, the electrogenic Na+-HCO3− cotransport is activated at the basolateral domain providing the cell with HCO3−. In addition, cholangiocyte fluid absorption favoring by apically located Na+-H+ exchanger (NHE3 isoform) is inhibited. Bombesin via an unknown pathway, but not cAMP, cyclic 3′,5′-guanosine monophosphate (cGMP), or intracellular concentration of Ca2+ ([Ca2+]i) signaling pathways, increases activity of apical Cl− channels and the Cl−-HCO3− exchanger, resulting in bicarbonate and water secretion. The transport processes on the apical membrane domain are also counterbalanced with ion transport on the basolateral plasma membrane domain by activation of Na+-HCO3− cotransporters and K+ channels. Vasoactive intestinal peptide (VIP) stimulates bicarbonate-rich cholangiocyte secretion, presumably with the involvement of the same transporters/exchangers/channels via an unidentified but apparently the cAMP-independent pathway. Nucleotides likely activate Ca2+-sensitive Cl− channels and subsequent HCO3− and water secretion. Corticosteroids regulate cholangiocyte secretion by increasing the number of the apical Cl−-HCO3− and basolateral NHE1 exchangers. Plus signs indicate activation; minus signs indicate inhibition. GcR, corticosteroid receptor; P2X and P2Y, nucleotides receptors; SR, secretin receptor.

cell membrane and establishes a Cl− gradient favoring activation of Cl−-HCO3− exchanger, which drives bicarbonate secretion in ductal bile (30,31,33,34,161–164). Secretin also stimulates cholangiocyte secretion through insertion of CFTR and other functionally related proteins from intracellular compartments into the apical plasma membrane (210,211,270–272). The secretin signal in cholangiocytes is inactivated by protein phosphatases 1, 2A, or both (164). These phosphatases dephosphorylate the regulatory domain of CFTR and promote conformational changes that lead to occlusion of the Cl− conductance pathway, thus restoring the basal quiescent state. Thus, ductal bile secretion stimulated by secretin is regulated at the level of CFTR by a balance between the activities of kinases (inducing activation) (164) and phosphatases (causing inactivation) of this channel (273).

An additional mechanism that could contribute to secretininduced choleresis is NHE3 expressed on the cholangiocyte apical plasma membrane (166) (see Fig. 59-8). NHE3 functions to stimulate absorption of fluid from the biliary lumen, thus counterbalancing fluid secretion in normal resting bile duct epithelia. In the postprandial phase, PKA, activated by secretin and cAMP, inhibits NHE3-mediated fluid absorption; as a result, net fluid secretion induced by secretin is increased (166). Bombesin In rat IBDUs, an endogenous neurotransmitter, the tetradecapeptide bombesin (also known as gastrin-releasing peptide), directly stimulates cholangiocyte fluid and bicarbonate secretion via unidentified but cAMP, cGMP, [Ca2+]i, and microtubule-independent mechanisms (159,274,275)

PHYSIOLOGY OF CHOLANGIOCYTES / 1519 (see Fig. 59-8). In vivo, bombesin is likely to have both direct and indirect effects on ductal bile secretion, the latter by inducing the release of other secretagogues (276–279). Bombesin-stimulated cholangiocyte secretion is mediated by an increase in Cl−-HCO3− exchanger activity, coupled to apical Cl− channels, and is counterbalanced by the electrogenic Na+-HCO3− cotransporter and K+ channels on the basolateral plasma membrane domains (159,279).

Basolateral (blood)

Secretin

Apical (bile)

cAMP

SR

+ Acetylcholine

PKA

+

Cl−

Cl−

HCO3−

H2O

M1,3

Cilium [Ca2+]i

Vasoactive Intestinal Peptide VIP, a 28-amino-acid neuropeptide thought to be the principal noncholinergic, nonadrenergic neurotransmitter in the gastrointestinal tract, stimulates cholangiocyte bicarbonate-rich secretion via unidentified but apparently cAMP-independent pathways (280–283) (see Fig. 59-8). In rat IBDUs, VIP is a more potent stimulus of biliary secretion than either secretin or bombesin (283). The VIP receptors on cholangiocytes have not yet been identified, but functional studies with a specific competitive VIP antagonist (283) and observed high binding of 125I-labeled VIP to nonparenchymal cells in the rat liver (284) suggest the potential expression of VIP receptors in cholangiocytes. Corticosteroids Rat cholangiocytes express glucocorticoid receptors. Activation of these by dexamethasone or budesonide in pharmacologic doses causes increased biliary bicarbonate secretion in vivo (see Fig. 59-8). In IBDUs isolated from rats treated with dexamethasone or budesonide, cholangiocyte bicarbonate secretion was increased because of the upregulation of two exchangers critical for ductal bile formation, that is, apical Cl−-HCO3− exchanger and basolateral NHE1 (170).

Potentiation of Secretin-Stimulated Cholangiocyte Secretion There are regulatory factors (i.e., a parasympathetic neurotransmitter, ACh, and adrenergic agonists) that do not influence cholangiocyte basal HCO3− secretion by themselves, but significantly potentiate cholangiocyte bicarbonate secretion initially stimulated by secretin (Fig. 59-9). Acetylcholine The ACh-induced potentiation of secretin-stimulated bicarbonate secretion may result from activation of muscarinic (i.e., M1, M3, or both) receptors expressed on cholangiocyte basolateral plasma membranes, followed by an increase of [Ca2+]i (42,239,285,286) (see Fig. 59-9). In cholangiocytes, ACh does not act via a Ca2+-activated PKC, but increases cAMP twofold, presumably by activation of Ca2+-sensitive AC isoforms (285). In isolated bivascularly perfused rat liver, arterial infusion of ACh also further increases secretin-stimulated

Phenylephrine

α/β

FIG. 59-9. Potentiation of secretin-stimulated bicarbonaterich ductal bile secretion. Acetylcholine and phenylephrine induce an increase in intracellular concentration of Ca2+ ([Ca2+]i) followed by further activation of adenylyl cyclase, resulting in an increase of cyclic 3′,5′-adenosine monophosphate (cAMP) and maximal stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR)/Cl−-HCO3− complex. Plus signs indicate activation; minus signs indicate inhibition. α/β, adrenergic receptor; M1/M3, muscarinic acetylcholine receptor; PKA, protein kinase A; SR, secretin receptor.

bicarbonate secretion (287). An opposite reaction, that is, a decrease of secretin-stimulated bile flow and bicarbonate secretion, was observed in cholangiocytes isolated from the liver of bile duct–ligated (BDL) rats with total vagotomy, additionally supporting the role of parasympathetic innervation and its neurotransmitter, ACh, in potentiation of secretin-stimulated bicarbonate secretion (65,288). Adrenergic Agonists Rat cholangiocytes and Mz-ChA-1 cells (a human cholangiocarcinoma cell line) express α1-, α2-, β1-, and β2-, and α2A-, α2B-, and α2C-adrenoreceptors, respectively (289–291). Activation of α1-adrenergic receptors by phenylephrine, an α1-adrenergic agonist, in BDL rats in vivo resulted in potentiation of secretin-stimulated bile flow and bicarbonate secretion (see Fig. 59-9). In in vitro models, phenylephrine alone did not alter basal cAMP levels in cholangiocytes or expansion of the lumen of enclosed IBDUs (a reflection of cholangiocyte secretion), but did significantly potentiate secretin-stimulated cAMP levels and IBDU expansion. The effects of phenylephrine on cholangiocyte secretion were abolished by benoxanthian hydrochloride, an α1-adrenergic antagonist, additionally suggesting the importance of adrenergic regulation in mechanisms of ductal bile formation. Adrenergic regulation of cholangiocyte functions in BDL rats occurs through a Ca2+-dependent, PKC-mediated amplification of the cAMP signaling pathway (289,291). Similarly, stimulation of α2-adrenoreceptors in Mz-ChA-1 cells causes up-regulation of cAMP levels (290). In contrast, degeneration of adrenergic innervation in BDL rats induced by

1520 / CHAPTER 59 6-hydroxydopamine results in a decrease of secretinstimulated choleresis and cholangiocyte cAMP levels (289,292).

Inhibition of Basal and Secretin-Stimulated Cholangiocyte Secretion Secretin-stimulated cholangiocyte secretion may be terminated or inhibited by a number of regulatory factors that include somatostatin, gastrin, dopaminergic agonists, and ET (Fig. 59-10). Somatostatin Somatostatin is a cyclic tetradecapeptide produced by neurons and secretory cells in the central and peripheral nervous systems and the gastrointestinal tract (293). In the human, dog, rat, and mouse, somatostatin inhibits both basal and secretin-stimulated ductal bile secretion (264,271, 293–297) (see Fig. 59-10). A wide array of physiologic effects of somatostatin in the body is mediated by five somatostatin receptor subtypes (sst1-5) (293,298). Four of them (i.e., sst1-4) are expressed in rat and mouse cholangiocytes (97,264,271).

In cholangiocytes, somatostatin inhibits expression of secretin receptors, secretin-stimulated bile flow, bicarbonate secretion, and insertion of exocytotic vesicles into apical membranes through interaction with sst2 receptors, followed by activation of the cGMP- and by inhibition of the cAMPsignaling pathways (97,264,271). In addition, enhanced ductular fluid reabsorption was suggested as one of the major mechanism of somatostatin-induced anticholeresis in dogs (294) and mice (264). Indeed, in IBDUs isolated from normal mice, somatostatin and L-779976, a selective somatostatin analog specific for sst2, not only inhibited secretinstimulated ductal fluid secretion, but also directly induced ductal fluid absorption; in sst2-knockout mice, these effects of somatostatin were diminished (264). In cholangiocytes isolated from wild-type mice, somatostatin decreased secretin-stimulated cAMP levels and increased intracellular cGMP levels; in contrast, in sst2 knockout mice, somatostatin did not influence cAMP and cGMP levels (264). Thus, somatostatin may regulate ductal bile formation by two mechanisms: (1) inhibiting secretin-stimulated cholangiocyte bicarbonate and fluid secretion, and (2) directly stimulating cholangiocyte fluid absorption via unidentified mechanisms (264,294). Gastrin

Apical (bile)

Basolateral (blood) Gastrin

CCK

[Ca2+]i

Somatostatin

sst2

cGMP

Secretin

PKC-α −

− SR

cAMP

+

Dopaminergic agonists

ETA

D2

[Ca2+]i − [Ca2+]i

Cilium

+

−

Endothelin-1

H2O

PKA

Cl−

PKC-γ

Cl− HCO3− H2O

Gastrin, a linear peptide secreted by G cells in the stomach (266), inhibits secretin-stimulated cholangiocyte secretion in normal and BDL rats (298–301). The inhibitory effect of gastrin on secretin-stimulated cholangiocyte secretion occurs through activation of the cholecystokinin B/gastrin receptors, followed by membrane translocation and activation of the Ca2+-dependent PKCα and ablation of the stimulatory effect of secretin on cAMP synthesis (300) (see Fig. 59-10). Gastrin also induces membrane translocation of PKCα in Mz-ChA-1 cells (302). In addition, effects of gastrin on cholangiocyte functions in BDL rats are associated with increased expression of PKCα, PKCβ1, and PKCβ2 (301). Dopaminergic Agonists

FIG. 59-10. Inhibition of secretin-stimulated bicarbonate-rich ductal bile secretion. Somatostatin inhibits cholangiocyte bicarbonate-rich secretion via suppression of the cyclic 3′,5′adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway, and directly stimulates cholangiocyte water absorption via an unidentified mechanism. Gastrin inhibits cholangiocyte secretion via the intracellular Ca2+ ([Ca2+]i)/ PKCα signaling pathway ablating the stimulatory effect of secretin on cAMP synthesis. Activation of dopaminergic receptors results in activation of the [Ca2+]i/PKCγ signaling pathway and inhibition of secretin-stimulated cAMP levels and PKA activity. Endothelin-1 inhibits cholangiocyte bicarbonate-rich secretion via a Ca2+-dependent inhibition of adenylyl cyclase activity. Plus signs indicate activation; minus signs indicate inhibition. CCK, cholecystokinin-B/gastrin receptor; cGMP, cyclic 3′,5′-guanosine monophosphate; D2, dopaminergic receptor; ETA, endothelin receptor; SR, secretin receptor; sst2, somatostatin receptor.

Rat cholangiocytes express one of three known dopaminergic receptors (i.e., D2) on their basolateral plasma membrane domain through which the D2 dopaminergic receptor agonist, quinelorane, induces an increase in intracellular IP3 and [Ca2+]i levels, resulting in inhibition of secretin-stimulated cholangiocyte secretion (see Fig. 59-10). The effect of quinelorane in cholangiocytes is realized through activation of PKCγ and inhibition of secretin-stimulated cAMP levels and PKA activity (303). Endothelin Cholangiocytes express both ET receptors (i.e., ETA and ETB). ET-1, a 21-amino-acid polypeptide with multifunctional properties (304,305), inhibits secretin-stimulated cholangiocyte secretion by selectively interacting with ETA

PHYSIOLOGY OF CHOLANGIOCYTES / 1521 receptors through IP3- and Ca2+-dependent inhibition of AC activity (131) (see Fig. 59-10).

on their apical plasma membrane domain through which bileborne regulatory factors (i.e., bile constituents, osmolarity, and flow) can regulate ductal bile formation (Fig. 59-11).

Regulation by Bile-Borne Factors

Bile Constituents Adenosine Triphosphate and Other Nucleotides

The chemical composition, osmolarity, and flow rates of bile continuously percolating through intrahepatic bile ducts vary over time and can significantly affect cholangiocyte functions. Cholangiocytes posses a number of receptors and channels

ATP is currently recognized not only as a ubiquitous enzyme cofactor and the major energy source in the cell, but also as an important autocrine/paracrine signaling molecule Apical (bile)

Basolateral (blood) Secretin

cAMP

SR

− +

Cl−

+

PKA

Cl−

HCO3−

−

H2O

−

Cl−

+

PKC-α

t-ASBT

[Ca2+]i

BAH Bile acids

Bile acids

BA− + H2CO3

Glucose

BAH + HCO3− ASBT

SGLT1

H2O

Amino acids

− Secretin SR

cAMP

PKA

[Ca2+]i

PKC-α

IR

Insulin

+

Cl− Cl−

HCO3−

H2O Cl−

Bile flow

PC1

PC2 Ca2+

P2X

Nucleotides P2Y

Bile osmolality ?

Ca2+

TRPV4

FIG. 59-11. Regulation of ductal bile formation by bile-borne factors. Unconjugated bile acids (BA−) may stimulate bicarbonate-rich choleresis and inhibit secretin-stimulated cholangiocyte secretion. Conjugated bile acids can both stimulate and inhibit secretin-stimulated cholangiocyte secretion. Glucose and amino acids absorbed by cholangiocytes via sodium-dependent glucose transporter SGLT1 and unidentified amino acid transporters, respectively, drive aquaporin mediated water absorption. Nucleotides, luminal bile flow, and bile osmolarity may regulate cholangiocyte secretion by increasing [Ca2+]i, followed by activation of Ca2+-regulated Cl− channels and Cl−/HCO3− exchanger. Insulin may regulate ductal bile formation by increasing [CA2+]i, followed by inhibition of secretin-stimulated cholangiocyte secretion. ASBT, apical sodium-dependent bile acid transporter; BAH, protonated unconjugated bile acids; cAMP, cyclic 3′,5′-adenosine monophosphate; IR, insulin receptor; PKA, protein kinase A; PKC, protein kinase C; SR, secretin receptor.

1522 / CHAPTER 59 that in the liver might be involved in hepatocyte-tocholangiocyte and cholangiocyte-to-cholangiocyte signaling through activation of P2X and P2Y receptors (158,169, 177,238,247,306–311). As mentioned earlier, P2X receptors (P2X1-7) are extracellular, ATP-gated, calcium-permeable, nonselective cation channels, the stimulation of which by ATP results in an increase of [Ca2+]i (240–244). Four of the seven known P2X receptors (i.e., P2X2, P2X3, P2X4, and P2X5) are expressed in Mz-Cha-1 cells, a human cholangiocarcinoma cell line (245), and four P2X receptors (i.e., P2X2, P2X3, P2X4, and P2X7) are expressed in normal rat cholangiocytes (245–247). P2Y receptors (P2Y1,2,4,6,11-14) belong to the family of G protein–coupled receptors with seven transmembrane domains and extracellular N and C termini. Activation of most P2Y receptors (i.e., P2Y1,2,4,6,11) leads to stimulation of phospholipase Cβ, mobilization of Ca2+, and activation of PKC (240–244). Cholangiocytes express four (i.e., P2Y1, P2Y2, P2Y4, and P2Y6) of the six cloned, molecularly distinct P2Y receptors in mammals (i.e., P2Y1,2,4,6,11,12) (158,247, 310,311). Functional studies suggest that P2X and P2Y receptors are expressed on both cholangiocyte basolateral and apical plasma membrane domains (142,156,218,293). In rat cholangiocytes, the P2X4 receptor is potentially a primary effector for regulation of Cl− secretion by ATP (246). At the cholangiocyte apical plasma membrane, P2Y receptors are equally activated by ATP and other nucleotides (i.e., ADP, UTP, and UDP) (158,247,310). In contrast, the basolateral P2Y receptors are activated with an agonist specificity of ADP ≥ ATP ≥ UTP (158,310). These data suggest that P2Y receptors are expressed heterogeneously within the cholangiocyte plasma membrane, and that cholangiocyte functions may be differentially regulated by different nucleotides through activation of different subtypes of P2Y receptors. Expression of the multiple P2X and P2Y receptors in cholangiocytes, their distribution within cholangiocyte plasma membranes, and activation by a variety of nucleotides, suggest that P2 receptors represent a highly specific mechanism of regulation of ductal bile formation. Indeed, activation of apically located P2Y receptors in rat cholangiocytes causes rapid and substantial increases in membrane Cl− permeability, favoring efflux of Cl− from the cell into the lumen, presumably through Ca2+-activated Cl− channels (158,177,310). In rat IBDUs, basolateral and luminal activation of cholangiocyte P2Y receptors results in an increase of [Ca2+]i and in biliary bicarbonate secretion, suggesting the importance of this mechanism in regulating ductal bile formation (239,247) (see Figs. 59-8 and 59-11). Interestingly, basolateral activation of P2Y receptors by ATP and other nucleotides is less effective in increasing [Ca2+]i compared with their apical activation (247). The exact mechanism of regulation of cholangiocyte secretion by nucleotides is unknown; however, it is likely that nucleotides activate Ca2+-sensitive Cl− channels and subsequent Cl-HCO3− exchange (177,247).

Bile Acids Both conjugated and unconjugated bile acids are involved in regulation of ductal bile formation (see Fig. 59-11). A small amount of the unconjugated bile acids secreted by hepatocytes in an anionic form are protonated in bile, passively enter cholangiocytes across their apical plasma membrane, and, during this process, stimulate bicarbonaterich choleresis (181). Stimulation of a bicarbonate-rich choleresis by unconjugated bile acids may also result from an increase in [Ca2+]i and Cl− efflux through Cl−channels, as was observed in Mz-ChA-1 human cholangiocarcinoma cells exposed to ursodeoxycholic acid (UDCA) (312). In cholangiocytes of BDL rats, UDCA inhibits secretin-stimulated cholangiocyte secretion through an increase in [Ca2+]i, followed by activation of PKCα (313). In a model of BDL rats, where the number of cholangiocytes dramatically increases because of cholangiocyte proliferation, conjugated bile acids (i.e., taurocholic and taurolithocholic acids) interact with cholangiocytes, increasing DNA synthesis, secretin receptor gene expression, cAMP levels, and Cl−-HCO3− exchanger activity, suggesting their ability to stimulate ductal bile secretion (314–318). Two other conjugated bile acids, that is, tauroursodeoxycholic (TUDCA) and taurohyodeoxycholic (THDCA) acids, induce choleretic effects in normal and BDL rats with a greater efficiency in BDL rats; TUDCA-induced hypercholeresis was associated with an increased bicarbonate secretion, whereas THDCA increased biliary secretion of phospholipids (319). TUDCA and THDCA decrease cAMPdependent cholangiocyte secretory activity as evidenced by decreased secretin receptor gene expression, secretinstimulated cAMP levels, and Cl−-HCO3− exchanger activity (318,319). Regulation of cholangiocyte secretion by TUDCA occurs through activation of the Ca2+-dependent PKCα (318) (see Fig. 59-11). In a model of bile acid–fed rats, different conjugated bile acids up-regulate or down-regulate the expression of bile acids transporters, that is, ASBT and t-ASBT, additionally suggesting their functional importance in regulation of cholangiocyte functions (288,313,314,318,320). Glucose The physiologic significance of the absorption of biliary glucose by SGLT1 expressed on the cholangiocyte apical plasma membrane (see Glucose Transport section earlier in this chapter) is likely related to regulation of ductal bile formation (see Fig. 59-11). Indeed, glucose actively absorbed from bile by cholangiocytes can provide osmotic gradients favoring water transport from bile into cells, that is, absorption (190–192). In vivo studies showed that as the amount of absorbed biliary glucose increased after infusion of D-glucose into the femoral or portal veins, bile flow decreased (190,192). In the isolated rat liver, the rate of bile secretion decreased by about one fifth when it was perfused with solutions containing

PHYSIOLOGY OF CHOLANGIOCYTES / 1523 15 mM D-glucose (191). In contrast, as the absorption of biliary glucose decreased after phlorizin, an inhibitor of the glucose transporter SGLT1, bile flow increased (190,192). Studies using the segmented retrograde intrabiliary injection technique in rats also demonstrated that phlorizin inhibited absorption of biliary glucose and moderately increased bile flow (321). Direct studies using the microperfused rat IBDU suggest that cholangiocytes rapidly absorb glucose from the perfusate via SGLT1, followed by the AQP-mediated absorption of water (192). This observation is consistent with studies demonstrating that the osmotic water permeability of perfused proximal kidney tubules, which actively absorb D-glucose by SGLT2, was reduced in AQP1 knockout mice by 80% (322). The importance of AQP1 in water movement across cell membranes in response to transported glucose also was demonstrated in experiments using expression of AQP1 and SGLT1 in Xenopus oocytes (323). The results suggested that although SGLT1 has several different roles in water transport (194,195,324), here it is principally involved in generating an osmotic driving force for AQP-mediated water transport. Thus, in cholangiocytes, absorption of glucose and glucose-stimulated water absorption may represent one of important mechanisms for controlling the chemical composition of bile. Amino Acids Amino acids present in bile are absorbed by cholangiocytes, potentially favoring water absorption. Absorbed biliary amino acids may generate osmotic gradients of 3 to 12 mOsm (41), drive water absorption, and thus be involved in regulation of ductal bile formation (see Fig. 59-11). Bile Osmolarity The regulation of ductal bile formation by changes in bile osmolarity has not yet been demonstrated experimentally. However, rat and mouse cholangiocytes express the osmoreceptor TRPV4 (N. F. LaRusso, unpublished observation), suggesting that cholangiocyte functions related to ductal bile formation can be affected by bile tonicity (see Fig. 59-11). Bile Flow The cholangiocyte apical plasma membrane is continuously exposed to mechanical forces generated by bile flowing through the intrahepatic bile ducts. It has become clear that cholangiocytes can sense luminal bile flow via primary cilia expressed on their apical plasma membrane domain (112,233,255) by mechanisms similar to those described in Madin–Darby canine kidney cells and cultured mouse kidney collecting duct epithelial cells (248,325–327). Cholangiocyte cilia express two proteins (i.e., polycystin-1 and -2) that can act as a mechanoreceptor and a Ca2+ channel, respectively. Increasing the flow rate of the perfusate through

the lumen of IBDUs causes a cilia-mediated increase in cholangiocyte [Ca2+]i (255) (see Fig. 59-11). Importantly, increased luminal flow rates also result in cilia-dependent inhibition of forskolin-stimulated cAMP signaling in cholangiocytes, suggesting that luminal mechanical stimuli can affect both the [Ca2+]i and cAMP intracellular signaling pathways critically involved in regulation of ductal bile formation (233). Luminal flow also induces in cholangiocytes a ciliamediated increase in pHi that is a reflection of Cl− and HCO3− transport (255), suggesting that luminal mechanical flow stimuli transmitted into cholangiocytes by cilia can activate the [Ca2+]i second messenger pathway, followed by Ca2+dependent regulation of Cl− transport. As mentioned earlier (see K+ Channels in the Ion Transport (Apical) section), cholangiocytes possess a dense population of Ca2+-activated Cl− channels, and the current density of the Ca2+-activated Cl− conductance is approximately twofold greater than the cAMP-activated Cl− conductance (148,150,153). Thus, cholangiocyte cilia may be an integral part of signaling machinery located on the apical cholangiocyte plasma membrane that monitors changes in bile flow (and perhaps also alterations in bile osmolality and chemical composition) within intrahepatic bile ducts and adjusts cholangiocyte functional response to such changes. We speculate that bile flow in intrahepatic bile ducts is pulsatile, and that its changes will alter the mechanical forces to which cholangiocytes are exposed. Because the cholangiocyte cilium contains a mechanoreceptor, polycystin-1, and a Ca2+ channel, polycystin-2, proteins thought to form a functional complex when a primary cilium is bent (248–254), luminal bile flow may bend cholangiocyte cilia, resulting in an increase in [Ca2+]i. In turn, this may regulate apically located channels, exchangers, and transporters involved in ductal bile formation.

Vesicular Trafficking in Mechanisms of Ductal Bile Formation The regulated targeted trafficking of vesicles containing cholangiocyte transport proteins from an intracellular compartment to the apical plasma membrane and their exocytotic insertion/endocytic retrieval into and out of membrane (i.e., recycling regulatory mechanism) in response to choleretic/cholestatic stimuli is one of the important mechanisms controlling cholangiocyte ion and water transport. Recycling regulatory mechanisms have been demonstrated in a number of different cell types including the following: (1) insulin-induced insertion of the glucose transporter, GLUT-4, into the plasma membrane of adipose and muscle cells (328); (2) insertion of AQP2 in the apical plasma membrane of the epithelial cells of cortical collecting ducts in response to vasopressin (329–331); and (3) insertion of H+,K+-ATPase in the plasma membrane of gastric parietal cells in response to gastrin and other secretagogues (332).

1524 / CHAPTER 59 The cholangiocyte plasma membrane is a highly dynamic structure; the basal rate of total exocytosis/endocytosis in Mz-ChA-1 cells and apical exocytosis/endocytosis in rat cholangiocyte monolayers is equal to approximately of 1.3% to 1.6% plasma membrane per minute (119,333). These processes are under the control of the cAMP signaling pathway; when cholangiocytes are stimulated by the cAMP analog, CPT-cAMP, exocytosis/endocytosis increases; in contrast, a PKA inhibitor, Rp-cAMPs, decreases basal exocytosis/ endocytosis (119). In cholangiocytes, recycling regulatory mechanisms respond to secretin, somatostatin, and changes in cell volume (210,211,270–272,333). In short-term cultures of normal rat cholangiocytes, secretin stimulates exocytosis via the cAMPsignaling pathway (270). In contrast, somatostatin decreases secretin-induced exocytosis (254). In both in vitro and in vivo experimental models, secretin stimulates insertion of vesicles containing AQP1 into the apical plasma membrane to facilitate the osmotic movement of water and, in turn, the elaboration of ductal bile (210,211). Importantly, in the basal state, cholangiocytes contain a subpopulation of vesicles in which AQP1 is sequestered with the chloride channel, CFTR, and with the Cl−-HCO3− exchanger, AE2. In response to choleretic stimuli (i.e., secretin, dibutyryl cAMP) these proteins move together from an intracellular location to the apical cholangiocyte plasma membrane with the involvement of molecular motors (i.e., dynein and kinesin) (272). In the apical plasma membrane, they form a microdomain transporting complex that promotes Cl−, HCO3−, and water transport into the lumen of intrahepatic bile ducts, resulting in an increase of net ductal bile secretion. Thus, secretin-induced cholangiocyte secretion may occur via exocytotic insertion of transporters/channels from intracellular vesicles into the apical plasma membrane. The inhibitory effect of somatostatin on secretin-induced cholangiocyte secretion may occur by endocytic retrieval of transporters/channels from the plasma membrane. These processes involve the intracellular cAMP and cGMP signaling pathways (264,270,272). The mechanisms by which vesicles containing CFTR, AE2, and AQP1 are targeted to the cholangiocyte apical plasma membrane, and how they dock and fuse into the membrane, are unknown. One of these transport proteins (i.e., CFTR) contains a PDZ-interacting domain and two C-terminal regions that are required for its functional expression on the apical plasma membrane (334,335), and it is likely that in cholangiocytes, polarization of CFTR to the apical plasma membrane occurs by a PDZ-dependent mechanism. How AE2 and AQP1, which do not contain PDZinteracting domains, are targeted to the cholangiocyte apical plasma membrane remains unclear.

Functional Heterogeneity of Cholangiocytes Most of the functional events described earlier occur in large, but not in small, cholangiocytes, supporting the concept of functional heterogeneity of biliary epithelia.

Large cholangiocytes express all necessary functional elements, such as specific receptors, CFTR, and the Cl--HCO3− exchanger, and respond to secretin and somatostatin with changes in cAMP and cGMP levels and Cl−, HCO3−, and water secretion (3,27,51,92,94–96,264). The lack of expression of secretin and somatostatin receptors, CFTR, the Cl--HCO3− exchanger, and ASBT in small cholangiocytes suggests that this portion of the biliary tree may be functionally passive in ductal bile formation (55,92). However, developments have suggested otherwise. Indeed, small cholangiocytes express functionally important proteins such as annexin V (138), ETA and ETB receptors, AQP1, and AQP4, suggesting their potential ability to be involved in mechanisms of ductal bile formation. In addition, small cholangiocytes are able to change their biochemical phenotype depending on the circumstances. For example, chronic feeding of bile acids induces expression of ASBT in rat small cholangiocytes that normally do not express this transporter (317), thus transforming them into functionally active cells with regard to bile acid transport. However, the major specific functions of small cholangiocytes remain to be determined.

INTEGRATED MODEL OF DUCTAL BILE FORMATION Ductal bile formation is a result of cholangiocyte secretion and absorption that occur with the involvement of numerous transporters, exchangers, and channels functioning in a coordinated fashion on the apical and basolateral plasma membrane domains. Cholangiocyte secretion is primarily caused by Cl− and HCO3− transport across the apical plasma membrane into the lumen of intrahepatic bile ducts, whereas cholangiocyte absorption is primarily caused by bile acids, glucose, and amino acid transport from bile across the apical membrane into the cell. In both cases, water follows the osmotic gradients established by secreted or absorbed osmotically active molecules. As discussed earlier and depicted in Figures 59-5, 59-8, 59-9, 59-10, and 59-11, secretin and other hormones initiate ductal bile formation by interacting with their receptors, and via different intracellular signaling pathways, they induce extrusion of Cl ions with subsequent depolarization of the cell membrane. Opening of Cl− channels induces a Cl− gradient favoring the activation of the apically located Cl−-HCO3 exchanger, resulting in a bicarbonate extrusion. These events on the apical plasma membrane require the coordinated functions of transporters, exchangers, and channels on the basolateral plasma membrane domain (see Fig. 59-5). Basolateral Na+-K+-2Cl− cotransport and Cl−-HCO3− exchange (the latter possibly linked to NHE) function as active Cl− uptake mechanisms that maintain the concentration of intracellular Cl− at greater than electrochemical equilibrium. An increase in apical membrane permeability to Cl− also results in activation of basolateral membrane K+ channels, SK2, through which K+ taken up by the Na+-K+ -2Cl− cotransporter and Na+,K+-ATPase is recycled back.

PHYSIOLOGY OF CHOLANGIOCYTES / 1525 The lumen-negative transepithelial electrical potential generated by these processes may drive passive paracellular transport of Na+ from blood into the lumen, potentially with respective amount of water. The electrogenic Na+-HCO3− exchanger, which is activated at the cholangiocyte basolateral domain as a consequence of depolarization induced by Cl− efflux, counteracts bicarbonate excretion, thus contributing to maintenance of concentration of intracellular HCO3−. The carbonic anhydrase II, an enzyme that catalyzes hydration of carbon dioxide to bicarbonate and hydrogen ions, is an additional mechanism controlling the concentration of intracellular bicarbonate. Cholangiocyte water secretion in response to osmotic gradients established by secreted HCO3− occurs through water channels, AQPs, which are constitutively present in both the basolateral and apical plasma membrane domains or are inserted into the apical membrane from internal cytoplasmic vesicles in response to choleretic stimuli. It has become evident that ductal bile formation may be initiated or modulated not only by activation of specific receptors on the cholangiocyte basolateral plasma membrane domain, but also by activation of regulatory mechanisms linked to the cholangiocyte apical plasma membrane domain. Here, we introduce a concept that cholangiocyte secretion may be initiated or modulated at the apical plasma membrane by bile-borne regulatory factors, for example, by nucleotides, bile osmolarity, and bile flow (see Fig. 59-11). Activation of P2X and P2Y receptors, TRPV4, and a ciliaryassociated mechanosensory polycystin-1/2 complex that are linked to the intracellular Ca2+ and cAMP signaling pathways may result in stimulation of Ca2+-regulated Cl− channels or in modulation of cAMP-dependent cholangiocyte secretion. Changes in efflux of Cl− and HCO3− into the lumen of intrahepatic bile ducts in response to luminal regulatory factors regulate basolateral transport of ions in a coordinated fashion as described earlier. Apically located SGLT1, ASBT, and amino acid transporters allow for absorption of solutes from bile into cholangiocytes, processes that may inhibit the secretory function of cholangiocytes by unidentified mechanisms, thus shifting cholangiocyte secretion to cholangiocyte absorption (see Fig. 59-11). However, taking into account the anatomic and functional heterogeneity of the intrahepatic biliary ductal system, we cannot exclude that different populations of cholangiocytes may be simultaneously involved in secretory and absorptive processes in biliary epithelia.

CONCLUDING REMARKS In this chapter, we have attempted to summarize recent information on the physiology of cholangiocytes, a rapidly evolving field receiving increasing attention from investigators with interests ranging from molecular and cellular biology to pathophysiology. Indeed, the application of novel techniques to the study of cholangiocyte biology has led to a rapid expansion of our knowledge in the field of

cholangiocyte physiology. The molecular, biochemical, and functional characteristics of many cholangiocyte transport proteins and numerous receptors expressed on both the basolateral and apical plasma membrane domains, and the existence of well-established intracellular signaling mechanisms that functionally link specific receptors and transport proteins, unequivocally suggest that the intrahepatic biliary ductal system is not a simple conduit for bile secreted by hepatocytes, but rather is an organ within an organ, responsible for the final determination of the physical, chemical, and physiologic properties of bile. As with any physiologic function, ductal bile formation is a highly regulated process. Cholangiocyte secretory/absorptive activity is regulated by a number of hormones and other regulatory factors acting via their receptors expressed on the basolateral plasma membrane. Importantly, in the last several years it has become more evident that cholangiocyte functional activity also is regulated by a number of factors affecting the apical pole of these cells. It is currently unclear how the regulatory mechanisms that occur on the opposite poles of the cholangiocyte are coordinated, and what mechanisms predominate in the resting state or when the functional activity of the liver is increased. To answer these and other questions, the physiology of cholangiocytes continues to grow extensively as a field of increasing attention and importance.

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (grant DK24031, N.F.L.) and the Mayo Foundation.

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309. Roman RM, Fitz JG. Emerging roles of purinergic signaling in gastrointestinal epithelial secretion and hepatobiliary function. Gastroenterology 1999;116:964–979. 310. Salter KD, Fitz JG, Roman RM. Domain-specific purinergic signalng in polarized rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 2000;278:G492–G500. 311. Dranoff JA. Purinergic regulation of bile ductular secretion. In: Alpini G, Alvaro D, Marzioni M, LeSage G, LaRusso N, eds. The pathophysiology of the biliary epithelia. Georgetown: Lamdes Bioscience, Eurekah, 2004;41–50. 312. Shimokura GH, McGill JM, Schlenker T, Fitz JG. Ursodeoxycholate increases cytosolic calcium concentration and activates Cl− currents in a biliary cell line. Gastroenterology 1995;109:965–972. 313. Alpini G, Glaser S, Alvaro D, Ueno Y, Marzioni M, Francis H, Baiocchi L, Stati T, Barbaro B, Phinizy JL, Mauldin J, Lesage G. Bile acid depletion and repletion regulate cholangiocyte growth and secretion by a phosphatidylinositol 3-kinase-dependent pathway in rats. Gastroenterology 2002;123:1226–1237. 314. Alpini G, Glaser S, Francis H, Marzioni M, Venter J, Phinizy J, LeSage G. Bile acids interactions with cholangiocytes. In: Alpini G, Alvaro D, Marzioni M, LeSage G, LaRusso N, eds. The pathophysiology of the biliary epithelia. Georgetown: Lamdes Bioscience, Eurekah, 2004;112–126. 315. Alpini G, Glaser S, Robertson W, Phinizy JL, Rodgers RE, Caligiuri A, LeSage G. Bile acids stimulate proliferative and secretory events in large but not small cholangiocytes. Am J Physiol 1997;273: G518–G529. 316. Alpini G, Glaser SS, Ueno Y, Rodgers R, Phinizy JL, Francis H, Baiocchi L, Holcomb LA, Caligiuri A, LeSage GD. Bile acid feeding induces cholangiocyte proliferation and secretion: evidence for bile acid-regulated ductal secretion. Gastroenterology 1999;116: 179–186. 317. Alpini G, Ueno Y, Glaser SS, Marzioni M, Phinizy JL, Francis H, Lesage G. Bile acid feeding increased proliferative activity and apical bile acid transporter expression in both small and large rat cholangiocytes. Hepatology 2001;34:868–876. 318. Alpini G, Baiocchi L, Glaser S, Ueno Y, Marzioni M, Francis H, Phinizy JL, Angelico M, Lesage G. Ursodeoxycholate and tauroursodeoxycholate inhibit cholangiocyte growth and secretion of BDL rats through activation of PKC alpha. Hepatology 2002;35: 1041–1052. 319. Baiocchi L, Alpini G, Glaser S, Angelico M, Alvaro D, Francis H, Marzioni M, Phinizy JL, Barbaro B, LeSage G. Taurohyodeoxycholateand tauroursodeoxy-cholate-induced hypercholeresis is augmented in bile duct ligated rats. J Hepatol 2003;38:136–147. 320. Kip NS, Lazaridis KN, Masyuk AI, Splinter PL, Huebert RC, LaRusso NF. Differential expression of cholangiocyte and ileal bile acid transporters following bile acid supplementation and depletion. World J Gastroenterol 2004;10:1440–1446. 321. Olson JR, Fujimoto JM. Demonstration of a D-glucose transport system in the biliary tree of the rat by use of the segmented retrograde intrabiliary injection technique. Biochem Pharmacol 1980;29: 213–219. 322. Schnermann J, Chou CL, Ma T, Traynor T, Knepper MA, Verkman AS. Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice. Proc Natl Acad Sci U S A 1998;95:9660–9664. 323. Zeuthen T, Meinild AK, Loo DD, Wright EM, Klaerke DA. Isotonic transport by the Na+-glucose cotransporter SGLT1 from humans and rabbit. J Physiol 2001;53:631–644. 324. Duquette PP, Bissonnette P, Lapointe JY. Local osmotic gradients drive the water flux associated with Na(+)/glucose cotransport. Proc Natl Acad Sci U S A 2001;98:3796–3801. 325. Praetorius HA, Spring KR. Bending the MDCK cell primary cilium increases intracellular calcium. J Membr Biol 2001;184:71–79. 326. Praetorius HA, Spring KR. Removal of the MDCK cell primary cilium abolishes flow sensing. J Membr Biol 2003;191:69–76. 327. Praetorius HA, Spring KR. The renal cell primary cilium functions as a flow sensor. Curr Opin Nephrol Hypertens 2003;12:517–520. 328. Bryant NJ, Govers R, James DE. Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 2002;3:267–277. 329. Knepper MA, Inoue T. Regulation of aquaporin-2 water channel trafficking by vasopressin. Curr Opin Cell Biol 1997;9:560–564. 330. Brown D. Targeting of membrane transporters in renal epithelia: when cell biology meets physiology. Am J Physiol Renal Physiol 2000;278: F192–F201.

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334. Moyer BD, Denton J, Karlson KH, Reynolds D, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, Stanton BA. A PDZ-interacting domain in CFTR is an apical membrane polarization signal. J Clin Invest 1999;104:13533–13561. 335. Milewski MI, Mickle JE, Forrest JK, Stafford DM, Moyer BD, Cheng J, Guggino WB, Stanton BA, Cutting GR. A PDZ-binding motif is essential but not sufficient to localize the C terminus of CFTR to the apical membrane. J Cell Sci 2001;114:719–726.

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CHAPTER

60

Gallbladder Function Sum P. Lee and Rahul Kuver Gallbladder Morphology, 1536 Techniques to Study Gallbladder Function, 1536 Electrolyte and Water Transport, 1537 NaCl Transport: Na+-H+ and Cl−-HCO3− Exchange, 1537 Water Transport, 1540 Regulation of Electrolyte and Water Transport, 1540 Electrolyte and Water Transport during Gallstone Formation, 1542 Absorption versus Secretion, 1544 Biliary Cholesterol Absorption, 1544 Cholesterolosis of the Gallbladder, 1545 Cellular Mechanisms of Cholesterol Absorption and Efflux, 1546 Bilirubin and Xenobiotic Transport, 1548 Bile Salt Transport, 1549

Transport of Amino Acids and Sugars, 1549 Mucins, 1549 Regulation of Mucin Secretion, 1550 MUC Gene Expression, 1551 Gallbladder Mucins and Gallstone Formation, 1551 Protein Absorption and Secretion, 1551 Bacterial Infection, Inflammation, and Gallbladder Function, 1551 Infection and Gallstone Pathogenesis, 1552 Cytokines, 1552 Nitric Oxide, 1552 Cholesterol Crystals, 1552 Oxysterols, 1553 Summary, 1553 References, 1553

The principal function of the gallbladder is to store and concentrate bile, a fact appreciated in the seventeenth century when Diemerbroek (1) wrote that bile enters the gallbladder to “acquire greater strength and digestive power.” Bile formation occurs at the canalicular membranes of hepatocytes, a process driven by an array of adenosine triphosphate (ATP)– dependent transport proteins (2). A significant portion of this hepatic bile enters the gallbladder, where it is stored and concentrated between meals. The composition of bile is changed by absorption and secretion by the gallbladder mucosa. Because of the low level of most organic bile constituents in the extracellular space and in plasma, concentration gradients of at least 10,000:1 are estimated to be present across the gallbladder epithelium (3). This epithelium

therefore represents a barrier of considerable integrity. In addition to restricting transepithelial diffusion of many substances, the gallbladder epithelium also facilitates net absorption or secretion of other substances. This requires high selectivity of the epithelium to passage of individual bile constituents. The primary barrier regulating gallbladder permeability is located in the epithelium, rather than in the subepithelial layers. The gallbladder epithelial cell plays a central role in the transport of water and electrolytes, the acidification of bile, the absorption of cholesterol and other biliary lipids, and the absorption of bile pigments, amino acids, and sugars. In addition, the gallbladder epithelial cell secretes mucins, cytokines, fluid, and electrolytes constitutively and when stimulated. Conversely, gallbladder epithelial cell functions are influenced by biliary constituents, such as bile salts and cholesterol (including oxidized species of cholesterol). As a consequence of its central role in these transport processes and its responsiveness to biliary constituents, the physiology of the gallbladder epithelial cell is discussed in detail in this chapter. Previous reviews of gallbladder mucosal function (3–5) summarize progress in this field.

S. P. Lee and R. Kuver: Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Seattle, Washington 98195. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1535

1536 / CHAPTER 60 An important theme of the current review is that proteins that mediate absorption and secretion by the gallbladder epithelial cell, including their regulation and mechanisms of action, are beginning to be characterized. Bile is likely to remain in the gallbladder for several hours until it is evacuated to the intestine after meal-stimulated neurohormonal signals that control gallbladder contraction. The hormone cholecystokinin (CCK) is the most potent of these signals. Ejection of gallbladder bile into the duodenum and regulation of hydrostatic pressure in the biliary tract are additional functions of the gallbladder. The gallbladder smooth muscle cell is the effector cell for gallbladder contraction. Neural control of the gallbladder and gallbladder motility are not discussed separately in this chapter because this topic is covered in Chapter 32. Much of the impetus behind the research into gallbladder physiology has been provided by diseases attributed to gallstones because of their high prevalence, clinical significance, and economic impact (6). In particular, the high prevalence of cholesterol gallstones in Western society has spurred research aimed at elucidating the pathogenesis of gallstone formation. Disparate predisposing factors contribute to gallstone formation, including hypersecretion of cholesterol by hepatocytes into bile and abnormal gallbladder function (7). Gallbladder functions gone awry, such as mucin hypersecretion and increased cholesterol absorption leading to gallbladder smooth muscle dysfunction with consequent impaired gallbladder motility, are thought to be important contributors to gallstone pathogenesis. Gallbladder functions discussed in this chapter are framed within the context of their roles in cholesterol gallstone pathogenesis. Other diseases in which the gallbladder plays a role, such as cystic fibrosis (CF), pigment gallstone formation, cholecystitis, and gallbladder cancer, have also provided insights into normal gallbladder function. Despite its many functions, the gallbladder is a dispensable organ. This fact is illustrated by the absence of this organ in many mammalian species, such as the rat, pigeon, and deer (8), an observation that Aristotle discussed at length in his treatise on animal anatomy written circa 350 BC. In these animals, the biliary ductal system and perhaps a continuous feeding pattern compensate for the absence of a gallbladder. Humans also adjust quite readily to the absence of a gallbladder. This is illustrated by the statistics of surgical practice in the United States, where more than 500,000 cholecystectomies are performed per annum. Its disposability notwithstanding, the physiology of this remarkable organ has given us many insights into the complexities of epithelial and smooth muscle function, many of which have relevance to other polarized epithelia and smooth muscle compartments of the body.

GALLBLADDER MORPHOLOGY The gallbladder mucosa is lined with a single layer of columnar epithelial cells that, in many species, secondarily form prominent folds that can reach into the muscular layer,

forming Rokitansky–Aschoff sinuses. A characteristic trait of gallbladder epithelial cells is their high degree of structural polarization, as evidenced by the asymmetric distribution of intracellular organelles and by the organization of the plasma membrane, which is divided into distinct apical and basolateral domains. The apical membrane (facing the lumen) contains microvilli, which are more or less prominent in different species (8). The basolateral membrane (facing the serosa) has a basal region that is anchored to the basement membrane by hemidesmosomes and a lateral region that is frequently interdigitated with the membranes of neighboring cells, forming convoluted lateral intercellular spaces capable of distending to accommodate absorbed fluid. Tight junctions separate the two membrane domains and join epithelial cells together to form a sheet, creating a selective permeability barrier and preventing water-soluble molecules from leaking between cells. These junctions consist of a zonula occludens, which contain points of contact between the outer leaflets of the plasma membranes of the apical and basolateral domains, and a zonula adherens, in which adjacent membranes are parallel and bridged by filaments. Gap junctions connect adjacent cells in the gallbladders of certain species, such as Necturus maculosus (9). Tight junctions also separate the apical plasma membrane from the basolateral plasma membrane, which allow these membrane domains to remain functionally distinct (10). Functionally and morphologically, gallbladder epithelial cells are related to intrahepatic and extrahepatic biliary ductal epithelial cells, but retain unique characteristics (11), in much the same way that small and large intrahepatic cholangiocytes are related yet distinct cell types (12). The subepithelial layer of the gallbladder contains the basement membrane, smooth muscle, blood vessels, and serosa. The basement membrane consists of proteoglycans (mainly heparan sulfates), type IV collagen, and laminin (10). Transport across the epithelium in vivo occurs into or from the underlying capillaries; the blood compartment thus functions as a sink for water and solutes absorbed from bile. Whether a similar sink phenomenon exists for cholesterol absorbed from bile remains an open question, given the clinical syndrome of cholesterolosis of the gallbladder, wherein cholesterol is deposited beneath the mucosa (13).

TECHNIQUES TO STUDY GALLBLADDER FUNCTION The study of gallbladder physiology has benefited from the use of a variety of model systems, both in vivo and in vitro, obtained from almost two dozen animal species. One limitation of these models is that variations in cellular physiologic mechanisms do not allow generalizations that pertain to human gallbladder function. Conversely, human gallbladder specimens available for investigation usually are obtained from patients undergoing cholecystectomy for clinically evident disease, thus the relevance to normal human gallbladder function remains open to interpretation. Classical physiologists

GALLBLADDER FUNCTION / 1537 used in vitro preparations of excised gallbladders from animal models to study gallbladder mucosal function. For example, Diamond (14–17) used whole excised gallbladders from rabbits. The gallbladder from the amphibian Necturus maculosus has been studied intensively with respect to water and electrolyte transport (18,19) using patch-clamp and Ussing chamber techniques. Of the animal models used, the prairie dog deserves to be singled out because it has proved to be an excellent model for human cholesterol gallstone pathogenesis. Prairie dog hepatic and gallbladder bile compositions are similar to those of humans. The gallbladder epithelium of the prairie dog is electrogenic and resembles that of humans. Furthermore, prairie dogs maintained on a cholesterol-rich diet experience development of cholesterol gallstones in a manner that recapitulates events that occur in humans with cholelithiasis. Various in vitro cell culture systems of gallbladder epithelial cells have provided insights into gallbladder mucosal function. The isolation of gallbladder epithelial cells and their use in monolayer and three-dimensional cell cultures have been reviewed (20). Primary and long-term cultures of canine (21), bovine (22), guinea pig (23), prairie dog (24), and human (25–28) gallbladder epithelial cells have been reported. The versatility of these in vitro models is illustrated by the well-differentiated dog gallbladder epithelial cells reported by Oda and colleagues (21), which have been used to study the mechanisms of mucin secretion (29,30), carcinogenesis (31), apoptosis (32), and cholesterol transport (33). Mouse gallbladder epithelial cell culture models have provided unique insights given the availability of knockout and transgenic mice (11,34–36). In addition, cell lines from gallbladder carcinoma have been used for functional and morphologic studies (37–40). Tissue culture explants also have been used for physiologic studies of the gallbladder, such as water and electrolyte transport. Ex vivo arterially perfused preparations of whole gallbladders have provided insights into gallbladder transport physiology. This is exemplified by the work of Corradini and colleagues (41,42), who used human and pig gallbladders to study lipid absorption. Selective breeding of mice with strain-specific phenotypic characteristics has been used to elucidate the mechanisms of cholesterol gallstone pathogenesis. These quantitative trait locus (QTL) analyses have showed a number of Lith genes that determine gallstone susceptibility in mice (43,44). This technique has led to the identification of specific mediators of abnormal gallbladder function, such as mucin hypersecretion, that are linked to gallstone formation (45). Gallbladder motility can be studied in vivo; indeed, transabdominal ultrasound and nuclear medicine biliary scan are time-tested tools to assess gallbladder function in the clinical setting. In the laboratory, several methods are used to study gallbladder smooth muscle function, including receptor binding studies using radioimmunoassays, light microscopy on isolated gallbladder smooth muscle cells, tensiometry studies on muscle strips, and whole gallbladders (see review by Portincasa and van Berge Henegouwen [46]).

ELECTROLYTE AND WATER TRANSPORT Electrolyte and water transport has been studied intensively in gallbladder epithelia. Although pertinent to the understanding of the processes underlying bile concentration in the gallbladder, the extensive nature of these studies point to the value of the gallbladder as a model system for studying isosmotic fluid absorption, thereby providing insights into similar processes occurring in more complex organ systems such as the kidney and small intestine. This field has been reviewed extensively (4,5,47). Reuss (9) has provided a comprehensive analysis of salt and water transport in gallbladder epithelium, focusing on studies performed on Necturus gallbladder. NaCl Transport: Na+-H+ and Cl−-HCO3− Exchange Early studies on the gallbladder epithelium showed a relatively leaky epithelium with a high rate of water absorption (15–17,48). Both transcellular and paracellular conductance pathways contribute to this leakiness. Apical membrane entry of sodium and chloride and the basolateral membrane extrusion of both of these ions are integral components of this process. The gallbladder epithelium absorbs NaCl and water in near-isosmotic proportions. This results in concentration of the impermeant components of bile in the lumen of the gallbladder. The presence of sodium in the lumen is necessary for fluid absorption, because bathing of the gallbladder in vitro in a balanced buffer solution that contains potassium substituted for sodium resulted in the cessation of net volume and electrolyte absorption. However, the requirement for the chloride ion is not absolute. Sodium and chloride entry at the apical membrane occurs via an electroneutral process. The passive permeability of the luminal cell membrane to sodium ion is too low to account for the observed transepithelial active sodium flux by diffusional entry alone (49). Nondiffusional sodium entry has been postulated to occur by a one-to-one electrically neutral coupled entry mechanism for sodium and chloride (50). Such a carrier-mediated mechanism would facilitate charge neutralization with chloride entry against an electrochemical gradient (51). Na+-H+ Exchanger One such carrier-mediated mechanism involves the Na+-H+ exchanger (NHE). NHE is one of the major sodium absorptive pathways in the gallbladder. NHE isoforms have been demonstrated in the gallbladder epithelium. Human gallbladder mucosa contains messenger RNA (mRNA) of isoform 3 of the NHE (NHE3) (52). In situ hybridization experiments in human gallbladder demonstrated that NHE3 mRNA was strictly localized to gallbladder epithelial cells (53). Maximal increases of intracellular cyclic adenosine monophosphate (cAMP) reduce apical membrane NHE in Necturus gallbladder (54), as well as in the guinea pig gallbladder (55). These findings are consistent with a major role of NHE3,

1538 / CHAPTER 60 because NHE3 activity is decreased by an increase in intracellular cAMP, which is unlike that observed with NHE1 and NHE2. In prairie dog gallbladder, mRNA for NHE1, NHE2, and NHE3 are present, with NHE2 and NHE3 being involved in apical sodium uptake and transepithelial sodium absorption (56). In primary cultures of prairie dog gallbladder epithelial cells, NHE1, NHE2, and NHE3 accounted for approximately 6%, 66%, and 28% of total NHE activity, respectively (57). In the bovine gallbladder epithelium, an apical NHE3 and a basolateral NHE1 were demonstrated (58). H+ secretion occurs at the apical membrane of the gallbladder epithelial cell, although this is quantitatively less important than NaCl absorption. Using Ussing chambers, Plevris and colleagues (59) showed that human gallbladder mucosa is capable of acidifying physiologic solutions in vitro. The [H+] on the mucosal side of the gallbladder tissue increased, whereas the [H+] in the serosal compartment decreased simultaneously, suggesting transfer of H+ from the serosal to the mucosal side of the tissue. The concomitant decrease of bicarbonate concentrations with increased PCO2 in the mucosal side indicates that the acidification was not simply caused by absorption of bicarbonate. Acidification was abolished when the gallbladder epithelium was exposed to sodium-free solution or in the presence of amiloride, a specific Na+-H+ transport inhibitor, in the mucosal compartment, suggesting that H+ secretion in the human gallbladder depends on NHE. Furthermore, regulation of the rate of apical membrane NHE can occur with changes in extracellular pH (60). Intracellular pH, however, has a greater effect than extracellular pH on NHE activity. Intracellular pH maintains the NHE activity at ~30% of the maximal rate, accounting for basal rates of salt absorption. Cl−-HCO3− Anion Exchanger In several species, exposure to HCO3−-CO2–buffered solutions increases the rate of fluid absorption (48,61). This effect is caused by stimulation of NaCl absorption. Exposure to HCO3−-CO2 stimulates NaCl entry across the apical membrane, as indicated by increases in cell volume and cell sodium and chloride contents (62,63). Bicarbonate also is required for the maintenance of intracellular H+, which, in turn, maintains NHE. Together, this indicates that a counter ion transport of Na+-H+ and Cl−-HCO3− is operative at the apical membrane, accounting for the electrical neutrality of this process (64). The most likely mechanism is stimulation of both cation and anion exchangers (NHE and AE2) at the apical membrane. However, the sodium and chloride fluxes across the apical membrane may be dissociated under appropriate experimental conditions, indicating that these transporters are not necessarily linked (65,66). The AE2 Cl−-HCO3− anion exchanger was demonstrated using specific antibodies in the apical membrane of human gallbladder epithelial cells (67) and calf gallbladder membrane vesicles (58). In other cell types, Cl−-HCO3− exchange is exquisitely sensitive to intracellular pH. This exchanger is

more active in physiologic solutions with greater bicarbonate concentrations. Increases in intracellular cAMP reduce Cl−-HCO3− exchange by about 50% (68). This effect results from the reduction of the apparent Vmax without effect on the apparent Km for external chloride ion. Carbonic anhydrase IV is expressed at high levels on the apical plasma membrane of human gallbladder epithelial cells (69). This enzyme, in concert with cytoplasmic carbonic anhydrase II, may participate in acidification of bile by mediating bicarbonate reabsorption, which is observed in the gallbladder epithelium in most species (48). Decreased expression of carbonic anhydrase II in gallbladder epithelium has been suggested to be a pathogenetic factor in cholesterol gallstone formation (70). Apical Plasma Membrane Chloride Conductance Increases in intracellular cAMP produce cell membrane depolarization that is caused by increases in apical membrane chloride conductance, an effect that becomes more pronounced on reduction of chloride in the mucosal solution (71,72). In Necturus gallbladder epithelial cells, the cAMPinduced reduction in intracellular chloride is caused by the combined effects of inhibition of Cl−-HCO3− exchange and stimulation of apical chloride conductance (19). The Necturus gallbladder epithelial cell apical membrane lacks a Ca2+activated chloride conductance (73); therefore, the apical plasma membrane chloride conductance appears to be caused by a cAMP-activated chloride conductance. Cystic Fibrosis Transmembrane Conductance Regulator There is evidence that a homologue to cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for much of this apical chloride conductance. In the Necturus gallbladder epithelium, an apical membrane chloride channel stimulated by cAMP-dependent phosphorylation, consistent with CFTR, was reported (74). This protein cross-reacted with anti-CFTR antibodies, and isolated cells displayed a large apical membrane chloride conductance and membrane depolarization after forskolin-stimulated increases in intracellular cAMP (75). Extracellular ATP also stimulated an apical chloride conductance that was mediated by cAMP, but not by intracellular calcium (76), likely caused by the CFTR homologue. Subsequent studies showed the presence of cftr protein in normal cultured mouse gallbladder epithelial cells (34,36). Gallbladders from cftr−/− mice lacked the cAMP-induced chloride current observed in normal mouse gallbladder. In fluid transport measurements, normal and cftr−/− gallbladders were equally active in basal resorption. The addition of forskolin, which activated cftr, resulted in net fluid secretion in normal mouse gallbladder. In contrast, cftr−/− gallbladders were unable to secrete fluid, whereas a complete inhibition of resorption by forskolin was observed. Therefore, in normal mouse gallbladder epithelium, cAMPinduced fluid secretion involves simultaneous inhibition

GALLBLADDER FUNCTION / 1539 of apical sodium chloride resorption and activation of cftr (36). CFTR mRNA and protein were detected in human gallbladder tissue sections predominantly localized to the apical membrane (77,78). CFTR mRNA and protein also were detected in cultured human gallbladder epithelial cells. Chloride efflux in these cells could be stimulated by intracellular calcium and more intensely by cAMP. Vasoactive intestinal peptide (VIP) and secretin stimulated chloride efflux in vitro (78). In Ussing chamber experiments, human CF and normal gallbladder epithelium were compared with respect to electrogenic anion secretion. Electrogenic anion secretion occurred in human gallbladder mucosa under basal conditions and was stimulated by a cAMP-dependent pathway mediated by CFTR, as well as by exogenous ATP via a CFTR-independent pathway. The latter pathway is upregulated in CF gallbladders and involves P2Y2 purinoreceptors and intracellular calcium (79). The intestinal peptide guanylin, which is similar in structure to the heat-stable enterotoxins produced by enterotoxigenic strains of Escherichia coli, activates CFTR-mediated chloride efflux (80). The pathway leading to chloride secretion involves binding of guanylin and activation of an apical plasma membrane guanylate cyclase C (GC-C), intracellular production of cyclic guanosine monophosphate (cGMP), and activation of the cGMP-dependent protein kinase II (cGKII) (81). Guanylin and its affiliated proteins, GC-C, and cGKII are expressed in the human gallbladder. Guanylin is localized to secretory epithelial cells of the gallbladder and is present in the bile, whereas GC-C, cGKII, and CFTR are confined exclusively to the apical membrane of the gallbladder epithelial cells. Functional studies in a biliary cancer cell line show that guanylin is a specific regulator of chloride secretion (82). These studies suggest that guanylin released apically into bile by human gallbladder epithelial cells have the capability to regulate chloride secretion in a paracrine/luminocrine manner. Bicarbonate Secretion and Cystic Fibrosis Transmembrane Conductance Regulator Function CFTR has been implicated in bicarbonate secretion. In mouse gallbladder, agents that increase cAMP cause an increase in short-circuit current. The direction of the current and its insensitivity to amiloride suggest that it is caused by the secretion of anions (83), with transport of bicarbonate from the serosal to apical direction being the most likely candidate. The presence of CFTR was essential for this response to forskolin, with neither cftr−/− nor CF ∆F508 mice showing a significant response (84). Interestingly, restoration of the forskolin-stimulated bicarbonate secretory response in murine gallbladder occurred with in vivo intratracheal gene transfer of the human CFTR complementary DNA (cDNA) (85). However, bicarbonate secretion in CF gallbladder does not necessarily require the restoration of CFTR function, because agents that increase intracellular calcium concentrations also increase bicarbonate secretion by activating

calcium-sensitive chloride channels. Uridine triphosphate (UTP) via activation of P2Y2 receptors also stimulates bicarbonate secretion in cftr−/− mice (86). CFTR has a small bicarbonate conductance, less than for chloride (87). About two thirds of the current remains when the Cl−-HCO3− exchanger is blocked, suggesting that the exit route for bicarbonate is via CFTR in murine gallbladder (84). Regulation of the activation of the Cl−-HCO3− exchanger by cAMP can require the presence of CFTR in other cell types such as fibroblasts and submandibular and pancreatic duct cells (88,89). Studies in Xenopus oocytes suggest that CFTR exists in either a bicarbonate-permeant or -impermeant form, depending on the external chloride concentration (90). Because these results were not obtained in gallbladder epithelial cells, the relation between CFTR and the Cl−-HCO3− anion exchanger in the gallbladder remains unclear, especially because the net result of apical membrane ion fluxes is acidification, rather than alkalinization of the luminal fluid, which is in contrast with the situation in the pancreatic ducts or the submandibular glands. Na+,K+-ATPase The Na+,K+-ATPase pump mediates basolateral membrane sodium exit. The importance of sodium transport was demonstrated by the use of the pump inhibitor ouabain, which prevented net water flow (91). Replacement of sodium in the mucosal fluid with potassium salts also prevented water absorption. The activity of the Na+,K+-ATPase pump correlates directly with the rate of fluid transport (51). Its activity, however, is not saturated under in vitro conditions and is dependent on the intracellular sodium levels, which, in turn, depend on sodium entry across the luminal membrane, the rate-limiting step for transepithelial sodium transport (49). Basolateral Membrane Chloride Transport The mechanism of chloride transport from cell to basolateral solution appears to result from both conductive transport and electroneutral KCl cotransport (19,66). In low bicarbonate media, the basolateral membrane chloride conductance is small, that is, ~6% of the basolateral conductance (92). In 10 mM HCO3−/1% CO2, about 50% of the total conductance of the basolateral membrane is due to chloride (93). Decreasing extracellular pH increases basolateral chloride conductance and causes cell membrane depolarization (93). Basolateral membrane chloride conductance, however, is insensitive to intracellular pH changes. Potassium Conductive Pathways The apical and basolateral cell membranes in amphibian and mammalian gallbladders are predominantly potassium conductive. As a result, the potassium equilibrium potentials across these membranes determine both the cell membrane voltages and the electrical driving forces for ion transport. In Necturus and rabbit gallbladder, the apical membrane provides

1540 / CHAPTER 60 a pathway for potassium secretion (94). Depolarization of the apical membrane in Necturus gallbladder epithelial cells activates an apical potassium conductance. Apical potassium conductance is increased by intracellular calcium levels and decreased by external H+ (95,96). In patch-clamp studies in Necturus gallbladder, the most commonly observed potassium channel is a large-conductance (~200 pS) maximum potassium channel, which is largely inactive at the resting membrane voltage, and activates steeply with depolarization or increase of intracellular calcium (97). Maximum potassium channels account for no more than 20% of the resting apical membrane conductance. They account for the voltage- and calcium-sensitive components of apical membrane conductance and are sensitive to decreases in intracellular pH (98). In guinea pig gallbladder epithelial cells, a calcium-activated apical membrane potassium conductance was elicited using patch-clamp techniques. Reverse transcriptase-polymerase chain reaction showed mRNA consistent with a member of the BK family of calciumactivated potassium channels (99). The basolateral membrane also has a potassium conductive pathway, across which part of the potassium transported inward by the Na+,K+-ATPase pump is recycled. This conductance is voltage insensitive (95). Basolateral membrane potassium channels are activated by intracellular calcium (100). Acidification of the serosal bath solution leads to a decrease in the basolateral potassium conductance that is concomitant with an increase in the basolateral chloride conductance.

Water Transport In humans, bile is concentrated 8- to 10-fold in the gallbladder (8). Concentration within the gallbladder is a consequence of the removal of water from the lumen; this concentration of hepatic bile is a result of one of the highest rates of water absorption reported. The rate of fluid absorption has been measured in several species and ranges from 5 to 80 µl/cm2 per hour (9). Transport rates are greater in mammals than in other vertebrates. Water transport by gallbladder epithelium has characteristics similar to those in the proximal renal tubule and the small intestine. Water is absorbed isosmotically; that is, the osmolality of the absorbed solution is equivalent to that in the gallbladder lumen. Water absorption can occur against its chemical gradient, and net water transport is coupled osmotically to NaCl transport in the same direction (see review by Reuss [9]). The precise mechanism of water transport is unknown, but the predominant view is that salt transport causes small osmotic gradients across both cell membranes, making the cell interior hyperosmotic to the mucosal solution and hypoosmotic to the fluid in the lateral spaces. Models to explain flow of water isosmotically toward the subepithelial space, given these osmotic considerations, have been reviewed extensively (9,19,101). Despite these theoretical considerations, the molecular basis for isosmotic transepithelial water transport in the gallbladder remains undefined.

Aquaporins are membrane proteins that facilitate water movement across biological membranes. Aquaporin-1 is widely expressed in epithelial and endothelial cells, and microarray analysis has showed high levels of mRNA expression in the human gallbladder (102). Aquaporin-1 and -4 have been reported to play a role in water transport in cholangiocytes (103,104). The functional relevance of aquaporin-1 and other aquaporins in gallbladder epithelial cell water transport remains to be determined.

Regulation of Electrolyte and Water Transport In the steady state, changes in the rate of transepithelial transport must involve parallel alterations of the rates of salt entry across the apical membrane and salt extrusion across the basolateral membrane. The rate of transepithelial ion absorption is the result of integrated activity of transporters in the apical and basolateral cell membranes. Figure 60-1 illustrates the localization and functions of the transport proteins involved in ion transport in the gallbladder epithelial cell. A variety of mediators affect the rate of electrolyte and water transport by the gallbladder. Some of these could act to regulate gallbladder water and electrolyte transfer under physiologic conditions by modification of NaCl influx, active sodium extrusion, and/or junctional permeability. These adaptive mechanisms prevent large changes in cell volume or ion content, or both, in response to primary stimulation or inhibition of transport at either membrane domain. In gallbladder epithelium, the best understood regulatory mechanisms involve intracellular factors such as pH, calcium, and cAMP. Changes in intracellular levels of these and other agents mediate the effects of peptides, hormones, and neurotransmitters on salt transport. For example, secretin, glucagon, and VIP inhibit fluid absorption by the gallbladder mucosa, whereas CCK and gastrin are without effect (see review by Svanvik [5]). Of the regulatory peptides mentioned earlier, only VIP and secretin are able to modify gallbladder fluid transport at physiologic concentrations. Other peptides, however, may act to potentiate or inhibit the effects of other regulatory factors. Cyclic Adenosine Monophosphate Secretin, VIP, as well as a number of other agents such as prostaglandins, bradykinin, and vasopressin increase intracellular cAMP levels, which decreases the rate of fluid absorption by the gallbladder and, in some species, can elicit net secretion. The predominant effects of cAMP on gallbladder epithelial cells are exerted at the apical membrane and consist of activation or insertion, or both, of chloride channels, such as CFTR, and inhibition of the apical NHE and Cl−/HCO3− exchanger. The observation that transepithelial fluid transport is abolished with maximal intracellular cAMP levels (71) implies that net transepithelial salt transport must be abolished as well. Therefore, in steady state, net sodium and chloride fluxes at each membrane are either zero

GALLBLADDER FUNCTION / 1541 APICAL

H 2O Na+

Paracellular

Cl−

ATP/UTP

AE2

NHE

Transcellular

CFTR cAMP

K+ H+

Na+

HCO−3

K+

Ca++

Ca++

Cl−

K+ Cl−

P2Y2

Guanylin +

CFTR cAMP

HCO−3 Cl−

Tight junction

Cl−

Ca++ ATP ase

Basolateral

K+

FIG. 60-1. Localization and function of ion transporters in the gallbladder epithelial cell. The Na+-H+ exchanger (NHE), the Cl−-HCO3− exchanger (AE2), the maximum potassium channel, cystic fibrosis transmembrane conductance regulator (CFTR), and the calcium-activated chloride channel belonging to the P2Y2 purinoreceptor class are the major ion transporters identified on the apical plasma membrane of gallbladder epithelial cells. On the basolateral plasma membrane, Na+,K+-ATPase, a calcium-dependent potassium channel, a potassium-chloride cotransporter, and a chloride channel have been identified. Water is absorbed by paracellular and transcellular routes and is coupled to NaCl absorption. ATP, adenosine monophosphate; cAMP, cyclic adenosine monophosphate; UTP, uridine triphosphate.

or balanced by a counter flux of another ion. For instance, net sodium absorption might be balanced by potassium, and chloride could be balanced by bicarbonate secretion (see review by Reuss and colleagues [19]). The three isoforms of endothelin (ET), ET-1, -2, and -3 are potent contractile agonists for smooth muscle in a variety of tissues including the gallbladder. Human gallbladder epithelial cells in primary culture secrete ET. Secretion was increased by physiologic concentrations of CCK (26). In freshly isolated gallbladder epithelial cells, preproET-1, -2, and -3 mRNA were detected, and ET-1 protein was identified. The cells also displayed ET receptor mRNA and highaffinity binding sites for ET-1, mostly of the ETB type. Electrogenic anion secretion across intact gallbladder mucosa was stimulated by forskolin, secretin, and exogenous ATP, as assessed by short-circuit current increases in Ussing-type chambers. ET-1 inhibited forskolin- and secretin-induced changes in short-circuit current without affecting baseline short-circuit current or ATP-induced changes. Accordingly, ET-1 significantly reduced the accumulation of intracellular cAMP elicited by forskolin and

secretin, and this effect was abolished by pertussis toxin. Therefore, ET-1 is synthesized and inhibits, via a Gi protein–coupled receptor, cAMP-dependent anion secretion in human gallbladder epithelium (105). Neural Control Electrolyte transport is also under neural control. Necturus gallbladder epithelial cells express muscarinic receptors on the basolateral membrane. Receptor activation by acetylcholine causes phospholipase C activation and increase of intracellular calcium levels and inositol 1,4,5triphosphate. This then leads to activation of potassium channels (106). Acetylcholine also inhibits adenylate cyclase, thereby abrogating cAMP-induced inhibitory effects on fluid absorption (107). In feline gallbladder, the net absorption of calcium and fluid was significantly enhanced by stimulation of the splanchnic nerves. Intravenous infusion of VIP increased pH and the [Ca2+] × [CO3(2−)] ion product significantly in the buffer during passage through the gallbladder lumen.

1542 / CHAPTER 60 Moreover, the basal fluid absorption was reversed to a net fluid secretion. In view of the presence of noradrenergic and VIP-immunoreactive nerve fibers in the gallbladder wall and VIP receptors on gallbladder epithelial cells, these results suggest that neural control mechanisms influence the transport of calcium by the gallbladder mucosa (108). Control of Cell Volume and Tight Junction Permeability Cell volume changes occur after swelling in many polarized transporting epithelia. The acute component of this regulation is mediated by conductive losses of potassium and chloride. In Necturus gallbladder epithelial cells, swelling-induced activation of plasma membrane potassium channels is paralleled by inhibition of chloride channels, which is not conducive to cell volume changes acutely. Necturus gallbladder epithelial cells therefore do not acutely regulate their volume after hyposmotic swelling (109). Tight junction regulation in the gallbladder epithelium is a determinant of the contribution of the paracellular pathway of electrolyte and water transport. In comparison with the regulation of electrolyte transport in the apical and basolateral membranes of gallbladder epithelial cells, relatively little is known about regulation of the paracellular permeability pathway. In Necturus gallbladder, ~30% of the mucosa-toserosa flow of water has been estimated to pass via this route, whereas the serosa-to-mucosa flow is much less likely to follow the paracellular route (110). Tight junctions in the Xenopus gallbladder epithelium were opened by stimulation of protein kinase A (PKA) by forskolin and theophylline, as well as by stimulation of protein kinase C (PKC) by phorbol dibutyrate. The paracellular pathway activated by PKA or PKC did not discriminate between small anions and cations. The PKA effects were mediated by PKC (111).

Electrolyte and Water Transport during Gallstone Formation Increased absorption of electrolytes and water across the gallbladder epithelium occurs during the early stages of experimentally induced gallstone formation. In the prairie dog model, this occurs in an otherwise healthy gallbladder mucosa because of exposure to lithogenic bile (112). Moreover, altered transepithelial absorption has been implicated in the pathogenesis of both cholesterol and pigment gallstones. Increased gallbladder absorption may contribute to gallstone formation by increasing the concentration of nonabsorbable constituents of bile. These constituents may serve as either components of gallbladder sludge, a precursor of gallstones, or as pronucleating factors promoting cholesterol nucleation and growth. As gallstone formation proceeds, a significant decrease in net sodium absorption occurs, primarily because of an increase in serosa-to-mucosa sodium flux, with loss of the normal sodium gradient. This finding is consistent with the down-regulation of the Na+,K+-ATPase observed in animals with gallstones (113).

Role of Calcium Gallbladder bile concentrations of total and ionized calcium are increased in numerous experimental models of both cholesterol and pigment gallstones. Calcium salts are found in the matrices of many cholesterol stones (114), with precipitates of calcium bilirubinate, carbonate, or phosphate serving as nidi for cholesterol crystallization (115). Biliary calcium thus plays a structural role in the formation of gallstones and contributes to the nucleation of cholesterol crystals by serving as a pronucleating agent (116). Therefore, Scheeres and colleagues (117) investigated whether extracellular calcium modulates gallbladder absorption. They demonstrated that in the rabbit gallbladder, alterations in serosal extracellular calcium did not significantly affect absorption. The calcium channel antagonist verapamil, however, significantly decreased absorption, suggesting that calcium channels may mediate this process (118). Changes in extracellular calcium affected ion transport across the gallbladder epithelium of the prairie dog. Cates and colleagues (119) demonstrated—by exposing prairie dog gallbladder to dantrolene, which traps calcium ions within intracellular organelles, and nickel, which prevents influx of extracellular calcium—that the effects of extracellular calcium on prairie dog gallbladder ion transport are mediated by changes in intracellular calcium. In support of this view, apical and basolateral calcium channels were demonstrated in the prairie dog gallbladder epithelium.120 Gallbladder apical calcium channels would allow biliary calcium entry into the cell, and therefore could represent an important regulatory pathway for gallbladder ion transport during gallstone formation. Subsequent studies elucidated the mechanisms by which changes in intracellular calcium regulates gallbladder ion transport. Prairie dog gallbladders mounted in Ussing chambers were exposed to trifluoperazine, a potent antagonist of Ca2+/calmodulin, a receptor protein in the intracellular calcium second messenger system. In addition, the ion transport effects of increased extracellular calcium were determined in the presence of Ca2+/calmodulin inhibition. Inhibition of Ca2+/ calmodulin resulted in an increase in net sodium and water absorption. Increasing luminal calcium could reverse the effects of trifluoperazine. These results supported the notion that Ca2+/calmodulin regulates basal absorption in the prairie dog gallbladder (120). This effect of Ca2+/calmodulin was diminished when the prairie dogs were fed cholesterol (121). Moreover, when the intracellular calcium concentration in prairie dog gallbladder was increased by the calcium ionophore A23187, mucosa-to-serosa chloride flux was inhibited and serosa-to-mucosa flux of sodium was stimulated, resulting in increased net chloride secretion and decreased net sodium absorption. A23187 thus converted gallbladder water absorption to secretion. The effect of A23187 was delayed by pretreatment with indomethacin, suggesting that prostaglandin-dependent mechanisms were operative (122). PKC also plays a role in the regulation of gallbladder ion transport in this model. Activation of PKC by phorbol esters

GALLBLADDER FUNCTION / 1543 resulted in inhibition of sodium absorption and a stimulation of chloride secretion. Similar effects were seen with serotonin, which mediates its effects through the phosphoinositide/ PKC pathway. Pretreatment of the tissues with H-7, a potent inhibitor of PKC, blocked the inhibitory effect of phorbol esters on ion transport. Because of the changes in sodium and chloride transport, mucosa-to-serosa water flux was diminished, whereas serosa-to-mucosa flux remained essentially the same, causing a change from net water absorption to secretion (123). Therefore, in the prairie dog gallbladder, intracellular calcium mediates its regulation of gallbladder ion transport through the Ca2+/calmodulin complex and PKC. The results suggest that Ca2+/calmodulin regulates gallbladder ion transport at basal intracellular calcium levels, whereas PKC regulates transport at increased calcium levels. This was investigated in a study that sought to correlate gallbladder sodium and chloride fluxes with biliary lipid composition during the various stages of gallstone formation. Prairie dogs were fed a standard or cholesterol-rich diet. Hepatic and gallbladder bile was analyzed for lipids and calcium. Animals were designated precrystal, crystal, or gallstone based on the absence or presence of crystals or gallstones, respectively. Gallbladders then were mounted in Ussing chambers, and unidirectional sodium and chloride fluxes were measured. Net sodium absorption was increased during the precrystal stage, but decreased during the gallstone stage because of increased mucosa-to-serosa and serosa-tomucosa flux, respectively. Increased serosa-to-mucosa flux of both sodium and chloride characterized the crystal stage. Biliary lipids (total bile acids, phospholipids, and total lipids) increased progressively during various stages of gallstone formation and correlated positively with fluxes of sodium and chloride. A significant positive correlation between sodium and chloride fluxes and calcium concentration also was observed (124). In summary, this series of studies in the prairie dog model illustrates the central role of both intracellular and extracellular calcium on gallbladder electrolyte transport. As gallbladder bile progresses from the precrystal stage into the crystal stage and finally to the gallstone stage, changes in biliary lipid concentration and biliary calcium levels are correlated with a change from a state of net sodium and water absorption to that of chloride and water secretion. Intracellular calcium signaling networks involving Ca2+/calmodulin kinase and PKC play key roles in these alterations in sodium chloride and water fluxes. These concepts are illustrated in Figure 60-2. Octreotide, the synthetic somatostatin analogue that is known to be lithogenic, exerts this effect both by inhibition of gallbladder contractility and by direct effects on gallbladder electrolyte and water transport. Prairie dog gallbladders mounted in Ussing chambers and exposed to octreotide displayed increased net sodium and water absorption, and converted the gallbladder from a state of chloride secretion to that of chloride absorption by increasing mucosa-toserosa fluxes (125). Gallstone formation during octreotide therapy has been linked to increased gallbladder bile calcium concentration. Under basal conditions, normal prairie dog

Na+ H+ CI− HCO−3

Ca++

H2O

APICAL

−

AE2

NHE −

−

Phosphorylation

PKC

+

Ca++ -Calmodulinkinase + Ca++

PIP3

DAG + IP3

Receptor PLC G protein

Basolateral Ca++

FIG. 60-2. Central role of intracellular and extracellular calcium in the prairie dog gallbladder epithelial cell. Calcium channels are present on both apical and basolateral plasma membranes. Intracellular calcium concentrations are increased in response to signals generated after receptor activation, phospholipase C (PLC) activation, and the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Increased intracellular calcium activates cellular kinases such as the calcium/calmodulin kinase II and protein kinase C (PKC). Phosphorylation mediated by these kinases inhibit Na+-H+ (NHE) and Cl−-HCO3− exchangers (AE2), which, in turn, leads to diminished fluid secretion, and in certain situations, to fluid secretion. This change is observed as prairie dogs are fed a high-cholesterol diet and cholesterol crystals develop in their gallbladder biles. PIP3, phosphatidylinositol 4,5-bisphosphate.

gallbladder absorbed mucosal calcium. Serosal octreotide converted the gallbladder from a state of basal calcium absorption to one of net calcium secretion by stimulating serosa-to-mucosa calcium flux. Octreotide also increased net sodium absorption by stimulating mucosa-to-serosa sodium flux (126). Role of Gallbladder Bile Acidification Acid secretion by the human gallbladder has important implications for gallstone formation, because changes in the pH of bile are of critical importance in influencing the solubility of calcium in bile (127). This was illustrated in bile sampled from obese patients undergoing rapid weight loss after gastric bypass surgery. Even though calcium concentrations increased over time as gallbladder bile was concentrated, the saturation index of CaCO3 decreased, in association with increased acidification of gallbladder bile (128). Whether a defect in acidification by the gallbladder mucosa actually

1544 / CHAPTER 60 contributes to gallstone formation is less clear. Biliary acidification and calcium concentrations were not altered in patients with cholesterol stones (129). In pigment gallstone formation, however, both calcium and bilirubin solubility decreases with increasing pH. This is relevant for brown and black pigment stones, because both types contain predominantly calcium bilirubinate. One proposed sequence of events leading to pigment stones invokes defective acidification of gallbladder bile. In this scenario, gallbladder stasis or bile infection allows the hydrolysis of bilirubin diglucuronide to its less soluble counterparts. A defect in gallbladder acidification, perhaps related to inflammation-mediated changes in gallbladder epithelial cell function, perturbs calcium and bilirubin solubility. Mucus secreted by the gallbladder epithelium buffers hydrogen ions, increasing the pH of bile and contributing to calcium bilirubinate precipitation (7).

fluid could have come from the bile ducts, and therefore the interpretation of de novo gallbladder secretion was challenged (135). Glickerman and colleagues (136) then investigated a patient with multiple bile duct strictures whose gallbladder was excluded from the extrahepatic ducts. This permitted separate analysis of gallbladder and hepatic secretions. The authors confirmed that the gallbladder produces a clear, colorless fluid. Although hepatic bile flow was continuous, gallbladder drainage was intermittent, occurring only after meals, and the volume was variable. The gallbladder fluid was rich in protein, with mucin accounting for more than 60% of the protein. The fluid had no bilirubin, bile salts, cholesterol, or phospholipids, and it had the ionic profile of an extracellular fluid. The secretion was alkaline and contained abundant bicarbonate.

BILIARY CHOLESTEROL ABSORPTION Absorption versus Secretion The gallbladder is conventionally regarded as an absorptive organ, but reports as early as 1887 indicated that it was also capable of secretion. In that year, the phenomenon of gallbladder secretion was reported in two patients who had undergone cholecystostomy (130). Studies in monkeys indicated that gallbladder secretion occurred under physiologic conditions, such as after feeding (131). Prostaglandins, prostacyclin, and gastrointestinal hormones can induce net fluid secretion, as can inflammation (see review by Svanvik [5]). Human gallbladder mucosa produces prostaglandins. The amount of prostaglandins produced by inflamed gallbladder muscle and mucosal cells increases with the severity of the inflammatory process (132). Prostaglandins alter the normal process of water absorption by the gallbladder mucosa and induce net water secretion. In prairie dog gallbladders mounted in Ussing chambers, inhibition of prostaglandin synthesis by treatment with indomethacin stimulated sodium and chloride absorption. These effects were rapidly reversed by prostaglandin E2 (PGE2). PGE2 promoted chloride secretion and decreased mucosal sodium absorption at concentrations found in the gallbladder bile of animals with gallstones (133). Studies by Igimi and colleagues (134) showed that the gallbladder also secretes water and electrolytes into the lumen during periods of digestion. They studied patients who had recovered from a percutaneous transhepatic gallbladder drainage performed for acute cholecystitis. After an overnight fast, gallbladder bile was dark brown and had a wide scatter in the lipid composition. Two hours after a meal, the gallbladder bile was opalescent white and had the composition of an extracellular fluid. In addition, dog gallbladder epithelial cells absorbed sodium at baseline, but the addition of secretin resulted in a reversal of sodium flux and net sodium secretion. These studies established that secretion is a physiologic function of the gallbladder mucosa. These observations were made in patients with patent bile ducts communicating with the gallbladder. Theoretically, the

The gallbladder absorbs cholesterol from bile (see review by Einarsson and colleagues [137]). This was initially demonstrated in guinea pig gallbladder (138). Subsequently, cholesterol uptake by human gallbladders obtained at cholecystectomy was studied using Ussing chambers. When real and artificial biles labeled with [14C]-cholesterol were incubated with human gallbladder tissue, all gallbladders absorbed cholesterol. Recovery of the absorbed cholesterol from the tissue showed that about 4% was esterified over 60 minutes. In artificial bile the rate of absorption of cholesterol increased as the cholesterol saturation index increased, but became constant once supersaturation was achieved. In contrast, supersaturated real bile permitted greater absorption of cholesterol, possibly because of enhanced cholesterol solubilization. Preincubation of gallbladder tissue in sodium cyanide caused a 30% reduction in cholesterol uptake, indicating a partial “active” component to the absorptive process. There were no pronounced differences in the rate of cholesterol absorption as gallbladders became more diseased, but there was a reduction in the amount of cholesterol ester formed (139). A significant portion of the absorbed cholesterol was effluxed into the serosal fluid. Biliary bile acid composition also played a role, because an increase in dihydroxy bile acids increased gallbladder permeability to cholesterol (140). These early studies were prescient in that several themes regarding cholesterol absorption by gallbladder epithelial cells were identified. First, the finding that biliary cholesterol absorption was dependent on the cholesterol saturation index indicated that cholesterol flux at the apical plasma membrane was dynamic and perhaps able to be regulated. At the apical plasma membrane, the extent to which cholesterol transport was driven by physical–chemical interactions versus protein-mediated transport functions was an important question. That the former mechanism was important, was demonstrated in monolayer cultures of dog gallbladder epithelial cells. Cholesterol in the apical membrane of the cells exchanged readily with that in bile, but only in the

GALLBLADDER FUNCTION / 1545 presence of bile salts. The rate of exchange was dependent on the concentration and species of bile salts, with hydrophobic bile salts exerting a greater effect on cholesterol flux (141). The gallbladder epithelial cell therefore was equipped to respond to the biliary milieu by alterations in cholesterol uptake and efflux. Second, the issue of whether these cholesterol absorption and efflux mechanisms were normal functions of the gallbladder that were disrupted in patients with gallstone disease was raised. Theoretically, if the rate of cholesterol absorption exceeds that of the other biliary lipids, then gallbladder bile would become less saturated with cholesterol. To address this question, Corradini and colleagues (142) measured biliary lipids and pigment content in fasting gallbladder bile samples obtained from gallstone-free control subjects and from four study groups—multiple and solitary cholesterol gallstone patients and morbidly obese subjects with and without gallstones. Bile salt and pigment content were significantly greater in gallstone-free control subjects than in all other study groups, suggesting more effective gallbladder mucosal fluid absorption in control subjects. Correlation plot analyses of biliary lipids showed lower concentrations of phospholipids and cholesterol than expected from the index bile salt concentrations. These findings were more pronounced in gallstone-free control subjects and were interpreted as evidence of more efficient gallbladder absorption of biliary lipids in control subjects. Therefore, it appeared that efficient gallbladder mucosal absorption of both fluid and lipids from bile is a normal physiologic process that is impaired during cholesterol gallstone disease (142). Further studies in an in vitro intraarterially perfused gallbladder model strengthened this hypothesis. Isolated pig and human gallbladders were perfused through the cystic artery to maintain organ viability. A standard pooled natural bile, radiolabeled with [3H]cholesterol and [14C]phosphatidylcholine, was instilled in the lumen via a cystic duct catheter. Changes in bile volume and lipid concentrations were monitored at time intervals to evaluate the disappearance of lipids from bile caused by gallbladder absorptive function. In human gallbladder, 23% of cholesterol and 32% of phosphatidylcholine, but only 9% of bile salts, disappeared from bile in 5 hours. Consequently, at the end of the experiments, cholesterol and phospholipid molar percentages were significantly reduced, whereas the bile salt molar percentage was significantly increased with respect to values at the beginning of the studies. The results supported the concept that the human gallbladder modifies the relative composition of biliary lipids in such a way as to increase cholesterol solubility in bile (41). Subsequent studies showed that this ability to desaturate biliary cholesterol from gallbladder bile was dysfunctional in gallbladders from patients with gallstones. The in vitro intraarterially perfused gallbladder model was used to compare the absorption rates of lipids from standard bile by gallbladders obtained from patients with and without cholesterol gallstones. Normal gallbladders, but not cholesterol gallstone gallbladders, significantly reduced cholesterol and

phospholipid levels and increased bile salt molar percentages in bile by selective cholesterol and phospholipid absorption over a 5-hour period (42). Therefore, the work of Corradini and colleagues (42) supports the notion that a normal function of the gallbladder is to absorb cholesterol from bile. This appears to be an effective mechanism to desaturate biliary cholesterol that becomes dysfunctional in gallbladders with gallstones.

Cholesterolosis of the Gallbladder The third issue raised by the initial observations was the fate of the cholesterol absorbed by the gallbladder epithelial cell. Esterification of cholesterol occurs in gallbladder epithelial cells, and it could account for a portion of the absorbed cholesterol (see later). A bidirectional trafficking of cholesterol could allow some of the cholesterol to exit the cell at the apical plasma membrane, but this mechanism would not predominate in the situation where gallbladder biliary cholesterol was supersaturated. Finally, efflux mechanisms at the basolateral membrane could exist, as had been demonstrated in the early studies by Jacyna and colleagues (139). One implication of a basolateral cholesterol efflux pathway was whether such a pathway was responsible for the clinical phenomenon of cholesterolosis of the gallbladder. A morphologic description of gallbladder cholesterolosis was provided by Satoh and Koga (13), who analyzed surgically removed gallbladders. Lipid droplets were found not only in the submucosa, but also in the infranuclear cytoplasm of epithelial cells. Macrophages often were present between the epithelial cells, which were filled with lipid droplets and became foam cells. The authors proposed that free cholesterol absorbed by gallbladder epithelial cells became esterified in the endoplasmic reticulum, and thus appeared as lipid droplets. They proposed that lipid droplets were released into the intercellular space and phagocytosed by macrophages. Macrophages filled with lipid droplets became too large and rigid to pass through the endothelium of lymph vessels, and those large “foam cells” accumulated in the submucosa (13). Excess cholesterol deposition in the submucosa would also potentially alter smooth muscle cell function, leading to deleterious effects on gallbladder motility. The pig in vitro intraarterially perfused gallbladder model confirmed such a serosal efflux pathway for cholesterol absorbed by the gallbladder epithelium. Gallbladder mucosa lipid absorption rates were measured with and without plasma lipoproteins perfusing the vascular tree. In addition, human gallbladder fragments mounted in Ussing chambers and incubated with plasma lipoproteins at different concentrations in the serosal side were studied. Total lipoproteins and high-density lipoprotein (HDL) increased the release of biliary cholesterol and phosphatidylcholine in plasma and decreased the tissue accumulation of cholesterol absorbed from bile. The scavenger effect of plasma lipoproteins on cholesterol absorbed from bile was concentration dependent (143).

1546 / CHAPTER 60 A relation between excess cholesterol deposition in the submucosa of the gallbladder and impaired gallbladder motility has also been demonstrated in human gallbladders with gallstones. Plasma membranes of gallbladder muscle were purified and measured for cholesterol content and cholesterol/phospholipid molar ratio. The maximal contraction induced by CCK was significantly less in gallbladders with cholesterol stones than in those with pigment stones. The membrane cholesterol content and cholesterol/phospholipid molar ratio were significantly greater in gallbladders with cholesterol stones than in those with pigment stones. Membrane anisotropy was also higher in cells from cholesterol gallstone gallbladders than in cells from gallbladders with pigment stones, reflecting lower membrane fluidity in gallbladders with cholesterol stones. After muscle cells from cholesterol stone gallbladders were incubated with cholesterolfree liposomes for 4 hours, CCK-induced contraction, membrane cholesterol content and cholesterol/phospholipid ratio, and membrane fluidity returned to normal levels. These studies demonstrated that gallbladder muscle from patients with cholesterol stones has impaired muscle contractility. These abnormalities are corrected by removing the excess cholesterol from the plasma membranes (144). The gallbladder epithelial cell also has the capability to synthesize and esterify cholesterol through the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and acyl coenzyme A:acyltransferase (ACAT), respectively (145). The HMG CoA reductase activity was considerably less in the gallbladder mucosa than in liver tissue. The ACAT activity in the gallbladder mucosa was, in contrast, several times greater than corresponding activity in the liver. In cholesterolosis, the gallbladder mucosa was characterized by a fivefold increase in esterified cholesterol and normal contents of free cholesterol. In the gallbladder mucosa, the HMG CoA reductase activity was similar in patients with and without cholesterolosis. The ACAT activity of the gallbladder mucosa was increased in subjects with cholesterolosis. A positive correlation between the cholesterol saturation of bile and the content of esterified cholesterol in the gallbladder mucosa was evident (146). This study suggests that increased absorption of cholesterol from bile, followed by increased esterification, is the predominant mechanism of cholesterolosis of the gallbladder.

Cellular Mechanisms of Cholesterol Absorption and Efflux Many studies have focused on the cellular and molecular mechanisms of cholesterol uptake and efflux in gallbladder epithelial cells. Because the apical plasma membrane of gallbladder epithelial cells are exposed in a sustained manner to some of the highest cholesterol concentrations in the body, cholesterol handling mechanisms at this interface must be highly adapted to handle cholesterol flux. To what extent these events are dependent on physical-chemical forces that are subject to thermodynamic and detergent effects and how

much are caused by protein-mediated processes is an active area of investigation. Although the classical view that physicalchemical events are paramount was accepted until recently, emerging data indicate that specific cholesterol binding and transporting proteins are present in the apical plasma membrane of gallbladder epithelial cells. Figure 60-3 illustrates the localization of such proteins in gallbladder epithelial cells and their putative functions. Such proteins are emerging as important components of the cellular machinery that mediate not only absorption and efflux of cholesterol from gallbladder epithelial cells, but also cholesterol flux at other sites where polarized epithelial cells interact with bile, such as the canalicular membrane of the hepatocyte and the apical membrane of the villous enterocyte. Apolipoproteins Apolipoproteins are serum proteins that mediate carriage of cholesterol and other lipids in the serum. As such, they form the protein components of lipoprotein particles such as HDL and low-density lipoprotein (LDL) in serum. Apolipoproteins also have been identified in bile, with apolipoprotein A-I (ApoA-I), the predominant apolipoprotein found in HDL, found at the highest concentrations (147). ApoA-I inhibits cholesterol crystallization in bile, albeit at supraphysiologic concentrations (148). At physiologic concentrations, ApoA-I enhanced the transfer of phospholipid and cholesterol from the mucosal to the serosal side of human gallbladder epithelial cell monolayers. These cells appeared to bind ApoA-I reversibly in a dose- and timedependent manner, suggesting interaction with an apical plasma membrane receptor. Cultured human gallbladder epithelial cells also secreted ApoA-I basolaterally, which was greatly increased by apical exposure to model bile solutions. This study therefore suggested a mechanism involving ApoA-I whereby cholesterol and phospholipids could be removed from bile by gallbladder epithelial cells (149). Subsequent work with dog gallbladder epithelial cells demonstrated that these cells enhance basolateral cholesterol efflux when ApoA-I is applied to the apical plasma membrane. Furthermore, these cells synthesize and secrete both ApoA-I and ApoE into the basolateral compartment of confluent polarized cells, a process that is enhanced when cells are stimulated by ligands for the nuclear hormone receptors liver X receptor (LXR)α and retinoid X receptor (RXR) (150). ApoJ also is expressed at high levels in gallbladder epithelial cells, with secretion directed apically. ApoJ has been postulated to play a role in cholesterol absorption mediated by the multifunctional receptor megalin (151). Scavenger Receptor Type B Class I Scavenger receptor type B class I (SR-BI) is a multifunctional receptor capable of binding a wide array of native or modified lipoproteins, phospholipids, or bile acid micelles. In the hepatocyte, an important ligand for SR-BI is HDL, in which ApoA-I is the predominant apolipoprotein.

GALLBLADDER FUNCTION / 1547

Apo A-I

APICAL

2 3

1

Megalin

SR-BI

ABCG5 ABCG8

Apo J

HMG CoA reductase

ACAT

7 Cholesterol Cholesterol ester

8

Oxysterol

6 LXR/RXR

4 ApoE

5

ABCA1 Basolateral

ApoA-I

FIG. 60-3. Localization and putative function of transporters involved in cholesterol and phospholipid flux in the gallbladder epithelial cell. A model encompassing various functions for these transporters is proposed: (1) Biliary apolipoprotein A-I (ApoA-I) interacts with apical plasma membrane scavenger receptor type B class I (SR-BI). Binding could initiate internalization of ApoA-I, which, in turn, could initiate downstream events that influence cholesterol uptake and transcellular transport of cholesterol (149). (2) Cholesterol uptake proceeds via an ApoJ/megalin interaction, with apically secreted ApoJbinding cholesterol, followed by binding to megalin with subsequent uptake, as hypothesized by Erranz and colleagues (151). (3) ABCG5/ABCG8 expressed on the apical plasma membrane mediates apical cholesterol efflux analogous to the role of this heterodimer on the canalicular membrane of the hepatocyte and the apical plasma membrane of the villous enterocyte (155,156). (4) ABCA1 mediates basolateral plasma membrane cholesterol and phospholipid efflux (33). (5) Endogenously synthesized ApoE and ApoA-I, secreted basolaterally, potentiates ABCA1-mediated basolateral cholesterol efflux (150). (6) LXRα/retinoid X receptor (RXR) ligands act as intracellular sensors for cholesterol and up-regulate ABCA1 and ABCG5/ABCG8 expression and/or localization accordingly when intracellular free cholesterol levels are increased (33,150,156). (7) Intracellular free cholesterol is esterified via acyl coenzyme A:acyltransferase (ACAT), forming cholesterol esters that then remain in the cell or are secreted basolaterally, contributing to cholesterolosis of the gallbladder (13,145). (8) Cholesterol synthesis also takes place in the gallbladder epithelial cell, involving 3-hydroxy-3methylglutaryl coenzyme A (HMG CoA) reductase (145).

Therefore, given the presence of ApoA-I in bile, SR-BI expression at the apical plasma membrane of gallbladder epithelial cells was postulated. This was subsequently demonstrated in human, mouse, and dog gallbladder epithelial cells (150,152,153). In one study, sr-bi expression levels did not correlate with gallstone formation in a mouse model (152). In another study, an inverse relation was observed between biliary cholesterol concentration and SR-BI expression in murine gallbladder mucosa. However, gallbladder wall cholesterol content and gallstone formation were not dependent on SR-BI expression, as shown by comparing normal and sr-bi–deficient mice fed a lithogenic diet (153).

SR-BI expression levels therefore do not directly affect cholesterol deposition in the gallbladder or cholesterol gallstone formation. The functional role of SR-BI with respect to cholesterol transport on the apical plasma membrane of gallbladder epithelial cells remains undefined, although a direct interaction with biliary ApoA-I remains plausible (150). ABCA1 ABCA1 is the cholesterol/phospholipid efflux protein that is defective in patients with Tangier disease and familial HDL deficiency. These patients cannot form HDL,

1548 / CHAPTER 60 and therefore have a defect in reverse cholesterol transport, whereby cholesterol is mobilized from peripheral sites and returned to the liver. Because ABCA1 is expressed in the sinusoidal membrane of hepatocytes and the basolateral membrane of enterocytes, it appeared to be a good candidate for playing a role in efflux of cholesterol and phospholipids at the basolateral plasma membrane of gallbladder epithelial cells. ABCA1 was expressed on the basolateral plasma membrane of polarized cultured gallbladder epithelial cells. Furthermore, a basolateral cholesterol efflux mechanism with the characteristics of ABCA1 was demonstrated in these cells (33). Ligands for LXRα and RXR stimulated basolateral cholesterol efflux and induced ABCA1 expression, which is consistent with the known effect of these nuclear hormone receptors on ABCA1 expression and function. ABCG5/ABCG8 When a mixture of bile acids and cholesterol was infused into damaged dog gallbladders, the cholesterol concentration of the bile increased, whereas the bile acid concentration decreased, suggesting that the gallbladder mucosa was capable of releasing cholesterol into bile (154). Although this experiment likely demonstrates cholesterol efflux related to cytotoxicity, more recent evidence suggests that an apical plasma membrane cholesterol efflux mechanism in normal undamaged gallbladder epithelial cells may be present. The bidirectional trafficking of cholesterol in gallbladder epithelial cell monolayers (141), as well as a subtle increase in apical cholesterol efflux after LXRα/RXR ligand treatment (33), suggest such a mechanism. Either of the half-transporters ABCG5 and ABCG8 is mutated in patients with sitosterolemia. These patients lose the ability to discriminate between cholesterol and noncholesterol neutral sterols, such that marked increases in absorption of the latter type of sterols occurs. This is in marked contrast with persons without this condition, who assiduously prevent intestinal absorption of noncholesterol sterols. Studies in ABCG5 and ABCG8 transgenic and knockout mice have shown that these half-transporters are intimately involved in preventing plant sterol absorption in the villous enterocyte and mediating cholesterol efflux at the canalicular pole of the hepatocyte. In both these cell types, an apical localization of ABCG5/ABCG8 function was shown (155). Because the apical plasma membrane of gallbladder epithelial cells also interacts with biliary cholesterol, an apical plasma membrane functional role for ABCG5/ABCG8 in this cell type appeared plausible. Human gallbladders express ABCG5 and ABCG8 mRNA. Cultured murine gallbladder epithelial cells also express ABCG5 and ABCG8 mRNA and protein, as do cultured dog gallbladder epithelial cells. Interestingly, treatment with model bile containing supersaturating concentrations of cholesterol, or treatment with LXRα/RXR ligands, did not lead to differences in expression of ABCG5 or ABCG8 in the murine or the canine cells. The subcellular localization of ABCG5 and ABCG8 did show alterations, with predominantly intracellular localization at baseline and predominantly apical localization after

treatment with model bile or a synthetic LXRα ligand (156). Another report has confirmed that ABCG5/ABCG8 is expressed in the apical plasma membrane, as well as in intracellular compartments in human gallbladders (155). The functional role of ABCG5/ABCG8 in mediating cholesterol transport events at the apical plasma membranes of gallbladder epithelial cells is currently under investigation. Megalin and Cubilin Megalin, a member of the LDL class of cell-surface receptors, works in conjunction with cubilin, a large peripheral membrane protein, to mediate endocytosis of numerous ligands including HDL/ApoA-I. Megalin and cubilin are expressed by human and mouse gallbladder epithelia. Megalin and cubilin expression levels were sensitive to bile acid levels in the diets of mice, an effect that appeared to be mediated by the bile acid nuclear hormone receptor farnesoid X receptor (FXR), which itself is expressed in gallbladder epithelial cells. Megalin, but not cubilin, also was strongly up-regulated after feeding a lithogenic diet. Therefore, megalin and cubilin could play a role in apical plasma membrane lipid uptake in gallbladder epithelial cells (151).

BILIRUBIN AND XENOBIOTIC TRANSPORT Gallbladder absorption of unconjugated bilirubin from bile proceeds much faster than absorption of the pigment in conjugated form. Metabolic inhibitors do not reduce bilirubin absorption even though fluid absorption is reduced. Absorption is linearly related to the concentration of the pigments in the luminal solution, which indicates that loss of this pigment from the gallbladder proceeds by simple diffusion (157). This finding is supported by the lack of expression of the unconjugated bilirubin transporter multidrug resistance protein 1 (MRP1) in gallbladder epithelial cells (158,159). Many endogenous and exogenous organic compounds, including xenobiotics, are conjugated in hepatocytes to allow biliary excretion to occur. These compounds find their way into the gallbladder, where concentration of bile would increase their concentrations. High concentrations of such compounds could theoretically pose risks to the gallbladder epithelium. There is evidence that the gallbladder epithelial cell possesses defense mechanisms directed toward such potential cytotoxins. For example, the guinea pig gallbladder is relatively impermeable to highly ionized organic compounds such as sulfobromophthalein and iodipamide (160). Expression of MDR1, MRP2, and ABCG2 on the apical plasma membrane of gallbladder epithelial cells suggests that the capability to efflux xenobiotics is present (67,158,161). MRP3, the transporter responsible for efflux of conjugated anionic compounds at the sinusoidal membrane of hepatocytes, also is expressed on the basolateral plasma membrane of gallbladder epithelial cells, permitting efflux of these anionic compounds back into the plasma compartment (158). Therefore, analogous to the situation in hepatocytes, gallbladder epithelial cells are capable of protecting themselves

GALLBLADDER FUNCTION / 1549 from cellular accumulation of potentially toxic compounds. Although a xenosensor mechanism using the nuclear hormone receptors PXR and constitutive androstane receptor (CAR) is also likely to be present in the gallbladder epithelial cells, this has not been directly demonstrated.

Na+

cAMP

ASBT (+)

BILE SALT TRANSPORT Although the gallbladder mucosa is subject to injury by exposure to unconjugated and conjugated bile salts in pure form, this detergent effect is abrogated by the concomitant presence of cholesterol, phospholipid, and bilirubin in bile (4). Bile salts can also directly affect gallbladder epithelial cell function without causing cellular injury, with hydrophobicity a key determinant of these effects. Mechanisms for uptake of conjugated bile acids exist in human gallbladder epithelial cells, because these cells express the apical sodium-dependent bile acid transporter (ASBT) and the organic anion transporting polypeptide (OATP-A). Expression of these transporters was associated with sodium-dependent and -independent uptake of taurocholate, with sodium-dependent uptake being significantly greater than sodium-independent uptake (162). Bile salts also influence anion flux in gallbladder epithelial cells. The effects of taurochenodeoxycholate (TCDC) and tauroursodeoxycholate (TUDC) were compared with respect to chloride efflux. TCDC and TUDC stimulated chloride efflux in a sodium-dependent fashion at similar rates (162). Bile salts also influenced the cAMP signaling pathway in human gallbladder epithelial cells. TCDC and TUDC increased forskolin-induced cAMP accumulation. This was abrogated after PKC inhibition. cAMP-dependent chloride secretion was significantly increased by TCDC and TUDC. Therefore, bile salts potentiate cAMP production in the human gallbladder epithelium via PKC regulation of adenylyl cyclase activity (163). Gallbladder epithelial cells also express the nuclear hormone receptor FXR, which is activated by hydrophobic bile acids such as chenodeoxycholic and cholic acids at physiologic concentrations (151). The presence of this receptor in this cell type suggests that bile salt regulation of gallbladder epithelial cell function occurs at the transcriptional level. Bile salts, transported into the cell by ASBT, can potentially affect a myriad of gallbladder epithelial cell functions via FXR activation. Although conceptually appealing, specific studies to address FXR regulation of gallbladder epithelial cell function currently have not been published. Figure 60-4 illustrates the effects of bile salts on gallbladder epithelial cell function.

TRANSPORT OF AMINO ACIDS AND SUGARS There is active absorption of amino acids and sugar by the dog, guinea pig, and human gallbladder (164). The unidirectional transepithelial flux of glycine from mucosa to serosa is several-fold greater than the oppositely directed flux, and tissue accumulation of glycine follows saturation kinetics. Uptake of sugars and amino acids is reduced by tissue

APICAL

Conjugated BA

CI−

TCDC TUDC

FXR/RXR

Basolateral

FIG. 60-4. Effects of bile salts on gallbladder epithelial cell function. Conjugated bile salts enter the cell from bile via the sodium-dependent apical bile acid transporter (ASBT). Hydrophobic bile acids, such as chenodeoxycholic and cholic acids, are ligands for the nuclear hormone receptor farnesoid X receptor (FXR). Taurochenodeoxycholate (TCDC) and tauroursodeoxycholate (TUDC) increase forskolininduced cyclic adenosine monophosphate (cAMP) accumulation, and increase cAMP-dependent chloride secretion (163).

incubation in sodium-free bathing solutions or by exposure to metabolic inhibitors or ouabain. This suggests that the gallbladder mucosa has the capacity to conserve much of the sugar and amino acids present in hepatic bile by an active transport mechanism (4).

MUCINS Gallbladder epithelial cells secrete mucins, which form a gel that protects the apical surfaces of these cells against noxious constituents of concentrated bile such as bile salts and lysophosphatidylcholine. Gallbladder mucins are heavily sulfated, but with scarce sialic acid residues (165). Besides having a protective function, gallbladder mucin can, in certain situations, stimulate cholesterol nucleation, crystal growth, and crystal aggregation, thereby contributing to the pathogenesis of gallstone formation. Lithogenic diets induce hypersecretion of gallbladder mucin (166,167), a result that precedes the detection of gallstones. Because of this pathogenetic role, the mechanisms and regulation of mucin secretion by gallbladder epithelial cells has been investigated intensively. Figure 60-5 provides an overview of the regulation of mucin secretion by the gallbladder epithelial cell.

1550 / CHAPTER 60 Mucin gel

Conjugated BA Na+

Mucin exocytosis

ATP P2u Receptor ASBT TCDC TC

APICAL

CFTR

Mucin granules

CI−

CaMKII PKC Ca++

The same secretagogues noted to increase intracellular cAMP also led to an increase in mucin secretion. Whether an increase in cAMP also stimulates mucin secretion in the human gallbladder is controversial. Prostaglandins, adrenaline, and isoproterenol caused an increase in cAMP in one culture system (28), but forskolin, secretin, and VIP did not significantly stimulate mucin secretion in another (170). Furthermore, cAMP, intracellular calcium, or PKC agonists did not accelerate mucin secretion by cultured mouse gallbladder epithelial cells (35).

cAMP

Role of Intracellular Calcium

VIP Adrenaline Isoproterenol Prostaglandins

Basolateral

FIG. 60-5. Regulation of mucin secretion in gallbladder epithelial cells. Mucin granules are released from the apical plasma membrane of gallbladder epithelial cells constitutively and after stimulation with specific agonists. Extracellular signals such as prostaglandins, vasoactive intestinal peptide (VIP), isoproterenol, and adrenaline increase intracellular cyclic adenosine monophosphate (cAMP) levels and accelerate mucin secretion in certain species. Conjugated bile salts, acting via an intracellular calcium signaling pathway, also induce accelerated mucin secretion. P2u purinergic receptors also act via the intracellular calcium pathway to induce mucin secretion. Finally, there is evidence that cystic fibrosis transmembrane conductance regulator (CFTR) is present on intracellular mucin granules in dog gallbladder epithelial cells. One proposed mechanism involves activation of such channels leading to influx of chloride ions and water, disruption of cationic shielding of mucins, and rapid hydration of mucin granule contents after exocytosis (174). ATP, adenosine triphosphate; BA, bile acid; CaMKII, calmodulin-dependent protein kinase II; PKC, protein kinase C; TCDC, taurochenodeoxycholate.

Regulation of Mucin Secretion Role of Cyclic Adenosine Monophosphate Mucin secretion in the gallbladder occurs at a low basal level in the fasting state and at a rapid rate after exposure to CCK and cholinergic agents (168,169). Compounds that cause an increase in intracellular cAMP (VIP, adrenaline, isoproterenol, prostaglandins) cause an increase in mucin secretion in the dog and guinea pig gallbladder, but not in the prairie dog gallbladder. Secretagogue-stimulated mucin secretion in dog gallbladder epithelial cells proceeds via a cAMP signal transduction pathway (29). Intracellular cAMP levels increased in response to PGE2, PGE1, VIP, epinephrine, and isoproterenol, with PGE2 showing the greatest effect.

In primary cultures of human gallbladder epithelial cells, ionomycin and phorbol-12-myristate 13-acetate increased mucin secretion. The effects of these agents were additive and were mediated by a calcium-dependent pathway implicating Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaMKII) and by the activation of PKC, respectively. Mucin secretion was stimulated by extracellular ATP via P2U receptors, leading to cytosolic calcium increase, and PKC via cytosolic calcium increase and Ca2+/CaMKII activation. Therefore, mucin secretion in human gallbladder is regulated predominantly by calcium-dependent pathways (170). In addition, ATP, UTP, uridine diphosphate, and adenosine diphosphate stimulated mucin secretion, whereas extracellular adenosine had no effect implicating P2u purinergic receptors (170). Role of Bile Salts Model biles increased mucin secretion in dog gallbladder epithelial cells. Taurocholate in model bile (but not cholesterol or phospholipid) caused a dose-dependent increase in mucin secretion, suggesting that bile salt was the bile component responsible for the stimulatory effect. Only the more hydrophobic bile salts TCDC and taurodeoxycholate, but not the more hydrophilic bile salts taurocholate (TC) and TUDC, stimulated mucin secretion (30). A direct interaction between these hydrophobic bile salts and the apical plasma membrane may have led to increased mucin secretion, because activation of canonical signal transduction pathways such as cAMP, PKC, and intracellular calcium did not appear to be involved (171). Subsequent studies also showed that the bile salt–mediated effects on mucin secretion were not caused by their detergent properties (172). The results with the dog gallbladder epithelial cells on bile salt–induced mucin secretion suggested that an uptake mechanism for conjugated bile salts was present in gallbladder epithelial cells, which was subsequently shown to be the case in human gallbladder epithelial cells (162). TCDC stimulated mucin secretion in cultured human gallbladder epithelial cells in a sodium-dependent fashion. Both TCDC and TUDC were efficiently transported in biliary epithelial cells, as assessed by competitive uptake experiments. However, as compared with TCDC, TUDC induced significantly lower mucin secretion. PKC down-regulation caused

GALLBLADDER FUNCTION / 1551 a 70% reduction in TUDC-induced mucin secretion, but did not affect TCDC-induced secretion, which was mediated predominantly by Ca2+/CaMKII activation. These results provided evidence that bile salts are transported mainly via ASBT into human gallbladder epithelial cells and stimulate mucin secretion in these cells (162). PKC also appeared to play a role in bile salt–mediated acceleration of mucin secretion. Role of Cystic Fibrosis Transmembrane Conductance Regulator CFTR also has been implicated in the regulation of mucin secretion by the gallbladder epithelium, as well as other epithelia. A fourfold increase in constitutive mucin secretion was reported in cultured dog gallbladder epithelial cells that overexpressed CFTR (173). However, in cultured mouse gallbladder epithelial cells, no evidence for a link between mucin secretion and CFTR activity was found (35). Immunofluorescence microscopic studies showed intracellular colocalization of mucins and CFTR. This suggested that CFTR was present in the mucin granules. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. These observations suggested that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis, and hydration of mucus (174).

MUC Gene Expression MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 are expressed in fetal gallbladder and adult human gallbladder, whereas MUC4 is not. In contrast, MUC4 is strongly expressed in gallbladder adenocarcinomas (175). Northern blot studies demonstrated the presence of MUC1, MUC3, MUC4, MUC5AC, and MUC5B mRNA in cultured human gallbladder epithelial cells, whereas MUC2 mRNA was barely detectable (176). Vandenhaute and colleagues (177) determined the pattern of expression of the different human mucin genes in biliary epithelial cells, including gallbladder epithelial cells, by means of Northern blotting and in situ hybridization. Cells showed strong mRNA expression of MUC3, MUC6, and MUC5B and weak expression of MUC1, MUC5AC, and MUC2. No expression of MUC4 and MUC7 was observed. Of the mucin genes expressed in the adult human gallbladder, MUC5B appears to be present consistently at high levels (178,179).

Gallbladder Mucins and Gallstone Formation The mucous gel overlying the gallbladder mucosa can act as a nidus for stone formation (180). Prevention of gallstone formation by aspirin in the prairie dog model was postulated

to occur because of inhibitory effects on mucin secretion (181). In humans, the significance of aspirin and other inhibitors of prostaglandin formation in the prevention of gallstone formation is less clear. However, some studies point to a possible inhibitory effect of drugs such as aspirin on mucin secretion by the gallbladder mucosa (182). Studies in mouse models have provided further evidence supporting a role for mucous hypersecretion in the pathogenesis of cholesterol gallstones. QTLs affecting gallstone phenotypes were identified by linkage analysis. A gene locus (Lith 3) on chromosome 15 that controls mucin accumulation harbors the mucin gene Glycam1, which was expressed in gallbladder epithelia. Therefore, gallstone and mucin loci colocalized with potential QTLs affecting the formation of cholesterol crystals (45). A role for the MUC1 gene product also has been demonstrated in murine gallstone formation. Muc1-deficient (muc1−/−) and wild-type mice were fed a lithogenic diet for 56 days. Muc1−/− mice displayed significant decreases in total mucin secretion and accumulation in the gallbladder, as well as retardation of crystallization, growth, and agglomeration of cholesterol monohydrate crystals. At 56 days of feeding, gallstone prevalence was decreased by 40% in muc1−/− mice. However, cholesterol saturation indices of gallbladder bile, hepatic secretion of biliary lipids, and gallbladder size were comparable in muc1−/− and wild-type mice (183).

PROTEIN ABSORPTION AND SECRETION A fervent interest in identifying factors in bile that could serve as pronucleating or antinucleating factors was a focus of gallstone research until the 1990s. This work led to the study of protein absorption from, and secretion into, bile by gallbladder epithelial cells (see review by Klinkspoor and Lee [3]). Albumin absorption from bile into gallbladder epithelium was described (184). Electron microscope studies using horseradish peroxidase showed a blood-to-bile pathway for serum proteins. An early study suggested that a significant amount of IgA could be added to bile during its storage in the gallbladder (185). The identity of specific proteins synthesized and secreted by gallbladder epithelial cells remains an area of active investigation (186). As the experience with ETs, cytokines, and guanylin demonstrates, many of the functional implications of such secreted proteins are being characterized.

BACTERIAL INFECTION, INFLAMMATION, AND GALLBLADDER FUNCTION Clinical conditions that lead to acute or chronic infection in the gallbladder have provided the rationale to study the implications of inflammatory mediators on gallbladder epithelial cell function. Some of the early studies in this area involved the role of arachidonic acid metabolites on gallbladder epithelial cell function, including their roles in fluid

1552 / CHAPTER 60 and electrolyte transport and mucin secretion. More recent studies suggest that the gallbladder epithelial cell directly participates in the inflammatory response, for example, by the synthesis and secretion of cytokines. Furthermore, bacterial infection and inflammation appears to have direct implications for gallstone pathogenesis.

Infection and Gallstone Pathogenesis The role of infection in the pathogenesis of gallstones has long been controversial (187). Bacterial infection has been associated with the formation of brown pigment stones, with positive bile cultures implicated in only a minority of cholesterol gallstones (188). Bacterial microcolonies were identified by electron microscopy in pigmented areas of mixed pigment/cholesterol gallstones (189). More recent evidence using in vitro amplification of bacterial DNA sequences from cholesterol-containing gallstones showed that bacteria were implicated in mixed stones containing less than 90% cholesterol by weight (190). Bacterial DNA sequences from E. coli or Pseudomonas species were identified in all brown and mixed cholesterol stones examined, but in only one of seven pure cholesterol stones (191). Using similar molecular techniques, investigators have isolated several Helicobacter species from human gallbladder tissue, although these sequences were found in gallbladders with and without gallstones (192). Biofilms containing bacteria can form on inert surfaces such as gallstones, allowing bacterial colonization without the induction of inflammation within the gallbladder. One clinical situation where this is relevant is with Salmonella typhi infection, wherein a carrier state can develop in the gallbladder. Individuals with gallstones are more likely to become typhoid carriers, and antibiotic treatments are often ineffectual against Salmonella typhi in carriers with gallstones. In the Salmonella model, efficient biofilm formation on gallstones was shown to be dependent on the presence of bile (193).

inflammation, communicating with each other and with immune system cells such as polymorphonuclear leukocytes and lymphocytes. Furthermore, the link with mucin secretion suggests a role for such cytokines in accelerating cholesterol crystallization and gallstone formation. Intraluminal bacterial LPS and IL-1 stimulated gallbladder inflammation in the guinea pig gallbladder, which was inhibited by both IL-1 receptor antagonist and indomethacin. LPS-stimulated gallbladder inflammation was manifested by increased myeloperoxidase and PGE2 release and by water secretion into the lumen (196). Therefore, bacterial infection was shown to induce gallbladder inflammation via a pathway that involved IL-1 and prostaglandins. In cultured human gallbladder epithelial cells derived from a welldifferentiated gallbladder carcinoma, similar roles for IL-1 and prostaglandins in mediating bacterial LPS-mediated inflammation were described. LPS, PGE2, IL-1α, and TNF-α decreased mucosa-to-serosa and net sodium and chloride fluxes and increased serosa-to-mucosa movement of sodium and unmeasured ions, inducing a secretory phenotype with respect to gallbladder epithelial cell fluid and electrolyte transport (197).

Nitric Oxide Although nitric oxide has been mainly studied as a mediator of gallbladder motility, it also has been shown to play a role in mediating fluid secretion in inflamed gallbladders. In a cat model of cholecystitis, fluid secretion in inflamed gallbladder was reversed to a net absorption in response to the nitric oxide synthase blockers Nω-nitro-L-arginine and aminoguanidine, and formation of nitrate was reduced. The effects were reversed by L-arginine. Increased levels of inducible nitric oxide synthase in inflamed gallbladder were shown by immunoblotting, by immunofluorescence (mainly in macrophages), and by calcium-independent [3H]citrulline formation from [3H]arginine. The nitric oxide synthase blockers had no effect on fluid transport in normal gallbladders (198).

Cytokines Cholesterol Crystals Bacterial lipopolysaccharide (LPS) stimulates mucin secretion in cultured dog gallbladder epithelial cells (194), suggesting that inflammation may initiate mucin hypersecretion that could then predispose to cholesterol crystallization and stone formation. Mouse gallbladder epithelial cells produce mRNA for several cytokines and chemokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 2, and intercellular adhesion molecule. mRNA levels of these cytokines and chemokines are altered after exposure to bacterial LPS (195). These findings suggest that gallbladder epithelial cells can serve a paracrine-autocrine function during

Cholesterol monohydrate crystals can stimulate gallbladder inflammation. Crystalline cholesterol monohydrate, LPS, lysolecithin, polystyrene latex spheres (noninflammatory particles), and saline were instilled into guinea pig gallbladders after cystic duct ligation. Crystalline cholesterol, LPS, and lysolecithin caused a significant reduction in mucous layer thickness, reversed water absorption to secretion across the gallbladder mucosa, caused significant increases in myeloperoxidase and IL-1 in gallbladder tissue, and caused significant increases in PGE2 and PGF1α in luminal fluid. Polystyrene latex particles caused no difference in outcomes compared with saline controls (199).

GALLBLADDER FUNCTION / 1553 Oxysterols Oxidized forms of cholesterol, or oxysterols, have been implicated in the pathogenesis of atherosclerosis, with a variety of cellular effects demonstrated on vascular endothelial cells and macrophages. Several oxysterol species, including cholesta-4,6-diene-3-one and cholest-4-ene-3-one, were identified in bile and stone samples. Oxysterol levels were markedly greater (as percentage of total sterols) in pigment gallstones, where bacterial DNA is most abundant (200). Biliary infection was proposed to be involved in the biogenesis of oxysterols in bile through the production of reactive oxygen species from activated leukocytes (201). These observations suggest biliary oxysterols are associated with the presence of bacteria and may play a role in the pathogenesis of pigment gallstones. Biliary and other oxysterols also influence gallbladder epithelial cell function. In the cultured dog gallbladder epithelial cell model, cellular functions such as mucin secretion and cell proliferation were altered by oxysterol treatment (202). Apoptosis also was induced by certain biliary oxysterols, an effect that was abrogated by hydrophilic, but not hydrophobic, bile salts (203). Biliary oxysterol-induced apoptosis proceeded via a mitochondrial-dependent pathway (32).

for specific cations and anions have been identified and characterized. Despite these advances, gaps remain in our understanding of the mechanisms of gallbladder epithelial cell function and regulation. Some of these gaps, such as the role of aquaporins in water transport in the gallbladder and the role of nuclear hormone receptors in the transcriptional control of gallbladder function, have been pointed out in this chapter and represent areas of future research. Finally, this review has highlighted the important role that gallstone pathogenesis has played in providing the impetus for research in this field. In the era of laparoscopic cholecystectomy, the continued importance of studying gallbladder physiology should not be overlooked. The gallbladder provides an ideal model system to study a variety of cellular transport systems, the features of which are shared with polarized epithelial cells from its neighbors, the liver, the small intestine, and the kidneys. In particular, the interactions that take place at the apical plasma membrane of gallbladder epithelial cells with bile are likely to have relevance for protein-mediated and physical chemical events that occur at the canalicular membrane of the hepatocyte and the apical membrane of the villous enterocyte. The uniform nature of the gallbladder epithelium, the extremely concentrated bile encountered at this interface, and the dynamic nature of the relation between the gallbladder epithelial cell and bile are features that can be exploited in the experimental setting.

SUMMARY The main functions of the gallbladder traditionally were thought to consist of storage and concentration of hepatic bile and ejection of this bile in response to neurohormonal signals. This simplified view of the gallbladder has given way to a more complex and nuanced view of gallbladder function. The contemporary view also includes modification of biliary lipid concentrations and secretion of a variety of proteins that subserve diverse functions that affect not only the gallbladder mucosa itself, but also other gallbladder compartments such as inflammatory and smooth muscle cells. More importantly, an appreciation of the dynamic nature of the relation between the gallbladder and biliary constituents has emerged. Cholesterol and bile salts are actively transported by the gallbladder epithelial cell, and these lipids or their derivatives influence gallbladder epithelial cell function in myriad ways. In addition, mechanisms to extrude potentially noxious biliary constituents are present on the apical plasma membrane. By these means, the gallbladder keeps attuned to and responds to the biliary environment. In this paradigm, the gallbladder appears as an astute organ that actively communicates with and participates in controlling the harsh environment that exists beyond the confines of its apical plasma membrane. The machinery underlying gallbladder epithelial cell function, from intracellular signaling pathways to plasma membrane transport events, is beginning to be understood at the cellular and molecular level. For example, specific transporters for cholesterol and phospholipid, for bile salts, and

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Tight Junctions and the Intestinal Barrier Thomas Y. Ma and James M. Anderson Intrinsic and Extrinsic Elements of the Barrier, 1560 Intestinal Epithelial Barrier and Transcellular and Paracellular Transport, 1560 Transport Pathways, 1560 Apical Junction Complex, 1561 Barrier Properties: Resistance, Flux, and Permselectivity, 1562 Protein Components of the Tight Junction, 1565 Transmembrane Proteins, 1565 PDZ-Containing Scaffolding Proteins, 1566 Signaling Proteins, 1566 Polarity Complexes, 1566 Claudins, 1567 Regulation of Intestinal Epithelial Tight Junction Barrier, 1568 Cytochalasins and the Intestinal Tight Junctional Barrier, 1569

Luminal Osmolarity and Solvent Drag Effect, 1571 Na+-Nutrient Cotransport and Physiologic Regulation, 1573 Cytokines, 1576 Infectious Pathogen–Induced Alteration of Intestinal Epithelial Tight Junction Permeability, 1579 Clinical Disorders of Intestinal Tight Junction Barrier Defect, 1582 Clinical Assessment of Intestinal Tight Junction Barrier Defect, 1583 Permeability Index and Celiac Disease, 1583 Crohn’s Disease and Intestinal Tight Junction Barrier Defect, 1584 Nonsteroidal Anti-inflammatory Drugs and Intestinal Permeability, 1586 Other Intestinal Permeability Disorders, 1586 References, 1586

The primary function of the gastrointestinal tract is to digest and absorb nutrients. To accomplish this, it must maintain a barrier between the luminal environment, technically a space outside the body, and the internal environment of the body; and it must selectively absorb and secrete nutrients, solutes, and water across the barrier. Separation of tissue spaces throughout the gastrointestinal tract is accomplished by continuous sheets of polarized columnar epithelial cells. An exception exists in the esophagus, which is sealed by a nonkeratinizing squamous epithelium. Epithelial barriers are selective and capable of excluding potentially noxious

luminal contents, such as gastric acid and colonic bacteria, while at the same time are capable of directional absorption and secretion of large volumes of solutes and water. Material can pass from one side of the epithelium to the other along one of two routes, either across cells or the space between them, referred to as the transcellular and paracellular pathways, respectively. The connection between individual cells is created by a series of intercellular junctions, the tight junction (TJ) being the most important for defining the characteristics of the paracellular barrier and its selectivity. The specific characteristics of epithelial barriers vary widely throughout the gastrointestinal tract, matched to the transport functions of each organ. However, in all cases, disruption of the barrier leads to a loss of normal transport and inflammation due to tissue damage or antigen exposure. This chapter focuses primarily on the role of the TJ in the intestinal barrier. We begin with the role of the TJ and paracellular pathway in normal transport. A large number of proteins have been identified as components of the TJ, and the function of several are now well defined. This allows interpretation of the barrier’s physiologic properties on

T. Y. Ma: Division of Gastroenterology and Hepatology, Departments of Medicine, Cell Biology and Physiology, University of New Mexico, Albuquerque, New Mexico 87131. J. M. Anderson: Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1559

1560 / CHAPTER 61 a strong cellular and molecular foundation. The TJ barrier is highly regulated, and we review mechanisms and physiologic relevance for the gastrointestinal tract. Finally, we review some of the intestinal disorders that have an associated defect in intestinal TJ barrier. We have taken a comprehensive perspective, but have not covered all topics in equal depth. In some cases, the reader is referred elsewhere for a more complete presentation, particularly of current controversies and unresolved issues.

Mucosa

Transcellular pathway Rap

Paracellular pathway RTJ

RLIS Rbl

Serosa

INTRINSIC AND EXTRINSIC ELEMENTS OF THE BARRIER The term epithelial barrier function is often used to describe all the mechanisms contributing to homeostasis of the epithelial barrier. The single layer of continuous epithelial cells and their intercellular junctions constitute the intrinsic elements of the barrier. The magnitude of this barrier is most often measured as the transepithelial electrical resistance (TER) and the permeability to paracellular markers, such as mannitol and inulin. TER correlates with the ability to separate ionic charge across the epithelia, reflected in either a transepithelial electrical potential difference or the current that creates the potential, measured experimentally as the short-circuit current (Isc). Extrinsic elements include the innate and acquired mucosal immune system, protective secretion of mucus, bicarbonate, IgA, and antimicrobial peptides, as well as mechanism of epithelial repair or restitution (1). The contribution of each element varies along the gastrointestinal tract, with mucus secretion being the most constant along the entire length from mouth to anus. Our goal in this chapter is to focus predominantly on the intrinsic barrier of epithelial cells and TJs in health and disease.

INTESTINAL EPITHELIAL BARRIER AND TRANSCELLULAR AND PARACELLULAR TRANSPORT Transport Pathways Throughout the gastrointestinal tract, transport of electrolytes, solutes, and water across epithelia occurs across both transcellular and paracellular pathways (Fig. 61-1). The transcellular route for hydrophilic molecules, for example Na+, Cl−, and glucose, is governed by the profile of membrane pumps, carrier, and channels expressed in a particular cell type. The passive movement across the lipid component of the membrane is limited for charged and hydrophilic molecules. For example, the electrical resistance across model lipid membrane bilayers is in the range of 106 to 109 Ω • cm2, whereas the resistance across real membranes in the gastrointestinal tract is 3 to 4 orders of magnitude less (2), reflecting facilitated conductance through protein-based channels (Table 61-1). The profile of conductance proteins differs among epithelia, explaining their unique functions. Individual transporters also show a polarized distribution to either the

FIG. 61-1. Equivalent electrical circuit model of the intestinal epithelial cell layer. Only resistive elements are shown. Series resistance across the transcellular pathway is the sum of individual resistance across the apical (Rap) and basolateral membranes (Rbl). These are in parallel with resistances of the tight junction (RTJ) plus the lateral intercellular space (RLIS). The RLIS is small, the membrane resistances are usually high, and the epithelial resistance is governed by resistance of the tight junction.

apical or basolateral membrane surface as the basis of directional transport. For example, the apical H+,K+-ATPase of gastric parietal cells is responsible for secreting hydrochloric acid within the stomach. Na+-dependent bile acid transporters are positioned on the sinusoidal surface of the hepatocyte and the apicoluminal surface in the ileum to produce the enterohepatic circulation of bile salts. The cystic fibrosis transmembrane regulator (CFTR), a chloride channel, is positioned on the apical surface of biliary, pancreatic, and intestinal surfaces to bring about luminal Cl− secretion, which is followed by Na+ and water secretion. Primary transcellular transport is “active,” powered by adenosine triphosphate (ATP) hydrolysis to move ions against an electrical or concentration gradient. The prime example is the ubiquitously expressed Na+,K+-ATPase, which moves three Na+ ions out the basolateral surface in exchange for two K+ ions, with the net effect being to generate an inwardly directed Na+ and outwardly directed K+ gradient and negative intracellular electrical potential. The high membrane conductance for K+ and its exit from the cell further enhances the intracellular negative electrical potential. These electrical and chemical gradients are then used in “secondary” active transport to couple energetically unfavorable uphill movement of nutrients, such as glucose or amino acids, to the downhill movement of Na+ through, for example, the Na+coupled glucose cotransporter (SGLT-1) of the jejunum. As a final generalization, the characteristics of transcellular transport are highly regulated by short-term signals, for example, hormone-stimulated bicarbonate secretion from pancreatic ducts, and by long-term transcriptional control, for example, aldosterone-stimulated expression of the Na+, K+-ATPase. We outline these features of transcellular transport before proceeding with a detailed discussion of the paracellular pathway to highlight the sharp distinction to paracellular transport, which is passive, nonrectifying, and does not appear to be as highly regulated at least by physiologic stimuli.

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1561 TABLE 61-1. Electrical characteristics of some epithelia Epitheliuma

Species

Rcellb

Rparacellular

PNa/PClc

Proximal tubule Gallbladder Duodenum Jejunum Ileum Distal colon Gastric fundus Urinary bladder

Dog Rabbit Rat Rat Rabbit Rabbit Mouse surface Crypt Necturus Rabbit

— 229 — 67 115 730 132 429 2826 160,000

6–7 21 98 51 100 385 3200 — 10,573 300,000

1.4 3.3 — 10.0 2.5 0.6 — — — —

Cell linesd Caco-2 LLC-PK1 MDCK

Human colon Pig prox. tubule Dog

125–250 100 60–4000

— — —

3.0 0.6 10.0

aAll

values can be found in Powell (14). resistance values are measured in Ω • cm2. c Permeability ratio of Na+ versus Cl−. P /P in free solute is 0.66. Paracellular pathways with ratios greater than this value Na Cl are more permeable for Na+ than Cl−, that is, cation selective. d Values for cell lines are the personal observations of Dr. C. Van Itallie. MDCK, Madin–Darby canine kidney. b Electrical

Apical Junction Complex The paracellular barrier to material movement coincides with continuous cell–cell contacts located at the apical end of their lateral surfaces (Figs. 61-2 and 61-3). The earliest histologic description of what we now refer to as the “apical junction complex” comes from the late nineteenth century. When sections of small intestine were stained with vital dyes, a distinct intercellular density was observed between cells at the apical end of the lateral space. The English literature referred to this as the terminal bar; other names indicate an assumed role in intercellular adhesion, for example, “Schlussleiten” and “bandelettes de fermeture.” The first speculation about a barrier function is attributed to Bonnet in 1895 (3). After examining several different gastrointestinal tissues obtained from an executed man, he concluded that the terminal bar was a general feature of all epithelia and might play a role in segregating the distinct fluid compositions found in different regions of the gastrointestinal tract. With the first ultrastructural images of intestinal epithelia in 1963 (4), the apical junction complex was shown as a set of morphologically distinct junction types (see Fig. 61-2). Each of these function in cell–cell adhesion and signal transduction and provide links to the cytoskeleton. The TJ is invariably the most apical. It appears along the apical to basal axis, in transverse sections, as a series of close cell–cell contacts, or “kisses.” In freeze-fracture images (see Fig. 61-3A), the contacts are revealed as continuous rows of transmembrane protein particles. Actin filaments terminate on the plasma membrane directly at the contacts, participate in the regulation of the TJ barrier (5), and are known to bind the peripheral membrane scaffolding proteins zonula occluden 1 (ZO-1) (6) and cingulin (7) (Table 61-2). Below this is the adherens junction (AJ), the location of cadherin, the intercellular adhesion molecule and its cytoplasmic binding partner β-catenin, and extensive attachments to a ring of perijunctional

actin filaments. The importance of cadherin in adhesion and maintaining the differentiated cell phenotype is underscored by its frequent mutation as a final step facilitating metastasis of colon cancer (8). β-Catenin has a second role in the nucleus where it signals cell growth and adenoma formation unless degraded by interacting with the adenomatous

Tight

Adherens

Desmosome Gap

FIG. 61-2. Junction types within the apical junction complex between intestinal epithelial cells. (Left) Two columnar epithelial cells with apical brush border typical of the small intestine. A thick band of perijunctional actin and myosin filaments connected to the tight and adherens junctions are typical of intestinal epithelial cells. (Right) Tight junction contacts are further magnified, showing rows of claudin strands adhering between adjacent cells to seal the paracellular space.

1562 / CHAPTER 61

A

B

C

FIG. 61-3. (A) Freeze-fracture electron microscopic replica of the tight junction region of mouse jejunum, showing the interconnected network on claudin-based strands crossing the membrane. Continuous rows of claudins from adjacent cells adhere and seal the paracellular space. A few apical microvilli are visible above the barrier contact zone. (B) Transmission electron micrograph of the apical junction complex region of two adjacent mouse mammary epithelial cells, rotated at 90 degrees around a vertical axis to the image in A. Lanthanum hydroxide (black) was added to the basolateral side; it freely diffuses through the intercellular space until it is partially blocked at the tight junction from reaching the apical side. The tight junction is recognized as a region of close cell–cell apposition. Microvilli are seen on the apical surfaces. (C) Immunofluorescent microscopic localization of the tight junction protein zonula occluden 1 (ZO-1) in mouse distal colon. The epithelial surface is at the top, and two crypts are visible descending toward the bottom of the image. ZO-1 at the tight junction is visible at the apical end of the lateral cell contacts, from crypt to surface.Tangential sectioning in the crypt shows the continuous circumferential location of tight junctions around each cell. Scale bar = 10 µm. (A, B: Reproduced from Cereijido M, Anderson JM. Introduction: Evolution of Ideas on the Tight Junction. Cereijido M, Anderson JM, eds. Tight junctions. 2nd ed. Boca Raton, FL: CRC Press, 2001;1–18, by permission. C: Courtesy of J. Holmes, University of North Carolina.)

polyposis coli (APC) protein. Human mutations in APC protein leave β-catenin free to signal cell growth and transformation into adenomatous polyps (9). Below the AJs are desmosomes, the transmembrane proteins of which, although homologous to cadherin, are linked to intermediate filaments, not actin. Desmosomes serve to protect the alimentary epithelia from shear-induced damage. Gap junctions allow transfer of small signaling molecules, such as Ca2+ and inositol 1,4,5-trisphosphate, between adjacent cells and coordinate epithelial functions such as secretion and exocytosis (10). They can be positioned at any depth along the lateral surface and often are found within the TJ strands. Viewed by freeze-fracture election microscopy (see Fig. 61-3A) the TJ barrier coincides with a network of transmembrane strands. In unfixed tissue, the strands often appear as rows of individual particles, now known to be a family of transmembrane adhesion molecules called claudins (11). The Latin root, claudere, means “to close.” Rows of claudins from each cell meet in the intercellular space forming adhesive contacts and a semipermeable seal. The complexity (number and cross-linking) of strands differs among various tissues. It was long thought that the number of strands correlated with the resistance of the barrier (12). Consistent with this, in the small intestine, the complexity of strands increases at the crypt to villus transition (13). Since discovery of the barrier-forming proteins, this structure-function correlation

has been called into question; the molecular species of claudin in a particular junction may be as important as the number of strands.

Barrier Properties: Resistance, Flux, and Permselectivity Barrier properties of the TJ are characterized by their magnitude and selectivity. The overall level of the TJ barrier is quantified by either its TER or transepithelial flux of fluid phase passively absorbed hydrophilic markers such as mannitol. Permselectivity refers to the ability to discriminate ionic charge and molecular size (see Table 61-1). Early fixation methods for electron microscopy caused the external membrane surfaces of adjacent cells to fuse at the TJ contact points obliterating the extracellular space (see Fig. 61-3B). This unfortunately led to the misconception that the TJ was a complete and impermeable barrier. Further reinforcing the absolute quality of the barrier were electron microscopic studies showing the inability of electron-dense proteins, such as hemoglobin and colloidal lanthanum, to pass through the TJs (see Fig. 61-3B). We now know these molecules are too large to pass through the TJ pores under normal conditions. Together, these powerful visual images led to the misleading and inappropriate term tight junction (TJ).

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1563 TABLE 61-2. Proteins located at the tight junction Category

Protein

Function

Transmembrane

Claudin(s) Occludin JAM(s) CAR ZO-1 ZO-2 ZO-3 MUPP1 ASIP/PAR-3 PAR-6

Barrier and pore selectivity Signaling scaffold, adhesion Adhesion? Coxsackie virus receptor MAGUK, binds occludin, claudin, ZAK, JAM, actin, ASIP, ZONAB Binds ZO-1, actin, claudins, fos, jun, CEBP Binds ZO-1, actin, claudins 13 PDZs and binds claudins Atypical PKC binding protein Cdc42-Par6-Par3-aPKC interaction required for polarity and junction formation aka Discs-lost aka Crumbs polarity protein Binds and phosphorylates ZO-1 Binds polarity proteins PAR-3, PAR-6, interaction required for junction assembly Occludin phosphorylation blocks ZO-1 binding Binds occludin Tumor suppressor binds MAGI-2 and 3 Binds aPKC, disassembles junction ErbB-2 activator

62 333 334 34 6 335 64 336

HuASH1 CEBP Fos, Jun Rab3B

Drosophila ash1 homolog Mutants inhibit LDLR delivery

340 335 335 50

Rab13 Gαi2 AF6 GEF-H1 Sec6/8 VAP33 Cingulin

Mutants inhibit claudin-1 delivery Binds SH3 of ZO-1 Binds to Ras, ZO-1 and actin Guanine nucleotide exchange factor influences permeability Exocyst complex Binds occludin and v-SNAREs Binds ZO-MAGUKs, JAM-1, actin

50 51 52 53 341 342 343

PDZ-scaffolding

Polarity

Kinases

Phosphatases Transcription factors

GTP-binding proteins

Vesicle targeting Other

PATJ Pals-1 ZAK aPKC src yes PTEN PP2A ZONAB

References

337 337 337 338 337 49 44 339 59 56

ASIP, Agouti signaling protein; CAR, constitutive androsterone receptor; GEF, guanine exchange factor; GTP, guanosine triphosphate; JAM, junctional adhesion molecule; LDLR, low-density lipoprotein receptor; MAGI, membrane-associated guanylate kinase with inverted orientation; MAGUK, membrane-associated guanylate kinase; MUPP, multi-PDZ domain protein 1; Pals, protein associated with Lin-7; PAR, protease-activated receptor; PATJ, Pals1-associated tight junction protein; PKC, protein kinase C; PP2A, protein phosphatase 2A; PTEN, phosphatase and tensin homolog; SH3, Src homology 3; v-SNARE, vesicular soluble N-ethylmaleimide–sensitive factor attachment protein receptor; ZO, zonula occluden, ZONAB, ZO-1–associated nucleic acid binding protein.

Electrical Resistance Epithelia are classified as ranging from “tight” to “leaky” based on their overall electrical resistance (14,15); most epithelia of the gastrointestinal tract are leaky or only moderately tight (see Table 61-1). In practice, these values are derived by mounting tissue with the mucosal and serosal surfaces facing electrically isolated fluid-filled chambers with current and voltage electrodes on both sides. This is the standard Ussing chamber configuration. An equivalent circuit of the epithelium includes the transcellular pathway represented by series resistances of the apical and basolateral membranes in parallel with the paracellular pathway (see Fig. 61-1). The paracellular resistive elements include TJ and the lateral intercellular space in series. In practice, the contribution of latter is small and usually ignored. Because membrane resistances are generally high, it is the variation in TJ resistance that determines whether an epithelium is leaky or tight. Some authors define leaky as when more than half of the overall conductance is paracellular (16). Others use numeric ranges with leaky up

to about 100 Ω • cm2 and intermediate up to several hundred. The tightest epithelia can exceed 100,000 Ω • cm2. Electrophysiologic studies before about 1960 reinforced the misconception that the TJ barrier was impermeant. Most early studies were performed on what we now refer to as “tight” epithelia, such as frog skin and urinary bladder, where the contribution of paracellular to total conductance is small. These tissues have large Isc (in the order of 100 µA/cm2) and high electrical resistances (>1500 Ω/cm−2). Isc is the current that must be experimentally passed to balance off the current being generated by the epithelium. It was obvious to investigators when the tissue lost viability because both the resistance and Isc declined. In contrast, so-called leaky epithelia such as the gallbladder and small intestine (see Table 61-1) always showed a low resistance. They were assumed to be more fragile and susceptible to damage during preparation. Despite this bias, a growing literature during the 1960s carefully characterized the high-conductance paracellular pathway across leaky epithelia, such as the gallbladder, as showing

1564 / CHAPTER 61 charge and size selectivity. This would not be expected if the tissue was simply damaged and the “shunt” pathway was through free solution. The localization of the shunt pathway to the intercellular space was finally demonstrated by Fromter and Diamond in 1972 (17) using conductance scanning methods. By passing a microelectrode over the gallbladder epithelial surface, a high-conductance shunt could be demonstrated at the intercellular junctions (17). Use of smaller electron microscopic tracers, such as ionic lanthanum also allowed ultrastructural visualization of permeation through the TJ. These studies led to acceptance of the existence of “tight” and “leaky” epithelia (17). Subsequent studies led to our model (which is detailed later) of TJs as barriers containing molecularly different pores that discriminate based on size and ionic charge. Epithelia of the gastrointestinal tract display a range of resistance: leaky (e.g., gallbladder and small intestine), moderately tight (colon and gastric antrum), and tight (gastric fundus and esophagus). A characteristic of leaky epithelia is their ability to move large amounts of isosmotic fluids. Typically, the cells produce active directed movement of an ion, for example, apical secretion of Cl− through CFTR, and a counter ion and water passively follow through the paracellular pathway. One of the leakiest epithelia in the gastrointestinal tract is found in the sublingual gland, 15 Ω • cm2, correlating with its ability to rapidly produce large volumes of saliva. Likewise, the entire small intestine is quite leaky to accommodate the massive bidirectional fluid movements of absorption and secretion. For example, on an average day, up to 8 L fluid will enter the adult human gut from mouth to the terminal ileum. This is derived from oral ingestion and secretions from salivary glands, pancreas, biliary tract, and the gut epithelium itself. Only 2 L or less pass from the ileum to colon, meaning that 6 L are reabsorbed by the small bowel. These high fluxes are required to dilute complex nutrients (e.g., glycogen, protein) as they are digested to subunits (e.g., monosaccharides, amino acids) and absorbed. In contrast with leaky epithelia, tightness implies the ability of an epithelium to maintain steeper ionic and osmotic gradients between tissue compartments and use them for extracting electrolytes and water. The intestine shows a modest increase in tightness in the distal colon and rectum, reflecting their ability to remove salt and water from the lumen to form solid stool. TJs also increase in tightness along the crypt to villous surface/axis. In reality, these statements are oversimplifications, and in practice, it may be difficult to separate the transcellular from paracellular resistances. Studies on mouse distal colon provide a cautionary example. The overall resistance of the surface epithelium is about 118 Ω/cm−2 and is classified as moderate. However, using impedance analysis, the different elements can more readily be separated demonstrating that the paracellular resistance is high, 3200 Ω/cm−2. This is an example where the TJ strongly resists back-leak of salt and water extracted across the cells, yet the epithelium does not appear tight because of the high membrane channel conductance (18). A further complication is introduced by variations in cell

number per unit area and the degree of infolding between adjacent cells. Both factors influence the linear length of junction per area and provide more opportunity for conductance. Permselectivity In addition to variation in electrical resistance, the TJ also shows discrimination for ionic charge. When considering pathologic changes in the gastrointestinal barrier, it is important to consider selectivity. If the epithelial layer is grossly damaged and denuded of the surface cells, then all discrimination may be lost because of large gaps in the epithelial surface. In contrast, some conditions may leave TJs intact and able to discriminate, but change their resistance and selectivity. Charge Discrimination Experimentally, charge selectivity is most often measured as the permeability ratio for sodium to chloride (PNa+/PCl−) (see Table 61-1). This ratio varies among tissues and cultured epithelial cell lines by about 30-fold (19); most epithelia in the body are cation selective including all those currently documented in the gastrointestinal tract (14). As discussed later in this chapter, it is now known that the many different members of the claudin family create charge-selective pores at the junction. Although all TJs show charge selectivity, tissue-specific differences are only meaningful in leaky epithelia, where there exists sufficient ion movement to make paracellular discrimination relevant. The level of discrimination is rather low compared with transmembrane K channel, which can discriminate 1000-fold between the monovalent cations Na+ and K+ and much more against anions. What is the relevance of paracellular charge selectivity? Transport begins with the active transcellular movement of ions down their electrochemical gradients. For example, when apically positioned chloride channels open, Cl− will exit into the gut lumen down its electrochemical gradient. The net movement of negative charge is followed by paracellular efflux of Na+, then by osmatically driven water efflux, resulting in fluid secretion. The ability to allow paracellular Na+ movement and restrict mucosal-to-serosal Cl− flux contributes to net secretion. In a converse situation, transcellular Na+ transport may be followed by paracellular Cl−. In general, these issues are not studied in detail for the gastrointestinal tract. All regions of the gut show complex gradients in absorption and secretion along the crypt to villous surface/axis. The role of osmotic gradients on water flux and paracellular permeability of water-soluble molecules is discussed in greater detail later in this chapter. Secretion from crypts often is based on Cl− secretion, villous-surface absorption is based on Na+ absorption. Theoretically, one would expect villoussurface junctions to restrict Na+ and crypt junctions to permit Na+ movement, yet local differences in charge selectivity have not been studied in detail. The relevance of paracellular charge selectivity in the intestine is highlighted by studies performed in patients with cystic fibrosis (CF), which suggest that charge selectivity

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1565 can adapt to counterbalance the primary defect in Cl− secretion (20). Mutations in the CFTR chloride channel lead to diminished chloride secretion into the gut lumen. In theory, unopposed Na+-dependent absorption in the opposite direction leads to dehydration of luminal contents, as seen in the neonatal presentation of meconium ileus and bowel obstruction. However, adult patients with CF do not hyperabsorb luminal fluid. Compared with normal intestine, their paracellular pathway is adapted to show a greater than normal discrimination against Cl−. Thus, transcellular Na+ absorption is not followed by the normal level of paracellular Cl− movement, and fluid absorption is reduced (20). Although not well studied, in other situations, the overall paracellular electrical resistance may be physiologically regulated. For example, aldosterone induces a greater resistance in TJs of the rabbit distal colon (21). It is easy to rationalize how this would contribute to overall Na+ absorption by complementing enhanced transcellular transport brought about by transcriptional increases in Na+,K+-ATPase and epithelial Na+ channel. Currently, there are few published examples of physiologically regulated changes in resistance or charge selectivity. The majority of documented changes occur in pathologic situations. Size Discrimination The TJ behaves as a barrier perforated by relatively large aqueous pores with a distinct size cutoff (15,22,23). A wide range of pore sizes have been reported ranging from about a 4- to 40-Å radius. Caution is appropriate when interpreting this literature; in some cases, the tissue may have been damaged or the tracer took a transcellular route. In general, the apparent permeability of hydrophilic nonelectrolyte markers is inversely related to size, down to an inflection, or cutoff, characteristic for each tissue (14). The range for size cutoff is relatively narrow among epithelia (≈7–15 Å), whereas some endothelial TJs permit larger solutes (40–60 Å) (24). The existence of larger pores in endothelia remains uncertain because some of these studies were preformed on “discontinuous” endothelia that normally have intercellular breaks or others that have highly active endocytosis, such as vessels of the blood–brain barrier. An early landmark study using nitrogenous cations demonstrated pore diameters of 8.8 Å in rabbit and 16.2 Å in frog gallbladder (25). Pores of this size would allow three or four water molecules to exist alongside a Na+ ion within the smallest cross section of the pore. These conclusions are consistent with more recent transmonolayer flux studies performed on cultured intestinal epithelial cell lines grown on permeable supports. Here, the transepithelial flux of a continuous series of polyethylene glycol (PEG) molecules was simultaneously determined after separation by liquid chromatography and quantified by mass spectroscopy. It was suggested that human colonic cell lines Caco-2 and T84 may have an upper cutoff in the range of 8.6 to 9.0 Å in diameter (26). However, other studies have shown small-intestinal tissue (in vivo and ex vivo) and T84 and Caco-2 intestinal monolayers to be permeable to large paracellular probes such

as inulin (5000 g/mol molecular weight), although to a much lesser extent (2–3 orders less) than to the smaller probe mannitol (343 g/mol molecular weight) (27–31). Thus, there may be a small population of TJ pores with larger functional pore size that allows paracellular permeation of larger molecules. The permeability differences among the monovalent alkalimetal cations are small. The low discrimination for similarly charged, but different sized, cations is consistent with a TJ pore size that is sufficiently large compared with the molecular diameter of cations (14). Although larger and less discriminating, paracellular pores share with transmembrane ion channels a similar ion concentration transport characteristics including dependence on and competition between different transported molecules (23).

PROTEIN COMPONENTS OF THE TIGHT JUNCTION Beginning in 1986 with the identification of ZO-1 as the first TJ protein (32), there has been continuous growth to almost 40 distinct proteins or protein families (see Table 61-2). Surprisingly, this number far exceeds the known components of other intercellular junctions, such as adherens, desmosomes, and gap junctions (see Fig. 61-2). The review of protein categories in Table 61-2 shows significant functional complexity, including proteins involved in cell polarity, signaling, gene transcription, cytoskeletal assembly, and even targeted delivery of vesicles containing basolateral membrane proteins. As a reference, Table 61-2 catalogs the known proteins, however in the following sections only those proteins of current interest to gastrointestinal physiology and disease are discussed. Some are described in more detail when regulation and pathology of the barrier are reviewed. To facilitate presentation, we divide proteins into distinct structural and functional categories including the transmembrane, cytoplasmic scaffolding, signaling and cell polarity proteins, and a group currently without clear functions. We discuss the claudins separately and in the greatest depth because of their uniquely important role in creating the barrier. Several junctional proteins bind to filamentous actin; the important role for actin and myosin in controlling junction permeability is discussed later in this chapter.

Transmembrane Proteins Several transmembrane proteins are found in the TJs; their functions include barrier formation, cell–cell adhesion, and regulation of immune cell transmigration. Claudin(s) and occludin are components of the TJ strands that form the actual barrier (see Fig. 61-2), whereas others such as junctional adhesion molecule-1 (JAM-1) (33), constitutive androsterone receptor (CAR) (34), and connexins-32 are located around the strands. Current studies suggest members of the JAM family play a role in immune cell trafficking across the intestinal

1566 / CHAPTER 61 epithelium (33). JAM-1 and -4 are expressed in junctions of the gastrointestinal epithelial cells; JAM-2 is expressed on endothelial cells. JAM-3 is found on T cells where it functions as a counterreceptor for JAM-1 on epithelial cells promoting lymphocyte transmigration through the junction (35,36). Like other members of the IgG superfamily, they are capable of homophilic adhesion through contact of the variable-type immunoglobulin domains. Although studies in endothelia suggest they promote immune cell trafficking, their exact role in the intestinal epithelia remains unclear. Their levels are decreased, not increased, in junctions closest to crypt abscesses in inflammatory bowel disease (37). CAR is a related IgG superfamily member and a receptor for the Coxsackie adenovirus (34). Occludin is a tetraspan protein of approximately 65 kDa concentrated in the claudin-based junction strands. Although a homophilic adhesion molecule (38), it cannot by itself create the strands (11). It is a unique protein, not a member of protein family, and no obvious homologues have been observed outside the vertebrata. The N and C termini are within the cytoplasm, and the C terminus binds directly to the scaffolding protein ZO-1 (6). Phosphorylation of the cytoplasmic tail correlates with focused location within the TJ, whereas nonphosphorylated occludin is on the lateral cell membrane (39,40). Defining a function for occludin remains an elusive goal. When expressed in cultured epithelial monolayers, it induces a small increase in transmonolayer electrical resistance (41). However, occludin-deficient knockout mice are viable, have TJs, and have a complex phenotype including chronic gastric inflammation and hyperplasia (42). Occludin binds to several signaling proteins, and its main function may be to recruit and coordinate signaling pathways at the junction. For example, it binds the transforming growth factor-β (TGF-β) type II receptor, localizes it to the junction, and enhances the effect of TGF-β to disassemble the junction and cause an epithelial-to-mesenchymal phenotypic change (43). It also binds the nonreceptor tyrosine kinase c-yes (44), although the functional implications are unknown.

PDZ-Containing Scaffolding Proteins The interface between the transmembrane proteins and most cytoplasmic components is formed by a set of scaffolding proteins with multiple PDZ domains. PDZ domains are protein-binding modules that recognize target sequences at the extreme C termini of transmembrane proteins. They are well characterized for their ability to cluster signal transduction complexes at specialized membrane contacts, such as synapses (45). PDZ proteins at the TJ include the ZO-MAGUK (membrane-associated guanylate kinase) proteins ZO-1, -2, and -3; the MAGUK relatives membrane-associated guanylate kinase with inverted orientation (MAGI)-1, -2, and -3; a protein with 13 PDZ domains called MUPP1 (multi-PDZ domain protein 1); and several of the polarity components cited later including protease-activated receptor 3 (PAR-3), PAR-6, protein associated with Lin-7 (PALS1), Pals1-associated tight junction protein (PATJ), scribble, and Discs-large

(see Table 61-2). Most of these proteins contain several PDZ domains and bind the tails of claudins and JAMs. Some, such as the ZO-MAGUKs, have additional protein-binding modules (Src homology 3 and guanylate kinase domains) known to bind additional targets. The undersurface of the junction had been likened to a Velcro-like interface with extensive PDZ-mediated cross-linking.

Signaling Proteins A large number of signaling pathways are known to regulate the barrier, although the specific protein targets and mechanisms are poorly defined. Several of cytoplasmic proteins are believed to transduce these signals to influence junction assembly or barrier state. Conversely, cell-to-cell contacts at the junction may induce signals within the cell. Many excellent reviews on this topic are available (46–48). Some signaling proteins are not unique to the junction, but are also found in other compartments of the cell. For example, the nonreceptor tyrosine kinases c-src (49) and c-yes both induce junction disassembly; c-yes binds directly to occludin (44). Several are controlled by guanosine triphosphate (GTP) metabolism. For example, Rab3b and Rab13 (50) are required for vesicle delivery; others are required for junction assembly (51–53). A role for protein kinase C (PKC) isoforms has been proposed in both assembly and disassembly of the junction and changes in resistance (54,55). In intestinal epithelial cells, ligation of the Toll-like receptor-2 with a bacterial cell wall product induces an increase in transmonolayer electrical resistance. This requires activation of PKCα and PKC∆ and correlates with focusing of ZO-1 at the junction (44). Presumably, this represents an innate protective response of the epithelium to luminal bacteria. Poorly understood are the roles of several transcription factors that are located at the junction or bind junction proteins. One of these, ZO-1–associated nucleic acid binding protein (ZONAB) (56), suggests a signaling theme similar to the β-catenin/lymphoid enhancer factor-1 pathway, which is located at AJs. ZONAB is a Y-box transcription factor with nuclear localization and transcriptional activity that are inhibited by binding to ZO-1. ZO-1 appears to titrate ZONAB out of the nucleus, where it would associate with cell division kinase 4 to enhance proliferation. It is proposed that the state of junction contacts regulate ZO-1 levels and its ability to influence ZONAB activity. Similar to the β-catenin pathway, one would expect this pathway to be activated in gastrointestinal cancers, but this has not been investigated.

Polarity Complexes Studies in an invertebrate model system have showed several highly conserved proteins required for generating different aspects of polarity, including asymmetric cell division in the embryo and anteroposterior gradients in body morphology. Best understood are three multiprotein complexes called

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1567 PAR-3/aPKC/PAR-6, Crumbs/PALS1/PATJ, and Scribble/ mDlg/mLgl. These are also required for mammalian epithelial cell polarity and TJ assembly. They all act at points upstream of the localization and assembly of TJ proteins. The activity of some is regulated by known promoters of junction assembly, such as active Cdc42 and Rho (57). Some bind directly to proteins that will remain permanent components of the junction (58). aPKC phosphorylates several junctional proteins, such as ZO-1, occludin, and claudin-1 (59), and this is under negative regulation by protein phosphatase 2A. Because the TJ defines the boundary between the apical and lateral cell domain, it is not surprising that the polarity machinery resides here. The details of assembly are under intense investigation (60,61), but a role for polarity proteins in human pathology remains unknown.

Claudins The discovery of claudins in 1998 significantly advanced our understanding of the TJ barrier. Claudins were first shown to have the ability to form strands (62) and confer cell-to-cell adhesion (63). Subsequent work clearly implicated this large protein family in creating the selective barrier properties. Claudins are tetraspan proteins, ranging from 20 to 25 kDa, and are recognized by a conserved amino acid motif in the first extracellular loops (W-GLW-C-C). There is no direct evidence that the cysteines form a disulfide bond, but it appears possible given the oxidizing extracellular environment and their total conservation among claudins. The first extracellular loops range from 41 to 55 amino acid residues, the second 10 to 21 amino acid residues, and the cytoplasmic tails 21 to 44 amino acid residues. They all end in PDZbinding motifs, which bind PDZ domains in the cytoplasmic scaffolding proteins ZO-1 (64) and MUPP1 (51,65) and possibly other proteins. ZO-1 has 3 PDZ domains and MUPP1 has 13, suggesting they can link several claudins together along the strand. Some mammalian claudins, for example, claudin-1, appear rather ubiquitously expressed, whereas others are restricted to specific cell types (66,67) or periods of development (68,69). The locations of several claudins have been documented along the gastrointestinal tract. Each shows unique crypt to villous surface expression patterns. Presumably, these confer regional gradients of resistance and charge selectivity, but the physiologic implications remain unexplored (70). Claudin gene families are found across a wide range of multicellular animals. The human and mouse genomes contain at least 24 (71). The puffer fish Takifugu contains 56 claudin genes, although this animal has other examples of gene expansions that lack obvious biological significance (72). The barrier-forming junctions of invertebrates, septate junctions, are structurally quite different from TJ; thus, it was surprising to find claudin-like proteins in both flies and worms and to learn that they are required to form epithelial barriers. Drosophila has six claudin sequences (73–75). Two of them, Megatrachea (Mega) (73) and Sinuous (Sinu) (75), are expressed at the barrier-forming septate junctions; mutations in Mega disrupts the intercellular barrier. Mutations of

either result in developmental defects in the size and shape of the tracheal epithelium. Although only 16% are identical to human, claudin-1 in the fly proteins contain the conserved W-GLW-C-C motif. Five claudin-like sequences have been identified in Caenorhabditis elegans (76), and mutation of “claudin-like protein 3” results in disruption of the barrier between epithelial cells of the intestine. Many lines of evidence point to claudins as the basis for the selective size, charge, and conductance properties of the paracellular pathway. This has been most directly tested by introducing selected claudins in cultured epithelial cell models. The first such experiments were conducted in lowresistance Madin–Darby canine kidney (MDCK) type I cells. Expression of claudin-1 resulted in an increase in TER (77,78), which was initially interpreted to result from enhanced cell– cell adhesion. In contrast, expression of claudin-2 in highresistance MDCK type I monolayers made them leakier (79). This was interpreted as resulting from claudins being unable to adhere to the endogenous claudins and creating breaks in the junction. Transepithelial resistance is experimentally measured from the current of charged solutes moving through the junction under an imposed electrical field. Thus, the changes after expression of claudin-1 and -2 might be caused by their contributing different ion permeabilities from the background, not that they close or create breaks in the paracellular space, respectively. This possibility was first demonstrated for claudin-4. When expressed under a regulated promoter in MDCK cells, it induced a dose-dependent increase in resistance. Using dilution potentials to characterize charge selectivity, the resistance increase was completely explained by discrimination against cations (80). These results suggested that claudin-4 creates channels that discriminate against cations. Subsequently, similar discrimination against cations relative to anions was observed for claudin-8 (65,81), and the opposite charge selectivity was demonstrated for claudin-2 (82). These results support a model where claudins create charge-selective pores. A role in charge selectivity has been further confirmed by performing charge-reversing mutations within the extracellular sequences. Replacing negative with positive charges has the predicted effect of enhancing anion and reducing cation permeability (83–85). It appears that pore selectivity is controlled by the net charge on a key set of residues in the first loop. Currently, human diseases are known to result from mutations in genes encoding claudin-1 (86), -14 (87), and -16 (66,88) and the cytoplasmic claudin-binding scaffold ZO-2 (89) (Table 61-3). Deletion of claudin-1 has been reported in a handful of neonates presenting with a sclerosing, cholangitislike picture and hypertrophy of the skin. The reasons for biliary destruction are obscure based on available histology. A mouse model with deletion of claudin-1 dies shortly after birth from rapid evaporative water loss from the skin. They do not live long enough to investigate whether they recreate the biliary pathology (90). The claudin-16 mutations are the easiest to rationalize as channel defects. Claudin-16 is largely restricted to the kidney, where it is found in TJs in the thick ascending loop of Henle (66). This segment of the nephron develops an intraluminal

1568 / CHAPTER 61 TABLE 61-3. Inherited human diseases of tight junctions Gene

Disease

Pathology/Mechanism

Cln-1 Cln-14 Cln-16 PMP22

Neonatal sclerosing cholangitis Deafness, DFNB29 Hypomagnesemia (HHN) Peripheral polyneuropathies Charcot–Marie– Tooth type 1A Dejerine–Sottas syndrome Familial hypercholanemia

Obliteration of large bile ducts Cochlear hair cell degeneration Defective renal Mg2+ reabsorption Defective myelin barriers

86 87 66 94

Reduced claudin binding

89

ZO-2

positive electrical potential, which drives cations, such as Mg2+, back through the TJ to the blood. Total body Mg2+ homeostasis is largely controlled by passive electrodiffusion at this site. In 1998, positional cloning identified claudin-16 (paracellin-1) as the gene responsible for a rare Mg2+ wasting disease called familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHN) (66). It presents in childhood with weakness and seizures caused by low blood Mg2+ levels and later progresses to renal failure because of interstitial calcium deposition. Urinary Mg2+ and Ca2+ losses appear to be caused by the inability of TJs in the thick ascending loop of Henle, in the absence of claudin-16, to allow paracellular reabsorption of cations from the tubule. A distinct form of abnormal renal calcium loss, grouped under idiopathic hypercalciuria, was shown to result from novel mutations in the claudin-16 gene, which interrupts binding to the PDZ domain of ZO-1 (88). In contrast with the symptoms of FHHN described earlier, the patients display self-limited childhood hypercalciuria. When the mutant protein was tested in cultured cells, it no longer localized to TJs, but accumulated in lysosomes. The reason for distinct clinical syndromes resulting from null versus targeting-defective mutants is unclear. Claudin-14 mutations are more difficult to rationalize as channel defects. They were first identified as the cause of nonsyndromic recessive deafness DFNB29 in two large consanguineous Pakistani families (87). Immunolocalization demonstrated claudin-14 in TJs of the sensory epithelium of the organ of Corti. It was initially assumed claudin-14 serves as a paracellular barrier to maintain the high extracellular K+ concentration found in the endolymph compartment, which is required for hair cell depolarization after mechanical stimulation. Loss of the ion gradient because of potassium channel mutations is known to cause deafness (91). Consistent with this proposed function, when expressed in MDCK cells, claudin-14 forms TJs of low-cation permeability (92). However, claudin-14 null mice have a normal endocochlear potential, but become deaf because of progressive degeneration of cochlear outer hair cells (92). It remains unresolved whether cell degeneration is secondary to a TJ barrier defect. Human TJ diseases have now extended beyond the claudins to the cytoplasmic scaffolding protein ZO-2. A point mutation in the PDZ domain of ZO-2 is the basis of Amish familial hypercholanemia (88), a bile secretory defect characterized

References

by increased serum bile acid concentrations, itching, and fat malabsorption. This mutation disables claudin binding to the PDZ domain. Conceivably, interruption of this connection disrupts the TJ barrier and allows back-leak of bile through the TJ with accumulation of serum bile acids. Morphologic changes are observed in hepatocyte TJs, although these are nonspecific and are seen in many forms of liver injury. Mutations in a bile acid–conjugating enzyme enhance the penetrance of disease in patients homozygous recessive for the ZO-2 mutations. This was the first example of a polygenic human disease in which TJs were involved. It appears likely that other TJ diseases influencing transport and drug pharmacokinetics will be discovered in the future. Several claudin mouse knockouts provide useful information about claudin function. The most informative about size selectivity is that of claudin-5 (93). Endothelial TJs in the brain express at least claudin-5 and -12. They are among the tightest endothelia in the body and the structural basis of the blood–brain barrier. Claudin-5 null mice are born with normal-appearing TJs in brain endothelia, but they die within several hours. Vascular perfusion studies show that null mice retain size discrimination, but allow slightly larger markers to pass, allowing passage of a 562-Da marker, yet still restricting a 1862-Da marker. Claudin-14 null mice phenocopy the human deafness syndrome (92). Other mutations are less relevant to the gastrointestinal tract, but can be found in the following references: claudin-11 (67), peripheral myelin protein 22 (PMP-22) (94,95), and claudin-6 (96).

REGULATION OF INTESTINAL EPITHELIAL TIGHT JUNCTION BARRIER A number of important advances have been made in the past quarter century to provide new insights into the regulation of the intestinal epithelial TJ barrier. Despite these advances, the precise intracellular mechanisms involved in the regulation of the intestinal TJ barrier remain largely undefined. Common themes that have emerged in these studies are the rapid dynamic nature of the intestinal TJ barrier regulation and the intricate interaction of cytoskeletal-TJ proteins in the regulation of the intestinal TJ barrier. Although alterations in TJ protein localization and expression are likely to be important in the modulation of the TJ barrier function, direct

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1569 experimental support for these alterations in the acute regulation of intestinal TJ barrier is lacking. An important reason for the lack of experimental evidence is the inability to selectively inhibit the changes in TJ protein expression and/or localization during acute modulation of the intestinal TJ barrier. This is an important area for future investigation. The intestinal TJ barrier is rapidly regulated in response to extracellular factors that activate various intracellular signaling pathways. As the fluid composition of the luminal and serosal compartments undergo continual change during the various digestive and interdigestive phases and vary depending on the types and amounts of food consumed, types of medication ingested, luminal bacterial composition and the bacterial load, the type and amount of digestive and proteolytic enzymes secreted into the lumen, the inflammatory state of the intestinal mucosa and the amount of proinflammatory cytokines and inflammatory mediators present, and the ionic and solute content of the luminal and serosal fluid, the intestinal TJ barrier also undergoes continual change in response to the changes in the extracellular fluid composition. Because the intestinal TJ barrier is modulated by wide-ranging pharmacologic, physiologic, microbial, and inflammatory factors, this section focuses on those factors that have historical importance (such as the cytochalasins and the luminal osmolarity) and those factors that have been more extensively studied and for which the mechanisms have been supported by experimental studies. An important emerging concept is the role of myosin light chain kinase (MLCK) and the cytoskeletal-TJ protein interaction as a unifying mechanism leading to the modulation of the intestinal TJ barrier. For many of the intestinal TJ barrier–modulating factors, MLCK induced perijunctional actin-myosin contraction, and the subsequent cytoskeletal-TJ protein interaction (possibly mediated via the actin/ZO-1 binding interaction) appears to be the common mechanism leading to the functional and morphologic opening of the intestinal TJ barrier. Despite important advances, major gaps in the understanding of the intracellular processes that regulate the intestinal TJ barrier remain and provide an important opportunity for future investigations in this area.

Cytochalasins and the Intestinal Tight Junctional Barrier Cytochalasins have been widely used as pharmacologic agents to examine the actin-dependent cellular functions and were among the first agents to be used to study the role of cytoskeletal elements in the epithelial TJ barrier modulation (28,97–104). Cytochalasins disrupt actin filaments by several mechanisms including direct severing of actin filaments, inhibition of actin subunit polymerization, and induction of reactive cellular responses (97,100,105–108). The cytochalasin disruption of actin microfilaments produces a morphologic alteration in intestinal TJs and an increase in intestinal TJ permeability in both the ex vivo and in vitro intestinal epithelial systems (28,29,97–100). The cytochalasin D treatment

of Ussing chamber–mounted guinea pig small-intestinal tissue produces a concentration-dependent (0.1–20 µg/ml) decrease in TER (100). The decrease in small-intestinal tissue TER directly correlated with an increase in Na+ and mannitol flux, confirming an increase in intestinal epithelial TJ permeability (100). The dual-flux studies indicated that the increased permeation through the paracellular pathways fully accounted for the increase in mannitol and Na+ flux (100). The cytochalasin D increase in intestinal TJ permeability was also accompanied by a marked condensation or aggregation of microfilaments in the perijunctional actin-myosin ring especially in the regions of multicellular contacts. The condensation of the perijunctional microfilaments produced a “pulse-string”–type contraction of the brush borders of surface intestinal epithelial cells with a bulging or convex appearance of the epithelial surface (Fig. 61-4), a decrease in the number of microvilli at the junctional contact areas, and a disturbance in the distribution, decrease in the number, and loss of meshwork-like organization of TJ strands (Fig. 61-5) (100). These findings suggested that the cytochalasin D–induced increase in intestinal TJ permeability was mediated by a centripetal tension generated by the contraction of the actin/myosin ring at the level of the TJs. The cytochalasin D–induced aggregation of perijunctional microfilaments, contraction of the perijunctional actin/myosin ring, alterations in TJ strands, and increase in TJ permeability were inhibited by energy depletion by 2,4-dinitrophenol, indicating the energy requirement in these processes (100). Similarly, cytochalasin (B and D) treatment of filter-grown intestinal epithelial monolayers Caco-2 and T84 cells also produced an acute decrease in TER and an increase in epithelial permeability to paracellular markers mannitol and inulin (Fig. 61-6) (28,97,98). In contrast, the disintegration of Caco-2 microtubules with tubulin depolymerizing agent colchicine did not have any effect on the Caco-2 TJ barrier function or junctional localization of TJ proteins, suggesting that intact microtubules are not required for the acute maintenance of the intestinal epithelial TJ barrier function (98). The cytochalasin B–induced increase in Caco-2 TJ permeability was rapidly reversible (within hours) after the cytochalasin B removal and was also mediated by an energy-dependent process, indicating that the cytochalasin effect was not caused by permanent cell damage or cell death, but rather a rapidly reversible process (97,98). The cytochalasin B–induced increase in Caco-2 TJ permeability also correlated with sequential changes in perijunctional actin and myosin filaments (Fig. 61-7). In Caco-2 monolayers, actin and myosin filaments are localized in a beltlike manner surrounding the apical junctional area. Cytochalasin treatment produces a rapid energy-independent severing of actin filaments into small fragments (early-phase response). Within the first minute of cytochalasin B treatment, the perijunctional actin filaments are severed, become fragmented, and are present diffusely in the cytoplasm at the level of the TJs (see Fig. 61-7). This early-phase fragmentation is then followed by an energy-dependent process in which the severed actin fragments reorganize to form large cytoskeletal

1570 / CHAPTER 61

A

B

C

D

FIG. 61-4. Scanning electron micrographs of vehicle control (A, B) and cytochalasin D (CD)– exposed (C, D) (10 ng/ml, 60 min.) mucosal sheets. The three villous ridges display smooth surfaces with intermittent linear folds. As seen in B, control villi are covered by polygonal absorptive cells with flat apical surfaces. In contrast, CD-exposed tissues display a cobblestone-like appearance of the villous surfaces (C). Higher magnification (D) shows this cobblestone effect is caused by purse-string contraction of the brush borders of individual absorptive cells, resulting in a convex apical absorptive cell surface and flaring of microvilli. Scale bars = 20 µm. (Reproduced from Madara [100], by permission.)

aggregates containing actin and myosin filaments (latephase response) (97,108). By 15 to 30 minutes of cytochalasin B treatment, the fragmented actin and myosin filaments coalesce to form large cytoskeletal clumps or “foci” near the perijunctional areas (see Fig. 61-7). It is likely that these macrocytoskeletal aggregates also contained TJ proteins; however, direct evidence for this is lacking (97). Similar effects of cytochalasin D on actin filament breakage and cytoskeletal clump formation (containing actin, myosins, and tropomyosins) were also demonstrated in the African green monkey kidney cells (BSC1cells) (108). Because actin-myosin contraction in smooth muscle and other cell types is mediated by MLCK activation, the possibility that the cytochalasin-induced increase in Caco-2 TJ permeability is also mediated by MLCK-activated actinmyosin interaction was considered (97). The mechanism of MLCK-induced activation of actin-myosin contraction is discussed in detail in Chapter 19. In brief, MLCK catalyzes MLC phosphorylation leading to the activation of Mg3+myosin ATPase, which hydrolyzes ATP to generate the mechanical energy needed for the actin-myosin contraction. The cytochalasin-induced alteration in actin and myosin filaments and increase in Caco-2 TJ permeability were accompanied by an increase in MLCK activity (Fig. 61-8) (97). The inhibition of cytochalasin-induced MLCK activation by MLCK inhibitors (ML-7, ML-9, KT-5926) prevented the

late-phase cytoskeletal clump formation and the increase in Caco-2 TJ permeability, but not the early-phase severing of actin filaments, suggesting that MLCK activation mediated the formation of the cytoskeletal clumps and the subsequent increase in Caco-2 TJ permeability (97). The cytochalasin severing of actin filaments has been proposed to be the triggering event for the MLCK activation (97). Consistent with this possibility, villin-induced severing of actin filaments has been shown to modulate Mg2+-myosin ATPase activity and actinmyosin interaction (109). The requirement of metabolic energy and mechanical contraction of actin-myosin filaments in the TJ barrier opening was supported by studies showing that Mg2+-myosin ATPase inhibitor (2,3-butadione monoxime) and metabolic inhibitors (sodium azide, 2,4-dinitrophenol) inhibit the cytoskeletal clump formation (late-phase response), the alteration in junctional localization of ZO-1 proteins, and the increase in TJ permeability, but not the severing of the actin filaments (early-phase response). In aggregate, these studies suggested that the cytochalasin-induced increase in intestinal TJ permeability was mediated by cytochalasininduced activation of MLCK, which causes aggregation of perijunctional microfilaments and the contraction of the perijunctional actin-myosin ring, which, in turn, leads to a pulse-string–type contraction of the cell membrane and centripetal-tension–generated opening of the intestinal TJ barrier (97,100).

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1571 Resistance (ohm-cm2)

340 Control 320

300

Cyto-B

280 0

A

Resistance (ohm-cm2)

380

2 4 6 8 10 Time (min)

Control

340 300 Cyto-B 260

220 0

20

A

40

Cyto-B

0.008 Mannitol flux (nmol/cm2)

60

Time (min)

0.007 0.006 Control 0.005 0.004

B FIG. 61-5. Freeze-fracture replicas of villous absorptive cell occluding junctions. (A) Control junctions are composed of a netlike mesh of cross-linked strands or grooves. Perijunctional microvilli are densely aligned above the junction. Scale bar = 0.1 µm. (Shadow angle is approximately left to right.) (B) Junction exposed to 10 µm/ml cytochalasin D for 40 minutes. Junction is composed of an irregular array of strands that underlie occasional broad protrusions of the apical membrane (arrowheads). Geometric irregularities produced by such protrusions result in a fracture plane that only focally includes the apical-most strand (straight arrows). Many perijunctional microvilli are lost and intramembrane particles penetrate into the incompletely isolated intrajunctional compartments (curved arrow). Scale bars = 0.1 µm. (Shadow angle is approximately left to right.) (Reproduced from Madara [100], by permission.)

Luminal Osmolarity and Solvent Drag Effect The relationship between luminal osmolarity, intestinal water flux, and intestinal paracellular permeability has been examined extensively in vivo by perfusing the rat small intestine with perfusate solutions having varying osmolarity (30,110–115). The rate of intestinal absorption of various sized paracellular markers including mannitol, PEG 400, PEG 900, and inulin was linearly related to the increasing concentration of the permeability markers, consistent with a passive uptake mechanism (30,110,112–115). In the in vivo rat intestinal perfusion studies, changing the luminal perfusate pH (from 6.0 to 7.5), varying the unstirred water layer resistance by increasing the luminal flow rate

0

B

20

40 Time (min)

60

FIG. 61-6. Effect of cytochalasin B (Cyto B) (5 µg/ml) on Caco-2 epithelial resistance and paracellular permeability. (A) Cyto B (5 µg/ml) effect on Caco-2 epithelial resistance expressed in Ω /cm−2. Inset is a magnified view of the early time course. (B) Cyto B (5 µg/ml) effect on mucosal-to-serosal flux of paracellular marker mannitol expressed in nmol/cm2. Values are means ± standard error. n = 4. (Reproduced from Ma and colleagues [97], by permission.)

(from 1 to 3 ml/min), or disruption of the mucous layer by treatment with the mucolytic agent acetylcysteine did not affect the intestinal flux rates of the paracellular markers (30,112–116). In a leaky or low-resistance epithelium such as the small intestine, a junctional or paracellular pathway is the major permeation pathway for the passive water and ion flux (30, 31,112,115). The passive water flux may be bidirectional, and the direction of the net water flux is regulated by the osmotic gradient or hydrostatic pressure across the intestinal TJ barrier. A linear relationship exists between decreasing luminal osmolarity and increasing paracellular water flux. The contribution of solvent drag on the absorption of paracellular markers may be assessed by manipulating the water flux by changing the osmolarity of the luminal perfusate solution (30,113,117). Decreasing the luminal osmolarity from 600 to 225 mOsm results in an osmotic gradient–dependent mucosalto-serosal water flux (Fig. 61-9) (30,112,113). As the water rapidly moves across the junctional or paracellular pathways,

1572 / CHAPTER 61

A

B

C

D FIG. 61-7. Effect of cytochalasin B (Cyto B) (5 µg/ml) on perijunctional Caco-2 actin microfilaments. Caco-2 F-actin filaments were labeled with fluorescein-conjugated phalloidin. The sequential effect of Cyto B (5 µg/ml) on Caco-2 actin microfilaments at time 0 (A) and 1 (B), 15 (C), and 30 minutes (D) is shown (original magnification ×80). By 1 minute of Cyto B exposure, perijunctional actin filaments were fragmented and present diffusely throughout the cytoplasm. By 15 to 30 minutes of Cyto B exposure, actin fragments coalesced to form large actin clumps or “foci” near the perijunctional areas. (Reproduced from Ma and colleagues [97], by permission.)

hydrophilic solutes in the luminal solution are also carried along via a solvent drag (30,31,113,117). There is a direct correlation between increasing intestinal water flux and increasing flux of the hydrophilic solutes via the solvent drag (Fig. 61-10). The relative contribution of diffusive and convective (or solvent drag effect) component to the passive transport of permeability probes and solvent drag reflection coefficient sf (an indicator of dependency on solvent drag effect) may be calculated using Fick’s law and the modified Kedem– Katchalsky equation of solvent drag affect (31,117). Fick’s first law expresses the flux rate JD at which a given solute diffuses across a semipermeable membrane as diffusional coefficient PD:

semipermeable epithelium by solvent drag is expressed quantitatively by a modified Kedem–Katchalsky equation (31):

PD = −JD/(C2 − C1),

The relative contribution of diffusion (JD) and solvent drag (JSD) on total passive solute flux (JS) may be determined using the combined equation and the solute and solvent flux data obtained during changes in luminal osmolarity (31,117).

where C1 and C2 are the concentrations of solute on the luminal and serosal side, respectively. The solute flux across the

JSD = JV (1 − σf) ([C1 + C2]/2), where JSD is the solute flux rate by solvent drag, (1 − σf) is the coefficient of solvent drag, JV is the solvent flow rate, and σf is the solvent drag reflection coefficient. Combining the above two equations provides an equation describing the relative contribution of diffusion and solvent drag to the net passive solute flux, JS, across a porous epithelium. JS = JD + JSD JS = −PD∆C + JV (1 − σf) ([C1 + C2]/2)

0′

2′

5′

10′

30′

FIG. 61-8. Effect of cytochalasin B (Cyto B) on Caco-2 myosin light chain kinase (MLCK) activity. Caco-2 monolayers were exposed to Cyto B for increasing time periods (0–30 minutes). Subsequently, Caco-2 monolayers were lysed, and Caco-2 MLCK was immunoprecipitated. The activity of the immunoprecipitated MLCK was determined by in vitro kinetic measurement of MLC phosphorylation. Phosphorylated MLC (P-MLC; ~19.5 kDa) was separated by 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis, stained with Coomassie blue solution, and autoradiographed. Cyto B produced a timedependent activation of Caco-2 MLCK with the peak activation occurring between 5 and 10 minutes after Cyto B exposure. (Reproduced from Ma and colleagues [97], by permission.)

Water flux (ml/100 cm-hr)

20 P – MLC

y = 23.0-0.50x r = 0.98

10

0

−10 100

300 500 Luminal osmolarity (mosmol/l)

700

FIG. 61-9. Relation between small-intestinal luminal osmolarity and water flux. (Reproduced from Ma and colleagues [30], by permission.)

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1573

Inulin flux (101 nmol/100 cm-hr)

8

y = 3.11 + 0.24x

r = 0.97

4

0 −10

0 10 Water flux (ml/100 cm-hr)

20

FIG. 61-10. Relation between jejunal water flux and inulin flux (n = 3–9 rats). Jejunal water flux was varied by changing osmolarity of luminal perfusate. Plotted values are mean of water and inulin absorption. (Reproduced from Ma and colleagues [30], by permission.)

At luminal perfusate osmolarity of 300 mOsm, solvent drag accounted for approximately 60% of total small intestinal flux for PEG 400 and mannitol (113,114). Compared with the smaller permeability markers, solvent drag contribution for inulin flux was much less and accounted for only 10% to 15% of the total small intestinal flux (30). The physiologic contribution of solvent drag to the luminal-to-serosal solute flux is continually changing as the intestinal luminal osmolarity undergoes continual changes depending on the luminal osmotic load and the fluid secretory states after and between meals. The close dependence of intestinal flux rates of hydrophilic solutes on solvent drag suggested that the changes in the direction and the extent of water flow could have a significant impact on intestinal permeability. Consistent with such possibility, exogenously added or endogenously produced secretagogues significantly affected the intestinal flux rates of the paracellular markers by altering the water flux rates (30,111,114). The addition of a known fluid secretagogue 16, 16-dimethyl prostaglandin E2, to the luminal perfusate solution caused a marked decrease in intestinal water flux and a corresponding decrease in solute flux rates (30,111). Conversely, addition of cyclooxygenase inhibitors that inhibit the endogenous production of prostanoids caused an increase in water and solute flux rates (30,111). Similarly, other fluid secretogogues including dibutyryladenosine-3′,5′-cyclic monophosphate, aminophylline, taurocholic acid, and chenodeoxycholic acid also caused a net decrease in water absorption and a decrease in luminal-to-serosal flux of the paracellular markers (30,111–114). Thus, pharmacologic agents and endogenously produced water secretagogues that affect water flux have important modulating effects on intestinal permeability by affecting the solvent drag effect. The potential clinical relevance of solvent drag in absorption of water-soluble drugs was suggested by the studies in which the administration of atenolol and hydrochlorothiazide in

high osmotic solution decreased the intestinal absorption of these drugs (118). Correlating with the in vivo studies, the exposure of the mucosal surface of guinea pig jejunum mounted on Ussing chamber to a high osmotic load (600 mOsm) caused a 64% increase in TER (101). The mucosal exposure to high osmotic load caused a collapse of the paracellular spaces, focal subjunctional lateral membrane evagination, and an increase in intestinal TJ strand count and depth on freeze-fracture analysis (Fig. 61-11) (101). The observed morphologic changes were consistent with the enhancement of the intestinal TJ barrier function. Similarly, in a Necturus gallbladder tissue mounted in Ussing chamber, increasing the osmotic load of the mucosal bathing solution caused a net increase in serosal-to-mucosal water flux with collapse of the intercellular spaces (116). Conversely, decreasing the mucosal osmotic load caused a net increase in mucosal-to-serosal water flux with dilatations of intercellular space and focal separation of TJs (116). Increasing paracellular water absorption leads to an engorgement of intercellular space including the intercellular space between the TJs, presumably leading to the physical separation of the TJ barrier (101,116). The precise mechanisms involved in the separation of the TJ barrier during fluid absorption need further clarification. Further evidence of physiologic importance of solvent drag effect on paracellular permeation of water-soluble molecules stems from the in vivo studies comparing small-intestinal and colonic permeability to paracellular markers PEG 400, PEG 900, mannitol, and inulin (30,110–115). These in vivo perfusion studies have consistently shown the colon to be more permeable than the small intestine (30,110–115). This was a somewhat surprising observation given that the TER of Ussing chamber–mounted colonic tissues have greater TER than the small-intestinal tissues (see Table 61-1), and the TJ barrier would have been expected to be less permeable in the colonic epithelium. In the in vivo perfusion studies, the water flux rates were markedly greater (about fivefold to sixfold greater) in the colon than in the small intestine under the same perfusate and osmotic conditions (30,110–115), and the contribution of solvent drag in the colonic absorption of the permeability probes also was markedly greater than in the small-intestinal absorption, accounting for about 90% of the PEG 400 absorption (30,110–115). Thus, these studies serve as an important example demonstrating that intestinal TJ permeability is not strictly regulated by the relative tightness of the TJ barrier as assessed by functional parameters such as TER, but also is regulated by other factors including the solvent drag effect. Na+-Nutrient Cotransport and Physiologic Regulation In 1987, Pappenheimer and coworkers (31,119,120) advanced a novel concept that the activation of Na+-nutrient cotransport induces a physiologically regulated modulation of the intestinal TJ barrier. In three accompanying articles (31, 119,120), these investigators provided the initial experimental

1574 / CHAPTER 61

A

B

C FIG. 61-11. Freeze-fracture replicas of absorptive cell tight junctions (A). Tight junction from control mucosal sheets displaying uniform depth and composition (B, C). Tight junctions from mucosal sheets exposed to 600 mOsm mucosal buffer for 20 minutes. Focally, junctional depth and strand counts (arrowheads) are large. In addition, the perijunctional apical membranes (asterisks) associated with these junctional areas are bulging and relatively devoid of microvilli. Thus, these areas may correspond with electron microscopic images such as those in Figure 61-5. Original magnification ×70,000. 3CJ, a three-cell junction. (Reproduced from Madara [101], by permission.)

evidence demonstrating that the activation of Na+-nutrient cotransport results in a physiologically regulated opening of the intestinal TJ barrier and an increase in paracellular flux of hydrophilic solutes. These studies formed the foundation for the subsequent studies aimed at delineating the intracellular mechanisms that mediate the physiologically regulated

modulation of the intestinal TJ barrier (121–127). In these studies (31,119,120), the perfusion of isolated rat or hamster intestinal segments with perfusate solution containing glucose or amino acids (alanine or leucine; 25 mM) produced a significant decrease in the junctional resistance, an increase in paracellular permeability, and an increase in water flux. It was calculated based on the rate of water absorption, clearance of the paracellular markers, and coefficient of osmotic flow, Lp, that solvent drag through the junctional or paracellular pathways was the principal mechanism of intestinal absorption of glucose or amino acids in the small-intestinal lumen, when the luminal concentrations of glucose and amino acids exceed the maximal transcellular uptake by the active transporter (31,119,120). The active transport of glucose via the transcellular pathways reaches its maximal capacity (Vmax) at luminal glucose concentration of 10 to 15 mM (120,121). However, intestinal absorption of glucose continues to increase with increasing luminal glucose concentration greater than 15 mM via the solvent drag through the paracellular pathway (121). It has been estimated that between 60% and 90% of glucose transport in the small intestine after a normal meal (where the luminal glucose concentration range between 50 and 300 mM) may be mediated through the solvent drag (31,121,122). The true contribution of solvent drag and paracellular pathways in glucose transport remains controversial, and some investigators (141,142) have argued that the contribution of paracellular transport is much smaller than that proposed by Pappenheimer (122). The effect of luminal glucose on junctional resistance in hamster small intestine has been estimated by steady-state transepithelial impedance measurements (31). The addition of glucose or amino acids (alanine or leucine) to the luminal perfusate produced a twofold to threefold decrease in the junctional resistance as estimated by the impedance measurements (31). The decrease in junctional resistance was associated with an increase in junctional flux of water-soluble solutes including glucose and amino acids and paracellular markers inulin and mannitol (31). Using ferrocyanide as a nonabsorbable marker, investigators have estimated that about 50% of fluid absorption at 25 mM luminal glucose concentration was paracellular. From these studies, it was concluded that solvent drag was a major mechanism of intestinal absorption of water-soluble nutrients (including glucose and amino acids) when the luminal glucose or amino acid concentrations were sufficiently high (31). Pappenheimer and coworkers (31,121,122) have proposed that the Na+-coupled transport of solutes from the intestinal lumen to the intercellular space produces an osmotic gradient across the intestinal TJ barrier, leading to osmotic gradient–generated bulk water flow through the junctional pathway with accompanying solvent drag of hydrophilic solutes. In addition to the functional changes in the intestinal TJ barrier, luminal glucose or amino acid also causes a morphologic change in the cellular structure that correlates with the functional change in the TJ barrier (31,121,122). The addition of glucose or amino acid to the luminal perfusate solution results in a distension of the intercellular space and formation of focal TJ dilatations (119). Accompanying the changes in

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1575 the intercellular space are large protuberances or dilatations within the TJ strand meshwork and a decrease in the number of TJ strands, morphologically correlating with the observed decrease in intestinal TJ barrier function (119). The glucoseinduced decrease in TJ barrier function and anatomic alterations in TJs were also accompanied by “condensation” of perijunctional actin-myosin ring, indicating alteration in cytoskeletal components (119). It was proposed that the activation of Na+-coupled nutrient transport induces a contraction of the perijunctional actin-myosin ring, resulting in a contractile tension–generated pulling apart of the apical membrane and the TJ complex and a functional opening of the TJ barrier (119,121,122). The role of Na+-glucose cotransporter activation in the luminal glucose–induced increase in intestinal TJ permeability was demonstrated by the studies showing that a specific inhibitor of Na+-glucose cotransporter phlorizin inhibits the glucose-induced increase in paracellular permeability and morphologic alterations in TJs (124,128). In addition, replacing the Na+ in the luminal solution with choline also prevented the increase in intestinal TJ permeability and morphologic alterations (124,128). These studies indicated that the activation of Na+-nutrient cotransporter is the initiating event leading to the physiologic regulation of the intestinal TJ barrier. In subsequent studies, the mechanisms that mediate the Na+-glucose cotransport–activated modulation of perijunctional actin-myosin filaments and intestinal TJ barrier were further delineated (129–134). Using an in vitro intestinal epithelial model system consisting of filter-grown Caco-2 intestinal epithelial cells, Turner and colleagues (129) showed that the Caco-2 cells transfected with DNA encoding the intestinal Na+-glucose cotransporter SGLT1 exhibited a physiologic Na+-glucose cotransport. The activation of Na+-glucose cotransport by luminal addition of glucose (25mM) produced a decline in transepithelial resistance in the SGLT1-transfected cells, and the addition of phlorizin (a Na+-glucose cotransport inhibitor) caused an increase in TER (130). The activation of SGLT1 produced a twofold increase in MLC phosphorylation, and MLCK inhibitors ML-7 and ML-9 prevented the SGLT1-mediated decline in TER (130). In aggregate, these studies suggested a causal relation between SGLT1-mediated MLCK activation and subsequent MLC phosphorylation and actin-myosin contraction–mediated increase in intestinal TJ permeability (130). Further insights into the mechanism of Na+-nutrient cotransport modulation of intestinal TJ barrier were derived from enterocyte cell volume studies (135,136). The effect of Na+-nutrient cotransport on enterocyte cell volume was studied using the villous epithelial cells isolated from the guinea pig jejunum (135,136). There is an initial increase (within 30 seconds) in cell volume after activation of Na+-glucose transport, which is followed shortly by a rapid cell shrinkage (complete in 2 minutes). The cell shrinkage after the initial Na+-nutrient–induced swelling has been shown to be due, in part, to the activation of K+ and Cl− conductive pathways (137, 138). Using a modest hypotonic dilution (5–7%) to induce an increase in cell volume, investigators found that after an initial cell acidification, the cell shrinkage was accompanied

by a rapid increase in intracellular pH (pHi, or cell alkalinization) (135,136). The alkalinization and cell shrinkage response was blocked by an inhibitor of Na+-H+ exchanger (NHE), 5-(N-methyl-N-isobutyl) amiloride, suggesting that cell alkalinization and shrinkage required NHE activation (136). In subsequent studies, Turner and Black (132) examined the role of NHE in Na+-glucose cotransport–induced increase in intestinal epithelial TJ permeability in SGLT1transfected Caco-2 cells. The activation of Na+-glucose cotransport produced an increase in pHi. The increase in pHi was inhibited by a preferential NHE3 inhibitor S-3226, but not HOE-694 (a preferential NHE1 and NHE2 inhibitor), suggesting that NHE3 was the NHE isoform responsible for the alkalinization response after the initiation of Na+glucose transport (132). The addition of preferential NHE3 inhibitors in Caco-2 cells undergoing active Na+-glucose cotransport also produced an increase in TER. The increase in TER by NHE3 inhibition appeared to be related to the initial cellular acidification process because alkalinization of the cytoplasm with NH4Cl (5 mM) prevented both the cytoplasmic acidification and the increase in TER (132). The inhibition of NHE also produced an inhibition of Na+glucose cotransport–induced increase in MLC phosphorylation, suggesting a role in the modulation of MLCK activity. These studies indicated that NHE3 activation mediates the Na+-glucose cotransport modulation of cell alkalinization and shrinkage, and the subsequent activation of MLCK and alteration in intestinal TJ barrier (132,133). Previous studies have also identified some of the intracellular mechanisms that mediate the Na+-glucose cotransport– induced activation of NHE3 activity. These studies have shown NHE3 activation to be mediated by p38 mitogenactivated protein kinase (MAPK), Akt-2, and ezrin activation (134,139,140). The Na+-glucose cotransport increase in NHE3 activation was associated with activation of p38 MAPK and of cytoskeletal linker protein ezrin (134). The inhibition of p38 MAPK inhibited both the ezrin phosphorylation and the NHE3 activation. The N-terminal dominantnegative ezrin also inhibited the NHE3 activation, but did not inhibit the p38 MAPK activation, suggesting that p38 MAPK mediated the activation of ezrin phosphorylation and ezrin activation mediated the NHE3 recruitment and activation (134). The ezrin phosphorylation was also mediated by p38 MAPK activation of Akt-2 (139). The Na+-glucose activation of Akt-2 was also inhibited by the p38 MAPK inhibitors. The in vitro phosphorylation studies demonstrated that the activated Akt-2 directly phosphorylates ezrin at threonine 567 in an ATP-dependent manner (139). These studies show that Na+-glucose cotransporter activation of NHE3 is mediated by p38 MAPK activation of Akt-2, which, in turn, phosphorylates ezrin (a cytoskeletal linker protein), which is involved in membrane recruitment and activation of NHE3. The investigation into the specific MLCK isoforms (MLCK1 or MLCK2) that mediate the Na+-nutrient increase in intestinal TJ permeability suggested MLCK1 to be the isoform involved (140). MLCK1 isoform expression correlated with the crypt-to-villus differentiation of enterocytes (140). The expression of MLCK1 isoform was limited to the

1576 / CHAPTER 61 absorptive villous enterocytes and correlated with the development of Na+-nutrient cotransport–dependent TJ regulation in the villous enterocytes (140). MLCK1 isoform localized at the perijunctional actin-myosin ring, and the inhibition of MLCK1 expression by small interfering RNA (siRNA) caused a decrease in intestinal epithelial TJ permeability. Thus, it appeared that MLCK1 was the MLCK isoform that mediated the Na+-nutrient cotransport regulation of intestinal TJ permeability (140). To demonstrate the potential clinical relevance of Na+glucose transport on intestinal permeability in human beings, the effect of glucose (277 mM) on intestinal permeability was assessed using creatinine as the paracellular marker (131). The oral administration of 200 ml of 277 mM glucose (vs mannitol) solution containing 0.8% weight/volume creatinine as the paracellular marker caused a significant increase in urinary recovery of creatinine (55% ± 4% vs 38% ± 9% creatinine recovery for glucose vs mannitol containing solution, respectively), indicating that high concentrations of luminal glucose cause an increase in intestinal permeability in humans. In summary, the activation of Na+-glucose cotransport results in an enterocyte uptake of Na+ and glucose. The enterocyte Na+-glucose uptake produces an initial increase in cell swelling, followed shortly by cell shrinkage. These changes in cell volume are accompanied by an initial decline in pHi, followed by an increase in pHi. The increase in pHi activates NHE3. The NHE3 recruitment and activation are mediated by p38 MAPK activation of Akt-2 and ezrin phosphorylation. The NHE3 activation induces a downstream activation of MLCK, which leads to MLC phosphorylation, actin-myosin contraction, and contractile force–generated retraction of the apical membrane and the TJ complex culminating in the opening of the intestinal TJ barrier. In addition, Na+ and glucose transport into the intercellular space via Na+,K+-ATPase and by facilitated diffusion of glucose leads to an increase in Na+ and glucose concentration in the intercellular space. Increasing Na+ and glucose concentrations results in an osmotic gradient across the TJ barrier and an osmotic gradient–driven mucosalto-serosal bulk water flux and flux of hydrophilic molecules by solvent drag effect. The bulk water flux into the intercellular space causes a distention and dilatation of the intercellular space, presumably causing a mechanical separation of the TJ barrier by the water influx. Thus, Na+-glucose cotransport–regulated opening of the intestinal TJ barrier appears to be mediated by the mechanical tension generated internally by MLCK-activated actin-myosin contraction, and possibly by the mechanical force generated externally by the osmotic gradient–induced water in-flux into the intercellular space causing physical separation of the TJ seal.

Cytokines In addition to their known effects on the modulation of the immune system, cytokines and other inflammatory mediators may modulate intestinal inflammation by their regulatory actions on the intestinal TJ barrier. The number of cytokines

and inflammatory mediators including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-4, IL-6, IL-12, IL-13, insulin, insulin-like growth factor, and hepatocyte growth factor induce an increase in intestinal TJ permeability, whereas some anti-inflammatory cytokines and growth factors including IL-10, TGF-β, and epidermal growth factor appear to have a protective action in maintaining the intestinal TJ barrier function (143–191). Since the first report by Madara and Stafford showing that IFN-γ causes an increase in TJ permeability in T84 epithelial cells (191), a number of investigators have shown that a wide variety of cytokines and inflammatory mediators also affect the intestinal TJ barrier (143–190). The cytokine-induced increase in intestinal TJ permeability has been proposed as an important mechanism contributing to intestinal inflammation by allowing increased paracellular permeation of toxic luminal antigens (129,146, 161,162,191). Despite increasing interest in the role of proinflammatory cytokines in the modulation of the intestinal TJ barrier, the intracellular mechanisms that mediate the cytokineinduced regulation of intestinal TJ barrier remained poorly understood. However, several recent reports have provided some new insights into the possible mechanisms involved (143,146,192). In the initial report by Madara and Stafford (191), IFN-γ did not have an acute effect on T84 epithelial TJ permeability (24 or 48 hours), but caused a delayed increase in T84 TJ permeability at the 72-hour period (191). The delayed effect of IFN-γ on T84 TJ permeability suggested that the IFN-γ effect was not caused by an acute intracellular signaling regulation of the T84 TJ barrier, but was related to an alteration in the protein expression (191). In addition, the IFNγ–induced increase in T84 TJ permeability was not related to cell death or epithelial damage causing large epithelial gaps, but was caused by a reversible alteration in T84 TJ barrier function (188,191). The IFN-γ decrease in T84 TJ barrier function was associated with an increase in degradation of TJ protein ZO-1, a decrease in ZO-1 protein synthesis, and a disturbance in the junctional localization in ZO-1 proteins (188). The alteration in ZO-1 localization also was closely linked to the perturbation in the apical actin organization, suggesting the possibility that actin alteration may be involved in the IFN-γ–induced TJ barrier modulation (188). Although these studies provided some descriptive information regarding the IFN-γ effect on the intestinal epithelial TJ barrier function and the TJ proteins, the intracellular mechanisms involved have yet to be delineated. TNF-α is a prototypical cytokine that has been shown to play a central role in the intestinal inflammation in Crohn’s disease and other inflammatory conditions (192,193). The importance of TNF-α in the intestinal inflammation of inflammatory bowel disease has been well validated by clinical and animal studies showing the effectiveness of anti–TNF-α antibody in the treatment of severely active Crohn’s disease and in animal models of intestinal inflammation (192–196). TNF-α–induced increase in intestinal TJ permeability has been proposed as an important proinflammatory mechanism (146,161,162,193). TNF-α at physiologically

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1577

Epithelial resistance (ohm-cm2)

500

400

300

200 0 1 10 50 100 TNF-α concentratin (ng/ml)

FIG. 61-12. Effect of increasing concentrations of tumor necrosis factor-α (TNF-α; 0, 1, 10, 50, and 100 ng/ml) on Caco-2 epithelial resistance (Ω/cm−2). Filter-grown Caco-2 monolayers were treated with TNF-α for the 48-hour experimental period. TNF-α produced a concentration-dependent decrease in Caco-2 epithelial resistance. Data represent means ± standard error of epithelial resistance (n = 4). *p < 0.01 versus control (0 hour). (Reproduced from Ma and colleagues [146], by permission.)

relevant concentrations (1–10 ng/ml) causes an increase in intestinal epithelial TJ permeability in both in vivo and in vitro intestinal epithelial model systems (including Caco-2, T84, and HT29 intestinal epithelial cells) (Fig. 61-12) (18,143,146,148,169,171,176,182,184). Several reports have provided new insights into the possible mechanisms involved (143,146,201). These reports indicated that the TNF-α–induced increase in Caco-2 TJ permeability was mediated by nuclear factor (NF)-κB activation (146). The TNF-α–induced increase in Caco-2 TJ permeability was associated with an increase in NF-κB activation and nuclear translocation of NF-κB p65, and the inhibition of NF-κB activation with NF-κB inhibitors (curcumin and triptolide) prevented the TNF-α–induced increase in Caco-2 TJ permeability. The NF-κB inhibitors also prevented the TNFα–induced down-regulation of ZO-1 protein expression and

A

B

disturbance in junctional localization (Fig. 61-13). These studies indicated that the TNF-α–induced increase in Caco2 TJ permeability required NF-κB activation (146). Because TNF-α is also known to induce apoptosis in various cell types, apoptosis as a possible mechanism of TNF-α increase in Caco-2 TJ permeability had been considered. In Caco-2 cells, TNF-α do not induce apoptosis or necrosis, indicating that apoptosis or cell death is not the mechanism responsible for the TNF-α increase in Caco-2 TJ permeability (146,182). The possibility that apoptosis may be the mechanism involved in the TNF-α–induced increase in TJ permeability in other epithelial cell types including HT29/B6 colonic epithelial cells and LLC-PK1 renal epithelial cells has been suggested by studies showing that TNF-α induces an increase in apoptosis in these cell types (18,197). Although it is possible that the TNF-α increase in apoptosis may have contributed to the overall increase in TJ permeability in these cells, the low rates of apoptosis (1–4%) observed in these cell types after TNF-α treatment are unlikely to explain the increase in epithelial TJ permeability (18,197). For instance, in T84 cells, IFN-γ– and TNF-α–induced increase in TJ permeability was also associated with about a twofold to threefold increase in apoptosis (148). However, the inhibition of TNF-α–induced increase in apoptosis by caspase inhibitor Z-Val-Ala-Aspfluoromethylketone (ZVAD-fmk) did not affect the increase in TJ permeability, indicating that the TNF-α–induced apoptosis was not the mechanism involved in the increase in intestinal epithelial TJ permeability (148). Consistent with the lack of involvement of apoptosis as a mechanism of intestinal epithelial TJ barrier disruption, it had been demonstrated that during normal physiologic sloughing of dying intestinal epithelial cells at the villous tip, adjacent cells rapidly stretch and extend to reestablish the TJ barrier as the dead cell extrudes (198). The TJ elements of adjacent epithelial cells proliferate at the point of the contact between the adjacent cells to rapidly reestablish the TJ barrier in a “zipper”-like manner (Fig. 61-14) (198). As the paracellular barrier is maintained throughout the cell extrusion process, the epithelial barrier function to macromolecules is maintained (198). Similarly, in the in vitro model

C

D

FIG. 61-13. Effect of tumor necrosis factor-α (TNF-α) on junctional localization of zonula occluden 1 (ZO-1) proteins. The junctional localization of ZO-1 proteins in filter-grown Caco-2 monolayers was assessed by immunofluorescent antibody labeling. (A) Untreated or control Caco-2 monolayers. (B) TNF-α treatment for 48 hours. (C) Curcumin and TNF-α treatment for 48 hours. (D) Triptolide and TNF-α treatment for 48 hours. TNF-α produced a progressive disturbance in ZO-1 protein localization at the cellular borders, with disruption in ZO-1 continuity and gaplike appearance at the points of multiple cell contacts (B, arrows). Curcumin and triptolide prevented the TNF-α–induced alteration in junctional localization of ZO-1 proteins. (Reproduced from Ma and colleagues [146], by permission.)

1578 / CHAPTER 61 TJ strands TJ kisses Extruding cell Adjacent cell (fracture face, (cross-section) plasma membrane) Terminal web

Lyosome

FIG. 61-14. Model of cell extrusion from intestinal epithelia. TJ, tight junction. (Reproduced from Madara [198], by permission.)

system consisting of HT-29/B6 colonic epithelial monolayers, artificially induced single-cell defect resulted in a rapid sealing of the epithelial defect (within minutes) by flattening and extension of the adjacent cells and rapid reformation of the TJ barrier (199). The reformation of the TJ barrier seal was dependent on MLCK-activated actin-myosin interaction (199). It was suggested that the TJ barrier formation may be

driven by a contractile “pulse-string” mechanism in which the perijunctional ring of actin and myosin filaments contract to generate the mechanical force to induce a rapid closure of the epithelial wound and to reestablish the functional TJ barrier (199,200). TNF-α–induced increase in intestinal TJ permeability also required prolonged exposure, suggesting that the TNF-α

MLCK β-actin Time (hrs)

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6

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A 5

Mannitol flux (nmol/h-cm2)

Epithelial resistance (ohm-cm2)

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400

3

2

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24 Time (h)

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FIG. 61-15. Time-course effect of tumor necrosis factor-α (TNF-α) on Caco-2 myosin light chain kinase (MLCK) protein expression, transepithelial electrical resistance (TER), and paracellular permeability. Filter-grown Caco-2 monolayers were treated with TNF-α (10 ng/ml) for increasing time periods (0–48 hours). The Caco-2 MLCK protein expression was determined by Western blot analysis. The effect of TNF-α (10 ng/ml) on Caco-2 TER and mucosal-to-serosal flux of paracellular marker mannitol (10 µmol/ml) was measured sequentially over the 48-hour experimental period. (A) Time-course effect of TNF-α on Caco-2 MLCK protein expression (β-actin was used as an internal control for protein loading). (B) Time-course effect of TNF-α on Caco-2 epithelial resistance (means ± standard error [SE]; n = 4). (C) Time-course effect of TNF-α on mucosal-to-serosal mannitol flux (means ± SE; n = 4). (Reproduced from Ma and colleagues [143], by permission.)

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1579 modulation of TJ barrier was mediated by a new protein synthesis (143). Because MLCK has been shown to play a central role in a wide variety of pharmacologically and physiologically induced alterations in intestinal TJ barrier, the role of MLCK protein expression was considered (143). TNF-α caused a time-dependent increase in Caco-2 MLCK protein expression, which correlated sequentially with the increase in Caco-2 paracellular permeability to mannitol and inulin and a decrease in Caco-2 TER (see Fig. 61-15). The increase in MLCK protein expression also correlated with an increase in MLCK activity. The inhibition of TNF-α increase in MLCK protein expression by cycloheximide prevented the increase in MLCK activity and Caco-2 TJ permeability (143). In addition, inhibition of MLCK activation by MLCK inhibitors ML-7 and ML-9 also prevented the TNF-α increase in Caco-2 TJ permeability, indicating the requirement of MLCK activation in the TNF-α increase in Caco-2 TJ permeability (143). Consistent with the role of MLCK-mediated actin-myosin contraction in the Caco-2 TJ barrier opening, Mg2+-myosin ATPase and metabolic energy inhibitors also prevented the TNF-α increase in Caco-2 TJ permeability. These studies demonstrated that the TNF-α– induced up-regulation of MLCK protein expression and subsequent MLKC activation mediated the TNF-α increase in Caco-2 TJ permeability (143). In subsequent studies, the molecular mechanisms involved in the TNF-α increase in Caco-2 TJ permeability also were investigated (201). To delineate the molecular mechanisms involved in the TNF-α–induced up-regulation of MLCK protein expression, investigators identified and cloned the MLCK promoter region and validated the functional activity of the promoter (201). In these studies, it was shown that the TNF-α increase in MLCK protein expression was associated with an increase in MLCK promoter activity and an increase in mRNA transcription, and the inhibition of MLCK mRNA transcription (by actinomycin D) prevented the increase in Caco-2 TJ permeability (143,201). These studies indicated that the TNF-α–induced increase in MLCK protein expression, MLCK activity, and Caco-2 TJ permeability was mediated by an increase in MLCK promoter activity and MLCK transcription (201). Using the deletion constructs generated from the full-length MLCK promoter region, investigators also delineated the regulatory site on the MLCK promoter responsible for the increase in MLCK activity. It was demonstrated that the upstream κB binding region GGAGCTTCCC (−84 to −75) was the regulatory site that mediated the TNF-α–induced up-regulation of the MLCK promoter activity. Electrophilic mobility shift assay studies indicated that the NF-κB p65/p50 dimer binding to the upstream κB binding site up-regulated the MLCK promoter activity (201). In further support of NF-κB as the transcription factor that up-regulates the MLCK promoter activity, NF-κB inhibitors curcumin and pyrrolidine dithiocarbamate prevented the TNF-α–induced increase in MLCK promoter activity and the subsequent increase in MLCK mRNA expression, MLCK protein expression, MLCK activity, and increase in Caco-2 TJ permeability (201). The siRNA

silencing of NF-κB p65 expression also prevented the TNF-α increase in MLCK promoter activity, further validating the role of NF-κB p65/p50 dimer in the regulation of the MLCK promoter activity (201). In aggregate, these studies provided new insights into the intracellular and molecular mechanisms involved in the TNF-α regulation of intestinal TJ barrier (143,146,201). These studies suggested that the TNF-α– induced NF-κB activation results in following sequence of events: (1) cytoplasmic-to-nuclear translocation of NF-κB p65/p50 dimer; (2) nuclear-translocated NF-κB p65/p50 dimer binds to the upstream κB binding motif on the MLCK promoter and activates the MLCK promoter; (3) MLCK promoter activation leads to an increase in MLCK transcription and protein expression; (4) increase in MLCK protein expression leads to an increase in MLCK activity and activation of MLCK pathways; and (5) MLCK-activated contraction of perijunctional actin-myosin filaments then leads to a contractile tension–generated opening of the TJ barrier.

Infectious Pathogen–Induced Alteration of Intestinal Epithelial Tight Junction Permeability Intestinal TJ barrier disruption by infectious pathogens has been postulated to play an important role in the pathophysiology of infectious diarrhea. In contrast with secretory diarrhea where the pathogen induces Cl− (Na+ and water) secretion, disruption of the TJ could induce diarrhea by eliminating the electrochemical gradient required for absorption and by enhancing inflammation and prosecretory inflammatory mediators by permitting bacteria and antigens to breach the epithelial barrier. A number of infectious agents and toxins elaborated by the enteric pathogens have been shown to cause a disturbance of the intestinal epithelial TJ barrier. In general, pathogens exert their effects either directly through binding to the intestinal epithelial cell or indirectly though the actions of a secreted toxins. Whether direct or indirect, their mechanisms of action can be divided into those that affect the perijunctional actomyosin ring with subsequent perturbation of the TJ barrier, and those that target the activity of specific TJ proteins. The best-studied infectious agents that affect the TJ barrier include enteropathogenic Escherichia coli (EPEC), Clostridium difficile, Vibrio cholerae, Bacteroides fragillis, Clostridium perfringens, and rotavirus (202–231). Tables 61-4 and 61-5 provide a summary of various effects of infectious agents and the factors secreted by the pathogens (212). The intracellular processes involved in the EPEC modulation of intestinal TJ barrier have been among the most extensively studied using the cultured intestinal epithelial cell models (202–206). They involve both effects on the cytoskeleton and on the specific proteins occludin and ZO1. Infection of T84 cells with EPEC (202,203) is associated with an increase in intracellular Ca2+, increase in MLC phosphorylation, phosphorylation of ezrin, dephosphorylation of occludin, and increase in transepithelial migration of

1580 / CHAPTER 61 TABLE 61-4. Enteric pathogens that modify epithelial barrier function Organism

Cell line

Pathophysiologic outcome

Structural modification of TJ

Clinical relevance

Diffusely adhering Escherichia coli

Caco2/TC7 T84

Enteropathogenic E. coli

T84 MDCKI

Disorganization of occludin and ZO-1, but not E-cadherinb ZO-1 staining diminished and disrupted, but E-cadherin unaffected; minor disruption of F-actin Intact apical actin filaments and ZO-1 staining (MDCK)

Associated in some studies with diarrheal disease

Enterohemorrhagic E. coli c

No change Rt (up to 5 hours) Increased paracellular permeabilitya Decrease in Rt (onset at 12 hours) Decrease in Rt (onset at 2 hours or later in all studied cell lines)

Caco-2

Rotavirus

Caco-2

Salmonella typhimurium

MDCK II Caco-2 T84

No change in Rt (up to 24 hours); increased paracellular permeabilitya Decrease in Rt by 15 minutes in MDCK II cells; decrease in Rt by 1 hour in Caco-2 cells; no change in Rt for up to 3 hours in T84 cells

No gross TJ change, but F-actin disorganized and, after 18 hours, ZO-1 staining focally diffused (T84; by TEM) Disorganization of occludin, but not E-cadherinb Apical pole contraction with clustering of F-actin, E-cadherin, and ZO-1 at sites of bacterial attachment (MDCK II cells); no gross TJ change (Caco-2; by TEM)

Leading cause of infectious hemorrhagic colitis; food-borne pathogen; associated with HUS Serious cause of acute and persistent diarrhea in young children (≤3 years old)

Major cause of infantile diarrhea globally Major cause of food-borne disease; Salmonella spp may infect up to 1% of population annuallyd

aDemonstrated

with [3H] mannitol. the actin stabilizer jasplakinolide, disruption of the normal occludin and zonula occluden 1 (ZO-1) structure was shown to be independent of the F-actin. cPurified Shiga toxins 1 or 2 do not decrease R . In addition, Shiga toxin–negative enterohemorrhagic E. coli strains do not t differ from wild-type enterohemorrhagic E. coli strains in their effects on T84 monolayer permeability, indicating that Shiga toxins do not play a role in the decreased Rt caused by these strains. dS. typhimurium is predicted to account for ~25% of food-borne Salmonella infections. Both S. choleraesuis and S. enteriditis have been reported to decrease Rt by 2 to 4 hours in MDCK I monolayers without gross tight junction (TJ) disruption by transmission electron microscopy (TEM) or change in E-cadherin staining. HUS, hemolytic uremic syndrome; MDCK I and II, Madin–Darby canine kidney cells forming high- (I) and low-resistance (II) monolayers; Rt, transepithelial electrical resistance. Reproduced from Sears CL. Molecular physiology and pathophysiology of tight junctions v. assault of the tight junction by enteric pathogens. Am J Physiol Gastrointest Liver Physiol 2000;279:G1129–G1134, by permission. bUsing

polymorphoneutrophils (204). Phosphorylation of occludin is required for its proper TJ localization. Exposure to EPEC results in dephosphorylation of occludin, its movement to a cytoplasmic location, and a decrease in barrier resistance (218). It has been speculated that the EPEC-induced increase in intracellular Ca2+ is responsible for the activation of MLCK, which enzymatically phosphorylates MLC and activates Mg2+-myosin ATPase. This leads to the contraction of the perijunctional actin-myosin filaments and retraction of enterocyte membrane and TJ complex, culminating in opening of the TJ barrier (205). Although the specific sequence of events has yet to be confirmed experimentally, inhibition of MLC phosphorylation by an MLCK inhibitor (ML-9) prevents the EPEC-induced increase in T84 TJ permeability, supporting the central role of actomyosin contraction in the pathophysiology (205). Ezrin phosphorylation may also be involved in the cytoskeletal-induced TJ retraction process and stimulation of transepithelial migration of polymorphoneutrophils (205,206).

C. difficile also appears to work through altering the actomyosin cytoskeleton, but by inactivating the small GTPbinding proteins Rho, Rac, and Cdc42, not through activation of MLCK (231–234). The Rho family of small GTPases is important in regulating many aspects of actin filament dynamics, which control cell shape and cell–cell junctions. By switching between the GDP- and GTP-bound states, they control the activity of proteins that control the location of actin attachments and filament assembly and disassembly. Both toxins A and B elaborated by C. difficile inactivate Rho family GTPs by covalently attaching glucose derived from UTP-glucose. Their inactivation leads to degradation of perijunctional actin and barrier failure (233,234). Both B. fragilis and V. cholera secrete proteases that degrade specific TJ proteins as their presumed mechanism of action (212–214,220). The B. fragilis enterotoxin, also known as fragilysin, is a 20-kDa metalloprotease. Exposure of human colonic cell lines and native colonic tissue to the

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1581 TABLE 61-5. Enteric pathogen virulence factors that alter tight junctional proteins or modify epithelial barrier function, or both Organism

Virulence factor

Bacteroides fragilis

Metalloprotease toxin

Cell line/tissue studied T84,Caco-2, HT-29, HT-29/C1

Proposed mechanism Cleavage of E-cadherin

MDCK

Human colon

Clostridium difficile

Toxin A

T84, human colon, rabbit ileum

Monoglucosylation of Rho at threonine 37

Toxin B

T84, Caco-2, human colon

Clostridium perfringens

Enterotoxin

MDCK I

Cleavage of claudins 3 and 4, but not 1 and 2

Escherichia coli

Cytotoxic necrotizing factor 1

Caco-2

Deamidation of Rho at glutamine 63

Helicobacter pylori

Vacuolating toxin

T84

Unknown

MDCK I

Listeria monocytogenes

Internalin

Caco-2

Binds to E-cadherin to mediate bacterial cellular invasion; Proline 16 of E-cadherin is critical to E-cadherin/ internalin interaction

Vibrio cholerae

zot

Rabbit ileum

Activation of protein kinase C

Pathophysiology and TJ structure

Clinical relevance of pathogen

Decrease in R t (onset in ~15 minutes)

Associated with community-acquired diarrheal disease in children and adults

Absent E-cadherin, dissociation of occludin and ZO-1 from TJ Gross separation of some TJs and ZO junctions Decrease in R t (toxins A and B onset in 2 and 4+ hours, respectively, in T84 monolayers)a Loss (toxin A) or flocculation (toxin B) of apical F-actin with intact TJ (EM) Decrease in R t (onset by 4 hours); loss of claudins and TJ strands (freezefracture EM) Decrease in R t (onset after 1 hour); enhanced actin filament formationb Decrease in R t (onset by 1 hour)

No changes in occludin, ZO-1, cingulin, or E-cadherin observed Unknown

Decrease in R t (onset by 1 hour); loss of TJ strands (freeze-fracture EM)

Only clearly documented cause of infectious nosocomial diarrhea

Food-borne, toxin-mediated, noninflammatory diarrheal disease Limited case reports of clinical illnesses, including diarrheal disease Associated with gastritis, gastroduodenal ulcer, gastric adenocarcinoma, and MALT lymphoma

Food-borne systemic disease (sepsis, meningitis) in immunocompromised hosts with 25% mortality; short-lived febrile diarrheal illness in immunocompetent hosts Classic epidemic secretory diarrheal disease

Continued

1582 / CHAPTER 61 TABLE 61-5. Enteric pathogen virulence factors that alter tight junctional proteins or modify epithelial barrier function, or both—cont’d Organism

Virulence factor Hemagglutinin/ proteasec

Cell line/tissue studied

Proposed mechanism

MDCK I

Cleavage of occludin

Pathophysiology and TJ structure

Clinical relevance of pathogen

Decrease in R t (onset after 1 hour); occludin degradation; ZO-1 and F-actin reorganization

Oral zot treatment has been proposed as a strategy to enhance drug absorption (e.g., insulin)

aToxin

B reduced Caco-2 monolayer Rt more rapidly. specific information on tight junctional (TJ) proteins reported. cData derived from studies of crude culture supernatants of a mutant V. cholerae strain, CVD 110, which is positive for hemagglutinin/proteinase. Studies using purified hemagglutinin/proteinase are not available. EM, electron microscopy; MALT, mucosal-associated lymphoid tissue; MDCK, Madin–Darby canine kidney; Rt, transepithelial electrical resistance; ZO, zonula occludens; zot, zonula occludens toxin. Reproduced from Sears CL. Molecular physiology and pathophysiology of tight junctions v. assault of the tight junction by enteric pathogens. Am J Physiol Gastrointest Liver Physiol 2000;279:G1129–G1134, by permission. bNo

enterotoxin results in disassembly of the perijunctional actin and disruption of the TJ barrier. Among it substrates is the AJ protein E-cadherin. Because the integrity of TJs is dependent on proper AJ function, loss of the key AJ adhesion molecule is presumed to affect the TJ barrier. V. cholera secretes a metalloprotease called hemagglutinin protease or HA/P, which among other substrates, hydrolyzes the extracellular domains of occludin. In addition to HA/P, V. cholera expresses several other toxins such as the classical cholera toxin A (ctxA), which induces Cl− secretion. When all but HA/P are genetically deleted, bacteria can still disrupt perijunctional actin and the TJ barrier. A causal connection is supported by the observation that application of a specific bacterial metalloprotease inhibitor prevents occludin cleavage and actin disruption. Another TJ-directed toxin in the arsenal of cholera is zot (zonula occludens toxin). Application of purified zot leads to disruption of perijunctional actin and the barrier with activation of PKC as an intermediate. Zot alone is not cytotoxic, and the effects on actin are reversible. The normal intestine produces a zot homolog that has been termed zonulin. There is some evidence that zonulin is an endogenous physiologic modulator of TJs, leading to the speculation that cholera has coopted this system to enhance TJ permeability (227). C. perfringens represents an interesting example where the toxin does not specifically target the TJ, but uses a TJ protein as its cell-surface receptor on a path to inducing cytolysis. The C. perfringens enterotoxin is a common cause of diarrheal food poisoning and less commonly antibiotic-associated diarrhea. The toxin is a 35-kDa polypeptide with a C-terminal domain that binds to several different claudins, notably 3 and 4, and an N-terminal domain required for toxicity. Binding results in formation of a large protein complex that includes claudins and occludin and a coincident increase in ionic permeability across the plasma membrane (223). It is hypothesized that the toxin induces claudins to form a transmembrane pore and to allow cytotoxic levels of calcium to enter. There has been speculation that C. perfringens enterotoxin could be

used as a chemotherapeutic agent for gastrointestinal malignancies that overexpress claudin-4, such as pancreatic cancer (229). This appears doubtful given the wide distribution of claudin-4 throughout the body. Rotaviruses infect epithelial cells of the small intestine and induce diarrhea without obvious histologic tissue damage at early stages of infection. Rotavirus infection of cultured Caco-2 cells causes a decrease in barrier function coincident with redistribution of claudin-1, occludin, and ZO-1. The mechanism of TJ disruption is unclear, but has been speculated to result from decreased ATP levels (207). In vivo, the rotavirus-induced diarrhea also was associated with an increased intestinal permeability to lactulose in infants (209, 211). The defective TJ barrier function may contribute to diarrhea by allowing paracellular leakage of intestinal fluid into the lumen (or allowing increased penetration).

CLINICAL DISORDERS OF INTESTINAL TIGHT JUNCTION BARRIER DEFECT A number of intestinal disorders have been shown to have an associated defect in intestinal TJ barrier as evidenced by an increase in intestinal permeability. An important question that remains to be resolved is whether the intestinal TJ barrier defect represents a primary pathogenic factor of the disease leading to the intestinal inflammation, or whether the defect is secondary to the epithelial damage resulting from intestinal inflammation. In either case, the defect in intestinal TJ barrier is likely to contribute to intestinal and systemic inflammation by allowing increased paracellular permeation of toxic luminal antigens and other harmful luminal agents that are normally excluded (162,235,245,279–281). This section discusses the intestinal TJ barrier defect and the possible implications of this defect in those permeability disorders that have been studied the most including celiac disease, Crohn’s disease, and nonsteroidal anti-inflammatory drug (NSAID)–associated enteritis.

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1583 Clinical Assessment of Intestinal Tight Junction Barrier Defect Intestinal epithelium serves a dual role (237,245,279,282). Intestinal epithelial cells regulate the uptake of nutrients, while at the same time they act as a barrier to the permeation of potentially harmful substances present in the intestinal lumen. This selective barrier function has been referred to as “intestinal permeability.” In clinical studies, the term intestinal permeability refers to the intestinal barrier function to passively absorbed water-soluble markers. Intestinal permeability has been defined as “the ability of medium and large sized water-soluble compounds to passively traverse the intestinal epithelial layer through paracellular tight-junctional areas” (245,279–281). Intestinal permeability studies assess the relative leakiness of the intestinal TJ barrier or paracellular pathways to passive permeation by water-soluble probes. For usage in clinical studies, permeability markers must be hydrophilic and passively absorbed, inert and nontoxic, not metabolized or endogenously produced, rapidly and completely excreted in urine, and should be measured easily (279,280, 282,287). The most commonly used permeability probes to assess the small-intestinal permeability in humans include PEG 400, mannitol, rhamnose, lactulose, cellobiose, and 51CrEDTA (279–281). Less commonly, creatinine and inulin also have been used. Because of their hydrophilicity, permeability probes permeate poorly across the bilipid enterocyte membrane, but are able to permeate across through the aqueous TJ or paracellular pathways. The intestinal permeation rates of the probes are directly related to their molecular size and the functional pore size of the TJs (279,280,282,289). All of the commonly used probes are relatively inert and nontoxic. Of the permeability probes mentioned, only PEG 400 and 51Cr-EDTA are not metabolized or endogenously produced. The sugar probes including mannitol, rhamnose, lactulose, cellobiose, and inulin are degraded by intestinal bacteria, and creatinine is produced endogenously. The permeability probes are excreted in a variable manner depending on their body distribution and urinary excretion. All of the above probes are measured with relative ease in urine. The intestinal permeability studies are intended to assess the “leakiness” of the intestinal TJ barrier in humans. Typically, in clinical studies, permeability markers are ingested orally after an overnight fast, and urine is collected for varying periods (between 6 and 24 hours). Previous studies have demonstrated no significant difference in the reliability of the permeability test whether a 6- or a 24-hour urinary collection period was used. Currently, the most commonly used collection period in clinical studies is 6 hours. The amount of permeability probe excreted in the urine is then used as an indirect measurement of intestinal TJ permeability.

Permeability Index and Celiac Disease A permeability index lactulose/mannitol or large probe/ small probe urinary excretion ratio (permeability index =

% urinary excretion of larger probe/% urinary excretion of smaller probe) has been used by investigators as an index of intestinal permeability in clinical studies (252,253,255,257, 263). For this purpose, most commonly used large probes are disaccharides lactulose and cellobiose, and 51Cr-EDTA and small probes are monosaccharides mannitol, rhamnose, and D-xylose. Historically, the usage of large-to-small probe urinary excretion ratio (including cellobiose/mannitol, lactulose/mannitol, or 51Cr-EDTA/mannitol excretion ratio) has been shown to be more sensitive in the detection of intestinal permeability disorders than when the excretion rates of probes were used alone (242,252–255,259,261–263). In patients with celiac disease, intestinal permeability to smaller probes including mannitol and L-rhamnose is decreased, correlating with a decrease in the net intestinal absorptive surface area resulting from the immune-mediated destruction of the villous surface (259–263). The decrease in intestinal permeability to mannitol and L-rhamnose is present during the active exacerbations of celiac disease, and the permeability changes resolve with the improvement of the disease after the removal of gluten from the diet (245–249, 251). Paradoxically, intestinal permeability of larger probes including lactulose, cellobiose, and 51Cr-EDTA increases with increasing mucosal damage and villous atrophy (259–263). Comparison of the sensitivity of the permeability index versus intestinal permeability measurements of the individual permeability markers in the detection of celiac disease indicated that the permeability index was more sensitive than the individual permeability measurements (251–255). The higher sensitivity of the permeability index is because of the inclusion of mannitol (or small-probe) permeation rates into the equation, which provides an indirect accounting of the decrease in the mucosal absorptive surface area (279). As discussed earlier in this chapter, in healthy intestinal epithelium, the effective pore size of the intestinal TJs are such that the smaller sized probes (<8–9 Å) are able to readily move across the intestinal TJ pores (289). In the healthy human intestinal epithelium, the intestinal permeation rates of larger probes (lactulose and 51Cr-EDTA) are about 50- to 100-fold less than the smaller probes (mannitol, PEG 400, and L-rhamnose) (289). Because the villous absorptive surface is damaged in patients with celiac disease as a result of gluteninduced intestinal inflammation, the absorptive surface area markedly decreases. The inflammation-induced damage of the mucosal surface also causes morphologic and functional alteration of the TJs (250). The differences in intestinal permeability to large-sized versus small-sized (<8 Å) permeability probes in celiac disease (and also in Crohn’s disease) may be explained by the increase in the functional TJ pore size during the disease states and the simultaneous decrease in the absorptive surface area resulting from the mucosal damage (Fig. 61-16) (279). The smaller probes (rhamnose, mannitol, and PEG 400), having small cross-sectional diameter (<7 Å), readily permeate across the normal intestinal epithelium and have high rates of urinary recovery (range is 10–30% urinary recovery per 6-hour collection period) in healthy individuals (279,289). In contrast, the larger probes

1584 / CHAPTER 61 Large probe Small probe

Paracellular pore

Paracelluar pore

A

B

FIG. 61-16. Proposed model of alteration of paracellular pathways or “pores” in Crohn’s disease. In healthy intestine (A), absorptive surface is relatively large (villous adaptation), and the paracellular opening or pores are “moderate” in size. The smaller probes (e.g., mannitol and polyethylene glycol [PEG] 400) readily permeate (urinary recovery rate of 10–30%/6 hours), whereas larger probes (e.g., lactulose) are mostly excluded (urinary recovery rate of 0.2–0.4%/6 hours) from permeation across the healthy intestinal epithelia. In diseased states (B), the overall surface area of absorptive surface may be diminished, but the paracellular pores are larger either as a primary defect or secondary to intestinal inflammation. The enlarged paracellular pores allow penetration of larger probes. The permeation of smaller probes also are increased, but to a much smaller extent. (The decrease in absorptive surface area negates the increase in permeation of smaller probes.) Solid squares indicate large probe; small solid circles indicate small probe. (Reproduced from Ma [279], by permission.)

(lactulose and 51Cr-EDTA) are mostly excluded by the healthy intestinal epithelium and have low rates of urinary recovery in healthy individuals (range is 0.2–0.7% urinary recovery per 6-hour collection period) (289). There is about a 50- to 100-fold difference in the relative intestinal permeability of small versus large permeability probes in humans (279). As the smaller probes permeate across the intestinal TJ barrier relatively unrestricted in the healthy intestinal epithelium, the increase in the TJ pore size during the disease states has a relatively minor impact in the TJ flux rates of the smaller probes. In active celiac disease, there is extensive destruction of the absorptive villous surface and almost complete denudation of the villous surface, resulting in a marked decrease in the overall absorptive surface area. Concurrently, the functional pore size of the TJs in the remaining epithelial surface is markedly increased (see Fig. 61-16). Presumably, the permeation rates of smaller probes are much more affected by the decrease in the absorptive surface area than by the increase in the TJ pore size, thus the net decrease in the permeation rates of the smaller probes. In contrast, as the larger probes are mostly excluded by the normal intestinal TJ barrier, their permeation rates are much more affected by the increase in the TJ pore size during the disease states and less by the changes in the absorptive surface area, and thus the net increase in the intestinal permeability to the larger probes (see Fig. 61-16). The alterations in intestinal TJ permeability in celiac disease closely correlate with the extent of the mucosal damage and villous atrophy induced by the gluten ingestion (248,257). The alteration in intestinal TJ permeability normalizes with the removal of gluten from the diet and the subsequent villus

regeneration (248,257,260,262). After gluten withdrawal induced normalization of intestinal permeability, an exposure to a single oral dose of gluten causes an increase in intestinal permeability (260). A TJ-modulating protein zonulin has been recently discovered by Fasano and collegues (290–294). They (290–294) have implicated zonulin as a possible factor mediating the gliadin-induced increase in intestinal TJ permeability in celiac disease.

Crohn’s Disease and Intestinal Tight Junction Barrier Defect Crohn’s disease is a chronic inflammatory bowel disease of unknown cause characterized by recurrent bouts of exacerbations (256). Clinical presentation during acute exacerbations include abdominal pain, diarrhea, weight loss, and bloody stool. Patients with Crohn’s disease as a group have an increase in intestinal permeability (242–244,279–281). As early as 1963, Gryboski and colleagues (258) found patients with Crohn’s disease to have an abnormal increase in intestinal permeability to lactulose and sucrose. Subsequently, numerous other investigators have shown patients with Crohn’s disease to have an increase in intestinal TJ permeability to a wide variety of permeability probes including 51Cr-EDTA, lactulose, cellobiose, and PEG 400 (242–244,308–314). Similar to celiac disease, clinical studies consistently have shown patients with Crohn’s disease to have an increase in intestinal permeability to the larger permeability probes and either a decrease or no change in intestinal permeability to the

TIGHT JUNCTIONS AND THE INTESTINAL BARRIER / 1585 smaller probes (279). As discussed in the section on celiac disease, the probe size–related differences in intestinal permeability in Crohn’s disease also appear to be related to the increase in the pore size of the TJ barrier having greater contribution to the increased TJ permeation of larger probes and the decrease in the intestinal absorptive surface (as a result of intestinal inflammation) having a greater contribution to the decrease in the absorption of smaller probes (see Fig. 61-16) (279). Although it is well established that patients with Crohn’s disease have an abnormal increase in intestinal TJ permeability, the significance of the defective intestinal TJ barrier in the pathogenesis of intestinal inflammation of Crohn’s disease remains unclear. It had been proposed by number of investigators that the defect in intestinal TJ barrier may represent a genetic disorder of Crohn’s disease, which predisposes susceptible individuals to development of Crohn’s disease later in life when exposed to the appropriate environmental factors (162,235,237,240,279,288,289,308). Another possibility may be that the increase in intestinal TJ permeability is secondary to the intestinal inflammation and plays a secondary role in promoting intestinal inflammation by allowing increased antigenic penetration (279–281). In support of the first possibility, clinical studies have consistently shown that healthy first-degree relatives of patients with Crohn’s disease (an at-risk group for development of Crohn’s disease) have preexisting intestinal permeability defect before any clinical evidence of Crohn’s disease (279–282,308). The first-degree relatives of patients with Crohn’s disease have about 30 to 100 times greater risk for development of Crohn’s disease compared with the normal population and have about a 10% lifetime risk for development of Crohn’s disease (256). Intestinal permeability studies in healthy first-degree relatives of patients with Crohn’s disease consistently have shown that between 10% and 30% of the relatives have an abnormal increase in intestinal permeability (241–244,288,308,312, 315–317), suggesting the possibility that the first-degree relatives with the increase in intestinal TJ permeability may be the ones who are genetically predisposed to development of Crohn’s disease later in life when exposed to appropriate environmental stresses. Consistent with this possibility, oral administration of a known intestinal permeability stressing agent, aspirin (325 mg), caused a significantly greater increase in intestinal permeability in the healthy first-degree relatives compared with the normal control subjects (318–320). A causal relation between increased intestinal permeability and immune activation was demonstrated in a study by Yacyshyn and Meddings (295), which showed that all clinically healthy first-degree relatives with increased intestinal permeability had increased CD45RO expression in B cells (an indicator of immune activation) (245,296), whereas none of the first-degree relatives with normal intestinal permeability had increased CD45RO expression, indicating that the increase in intestinal permeability was responsible for the immune activation. In addition to the functional abnormality in the intestinal TJ barrier function, patients with Crohn’s disease also have morphologic alterations in the intestinal

TJ complexes (297–301). The examination of the healthy, uninvolved areas of small-intestinal tissue obtained from patients with Crohn’s disease showed alteration in the TJs characterized by irregular distribution, fragmentation, and aberrant TJ strands (236,297–301). The role of defective intestinal TJ barrier in the intestinal inflammation of Crohn’s disease is further supported by studies showing that the relapse of Crohn’s disease is often preceded by drugs or conditions that affect the intestinal TJ barrier, such as NSAID usage, acute gastroenteritis, and alcohol binges (302–305). Together, these studies form the basis for the “leaky gut” hypothesis that the defect in the intestinal TJ barrier in patients with Crohn’s disease may predate the development of Crohn’s disease and may be an important causative factor contributing to the pathogenesis of Crohn’s disease (245,279–281, 288,308) (Fig. 61-17). More investigations are required to further define whether the defective intestinal TJ barrier is truly a primary causative factor of Crohn’s disease. An important long-term study would be to follow the first-degree relatives of patients with Crohn’s disease who have an abnormal increase in intestinal permeability to determine whether Crohn’s disease eventually develops in these individuals. In support of the possibility that the increase in intestinal TJ permeability in Crohn’s disease is secondary to intestinal inflammation, a number of proinflammatory cytokines and inflammatory mediators that are produced during active inflammation of Crohn’s disease have been shown to cause an increase in intestinal TJ permeability (see earlier discussion).

Defective intestinal epithelial barrier Exposure to environmental or endogenous permeability stressing factors Increase in intestinal permeability "leaky gut"

Intestinal penetration of luminal antigens

Activation of immune response

Intestinal inflammation

FIG. 61-17. Proposed model of “leaky gut” hypothesis. The proposed primary defect of intestinal epithelial barrier function (increased paracellular permeability) allows increased mucosal penetration by toxic luminal antigens (including infectious agents). The antigenic penetration activates immunologic and inflammatory responses, which produce epithelial injury and further damage of the intestinal epithelial barrier function. Exposure to environmental or endogenous factors that increase intestinal permeability, such as nonsteroidal anti-inflammatory drugs, may act as a trigger for the onset or exacerbation of Crohn’s disease in a susceptible population. (Reproduced from Ma [279], by permission.)

1586 / CHAPTER 61 A direct correlation exists between the degree of active intestinal inflammation and an increase in intestinal permeability, and improvement of intestinal inflammation in response to medical treatment is associated with a normalization of intestinal permeability (238,239,306). In addition, increase in intestinal permeability has been shown to be an excellent predictor of clinical relapse (238,239,306). Thus, the presence of intestinal inflammation in Crohn’s disease appears to be an important factor affecting the intestinal TJ barrier function in Crohn’s disease.

Nonsteroidal Anti-inflammatory Drugs and Intestinal Permeability NSAIDs are widely used as therapeutic agents for antiinflammatory purposes and for pain control. Important adverse effects of NSAIDs are related to its damaging effects on the gastrointestinal mucosal surface, leading to ulcers and gastrointestinal bleeding (270,274,275,277). Chronic and acute NSAID therapy is associated with high prevalence of small-intestinal inflammation and ulcers (277,307). In autopsied cases, 10% of the NSAID users (chronic and occasional users) had small-intestinal ulcers (307). NSAIDs are known to cause an acute increase in intestinal permeability in humans and in animals (273–278). Acute ingestion of NSAIDs (including aspirin, indomethacin, and ibuprofen) causes a significant increase in small-intestinal permeability in humans (276–278). The NSAID increase in small-intestinal permeability may be mediated by systemic effects because rectal administration also causes an increase in intestinal permeability (277,278). Since the NSAID-induced increase in intestinal TJ permeability precedes any detectable injury to the intestinal mucosal surface, the increase in intestinal TJ permeability does not appear to be related to the damage to the mucosal surface (272,274–277). Since the increase in intestinal permeability precedes the damage to the mucosal surface, it was proposed that the increase in intestinal TJ permeability may be an important pathogenic factor leading to the NSAID-induced small-intestinal inflammation (272,282). The mechanisms that mediate the NSAID effect on intestinal permeability remain unclear; however, it appears that its effect on cyclooxygenase inhibition is important as the potency of the NSAID effect on intestinal permeability was directly related to the drug potency to inhibit cyclooxygenase (276,277), and the NSAID effect was attenuated by prostaglandin analogs (273). As discussed earlier, it has been shown in rat intestinal perfusion studies that the NSAID effect on increasing intestinal permeability was related in part to an increase in the solvent drag effect (30,111). The effect of NSAIDs on intestinal permeability is likely to be caused by a combination of effects including modulation of intestinal fluid fluxes (increased net fluid absorption) and the disruption of intestinal TJs. The possibility that the NSAID action on intestinal TJ barrier may be mediated by inhibition of metabolic energy (or ATP depletion) has been hypothesized, but direct experimental evidence is lacking (269,271).

Because a single dose of NSAIDs causes an acute increase in intestinal permeability in humans, NSAIDs have been widely used as a “permeability stressing” agent in clinical studies (276–278,318–320). As an example, administration of aspirin as a permeability stressing agent in patients with Crohn’s disease and their healthy relatives caused a significantly greater increase in intestinal permeability in the patients with Crohn’s disease and their relatives than in the healthy control subjects (318–320). Thus, NSAIDs are useful as a permeability stressing agent to study the susceptibility of the intestinal TJ barrier to external stressing agents in clinical studies.

Other Intestinal Permeability Disorders A number of other clinical conditions have been shown to have an associated increase in intestinal TJ permeability including sepsis, critically ill, burn injury, heat stress, alcoholic liver disease, irritable bowel syndrome, and various infections syndromes (149,264,265,267,268,283–286,321–332). In each of these clinical conditions, the breach in the intestinal TJ barrier has been proposed to play an important pathogenic role in promoting intestinal or systemic inflammation. For example, in alcoholic liver disease, the defective intestinal TJ barrier has been proposed as the primary pathogenic factor allowing epithelial penetration of bacterial endotoxins that induce inflammatory reaction in the liver (264–266, 268). Similarly, in patients with burn injury and those who are critically ill, the leaky intestinal TJ barrier allows bacterial translocation and antigenic penetration, leading to sepsis and systemic inflammation (277,278,283,329). Many studies have also suggested that the stress-induced alteration of the intestinal TJ barrier may play a prominent role in immune activation in irritable bowel syndrome (239,240). Although the precise mechanisms that modulate the intestinal TJ barrier in these clinical conditions remain to be further elucidated, the rapidly expanding investigations into these areas are likely to provide innovative and important new insights into the intracellular and molecular mechanisms involved in the modulation of intestinal TJ barrier and establish a causal relation between the TJ barrier defect and the development of various inflammatory diseases in the not too distant future.

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Protein Sorting in the Exocytic and Endocytic Pathways in Polarized Epithelial Cells V. Stephen Hunt and W. James Nelson Cytoarchitecture of the Polarized Epithelial Cell, 1595 Cytoarchitecture of the Polarized Epithelial Cell, 1596 Pre–Trans-Golgi Network Protein Traffic, 1597 Protein Sorting at the Trans-Golgi Network and Beyond, 1597 Selective Retention, 1599 Summary, 1599 Cell Biology Toolkit, 1600 Molecular Mechanisms of Polarized Traffic, 1600 Sorting Sequences and Examples, 1600 Vesicle Budding: Coats and Adaptors, 1605

Role of Lipids and Proteolipids in Protein Sorting and Transport, 1610 Vesicle Motility: Role of the Cytoskeleton and Motors, 1612 Regulators of Compartmental Identity: The Rab GTPases, 1615 Vesicle Fusion: The SNAREs, 1616 Membrane Hot Spots: The Spatial Regulation of Exocytosis, 1617 Conclusion, 1618 References, 1619

The asymmetric localization of proteins to different cell surfaces is vital for epithelial cell function throughout the gastrointestinal (GI) tract. The epithelial cells lining the GI tract separate the outside environment in the lumen from the inside of the organism, functioning as barriers to harmful stimuli, vectorial transporters of nutrients and waste, and polarized secretors of proteins, hormones, and lipids. In general, cellsurface proteins are restricted to either the apical plasma membrane, which faces the intestinal lumen, or the basolateral plasma membrane, which contacts surrounding cells and basal lamina and faces the interstitium. Intracellular protein sorting is necessary for the establishment and maintenance of this structural and functional asymmetry at the cell surface. The physiology of regulated secretion is covered in some detail elsewhere (see Chapters 48, 53, 55) in this textbook and is beyond the scope of this chapter. We focus on constitutive

protein trafficking in the polarized epithelial cell. We provide a basic overview of the cytoarchitecture, membrane compartments, and routes of protein trafficking in intestinal epithelial cells. Because much of the progress in dissecting the molecular sorting pathways has resulted from in vitro model systems, we also provide a brief overview of some of the tools used to study protein trafficking. The final sections provide a more detailed examination of the molecular machinery and mechanisms involved in protein sorting and trafficking. Examples are presented throughout the chapter to highlight mechanisms of protein sorting.

V. S. Hunt and W. J. Nelson: Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

CYTOARCHITECTURE OF THE POLARIZED EPITHELIAL CELL A discussion of the mechanisms of protein sorting in polarized epithelial cells requires an understanding of the architecture of this specialized cell (Fig. 62-1). Although polarized epithelial cells throughout the GI tract differ in their precise morphology, for the sake of identifying basic principles, we discuss cytoarchitecture in terms of a generic simple polarized epithelial cell such as represented by the enterocyte.

1595

1596 / CHAPTER 62

AJC CE/SAC

TGN trans-Golgi medial-Golgi cis-Golgi ERGIC ER

Microtubule

Cytokeratin

Actin

FIG. 62-1. Cytoarchitecture of the polarized simple epithelial cell. The cytoarchitecture of polarized epithelial cells of the gastrointestinal tract has functional significance. The microtubules are arrayed in a longitudinal pattern along the apicobasal axis with their minus ends anchored at the apical end of the cell. Several intracellular membrane compartments are dependent on anchoring to microtubules for fidelity and localization, including the common endosome/subapical compartment (CE/SAC) and the trans-Golgi network (TGN). Actin bundles comprise the core of the brush-border microvilli, and an actin ring is located at the apicolateral border. Cortical actin is a chief component of the submembrane cytoskeleton and is particularly dense at the lateral membrane. Cytokeratins are localized to the terminal web region just below the apical membrane and mediate attachment of the microtubule minus ends. The apical junctional complex (AJC) helps to mediate adhesion between adjacent cells and is composed of the tight junction and adherens junction proteins. ER, endoplasmic reticulum.

Cytoarchitecture of the Polarized Epithelial Cell Overview of Cell Junctions About a half century has passed since Farquhar and Palade (1) first described the beautiful ultrastructure of the tripartite junctional complexes in epithelial cells. They identified three separate “elements”—the zonula occludens (ZOs; tight junction), zonula adherens (adherens junction), and macula adherens (desmosome)—that are organized in a stereotypic manner. The tight junction is located at the apex of the lateral membrane. It functions both as a barrier to transepithelial

passage of ions and other solutes (gate function), and to restrict lateral diffusion of membrane proteins between the apical and basolateral membrane domains (fence function). The identification and characterization of the molecular components of the tight junction, including membrane proteins (occludin and claudins), cytoskeleton-associated proteins (ZO-1, -2, and -3), and signal transduction proteins (Rho family GTPases, protease-activated receptor 3 [PAR-3] and -6, and protein kinase C [PKC] isoforms), led to the realization that, in addition to maintaining cell polarity, the tight junction plays multiple roles in cell signaling (2)

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1597 and interacts with components of the protein transport and sorting apparatus (e.g., rab13, exocyst complex) (3–5). The adherens junction is located just below the tight junction at the upper aspect of the lateral membrane. It, too, is composed of a number of transmembrane and associated proteins with functions that are critical in the maintenance of cell polarity (6). The transmembrane protein E-cadherin mediates Ca2+dependent adhesion between adjacent cells and initiates and maintains diverse regulatory pathways involved in the development and maintenance of epithelial cell polarity (7). The third “element,” desmosomes, are composed of desmosomal cadherins and associated intermediate filament (IF) attachment proteins and are localized at many spots down the basolateral membrane. They function to maintain strong intercellular adhesion and to resist shearing stress, thereby preserving the structural integrity of the epithelium (8). Cytoskeleton The apical surface of enterocytes is covered by long membrane protrusions termed microvilli. Actin bundles form the core of these apical microvilli. A dense ring of actin cables circumscribes the cell at the apex of the lateral membrane (9,10). This actin ring is associated with cell adhesion junctions and aids both in junctional stability and in actinmyosin–based contraction of the apex of the cell, for example, during wound healing (11–13). Actin also forms a cortical meshwork underneath the entire plasma membrane. At the lateral membrane, this actin meshwork is associated with cell junctional proteins, some of which may be integrated into an ankyrin/fodrin-based submembrane cytoskeleton (10,14). Microtubules are oriented in a longitudinal array of bundles oriented in the apicobasal axis of the cell adjacent to the lateral membrane domain, with their minus ends directed toward the apical surface and plus ends toward the basal membrane; there are also subapical and basal meshworks of short, randomly oriented microtubules (15). This microtubule orientation is important in establishing an axis of cell polarity, positioning of subcellular compartments and organelles, and providing directionality for microtubuledependent vesicle transport. The subapical terminal web region of polarized intestinal epithelial cells contains cytokeratin (CK) IFs CK8, CK18, and CK19 (16). Microtubule minus ends and microtubule organizing centers are embedded in this subapical meshwork of CKs (17,18). Two other IF proteins, plectin and desmoplakin, are restricted to areas underlying the lateral plasma membrane, where they mediate anchorage of IFs to desmosomes (19–22). In cultured epithelial cells, another IF protein, vimentin, is distributed in a reticular network throughout the cytoplasm, with particular enrichment around the nucleus (20). Membrane Compartments Protein synthesis, targeting, and sorting to specific membrane compartments occur at distinct intracellular membrane compartments that have asymmetric distributions

in polarized epithelial cells (Fig. 62-2). The endoplasmic reticulum (ER) is located near the nucleus, which typically is positioned toward the base of the cell. The Golgi compartment is located more to the top half of the cell beneath the apical membrane. The trans-Golgi network (TGN), a tubulovesicular extension of the trans face of the Golgi apparatus, is located just apical to the trans-Golgi; it serves as a major site for sorting exocytic proteins into vesicles destined for different plasma membrane domains and organelles. Using fluorescent-labeled fluid-phase and protein markers, investigators have defined additional intracellular sorting compartments involved in protein trafficking pathways as apical (AEEs) and basolateral early endosomes (BEEs), apical recycling endosomes (AREs), common endosomes (CEs, also termed the subapical compartment [SAC]), and late endosomes (LEs) (23,24). The CE/SAC is a branching tubulovesicular network 60 to 100 nm in diameter positioned in the subapical region of the cell (24). As Figure 62-2 illustrates, the CE/SAC is situated at the center of the sorting apparatus, and it is perhaps second only to the TGN as a site of protein sorting. Many apical and basolateral proteins pass through the CE/SAC during exocytosis, during transcytosis, or after endocytosis and subsequent recycling (24). The ARE consists of a population of 100- to 200-nm cup-shaped vesicles located immediately beneath the apical membrane (24). Transcytotic and apical markers have been shown to pass sequentially through the CE/SAC and then the ARE in transit to the apical plasma membrane (24). Apical and basolateral proteins can also meet in the LE, on the pathway either to the Golgi or to destruction in the lysosome (24).

Pre–Trans-Golgi Network Protein Traffic Protein synthesis begins on free ribosomes in the cell cytoplasm. The mechanism for targeting proteins to the ER, and their transport through the ER and Golgi compartments, is beyond the scope of this discussion, but has been reviewed in the literature (25–27). Notable, however, is the fact that the ER and Golgi serve as sites for the addition and modification of complex oligosaccharide side chains to proteins, which form sorting signals for protein trafficking (see later discussion). Inside the ER lumen, the oligosaccharyl transferase complex transfers preassembled core oligosaccharides to the protein at Asn-X-Ser/Thr sequences, a process termed N-glycosylation. In addition, some proteins have oligosaccharide side chains added after translation to the -OH group of serine or threonine side chains, a process termed O-glycosylation. The Golgi compartment is a series of membrane “stacks” or cisternae that perform successive modifications of the oligosaccharide side chains.

Protein Sorting at the Trans-Golgi Network and Beyond Proteins are delivered from the trans-Golgi face to the TGN compartment, where they are sorted into membranous vesicles

1598 / CHAPTER 62

2c ARE

AEE

AJC 1b 1c

4b

4b

2b

AEE

CE/SAC

BRE

4b

4a

3b LE 3a 3c L

2a TGN

BEE

1a

BEE

trans-Golgi medial-Golgi cis-Golgi ERGIC ER

Nucleus

FIG. 62-2. The membrane compartments and routes of protein traffic. Proteins are sorted at multiple membrane compartments within the cell. Basolateral traffic can arrive: (1a) directly from the transGolgi network (TGN), (1b) from the common endosome/subapical compartment (CE/SAC), or (1c) from basolateral recycling endosomes (BREs). Apical traffic can arrive (2a) directly from the TGN, (2b) from the CE/SAC, or (2c) from the apical recycling endosomes (AREs). Proteins can arrive at the lysosome (L) (3a) directly from the TGN, (3b) from the CE/SAC, or (3c) from late endosomes (LEs). Proteins can arrive at the CE/SAC (4a) directly from the TGN or (4b) from plasma membrane endocytic traffic. The intracellular compartments through which both apical and basolateral traffic can pass are shaded in gray, and they include the TGN, LE, and CE/SAC. The TGN and CE/SAC are the primary protein sorting compartments within the cell. Proteins also can be sorted at the plasma membrane domains or the apical (AEEs) and basolateral early endosomes (BEEs) in the case of transcytotic traffic. AJC, apical junctional complex; ER, endoplasmic reticulum.

bound for either the lysosomes or the exocytic pathway to different plasma membrane domains, endosomes, or lysosomes. The TGN serves as the earliest membrane compartment for sorting of newly synthesized proteins into apical- or basolateral-specific trafficking pathways (28). Notably, however, this sorting process at the TGN is not restricted to polarized epithelial cells, because it is active in nonpolarized epithelial cells and in nonepithelial cell types (29,30). TGN sorting mechanisms are thus insufficient to explain the selective distribution of proteins in polarized epithelial cells. Post-TGN protein sorting to the apical and basolateral membrane domains primarily occurs via separate, albeit

overlapping, pathways. Some of the molecular players involved in apical and basolateral exocytosis function in both trafficking pathways (see detailed discussion in Molecular Mechanisms of Polarized Traffic section later in this chapter); however, the apical and basolateral pathways also contain pathway-specific homologues and, in some cases, unique protein species. As alluded to earlier, further post-TGN sorting can occur at a number of different membrane compartments including the plasma membrane and various endosomal compartments. A theme is emerging in which the membrane compartment performing the majority of the sorting for a given protein

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1599 depends on both the cell type and the protein of interest. For example, hepatocytes traffic the majority of newly synthesized apical proteins via transcytosis, with proteins first passing through the basolateral membrane (31). Madin–Darby canine kidney (MDCK) cells (a polarized renal epithelial cell line) traffic many apical proteins directly from the TGN to the apical membrane, thereby bypassing the basolateral membrane completely (32,33). Enterocytes and enterocyte-derived cell lines (e.g., Caco-2) are thought to fall somewhere in between these two phenotypes, with significant TGN (direct) and transcytotic (indirect) sorting of apical proteins occurring. Sorting of the p75 neurotrophin receptor provides a good illustration of these differences. Soluble forms of p75 (lacking a transmembrane domain) are secreted from the apical and basolateral domain of MDCK and Caco-2 cells, respectively (34,35). However, the membrane-bound form of p75 is trafficked to the apical membrane directly in MDCK cells, but indirectly via transcytosis through the basolateral membrane in Caco-2 cells (34,35). Another illustrative example is the trafficking of the complement protein gp80. In MDCK cells, gp80 is selectively secreted from the apical plasma membrane, indicating an apical sorting pathway (32). In hepatocytes, however, gp80 is secreted from the sinusoidal (basolateral) surface into the blood, presumably because the majority of apical traffic first passes through the basolateral domain (36). Albumin, however, is trafficked in part directly and in part indirectly to the apical surface in HepG2 cells (a hepatocyte-derived cell line), whereas in MDCK cells, glycosylphosphoinositol (GPI)-anchored proteins can travel via transcytosis to the apical surface (37,38). The intestinal brush-border protein lactase-phlorizin hydrolase (LPH) is trafficked directly to the apical membrane in Caco-2 cells (39). Thus, it is not possible to classify a given cell type simply by either the mechanisms or pathways of protein sorting. Specific proteins can be sorted in the TGN and trafficked directly to a given plasma membrane domain, or they can arrive at the surface indirectly via another membrane compartment, or they can use both direct and indirect pathways (40–42).

Selective Retention Some proteins may be sequestered from further sorting by selective retention and restriction of their lateral mobility in a given membrane domain. The first hint that plasma membrane proteins are restricted in their lateral membrane mobility came from a study demonstrating that an apical marker protein was polarized in MDCK cells lacking tight junctions (43). Subsequent analysis of the lateral mobility and extractability of basolateral and apical marker proteins in polarized MDCK cells showed that domain-specific protein immobilization could account for the maintenance of polarity of a substantial (30–90%) fraction of basolateral and apical proteins (44). Definitive proof of such a mechanism followed shortly. In polarized MDCK cells, Na,K-ATPase, which is

present on the basolateral membrane at steady state, can be targeted to both plasma membrane domains. However, Na,K-ATPase is rapidly internalized from the apical domain, although it is stabilized at the basolateral domain by binding directly to the ankyrin/fodrin-based submembrane cytoskeleton located beneath the lateral membrane (45). In polarized retinal pigment epithelial cells, the ankyrin/fodrin-based submembrane cytoskeleton is located instead beneath the apical plasma membrane domain, and Na,K-ATPase localizes to the apical membrane (46). The PrPc protein provides another example of selective membrane retention of a protein. PrPc is a ubiquitously expressed, GPI-anchored protein that has been identified as the agent responsible for transmissible spongiform encephalopathies on its conversion to the protease-resistant PrPsc form (47,48). Unlike every other native GPI-anchored protein studied, at steady state, PrPc is located at the basolateral membrane in polarized MDCK cells (49,50). However, newly synthesized PrPc is sorted to both the apical and basolateral membrane, but like the Na,K-ATPase, it is only stabilized at the basolateral domain (50). The basolateral membrane laminin receptor (LR), derived from its precursor protein (LRP), is the cell-surface receptor for PrPc (54), in addition to its role as a receptor for the extracellular matrix protein laminin (51–53). Two separate binding domains on LRP/LR and PrPc have been characterized, one of which is modulated by cell-surface heparin-sulfate proteoglycan (52). PrPc thus is stabilized on the outer leaflet of the basolateral membrane through its direct interactions with LRP/LR, which, in turn, is stabilized by binding to the extracellular matrix and cell-surface proteoglycans.

Summary A polarized epithelial cell has a specific orientation of cytoskeleton structures and membrane compartments. Multiple trafficking pathways regulate the establishment and maintenance of the steady-state localization of a plasma membrane protein to either the apical or basolateral membrane domain. The TGN is one of several membrane compartments that participate in the sorting of membrane and secreted proteins. Additional membrane compartments have evolved, perhaps in part to compensate for the increased number of sorted protein species and the reduced TGN membrane surface-to-volume ratio for sorting in eukaryotic cells. Cell types differ in the amount of sorting that occurs at each membrane compartment, whereas the ability of a given cell type to sort either directly or through endosomal recycling pathways depends on the protein being sorted. Selective retention at the plasma membrane also plays a role in the final localization of a given protein. Decades of research have delineated many of the sorting signals contained in the amino acid sequence of a protein that direct it toward the apical or basolateral surface (55). Many of the molecular players that regulate these sorting pathways are known, as are their mechanisms and sites of action (56,57). The Molecular

1600 / CHAPTER 62 Mechanisms of Polarized Traffic section later in this chapter explores how cell-type–specific expression of these players, in combination with their compartmental localization, determines the trafficking pathways and steady-state localization of a given protein in a given cell type.

with an emphasis on comparing and contrasting the apical and basolateral sorting and trafficking machinery. The celltype–specific expression of the individual players in combination with their compartmental localization determines the sorting mechanisms, trafficking pathways, and steady-state localization of a given protein in a given polarized cell type.

CELL BIOLOGY TOOLKIT Sorting Sequences and Examples Direct examination of primary tissue samples can provide a basic understanding of the steady-state distribution of proteins in polarized intestinal epithelial cells. However, a molecular understanding of how these proteins became localized to those sites requires in vitro experimental manipulation and observation. Table 62-1 outlines examples of the tools used to study protein sorting and trafficking in polarized cells. Because primary cells are both difficult and costly to obtain and are typically limited in their ability to propagate in culture, several polarized cell lines are used to mimic properties of polarized cells in the GI tract (58–65). The earliest examinations of protein trafficking pathways used viruses that bud specifically from one or the other plasma membrane domain, and though still in use, advances in molecular biology and cell transfection techniques now allow for expression of virtually any desired marker protein without the cytopathic effects of viral infection (66–69). Experimental study of the trafficking of representative apical and basolateral marker proteins in these model polarized cells has defined much of the molecular machinery responsible for protein sorting and transport pathways. Toxins have proved useful in defining endocytic protein trafficking pathways (70). Chemical inhibitors also have proved useful in dissecting protein sorting and trafficking pathways (71–82). As the molecular machinery is defined, more specific techniques, such as antibody depletion, proteinfragment overexpression, and RNA silencing are being used to further characterize the contributions of individual proteins to protein sorting and trafficking.

MOLECULAR MECHANISMS OF POLARIZED TRAFFIC Regulation at any stage in the trafficking of proteins through the exocytic or endocytic pathways could impart specificity of protein localization to a target membrane. Such regulation could occur at the time of budding of carrier vesicles from a membrane compartment, during trafficking along cytoskeletal tracks, upon tethering at a target membrane compartment, during fusion with the target membrane, or it could involve selective retention of the protein in a particular membrane. Protein sorting occurs at each of these stages, and a breakdown in the sorting machinery at any stage can lead to protein mistargeting with consequences for the normal function of the epithelium. The following sections outline important molecular players and mechanisms that participate in protein sorting and transport in polarized cells,

Several important sorting decisions are made throughout the life of a protein. For example, during exit from the TGN, a protein can be directed to either the apical or basolateral plasma membrane domain, or to endosomal compartments. At the endosomal compartments, a protein can be sorted to the lysosome, to either plasma membrane domains or in a few cases, back to the TGN. At the cell surface, a protein can remain at the cell surface or be internalized and trafficked through the endosomal sorting compartments. Although pathway-specific machinery may aid in the polarized distribution of proteins at the surface, intrinsic signals within the amino acid sequence or folded structure of a protein are needed to specify both the final localization of a protein and the path it takes to get there. This section examines some of the known signal sequences that play roles in these sorting decisions. (Because the body of data elucidating these signals is large, the reader is referred to an excellent review on this subject by Bonifacino and Traub [55] and the references therein.) Some of the machinery that interacts with these sequence-sorting motifs is examined in detail later in this chapter (see Vesicle Budding: Coats and Adaptors section). Two Common Classes of Cytoplasmic Sorting Signals A common type of sorting signal motif is a short, linear sequence of four to seven amino-acid residues within the cytoplasmic domain of transmembrane proteins (55). Usually only a few residues are absolutely critical to serve as a sorting signal, and these residues are generally charged amino acids or bulky hydrophobic amino acids (55). These critical residues can be used to separate sorting signals into classes, two of which are the “tyrosine-based” and the “dileucine-based” classes (55). Each of these classes, in turn, specifies a number of conserved signal sequences. The two most commonly identified tyrosine-based sorting signals are the NPXY and YXXϕ motifs (55), where X is any amino acid and ϕ is a bulky hydrophobic amino acid. NPXY signals generally are found within cytoplasmic domains containing 40 to 200 amino-acid residues, although they also have been found within the larger cytoplasmic domains of signaling receptors. The NPXY motif codes for rapid endocytosis of a subset of type I integral membrane proteins, including the insulin receptor, the epidermal growth factor receptor (EGFR) family, the low-density lipoprotein receptor (LDLR) family, megalin, and the β chains of integrins, and this motif is highly conserved throughout metazoan evolution (55). NPXY is the minimal motif shared by these

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1601 proteins and is necessary for proper internalization, but it alone is not sufficient for directing endocytosis in all cases (55). The YXXϕ motif is an even more common sorting motif than NPXY, and its evolutionary roots stretch as far back as the protists (55). The motif is commonly found even within cytosolic proteins, although most often in these cases the motif is folded within the internal structure of the protein, and thus is unavailable for interactions with the protein sorting machinery. The YXXϕ motif encodes a sorting signal for rapid endocytosis, lysosomal targeting, and basolateral targeting of type I, type II, and multispanning integral membrane proteins including lysosomal membrane proteins (e.g., the lysosome-associated membrane proteins [LAMPs] and endolyn), intracellular sorting receptors (e.g., the mannose phosphate receptors [MPRs]), TGN proteins (e.g., TGN38 and furin), and endocytic receptors (e.g., FcR, TfR, asialoglycoprotein receptor H1) (55). Although the final localization of these proteins may differ, in general they all traffic through the plasma membrane on their way to their final membrane destination. Although YXXϕ is the minimal motif shared by these proteins, the X residues and flanking amino acids often also contribute to the strength and specificity of the signal (55). For example, the YXXϕ signals involved in lysosomal targeting most commonly have a glycine residue immediately preceding the tyrosine residue (55). Perhaps even more important to the function of the YXXϕ signals is their position within the cytosolic domain of the protein. For example, endocytic YXXϕ signals are commonly 10 to 40 residues from the transmembrane domain, whereas lysosomal YXXϕ signals are commonly 6 to 9 residues from the transmembrane domain (55). This dependence on position in the cytoplasmic domain is required for interaction with the sorting machinery. The YXXϕ signals are known to bind to several vesicle coat proteins, and these binding partners are examined later in this chapter (see Vesicle Budding: Coats and Adaptors section). The two most commonly identified dileucine-based sorting signals are [DE]XXXL[LI] and DXXLL (55). Similar to the YXXϕ motif, the [DE]XXXL[LI] motif is found in many proteins, and its evolutionary roots also stretch as far back as the protists. The [DE]XXXL[LI] motif also has been shown to code for rapid endocytosis, lysosomal targeting, endosomal targeting, and basolateral targeting of a wide array of type I, type II, and multispanning integral membrane proteins. These proteins are localized to a variety of intracellular compartments, including the LE/lysosome (e.g., the Lysosome Integral Membrane Protein [LIMP] and Niemann-Pick [NPC] family of proteins), stimulus-responsive storage vesicles (e.g., glucose transporter 4 [GLUT4] and aquaporin 4 [AQP4]), and the basolateral plasma membrane (e.g., FcRIIB2, LDLR, and E-cadherin) (55). Similar to YXXϕ, [DE]XXXL[LI] is the minimal motif shared by these proteins, but the X residues and flanking amino acids often also contribute to the strength and specificity of the signal. For example, the [DE]XXXL[LI] signals involved in lysosomal targeting most commonly have an acidic residue at the fourth residue preceding the first leucine residue. Also similar

to the YXXϕ signal, the position of the [DE]XXXL[LI] signal within the cytosolic domain of the protein has functional importance. For example, similar to lysosomal YXXϕ signals, lysosomal [DE]XXXL[LI] signals are commonly 6 to 11 residues from the transmembrane domain. Similar to YXXϕ motifs, [DE]XXXL[LI] motifs are known to bind to several vesicle coat proteins; these binding partners are examined later in this chapter (see Vesicle Budding: Coats and Adaptors section). The DXXLL signals generally are found within one or two amino-acid residues from the carboxyl-termini of the protein, and the D residue generally is found within the context of a cluster of acidic residues (55). The DXXL motif is highly conserved throughout metazoan evolution (55). The DXXLL motif appears to code for incorporation into clathrin-coated vesicles at the TGN for proteins that cycle between the TGN and endosomes (55). Examples of proteins containing the DXXLL signal include the MPRs, LRP3, LRP10, and the Golgi-localized, Gamma-ear containing, Arf-binding (GGA) family of adaptor proteins (55). The GGA family of clathrin adaptor proteins has now been identified as the binding partner for DXXL motifs, and the GGAs share both structure and function with the other vesicle coat proteins discussed later in this chapter (see Vesicle Budding: Coats and Adaptors section) (83). Other Sorting Signals and Examples Although the cytosolic sorting signals discussed earlier are perhaps the best understood mechanistically and structurally, a number of other sorting signals have been identified as being important for protein sorting. One family of sorting motifs consists of clusters of acidic residues, which serve as sites for phosphorylation by casein kinase II (55). This signal is thought to regulate retrograde retrieval of proteins from the endosomal system to the TGN, which is complementary to the DXXL anterograde signal. Examples of proteins with this sorting signal are vesicle-associated membrane protein isoform 4 (VAMP-4), endotubin, and furin (55,84). Many of the MPR family members have a lysosomal avoidance signal that permits them to stay active through several cycles between the endosome and TGN (55). In addition to its well-characterized role in targeting proteins to the proteosome and lysosome for degradation, ubiquitination also has been shown to regulate the endocytosis of certain transmembrane proteins, including the growth hormone receptor, the EGFR, FcRγIIA, E-cadherin, and the epithelial sodium channel (ENaC) (55,85). Most of the characterized sorting signals mediating basolateral targeting of proteins correspond to cytosolic tyrosine and dileucine motifs described earlier. As mentioned previously, examples include the TfR and LDLR, which contain YXXϕ motifs, and E-cadherin and FcRII-B2, which contain the [DE]XXXL[LI] motif (55). Other basolateral proteins contain alternative sorting motifs and presumably interact with different sorting machinery. For example, transforming growth factor-α (TGF-α) contains neither tyrosine- nor

1602 / CHAPTER 62 TABLE 62-1. Tools for studying protein sorting in polarized epithelial cells Name

Cell-line origins

Species

Coat protein

Virus

Bud site

Influenza

Apical PM

Hemagglutinin (HA)

RSV

Apical PM

SV40

Apical PM

Protein G (RSV-G) SV40-CP

VSV

Basolatera l PM

Protein G (VSV-G)

Agent

Toxin

A: Cell lines and viruses Polarized cell lines MDCK

Renal tubule

Canine

LLC-PK1

Renal tubule

Porcine

Caco-2

Colonic adenocarcinoma

Human

HT-29

Colonic adenocarcinoma

Human

T84

Colonic adenocarcinoma

Human

SK-CO-15

Colonic adenocarcinoma

Human

WIF-B

Hepatomafibroblast fusion

Human/rat hybrid

HepG2

Hepatoblastoma

Human

Asymmetrically budding viruses

Toxins and chemical inhibitors Toxins Ricin

Shiga

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1603

Source

Target

Notable characteristics

High TEER, polarize in ~3 days, direct apical sorting predominant, well-characterized, single primary cilium, short apical microvilli Similar to MDCK, lack µ1B-subunit of AP-1B adaptor, resulting in mislocalization of basolateral markers Medium TEER, polarize in ~14 days, well-developed brush border in ~21 days, indirect (transcytotic) apical sorting predominant; most similar to absorptive enterocytes Similar properties to Caco-2, but more akin to intestinal goblet cells, rather than absorptive enterocytes; carbacholdependent mucin secretion; require glucose-free media to differentiate High TEER, polarize in ~5 days, indirect (transcytotic) apical sorting predominant; most similar to intestinal crypt cells High TEER; more similar to MDCK than Caco-2 cells; lack brush border and small intestine markers Well characterized, similar to primary hepatocytes; development proceeds through transitory simple epithelial cells stage resembling MDCK (~4 days) to hepatic morphology (~1–3 weeks) Well characterized, similar to primary hepatocytes and WIF-B

Castor bean

Ribosomes

Bacterial toxin

Ribosomes

General mechanism

References

58

59 60

61

62

63 64

65

Used extensively to study apical trafficking; HA now more commonly used as marker to eliminate cytopathic effects of viral infection Used to study apical trafficking through the ARE Used to study apical ER trafficking through the caveosome Used extensively to study basolateral trafficking; VSV-G now more commonly used as marker to eliminate cytopathic effects of viral infection

66

Used to study endocytic pathways; B-moiety binds cell-surface receptor and mediates transport; A-moiety inactivates ribosomes Used to study endocytic pathways; B-moiety binds cell-surface receptor and mediates transport; A-moiety inactivates ribosomes

70

68 67,69 66

70

Continued

1604 / CHAPTER 62 TABLE 62-1. Tools for studying protein sorting in polarized epithelial cells—cont’d Name

Cell-line origins

Species

Virus

Bud site

Coat protein

Agent

Toxin Cholera

Cytoskeletal disruptors 2,3-butanedione monoxime (BDM) Cytochalasin D Latrunculin A/B

Swinholide A Jasplakinolide Taxol Nocodazole Acrylamide

Other chemical disruptors Blebbistatin Brefeldin A

Wortmannin ARE, apical recycling endosome; cAMP, cyclic adenosine monophosphate; ER, endoplasmic reticulum; GEF, guanine nucleotide exchange factor; MDCK, Madin–Darby canine kidney; PI3K, phosphatidylinositol 3-kinase; PM, plasma membrane.

dileucine-based signal sorting motifs, but instead is trafficked to the basolateral membrane of MDCK cells via an interaction between eight juxtamembrane amino-acid residues on TGF-α and Naked 2 (NKD2), a receptor for Dishevelled (86). NKD2 is myristoylated at a specific glycine residue, and expression of a myristoylation-defective NKD2 is sufficient to block basolateral targeting of both NKD2 and TGF-α; basolateral targeting of both proteins is restored by silencing expression of this mutant NKD2 (86). Lumenal domains also have been identified as being critical for the basolateral targeting of certain proteins. The case of the prion precursor PrPc has already been presented, wherein PrPc is stabilized at the basolateral surface by binding to the laminin receptor and cell-surface glycosaminoglycans (50). Another example is the basolateral targeting of the GPP130 protein in MDCK cells, which requires a membrane-proximal lumenal domain (87). Thus, basolateral targeting is heavily reliant on the dileucine- and tyrosine-based sorting motifs, but

alternate sorting signals mediate basolateral targeting of some proteins. Elucidation of apical-specific signal sorting motifs has proved more difficult. Cytoplasmic sorting signals have been identified in several apical-targeted proteins. For example, a cytoplasmic 14-amino-acid sequence with a β-turn structure is required for apical membrane sorting of the ileal bile acid transporter (88). Rhodopsin contains a C-terminal sorting motif that interacts directly with the light chain of dynein, and this interaction is responsible for its apical sorting (89). Megalin, endotubin, and GLUT3 also contain specific apical cytoplasmic sorting motifs (90–93). Other apical proteins use cotransport mechanisms to arrive at the apical surface, including ErbB2 and the adenosine triphosphate (ATP)–binding cassette proteins ABCG5 and ABCG8 (94,95). Some apical proteins use specific N- or O-glycosylation signals for apical sorting (96,97). Examples include sucraseisomaltase, dipeptidyl peptidase IV, p75, ENaC, and some

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1605

Source

Target

Notable characteristics

Bacterial toxin

α-Subunit of Gs

Used to study endocytic pathways; B-moiety binds cell-surface receptor and mediates transport; A-moiety activates heterotrimeric Gs proteins, increasing cAMP

Synthetic

Actin

Fungal toxin

Actin

Marine Actin sponge toxin Marine Actin sponge toxin Marine Actin sponge toxin Pacific yew tree Microtubules Synthetic

Microtubules

Synthetic

Intermediate filaments

Synthetic Fungal toxin

Nonmuscle myosin II Arf1-GEFs

Bacterial toxin

PI3Ks

GPI-anchored proteins (34,35,98–101). Although the sorting machinery responsible for decoding glycosylation has yet to be fully characterized, it is thought to consist of specific lectin chaperone proteins such as vasoactive intestinal peptide 36 (VIP36) (102–104). The sorting of many apical proteins is thought to be dependent on segregation into specialized lipid subdomains called “rafts,” or associations with raft-associated proteins (105). The role of lipids and proteolipids in apical protein sorting are discussed later in this chapter.

Vesicle Budding: Coats and Adaptors Several mechanisms are involved in the budding of protein transport vesicles from membranes within the cell, some of which require classical coat proteins and specific fission factors and some that require neither. This section

General mechanism

References 70

Prevents Arp 2/3 binding to F-actin; prevents polymerization Binds both barbed and pointed ends of actin filaments; prevents polymerization Sequesters monomeric actin; prevents polymerization Stabilizes actin dimers and severs actin filaments; depolymerizes actin Stabilizes actin filaments; prevents depolymerization Stabilizes microtubules; prevents depolymerization Binds β-tubulin; depolymerizes microtubules Mechanism unknown; disassembles intermediate filaments

77 78 79 81 80 82 76 74,75

Selective inhibitor of NMMII

71

Loss of Arf1 activation results in loss of COPI and clathrinadaptor protein recruitment to membranes Nonselective inhibitor of PI3Ks

73

72

examines the mechanism for generating coated transport vesicles. Central to this process is the recruitment of a protein coat to the donor compartment membrane. These coat proteins aid in cargo recruitment, generation of membrane curvature, and in some cases, the vesicle scission process. There are at least three major classes of coated transport vesicles: COPI-coated vesicles, COPII-coated vesicles, and clathrin-coated vesicles. COPI-coated vesicles mediate intra-Golgi and retrograde Golgi to ER transport. COPIIcoated vesicles mediate anterograde ER to ERGIC and Golgi transport. Clathrin-coated vesicles mediate the later exocytic and protein recycling pathways. Although the classes of coat proteins differ in terms of their component proteins and their sites of action in the cell, they share many structural and mechanistic details that illustrate their common evolutionary origin and function. Because the vesicle budding machinery lies at the heart of protein sorting, the components and mechanisms are discussed here in detail.

1606 / CHAPTER 62 Coat-Based Budding Model Generation of protein transport vesicles is a highly coordinated process. Budding of a functional vesicle must fulfill at least three requirements (106). First, because vesicle budding at various membrane compartments in the cell involves different coat protein complexes, a given donor membrane must assemble the correct species of cytosolic coat proteins. Second, the budding process must incorporate the correct complement of proteins essential for trafficking and fusion of the vesicle at the acceptor membrane compartment. Third, the correct cargo proteins must be recruited to the vesicle budding site. In some cases, additional proteins may be required for scission of the budding vesicle from the donor compartment. A mechanistic model of vesicle budding has been proposed to explain how these requirements are met, and this section provides an overview of this model (106). Central to the model is the priming complex, an association of a primer protein, a small GTPase, and one or several subunits of the vesicle coat protein complex. The basic steps of coat formation are as follows. First, the primer protein in combination with a compartment-specific guanine nucleotide exchange factor (GEF) recruits a cytosolic, guanosine diphosphate (GDP)–bound GTPase to the donor membrane. The GEF catalyzes the exchange of GDP for guanosine triphosphate (GTP) on the GTPase, thereby activating the GTPase for further molecular interactions. Second, the primer protein in combination with the GTPase recruits additional coat protein components. The restricted overlapping localization of the primer protein and the activated GTPase ensures coat and compartment specificity. The primer protein could be the cargo protein itself or some functionally essential vesicle protein, such as a vesicular soluble N-ethylmaleimide–sensitive factor attachment protein receptor (v-SNARE). When the cargo protein is not itself the primer protein, it is recruited to the vesicle budding site via an association with some other member of the vesicle budding machinery. Cargo proteins and the priming complex serve as the nucleus for recruitment of additional priming complexes, coat proteins, and cargo. The GTPase is released from the priming complex on GTP hydrolysis, stimulated either by intrinsic GTPase activity or a GTPase-activating protein (GAP). The GDP-bound GTPase is then available for subsequent rounds of coat and cargo capture. The primer complex or cargo proteins may increase GTPase hydrolysis activity directly by interacting with the GTPase or indirectly by recruitment of or association with a GAP. Polymerization of the coat protein complexes either directly generates membrane curvature or aids in its generation by recruiting proteins which generate membrane curvature. Finally, membrane scission and vesicle release could be inherent to the mechanism of coat protein polymerization or could involve additional scission factors. The following sections provide a distillation of some of the key details of coated vesicle budding, as outlined in several excellent reviews (107–110). Coat Protein II–Mediated Budding The mechanism of vesicle budding from the ER is perhaps the best characterized of all vesicle budding reactions, and

therefore serves to illustrate the general mechanisms of vesicle budding. This section summarizes some of the key details in the field, and further details can be found in published reviews and the references therein (107,108). The Sec12 type II transmembrane protein is concentrated in specialized exit sites on the ER (transitional elements, tERs) through an association with the Sec16 scaffolding protein. Sec12 is a GEF for the Sar1 family of small Ras-like GTPases, and in concert with Sec16 serves to recruit Sar1-GDP to the tERs. Sec12 catalyzes the exchange of GDP for GTP on Sar1, activating the Sar1 GTPase and causing a conformational change of Sar1 leading to the insertion of its N-terminal α helix into the donor membrane. Sar1-GTP is now able to recruit the Sec23Sec24 subcomplex from the cytosol, which together with the Sar1-GTP constitute the “prebudding complex.” The Sec23 subunit associates directly with Sar1-GTP, whereas the Sec24 subunit participates in cargo recognition. X-ray crystallography has determined that a number of domains within the Sec23-Sec24 complex constitute a positively charged concave surface for membrane interaction and deformation. The membrane-associated prebudding complex then recruits the Sec13-Sec31 subcomplex, which is a flexible structure that polymerizes to form a meshlike scaffold on the outer surface of the prebudding complex. Although a mixture of Sar1-GTPγS, Sec23, Sec24, Sec13, and Sec31 are all that is needed to form COPII-coated vesicles from liposomes in vitro, cargo recruitment is likely to be physiologically essential for the budding reaction. Interactions between cytosolic signal sequences on the cargo protein and specific domains of the Sec24 subunit serve to stabilize the complex at the tER membrane. These signal sequences include diacidic motifs, short hydrophobic motifs, and dibasic motifs. In addition, three distinct signal sequence binding sites have been identified in Sec24: the A, B, and Arg342 binding sites. Clustering of coat proteins, particularly the Sec23-Sec24 subunit, at the bud site serves to deform the membrane resulting in the vesicle budding. No additional scission factors have been identified, leading to the conclusion that the COPII coat is itself sufficient to catalyze membrane scission. The Sec23 subunit serves as a GAP for Sar1, stimulating GTP hydrolysis and release of Sar1 from the membrane. The GAP activity of Sec23 is multiplied 10-fold by addition of the Sec13-Sec31 subunit, suggesting that GTP hydrolysis is coupled to formation of the full complement of coat proteins. Under the mechanism proposed earlier, Sar1-GDP then would be released to participate in further rounds of prebudding complex formation. This also provides a mechanism to ensure that nonproductive budding does not occur. In the absence of cargo protein, hydrolysis of GTP releases Sar1 and its associated coat proteins from the membrane, thus ending vesicle budding. Alternatively, the kinetics of GTP hydrolysis may be slower than the kinetics of vesicle budding, and GTP hydrolysis by Sar1 may function during vesicle uncoating. The uncoating mechanism is perhaps the least understood step in the process of COPII vesicle biogenesis, but it may involve additional regulatory factors not yet clearly defined.

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1607 Mechanisms involved in generating COPI-coated vesicles are remarkably similar to those of COPII vesicles. This section summarizes some of the key details in the field, and further details can be found in published reviews and the references therein (109,110). First, COPI coat formation begins with the recruitment and activation of Arf1 (ADP ribosylation factor), a Ras family small GTPase related to Sar1. Similar to Sar1-GTP, an N-terminal amphipathic helix on Arf1-GTP inserts into the membrane and may aid in generating membrane curvature during vesicle bud formation. Arf1-GTP simultaneously recruits a membrane-proximal F-subcomplex and a membrane-distal B-subcomplex, analogous to the Sec23-Sec24 and Sec13-Sec31 COPII subunits, respectively. The F-subcomplex consists of four proteins, β-, δ-, γ-, and ζ-COP, and the B-subcomplex consists of three proteins, α-, β′-, and ε-COP. Multiple signal sequence binding sites have been identified in both the B- and F-subcomplexes. An ArfGAP1 protein activates Arf1-GTP hydrolysis. Although some GTP hydrolysis may be involved in coat disassembly, evidence now supports the proposed mechanism outlined earlier. Inhibition of Arf1 GTPase activity does not result in coated vesicles that are unable to uncoat, but instead results in no vesicle formation (111). ArfGAP1induced hydrolysis promotes both cargo incorporation and formation of coated vesicles. These vesicles contain cargo protein, ArfGAP1, and coat proteins, but no Arf1 (111). This supports the model whereby GTPase activity releases the GDP-bound GTPase (e.g., Arf1-GDP) to participate in further rounds of coat and cargo recruitment (106). This mechanism also ensures that nonproductive budding does not occur, because in the absence of cargo protein, GTP hydrolysis releases Arf1 and the associated coat proteins from the membrane, thereby ending vesicle formation (112). A few mechanistic differences between COPII and COPI budding are worth noting (110). First, unlike Sar1, Arf1 is not specific for COPI assembly, but has multiple downstream effectors including clathrin adaptor proteins. Second, Arf1-GTP simultaneously recruits both the membraneproximal and -distal components of the coat, in contrast to the stepwise recruitment of components by Sar1-GTP. Third, Arf1 is regulated by several GAPs and GEFs, which are distributed on different membrane compartments. The subcellular localization of these GEFs and GAPs, like that of Sec12, dictates the sites of Arf1 activation and deactivation. Also notable is that many of the subunits of the COPI machinery share sequence homology with subunits of the clathrin adaptor protein complexes discussed earlier, indicating a common evolutionary origin. Clathrin-Mediated Budding Mechanisms involved in generating clathrin-coated vesicles are remarkably similar to those of the COP-coated vesicles. This section summarizes some of the key details in the field, and further details can be found in published reviews and the references therein (109,110). Similar to COPI-mediated vesicle budding, Arf family GTPases often are involved in recruiting membrane-proximal proteins from the cytosol. As in the other complexes, coat proteins have multiple signal

sequence binding sites for sequestration of cargo proteins. The membrane-proximal coat proteins are called cargo adaptor proteins, because they link the outer protein coat with the membrane and cargo proteins. The protein clathrin serves as the outer protein coat, a role analogous to the membrane-distal subcomplexes of the COP protein coats. Similar to COP-coated vesicle formation, coat proteins aid in the generation of highly curved membrane areas. In addition, similar structural motifs, for example, the β-propeller structure in clathrin, also are found in COP coat subunits. Indeed, sequence homologies exist between adaptor protein subunits and COPI subunits. Finally, coat polymerization and cargo protein recruitment are intrinsically linked and are interdependent. Significant differences between the clathrin-adaptor mechanisms of vesicle budding and the COP-mediated budding mechanisms should be noted, however (109). First, the membrane-proximal protein coat is not a single subcomplex of proteins. Instead, as would be expected for the multiple membrane compartmental sites of action, a number of adaptor protein complexes have evolved to share this function, thus ensuring compartmental and pathway specificity. Second, Arf-mediated recruitment of coat protein subunits is not the sole mechanism for assembling the membrane-proximal adaptor protein coat. Specific phosphoinositides (PIs) also serve this purpose. Also notable is that clathrin itself may not be necessary or involved in all vesicle budding reactions, but instead certain adaptor proteins appear to function without the outer clathrin coat. Clathrin coat assembly is also a more regulated process and involves several kinases, phosphatases, and accessory proteins. Vesicle uncoating is mediated by phosphorylation of adaptor subunits. The phosphatases involved are recruited to the vesicle by chaperone proteins such as Hsc70 and auxilin. Finally, accessory scission factors such as the dynamin family of GTPases are necessary for vesicle scission. The complexity of the vesicle budding apparatus and the adaptor protein complexes make an exhaustive review of every known component and mechanism beyond the scope of this chapter. Instead, the following section provides a more detailed examination of the original family of adaptor proteins (assembly polypeptides [APs]), because they are of central importance in protein sorting in polarized epithelial cells. In brief, however, the GGA family of adaptors and other alternative clathrin-coated vesicle adaptors have been the subject of several reviews (83,109,113). Many of the mechanisms of action of these alternative adaptors are proving to be similar to COP- and AP-mediated coated vesicle formation. Assembly Polypeptide Adaptor Protein Family The AP family of heterotetrameric adaptor protein complexes (114) consists of four members: AP-1, -2, -3, and -4 (56). In addition, AP-1 and -3 each have a tissue-specific isoform: AP-1b, an epithelial cell–specific isoform of AP-1, and AP-3b, a neuronal-specific isoform of AP-3. Although all

1608 / CHAPTER 62 four complexes evolved early (e.g., both plants and vertebrates express all four APs), many eukaryotes have lost one or more of the complexes (115,116). As mentioned previously, AP complexes bind to a variety of sorting signal sequences on cargo proteins, facilitating their incorporation into a budding vesicle at a donor membrane. The following section examines each of these complexes, their subunit components, and their roles in protein sorting and trafficking pathways (Fig. 62-3). The role of the AP-1a complex remains controversial. It is localized to both the TGN and endosomes and is implicated in both TGN-to-endosome traffic and retrograde endosometo-TGN traffic (56). Whereas Arf1 has been shown to recruit

AP-1a to membranes, AP-1 species also are capable of binding PI(4)P, a PI found primarily at the TGN (117). Loss of PI(4)P because of a knockout of the phosphoinositide kinase PI4KIIa causes AP-1 to become cytosolic (117). Clathrin binding, cargo protein binding, and vesicle uncoating are mediated by phosphorylation of specific AP proteins by protein phosphatase 2A (PP2A) (118). The AP-1a complex subunits are γ, β1, µ1A, and σ1 (109). The γ-subunit binds DFGxϕ and DFxDF motifs, and it is thought to function in AP-1a membrane binding via Arf1 and the recruitment of accessory factors to AP-1 complexes. The β1-subunit binds dileucine cargo motifs, [DE]xxxL[LI] and clathrin, and it is thought to function in membrane binding via Arf1, clathrin

AP-2 CL

AJC AP-1b CL AP-4

AEE

CE/SAC AP-3a CL

TGN

L

trans-Golgi medial-Golgi

BEE

AP-2 CL

cis-Golgi ERGIC ER

Nucleus

FIG. 62-3. Vesicle coat proteins and adaptors. Vesicle budding reactions depend on vesicle coat proteins and adaptor proteins both for deforming the membrane and sequestering cargo proteins. The assembly polypeptide (AP) family is one of the best-characterized families of adaptor coat proteins. AP-4 mediates budding of basolateral traffic from the Golgi. AP-1b mediates budding of basolateral traffic from the common endosome/subapical compartment (CE/SAC) and perhaps also the trans-Golgi network (TGN). AP-3a mediates traffic from the TGN or CE/SAC to the lysosome. AP-2 mediates endocytic traffic from both plasma membrane domains. AP-4 is the only family member that does not contain a binding motif for the coat protein clathrin (CL). AEE, apical early endosome; AJC, apical junctional complex; BEE, basolateral early endosome; ER, endoplasmic reticulum.

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1609 binding, accessory protein recruitment, and cargo protein recognition. The µ1A-subunit binds YXXϕ cargo motifs, and it is thought to function in cargo protein recognition and membrane interaction. The σ1-subunit binds [D/E]xxx[L/I] cargo motifs in complex with the µ1-subunit, and it is thought to function primarily in stabilizing the AP-1 core complex by mediating interactions between subunits. The AP-1b complex is an epithelial-specific isoform of the AP-1 complex (109). In the polarized epithelial cells in which it is expressed, AP-1b mediates a subset of basolateral membrane protein traffic either at the level of the endosome or TGN (56,119,120). AP-1b differs from the AP-1a complex in that an epithelial-specific homologue of the µ1-subunit, µ1B, takes the place of µ1A in the complex. The µ1B-subunit binds to a subset of Yxxϕ cargoes. The AP-1a and AP-1b complexes localize to the TGN and endosomes in Caco-2 and other polarized epithelial cells, but immunofluorescence analysis indicates that they do not have overlapping localization within a specific compartment, supporting the notion that they are involved in different sorting pathways (120). A recent reexamination of Rab8- and Cdc42mediated basolateral traffic indicates that these GTPases regulate basolateral traffic involving AP-1b (119). The AP-2 complex mediates endocytosis of membrane proteins at both the apical and the basolateral membranes (56,121). The AP+2 complex subunits are α, β2, µ2, and σ2 (109). AP-2 is unique from the other AP complexes in that Arf proteins do not assist in its recruitment to the membrane. Instead, phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for recruitment of AP-2 to the plasma membrane. Binding sites for PIP2 have been identified on both the α- and µ2-subunits of AP-2. A PIP phosphatase, synaptojanin, facilitates the release of AP-2 from the membrane by converting PIP2 to PI. The α-subunit binds DxF, FxDxF, and WVxF accessory protein signal peptide motifs, and it is thought to function in AP-2 membrane binding to PIP2 and in the recruitment of accessory factors to AP-2 complexes. The β2-subunit binds dileucine cargo motifs, [DE]xxxL[LI], and clathrin, and it is thought to function in clathrin binding, accessory protein recruitment, and cargo recognition. The µ2-subunit binds the YXXϕ cargo motif and the WVxF accessory protein signal peptide motif, and it is thought to function in cargo recognition, accessory protein interactions, and membrane interaction via PIP2. The σ1-subunit is thought to function primarily in stabilizing the AP-2 core complex by mediating interactions between subunits. The AP-3a complex is involved in sorting of membrane proteins to lysosomes and lysosome-related organelles (56). In nonpolarized cells, the AP-3a complex participates in the budding of basolateral proteins from the Golgi, although this remains to be confirmed in polarized cells (122). The AP-3a complex subunits are δ, β3A, µ3A, and σ3A (109). The δ-subunit is thought to function in the recruitment of accessory factors to AP-3 complexes. The β3A-subunit binds the [DE]xxxL[LI] dileucine cargo protein motif and clathrin, and it is thought to function in clathrin binding, accessory protein recruitment, and cargo recognition. The µ3A-subunit binds the YXXϕ

cargo motif, and it is thought to function in cargo protein recognition. The σ3A-subunit binds to [D/E]xxx[L/I] cargo motifs in complex with the µ3A-subunit, and it is thought to function primarily in stabilizing the AP-3 complex by mediating interactions between subunits. The AP-4 complex is ubiquitously expressed and mediates basolateral traffic from the TGN to endosomes or the basolateral plasma membrane (56,109). The AP-4 complex is unique in that clathrin-binding domains have not been identified, suggesting that AP-4–mediated budding is clathrin independent. The AP-4 complex subunits are ε, β4, µ4, and σ4 (109). The ε-subunit is thought to function in AP-4 membrane binding via Arf1 and in the recruitment of accessory factors to AP-4 complexes. The β4-subunit does not contain an obvious clathrin-binding domain. The σ4-subunit binds the YXXϕ cargo protein motif and other nonclassical cargo protein motifs, and it is thought to function in cargo protein recognition. The σ4-subunit is thought to function primarily in stabilizing the AP-4 complex by mediating interactions between subunits. In summary, the AP family of adaptor protein complexes functions in protein sorting during vesicle budding. Different AP family members regulate specific sorting pathways (see Fig. 62-3). The compartmental localization of GEFs and GAPs ensure the correct localization of Arf proteins and the recruitment of the correct AP complex to a specific membrane compartment. However, several lines of inquiry indicate that our current understanding of these vital components of the protein sorting machinery is still elementary. For example, it is now clear that Arf GTPases interact directly with and activate lipid-modifying enzymes, and that these lipid modifications may be more involved in AP recruitment than direct binding of AP proteins to Arf1 (123). Another member of the Arf family of GTPases, Arf-6, has been localized to the ARE and shown to mediate apical recycling traffic in several polarized cell types (124–127). In addition, 14 ArfGEFs and 16 ArfGAPs have been isolated thus far in mammalian cells, and their characterization is certain to offer new insights into the regulation of the protein sorting machinery (112,123). It is also clear that the vesicle budding process is a highly dynamic one, with adaptors and clathrin rapidly cycling on and off the membrane independently of each other (128). Finally, the isolation of “hybrid” clathrin-coated vesicles containing both GGA and AP adaptor protein complexes suggests further complications in our understanding of clathrin-coated vesicles and their mechanisms of protein sorting (129). Vesicle Scission The dynamins (1, 2, and 3) are part of a large superfamily of GTPases that function in membrane tubulation and scission (130). At least four splice variants of each of the three dynamins have been isolated in human tissue; for example, dynamin-2 exists as dynamin-2(aa), (ab), (ba), and (bb). Dynamin-2(bb) has been shown to mediate the release of specific apical but not basolateral traffic from the TGN in MDCK cells (131,132). A dominant-negative mutant of

1610 / CHAPTER 62 dynamin-2 (K44A) has been shown to inhibit endocytosis at both the apical and basolateral membranes in polarized MDCK cells (133). Dynamin also has been implicated in vesicle scission at the apical membrane in polarized acinar epithelial cells (134). In addition to clathrin-coated vesicle budding, dynamin is required for the budding of another vesicle type called caveolae (105). There are clathrin- and caveolae-independent vesicle budding mechanisms that do not use dynamin, including the COP-mediated vesicle budding reactions and pinocytosis (105,135). The serine-threonine kinase, protein kinase D (PKD), has been shown to be critical for organization of the TGN, Golgi, and protein sorting pathways. Overexpression of PKD or its pharmacologic activation causes a dramatic vesiculation of these structures, whereas blocking its kinase activity results in extensive tubulation because of a lack of membrane fission (136,137). PKD is recruited to the TGN by diacylglycerol (DAG), a signaling phospholipid (138). Although the precise mechanism of PKD-induced membrane fission remains unclear, it involves a coordinated cycle of PKD recruitment of membrane budding machinery and catalytic activation of downstream lipid-modifying enzymes, resulting in production of the fission-promoting lipid phosphatidic acid (139). PKD isoforms are necessary for vesicle scission and release of basolateral but not apical membrane proteins from the TGN in both polarized and nonpolarized cells (140,141). The small GTPase Cdc42 also mediates budding of basolateral but not apical traffic from the TGN in MDCK cells by stimulating actin polymerization and reorganization (142,143). Thus, similar to the adaptor proteins, vesicle scission factors differentially regulate budding in specific protein transport pathways.

Role of Lipids and Proteolipids in Protein Sorting and Transport Although the majority of research has focused on the roles specific proteins play in cellular physiology, the important roles played by lipids in processes such as protein sorting are becoming more apparent. Plasma membrane domains and intracellular membrane compartments are not of a uniform lipid composition. Indeed, even within a given area of membrane, the two leaflets of a membrane often are remarkably different in their composition. These differences are functionally significant in that the phospholipid composition affects membrane fluidity, the deformability and fissionability of the membrane, the dynamics of integral membrane diffusion or lipid-anchored proteins, and which membraneassociated proteins or enzymes will be recruited to a given membrane. The following section gives a brief overview of the roles of lipids and lipid-associated proteins in protein sorting and trafficking in polarized cells of the GI tract. Lipid Rafts and Proteolipids In vitro evidence indicates that lipids coexist in three different phases: the gel phase, the liquid ordered phase,

and the liquid disordered (fluid) phase (105). The gel phase is that phase in which lipids are relatively immobile; the fluid phase is the one in which lipids are highly mobile and loosely packed; and the liquid ordered phase is the intermediate phase in which the lipids exhibit high lateral mobility but also have some structure because of the packing of their fatty acids. These phases coexist in membranes in a temperature- and composition-dependent manner, which manifests as phase separation and membrane subdomain formation at physiologic temperatures because of lipid–lipid interactions of the fatty acid side chains (105). Biological membranes, however, do not exist in isolation from proteins, and proteolipid interactions influence the diffusion properties of a given membrane. Thus, certain proteins interact directly with specific lipid species, particularly cholesterol and sphingolipids, thereby creating a cluster or shell of specific lipids around the protein. These interactions are mediated by either the intrinsic lipid-binding properties of the transmembrane domain of the protein, lipid modifications of the protein (e.g., acyl chains of GPIanchored proteins or palmitoylated and myristoylated side chains of other proteins), or direct binding of certain membrane-associated proteins to specific phospholipids. In addition, protein–protein interactions can indirectly link or cluster proteins that do not interact with a given lipid species to those that do. This presents a model in which lipid–lipid, proteolipid, and protein–protein interactions all play a role in defining subdomains within a specific membrane (105). Cholesterol and sphingolipids are important for proteolipid interactions. Cholesterol is synthesized in the ER, whereas sphingomyelin and glycosphingolipids (GSLs) are synthesized in the Golgi. Cholesterol and sphingolipids are excluded from retrograde COPI-mediated Golgi/ER traffic, resulting in a progressively increasing concentration of these lipid species from the Golgi through the trafficking pathways to the plasma membrane (144). Cholesterol is a highly mobile lipid, the location of which within a membrane is largely determined by its high affinity for sphingolipids and saturated glycerophospholipids (145). These interactions dictate that as proteins and membranes pass through the exocytic pathway, increasing concentrations of cholesterol and sphingolipids can cause a coalescing effect at both the lipid–lipid and the proteolipid levels (144). Lipid rafts are best defined as liquid-ordered phase subdomains that are enriched with cholesterol, sphingolipids, and specific protein species, which, because of the lipid–lipid and proteolipid interactions, are not easily solubilized by nonionic detergents in the cold (105,144). These liquid-ordered phase rafts exist within the larger liquid-disordered fluid-phase membrane and can diffuse as a single unit within the larger membrane or can be anchored because of proteolipid or protein–protein interactions with the submembrane cytoskeleton. The apical membrane of polarized epithelial cells is highly enriched for GSLs and raft-associated proteins (144). Mutations that perturb the interaction of a given protein with lipid rafts can result in incorrectly sorting of proteins to the basolateral membrane (144). Conversely, protein

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1611 modifications that increase the interaction of a given protein with lipid rafts is sufficient in some cases to target basolateral proteins to the apical membrane (144). In addition, perturbing cholesterol biosynthesis results in a general decrease in apical traffic and incorrect sorting of some apical but not basolateral proteins (144). These results illustrate the importance of proteolipid interactions in the correct trafficking of many apical proteins (144). However, it is important to remember that protein–protein interactions play a role in clustering proteins within a membrane subdomain. Given that there are alternative protein sorting machinery and apical-specific signal sequences (see earlier), apical protein trafficking pathways are not reliant exclusively on proteolipid interactions between a given apical cargo protein and lipid subdomains (105,144,145). The MAL/BENE family of raft-associated proteolipids has been shown to be part of the sorting machinery for apical proteins, although the precise mechanisms for their action has yet to be understood (129,146–157). The annexins are a large evolutionarily-conserved multigene family that binds lipid rafts and negatively-charged phospholipids in a Ca2+dependant manner (158). Two family members, annexin II and annexin XIIIb, are necessary for the trafficking of some apical proteins (159–164). Similar to the MAL/BENE family of proteolipids, the precise mechanism by which annexins mediate apical traffic remains unclear, although evidence suggests they function as scaffolding proteins for recruiting apical cargo or stabilizing lipid rafts (158). Caveolae and the Caveolins Caveolae were first defined ultrastructurally in the early 1950s as flask-shaped invaginations of the plasma membrane (165). Similar to lipid rafts, caveolae are enriched for cholesterol, sphingolipids, and associated detergent-insoluble proteins (165). They can be defined as a subset of lipid rafts that function as mediators of membrane trafficking pathways, particularly endocytic pathways, that are clathrinindependent, dynamin-dependent, and sensitive to cholesterol depletion (165). A family of proteins, the caveolins, was subsequently localized to caveolae and shown to mediate their formation (166). Endocytosed caveolae traffic to a distinct caveolin-containing endosome, the caveosome (165,166). Whether a given protein traffics through the caveosome pathway or the AP-2/clathrin-mediated pathways impacts both the signaling characteristics of the protein and the membrane compartments to which it is ultimately delivered (165,166). For example, the apical monosialoganglioside GM1 serves as a receptor for the B-subunit of cholera toxin (CT), and the CT/GM1 complex is internalized by caveolae from which it is trafficked through the caveosome to the ER (167). Retrograde trafficking through the ER is a necessary step in toxin action (168). Cholesterol or GM1 depletion blocks caveolae-mediated internalization, resulting in uptake of the CT via clathrin-coated pits and the subsequent trafficking through the recycling endosomal system, where it is less able to elicit a functional response (168).

Other enterotoxins thought to share the caveolar pathway for trafficking to the ER include Shiga and the Escherichia coli heat-labile type I and type II toxins (168). In addition, a large number of viruses and bacteria exploit this alternate pathway to avoid being delivered to the lysosome (165). Several lines of evidence support a role for caveolae in endogenous protein sorting in polarized epithelial cells. In MDCK cells, the apical membrane contains homooligomers of caveolin-1, but does not contain caveolin-2 (169). The basolateral domain contains caveolin-1/2 heterooligomers and morphologically defined caveolae (169). Overexpression of caveolin-2 in MDCK cells results in an increased number of caveolae on the basolateral membrane, supporting a role for this protein in generating caveolae (170). In its homooligomeric state, caveolin-1 interacts with and serves as a scaffold for numerous lipid raft–localized proteins and the actin-binding protein filamin; it also stabilizes lipid rafts and negatively regulates endocytosis through caveolae (165). Anti–caveolin-1 antibodies selectively inhibit apical trafficking of hemagglutinin (HA), but not basolateral trafficking of Vesicular Stomatitis Virus G (VSV-G) protein in MDCK cells (169), perhaps because of the raft-stabilizing characteristics of HA acting at the TGN or Golgi (169). Paradoxically, ectopic expression of caveolin-1 and -2 in Caco-2 cells, which do not endogenously express caveolins, gave different results. Expression of caveolin-1 caused caveolae formation at the plasma membrane, whereas caveolin-2 was restricted to the Golgi and did not induce caveolae formation (171). However, ectopic expression of caveolin-2 increased recruitment of the apical protein, sucrose isomaltase, to lipid rafts, suggesting a function akin to caveolin-1 in MDCK cells in recruiting or stabilizing lipid raft–associated apical proteins (171). Caveolin-1 is expressed throughout the GI tract and is highly enriched in dynamic lipid-raft microdomains, called deep-apical tubules (DATs), in the apical brush-border region of enterocytes. DATs are 50 to 100 nm in diameter and project up to 1 µm into the cytoplasm (172). They contain markers for transcytosis, as well as resident brushborder enzymes, and function as a sorting hub in apical membrane trafficking (172). Biochemical and immunohistochemistry studies indicate that the vitamin D receptor colocalizes with caveolin-1 in apical caveolae in the intestine, although it remains to be tested whether this localization is functionally important (173,174). Caveolin-1 forms a complex with annexin-2 and is responsible for cholesterol uptake at the apical membrane from the intestinal lumen and its transport back to the ER, illustrating a role for caveolins in cholesterol trafficking in the intestine (163). The functional importance of caveolins in the GI tract is also suggested by the number of intestinal diseases that coincide with altered caveolin expression (175). For example, caveolin-2 is expressed on the apical surface of inflamed mucosa in patients with ulcerative colitis (UC), and caveolin-2 overexpression is increased in accordance with the disease activity of UC (176). Caveolin-1 levels are down-regulated in human colon tumors, and ectopic expression of caveolin-1 in colon

1612 / CHAPTER 62 carcinoma cell lines reduces cell tumorigenicity (177). Other studies found that caveolin-1 is overexpressed in human colon adenocarcinomas (178,179). It is now apparent that caveolin-1 expression is down-regulated in early stages of tumorigenesis, and then up-regulated in later stages (165). Clearly, our understanding of caveolins is incomplete, and elucidating their mechanisms and interacting proteins will aid our understanding of protein sorting and diseases of the GI tract. Phosphoinositides PIs belong to a class of phospholipids localized to the cytoplasmic face of cell membranes. They are derivatives of phosphatidylinositol that are phosphorylated at one or more positions of the inositol head group (180). PIs regulate membrane insertion and retrieval during exocytosis and endocytosis, respectively (180). As mentioned previously, the PKDmediated mechanism for budding of basolateral traffic from the TGN is intimately related to its stimulation of PI metabolism. Also, PIP2 is necessary for recruiting members of the AP-2 clathrin adaptor complex to the plasma membrane during endocytosis. Other proteins that are recruited by PIs to specific membrane compartments include Cdc42, dynamin, Arf1, and Arf6, all of which are participants in protein sorting and trafficking (180). The phosphoinositide 3-kinase (PI3K) Vps34p catalyzes the production of PI(3)P, and it has been shown to differentially regulate apical and basolateral membrane protein recycling in polarized hepatocytes and MDCK cells (181,182). Pharmacologic or immunologic blockade of PI(3)P production results in aberrant targeting of apical proteins from the plasma membrane to lysosomes, but not newly synthesized apical membrane proteins (181,182). These are just a few examples in which PIs can interact with the protein sorting and trafficking machinery. Phospholipids can be modified rapidly by multiple signaling pathways, and these proteolipid interactions enable rapid regulation of the protein trafficking pathways in response to a constantly changing environment.

Vesicle Motility: Role of the Cytoskeleton and Motors Vesicle transport in mammalian cells is both microtubule and actin dependent (183,184). For example, both actin and microtubules are required for postendocytic trafficking of the IgG receptor in MDCK cells (185). Evidence is accumulating for a two-stage process common to many cell types, with long-range transport carried out by microtubules and short-range transport at the cell cortex carried out by actin (186,187). As the sorting pathways have been uncovered at the molecular level, it has become apparent that the apical and basolateral trafficking pathways use different molecular motors, and this pathway-specific utilization of motor proteins aids in protein sorting (Fig. 62-4). Microtubule-Mediated Transport Early studies used microtubule depolymerizing drugs to indirectly examine the dependence of apical and basolateral

traffic on intact microtubule-mediated transport (see Table 62-1). Most studies concluded that apical traffic is more sensitive than basolateral traffic to microtubule disruption in polarized cells (15,188–190). Three-dimensional imaging of vesicle trafficking in live cells observed fusion of apical but not basolateral vesicles with the basal membrane in polarized cells treated with a microtubule depolymerizing drug (40). This also supports the notion that basolateral traffic is less sensitive to microtubule depolymerization. By comparison, neither basolateral nor apical vesicles normally fuse with the basal membrane in polarized cells, whereas both fuse with the basal membrane in nonpolarized cells (40). Others have observed sensitivity to microtubule disruption in the delivery of basolateral secretory, but not integral membrane proteins (191). Another study reported that both apical and basolateral membrane delivery from the TGN use microtubule-dependent transport, because the efficiency but not the fidelity of transport to both membrane domains was inhibited by microtubule depolymerization (192). All of these studies used toxins to disrupt the microtubule cytoskeleton. Unfortunately, pharmacologic destruction of the microtubule cytoskeleton affects not only exocytic trafficking, but also exerts profound changes on the entire organization and polarity of a cell. Confounding effects of these drugs may help to explain the conflicting results often noted in these early studies. Alternatively, because the studies often used different marker proteins, the disparate results may indicate that different proteins use separate transport pathways to arrive at the cell surface. Definitive support for microtubule-mediated transport awaited the characterization of microtubule motor proteins. Two large, evolutionarily distinct, motor superfamilies participate in microtubule-based vesicle trafficking: dyneins and kinesins (193). In isolated hepatocytes, cytoplasmic dynein staining exhibits a punctate vesicular pattern (194). Nocodazole treatment disrupts basolateral-to-apical transcytosis of a fluorescent bile salt derivative in these cells, indicating that microtubule-mediated transport is necessary for transcytosis and likely involves cytoplasmic dynein (194). Isolated zymogen granules from rat and chicken pancreas contain dynein heavy chain (DHC), dynein intermediate chain (DIC), and all of the components of the dynactin (dynein receptor) complex, but not kinesins, indicating that apical targeting of zymogen granules in the pancreas requires the dynein machinery (195). Similar results were found in polarized rabbit lacrimal acinar cells, with a stimulationdependent recruitment of dynein/dynactin to the Golgi, where it colocalized with apical vesicle fusion proteins (196). Overexpression of the p50/dynamitin subunit of dynactin inhibited this recruitment and blocked apical trafficking of these fusion proteins. The COOH-terminal tail of rhodopsin, a marker for apical traffic, binds directly to the 14-kDa dynein light chain (DLC) Tctex-1, but not to the Tctex-1 homologue RP3 (89). Tctex-1 and RP3 compete for binding to DIC, and overexpression of RP3 in MDCK cells disrupts rhodopsin trafficking to the apical membrane (89). Basolateral markers and other apical markers were not

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1613

2 AEE

1. Basolateral Kinesin I Kinesin II (KIF3A/B) Myosin Vc? Rab8 Rab13 Syntaxin 4 Mlgl SNAP23 TS-VAMP Exocyst PKD Cdc42

ARE 2

1 1

CE/SAC

BRE

2 BEE

1

2. Apical KIFC2/KIFC3 Dynein Myosin Ia Myosin Vb Myosin VI Rab11a Rab25 Rab3b? Syntaxin 3 TI-VAMP7 TI-VAMP8 Munc-18b Annexin II Annexin XIIIb MAL/BENE Lipid Rafts Glycosylation Dynamin II(bb)

TGN trans-Golgi medial-Golgi cis-Golgi ERGIC ER

Nucleus

FIG. 62-4. Protein sorting machinery in the apical and basolateral pathways. In addition to the direct sorting interactions of the vesicle coat proteins, polarized epithelial cells use pathway-specific motors, Rabs, soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), tethers, and fission factors, as listed on the left, to aid in protein sorting. AEE, apical early endosome; AJC, apical junctional complex; BEE, basolateral early endosome; BRE, basolateral recycling endosome; CE/SAC, common endosome/subapical compartment; ER, endoplasmic reticulum; PKD, protein kinase D; SNAP-23, synaptosomal-associated protein of 23 kDa; TGN, trans-Golgi network; TI-VAMP, toxin-insensitive vesicle-associated membrane protein isoform.

affected by this loss of Tctex-1 function (89). Thus, only a subset of apical markers uses this variant of the DLC. Immunodepletion of DIC or UV-vanadate depletion of DHC both inhibit apical trafficking of HA, whereas not affecting basolateral trafficking of VSV-G in MDCK cells (197). In summary, data from a diversity of cell types and proteins indicate that apical but not basolateral protein traffic is partially dependent on dynein-mediated transport. Immunodepletion of conventional kinesin inhibits both apical and basolateral transport in MDCK cells, supporting the notion that both pathways use microtubule-based transport (197). A nonconventional kinesin was shown to be necessary for trafficking of some apical but not basolateral proteins along microtubules (131). KIFC3 associates with lipid rafts, purified vesicles, and the apical protein annexin

XIIIb, and it is localized on vesicles immediately below the apical membrane in renal tubule epithelia and MDCK cells (198). Overexpression of a dominant-negative KIFC3 blocks trafficking of annexin XIIIb and HA to the apical surface (198). KIFC3 is also necessary for the proper positioning of Golgi in adrenocortical cells (199). The same study found that overexpression of dynamitin (akin to knockdown of dynein) had additive inhibitory effects in a KIFC3−/− background, suggesting the proteins may act in concert in the apical pathway. Reconstituted microtubule-mediated vesicle traffic using fluorescently labeled basolateral endocytic vesicles from rat liver showed that the vesicles exhibited microtubule (+)-end–directed and (−)-end–directed motility and fission, each of which could be selectively blocked pharmacologically (200); KIFC2 was subsequently

1614 / CHAPTER 62 identified as the microtubule (−)-end–directed motor, indicating that this motor may be involved in transcytotic apical trafficking pathways (201). In isolated endosomal populations purified from rat livers, early endosomes were found to contain KIFC2, kinesin I, and rab4, whereas LEs contained the kinesin II subunit KIF3A, rab7, dynein, dynactin, and dynamitin, implicating both kinesins I and II in microtubule (+)-end–directed (basolateral) motility (202). Kinesin II is a microtubule (+)end–directed motor that exists as a dimer of any two out of three possible subunits: KIF3A, KIF3B, and KIF3C. A kidney-specific knockout of the KIF3A subunit of kinesin II disrupted basolateral sorting of the EGFR but not the AQP2 channel in renal tubule cells, and it did not affect trafficking of apical markers (203). This suggests that kinesin II may function selectively in the basolateral trafficking of some proteins. Together, these data suggest that apical, basolateral, and transcytotic pathways in polarized epithelial cells use microtubule-based transport, that pathway-specific motors may aid in protein sorting, and that individual proteins in a given pathway may use separate microtubule motors (see Fig. 62-4). Actin-Mediated Transport Studies of actin-mediated transport have also used depolymerizing toxins to disrupt the actin cytoskeleton (see Table 62-1). Actin-based traffic pathways in polarized epithelial cells often proved remarkably resistant to these toxins with often conflicting conclusions (204,205). For example, one group demonstrated a dependence on actinmediated transport of basolateral trafficking in MDCK cells (206). Another group found that some apical proteins require actin-mediated transport, whereas other apical proteins did not, but instead were targeted directly to the apical membrane via microtubule-mediated transport (39). This alternate pathway may explain why perturbations of actinmediated transport have had such mild effects on apical trafficking in previous studies. Again, however, pharmacologic destruction of the actin cytoskeleton likely has multiple effects within the cell that may confound interpretation of results from these types of experiments. Stronger evidence for actin-mediated transport came with the identification of specific myosin motors that were restricted to the separate vesicle transport pathways. The isoform of “conventional” myosin found in epithelial cells, nonmuscle myosin II, does not appear to be involved in vesicle transport, but has instead a well-delineated role in contraction of the apical actin ring (207). Several unconventional myosins, in contrast, have been implicated in protein sorting and trafficking in polarized epithelial cells, including myosin VI, myosin Ia, and the myosin V family. Myosin VI assists in apical endocytosis at the intestinal brush border (208). Myosin Ia is an apical-specific motor localized to the subapical and brush-border regions that may play a role in a direct TGN to apical membrane transport pathway that bypasses the endosomal system (209,210). Type V myosins

are one of the best-characterized myosin families (211). Myosin Vb is restricted to the ARE and CE in polarized epithelial cells, where it has been shown to interact with the Rab11 family of small GTPases and to mediate vesicle trafficking to the apical membrane (212). The newly identified myosin Vc is present in polarized epithelial cells (213). Overexpression of the tail of myosin Vc in HeLa cells demonstrated an interaction with Rab8, the basolateralspecific Rab present in polarized epithelial cells, indicating that this motor may function in the basolateral pathway (213). Together, these data indicate that both apical and basolateral pathways in polarized epithelial cells use actinmediated forms of transport, and pathway-specific myosins may aid in protein sorting (see Fig. 62-4). Intermediate Filaments The role of CKs in organization of the apical pole is well documented. For example, CK8 null mice experience colitis, hyperplasia, diarrhea, and hepatic insufficiency (214,215). Colonic mistargeting of the apical Na-transporter, ENaC-γ, has been shown to be responsible for the diarrhea phenotype (214). Villous enterocytes in these mice lack apical CKs and lose apical expression of syntaxin-3, alkaline phosphatase, sucrose isomaltase, and cystic fibrosis transmembrane conductance regulator, whereas hepatocytes mistarget the apical marker HA4 to the basolateral membrane (215). Targeted nucleotide knockdown of CK19 in Caco-2 cells is sufficient to disorganize apical microvilli and microtubules and depolarize two apical proteins, while not affecting the basolateral polarity of the Na,K-ATPase (216). Forskolin treatment of Caco-2 cells quantitatively and qualitatively alters the phosphorylation of CK8, CK18, and CK19, resulting in their depolymerization and in morphologic changes such as shortening of the microvilli and decreased apical expression of specific hydrolases (217). Finally, cyclin B/p34cdc2(cdk1)-mediated phosphorylation of a single unidentified protein of a 160- to 220-kDa molecular mass present in γ-tubulin/CK19 multiprotein complexes is sufficient to release CK19 from the complex and causes the release of γ-tubulin and microtubules from apical IFs in permeabilized Caco-2 cells (218). These and other examples are consistent with the role IFs play in microtubule orientation and apical pole organization. Indirect evidence suggests that IFs may play more direct roles in protein sorting and trafficking as cytoplasmic scaffolds for organizing the protein sorting and transport machinery and as adaptor proteins on vesicles. Cytoplasmic IFs function as dynamic and multipurpose scaffolds for an ever-growing list of proteins in a wide variety of cell types (219). For example, vimentin IFs directly bind and serve as a reservoir for synaptosomal-associated protein of 23 kDa (SNAP-23) in fibroblasts (220). This is perhaps unsurprising, because vimentin has long been known to bind directly to ribosomes (221–223) and nucleic acids (224), and SNAP-23 is synthesized in the cytosol and recruited to membranes after translation (225). In addition, the motor proteins myosin,

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1615 kinesin, and dynein have been shown to bind vimentin, keratins, and neurofilaments, and to transport these IFs along actin and microtubule tracts (226,227). Myosin Va binding to neurofilaments is essential to establish the normal distribution, axonal transport, and axonal levels of myosin Va, as well as the proper numbers of neurofilaments in axons (228). Myosin V is in the terminal web of the intestinal brush border (229). Intriguingly, myosin V is remarkably resistant to extraction by high salt concentrations and ATP compared with myosins I, II, and VI, and detergent, in addition to ATP, was required for efficient solubilization of myosin V (229). This is consistent with the hypothesis that myosin V binds to IFs in the subapical region, rendering it more resistant to extraction. Myosin V is localized to a pericanalicular “ring” in WIF-B polarized hepatocytes, and this ring is sandwiched between the subapical actin layer and the ringlike zone of microtubule endings (230). This is consistent with the possibility that subapical CKs function to localize exocytic motors, and these motors may provide a direct link between the actin and IF cytoskeleton. Cytoskeleton-enriched canalicular membranes have been isolated that consist of the apical bile canaliculus compartment complete with membrane-attached pericanalicular actin filaments and the surrounding CK sheath (231). On stimulation with a “contraction solution,” the CK sheath remains unchanged, whereas actin and myosin II formed aggregates that appear to be attached to the CK filaments. This suggests that the IFs in the outer sheath (terminal web) stabilize the bile canaliculus and help to maintain its integrity during contraction, as suggested previously (232). Finally, an entire new family of myosin heavy-chain isoforms has been identified that colocalize with IFs and copurify with CKs (233). A role for IFs as adaptor proteins on vesicles is even more circumstantial. Isolated vimentin IFs have been found in association with vesicles (234). Moreover, purified vimentin filaments bind laterally to lipid vesicles, and cholesterol, cholesterol fatty acid esters, monoglycerides, diglycerides, and triglycerides efficiently bind to vimentin, and preferentially to vimentin filaments (235). When cholesterol was incorporated into phospholipid vesicles, the affinity of the liposomes for vimentin filaments increased considerably (235). The carbohydrate moiety of GSLs is localized in a punctate staining along vimentin IF networks in a variety of cell types, including MDCK cells (236). GSLs are concentrated in the apical plasma membrane of polarized epithelial cells and are one of the primary components of lipid rafts, which are thought to be essential for the transport of certain proteins to the apical surface (see earlier). Significantly, surface expression of GSLs is reduced in vimentin null cells (237), indicating that vimentin contributes to intracellular transport of GSLs and sphingoid bases perhaps by organizing subcellular organelles or by binding to proteins that participate in the transport process (238). In a primary culture of renal proximal tubule cells from vimentin-null mice, the Na-glucose cotransporter, SGLT1, was no longer incorporated into lipid rafts and was no longer functional in

the cells (239). Furthermore, vimentin has been coimmunoprecipitated with caveolin; vimentin and caveolin still cosedimented when lipid rafts were subjected to two sequential sucrose gradients of different density, and vimentin disappeared from lipid rafts after treatment of the cells with methyl-β-cyclodextrin, an agent known to disrupt lipid rafts (239). Another group analyzed the protein content of purified vesicles from insulin-treated 3T3-L1 adipocytes using mass spectrometry (240). In addition to the expected vesicle components, such as fusion proteins and the GLUT-4 channels, the authors also identified vimentin as being a vesicleassociated protein. The authors then confirmed the localization of vimentin on gradient-purified vesicles using immunoelectron microscopy. In combination with the aforementioned data indicating that vimentin directly associates with all three classes of motors and is transported on actin and microtubule networks, these results suggest that vimentin may function as an adaptor protein on cholesterol-rich vesicles. Because vimentin expression is limited to a subset of intestinal epithelial cells (241,242), perhaps CKs play a similar role as vesicle adaptors in the apical transport of lipid raft–containing vesicles and associated proteins. CKs exhibit both slow and bidirectional fast movements (226,243,244). Although intestinalspecific CKs remain to be tested in this regard, anomalous punctate staining has been observed with a CK19 monoclonal antibody (217).

Regulators of Compartmental Identity: The Rab GTPases The Rab family of prenylated GTPases regulates exocytosis at every stage of the trafficking process: from budding, to motor-driven transport, to tethering and fusion at the acceptor membrane (245). The basic Rab cycle is as follows: (1) Cytosolic pools of Rab-GDP are bound to GDP dissociation inhibitor (GDI) proteins that sequester the prenylated domain of the Rab, keeping the Rab in the cytosol; (2) GDI displacement factor (GDF) proteins on target membranes dissociate the GDP-bound Rab from GDI, which enables the Rab to bind to a target membrane; (3) the Rab encounters a GEF on the target membrane, which activates the Rab by promoting an exchange of GTP for GDP; (4) the activated Rab now binds to its downstream effectors and is stabilized on the membrane; (5) through either intrinsic activity or driven by an association with a GAP, the Rab hydrolyzes GTP to GDP; and (6) the GDP-bound form of the Rab is released from the membrane by GDI, which preferentially binds to GDP-bound Rab. This mechanism ensures that a specific Rab activity is localized to a subset of membranes, because both the effector molecule and the GEF must be present to stabilize the recruitment of the Rab to those membranes. A general picture is beginning to emerge in which Rab proteins mediate the interactions of specific motors, adaptors, tethers, and fusion proteins, thus serving a central role in protein sorting and traffic (246,247). This is perhaps

1616 / CHAPTER 62 best understood in budding yeast, in which the homologue of mammalian Rab proteins, Sec4p, is located on exocytic vesicles and regulates myosin (Myo2p)-driven transport along actin cables to the bud tip (248). A Sec4p-specific GEF and GAP regulate Sec4p activity (248). Sec4p on the vesicle also interacts with vesicle fusion proteins and recruits members of the exocyst complex, a protein complex thought to function in targeting and tethering exocytic vesicles at the cell membrane (248). Rab8 functions in basolateral trafficking and in cortical actin reorganization during exocytosis (249–251). Rab8 predominantly localizes to recycling endosomes (i.e., the CE/SAC) and affects only the basolateral traffic that uses the AP-1B adapter (119). The GEF, GAP, and GDI proteins that act on Rab8 have all been characterized, whereas the GDF for Rab8 either remains to be identified or perhaps is simply the various vesicle cargo proteins themselves (252–254). Rab8 may regulate actin-mediated trafficking through the mammalian Myo2p homologue, myosin Vc, although this interaction remains to be fully explored (213). It is unknown whether Rab8 recruits exocyst components to exocytic vesicles as reported for Sec4p in yeast. Additional regulators and downstream effectors have been identified for Rab8, but the precise roles this protein serves in basolateral exocytosis await further characterization (255,256). Another member of the Rab8 family, Rab13, is localized to the tight junction and exocytic vesicles, and it has been implicated in the regulation of basolateral protein trafficking and tight junction assembly (4,5). Rab3b is rapidly recruited to the apical pole on cell contact in polarized epithelial cells, and it has been hypothesized to be a regulator of apical or junctional protein traffic (257). The Rab11 family members Rab11a and Rab25 both function in apical trafficking through the ARE, although only Rab11a is thought to regulate traffic at the CE/SAC (258,259). Rab11a interacts with the myosin Vb motor in vesicle recycling to the apical membrane (260,261). Together, these data indicate that both apical and basolateral pathways in polarized epithelial cells are regulated by Rab GTPases, and pathwayspecific Rab proteins aid in protein sorting (see Fig. 62-4).

Vesicle Fusion: The SNAREs The soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) are thought to constitute the minimal molecular machinery necessary for membrane fusion (262). The general mechanism for their function is as follows: a target membrane SNARE (t-SNARE) from the syntaxin family of proteins is bound in cis with a t-SNARE from the SNAP-23/25 family proteins on the acceptor membrane; this heterodimeric complex binds in trans a vesicle SNARE (v-SNARE) from the VAMP family proteins to form a fusion complex (263). The original SNARE hypothesis postulated that the binding properties between the v-SNARE and its cognate t-SNAREs confer specificity of fusion between donor and acceptor membrane compartments (264). Thus, it was thought that the restriction of the each SNARE protein to

specific donor and acceptor membranes defined the membrane as belonging to given transport pathway. In most polarized epithelial cells including those of the intestine, syntaxin-3 is localized to the apical membrane, whereas syntaxin-4 is localized to the basolateral membrane (265–267). The t-SNARE, SNAP-23, complexes with both syntaxin-3 and -4 and localizes to both membrane domains (265,266,268). Cleavage of v-SNAREs with specific neurotoxins preferentially blocks basolateral traffic, suggesting at first that SNARE function was not essential for apical traffic (269). The subsequent demonstration that toxin-insensitive VAMP-7 mediates TGN-to-apical membrane traffic, whereas a toxin-sensitive VAMP mediates basolateral traffic, resolved this enigma (270). An additional toxin-insensitive VAMP homologue, VAMP-8/ endobrevin, has been shown to selectively cycle between the apical membrane and the recycling endosome, and it is thought to mediate this trafficking pathway (270,271). Additional SNAREs that may participate in protein trafficking in polarized epithelial cells include syntaxin-13, which is thought to mediate trafficking through early endosomes, syntaxin-7, which is thought to mediate traffic through the LE-lysosome pathway, and syntaxin-2a, which is localized to the apical membrane (271–273). Syntaxin-1a and -2b are localized to both membranes, and thus are less likely to participate in apical- or basolateral-specific trafficking pathways (274). How the t-SNAREs are themselves restricted to the separate membrane domains in polarized cells remains unclear. The transmembrane domain of the syntaxins may be important for their targeting to the plasma membrane (275). Syntaxin-4 is in a complex with the mammalian homologue of lethal giant larvae, Mlgl, at the lateral membrane, which is thought to stabilize syntaxin-4 at that site (276). Phosphorylation within a highly conserved region of Mlgl is necessary for its restriction to the lateral domain, and mutations in this domain lead to a loss of cell polarity and formation of epithelial cell–derived tumors (277). Mlgl is rapidly recruited to sites of cell–cell adhesion during cell polarization, where it interacts with multiple partners of the polarity signaling machinery (278). Thus, Mlgl may serve to link the cell polarity signaling machinery to the protein sorting and targeting machinery. On the apical membrane, both syntaxin-3 and VAMP-7 have been shown to be associated with lipid rafts (279). Interestingly, syntaxin-1a, which is found at both apical and basolateral membranes, is also associated with lipid rafts (274). However, syntaxin-1a, like syntaxin-4, contains five C-terminal tyrosine residues, one of which is not present in syntaxin-3 (274). This fifth tyrosine residue may be responsible for the codominant cell-surface expression of syntaxin-1a, and lack of it may help to explain syntaxin-3 expression solely at the apical membrane. A role for glycosylation of syntaxin-3 in apical sorting also has been suggested. Addition of either the tail domain of syntaxin-3 or an N-glycosylation signal to a basolateral transmembrane protein is sufficient to target it to the apical surface (280). GalNAcα-O-bn, a sialylation inhibitor, blocked the transport of syntaxin-3, annexin XIIIb, and the apical marker dipeptidyl peptidase IV to the apical surface in polarized enterocyte-like HT-29 cells (281), and the

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1617 proteins accumulated in the cytosol in vesicle-like puncta, indicating a block of either vesicle transport or docking/fusion at the acceptor membrane. Finally, VAMP-7 shares homology in its N-terminal sequence with the intestinal-specific apical protein annexin XIIIb, and similar to annexin XIIIb, associates with lipid rafts, suggesting a shared mechanism for apical targeting (270). Whether basolateral-specific v-SNAREs are competent to promote fusion with the apical t-SNAREs has not been reported. A combination of microtubule depolymerization, a situation that normally promotes basolateral fusion of apical vesicles, and addition of syntaxin-3–blocking antibodies demonstrated that apical vesicles do not fuse with the basolateral membrane in the absence of functional syntaxin-3 (40). These data support the hypothesis that SNARE-pairing confers specificity to protein trafficking pathways (see Fig. 62-4). However, basolateral traffic has been shown to occur specifically at the upper region of the lateral membrane, whereas syntaxin-4 localizes along the entire length of the lateral membrane (40). This suggests that some other factor or mechanism is necessary to direct the vesicle traffic to specialized sites of polarized membrane addition. Thus, although SNAREs may be necessary for conferring fusion competence in a given pathway, they are insufficient to explain site specificity of basolateral exocytosis. Multiple mechanisms and effector molecules, including the SNAREs and their regulatory proteins, are likely to play complementary roles in specifying the site of vesicle fusion at the plasma membrane (282). An inhibitory class of SNARE proteins, the i-SNAREs, prevent incorrect pairing and membrane fusion reactions (283). Munc-18-2/Munc-18b is an epithelial cell-specific homologue of the yeast Sec1 protein that is localized to the apical membrane in the intestine and other simple epithelial cells (284,285). Munc-18b is capable of binding syntaxin-1a, -2, and -3, but not syntaxin-4 (285,286). Overexpression of wild-type Munc-18b inhibits apical but not basolateral traffic (284). Interestingly, overexpression of a mutant Munc18b that can bind syntaxin-1a and -2, but not syntaxin-3, partially inhibited HA traffic to the apical surface of MDCK cells, but not Caco-2 cells (287). This confirmed earlier reports that Caco-2 cells, and presumably enterocytes, have little endogenous syntaxin-2, and apical traffic is predominantly mediated by syntaxin-3 (287). By contrast, it would appear that syntaxin-1a or -2 mediates apical traffic to some degree in MDCKII cells. The following section examines another putative SNARE-interacting protein complex, the exocyst. Further characterization of the SNAREs and SNARE-interacting proteins will aid our understanding of the protein sorting and trafficking pathways in polarized epithelial cells.

Membrane Hot Spots: The Spatial Regulation of Exocytosis Exocytic trafficking to the apical and basolateral domains is not uniformly spread over the entire surface of these

domains, but is instead restricted to subdomains. It was first noted about a quarter century ago that the apicolateral junction is a “hot spot” for integral membrane protein recycling in polarized epithelial cells (288). This is only one of the many examples of spatial regulation of exocytosis in epithelial cells. Other examples include targeted exocytosis during filopodial extension and targeted exocytosis during polarization in epithelial cells, where essentially all of the exocytic traffic is directed to sites of cell–cell adhesion during lateral membrane growth. Although studies from yeast, plants, worms, flies, and a number of mammalian cell systems have elucidated much of the molecular machinery involved in exocytosis, less is known about the spatial regulation of exocytosis (248,289–294). More recently, the molecular machinery responsible for targeted exocytosis has begun to be delineated (294). Remarkably, essentially identical machinery is found to function in polarized membrane addition in mammalian cells and budding yeast, illustrating a conserved primordial pathway for spatial regulation of exocytosis (6). A model is emerging for targeted secretion in which each cell type has adapted an evolutionarily conserved core of molecular players and mechanisms, including localized assembly of signaling complexes, cytoskeleton reorganization and recruitment, mobilization of further targeting components from intracellular pools, and finally targeted vesicle delivery to sites of membrane growth (6). The following sections highlight a few of the key players and mechanisms identified in yeast systems, as well as their putative homologous roles in targeted vesicle delivery in polarized epithelial cells. Actin Cables and the Formins The formins are an evolutionarily conserved family of multidomain scaffolding proteins that function in remodeling of the cytoskeleton (295). One of their most remarkable functions is the nucleation and polymerization of unbranched linear actin filaments called actin cables. Polarized exocytosis in yeast requires actin cables (296). Cable synthesis and assembly at the yeast bud tip are under the control of the formin family of proteins, the localization and function of which are, in turn, under the control of the Cdc42 and Rho family GTPases (297,298). In addition to serving as tracts for the recruitment of exocytic vesicles, the actin cables also serve as tracks for the recruitment of the microtubule (+)-ends (6). An intriguing area of inquiry will be to determine whether actin cables function similarly in polarized exocytic trafficking or microtubule recruitment during or after development of polarity in epithelial cells. E-cadherin is thought to regulate actin through the catenin family of proteins (α- and β-catenin) (299). In addition, E-cadherin–mediated cell adhesion is known to activate GTPases and signaling pathways responsible for regulating actin polymerization (299). α-Catenin recruits mammalian formin-1 to the adherens junction at sites of cell adhesion in mammalian keratinocytes (300). Akin to its role in yeast, mammalian formin-1 has been

1618 / CHAPTER 62 shown to polymerize radial actin cables at sites of cell–cell contact in mouse primary keratinocytes, which stabilize the adherens junction (300). It has long been recognized that cell polarization induced by E-cadherin–mediated adhesion results in the formation of a radial actin ring at the apicolateral boundary. Whether this ring is composed of forminmediated actin cables, and whether these cables serve as tracts for protein traffic, remains to be determined. The Exocyst The exocyst complex is among a growing family of heterologous proteins or protein complexes that are thought to function in membrane targeting, tethering, and SNARE protein regulation (301–303). Ultrastructurally, the only commonality in these complexes is that they are either elongated coil–coil proteins or multisubunit complexes (304). In addition to their known interactions with the SNARE family of proteins, many of the components of these complexes are cytosolic proteins that are recruited to specific membranes by compartment-specific Rab GTPases (305). An emerging theme is localization of part of the multisubunit complex to the donor membrane, whereas the remainder of the complex (the “landmark”) is localized at the acceptor membrane. The exocyst complex was first isolated in budding yeast, where it was shown to be necessary for polarized vesicle exocytosis (306). Mutants in any of the eight subunits of the complex (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p) resulted in the accumulation of vesicles under the membrane of the daughter bud (306). A similar phenotype is seen in mutants of Sec4p (the yeast homologue of mammalian Rab GTPases), and components of the exocyst complex are recruited to the vesicle through Sec4p (307,308). Other components of the exocyst complex are recruited to the site of exocytosis at the bud tip, where they interact with additional “landmark” proteins responsible for specifying the site for membrane addition, including the small GTPases, Rho1, and Cdc42p (309–312). In MDCK cells, cytosolic Sec6 and Sec8 were shown to be recruited rapidly to contacting lateral membranes on initiation of Ca2+-dependent, E-cadherin–mediated, cell–cell adhesion (313). Ca2+ depletion results in a loss of Sec6 and Sec8 from the lateral membranes (313). On full polarization of cells, Sec6 and Sec8 were predominantly localized to the apex of the lateral membrane region near the apical junctional complex, the site of polarized exocytosis (313). Function blocking anti-Sec8 antibodies selectively blocked vesicle trafficking to the basolateral membrane, but not to the apical membrane (313). Data suggest that the localization of the exocyst complex in polarized cells is caused by interactions with components of the apical junctional complex (3). Although the full complement of binding partners of exocyst components remains to be determined, several additional binding partners have now been identified in mammalian systems. A well-characterized functional interaction of exocyst components in mammalian systems is that among the small GTPase, RalA, and exocyst components

Sec5 and Exo84 (314–316). Disruption of this interaction using dominant-negative RalA caused the mistargeting of basolateral proteins to the apical plasma membrane, although this did not noticeably affect apical protein traffic (317). This supports a role for the exocyst in selective basolateral trafficking. Because Sec5 is localized to target membranes, whereas Exo84 is localized to donor membranes, RalA is thought to mediate the assembly of donor and target membrane exocyst components into a functional complex (315). Finally, the exocyst is likely to serve additional functions in mammalian systems, with putative roles identified in mediating microtubule dynamics and in protein sorting or biosynthetic processes at the TGN and ER (318–322). Accumulating evidence for an interaction between the exocyst and the ER translocon has led some to suggest a common regulatory circuit for protein synthesis and exocytosis (323). Septins The septins are a family of GTPase filament-forming proteins that are localized to sites of polarized membrane addition in yeast (324,325). Mutants in these proteins show defects in polarized exocytosis and cytokinesis (326). Septin localization and activation in yeast require Iqg1p and Cdc42p, respectively, two proteins with mammalian homologues (IQGAP and Cdc42) that are known to be rapidly recruited and activated at sites of E-cadherin–mediated adhesion (310,327,328). In mammalian cells, septins colocalize with both the actin and microtubule cytoskeletons and cell membranes, and they are thought to serve multiple functions (329). The septin protein Nedd5/Septin-2 colocalizes with actin, microtubules, and exocyst components in a variety of cell types (330,331). In PC12 cells, a model for neuroendocrine secretion, Nedd5/Septin-2, redistributes from the microtubule organizing center to the periphery on differentiation in a manner identical to the redistribution of exocyst components (322). Thus, septin filaments may function in concert with the exocyst and other downstream effectors of E-cadherin–mediated adhesion to specify the site for spatially regulated exocytosis. This hypothesis remains to be fully explored in polarized epithelial cells.

CONCLUSION Cell polarity and the asymmetric localization of plasma membrane proteins are vital features of epithelial cells throughout the GI tract in their role as interfaces between the body and the outside world. Intracellular protein sorting is necessary for the establishment and maintenance of this cellsurface asymmetry. Intracellular protein sorting occurs at multiple membrane compartments within a cell. Much of the molecular machinery responsible for protein sorting is known, as are many of the mechanisms and intracellular sites of action. Proteins are primarily sorted by signal sequences, which define their interactions with protein or lipid components of the sorting machinery. The cell type–specific expression of

PROTEIN SORTING IN THE EXOCYTIC AND ENDOCYTIC PATHWAYS IN POLARIZED EPITHELIAL CELLS / 1619 individual components of the sorting machinery, in combination with their compartmental localization, determines the trafficking pathways and steady-state localization of a given protein in a given cell type. Further exploration of this machinery, its expression patterns, and its mechanisms of action will aid our understanding and treatment of diseases of the GI tract.

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CHAPTER

63

Physiology of the Circulation of the Small Intestine Philip T. Nowicki Intestinal Microvascular Anatomy, 1627 Artery-Arteriolar Transition, 1627 Muscularis Microcirculation, 1628 Mucosal and Villous Microcirculations, 1629 Physiologic Considerations, 1629 Factors That Generate Vasoconstriction, 1630 Myogenic Response, 1631 Angiotensin, 1631 Endothelin, 1632 Somatostatin, 1633 Platelet-Activating Factor, 1634 Factors That Generate Vasodilation, 1634 Nitric Oxide, 1634 Nitric Oxide and Flow-Induced Dilation, 1635

Oxygen, 1636 Adenosine, 1639 Histamine, 1640 Glucagon, 1640 Postprandial Hyperemia, 1640 Sites of Postprandial Hyperemia, 1641 Constituents of Chyme Responsible for Intestinal Postprandial Hyperemia, 1641 Mechanisms of Postprandial Hyperemia, 1641 Acknowledgments, 1647 References, 1647

The principal function of the intestine, nutrient absorption, cannot be effectively carried out in the absence of synergy between parenchymal activity and local blood flow. The intestine achieves this pairing by the integration of local and systemic vasoregulatory mechanisms that tightly regulate perfusion. The purpose of this chapter is to present an overview of the circulation of the small intestine with a specific emphasis on nonneural, locally derived mechanisms of vascular control; neural regulation of the intestinal circulation is presented in Chapter 31. The discussion begins with an overview of intestinal microvascular anatomy, and then proceeds to factors that vasoconstrict or vasodilate the small-intestinal circulation; these later sections are limited to agents that appear to generate physiologically relevant vasoregulation; hence, the lists of agents is not exhaustive. The final section describes the phenomenon of postprandial

hyperemia, arguably the most important vascular response of the intestinal circulation insofar as it is requisite for nutrient absorption to occur.

INTESTINAL MICROVASCULAR ANATOMY The intestinal microcirculation has been elegantly described by Bohlen, primarily within the adult rat (1–3), although other species also have been studied (1,4,5). Because of the wealth of data available, this discussion focuses on the anatomy of the rat intestinal microcirculation with comparisons made to other species as warranted. Figure 63-1 presents a schematic representation of the intestinal microcirculation.

Artery-Arteriolar Transition P. T. Nowicki: Departments of Pediatrics and Physiology, Center for Cell and Vascular Biology, Columbus Children’s Research Institute, College of Medicine and Public Health, The Ohio State University, Children’s Hospital, Columbus, Ohio 43205. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Small arteries pierce the longitudinal and circular muscle layers and cause several arteriolar branches within the outer layer of the submucosa. These branches are designated as 1A because they represent the first vessels of arteriolar dimension that reside entirely within the intestine. 1A arterioles cause numerous 2A branches, which also remain entirely within

1627

1628 / CHAPTER 63

4V

LC

Longitudinal muscle

5A

Circular muscle CC

4A

5A

3A

3V IA

2V

4V

IV

SA SA

2A CV

Submucosa DA 2VM MC PC

Villus

FIG. 63-1. Microvascular anatomy of the rat small intestine. Small arteries (SA) pierce the muscularis layers from the mesentery and give rise to 1A (first order) arterioles within the submucosa, from which 2A (2nd order) arterioles arise. 3A (third order) arterioles branch from the 2A, descend into the mucosa, and thereafter become the single arteriole perfusing each villus; before this descent, however, a single 4A (fourth order) arteriole branches from each 3A. The 4A ascends into the muscularis, where it gives rise to 5A (fifth order) arterioles in the muscularis layers. The 5A arterioles give rise to capillaries in the circularis (CC) and longitudinalis (LC) muscle layers. In the villus, the terminal 3A gives rise to two distributing arterioles (DA) at the villous tip that descend toward the villous base, giving rise to capillaries (MC). Note that precapillary sphincter (PC) resides at the start point of each MC. MCs drain into 2VM, second order venules within the villus, which proceed to drain into a collecting venule (CV) within the mucosa. The CV drains into the 2V (second order) venule in the submucosa, which, in turn, enters the 1V (first order) submucosal venule, which, in turn, enters the small vein (SV) that exits the bowel wall. The muscularis CC and LC drain into separate 4V (fourth order) venules within each muscle layer, which, in turn, drain into a 3V (third order) venule. The 3V proceeds to the submucosa, where it enters the 2V. (Reproduced from Bohlen [1], by permission.)

the submucosa. These 2A arterioles interconnect with other 1A arterioles forming arteriolar-arteriolar shunts; in this context, the 2A arterioles form a network, or manifold, that serves to pressurize the terminal portion of the intestinal microcirculation that begins with the 3A arterioles.

Muscularis Microcirculation 3A arterioles are derived from the 2A arteriolar manifold and descend toward the mucosa; however, before penetration, each 3A causes a single 4A arteriole that turns upward and enters the muscularis layers. The 4A arteriole generates

5A branches within each muscle layer that run perpendicular to the muscle fibers. The 5A arterioles generate numerous capillaries that travel parallel to the muscle fibers. These capillaries do not appear to have a precapillary sphincter, that is, the single muscle fiber encircling the capillary at its arteriolar origin that governs capillary perfusion (6), suggesting that regulation of the perfused capillary density is limited within the muscularis. Capillaries in both muscle layers drain into 4V venules that run parallel to the muscle fibers; thereafter, 4V venules drain into 3V venules that descend back to the submucosa to join 2V venules therein. 2V venules form a dense, interconnected venous pool within the submucosa, which drains both the muscularis

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1629 Physiologic Considerations

and mucosal microcirculations. These venules empty into 1V venules, which then drain into small veins that exit the bowel wall into the mesentery.

From a functional standpoint, the intestinal arterial microcirculation can be subdivided into two regions. The first region extends from the mesenteric artery down to the 2A arterioles within the submucosa. This region represents the transition zone from artery to arteriole and generates the 2A arteriolar network, which serves to pressurize the terminal portion of the intestinal microvasculature. The second region, or terminal microvasculature, begins at the 3A arteriole and ends at the DA within the villus or the 5A arteriole within the muscularis, vessels that ultimately generate flow through the capillaries, or exchange vessels. The small artery (SA), 1A, and 2A vessels that constitute the first portion of the arterial circulation present variable resistors set in series (Fig. 63-2). The branch point that separates muscularis and mucosal circulations occurs at the 3A arteriole, a circumstance that causes parallel resistance circuits between the 3A-4A branch point and the merge of mucosal collecting venules and muscularis 3V venules. A substantial pressure reduction occurs from the SA to 2A arteriole; thus, pressure within the main mesenteric artery averages 100 mm Hg, whereas that in the 2A arteriole averages 40 mm Hg (Fig. 63-3). Hence, approximately 65% of total intestinal vascular resistance (i.e., resistance as determined from the main mesenteric artery to vein) occurs between the SA and 2A arterioles; that is, these vessels are the principal site of flow regulation into the intestine. The SA-1A-2A vessels are 500 to 700 µm from the villi, the site of nutrient absorption, and the main locus of gut O2 consumption, yet they dictate, in large part, the rate of intestinal perfusion. It is thus logical to presume that mechanisms exist to facilitate communication between the downstream events within the villi and these upstream vessels. Both neural (i.e., the enteric nervous system) and nonneural mechanisms of communication have been proposed; the nonneural

Mucosal and Villous Microcirculations Following the 4A branch point, the 3A arteriole proceeds into the glandular mucosa where it generates capillaries. Thereafter, each 3A arteriole enters a single villus and proceeds up to the villous tip where it forms two descending arterioles (DAs), which travel approximately two thirds of the distance back toward the base of the villus, generating multiple capillaries during their descent. The density of the capillary network is greater near the villous epithelium than at the center of the villus. Unlike the muscularis capillaries, a precapillary sphincter can be identified at the arteriolar origin of approximately 75% of villous capillaries. These sphincters can constrict and dilate independently of the 3A or DA arterioles; as is discussed further later, this capacity greatly enhances the regulatory capacity of the villous microcirculation (7,8). Multiple capillaries drain into a single second-order capillary venule (2VM), which, in turn, coalesces into a single collecting venule (M) at the base of each villus. Collecting venules from adjacent villi interconnect within the glandular mucosa, ultimately entering the 2V venules within the submucosa. An alternative pattern of the villous microcirculation is present in the rabbit. The terminal portion of the rabbit 3A arteriole travels up one side of the villus, and multiple capillaries are generated during the ascent. These capillaries drain into a single venule on the opposite side of the villus. This microvascular pattern may reflect the leaflike shape of the rabbit villus, which differs from the finger-like shape of the rat villus. A similar villous capillary pattern is present in human intestine (4).

Mucosa

3A

CV 2V 1V

SA

1A

2A 4V

4A

PV

PA Muscularis

FIG. 63-2. Schematic representation of the intestinal arteriolar microvasculature. The small artery (SA), 1A, and 2A vessels are resistors set in series and represent the principal site of resistance regulation within the intestinal circulation. They, in conjunction with the downstream 3A arterioles, serve as the “resistance vessels” of the metabolic model of local blood flow regulation; that is, the sites at which blood flow, and hence O2 delivery into intestine, is regulated. The mucosal and muscularis circulations exist in parallel from the 3A-4A branch point until the venular convergence in the 2V submucosal venules. This parallel arrangement facilitates independent regulation of pressure and flow within these gut layers. Abbreviations correspond to those used in Figure 63-1.

1630 / CHAPTER 63 2A (11)

1A (16)

50

Microvascular pressure (mm Hg)

40

3A (14)

DA (14)

35 5A (11)

LC, CC Muscularis (12) 4V (20)

20

2V (10) IV (11)

Mucosa MC 2VM (11) (8)

10

Venules

Arterioles

0

60

50

40

30

20

10

3

10

20

30

40

50

60

70

Inside diameter (microns)

FIG. 63-3. Microvascular pressures as a function of vessel diameter within the intestinal microvasculature. These data were collected in innervated rat intestine, and systemic pressure was 104 ± 4 mm Hg. Data are given as means ± standard error of the mean. The number in parentheses below each vessel designation denotes the number of observations made for that vessel type. The pressure decline from the systemic level to the 1A-2A arterioles (∼60 mm Hg) indicates that these vessels function as the principal site of resistance regulation within the intestine. The dotted line beginning at the 3A-DA represents the pressure/diameter within the mucosal circuit. The lower MC pressure reflects the presence of precapillary sphincters at the start point of these capillaries, as well as a lower pressure in 2VM villous venules compared with the circumstance in the muscularis microcirculation. Abbreviations correspond to those used in Figure 63-1. (Reproduced from Gore and Bohlen [2], by permission.)

mechanisms are discussed in subsequent sections of this chapter. The muscularis and mucosal microcirculations can be considered as parallel circuits (see Fig. 63-2). Resistance across these circuits is quite different. Thus, a substantial pressure reduction occurs between the 3A arteriole (32 mm Hg) and the 2VM villous capillary (13 mm Hg); in contrast, pressure within the muscularis capillary (24 mm Hg) is much greater. This disparity is likely because of two features. First, villous capillaries generally exhibit precapillary sphincters that might generate a pressure decrease from DA to capillary, whereas muscularis capillaries do not. Second, the collecting venule that immediately drains the villi forms a dense plexus before entering the 2V venules, whereas the 4V and 3V venules that drain the muscularis layers directly enter the submucosal 2V venule. Thus, although precapillary resistance is similar in both mucosal and muscularis vascular compartments, the postcapillary resistance is less in the mucosa so that the net resistance to flow is less across the mucosal circuit. This fact accounts for the 70:30 ratio of mucosal/muscularis blood flow present in rat intestine (2), a ratio also demonstrated in lamb (9) and piglet (10) intestines. The greater rate

of mucosal perfusion, and hence oxygen delivery, reflects its greater rate of oxygen utilization when compared with the muscularis layers. Also, the lower capillary pressure within villous capillaries facilitates their absorptive role; hence, a low vascular pressure would permit net absorption when interstitial oncotic pressure increases, as would occur during nutrient absorption. In contrast, muscularis capillaries likely exist in a state of constant filtration.

FACTORS THAT GENERATE VASOCONSTRICTION An important vasoconstrictor stimulus in the intestinal circulation is derived from sympathetic efferents that originate in the prevertebral celiac and mesenteric ganglia (11); indeed, existing data indicate that all neurogenic vasoconstriction of the submucosal arterioles (i.e., the 1A-2A arterioles) emanates from extrinsic sympathetic efferents (12). Discussion of neurogenic intestinal vasoconstriction is presented in Chapter 31. The remainder of this section discusses nonneurogenic vasoconstrictor mechanisms that are locally derived,

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1631 that is, that originate and end within the intestine and its attendant circulation.

Myogenic Response Smooth muscle contracts when stretched; hence, the stretch stimulus generated by increased intraluminal pressure contracts circumferential vascular smooth muscle, resulting in vasoconstriction. Conversely, reduction of intraluminal pressure leads to vasodilation. This process, first described by Bayliss in 1902 (13), is called the myogenic response. The mechanistic basis of the myogenic response appears to be multifactorial, but is yet to be fully established. A detailed description of these mechanisms is beyond the scope of this chapter; however, a review by Davis and Hill (14) provides excellent insight in these putative mechanisms. The myogenic response is present within the intestine and can be elicited by manipulating arterial pressure, venous pressures, or both (15–18). The intensity of the myogenic response responds inversely to vessel diameter; thus, the magnitude of vasoconstriction in response to a pressure stimulus is greatest in the smallest arterioles (19). This pattern is present within the intestinal microcirculation (20), although the strength of the myogenic response is also contingent on the microvascular site under consideration (21). Thus, 2A and 3A arterioles constrict in response to an increase in intravascular pressure; however, 5A arterioles perfusing the muscularis layers dilate in response to this perturbation. The physiologic relevance of the myogenic response in intestinal vascular regulation is incompletely determined, although three hypotheses have been offered (14). First, the myogenic response may contribute to generation of basal vascular resistance. The presence of a basal state of vasoconstriction, or vascular “tone,” is vital within the intestine insofar as the capacity to enhance blood flow via vasodilation is contingent on a preexisting state of relative vasoconstriction. It is mandatory that intestinal perfusion increases during nutrient absorption and this increase, generated by vasodilation within the intestine rather than by an increase in cardiac output, can only occur if the vessels therein maintain the physical capacity for vasodilation. Extrinsic neural stimuli (e.g., sympathetic efferents) contribute to basal vascular tone within the intestine; however, basal vascular tone is also present in fully denervated yet autoperfused intestine, and it is likely that myogenic-based vasoconstriction is the principal mechanism in the creation of nonneural basal vascular tone. Second, the myogenic response may participate in blood flow autoregulation, that is, the intrinsic process whereby regional blood flow remains relatively constant despite changes in vascular pressure (22). Thus, an increase in arterial inflow pressure would elicit a myogenic response, induce vasoconstriction, and hence dampen the effect of the increased pressure on flow (and vice versa). This process occurs to a limited extent in the intestine (18,23), although other factors also appear to contribute to autoregulation insofar as tissue metabolic rate (24) and rate of oxygen delivery (16) significantly alter the magnitude of the

myogenic response. Third, the myogenic response may serve to stabilize capillary pressure. This capacity would be critical in the mucosal villous microcirculation wherein maintenance of a relatively low capillary pressure is necessary to permit an absorptive state. Experimental evidence of this role is mixed and depends on how pressure across the intestine is perturbed, that is, by change in arterial or venous pressure. Capillary pressure is relatively well maintained during significant fluctuations of arterial pressure (17), but not so in response to change in venous pressure (21).

Angiotensin Angiotensin was initially described as a systemic pressor agent generated by the kidneys. The preproform of the active peptide, angiotensinogen, is cleaved by the enzyme renin that is generated within juxtaglomerular cells. This first cleavage generates the 10-aminoacid peptide angiotensin I (Ang I); thereafter, Ang I is cleaved within the lung microvasculature by angiotensinconverting enzyme to form the 8-residue peptide Ang II that functions as a humoral or systemic pressor agent by acting on several regional circulations, including the intestine. This action is mediated primarily by the angiotensin I (AT1) receptor, located on vascular smooth muscle. Although Ang I has modest vasoactive properties, the principal vascular effector is Ang II. AT1 receptors are present within the intestinal circulation, and infusion of Ang II generates sustained gut vasoconstriction without causing significant compromise of intestinal oxygenation unless the flow reduction is severe (25–27). However, Ang II has been implicated in the pathogenesis of nonocclusive ischemic colitis (28) and necrotizing enterocolitis (29), two diseases in which intestinal ischemia appears to play a pivotal role in pathogenesis. Thus, reduction of cardiac output (tamponade) reduces intestinal perfusion and compromises intestinal oxygenation, and these effects are significantly attenuated by pretreatment with the AT1 antagonist losartan. Similarly, intestinal ischemia generated by septic shock is mediated, in part, by Ang II; this gut vascular compromise appears to be an important factor in mucosal damage and subsequent bacterial translocation during sepsis, a common cause of death during the cascade of events that constitute sepsis (30). More recent studies have identified all the components of the renin-angiotensin system within elements of the vascular wall, including mesenteric vessels; in addition, it has become clear that Ang II does not function in solely a vasoconstrictor role within the gut (31,32). In part, this dichotomy of vascular function reflects the presence of AT2 receptors within the intestinal circulation (25,33). AT2 receptors are primarily located on the endothelium, and ligand binding thereto activates the endothelial isoform of nitric oxide synthase (NOS), thus generating the potent vasodilator nitric oxide (NO) (34). In this context, shear-stress activation of AT2 receptors by locally produced Ang II participates in the phenomenon

1632 / CHAPTER 63 of flow-induced dilation in mesenteric arterioles (see later description) (31). The relative proportion of AT1 and AT2 receptors is developmentally regulated: AT2 receptors predominant during early newborn life, whereas this pattern reverses during postnatal development into early adulthood (35); this circumstance, in concert with the developmental regulation of the endothelin (ET) vascular effector system and eNOS-derived NO, may explain the lower vascular resistance characteristic of newborn intestine (36).

Endothelin ET-1 is a member of a family of peptides that include ET-1, ET-2, and ET-3. ET-2 and ET-3 differ from ET-1 by two or five amino-acid substitutions, respectively. The physiologic roles of ET-2 and ET-3 remain ill defined; ET-1 is the dominant effector in regulation of vascular tone, being the most potent vasoconstrictor currently identified. ET-1 also serves as a potent mitogen. ET-1 produced by several cell types, including the vascular endothelium (from which it derived its name). ET-1 is transcribed as prepro-ET, which then undergoes posttranslational modification by means of two cleavage steps to generate the mature, vasoactive, 21-amino-acid peptide. These steps are not precisely regulated, nor is the final peptide ET-1 stored; hence, regulation of ET-1 production occurs at the level of transcription. Although it is constitutively produced, several factors enhance ET-1 transcription including hypoxia (37), oxidative stress (38), mechanical stretch (39), and NO (40). The mechanostimulus of shear stress generated by the flow of blood against the static endothelium also alters ET-1 production, although the effect appears to be different depending on the regional circulation, vessel type, and vessel size (39,41). ET-1 induces vasoconstriction by binding to ETA receptors present on vascular smooth muscle (42). In this context, the vascular effect of ETA is generated in a paracrine manner, with peptide production within the endothelium and site of action in adjacent vascular smooth muscle. ET-1–induced vasoconstriction is profound, even at physiologically relevant ligand concentrations. Also, this vasoconstriction is sustained, that is, it cannot be “washed out”; this effect may reflect the capacity of the receptor-ligand dyad to continue to initiate the signal transduction pathway that leads to constriction even after migration of the complex off the plasma membrane (43). These characteristics of ET-1–induced vasoconstriction may explain, in part, the apparent ability of ET-1–induced vasoconstriction to generate ischemic tissue damage (44). A second ET-1 receptor, termed ETB, is present on endothelial cells. ET-1 binding to endothelial ETB receptors activates the endothelial isoform of nitric oxide synthase (eNOS) in a Ca2+/calmodulin-dependent manner, and hence increases NO production (45). ETB-induced vasodilation serves to counterbalance ETA-induced vasodilation; hence, the net effect of ET-1 on vascular resistance is determined, in part, by the ratio of ETA and ETB receptors.

An important feature of ET-1 is its capacity to interact with other vascular effector systems, particularly vasoconstrictors. ET-1 activates the local renin-angiotensin system within mesenteric resistance vessels, thus increasing production of Ang II (43). Chronic infusion of subpressor doses of ET-1 enhances the contractile effect of Ang II; that is, they shift the Ang II dose–response curve leftward (47). A similar effect occurs with norepinephrine (48). ET-1 decreases expression of eNOS, and hence reduces the capacity of the endothelium to generate the vital vasodilatory stimulus NO (40). These interactions have the potential to greatly amplify the vasoconstriction generated by ET-1, both by enhancing the contractile efficacy of other constrictors and by reducing the presence of a counterbalancing vasodilator stimulus. ET-1 also acts in a proinflammatory role within the gut microcirculation. It enhances leukocyte adhesion (49), causes degranulation of resident mast cells (50), and increases vascular permeability within the mucosa (51). This latter effect may occur in association with platelet-activating factor (PAF) (52). Infusion of ET-1 into the mesenteric circulation at physiologically relevant concentrations (10−9 M, the Kd for both the ETA and ETB receptors) causes profound circulatory disturbance. Total intestinal vascular resistance significantly increases and blood flow decreases, compromising O2 delivery (53). The perfused capillary density, a marker of capillary perfusion, also decreases, indicating that ET-1 exerts an effect on the terminal microvasculature (villous 3A and DA arterioles), as well as on the precapillary sphincters that govern villous capillary perfusion. The means by which ET-1 compromises microvascular flow is dependent on the vessels under consideration. In proximal arterioles (i.e., the 1A-2A submucosal arterioles), ET-1 reduces flow by inducing vasoconstriction, and hence increasing resistance across this segment of the gut microvasculature. Further downstream, however, ET-1 reduces flow rate without a concomitant change in 3A arteriolar diameter. This change may reflect a loss of plasma volume insofar as ET-1 induces a significant increase in vascular permeability within the terminal microvasculature of the intestine. This effect on permeability is mediated via ETA receptors and appears to involve the copresence of PAF. Sustained infusion of ET-1 causes histopathologic damage consist with ischemia, and this effect is modestly attenuated by antioxidants, as well as by blockade of PAF (54). A role for ET-1 has been proposed for several intestinal diseases, as detailed in the following sections. Gut Strangulation Increase of the external pressure surrounding a gut loop, as might occur during intussusception or volvulus increases the venous concentration of ET-1, whereas blockade of ETA receptors attenuates the hemodynamic effects of strangulation (55,56). In addition, blockade of ETA receptors decrease the extent of intestinal mucosal injury after ischemia-reperfusion, an event that likely occurs as the strangulation process waxes and wanes during disease development. Hence, ET-1 may

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1633 participate in the intestinal damage coincident with clinical events such as intussusception or volvulus. Endotoxic Shock Endotoxic shock, the hemodynamic events that occur during systemic bacteremia and endotoxemia during systemic infection with gram-negative organisms, compromises the intestinal microvasculature (58). Lipopolysaccharide, a principal mediator of endotoxic shock, reduces microvascular flow rate, increases leukocyte sticking, and enhance microvascular permeability. These intestinal vascular events are significantly ameliorated by blockade of ETA receptors (59,58). Clearly, the intestinal hemodynamic consequences of endotoxic shock are generated by more that just ET-1; however, blockade of the ETA receptor may provide some relief under specific circumstances. Inflammatory Bowel Disease ET-1 immunoreactivity is increased in surgical biopsies of Crohn’s disease and ulcerative colitis (60), whereas pretreatment with bosentan, a nonselective ET-1 receptor antagonist attenuates inflammation and tissue damage in trinitrobenzene sulfonic acid–induced colitis in rats (61). Later work, however, failed to confirm the presence of ET-1 in human inflammatory bowel disease (62) and at present the putative role of ET-1 in this process remains unresolved. The role of ET-1 in the pathogenesis of inflammatory bowl disease, if any, remains unclear. Hirschsprung’s Disease Rats which demonstrate congenital aganglionosis are ETB receptor deficient (63). These lethal mutants manifest histological and physiological features consistent with Hirschsprung’s disease. Although these observations do not establish a cause-and-effect relation, they imply that the ETB receptor may participate in the pathogenesis of colonic aganglionosis. Neonatal Necrotizing Enterocolitis Neonatal necrotizing enterocolitis (NEC) is a disease unique to preterm human infants that entails the necrosis of bowel, particularly the ileum and ascending colon, that often requires surgical resection. The pathogenesis of NEC is not established, although ischemia and inflammation appear requisite for its development. ET-1 has been implicated as one of the mechanisms responsible for this ischemia. Mesenteric artery from newborns demonstrates a greater number of ETA than ETB receptors, predicting that the newborn intestinal circulation might be more prone to vasoconstriction and tissue hypoxia in response to ET-1 (36). This predication has been verified: Infusion of ET-1 into the newborn intestine compromises tissue oxygenation, mainly because it reduces capillary perfusion. Similar changes do not occur during ET-1

infusion into the intestine from older subjects (53). The ischemia that occurs in newborn intestine after an episode of ischemia-reperfusion, a phenomenon believed to participate in NEC pathogenesis, is essentially eliminated by pretreatment with the ETA receptor antagonist BQ610 (13,59). Most importantly, however, the tissue concentration of ET-1 in human preterm intestine resected for NEC is greater in areas of resected intestine that demonstrated histopathologic evidence of NEC compared with normal-appearing regions within the same specimen, whereas arterioles removed from these resection specimens demonstrate a significantly greater resting vascular resistance that is attenuated by blockade of ETA receptors (12,26). In addition, a significant inverse correlation exists between plasma ET-1 concentration and superior artery mesenteric blood flow velocity in preterm infants (64).

Somatostatin Somatostatin is a 14-amino-acid peptide initially identified as a hormone secreted by the hypothalamus that inhibited secretion of growth hormone from the anterior pituitary. Subsequently, it was determined that somatostatin also was secreted by δ cells within the islets of Langerhans, and that the peptide had complex gastrointestinal effects including regulation of insulin and glucagon secretion, regulation of gastrointestinal motility, and regulation of intestinal absorption and secretion. Immunoreactive somatostatin is located within enteric neuron cell bodies, as well as in nerve fibers in close proximity to vascular smooth muscle membranes (65). Somatostatin acts as a vasoconstrictor within the intestinal circulation, although the mechanism of action remains ambiguous. Infusion of somatostatin into the mesenteric artery (1 µg/kg/min for 10 minutes) increases intestinal vascular resistance and reduces blood flow; it also decreases the perfused capillary density, suggesting that it acts within upstream vessels, as well as in the terminal portion of the gut microvasculature (66). This vasoconstriction is not consequent to a direct effect of the peptide on vascular smooth muscle tone; rather, attenuation of vasodilatory stimuli such as glucagon has been proposed (67). The clinical relevance of somatostatin in intestinal vascular physiology is based on its vasoconstrictive properties. Octreotide, a stable analogue of somatostatin, has been used in the treatment of acute variceal and nonvariceal gastrointestinal hemorrhage (68,69). The vasoconstrictive effect of octreotide occurs within the submucosal 1A arterioles, rather than in the upstream conduit vasculature, which might suggest it to be ideally suited for the treatment of hemorrhage (70). However, octreotide causes a generalized mucosal ischemia and perfusion heterogeneity within the gut wall, suggesting that sustained perfusion in this area may cause ischemic damage to the intestinal mucosa (71). Octreotide also has been used to attenuate the intestinal hyperemia that occurs during the portal venous hypertension that accompanies cirrhosis, although local vasodilatory mechanisms within the intestinal

1634 / CHAPTER 63 circulation prevent sustained intestinal vasoconstriction during octreotide treatment.

systems with a particular emphasis on the roles of NO and oxygen, followed by other factors that induce intestinal vasodilation that is of physiologic relevance; as was the case in the previous section, the list of vasodilators is not exhaustive.

Platelet-Activating Factor PAF is an endogenous phospholipid mediator produced within the gut by a wide variety of cell types, including inflammatory and endothelial cells (72). Infusion of PAF causes intestinal ischemia, an effect that is mediated indirectly via the PAF-induced release of leukotriene C4 and norepinephrine (73). The site of PAF-induced vasoconstriction includes venules within the microcirculation (74). PAF, a proinflammatory mediator, also increases vascular permeability within the intestinal microvasculature. This effect may also be indirect and secondary to the generation of reactive oxygen species generated by the intestinal parenchyma, as well as by adherent leukocytes inasmuch as PAF increases leukocyte adherence to the microvascular venular endothelium (75,76). Release of PAF occurs in response to several stimuli, including hypoxia, ischemia, and endotoxin; moreover, gut ischemia generated in response to exogenously administered PAF releases more of the mediator, thus initiating a cycle that generates progressively worsening vascular pathophysiology (77,78). Several lines of evidence suggest that PAF-induced intestinal vascular dysfunction, including vasoconstriction, generates intestinal necrosis (77,79). PAF, infused into the mesenteric artery at physiologically relevant concentrations, causes mucosal necrosis; this effect is amplified by the coexistence of arterial hypoxia or endotoxemia. Conversely, intestinal tissue damage elicited by hypoxia or endotoxemia is attenuated by prior administration of PAF receptor antagonists (80,81). Similar to ET-1, PAF may be involved in the pathogenesis of NEC (82,83). The serum PAF concentration in infants with this disease is significantly greater than their agematched control subjects; furthermore, PAF-acetylhydrolase, the enzyme responsible for PAF inactivation, is decreased in affected infants. Pretreatment of newborn rat pups with a PAF receptor antagonist significantly reduces the development of NEC-like lesions in this animal model (84). These pups also demonstrate a greater capacity to generate PAF when compared with their older counterparts (79). The interactions between ET-1 and PAF suggest that these agents might conspire in the generation of intestinal vascular dysfunction, which generates regional, microvascular ischemia that participates in NEC pathogenesis.

FACTORS THAT GENERATE VASODILATION Partition of factors that generate vasodilation within the intestinal circulation into discrete entities has become increasingly difficult with the accumulation of evidence that they rarely function in a singular manner, but rather coalesce and overlap. This section reviews nonneural vasodilator

Nitric Oxide Biochemistry of Nitric Oxide NO is produced during the reduction of L-citrulline to L-arginine by NOS, in a reaction that requires NADPH and O2 as cofactors; in some instances, Ca2+ and calmodulin are also requisite (85). Three isoforms of NOS have been identified, differing substantially in transcriptional and posttranslational regulation, site(s) of expression, and regulation of activity: (1) neuronal, or nNOS; (2) inducible, or iNOS; and (3) endothelial, or eNOS. The latter isoform, eNOS, is principally involved in vascular regulation, and hence remains the focus of this discussion. Endothelium-, eNOS-derived NO generates its vascular effect by diffusion from the endothelium to adjacent vascular smooth muscle where it activates soluble guanylate cyclase. This action generates a serial cascade ultimately leading to vasodilation, as follows: generation of cyclic guanosine monophosphate, which leads to reduction of [Ca2+], which causes relaxation of vascular smooth muscle, which increases vessel diameter, that is, induces vasodilation (86). Molecular Biology of Endothelial Nitric Oxide Synthase and Nitric Oxide The essential role of eNOS, and thus of eNOS-derived NO, in intestinal vascular regulation warrants delineation of some salient features of eNOS transcription, translation, and activity. The potential relevance of these descriptions to intestinal vascular physiology is detailed in subsequent sections, wherein the putative role of NO in a variety of intestinal vascular responses, including postprandial hyperemia, are described. Regulation of Endothelial Nitric Oxide Synthase Transcription eNOS is constitutively expressed within endothelial cells; however, the basal transcription rate can be altered by a wide variety of stimuli. The human eNOS promoter is “TATAless” and includes several cis-regulatory elements including GATA, SP-1, activator protein-1, nuclear factor (NF)-1, the shear-stress response element, the sterol-regulatory element, and the hypoxia-inducible element (87,88). Several peptides, including estrogen (89), insulin (90), and vascular endothelial growth factor (91), enhance eNOS transcription; in contrast, the vasoconstrictor peptide ET-1 inhibits eNOS transcription (40). Protein kinase C (PKC) may participate in some of these interactions (92). Hydrogen peroxide, the relatively stable metabolite of the superoxide anion, stimulates eNOS transcription (93). The mechanostimulus of shear stress is also a critical stimulus for eNOS expression.

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1635 This latter response appears mediated by the tyrosine kinase c-Src, which acts to enhance eNOS transcription rate, as well as eNOS mRNA stability (94). The nuclear transcription factor NF-κB is also involved in this process (95). Posttranslational Modification of Endothelial Nitric Oxide Synthase Effective eNOS activity is contingent on its location on the plasma membrane. To this end, posttranslational modification of eNOS includes myristoylation and palmitoylation, alterations that permit trafficking of eNOS to cholesterolrich sites within caveolae, specialized microdomains within the plasma cell membrane. Caveolae appear to colocalize eNOS with elements of signal transduction pathways requisite for its function (e.g., peptide receptors and G proteins, Ca2+ channels); also, caveolae contain the scaffolding protein caveolin-1, which plays an important role in modulation of eNOS activity by altering accessibility of stimulants to stimulants (96). Regulation of Endothelial Nitric Oxide Synthase Activity Several factors alter eNOS activity. Protein–protein interactions among caveolin-1, calmodulin, and eNOS appear to alter availability of eNOS to signals within the caveolae; hence, a regulatory cycle exists, wherein Ca2+/calmodulin releases eNOS from the scaffolding domain of caveolin-1, thus permitting enzyme activity (97). Several agents, including muscarinic agonists, insulin, histamine, adenosine, estrogen, bradykinin, and vascular endothelial growth factor stimulate eNOS activity via their capacity to increase [Ca2+] (96). Alternatively, ET-1 reduces eNOS activity; interestingly, the opposite effect also occurs wherein NO inhibits ET-1 activity (98). This regulatory interaction among eNOS, NO, and ET-1 plays a pivotal role in setting basal vascular tone within the newborn intestinal circulation (36). Although the eNOS isoform was initially described as entirely Ca2+ dependent, it is now clear that enzymatic activity can be increased in the absence of a change in [Ca2+]. Thus, the mechanostimulus of shear stress increases eNOS activity via K+ channels in a response that requires tyrosine phosphorylation of the channel proteins (99). In addition, phosphorylation of eNOS at Ser-1177 increases eNOS activity in a Ca2+-independent manner, whereas phosphorylation at Thr-497 has the opposite effect (100). Several agonists, including shear stress (101) and estrogen (102), induce a coordinate effect on the relevant phosphorylase (PKA, PKB) and phosphatase (protein phosphatase 1 [PP1], PP2A) to enhance and reduce Ser-1177 and Thr-497 phosphorylation, respectively.

elements of the intestinal circulation. Thus, eNOS has been identified in virtually all portions of the intestinal circulation, including conduit arteries, the arteriolar microcirculation, and the venular circulation (103,104). As expected, eNOS is principally located within the endothelial layer of the intestinal vasculature. nNOS, which generates NO that functions as a neurotransmitter, is mainly present within the myenteric ganglia and participates in regulation of intestinal motor function; however, nNOS-containing nerve terminals are also present surrounding submucosal arterioles (105). Physiologic Effects Periarteriolar [NO] at submucosal 1A and 2A arterioles is greater than the dissociation constant for the NO-soluble guanylate cyclase reaction within vascular smooth muscle, suggesting that the perivascular [NO] is within a physiologically relevant range within the microcirculation (106). The perivascular [NO] also changes in direct proportion to the diameter of these arterioles; hence, an increase of flow velocity, and thus shear stress, increases both [NO] and diameter (106). Vasoconstriction of 1A and 2A submucosal arterioles by sustained sympathetic stimulation is attenuated by endothelium-, eNOS-derived NO; this effect is mediated by a reduction of arterial wall pO2 (30,107,108). Consistent with these observations, reduction of mucosal O2 availability by reducing the pO2 in the buffer suffusing the mucosal surface caused an increase in both periarteriolar and perivenular [NO]. Studies performed in denervated, autoperfused loops of small intestine, wherein hemodynamic observations were made of the entire intestinal circulation (i.e., from conduit artery to collecting vein), also indicate that NO participates in intestinal vascular regulation. Blockade of NOS activity causes a significant increase in intestinal vascular resistance, but has no effect on intestinal O2 uptake (109). Preservation of O2 uptake indicates that the effects of NOS blockade on vascular resistance were caused by a direct effect of NO on vascular tone (27). Similar findings have been reported for the SAs that lie immediately upstream of the gut intramural microcirculation, studied under in vitro conditions (110). Collectively, studies performed at several levels of the intestinal circulation indicate that NO contributes an essential, basal vasodilatory stimulus that participates in setting basal vascular tone, or resistance. NO also plays an essential role in generation of intestinal vasodilation during nutrient absorption, that is, postprandial hyperemia. This role is described later in this chapter (see the Postprandial Hyperemia section).

Nitric Oxide and Flow-Induced Dilation Nitric Oxide and Endothelial Nitric Oxide Synthase within the Intestinal Circulation Localization Anatomic and immunohistochemical studies demonstrate the presence of NOS within the intestine, particularly within

As first reported by Schretzenmayr (111), large conduit arteries, such as the femoral or mesenteric artery, dilate in response to an increase in flow rate in a phenomenon termed flow-induced dilation. This observation was later repeated in resistance arterioles, as well as within microvasculature under

1636 / CHAPTER 63 in vivo conditions (112,113). It was initially postulated that this mechanism served an important role in tissue oxygen delivery (114). Thus, metabolic-induced vasodilation within the downstream terminal vasculature increases flow rate through upstream conduit vessels; in turn, this increased flow rate induces dilation of the conduit arteries, particularly in the SAs immediately upstream of the microvasculature. The net consequence of these events is preservation or augmentation of oxygen delivery to the downstream microvasculature. In essence, this interpretation suggests that flow-induced dilation is an extension of metabolic vascular regulation described in the next section. Although the aforementioned concept has proved correct, later studies demonstrated that the principal stimulus of flow-induced dilation was actually wall shear stress, rather than flow per se (112). Wall shear stress is the mechanical force generated by the movement of flowing blood against the static endothelium and, in simple terms, is given by the following equation (115): τ = (4 • η • Q)/(r3 • π) where τ is wall shear stress, η is viscosity, Q is flow rate, and r is radius; more detailed explanations of the biophysics of wall shear stress are available (116). Hence, an increase in flow rate acts to increase wall shear stress; thereafter, “flow”-induced dilation subsequently serves to increase vessel radius and restores shear stress to baseline. Because shear stress is an inverse function of the third power of the radius, only a modest degree of vasodilation is necessary to preserve shear stress in the presence of flow variation. The mechanistic basis for flow-induced dilation has received considerable attention. Early work indicated that the vascular endothelium played a pivotal role in the response, as evidenced by its ablation after removal of the endothelium (117). Subsequent work confirmed this effect in several circulations, including the gut (112,113,118). Several studies report that endothelium-derived NO, generated by eNOS, is the vasodilator mechanism responsible for flow-induced dilation (119,120). Periarteriolar [NO] demonstrates a direct linear relation with flow rate in the 1A submucosal arterioles (106). Other candidates that might mediate flow-induced dilation include prostaglandins (120) and hydrogen peroxide (121,122). An intact endothelial cytoskeleton is requisite for the generation of flow-induced dilation, suggesting that the cytoskeleton plays a pivotal role in the mechanotransduction process (123). Flow-induced dilation occurs within the intestinal circulation and appears to play an important role in maintaining the increase in gut perfusion after feeding, that is, postprandial hyperemia. In rat mesenteric arcades, concomitant escalation of red cell velocity and wall shear stress induces significant vasodilation that fully develops within 1 minute, and that is sustained for the duration of the stimulus (124). Similar findings have been reported in newborn intestine; here, an increase in flow rate generates significant mesenteric artery vasodilation in an age-specific manner, the response being

greater in younger subjects (118). Vasodilation of the SAs immediately upstream of the microcirculation plays an important role in setting total intestinal vascular resistance (125,126); hence, their vasodilation is likely an essential response to the increased tissue metabolic demands associated with nutrient absorption (127). Oxygen Effects of O2 on Vascular Smooth Muscle Isolated blood vessels studied under in vitro conditions, as well as vascular smooth muscle myocytes, alter their contractile state in response to changes in environmental pO2. Early work by Pittman and Duling (128) demonstrated relaxation of strips of large conduit arteries in response to reduced buffer pO2 and noted that the effect was greater in thick-walled arteries, suggesting that the effect was contingent on O2 diffusion into the vessel wall, or on the pO2 within (or around) vascular smooth muscle myocytes. The opposite effect, that is, contraction in response to an increase in environmental pO2, also was reported (129). Studies in isolated, buffer-perfused resistance vessels (e.g., 100–300 µm) showed a similar pattern: vasodilation in response to reduced buffer pO2, and vice versa (130). In the gut microcirculation, suffusion of the mucosal surface with low pO2 buffer (∼3 mm Hg; normoxic buffer ∼42 mm Hg) increased the diameter of 1A, 2A, 3A, and DA arterioles (131). The mechanistic basis for this interaction includes a direct effect of pO2 on ion channels of vascular smooth muscle myocytes. The contractile state of vascular smooth muscle myocytes is determined, in large part, by the intracellular calcium concentration, or [Ca2+]vsm; hence, a decrease in [Ca2+]vsm causes relaxation, and vice versa. In this context, perivascular pO2 affects the “open-closed” status of Ca channels. Hypoxia inhibits voltage-dependent Ca-channel activity, an effect that reduces [Ca2+]vsm (132); also, pretreatment with the L-type Ca-channel antagonist nifedipine attenuates hypoxia-induced relaxation in vitro (133). Interestingly, the oxygen sensitivity of voltage-gated Ca channels is site specific: hypoxia decreased Ca channel activity in large conduit vessels, but increased this activity in small resistance arterioles (134). These actions would lead to hypoxic vasodilation in large arteries, but vasoconstriction in arterioles. Adenosine triphosphate (ATP)–dependent K+ channels also have been implicated in hypoxic relaxation. Blockade of these channels with glibenclamide reduces hypoxic vasodilation, whereas the effect of hypoxia on vascular tone can be mimicked with cromakalim, which opens ATP-dependent K+ channels (135). Endothelium-derived autocoids, including NO and prostaglandins, also modulate the response of isolated blood vessels to change in perivascular pO2. Perfusion of isolated conduit arteries with hypoxic buffer (pO2 ∼25 mm Hg) induced production of NO, whereas cultured endothelial cells demonstrated the same effect (136,137). Hypoxia-induced

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1637 vasodilation is blocked by pretreatment with L-arginine analogues that attenuate eNOS activity, and hence NO production (129,138). In the gut microcirculation, reduction of the buffer pO2 suffusing the mucosal surface by ∼15% increases 1A periarteriolar [NO] by ∼135%; a similar change, but of less magnitude, occurs at the 1V perivenular [NO] (139). This change in perivascular [NO] is associated with significant increases in arteriolar diameter, and hence flow rate, and these changes can be blocked by prior administration of an L-arginine analogue or by CO2 embolization to destroy endothelial cell function. Prostaglandin E2 (PGE2) and PGI2 production also are increased in response to a reduction in buffer pO2 in isolated conduit arteries, whereas pretreatment with the cyclooxygenase inhibitor indomethacin attenuated the response of isolated resistance vessels to reduced buffer pO2 (130,140). The roles for the endothelium-derived autocoids NO and prostaglandins in the intrinsic response of isolated blood vessels to pO2 support the hypothesis that the vascular endothelium functions as an oxygen-sensing system (141). This hypothesis states that the endothelium functions as a local chemoreceptor system, complementing and interacting with systemic regulatory factors (e.g., the carotid body chemoreceptors) to conserve O2 transport via its capacity to sense regional, microvascular, O2 homeostasis. The chemical nature of the signal sensed by the endothelium was initially proposed to be ATP, insofar as ATP is a sensitive indicator of O2 sufficiency and its binding to purinergic P2y receptors increases eNOS activity by increasing [Ca2+]ec (142). The source of this ATP does not appear to the endothelial cells themselves; thus, ATP is not generated by cultured endothelial cells in response to hypoxia (141). An alternative source may be red blood cells (RBCs). RBCs exposed to a low pO2 (∼14 mm Hg) increased ATP release (143); furthermore, isolated arterioles perfused with hypoxic buffer plus RBCs demonstrate vasodilation, whereas those perfused with a cellfree hypoxic buffer do not dilate (144). One interpretation of these data is that RBCs release ATP as they traverse the microcirculation, which, in turn, regulates endothelial autocoid production. Interactions between Parenchymal O2 Demand and Vascular O2 Supply Early studies suggested that local or intrinsic regulation of vascular resistance was linked to the state of parenchymal oxygenation. For example, autoregulation, defined as intrinsic modulation of vascular resistance to maintain blood flow in the face of variable systemic pressure, was noted to be significantly more effective when tissue metabolic rate, and thus O2 demand, was enhanced (145). Observations of this nature led the metabolic or vasodilator theory of local blood flow regulation that proposes that the principal controlled variable in local vascular regulation is tissue oxygenation; that is, that the moment-to-moment oxidative demands of the parenchyma dictate intrinsic regulation of its attendant vasculature.

Metabolic Theory Tissue oxygenation, or O2 consumption, is defined by the Fick equation: VO2i = Qi × (a − vO2)i where VO2i is gut O2 consumption, Qi is gut blood flow, and (a − vO2)i is the arteriovenous O2 difference across the gut, or the level of O2 extraction (8). The extracellular O2 transport designed to maintain VO2i can be partitioned into two phases: conductive and diffusive. The convective phase entails the transport of O2 through the conduit vasculature to the terminal gut microvasculature, that is, 3A and DA arterioles in the mucosa, and 5A arterioles in the muscularis. This convective O2 flux is actively regulated by resistance vessels that govern the rate of blood flow, and thus O2 delivery, defined as the product of flow and the arterial O2 concentration. The diffusive phase entails the passive flux of O2 from capillary-to-cell and is dictated by the laws of diffusion, that is, the capillary-cell pO2 gradient and capillary surface area available for diffusion. The latter term, also called the capillary exchange capacity, is actively controlled by precapillary sphincters, that is, vascular smooth muscle elements that govern perfusion of individual capillaries. Vasoregulation of resistance vessels and precapillary sphincters is determined by a vasodilatory feedback signal(s) produced by the gut parenchyma in proportion to its state of O2 sufficiency. The metabolic theory contends that local vascular regulation within the gut is directed to maintain VO2i, and that this regulation may involve changes in vascular resistance (conductive O2 transport) or the capillary exchange capacity (diffusive O2 transport), or both. Evidence of Metabolic Regulation in the Intestinal Circulation: Whole-Organ Studies Experimental verification of the metabolic theory within the intestinal circulation has been generated using bloodperfused, denervated loops of small intestine. Perturbation of gut O2 homeostasis has been achieved by altering the O2 demand of the tissue (i.e., increasing or decreasing tissue metabolic rate), or by altering O2 supply to the tissue. The metabolic theory predicts that perturbation of either term, that is, the gut O2 supply/demand ratio, will lead to local vascular regulation designed to restore VO2i to a level consistent with the momentary tissue metabolic rate. Increase of intestinal O2 demand has been achieved by three means: instillation of luminal nutrients, intraarterial infusion of 2,4-dinitrophenol to uncouple oxidative phosphorylation, and increase of tissue temperature. In response to these perturbations, blood-perfused, denervated loops of small intestine demonstrated vasodilation and increased Qi, as well as an increased capillary exchange capacity and (a − vO2)i (24,146–149). In contrast, reduction of intestinal O2 demand, achieved by induction of tissue hypothermia, increased gut vascular resistance and reduced capillary exchange capacity (148).

1638 / CHAPTER 63 at ∼26 mm Hg; the decline in villous pO2 reflects an increased O2 demand in response to the active cotransport of glucose and Na+. Submucosal 1A and 2A arterioles and villous 3A and DA arterioles all dilated to an equivalent extent in response to glucose, and blood flow through the entire microvasculature increased ∼200%. Although dilation of the villous 3A and DA vessels might be ascribed to a direct effect of reduced pO2, dilation of the submucosal 1A and 2A vessels cannot. Furthermore, a temporal dissociation in arteriolar dilation was noted: submucosal 1A and 2A arterioles dilated ∼2 minutes after the villous vessels, suggesting that diffusion of a vasodilator agonist from villi to submucosa might have occurred. The effects of increased O2 supply to the microcirculation have been studied by changing the pO2 of the buffer suffusing the exposed mucosa. Increase of buffer pO2 to ∼70 mm Hg caused vasoconstriction of all arterioles and significantly attenuated the vasodilatory response to glucose suffusion; in contrast, reduction of buffer pO2 to ∼5 mm Hg induced a generalized vasodilation and increased the vasodilatory response to glucose. Similar results were reported in skeletal muscle (154). Increase of buffer pO2 induced a generalized arteriolar vasoconstriction; in contrast, manipulation of pO2 at a specific periarteriolar site by microinjection of buffer

Direct manipulation of O2 supply has been generated by induction of arterial hypoxia or by reduction of blood flow. These perturbations reduced intestinal vascular resistance and increased, or stabilized, intestinal blood flow; also, the capillary exchange capacity, and hence (a − vO2)i, increased as O2 delivery declined (149–152). Conversely, O2 supply has been increased by direct regulation of intestinal blood flow by pump perfusion, a circumstance that eliminates resistance vessel regulation as a means to sustain VO2i. Pump-induced increase of intestinal blood flow increased O2 delivery, but had no effect on VO2i insofar as capillary exchange capacity maintained a reciprocal relation with flow rate (Fig. 63-4); this relation was maintained when tissue metabolic rate was independently manipulated by changing environmental temperature (148). Evidence of Metabolic Regulation: Microvascular Studies The effects of increased O2 demand have been studied in the intestinal microvasculature by suffusing glucose over the mucosal surface, inasmuch as this action stimulates the active cotransport of glucose and Na+ and increases tissue metabolic rate (131,153). Glucose suffusion decreased villous pO2 from ∼15 to ∼7 mm Hg, although submucosal pO2 remained steady

Submucosal [Na+] (µm)

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FIG. 63-4. Effects of mucosal glucose exposure on submucosal [Na+] and submucosal 1A arteriolar diameter. Data obtained in a rat prepared for direct observation of the mucosal-submucosal vessels. Glucose (100 mg/ml) was suffused over the mucosal surface, whereas 1A diameter and submucosal [Na+] were measured. Note the temporal correlation between 1A vasodilation and submucosal [Na+]. (Reproduced from Bohlen [248], by permission.)

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1639 with increased or reduced pO2 had no effect on arteriolar diameter at that site. Identity of the Metabolic Feedback Signal The identity of the metabolic feedback signal remains unclear; indeed, it is likely that several signals coexist and function in different capacities. Numerous candidates have been advanced woven together by two common threads: production in response to cellular hypoxia and the capacity to induce vasodilation; thus, lactic acid, H+, K+, adenosine phosphates (ATP, ADP), adenosine, CO2, and lactic acid have been proposed (155). More recently, NO has been suggested as an important mediator of metabolic vascular regulation. NO and adenosine are discussed in more detail in this chapter because of their potential relevance to the intestinal circulation.

Adenosine Adenosine is formed by the sequential hydrolysis of ATP and induces vasodilation by binding to A1 or A2A purinergic receptors (156). The vasodilatory effect of adenosine is significantly attenuated by removal of the endothelium or by blockade of eNOS activity with L-arginine analogues, indicating that eNOS-derived NO is the mechanism of adenosine-induced vasodilation (157); in addition, blockade of ATP-sensitive K+ channels reduces the vasodilatory effect of adenosine (158). Adenosine has been proposed as a candidate for the metabolic feedback signal described previously insofar as sequential hydrolysis of ATP occurs during increased metabolic activity (increased O2 demand) or during reduced O2 supply, as occurs during arterial hypoxemia, that is, during a reduction in the O2 supply/demand ratio. Adenosine is an intestinal vasodilator, although its effects within the gut circulation are complex (159,160). Infusion of adenosine into the mesenteric artery causes a net increase gut blood flow, that is, an increase in the rate of flow across the intestine generated by a decrease in vascular resistance; however, it also causes a redistribution of flow within the gut wall, such that muscularis flow increases, whereas mucosal flow decreases. This physiologic observation has a clear anatomic footing insofar as the muscularis and mucosal microcirculations run in parallel, a circumstance that facilitates independent resistance regulation across each layer (see Fig. 63-2). Importantly, blockade of adenosine receptors has no effect on resting, or basal intestinal vascular resistance, indicating that although infusion of exogenous adenosine alters gut hemodynamics, it is unlikely that adenosine participates in setting basal vascular tone within the gut. Infusion of adenosine also decreases intestinal metabolic rate. In part, this phenomenon might reflect adenosine-induced shunting of blood away from the mucosa insofar as this layer accounts for more than 80% of this intestinal O2 consumption. However, adenosine also depresses the metabolic rate within in vitro strips of gut muscularis or mucosa, indicating a direct

effect on O2 utilization not requisite on vascular O2 supply. This direct effect may be mediated via adenosine-induced NO production insofar as NO depresses mitochondrial respiration (161). These observations appear at odds with a role for adenosine as the metabolic feedback signal in the gut. Other studies, however, indicate that adenosine plays an important role in regulation of the intestinal circulation during nutrient absorption (162). Pretreatment with dipyridamole, theophylline, or adenosine deaminase to attenuate adenosine activity essentially eliminates the increase in both intestinal blood flow and O2 consumption that follows the placement of nutrients into the gut lumen (163). Moreover, the concentration of adenosine in the venous effluent of isolated, perfused gut loops increases follows nutrient instillation (164). This hemodynamic effect occurs, in part, within the submucosal 1A arterioles; thus, blockade of adenosine significantly attenuates dilation of 1A arterioles, which normally occurs during suffusion of the mucosal surface with a solution of oleic acid and glucose (165). These observations present a paradox when placed into the context of the aforementioned adenosine infusion studies that demonstrated shunting of blood away from the mucosa, the obvious site of nutrient absorption, and hence metabolic activity. In part, however, this paradox may reflect the difference between exogenous infusion of adenosine into the mesenteric artery versus endogenous production within parenchyma or blood vessels in response to nutrient absorption. Adenosine also participates in hemodynamic stabilization of intestinal perfusion during sustained stimulation of postsynaptic mesenteric (adrenergic) nerves. Nerve stimulation increases intestinal vascular resistance, an effect mediated by α-adrenergic receptors; this hemodynamic effect is transient, however, because even in the face of continued nerve stimulation, vascular resistance returns to baseline in a process termed autoregulatory escape (110,166). Blockade of adenosine attenuates this escape response (15). On a microvascular level, that is, within submucosal 1A arterioles, adenosine also participates in this response. Sympathetic stimulation acutely reduces flow rate, and hence removes an important stimulus for eNOS activation, that is, shear stress (167). NO production is maintained, however, by an increase in local adenosine production that acts to replace shear stress as a stimulus of eNOS activity. This process is mediated by a reduction in periarteriolar pO2 that occurs in response to the reduced flow rate (168). The source of this adenosine appears to be within 50 µm of the arteriole; hence, it is unlikely that it is derived from the surrounding intestinal parenchyma as might be predicted based on the metabolic theory. It also did not appear to be RBCs, a potential source of ATP for the arteriolar wall, because these cells transit the intestinal microcirculation (143), nor can venulararteriolar diffusion of adenosine explain the process, even though such diffusion has been described (169). By exclusion, the most likely site of ATP is the arteriolar wall itself, a conclusion that supports the concept that the vascular endothelium serves as an oxygen-sensing system (141).

1640 / CHAPTER 63 Histamine Histamine (β-aminoethylimidazole) is a naturally occurring amine with multiple sites of origin in the vasculature, including the vascular endothelium, subendothelial mast cells, and adventitial mast cells (170). Histamine exerts its vasoactive effects by binding to H1, H2, or H3 receptors, alone or in combination. H1 and H2 receptors are located on endothelial cells, whereas H3 receptors are located on postganglionic adrenergic nerve fibers (171). Histamine binding to H1 and H2 receptors causes an endothelium-dependent vasodilation, the final mechanism of action of which is activation of eNOS with subsequent production of NO (172). Activation of H1 receptors also up-regulates transcription of eNOS; thus, the H1 receptor–mediated vasodilation has both acute and sustained components, because it activates existing eNOS and increases eNOS expression (170). The H3 receptor functions in a presynaptic inhibitory role and most likely generates vasodilation by inhibition of adrenergic vasoconstrictor tone (171). Histamine acts as an intestinal vasodilator (173,174). Infusion of histamine into the mesenteric artery causes a biphasic vasodilation: a rapid, spikelike reduction of intestinal vascular resistance that fades, followed by a sustained vasodilation. The early phase is mediated by H1 receptors, whereas the sustained phase is mediated by H2 receptors. Activation of H1 receptors also increases vascular permeability and is one of several factors responsible for the edema formation characteristic of an acute allergic reaction, including that which occurs in the gut in response to food allergens. Systemic infusion of imetit, a selective H3 agonist, reduces intestinal vascular resistance and increases gut perfusion; it also increases the perfused capillary density, most likely by opening precapillary sphincters within the mucosal microcirculation (171). This observation is consistent with the existence of a constitutive adrenergic constrictor stimulus at 1A and 2A submucosal arterioles, as well as the 3A arterioles and precapillary sphincters within the mucosa. Notably, although infusion of exogenous histamine or its receptor-specific analogues causes intestinal vasodilation, selective blockade of H1, H2, or H3 receptors, alone or in combination, has no effect on intestinal hemodynamics under resting conditions, suggesting that histamine does not participate in setting basal vascular resistance within the intestinal circulation (171,175). Histamine participates in at least three vascular regulatory responses within the intestinal circulation. First, blockade of H1 receptors blunts the vasodilation that normally occurs after feeding, or, in the case of isolated perfused gut loops, subsequent to luminal instillation of nutrients (175). This effect is likely of physiologic relevance in the generation of postprandial hyperemia inasmuch as feeding causes the release of histamine from the stomach and intestine (176). In this context, it is possible that histamine released during degranulation of mast cells within the villi in response to nutrient absorption (or in response to the physical stimulus provided by a chyme bolus) stimulates sensory afferents within the

villi that project to submucosal interneurons innervating submucosal arterioles (12). These neurons project to submucosal arterioles to generate an acetylcholine-mediated activation of eNOS, with subsequent vasodilation. Second, blockade of H1 receptors attenuates pressure-flow autoregulation within the intestine (177). In this instance, autoregulation is defined as the capacity of the intestinal circulation to vasodilate in response to pressure reduction, that is, an ability to maintain a stable flow rate despite a lower perfusion pressure. Finally, H1 receptor blockade weakens “autoregulatory escape” from norepinephrine infusion (15).

Glucagon Glucagon is produced within the pancreas, but also by neuroendocrine cells within the intestinal mucosa (178). Intravenous infusion of pharmacologic doses of glucagon (5 µg/kg/min) induces generalized intestinal vasodilation (179). On a microvascular level, glucagon dilates submucosal 2A and villous 3A arterioles, but not submucosal 1A arterioles; the net effect of this vasodilation is an increased flow rate to the villi (180,181). These studies were performed using doses of glucagon designed to duplicate in vivo glucagon levels, and thus are likely to be physiologically relevant. The mechanistic basis for glucagon-induced intestinal vasodilation is unclear. In the renal microcirculation, arteriolar vasodilation is not contingent on NO or prostaglandin synthesis; also, the vasodilatory effects of glucagon are not endothelium dependent, implying a direct effect on vascular smooth muscle tone (182). The vasodilatory effects of glucagon participate in the intestinal hyperemic response to chronic portal hypertension (183). Generation of chronic portal hypertension reduces intestinal vascular resistance, and thus increases the basal rate of gut perfusion. The basis for this effect is multifactorial and includes both neural and humoral factors; one of the humoral factors is glucagon. Thus, the glucagon concentration within the mesenteric venous effluent in portal hypertensive rats is threefold greater than in control animals, whereas cross-perfusion between portal hypertensive and control subjects causes intestinal vasodilation in the latter (184). The vasoconstrictor effect of norepinephrine within the intestinal circulation is decreased in portal hypertensive rats, and this effect is localized to the 2A and 3A arterioles within the submucosa and mucosa (181). Notably, other locally produced vasodilatory stimuli, such as NO, contribute to portal hypertension-induced gut vasodilation, such as PGI2, and that prostaglandin, as well as NO, compromises the vasoconstrictor efficacy of norepinephrine within the gut microvasculature (185).

POSTPRANDIAL HYPEREMIA One of the earliest demonstrations that nutrients altered intestinal hemodynamics was reported in 1910 by Brodie

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1641 and colleagues (186), who observed an increase in intestinal perfusion after the placement of Witte’s peptone solution into an in vivo loop of jejunum. Thereafter, Herrick and colleagues (187) reported an increase in flow rate through splanchnic arterial trunks (celiac axis, superior and inferior mesenteric arteries) in conscious subjects after ingestion of a meal. Subsequently, it was established that gastrointestinal blood flow increases in response to feeding, or in response to placement of nutrients into contact with the gut mucosal surface in several species: humans (188), baboons (189), dogs (146), cats (190), rats (191), sheep (192), and pigs (193). This physiologic phenomenon has been termed postprandial hyperemia. This section discusses hemodynamic changes initiated by nutrients in the small intestine and the mechanisms, specifically those nonneural in nature, that may participate in this response.

Sites of Postprandial Hyperemia The response to feeding occurs in two phases (168). The first or anticipation-ingestion phase entails a generalized cardiovascular response generated by the anticipation of a meal and is mediated primarily by sympathetic neural activity (194). Entry of food into the stomach elicits a substantial (∼200%) increase in celiac blood flow secondary to a decrease in local vascular resistance; the mechanistic basis for this vasodilation is complex and involves locally derived vasoactive effector systems (195). The increase in gastric perfusion is short lived because flow rate begins to decline within minutes of eating, despite the continued presence of a large portion of the meal (>90%) in the stomach (196). The second or digestive-absorptive phase is a sustained intestinal vasodilation mediated entirely by local vasoactive systems described later in this section. The duration of the second phase can last from 2 to 6 hours depending on the composition and size of the meal, and local contact between luminal nutrients and the mucosal surface is requisite for this nutrient-induced vasodilation (197). A similar requirement for nutrient contact occurs in the colon (198). Hemodynamic changes are site specific within the intestinal wall; thus, nutrient-induced hyperemia is localized to the mucosal layer, with minimal change occurring in the muscularis layers (193,195,199).

Constituents of Chyme Responsible for Intestinal Postprandial Hyperemia The magnitude by which superior mesenteric artery (SMA) flow rate increases after a meal is determined, in part, by the energy content of the meal (200). Isovolumic liquid meals consisting of corn oil, skimmed milk, and glucose fed to healthy volunteers caused an increased SMA flow rate that peaked ∼30 minutes after feeding. The magnitude of the increase was 106% with an 800-kJ meal versus 240% with a 4800-kJ meal, and the duration of the postprandial hyperemia was also greater in subjects fed a high-energy meal.

Studies performed using vascularly isolated in vivo gut loops have demonstrated that predigestion, the addition of bile, or both are requisite to induce postprandial hemodynamic changes (201). Placement of standard dog food that had been predigested by the addition of pancreatic enzymes into a canine jejunal loop elicited a brisk increase in blood flow and tissue O2 uptake. Instillation of pancreatic enzymes or undigested food alone had no effect. Separation of the meal into solid and liquid phases followed by instillation of each phase into separate jejunal loops showed that the vasodilatory stimulus is located in the liquid phase. Addition of bile to the mixture enhanced the hemodynamic effects, although bile itself had no effect on jejunal hemodynamics (202). These observations indicate that the water-soluble end products of the digested meal provide the stimulus for postprandial hyperemia. The individual constituents of chyme, that is, carbohydrate, protein, and fat, have unique effects on intestinal hemodynamics (197,201,203). Instillation of an isotonic glucose solution (150 mM) increases flow rate and tissue metabolic rate within isolated in vivo jejunal loops. Placement of oleic acid, a long-chain fatty acid, or the monoglyceride monoolein has no effect on jejunal hemodynamics; however, the same fats solubilized with bile or sodium taurocholate cause a marked hyperemia that is greater in magnitude than that induced by isotonic glucose. In this context, it is interesting that bile, which by itself has no effect on jejunal hemodynamics, increases blood flow in the ileum, its site of absorption (204). Chou and colleagues (201) reported that instillation of amino acids at physiologic concentrations, either alone or in the combination, have no effect on intestinal blood flow. Others, however, have demonstrated an increase in both intestinal flow rate and O2 consumption in response to luminal placement of isosmotic L-alanine (205). Ingestion of a protein meal (i.e., lean beef) increases SMA blood flow, whereas jejunal instillation of protein predigested with pancreatic enzymes increased flow through an in vivo jejunal loop (197,206). The osmolarity of luminal nutrients also modulates their intestinal hemodynamic effect (207). The degree of jejunal hyperemia induced by the solute phase of a mixed meal predigested with pancreatic enzymes increased as a function of osmolarity over the range of 183 to 1000 mOsm/L; solutions with an osmolarity less than 100 mOsm/L had no effect on flow rate. However, luminal osmolarity itself is not a factor: Instillation of nonabsorbable polyethylene glycol at 1000 mOsm/L has no effect on gut hemodynamics.

Mechanisms of Postprandial Hyperemia The preponderance of evidence suggests that the mechanisms responsible for intestinal postprandial hyperemia are locally derived; that is, these mechanisms begin and end within the intestine and its attendant circulation. Furthermore, it is clear that postprandial hemodynamic changes require contact between luminal nutrients and the intestinal mucosa.

1642 / CHAPTER 63 Partition of the mechanisms, although useful for presentation here, is clearly artificial inasmuch as a complex interplay among them is likely the rule. That caveat established, the following sections detail putative nonneural mechanisms responsible for postprandial hemodynamic events. Metabolic Vascular Regulation The rate of intestinal O2 consumption, and hence tissue O2 demand, increases after luminal instillation of nutrients into an in vivo gut loop or after feeding in a conscious subject (193,205,208–213). The metabolic theory of local blood flow regulation (see earlier) predicts that vasodilation of resistance vessels or opening of precapillary sphincters, or both, should occur in response to this nutrient-induced increase in gut O2 demand. This predication has been verified experimentally. Mangino and Chou (213) and Gallavan Jr. and Chou (212) demonstrated a significant correlation between the magnitude of a nutrient-induced increase in O2 demand and the magnitude of postprandial hyperemia. In contrast, Granger and Norris (208) reported that the effects of a nutrient-induced increase in gut O2 demand on the gut circulation were dependent on baseline hemodynamics within the intestine. When the arteriovenous O2 content difference across the gut loop, or (a − v)O2i was less than 5 ml O2/dl, the extent of postprandial hyperemia was relatively small. The nutrient-induced increase in gut O2 demand was primarily met by an increase of (a − v)O2i. However, if the baseline level of (a − v)O2i was more than 5 ml O2/dl, the level of postprandial hyperemia was proportionately greater. In either instance, the requisite changes in local O2 transport were achieved so that the nutrient-induced increase in O2 demand was satisfied, as predicted by the metabolic theory. These findings were confirmed by Shepherd (209), who provided direct evidence of an increase in the perfused capillary density in response to nutrient instillation into a jejunal loop pump perfused at a constant flow rate, thus preventing a nutrientinduced increase in flow rate in response to increased gut O2 demand. The time course of changes in flow rate and (a − v)O2i after nutrient placement into an in vivo jejunal loop are dissimilar. During the first 30 minutes, flow rate significantly increases and (a − v)O2i declines slightly, although the increase in flow is proportionately greater than the decline in (a − v)O2i, thus the net effect is a marked increase in gut O2 consumption. Thereafter, the flow rate declines, nearly reaching baseline 60 minutes after food instillation; however, (a − v)O2i progressively increases during this time so that gut O2 consumption is maintained at a level significantly greater than baseline (214). These data are consistent with the metabolic theory that proposes separate interactions between a parenchymally derived vasodilatory feedback signal and either or both resistance and exchange vessels, so that postprandial increases in blood flow or (a − v)O2i can occur jointly or independent of the other (215). An important component of the metabolic theory is that a compromise of tissue oxygenation generates the vasodilatory

feedback signal that restores or enhances O2 transport to a level consistent with O2 demand (215). In this context, Bohlen (153) demonstrated a reduction in villous apex pO2 from ∼15 to ∼7 mm Hg during mucosal suffusion with isotonic glucose solutions from 25 to 500 mg/ml made isotonic with sodium bicarbonate; simultaneously, submucosal pO2 remained unchanged. Glucose suffusion induced a significant vasodilation of 1A, 2A, 3A, and distributing arterioles. Furthermore, increasing or decreasing the pO2 of the mucosal suffusion solution to increase or decrease tissue O2 availability blunted or enhanced, respectively, the glucoseinduced vasodilation (131). These data, however, are not entirely supportive of the metabolic theory. Submucosal pO2 remained unchanged during mucosal glucose suffusion; however, submucosal 1A and 2A arterioles dilated to the same extent as the villous 3A and DA arterioles. Also, active transport of glucose and sodium across the villous epithelium reaches steady state within 30 seconds after the onset of glucose exposure, whereas dilation of 3A and DA arterioles did not occur until 1.5 to 2.0 minutes after initiation of the glucose suffusion. Other observations of postprandial intestinal hemodynamics are not consistent with metabolic vascular regulation. Glucose absorption generates an increase in gut O2 demand and elicits both a hyperemia and an increase in the perfused capillary density (211). In contrast, absorption of 3-O-methylglucose, which does not increase gut O2 demand, still induces a modest increase in blood flow (216). Similarly, a solution of oleic acid and bile induces a brisk hyperemia (greater in magnitude than that generated by glucose), but does not increase gut O2 demand (211). As described previously, the nature of the vasodilatory feedback signal(s) crucial for metabolic vascular regulation has yet to be determined, although adenosine has been proposed as a likely candidate. Adenosine is a potent intestinal vasodilator (160,217,218). Adenosine is released into the venous effluent of vascularly isolated jejunal gut loops after luminal instillation with a predigested meal (164). The nonspecific adenosine antagonist theophylline attenuates nutrient-induced vasodilation, but only when the concomitant level of jejunal motility is increased; in contrast, the more specific blocking agent 8-phenyltheophylline decreases nutrient-induced vasodilation regardless of the state of motility (162). Similarly, dipyridamole and adenosine deaminase abolished nutrient-induced hyperemia and eliminated the normal increase in plasma adenosine within the jejunal venous effluent (163). Gastrointestinal Hormones Feeding induces the release of numerous gastrointestinal hormones, some of which have vasoactive properties. Two of these hormones have been evaluated as mediators of postprandial hyperemia: cholecystokinin (CCK) and neurotensin. CCK is present in 2 active forms of 8 and 58 amino acids, respectively, both generated by posttranslational modification of a 115-amino-acid preprohormone (219). CCK is produced

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1643 in neurons throughout the central and peripheral nervous systems, and by intestinal neuroendocrine cells (I cells) located within the jejunal mucosa (220). The peptide has multiple roles in digestion, including effects on gallbladder emptying and intestinal motility. Early studies suggested that CCK extracted from intestinal homogenates had vasodilatory proprieties within the intestinal circulation (190,221). Later studies, using synthetic CCK, suggested that infusion of the peptide at pharmacologic levels was necessary to generate intestinal vasodilation (222). Furthermore, the amount of CCK released in response to luminal instillation of oleic acid plus bile, a potent stimulus of postprandial hyperemia, was minimal (∼ 1 ng/min/100 g intestinal weight) and less than that needed to affect blood flow; also, the duration of release was quite brief (223). Neurotensin is a 13-amino-acid peptide produced within neuroendocrine cells (N cells) present throughout the intestinal tract, but primarily in the distal jejunum and ileum (224,225). Systemic infusion of neurotensin (0.125 ng/kg/min) reduced intestinal vascular resistance 30%, thus increasing gut perfusion (226). Intraarterial infusion of neurotensin into vascularly isolated terminal ileum caused vasodilation and a significant increase in ileal blood flow; it also increased the radii of large capillary pores (227). Similar studies using jejunal gut loops, however, failed to demonstrate a significant vasodilation in jejunal gut loops, suggesting that the neurotensin vasodilatory effect was region specific within the small intestine (222). Neurotensin is released in response to feeding, as evidenced by an increase in neurotensin-like immunoreactivity in human plasma 2 to 4 hours after eating (228). The presence of neurotensin also increases in the venous effluent of vascularly isolated ileum (226) and jejunum (229) after luminal instillation of a micellar lipid solution, an action that also induced vasodilation and hyperemia. However, the plasma concentration of neurotensin necessary to generate ileal or jejunal vasodilation was several orders of magnitude greater than that noted within the intestinal venous effluent after luminal fat instillation. Prostaglandins Under resting conditions, the arteriovenous gradient of prostaglandins across a vascularly isolated jejunal loop indicates intestinal production of PGI2, PGE2, thromboxane A2 (TXA2), and PGF2α, and intestinal prostaglandin production, particularly of PGI2 and PGE2, increases after feeding (230,231). Localization of prostaglandin production within the gut wall reveals heterogeneity; thus, TXA2 is the principal cyclooxygenase product produced within the mucosa, whereas generation of PGI2 is dominant in the muscularis (232). The site of intestinal prostaglandin production also includes the vascular endothelium, especially for PGI2 and TXA2 (233). Prostaglandins induce variable effects on intestinal vascular tone: PGE2 and PGI2 are vasodilators, whereas TXA2 and PGF2α are vasoconstrictors (234,235). Under resting conditions, inhibition of cyclooxygenase with indomethacin or mefenamic acid increases vascular

resistance and reduces blood flow in isolated jejunal loops (212). Despite this apparently vasoconstrictive effect, the magnitude of nutrient-induced hyperemia is significantly increased in the presence of cyclooxygenase inhibition. This paradoxical effect could reflect the vasoconstrictive effect of cyclooxygenase under resting conditions (236). The net effect of a vasodilator is determined, in part, by the initial state of resistance. If the basal vascular resistance is high, then the magnitude of vasodilation in response to locally derived adenosine may be increased. Alternatively, the effect of cyclooxygenase inhibition could be indirect. Some prostaglandins inhibit active transport of glucose, so that cyclooxygenase blockade increases both transport and metabolism of glucose within the intestine, processes that use O2 (237,238). Cyclooxygenase blockade during mucosal glucose suffusion thus increases the level of tissue O2 demand that accompanies this process. Interpreted within the context of the metabolic theory presented previously, this action could lead to a greater degree of postprandial hyperemia, the latter generated by the metabolic feedback signal. Another indirect means by which prostaglandins might indirectly alter postprandial hemodynamics is via their established interaction with histamine receptors (239). Although exogenous histamine acts as an intestinal vasodilator, blockade of histamine H1 and H2 receptors blunts postprandial hyperemia so that the prostaglandin effect might be mediated by reducing the inhibitory effects of histamine (175). A specific role for TXA2 in modulating postprandial hyperemia has been studied. Imidazole, a selective inhibitor of TXA2 synthesis, has no effect on resting vascular resistance or on the vasodilatory effects of luminal instillation of predigested food into an isolated jejunal loop. Intramesenteric artery infusion of TXA2 causes intestinal vasoconstriction that is more significant in the mucosal than the muscularis layers of the gut (240), an effect blocked by the specific TXA2 receptor antagonist SQ 29548 (241). Hence, despite the presence of TXA2 receptors within the mucosal microcirculation, neither the constitutive nor nutrient-induced release of TXA2 appears to participate in regulation of jejunal vascular resistance (230). However, imidazole increases jejunal O2 consumption under resting conditions and in response to nutrients, and this effect is not mediated by alteration of glucose transport or metabolism (242). Instead, the imidazole-induced increase in (a − v)O2i reflects a direct effect on capillary perfusion (i.e., on precapillary sphincters within the villi) insofar as the capillary filtration coefficient, Kf,c, a marker of the perfused capillary density, was increased after imidazole (243). The effects of prostaglandins on postprandial hyperemia also have been investigated within the intestinal microvasculature, specifically the 1A submucosal arterioles (244). The glucose-induced increase in arteriolar flow rate is unaffected by cyclooxygenase inhibition with meclofenamate or indomethacin. In contrast, the increase in arteriolar flow rate induced by a solution of oleic acid plus bile is significantly attenuated by cyclooxygenase inhibition. Arachidonate has no effect on submucosal arteriolar flow rate.

1644 / CHAPTER 63 However, arachidonate plus indomethacin caused a significant increase in this parameter, suggesting that a nonprostaglandin metabolite of arachidonate induces arteriolar vasodilation. In this context, epoxyeicosatrienoic acids, arachidonic acid metabolites produced by cytochrome P450 monooxygenase, induces intestinal vasodilation (245). Sodium-Induced Hyperosmolarity Sodium (Na+) is cotransported with simple sugars and many amino acids (246,247). This action generates a concomitant Na+-induced hyperosmolarity within the villus during glucose absorption (248), a circumstance that is greatly increased by the apparent existence of a countercurrent exchange system for Na+ within the villus such that a substantial osmolarity gradient is present from the villous tip to base under resting conditions that is substantially increased during glucose absorption (246,247). Interstitial Na+-induced hyperosmolarity can activate eNOS by altering the fluxes of Na+ and Ca2+ across the endothelial cell membrane (249). The process entails two steps: first, Na+ enters the endothelial cell via the Na+-K+-2Cl− cotransporter or via the Na+-H+ exchanger. Second, the Na+-Ca2+ exchanger extrudes the Na+ from the cell in favor of Ca2+ entrance, thus activating eNOS. It is therefore possible that Na+-induced hyperosmolarity generated within the villus during nutrient absorption can lead to arteriolar vasodilation. Perivascular [Na+] increases during glucose absorption within the villi and submucosa (248). An increase of ∼70 mM occurs in the upper half of the villi, whereas the increase in the submucosa is ∼35 mM. Submucosal and villous arterioles dilate when perivascular [Na+] increases during mucosal glucose suffusion; furthermore, dilation of the submucosal 1A arterioles occurs within the same time frame as the increase in submucosal [Na+] (see Fig. 63-4). Dilation of submucosal 1A and villous 3A arterioles is temporally disparate during mucosal glucose suffusion; that is, villous 3A arterioles reach steady-state dilation ∼1.5 minutes earlier than submucosal arterioles (153). If Na+-induced hyperosmolarity is responsible for dilation of submucosal 1A and 2A arterioles, then a mechanism must be present by which transport of Na+ or other vasodilator stimuli are transported from villi to submucosa. Simple passive diffusion is unlikely because the distance precludes timely movement (5). Two means by which transport of a chemical stimulus such as Na+ from villi to submucosa are venous blood and lymph, and both mechanisms have been demonstrated. The lymphatic system within the bowel wall is ideally suited for movement of chemical vasoactive stimuli from villus to submucosa (248). In rats, two to three lacteals are present within each villus, whereas a single lacteal is present in dog, cat, and rabbit intestine (Fig. 63-5). Villous lacteals empty directly into a dense submucosal plexus. Lymphatics from the muscularis layers do not drain into the submucosal plexus, but rather merge with large collecting lymph channels that exit the bowel wall. Lymphatics within the submucosa are in close contact with arterioles and venules and are highly

permeable, and the movement of lymph from villous lacteal to the submucosal lymphatic plexus is rapid and is primarily dependent on bowel motility (250). Mucosal suffusion with glucose or oleic acid plus taurocholate increases osmolarity of the lymph within the submucosal plexus (Fig. 63-6) (251). This hyperosmolarity, which is likely caused by Na+, can generate dilation of the submucosal arterioles. Thus, direct perfusion of submucosal lymphatics with media of varying [Na+] (and hence osmolarity) induced osmolarity-dependent dilation that was dependent, in part, on the generation of NO, as evidenced by the inhibitory effect of an L-arginine analogue that attenuated NO production (252). Production of NO by submucosal lymphatics also occurs in response to microiontophoresis of acetylcholine or bradykinin, agents that activate eNOS via augmentation of endothelial cell [Ca2+], confirming that the lymphatic endothelium is capable of producing eNOS-derived NO (253). Collectively, these observations indicate that Na+-induced hypertonicity generated within the villus can be transferred to the submucosal lymphatic plexus, and thereafter activate eNOS to generate NO at a site immediately proximate to the submucosal arterioles, causing vasodilation therein. Another potential route is the venules that drain the villi. The collecting venules within the villi drain into a dense mucosal plexus, which then drains into 2V and 1V venules within the submucosa (see Fig. 63-1). The 1V venules run in close proximity (<30 µm) to 1A arterioles, facilitating venule-to-arteriole diffusion (254,255). Mucosal suffusion with glucose or oleic acid plus taurocholate increases plasma osmolarity of blood within submucosal venules (Fig. 63-7). The role of the venular route of ascent and transfer of vasoactive stimuli from villi to submucosa can be confirmed by blocking movement of lymph. Under this condition, the submucosal arterioles still dilate in response to mucosal glucose suffusion (256). Both lymphatic and venular pathways thus contribute to the ascent of Na+-induced hyperosmolarity to the submucosa where it induces vasodilation. The cotransport of Na+ and glucose into the villus also plays a role in setting basal vascular tone within the intestine (156). The intestine constantly maintains a state of relative hyperosmolarity at the villous tip. Elimination of Na+ transport by isotonic replacement of the ion with mannitol in the mucosal bathing solution led to a significant increase in vascular resistance in 1A-2A arterioles, as well as in villous 3A arterioles, and a concomitant decrease in microvascular flow. These vascular effects were mediated by a loss of NO production. Substitution of mannitol for Na+ also reduced tissue O2 consumption. This latter effect likely reflected the large metabolic work necessary to maintain active Na+ cotransport, and hence the hypertonicity of the villous interstitium. Dilation of Upstream Small Mesenteric Arteries The SAs immediately upstream of the submucosal arteriolar plexus, that is, the terminal arteries of the mesenteric arterial arcade that pierce the muscularis, play an important role in setting overall vascular resistance across the intestine (256).

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1645 Lymphatic sinus Muscle layer

Submucosa

Lacteal Valve

Rat villus

Lacteal

Rabbit, dog and cat villus

FIG. 63-5. Anatomy of lymphatics within the intestinal wall. Two villi are shown: villous anatomy of rabbit, dog, and cat villi in which a single central lacteal is present; and villous anatomy of the rat, in which two to three lacteals are present. In all species, the villous lacteals drain directly into a dense submucosal plexus that is entirely devoid of valves. Although not shown, the submucosal lymphatic plexus lies in close proximity to 1A and 2A submucosal arterioles, providing an anatomic basis for lymphto-arteriole diffusion of vasoactive agents ascending from the villi via the lymphatics. The muscularis layers have a less dense lymphatic plexus that does not connect directly with the submucosal plexus. Lymphatic drainage from both areas merges to exit the bowel wall. (Reproduced from Unthank and Bohlen [250], by permission.)

10

360 Submucosal lymph osmolarity mOsm

8

7 14

8

11 8

340

320 14 300

280 0

35

65

125

250

Glucose concentraton mg %

550

5

20

Oleic acid mM

FIG. 63-6. Submucosal lymph osmolarity during mucosal nutrient exposure. Data were collected in rats. Submucosal lymph was collected by direct aspiration during suffusion of the mucosal surface with either glucose (25–550 mg %) or oleic acid + taurocholate (5 or 20 mM). Data are given as means ± standard error of the mean. The number above the bar denotes the number of observations. (Reproduced from Bohlen and Unthank [251], by permission.)

1646 / CHAPTER 63 360

10 7 9

Submucosal venular blood osmolarity in mOsm

340 9

12 9

10

320 16

300

280 0

35

65

125

250

Glucose concentraton mg %

550

5

20 Oleic acid mM

FIG. 63-7. Submucosal venular blood osmolarity during mucosal nutrient exposure. Data were collected in rats. Submucosal venular blood was collected by direct aspiration during suffusion of the mucosal surface with either glucose (25–550 mg %) or oleic acid + taurocholate (5 or 20 mM). Data are given as means ± standard error of the mean. The number above the bar denotes the number of observations. (Reproduced from Bohlen and Unthank [251], by permission.)

Dilation of these vessels is crucial to the maintenance of postprandial hyperemia. Three mechanisms could account for this dilation. First, conducted vasodilation, wherein cell–cell communication spreads vasodilation upstream from the microvasculature to SAs has been described in the hamster cremaster muscle (257). Second, dilation of the downstream microvasculature will increase flow rate through the upstream SAs, leading to flow-induced dilation of these SAs, as described earlier. Third, diffusion of vasoactive agents from vein to artery could occur. These agents could include dilators, such as NO, or agents that might stimulate vasodilator production by the arteriole, such as Na+ as described in the previous section. The possibility of conducted vasodilation, that is, cell–cell communication, has not yet been studied in the intestinal circulation. However, the latter two possibilities have received attention. Bohlen (258) measured perivascular [NO] at the SA and vein immediately upstream from the intestinal wall. At rest, perivenular [NO] exceeded periarterial [NO] by ∼50 nM; this difference increased to ∼85 nM during mucosal suffusion with glucose, whereas the artery dilated ∼17%. Downstream blockade of the vein to prevent venous-to-arteriolar diffusion reduced periarterial [NO] and reduced the glucose-induced arterial dilation by 50%. Superfusion of the mesentery with medium made hypertonic with Na+ to duplicate the glucose-induced increase in Na+ increased periarterial [NO] and induced vasodilation. It thus appeared that venous-to-arterial diffusion generated, in part, the upstream vasodilation noted during glucose-Na+ cotransport. Work from the same laboratory has demonstrated

the importance of NO-based, flow-induced dilation in these vessels. A mechanically forced increase in flow rate increased periarterial [NO] and caused vasodilation, whereas reduction of flow rate, generated by decreasing metabolic rate, reduced periarterial [NO] and caused vasoconstriction. Summary of the Putative Mechanisms of Postprandial Hyperemia The relative role of the aforementioned mechanisms in inducing and sustaining postprandial hyperemia is unknown. Under carefully controlled experimental conditions, the mechanistic basis for the vasodilation that follows mucosal suffusion with specific nutrients (e.g., glucose or oleic acid + taurocholate) appears to be clear. Although it is necessary to dissect the postprandial response in this manner to identify specific mechanisms, the in situ experience remains profoundly complex. However, three observations appear consistently: (1) nutrient absorption increases tissue metabolic rate, and hence O2 demand, and this process generates vasodilation; (2) transport of agents from the villi to the submucosa and the small mesenteric arteries via lymphatics and venules is requisite for upstream vasodilation; and (3) NO is an important final mediator of nutrient-induced vasodilation. This chapter has focused on nonneural mechanisms of intestinal vascular regulation insofar as neural regulation is covered elsewhere in this textbook. Notably, however, sensory afferents that ascend from the villi to interact with

PHYSIOLOGY OF THE CIRCULATION OF THE SMALL INTESTINE / 1647 submucosal ganglia of the enteric nervous system may also participate in postprandial hyperemia. Interaction between neural and nonneural mechanisms most likely generates the final response to luminal nutrients under in situ conditions.

ACKNOWLEDGMENTS The work was supported by grants HD25256 (National Institute of Child Health and Development) and DK65306 (National Institute of Diabetes and Digestive and Kidney Diseases).

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Sugar Absorption Ernest M. Wright, Donald D. F. Loo, Bruce A. Hirayama, and Eric Turk Overview, 1654 SGLT Gene Family, 1654 GLUT Gene Family, 1654 Absorption of Glucose, Galactose, and Fructose, 1654 SGLT1, GLUT2, and GLUT5 Are the Major Intestinal Sugar Transporters, 1656 SGLT1 Sugar Selectivity, 1656 GLUT Sugar Selectivity, 1657 SGLT1 Cation Selectivity, 1657 Transport Kinetics, 1658

SGLT1, 1658 Presteady-State Kinetics, 1660 GLUTs, 1661 Genetic Defects of Sugar Absorption, 1662 Glucose-Galactose Malabsorption, 1662 Fanconi–Bickel Syndrome, 1662 Fructose Malabsorption, 1662 Regulation of Sugar Absorption, 1663 Future Directions, 1663 Acknowledgments, 1663 References, 1664

Humans need to extract energy from their environment to grow and to function normally. Energy comes from food in the form of carbohydrate, fat, and protein, which before absorption by the small intestine must be processed and digested to monosaccharides, peptides and amino acids, and fatty acids. Glucose plays a central role in energy metabolism: Adults use ~250 g daily, and about half comes directly from carbohydrate in the diet, the remainder coming from either stored glycogen or gluconeogenesis. A major challenge is to ensure a constant supply of glucose to the brain through the blood–brain barrier (125 g/day); thus, the body goes to extreme lengths to ensure glucose homeostasis. Brain function is severely compromised if blood glucose declines to less than 3.5 mM, and bodily functions go awry over the long term if the regulation of blood glucose is impaired, for example, by diabetes. Perhaps surprisingly, glucose is not an essential nutrient, and this is best exemplified by people who are intolerant of glucose in their diet, for example, those with glucose-galactose malabsorption

(GGM) who live a full, healthy life on a carbohydrate-free diet (see later). In these cases, glucose is provided by gluconeogenesis. In the United States, a healthy adult consumes between 100 and 800 g carbohydrates, which provides more than half of the calories needed for normal activity. Starches account for about 50% of the carbohydrates per day, and the remainder consists of disaccharides, such as sucrose, lactose, and maltose. An ever increasing amount of fructose is consumed as this sugar is used as a sweetener in drinks and foods. Starches are digested to D-glucose, first by salivary and pancreatic amylases, and then by enzymes (isomaltase) anchored in the brush-border membrane of enterocytes. Brush-border enzymes, sucrase and lactase, hydrolyze sucrose and lactose to glucose, galactose, and fructose. Digestion and the absorption of the free monosaccharides are generally complete by the time the food (chyme) reaches the distal jejunum. Lactose in milk is the sole source of dietary carbohydrate in young children, and the premature introduction of other sugars (sucrose and fructose) into the child’s diet may produce diarrhea because of the poor absorption of fructose. In this edition of this textbook, we focus on the molecular mechanism of glucose, galactose, and fructose absorption across the human intestine. See the previous editions for a historical perspective, in-depth reviews of carbohydrate digestion, and the earlier literature on intestinal sugar absorption (1–3).

E. M. Wright, D. D. F. Loo, B. A. Hirayama, and E. Turk: Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1653

1654 / CHAPTER 64 OVERVIEW The challenge in absorbing dietary glucose, galactose, and fructose across the small intestine stems from the fact that these polar molecules permeate slowly, if at all, across the lipid bilayer of plasma membranes of the epithelial cells lining the small intestine. Furthermore, there is no compelling evidence for any significant absorption of sugars between the epithelial cells, that is, the paracellular pathway through the tight junctions, as exemplified by the malabsorption in subjects with mutant sugar transporter genes (see later). Thus, the bulk of sugar absorption is mediated by specific sugar transport proteins in the apical (brush-border) and basolateral membranes of the enterocytes lining the small intestine. There are two classes of sugar transport proteins, the facilitated glucose transporters or uniporters (GLUTs of the SLC2 gene family) (4) and the sodium-glucose cotransporters or symporters (SGLTs of the SLC5 gene family) (5). The uniporters simply facilitate the transport of glucose down the glucose concentration gradient, whereas the cotransporters use the potential energy stored in the Na+ (or H+) electrochemical potential gradient across the plasma membrane to drive uphill glucose transport (“active” transport).

SGLT GENE FAMILY The human genome contains 11 members of the sodium/ glucose cotransporter gene family (SLC5) (5). SGLT1 (SLC5A1), the founder member, is expressed at high density in the small intestine, and there is compelling evidence that this gene codes for the transporter responsible for the bulk of glucose transport across the brush-border membrane of the mature enterocytes (see later). SGLT2 is highly expressed in the kidney where there is evidence that it codes for the transporter largely responsible for the reabsorption of glucose from the glomerular filtrate. SGLT3 is expressed in cholinergeric neurons of the enteric nervous system, not enterocytes, where it behaves as a glucosensor (6). SGLT4 is widely expressed in the human body, including the intestine and kidney, and evidence suggests that this functions as a mannose transporter (7). SGLT5 expression is virtually limited to the kidney, but the function of this gene has not yet been established. SGLT6 is expressed in many different organs and tissues including the intestine, kidney, brain, and lung. At least in the case of rabbit kidney, there is evidence that SGLT6 functions as a brush-border Na+/myoinositol cotransporter, yet also transports glucose with a low affinity (8). The remaining five members of the SLC5 gene family code for myoinositol, iodide, biotin, choline, and short-chain fatty acid cotransporters. Given that membrane proteins are multifunctional (9), it is possible that genes other than SGLTs can function as sodium/glucose cotransporters in the intestine. Some credence to this is provided by the identification of a novel gene that codes for a rat renal brush-border Na/glucose (fructose) cotransporter (10). The gene coding for SGLT1 is located on chromosome 22 (22q13.1) (11). The organization of SGLT1-6 is similar with

15 exons, but there is considerable variation in the size of the introns. All code for membrane proteins of approximately 70 kDa with 14-transmembrane–spanning helices. More than 450 members of the SGLT (SSF) gene family are entered in the Swiss-Prot database, and approximately 230 of these share a common secondary architecture and a consensus sequence pattern [GS]-2(2)-[LIY]-x(3)-[LIVMFWSTAG](7)-x(3)[LIV]-[STAV]-x(20-G-G-[LMF]-x-[SAP] (9).

GLUT GENE FAMILY The human GLUT (SLC2) family of glucose transporters contains 13 members (4). GLUT1 (SLC2A1) was the first to be cloned, and this gene is highly expressed in red blood cells and the blood–brain barrier. GLUT2 (SLC2A2) is expressed in the intestine and kidney, where it plays an important role in facilitated glucose transport across the basolateral membrane. GLUT3 protein is located mainly in the brain, where it is responsible for glucose uptake into neurons, and GLUT4 is the major glucose transporter in adipocytes and skeletal and cardiac muscle. GLUT5 is not a glucose transporter, but instead is a fructose transporter that is expressed in the brush-border membrane of enterocytes. GLUT7 also has been detected in rat intestinal brush-border membranes, and oocyte expression studies indicate that this protein behaves as a high-affinity transporter for glucose and fructose (K0.5 60–300 µM) that does not accept galactose (12). Whereas considerable attention has been given to the physiologic significance of GLUT1-5, the importance of GLUT7 (as well as GLUTs 6, 8, 9, 10, 11, and 12) in intestinal sugar absorption has yet to be determined. The structure of GLUT proteins and other members of the gene family have been actively pursued. All appear to contain 12-transmembrane helices with both the N and C termini located in the cytoplasmic side of the plasma membrane. High-resolution atomic models have been derived from the electron diffraction pattern crystals of two bacterial members of the MFS family, the lactose permease and the glycerol 3-phosphate transporters (13,14), which are consistent with these two-dimensional structural models. Direct evidence for the oligomeric state of GLUTs is lacking.

ABSORPTION OF GLUCOSE, GALACTOSE, AND FRUCTOSE The epithelial cells of the small intestine use the SGLT1 and GLUT2 transporters in a clever way to actively absorb glucose and galactose from the gut lumen into the blood (Fig. 64-1). First, the SGLT1 is expressed exclusively in the brushborder membrane of the enterocyte. SGLT1 uses the Na+ electrochemical potential gradient across the brush border to drive the uphill transport of glucose and galactose from the gut lumen into the enterocyte. Two sodium ions accompany each sugar molecule transported across the brush border by

SUGAR ABSORPTION / 1655 Basolateral membrane Brush border membrane

Glucose

G6P

Na+

Glucose

Glucose

Glucose (Galactose)

Na+

Na/K Pump K+

Tight junction

Lateral intercellular space

FIG. 64-1. Model for glucose absorption across enterocytes. Glucose (and galactose) is accumulated within the cell from the gut lumen by the Na+/glucose cotransporter (SGLT1) in the brush-border membrane. The sodium electrochemical potential gradient across the brush-border membrane provides the energy for uphill sugar transport. The two sodium ions that enter the cell with each glucose molecule are pumped out across the basolateral membrane by the Na-K pump. The glucose (and galactose) that accumulates within the cell exits across the basolateral membrane into the blood either directly via the facilitated glucose transporter GLUT2 or indirectly by transport of glucose 6-phosphate into intracellular vesicles and the subsequent exocytosis. Glucose absorption is normal in GLUT2-deficient human and animal subjects, but is impaired in the absence of sugar phosphorylation.

SGLT1, and the sodium gradient across the membrane is maintained by the Na-K pump in the basolateral membrane. The kinetics of the pump are poised such that any increase in intracellular Na concentration triggers an increase in Na transport out of the cell across the basolateral membrane. The sugar that accumulates within the epithelium then passes into the blood across the basolateral membrane through a facilitated glucose transporter (GLUT2). There is net efflux of sugar from the cell as long as the intracellular glucose and galactose concentrations are greater than those in blood. In the steady state, the operation of SGLT1, GLUT2, and the Na-K pump results in the net transport of two Na+ ions and one glucose molecule across the epithelium. Two anions and water follow to maintain electroneutrality and osmotic balance (15). As much as 6 L water is linked directly, or indirectly, to glucose absorption, and this provides an explanation for the success of oral rehydration therapy to combat infectious diarrheas such as cholera (16). Fructose is transported across the brush border by GLUT5, another member of the GLUT family of uniporters. It is not transported by SGLT1 and this is best supported by the finding that fructose absorption is normal in those patients with defects in glucose transport across the brush border (patients

with GGM) (17). Fructose also is transported by GLUT2, which means that fructose transport across both the brush border and basolateral membranes of the enterocyte occurs by facilitated diffusion; that is, the rate and direction of net fructose transport depends strictly on the fructose concentration gradient across the intestinal epithelium. Fructose absorptive capacity is limited in both children and adults. In rats, expression of GLUT5 in the gut does not occur until weaning, which may account for fructose intolerance (toddlers’ diarrhea) (18); in adults, oral ingestion of 50 g fructose results in malabsorption symptoms in 50% of subjects in both the United States and the United Kingdom (19) (B. H. Hirst, personal communication). This is due in part to the low affinities of GLUT5 (Km 6–14 mM) and GLUT2 (66 mM) for fructose (20) and in part to the limited expression of GLUT5 (21). There is some evidence that there is a second, low-affinity glucose transporter in brush-border membranes in some animals, and it has been suggested that this is GLUT2 (22). It is proposed that GLUT2 is targeted to the rodent brushborder membrane under certain experimental manipulations, and that this results in an increase in both glucose and fructose absorption (see also Gouyon and colleagues [23]). The significance of such findings with respect to sugar absorption

1656 / CHAPTER 64 in humans is unclear, especially in light of the lack of significant glucose absorption in patients with GGM. Adult patients with GGM have negative glucose oral tolerance tests; that is, they show no increase in blood glucose levels after a test meal (17). These patients are intolerant of glucose (and galactose) in their diets, and symptoms of malabsorption are elicited by ingestion of as little as 6 g glucose (24).

BBM SGLT1

SGLT1, GLUT2, AND GLUT5 ARE THE MAJOR INTESTINAL SUGAR TRANSPORTERS Now that the Human Genome Project is nearing completion, the challenge is to determine the physiologic role(s) of each gene. What evidence is there for the roles of SGLT1, GLUT2, and GLUT5 in sugar absorption in the gut? The following evidence exists for the role of SGLT1 in sugar absorption: (1) the gene is expressed in the intestinal epithelium (25); (2) the open reading frame of the gene codes a 664-amino-acid membrane protein with a predicted weight (73 kDa) consistent with the brush-border Na/glucose cotransporter (26); (3) SGLT1 antibodies immunoreact with the brush-border membrane of mature enterocytes (Fig. 64-2); (4) the kinetics, sugar and cation specificity, and phlorizin sensitivity of the cloned transporter expressed in heterologous expression systems fit closely with those for sugar transport in native intestinal tissues, cells, and brush-border membranes (see later); (5) the inherited disorder GGM is caused by mutations in SGLT1 (17); and (6) in the sheep model, the levels of SGLT1 protein expression correlates closely with the level of Na/glucose transport over several orders of magnitude (27,28). The following evidence exists for the role of GLUT2 in sugar absorption: (1) the kinetics, specificity, and inhibition of glucose transport into basolateral membrane vesicles agrees closely with those for glucose transport by GLUT2 in heterologous expression systems (20,29); (2) GLUT2 is expressed in the intestinal epithelium; (3) GLUT2 antibodies immunoreact with the basolateral membranes of intestinal enterocytes (see Fig. 64-2); and (4) mice lacking functional GLUT2 genes in the small intestine are unable to absorb the model substrate 3-O-methyl-D-glucoside (30). The following evidence exists for the role of GLUT5 in sugar absorption: (1) the kinetics, specificity, and inhibition of fructose transport into brush-border membrane vesicles and across the brush-border membrane in intact tissues agree closely with those for fructose transport by GLUT5 in heterologous expression systems (see Wright and colleagues [1]); (2) GLUT5 is expressed in the small intestine (see Wright and colleagues [1]); (3) GLUT5 antibodies immunoreact with intestinal brush-border membranes (see Wright and colleagues [1]); and (4) there is a good correlation between the level of GLUT5 messenger RNA (mRNA) and protein in the small intestine and the level of fructose transport in rodents as a function of development and diet (21). Other sugar transporter genes, SGLTs and GLUTs, may also be expressed in the intestinal epithelium (see earlier),

BLM GLUT2

Nu

FIG. 64-2. Location of Na+/glucose cotransporter 1 (SGLT1) in brush-border membrane and glucose transporter 2 (GLUT2) of enterocytes lining the upper villus of rat small intestine. A villus from a rabbit intestine was stained with antibodies against the Na+/glucose cotransporter SGLT1 (yellow) and the facilitated glucose cotransporter GLUT2 (red). SGLT1 is exclusively localized to the brush-border membrane (BBM), whereas GLUT2 is confined to the basolateral membrane (BLM). The basally located nuclei (Nu) are stained with DAPI (4′,6′-diamino-2-phenylindole-2 hydrochloride). The antibodies were gifts from M. Kasahara (Teikyo University, Tokyo, Japan; anti-SGLT1) and W. Pardridge (UCLA, Los Angeles, CA; anti-GLUT2). (See Color Plate 33.) (Courtesy of M. P. Lostao and E. M. Wright, UCLA, Los Angeles, CA.)

but further studies are required to define any putative functional role in sugar absorption.

SGLT1 SUGAR SELECTIVITY Sugar selectivity has been carefully examined in Xenopus laevis oocytes expressing human SGLT1 (hSGLT1) using electrophysiologic assays, that is, sugar-induced inward current measurements. Control studies have demonstrated that the sugar currents are sodium currents and that the rate of sugar uptake is directly proportional to the sodium current (31,32). The advantage of the electrical assay over radioactive tracer assays (when they are available) is that the kinetics of lowaffinity substrates can be determined easily. Nontransported blockers of Na/glucose cotransport also have been identified by their effect on the glucose-induced currents and shifts in presteady-state kinetics (33–35). In previous studies, sugar transport selectivity has been inferred from competitive inhibition of glucose transport, but it is now obvious that

SUGAR ABSORPTION / 1657 inhibitors may not be themselves transported substrates. It is noted that the structural differences between substrates and inhibitors may be quite subtle (33–35). D-Glucose and D-galactose are the most abundant sugars in the diet and are transported by SGLT1 with identical apparent affinities, 0.5 mM, and maximum rates of transport (Jm) (36). The only structural difference between D-glucose and D-galactose is that the C4 hydroxyl group is in the axial, not equatorial, position in D-galactose. Neither fructose nor disaccharides interact with SGLT1. A hallmark of Na+/glucose cotransporters has been their restricted sugar selectivity, that is, the sugar must be a pyranose with an equatorial -OH at C2 (Fig. 64-3): the pentose homomorph of D-glucose, D-xylose, is a poor substrate (K0.5 ~100 mM); D-mannose with an axial -OH at C2 does not interact, and 2-deoxy-D-glucose is poorly transported (K0.5 ~110 mM with a Jmax identical to that for D-glucose). It is noted that 2-F-2-deoxy-D-glucose (FDG) is a good substrate for GLUTs, but is poorly transported by SGLT1. Another distinction between the substrates for SGLT1 and GLUTs is that α and β methyl-glucopyranosides are substrates for SGLT1, but not the GLUTs (29,37). Ethyl and propyl glucopyranosides are also substrates for SGLT1, but longer alkyl derivates are nontransported blockers. Using our approach, we have examined the interactions among a diverse array of glucopyranosides and hSGLT1 (33–35). A wide variety of aromatic β-glucopyranosides (phenyl, naphthyl, and indolyls) are transported; for example, indican is transported with high affinity, 60 µM, but with a low maximal rate, 14% of that for D-glucose. However, subtle changes in structure convert the glucopyranoside to either a blocker or a noninteracting species: p-nitrophenylD-glucopyranoside is a blocker, whereas phenyl- and p-hyroxyphenyl-D-glucopyranosides are substrates; and 5-Br3-indoyl-D-galactopranoside is a blocker, whereas indoxyl-βD-glucopyranoside (indican) is a high-affinity substrate. The best known inhibitor of Na+/glucose cotransport is phlorizin, a plant glucopyranoside. It is a specific, nontransported, competitive blocker SGLT1 with a Ki of 30 to 200 nM. Notably, phlorizin is actually transported by a mutant SGLT protein

6 4 5 3

2

expressed in oocytes as determined by [3H]-phlorizin uptakes and currents (K0.5 5 µM) (38). There is considerable interest among nutritionists in the ability of the intestine to absorb glucopyranosides from herbs and health foods and beverages, and the pharmaceutical industry is using phlorizin as a lead compound to develop specific inhibitors of the intestinal and renal SGLTs. The -OH group at C3 also appears to be important for sugar transport: 3-deoxy-D-glucose and D-allose (the -OH is axial) are poor substrates. 3-O-methyl-D-glucoside is transported with an apparent affinity an order of magnitude less than D-glucose (6 vs 0.5 mM). 3-O-methyl-D-glucoside has been a popular sugar for studies of intestinal absorption in rodents because it is transported by both SGLT1 and GLUT2 and is completely recovered in the urine (39). The hydroxyl at C6 appears to be somewhat less critical as 6-deoxy-Dglucose is transported by hSGLT1 with a K0.5 of 3 mM. As indicated above, it is well known that phlorizin is a potent, specific, competitive inhibitor of hSGLT1. At micromolar concentrations, this glucoside specifically inhibits intestinal Na+/glucose cotransport in various species (40), but at greater concentrations is also known to inhibit facilitated GLUTs. This has to be borne in mind when high concentrations of phlorizin are used as a pharmacologic tool to dissect out transport pathways.

GLUT SUGAR SELECTIVITY The functions of GLUT2 and GLUT5 have largely been resolved by expressing the genes in heterologous expression systems and measuring sugar transport (20). GLUT2 expressed in oocytes behaves as a low-affinity transporter for glucose (K0.5 17 mM), galactose (K0.5 92 mM), mannose (K0.5 125 mM), and fructose (K0.5 76 mM). Transport is blocked by cytochalasin B with an IC50 of 2 µM. In contrast, GLUT5 transports fructose with a K0.5 of 6 mM, but does not transport glucose. Fructose transport is not inhibited by cytochalasin B or phloretin. 3-O-methyl-D-glucoside, mannose, and 2-deoxy-D-glucose are also transported by GLUT2 with a similar affinity to that of D-glucose, but α-methyl-D-glucopyranoside is not transported. The most comprehensive data on sugar selectivity available are for native GLUT1 in human red blood cells (see Stein [37]). Competition experiments indicate that glucose, 1-deoxy-glucose, 2-deoxy-glucose, mannose, 3-deoxy-glucose, galactose, xylose, 6-deoxy-glucose, and 6-deoxy-galactose all interact with inhibitor constants in the range of 6 to 90 mM. In contrast, methyl-glucopyranoside and allose do not inhibit. Similar results were obtained for GLUT2 in rat basolateral membrane (29).

1

SGLT1 CATION SELECTIVITY FIG. 64-3. D-Glucose. The structure of D-glucose showing the position of each carbon atom (1-6), the ring oxygen (dark gray), and the position of each -OH group.

Sodium is the physiologic cation that drives sugar transport through SGLT1 with a K0.5 ~6 mM at physiologic sugar

1658 / CHAPTER 64 concentrations and membrane potentials. The affinity for protons is ~1000 times greater than sodium, 7 µM, but this comes at the cost of a lower affinity for sugar, 6 versus 0.3 mM (32,41). Proton/glucose cotransport may be important in the duodenum, where the intestinal mucosa is exposed to acid chyme containing high glucose concentrations. Lithium also drives SGLT1, but this is of little physiologic importance (42).

TRANSPORT KINETICS SGLT1 The cloning of SGLT1 and its overexpression in Xenopus laevis oocytes has allowed a detailed characterization of the kinetics of cotransport using electrophysiologic and tracer uptake methods (26). These studies have demonstrated that SGLT1 is electrogenic, with a stoichiometry of 2 Na:1 glucose. Cotransport occurs by an alternating access mechanism via a series of ligand-induced conformational changes (43,44). Two external Na+ ions bind first enabling glucose to bind, and then the substrates are transported simultaneously across the membrane. Na+/glucose cotransport is reversible; the direction of cotransport depends on the direction of the driving forces. There is an asymmetry in sugar affinity and specificity between the forward and reverse modes. SGLT1 exhibits a presteady-state current (or charge movement) with step changes in membrane potential in the absence of sugar. A six-state kinetic model with two voltage-sensitive steps—reorientation of the empty transporter between inward and outward facing conformations, and Na+ binding/ dissociation—can account for the kinetics of SGLT1. Simultaneous charge and fluorescence measurements have confirmed that the charge movements are associated with protein conformational changes. Steady-State Kinetics Stoichiometry Figure 64-4A shows the membrane current from an oocyte expressing hSGLT1 when the membrane potential was maintained at −50 mV using the two-electrode voltage clamp. An inward current was induced on addition of α-MDG (α-methyl-D-glucopyranoside, a nonmetabolized sugar) to the bathing NaCl buffer. The sugar-induced current is blocked by the SGLT1-specific inhibitor phlorizin. Experiments simultaneously monitoring the accumulation of 22Na+ and the net inward charge (obtained by integrating the sugar-coupled current record) showed a 1:1 correlation between the influx of 22Na+ into the oocyte and inward charge, indicating that the sugar-induced current is carried by Na+ ions. In addition, simultaneous determination of the uptake of radiolabeled α-MDG and inward charge showed a ratio of 1 α-MDG:2 inward charges. Thus, the stoichiometry of SGLT1 is two Na+ ions per glucose molecule (31). Protons (H+) and lithium (Li+) can substitute for Na+ in driving sugar transport (42).

Na+/glucose and H+/glucose cotransport has the same cation: sugar stoichiometry (2:1), maximal cotransport rate, and turnover rate (32). The constant inward charge caused by Na+ (or H+) in the presence of glucose indicates that Na+/glucose cotransport is a tightly coupled process. However, in the absence of sugar, there is an uncoupled Na+ current (Na+ leak, or uniporter mode) mediated by the transporter, which is blocked by phlorizin with the same sensitivity as cotransport (inhibitory constant Ki ~5 µM). The turnover rate (at 20°C) is 2 to 4 per second for the uniporter mode and 30 to 60 per second for Na+/glucose cotransport. The uniporter mode involves two Na+ ions (Hill coefficient for Na+ is 2) and a conformational change of SGLT1 because the activation energy is relatively high (19 kcal/mol for Na+ uniport vs 26 kcal/mol for Na+/glucose cotransport) (45). The current versus voltage (I-V) relation for Na+/glucose cotransport is shown in Figure 64-4B. The sugar-coupled current (or cotransport) saturated at large hyperpolarizing membrane voltages and decreased to zero at large depolarizing voltages. Thus, at lower voltages, the rate-limiting step for cotransport is dependent on voltage and becomes voltage insensitive as membrane voltage is made more negative. This dependence on voltage is caused by the increase in apparent Na+ affinity with negative membrane voltages. Reversibility and Asymmetry SGLT1 is reversible: the protein can transport Na+ and glucose out of the cell when Na+ and sugar gradients are reversed (46–48). An example of outward Na+/glucose cotransport is shown in Figure 64-4C. This experiment was performed using the giant excised patch method with the [Na+] gradient reversed ([Na+]out/[Na+]in changed from 100:10 to 10:500 mM). Addition of sugar (100 mM α-MDG) to the cytosolic surface resulted in an outward current. In contrast with the forward mode, the I-V relation of outward sugar transport decreased at large negative membrane voltages and saturated at large depolarizing voltages (see Fig. 64-4D). The direction of cotransport depended on the direction of the driving force. Negative membrane voltages favor transport in the forward mode, and positive voltages favor transport in the reverse mode. The half-saturation concentration (K0.5) or apparent affinity constant for Na+ is similar for forward and reverse transport (K0.5 for Na+ 艐 35 mM at 0 mV). In contrast, there is significant asymmetry with respect to the K0.5 for sugar. The K0.5 for sugar is 250-fold greater for the internal than that for the external membrane surface (at 0 mV, K0.5 for α-MDG is 0.15 mM for the forward and 35 mM for the reverse mode). In addition to sugar affinity, there is a difference in sugar specificity between the two membrane surfaces. The relative affinity for sugar cotransport in the forward direction by rabbit, rat, and human isoforms of SGLT1 is α-MDG ∼ D-glucose ∼ Dgalactose > 3-O-methyl-glucose (48). In contrast, in the reverse direction, the relative affinities (for rSGLT1) are α-MDG > D-galactose > 3-O-methyl-glucose > D-glucose >> L-glucose.

SUGAR ABSORPTION / 1659 −150

−100

−50

50

0

Vm (mV)

Choline Na

−200

Forward transport

−400

100 nA 1 min

Iin (nA) −600 αMDG

A

C 6 Reverse transport 4

3 pA 2 10 s Iout (pA)

Na −150

αMDG

B

D

−100 −50 Vm (mV)

0

50

FIG. 64-4. Forward and reverse cotransport by Na+/glucose cotransporter 1 (SGLT1). (A) Inward (forward) Na+/glucose cotransport. The experiment was performed on an oocyte expressing human SGLT1. Membrane potential was maintained at −50 mV using the two-electrode voltage clamp. Addition of α-methyl-D-glucopyranoside (α-MDG; 5 mM) to the external NaCl buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic]; pH 7.4) generates an inward current (downward deflection of the current trace). When substrates (Na+ and α-MDG) were removed from the bathing medium (choline Cl replacement), the current decreased to a level less than the baseline in Na+. (B) Outward (reverse) Na+/glucose cotransport. The experiment was performed (on rabbit SGLT1) using the giant excised patch (where the composition of the cytoplasmic side of the membrane could be controlled). Pipette or external solution contained 10 mM NaCl, 90 mM choline Cl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES; pH 7.5. Bath or internal solution contained 500 mM NaCl, 2 mM KCl, 1 mM CaCl2, 10 mM HEPES; pH 7.2. Addition of α-MDG (100 mM) to the bath solution induced an outward current. This outward current is blocked by phlorizin on the internal membrane surface (not shown). (C) The current-voltage (I-V) curve for forward cotransport. The experiment was performed using the giant excised patch. The sugarinduced current (for rabbit SGLT1) was obtained by subtracting the baseline current in 100 mM NaCl buffer from the total current when 10 mM α-MDG was added to the external medium. (D) I-V curve for reverse cotransport. The experiment was performed on rabbit SGLT1 with bath solution containing 500 mM NaCl as described in B. The outward current was induced by addition of 100 mM α-MDG to the bath solution. (A: Modified from Loo and colleagues [45], by permission; B–D: Modified from Eskandari and colleagues [48], by permission.)

Similar results have been obtained for hSGLT1: α-MDG > 3-O-methyl-glucose ~ D-galactose > D-glucose (47). The differences in sugar affinity and specificity for forward and reverse cotransport suggest differences in structure between outward- and inward-facing sugar-binding sites.

Kinetic Model The kinetics of SGLT1 can be largely accounted for by a simple six-state model based on the alternating access mechanism (Fig. 64-5). This model described Na+-coupled sugar transport as a series of ligand-induced conformational changes. SGLT1 was postulated to be negatively charged

1660 / CHAPTER 64 (2) reversibility; (3) kinetics of Na+ and sugar activation of cotransport; (4) kinetics of Na+ leak (K0.5 for Na+ is 2.5 mM); and (5) the phlorizin sensitivity of Na+ leak and Na+/glucose cotransport (Ki 艐 5 µM).

OUT +

+

+

Presteady-State Kinetics

C1

C2

C6

+

C5

C3

+

C4

+ IN

FIG. 64-5. Six-state kinetic model for Na+/glucose cotransporter 1 (SGLT1). The protein is negatively charged (valence –2). There are six conformations, the ligand-free (C1, C6), the Na+-bound (C2, C5), and the Na+- and sugar-bound (C3, C4) transporter on both sides of the membrane. Substrate binding is ordered with two Na+ ions binding to the transporter before glucose (C1 → C2), and the substrates are transported simultaneously via a conformational change of the fully loaded transporter (C3 → C4) to expose the bound substrates to the interior of the cell. After Na+ is released on the intracellular surface (C5 → C6), the empty transporter returns to the external membrane surface (C6 → C1). The conformational change of the empty transporter (C1 s C6) and Na+ binding (between C1 s C2) are sensitive to membrane voltage. (Modified from Parent and colleagues [44], by permission.)

(valence of −2), and membrane voltage was assumed to influence the following factors: (1) the reorientation of the ligand-free or empty carrier between inward- and outwardfacing conformations; and (2) the binding of external Na+ to a site in the membrane electric field. On the external membrane surface, two Na+ ions bind to the empty transporter (conformation C1), forming the complex C2 before the binding of the sugar molecule. Sugar is transported via a conformational change (C3 s C4) of the fully-loaded transporter (C3) between the external and internal faces of the membrane. After the dissociation of sugar and Na+ from the transporter at the internal surface, the empty transporter (C6) is returned to the external surface via another conformational change (C6 s C1). In the absence of sugar, the uncoupled Na+ flux through SGLT1 (uniporter or Na+ leak mode) is mediated by the conformational change (C2 s C5). In the presence of Na+ and sugar, phlorizin locks the transporter protein in one conformation (the phlorizin-bound form of C3). Computer simulations indicate that the six-state model for SGLT1 can largely account for the steady-state kinetics of both forward and reverse modes. The model can account quantitatively for the following properties of SGLT1: (1) electrogenicity and voltage dependence of cotransport in both directions;

The voltage dependence of the model arises from two sources: (1) reorientation of the empty carrier between inwardand outward-facing conformations; and (2) binding of external Na+ to a site in the membrane electric field. Thus, there is a predicted charge movement associated with the partial reactions C1 s C6 and C2 s C1 (44,49). Representative records of the transporter capacitance, or charge movements, of hSGLT1 observed with stepped jumps in membrane voltage (in the presence of Na+ but absence of sugar) are shown in Figure 64-6A. These presteady-state currents are blocked by phlorizin. When the current transients are integrated (at each voltage, V) to obtain the charge (Q) moved, the charge versus voltage relations (Q-V) could be fitted by a Boltzmann relation with a voltage-sensitivity factor (z) of 1.0 and a midpoint voltage (V0.5) of −50 mV. The Boltzmann relation indicates that there is a distribution for a moveable charge (of valence z) between two states (C2 and C6) depending on membrane voltage; at the V0.5, the transporter is equally distributed between the two states. The maximal charge (Qmax) provides a measure of the number of transporters expressed in the oocyte plasma membrane, and hence an evaluation of the transporter turnover number (1000–2000 sec−1 at 37°C). The presteady-state current is associated with the voltagedependent conformational changes (among C2, C1, and C6). Examination of the relaxation time constants of the presteadystate current at different external [Na+] have showed that the major voltage-dependence step is the conformational change of the empty transporter (C1 s C6) and the ratelimiting step is Na+ binding/dissociation (C2 s C1) (49,50). By combining presteady-state measurements with optical techniques using extrinsic fluorescent probes covalently bound to engineered cysteine residues in the transporter, we have confirmed that the presteady-state currents are associated with protein conformational changes (50–52). In these experiments, the substrate- and voltage-induced changes in SGLT1 conformation were monitored by the fluorescence of tetramethylrhodamine-6-maleimide (TMR6M)–labeled mutant hSGLT1 Q457C expressed in Xenopus oocytes. Voltage-jump experiments (in 100 mM NaCl buffer in absence of sugar) elicited parallel changes in presteady-state charge movement and fluorescence intensity (see Fig. 64-6B), and the time course of the change in fluorescence closely followed the presteady-state charge movement. Our studies indicate that there is a voltage-dependent conformation change in the empty transporter. Two Na+ ions bind in a highly cooperatively manner to the transporter before sugar, and sugar binding induces another conformational change of the transporter (52).

SUGAR ABSORPTION / 1661 There are at least two intermediate conformations (C1a, C1b) between C1 and C6 (C1 s C1a s C1b s C6). On the external surface, conformation C1 is metastable, and the effect of Na+ binding is to stabilize the transporter in the Na+-bound conformation (C2).

Q457C Vm (mV) +30

Charge

−150

+10 −90 −30

Summary −70

0 nA

−70

−30

−90 −150

+10

50 nA +30

A Fluorescence

20 ms Vm (mV)

SGLT1 works via an alternating access mechanism, with a strict order where two Na+ ions bind to the protein before glucose. The binding of the Na+ ions is a highly cooperative process. Cooperativity also is involved in the binding of sugar (because there is a 103-fold increase in the affinity of the transporter for sugar after Na+ is bound). SGLT1 is reversible and can mediate inward and outward Na+/sugar cotransport. Given the low affinity for glucose at the cytoplasmic surface, under physiologic conditions, there would be little or no sugar efflux mediated by SGLT1. The consequence of the functional asymmetry is that the transporter is poised to accumulate sugars in the intestine and kidney with high efficiency.

−150 −130

GLUTs

−110 −90 −70 −50 −30 −10 +10

B

+30 +50

∆F (au) 20 ms

FIG. 64-6. Presteady-state kinetics and simultaneous charge and fluorescence measurements. (A) Na+/glucose cotransporter 1 (SGLT1) presteady-state currents. The experiment was performed using the two-electrode voltage clamp on human SGLT1 Q457C labeled with tetramethylrhodamine-6maleimide. Membrane potential was held at −50 mV, and then stepped to a series of test voltages for 100 milliseconds before returning to the holding potential. The test voltage ranged between +50 mV and −150 mV in 20-mV steps. The current traces have been compensated for the capacitance of the oocyte plasma membrane. (B) Time course of change of rhodamine fluorescence intensity. The experiment was performed on the same oocyte as A. Depolarizing voltages decrease and hyperpolarizing voltages increase fluorescence intensity.

The conformational changes from C2 to C6 were studied in more detail by extending the time scale of the conformational changes (monitored by charge and fluorescence measurements) from microseconds to seconds using the cut-open oocyte and the two-electrode voltage clamps (50). The results indicate that representing the conformational change of the empty transporter between the inward- and outward-facing conformations by a single step (C6 s C1) is an oversimplification.

There are no comprehensive kinetic studies of either GLUT2 or GLUT5 apart from radiotracer uptake and competition experiments using the recombinant proteins expressed in heterologous expression systems such as Xenopus laevis oocytes. However, there have been intensive kinetic studies of the native glucose transporter (GLUT1) expressed in human red blood cells (see Stein [37]). Using intact red cells or ghosts, investigators have determined the kinetics of unidirectional sugar influx, efflux, net transport, and countertransport in the presence and absence of inhibitors. It is generally accepted that the results cannot be fit by a simple four-state alternating access model. One explanation invoked by several workers to explain the discrepancies is that the functional GLUT1 transporter is a homomultimer, and this appears to be consistent with the autosomal-dominant defect in glucose transport across the blood–brain barrier (53). However, in the case of GLUT2, the genetic disorder is an autosomal-recessive disorder (54). In contrast with SGLT1, progress in understanding the kinetics of sugar transport by GLUTs is limited by the lack of biophysical approaches to dissect out the partial reactions. Given the reputed high turnover of the transporter, 10,000 sec−1 (20,55), it has been a challenge to test kinetic models with the low time resolution of transport measurements. Quench-flow techniques have been used to measure glucose uptake into red blood cells over time intervals from 5 milliseconds to 8 hours (56). The results are modeled as a modified alternating access scheme with an occluded state where the sugar is trapped within the translocation pathway. We look forward to the results of further studies using quenchflow and other biophysical methods on GLUT1, as well as GLUT2 and GLUT5. From a physiologic perspective, it is

1662 / CHAPTER 64 essential to know the kinetics of sugar transport to evaluate the importance of immunologic detection of GLUTs in sugar absorption.

GENETIC DEFECTS OF SUGAR ABSORPTION Two rare autosomal-recessive disorders of carbohydrate malabsorption provide unique insights into the physiology of sugar transport: GGM (MIM 182380) and the Fanconi– Bickel syndrome (MIM 227810). GGM presents with severe life-threatening diarrhea during the neonatal period, which ceases immediately on removing the offending carbohydrates, glucose, galactose, and lactose from the diet (17). The diarrhea promptly resumes on feeding these sugars, but not on feeding with fructose. Patients with Fanconi–Bickel syndrome present with severe liver and kidney problems and impaired utilization of glucose and galactose. Intestinal glucose malabsorption is reported in some, but not all, of these subjects (54).

Glucose-Galactose Malabsorption A detailed clinical evaluation of the first U.S. child diagnosed with GGM demonstrated that there was malabsorption of glucose and galactose, whereas fructose absorption was normal, and laboratory studies with duodenal biopsies showed that the defect was caused by impaired transport of glucose and galactose across the brush-border membrane (see Wright and colleagues [17]). Thirty years after this study, we were able to examine this patient and found that her diet was completely carbohydrate free. In addition, we established that she inherited two different SGLT1 mutations from her mother and father (Cys355Ser and Leu147Arg), and that each mutation was in a completely defective transporter (24). We have screened 82 patients with GGM in 74 families and have identified the SGLT1 mutations that cause the defect in sugar absorption in all but 3 patients. Forty-six different mutations have been found in these 79 patients: 34 missense, 6 nonsense, 7 frameshift, and 7 splice site mutations (57). About 35% of the patients with GGM are complex heterozygotes (different mutations on each allele). In all but one case, the mutations produce either truncated proteins or proteins that are not delivered properly to the plasma membrane. The one exception, Gln457Arg SGLT1, trafficked normally to the brush-border membrane in the patient and to the plasma membrane of the expression system. In this case, the protein could bind glucose, but was unable to transport sugar across the plasma membrane. There is one comprehensive clinical study in the literature of an adult patient who was diagnosed with GGM after lifelong diarrhea (58). Sugar tolerance tests provided the basis for the diagnosis, and this was confirmed by direct measurement of sugar absorption from jejunal perfusion studies. Notably, in this patient, glucose failed to produce a change in the electrical potential difference across the small intestine, which again indicates the lack of Na/glucose absorption in

this patient. This was entirely consistent with our genetic analysis that showed that the SGLT1 protein was severely truncated in this subject. The complete intolerance of these two adult patients to carbohydrate together with the genetic analysis demonstrating the absence of functional SGLT1 proteins leads to our conclusion that SGLT1 plays a critical role in the intestinal absorption of glucose.

Fanconi–Bickel Syndrome GLUT2 was considered a candidate gene for defects in this syndrome given the clinical manifestations and the distribution of the gene in the body. The gene has been examined in 63 patients and 34 different mutations, and mostly truncated proteins have been found (54). Surprisingly, only 16% of the patients diagnosed with this syndrome have intestinal symptoms such as diarrhea. Hydrogen breath tests were conducted on one patient with a nonfunctional GLUT2 protein where no defects in either glucose or galactose absorption were observed (59). This indicates that GLUT2 is not required for intestinal sugar absorption, and that another pathway is involved in the exit of glucose from the enterocyte (see Fig. 64-1). This particular patient, as well as others, does present with severe renal glucosuria, and this suggests that GLUT2 is required for glucose transport across the renal tubule. The explanation for this conundrum emerges from sugar absorption studies on mice lacking GLUT2 (30). As in the patients with Fanconi–Bickel, oral tolerance tests with glucose were identical to those mice expressing GLUT2 in the small intestine. In both cases, phlorizin greatly reduced intestinal sugar absorption. However, in the absence of GLUT2, absorption was inhibited by a blocker of glucose-6phosphatase. Finally, the absorption of the nonmetabolized model substrate for GLUT2, 3-O-methyl-glucoside, was eliminated in the GLUT2-deficient mice. This led to the hypothesis that the alternative pathway to GLUT2 for exit of glucose from enterocytes across the basolateral membrane involved the accumulation of glucose-6-phosphate in the endoplasmic reticulum and release of glucose by exocytosis (see Fig. 64-1). Presumably, this pathway is not available in the renal proximal tubule.

Fructose Malabsorption Isolated fructose malabsorption (IFM) is a rare poorly defined disorder that is distinct from hereditary fructose intolerance (MIM 229600) caused by a deficiency of aldose B (18). GLUT5 is the best candidate gene for IFM, although no mutations currently have been found. There is a limited capacity to absorb fructose in both infants and adults. Interestingly, some 50% of healthy adults are fructose malabsorbers as judged by hydrogen breath tests (19). In young children, toddler’s diarrhea may be caused by overload of the

SUGAR ABSORPTION / 1663 capacity to absorb fructose from fruit juices because it is known in rodents that expression of GLUT5 in the small intestine does not occur until the time of weaning (21).

REGULATION OF SUGAR ABSORPTION In both humans and rodents, there is a gradient of sugar absorption along the length of small intestine from duodenum to ileum. The bulk of the ingested carbohydrate is digested and absorbed by the time the chyme normally reaches the distal jejunum. In addition to the gradient of activity along the intestine, there is a gradient of sugar transport activity from crypt to villus. As discussed previously (1), there are 6 to 9 crypts per villus, and the proliferative zone in the crypt supplies about 300 cells to each villus per day. Each new cell differentiates into mature enterocytes and goblet cells as they migrate up the villus at about 10 µm/hr. Transporter genes (SGLT1, GLUT2, and GLUT5) are transcribed in the lower villus, SGLT1 and GLUT5 are inserted into the brush-border membrane and GLUT2 into the basolateral membrane in the mature enterocytes. Given that there is evidence that the intestine can adapt to diet, surgical resection, and a variety of pathologic conditions, it has been a challenge to relate changes in sugar absorption to direct changes in the expression, density, or activity of SGLT1, GLUT2, and GLUT5. There is no evidence that the activity of the transporters is regulated directly, but it is clear that the transport activity can be regulated by the insertion and retrieval of transporters into the plasma membrane from intracellular pools (e.g., see Hirsch and colleagues [60]). In both heterologous expression systems and native tissues, the effects of agents that alter intracellular levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate produce modest effects (±25%) within 5 to 10 minutes. Perhaps the best studied case of regulation in humans is one where the expression of SGLT1, GLUT5, and GLUT2 was examined in patients with diabetes (61). The abundance of transporter mRNA in duodenal biopsies, the abundance of SGLT1 and GLUT5 protein in brush-border membrane vesicles, and the Na/glucose cotransport in brush-border vesicles were measured in both healthy subjects and patients with diabetes. The results demonstrated that in the patients with diabetes there was a threefold to fourfold increase in Na/glucose cotransport, SGLT1 and GLUT5 brush-border protein, and SGLT1, GLUT5, and GLUT2 mRNA abundance in the mucosa. Although the results are clear that there is an increase in the expression of monosaccharide transporters in subjects with non–insulin-dependent diabetes mellitus, it is essential to know if this actually translates into a threefold to fourfold increase in glucose absorption in these subjects. This would require quantitative measurement of glucose absorption in control subjects and patients with diabetes. Even larger increases in expression of SGLT are observed in a sheep model. Before weaning, the carbohydrates in the diet, milk, reach the small intestine where they are digested, and the hexoses, glucose and galactose, are absorbed in the

customary manner (see Fig. 64-1). However, after weaning and development of the rumen, the dietary carbohydrate is fermented in the rumen to short-chain fatty acids; that is, carbohydrates do not normally reach the small intestine in adult sheep. After weaning, there is a marked reduction in expression of SGLT1 in brush borders and the in rate of brush-border Na/glucose cotransport (27). Within 4 days after the introduction of 30 mM glucose or other hexoses into the small intestine of adult sheep, there is a 400-fold increase in expression of both brush-border SGLT1 abundance and Na/glucose cotransport after 3 to 4 days. Interestingly, infusion of sugars that are poor substrates for sheep SGLT1, for example, 2-deoxy-D-glucose and di(gluco-6-yl) poly(ethylene glycol) 600, also trigger the up-regulation of SGLT1 (62,63). This raises interesting questions about the mechanisms involved in sugar “tasting” in the small intestine. One intriguing possibility is that SGLT3 is this glucosensor (6).

FUTURE DIRECTIONS In this postgenomic era, our challenge is to identify the function and anatomic location of the sugar transporter genes expressed in the epithelial cells of the small intestine. It is no longer sufficient to simply record whether our favorite gene is transcribed and translated in the intestinal mucosa. First, we need to know the physiologic function of cloned membrane transport proteins in view of that transporters are multifunctional (9). Second, we need to determine the precise anatomic location of the protein using high-resolution imaging techniques, preferably with electron microscopy, because light microscopy even with the power of the confocal microscope does not allow one to conclude that a transporter is actually in a membrane. Promising techniques to eliminate the resolution gap between light and electron microscopy are now beginning to emerge (64–66). Third, given the location and density of a membrane protein, we need to determine that the kinetics is sufficient to quantitatively account for the rate and direction of transport across the membrane. Fourth, it is imperative to know the determinants of the transport protein density in a given membrane during development, changes in diet, and disease. This involves an understanding of the factors that control gene transcription, protein synthesis, and trafficking of the transporter to and from the brushborder and basolateral membranes. In the intestine, these factors are compounded by the dynamics of enterocyte maturation and apoptosis. Finally, this understanding of the molecular physiology needs to be applied to the question of sugar absorption in the whole intestine. To do so requires considerable effort to develop new noninvasive methods to evaluate sugar absorption in both human and animal subjects.

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (grants DK19567, DK44582, and DK44602).

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58. Phillips S, McGill D. Glucose-galactose malabsorption in an adult: perfusion studies of sugar, electrolyte, and water transport. J Dig Dis 1973;18:1017–1023. 59. Santer R, Hillebrand G, Steinmann B, Schaub J. Intestinal glucose transport: evidence for a membrane traffic-based pathway in humans. Gastroenterology 2003;124:34–39. 60. Hirsch JR, Loo DDF, Wright EM. Regulation of Na+/glucose cotransporter expression by protein kinases in Xenopus laevis oocytes. J Biol Chem 1996;271:14740–14746. 61. Dyer J, Wood S, Palejwala A, Ellis A, Shirazi-Beechey P. Expression of monosaccharide transporters in intestine of diabetic humans. Am J Physiol Gastrointest Liver Physiol 2002;282:G241–G248. 62. Dyer J, Barker P, Shirazi-Beechey P. Nutrient regulation of the intestinal Na+/glucose co-transporter (SGLT1) gene expression. Biochem Biophys Res Commun 1997;230:624–629. 63. Dyer J, Vayro S, King T, Shirazi-Beechey P. Glucose sensing in the intestinal epithelium. Eur J Biochem 2003;270:3377–3388. 64. Zampighi GA. Distribution of connexin50 channels and hemichannels in lens fibers: a structural approach. Cell Commun Adhes 2003;10 (4-6):265–270. 65. Zampighi GA, Kreman M, Lanzavecchia S, Turk E, Eskandari S, Zampighi L, Wright M. Structure of functional single AQP0 channels in phospholipid membranes. J Mol Biol 2003;325:201–210. 66. Lanzavecchia S, Cantele F, Bellon PL, Zampighi L, Kreman M, Wright E, Zampighi GA. Conical tomography of freeze-fracture replicas: a method for the study of integral membrane proteins inserted in phospholipids bilayers. J Struct Biol 2005;149:87–98.

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CHAPTER

65

Protein Digestion and Absorption Vadivel Ganapathy, Naren Gupta, and Robert G. Martindale Overview, 1668 Role of Gastric and Pancreatic Proteases in Protein Digestion, 1668 Role of Membrane-Bound and Cytoplasmic Peptidases in the Enterocyte in Protein Digestion, 1669 Sites of Protein Absorption, 1669 Generation of Driving Forces for Active Transport Systems in the Enterocyte, 1670 Entry of Protein Digestion Products into the Enterocyte across the Brush-Border Membrane, 1671 Amino Acid Transport, 1671 Peptide Transport, 1675 Fate of Absorbed Amino Acids and Peptides in the Enterocyte, 1677 Exit of Protein Digestion End Products across the Basolateral Membrane, 1677 Amino Acid Transport, 1677 Peptide Transport, 1680 Transport of Glutathione in the Small Intestine, 1681

Genetic Disorders of Intestinal Amino Acid and Peptide Transport, 1681 Hartnup Disease, 1681 Cystinuria, 1682 Lysinuric Protein Intolerance, 1682 Genetic Defects in Intestinal Peptide Transport, 1683 Nutritional, Clinical, and Pharmacologic Relevance of Intestinal Peptide Transport, 1683 Regulation of Intestinal Amino Acid and Peptide Transport, 1684 Developmental Regulation, 1684 Dietary Regulation, 1685 Hormonal Regulation, 1685 Diurnal Rhythm, 1686 Na+-H+ Exchanger and Regulation of Intestinal Amino Acid and Peptide Transport, 1686 Conclusions and Future Perspectives, 1686 References, 1687

Nutritional needs for amino acids in human and some animals are met by assimilation of dietary proteins in the small intestine. The process involves digestion of the proteins in the intestinal lumen to generate products of smaller size that are absorbable by the enterocyte. Interestingly, the end products of protein digestion in the intestinal lumen are not exclusively free amino acids, but rather a mixture of free amino acids and small peptides. The intestinal epithelium has efficient transport mechanisms to absorb from the lumen

not only free amino acids but also dipeptides and tripeptides. The absorbed small peptides, however, are digested intracellularly to free amino acids in the cytoplasm. Thus, the final stages of digestion of dietary proteins occurs within the enterocyte, and free amino acids then exit the cell to enter the portal circulation. This characteristic is unique to protein assimilation because in the case of carbohydrates, digestion in the intestinal lumen has to be complete to yield monosaccharides before absorption into the enterocyte can occur (1,2). There are no transport mechanisms in the enterocyte for carbohydrate digestion products other than monosaccharides. An additional feature that is unique to protein assimilation is the need for the presence of multiple transport systems in the intestinal epithelium to handle the digestion end products. Proteins are made up of 20 different amino acids that are distinct from each other in terms of their chemical structure, which determines their size, lipophilicity, and electrical charge. Because of their widely different physicochemical properties, a single transport system cannot handle all amino acids.

V. Ganapathy: Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912. N. Gupta: Department of Surgery, University of Virginia Health System, Charlottesville, Virginia 22908. R. G. Martindale: Department of Surgery, Oregon Health and Science University, Portland, Oregon 97239. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1667

1668 / CHAPTER 65 The situation is even more complicated in the case of peptides, where the possible number of chemically distinct forms is 400 for dipeptides and 8000 for tripeptides. Interestingly, however, as discussed later in the chapter, intestinal absorptive cells express multiple transport systems to absorb the 20 different amino acids from the lumen, but only a single transport system to absorb the 400 different dipeptides and 8000 different tripeptides. One of the most difficult tasks in the area of protein assimilation has been, and still is, to differentiate the multiple amino acid transport systems and study their characteristics individually, because the substrate specificities of these transport systems often overlap to a considerable extent. In addition, absorption of the digestion products from the lumen into blood across the intestinal epithelium requires transport systems in the brush-border membrane for entry into the cell and transport systems in the basolateral membrane for exit out of the cell. These two membranes express different sets of transport systems to facilitate vectorial transfer of these protein digestion products. Furthermore, the basolateral membrane has to express transport systems that will enable the cells to obtain amino acids from blood for cellular nutrition during the intervals between meals. This adds to the complexity of the overall process of protein assimilation and nutrition in the intestinal tract. An in-depth account of the subject of protein digestion and absorption, in terms of its history and development, can be found in a monograph written by Matthews in 1991 (3). Important advances have been made since then in this area, especially regarding the molecular aspects of the various transporters that participate in the absorptive process. This has greatly improved our understanding of the physiologic and pathologic aspects of assimilation of dietary proteins in the intestine at the molecular level.

OVERVIEW Figure 65-1 presents a scheme of intestinal assimilation of proteins. The daily intake of protein in the diet varies considerably in different parts of the world. A typical Western diet contains ~100 g protein per day. In addition to the proteins originating from the diet, salivary and gastrointestinal secretions contain a significant amount of protein (~35 g/day) that needs to be digested and absorbed in the gastrointestinal tract. Luminal digestion of these exogenous and endogenous proteins is carried out by gastric and pancreatic proteases. The resultant end products, mostly large peptides, undergo further hydrolysis by a variety of peptidases present in the brush-border membrane of the intestinal epithelium. Analysis of luminal contents after a protein meal has shown that amino acids are present in the lumen primarily in peptide form, rather than in free form (4,5). The peptides in the lumen consist mostly of two to six amino acids, and the concentrations of peptide-bound amino acids are as high as 80% of total amino acids. Free amino acids are absorbed into the enterocyte across the brush-border membrane via multiple amino acid transport systems. Small peptides, primarily those

Proteins (Diet, Gl secretions) Gastric and pancreatic proteases and peptidases

Amino acids

Large peptides

(~20%)

(~80%)

1

LUMEN 2

Amino acids

BLOOD

Dipeptides Tripeptides

3

4

Dipeptides Tripeptides

5

6

Amion acids (~90%)

Dipeptides Tripeptides (~10%)

FIG. 65-1. Overview of protein digestion and absorption in the gastrointestinal tract. (1) Brush-border peptidases; (2) brushborder amino-acid transport systems; (3) brush-border peptide transport system; (4) cytoplasmic peptidases; (5) basolateral amino-acid transport systems; (6) basolateral peptide transport system(s). GI, gastrointestinal.

consisting of two or three amino acids, are transported intact across the brush-border membrane via a specific peptide transport system. Notably, the protein digestion products enter the enterocyte primarily in the form of dipeptides and tripeptides. Transport of free amino acids contributes relatively less to the entry of protein digestion products into the enterocyte. Nonetheless, the protein digestion products enter the portal circulation mostly as free amino acids because of the efficient intracellular hydrolysis of peptides by cytoplasmic peptidases. Peptides that are resistant to cytoplasmic peptidases may be transported intact across the basolateral membrane, but the contribution of this route to the total absorption of protein digestion products is minimal.

ROLE OF GASTRIC AND PANCREATIC PROTEASES IN PROTEIN DIGESTION The gastric phase of protein digestion involves the protease pepsin. This enzyme is secreted by the chief cells of the stomach as an inactive precursor pepsinogen. This zymogen is activated by the acidic pH in the stomach lumen. High levels

PROTEIN DIGESTION AND ABSORPTION / 1669 of H+ cause a conformational change in pepsinogen and expose the catalytic active site that is responsible for protease action. The resultant catalytically active form of pepsinogen then acts on inactive pepsinogen and generates pepsin by limited proteolysis. Pepsin then acts on inactive pepsinogen to generate more pepsin by a process called autocatalysis. Pepsin is an acid protease that is optimally active under acidic conditions. Therefore, pepsin remains active in the stomach lumen and initiates digestion of dietary proteins. The end products of pepsin action on proteins are large polypeptides. When the stomach contents enter the small intestine, endocrine cells in the duodenum are exposed to an acidic pH that stimulates the secretion of the hormone secretin. This hormone acts on the pancreas and bile ducts to induce secretion of bicarbonate. Bicarbonate-rich pancreatic and biliary secretions reach the duodenum where they serve to rapidly neutralize the acid. Polypeptides and fat in the stomach contents act on endocrine cells in the duodenum to induce the secretion of another hormone cholecystokinin. This hormone induces the secretion of pancreatic fluid rich in digestive enzymes. Cholecystokinin also causes contraction of the gallbladder to release bile into the duodenum. Thus, the entry of stomach contents into the small intestine initiates processes in the duodenum that lead to the delivery of bicarbonate, bile, and pancreatic digestive enzymes to the intestine. Unlike pepsin, pancreatic digestive enzymes are optimally active at neutral pH. Therefore, neutralization of the acid pH by bicarbonate in the duodenum is critical for the activity of these enzymes. Pancreatic secretions contain several enzymes relevant to protein digestion. All of these enzymes are secreted as inactive precursors. These are trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidases. The first step in the activation of these zymogens is the activation of trypsinogen. This is mediated by an enzyme called enteropeptidase associated with the brush-border membrane of the intestinal epithelial cells. When trypsinogen comes in contact with the intestinal cells in the lumen, it is subjected to limited proteolysis. The resultant product is the active enzyme trypsin. Trypsin then acts on chymotrypsinogen, proelastase, and procarboxypeptidases and generates the active forms of these enzymes: chymotrypsin, elastase, and carboxypeptidases. Polypeptides that enter the small intestine from the stomach are acted on by these pancreatic enzymes to generate smaller peptides consisting of six to eight amino acids. Free amino acids are also generated to a small extent by the action of these enzymes. The specificity of pancreatic enzymes is determined by the nature of the amino acids that make up the peptide bonds in polypeptides. Trypsin acts on peptide bonds that are formed by the carboxyl group of cationic amino acids. Chymotrypsin prefers to hydrolyze peptide bonds that are formed by the carboxyl group of aromatic amino acids. Elastase hydrolyzes peptide bonds that are formed by the carboxyl group of small short-chain amino acids. This differential specificity of these proteases for peptide bonds in polypeptides makes the digestive process efficient. The end products of protein digestion by pancreatic proteases

consist predominantly of peptides with six to eight amino acids. Free amino acids comprise only a small fraction of the products of protein digestion by pancreatic proteases.

ROLE OF MEMBRANE-BOUND AND CYTOPLASMIC PEPTIDASES IN THE ENTEROCYTE IN PROTEIN DIGESTION The peptides arising from protein digestion by pancreatic proteases are subjected to further hydrolysis by peptidases associated with the brush-border membrane of enterocytes, and the products are released into the intestinal lumen. These peptidases are ectoenzymes, and their specificity is toward oligopeptides consisting of six to eight amino acids. The resultant end products consist predominantly of smaller peptides containing two to three amino acids. These dipeptides and tripeptides are transported into enterocytes via a specific transport system in the brush-border membrane. Once inside the cells, the small peptides are subjected to hydrolysis by cytoplasmic peptidases to release free amino acids (6,7). There are significant differences for chain length of the peptides that are recognized as substrates by brush-border peptidases and cytoplasmic peptidases in the enterocyte, the former preferring longer peptides consisting four or more amino acids and the latter preferring dipeptides and tripeptides. This makes sense because the brush-border peptidases act on longer peptides to generate dipeptides and tripeptides in the intestinal lumen. The peptide transport system in the brush-border membrane mediates the entry of these smaller peptides into the enterocyte where the cytoplasmic peptidases act on them to release free amino acids. The basolateral membrane of the enterocyte also possesses a number of amino acid transport systems that are responsible for the exit of amino acids from the cell into the portal circulation. Peptides that escape hydrolysis by cytoplasmic peptidases enter the portal circulation via a peptide transport system present in the basolateral membrane that is distinct from the brush-border membrane peptide transport system.

SITES OF PROTEIN ABSORPTION Even though the process of protein digestion in the gastrointestinal tract is initiated by pepsin in the stomach, absorption of the digestion products is negligible at this site. This is supported by the findings that total gastrectomy rarely results in significant disruption of the protein digestive and absorptive processes (8). The small intestine is the principal site of protein absorption. By the time the luminal contents enter into the ileocecal junction, absorption of proteins is almost complete. The colonic epithelium does possess an appreciable capacity to absorb protein digestion products, but the physiologic significance of this in the overall process of absorption of dietary proteins is questionable. It is possible that bacterial proteins are digested and absorbed to a significant extent in the colon.

1670 / CHAPTER 65 Within the small intestine, there are regional variations in the absorptive capacities for protein digestion products. The two groups of end products, namely, amino acids and small peptides (dipeptides and tripeptides), are absorbed at different rates in different sections of the small intestine. The absorptive capacity for dipeptides and tripeptides is greater in the proximal small intestine than in the distal small intestine (9–11), whereas in the case of amino acids, the absorptive capacity is greater in the distal small intestine than in the proximal small intestine (9–14). These reciprocal axial gradients in the activities of amino acid and peptide transport systems in the small intestine arise from variations in transport capacities, rather than in affinities along the length of the small intestine (11,14). The observed differential gradients in the transport capacities for amino acids and small peptides along the jejunoileal axis may have physiologic relevance and importance to the maintenance of optimal protein nutrition. Digestion of proteins in the intestinal lumen by pancreatic proteases releases primarily large peptides, which are not absorbable as such. It is the action of the membrane-bound peptidases in the brush-border membrane of the enterocyte that generates a major portion of the absorbable products, namely, amino acids, dipeptides, and tripeptides. Though these peptidases are present throughout the small intestine, their activities are much higher in the ileum than in the jejunum (15), implying that the ileal brushborder membrane is capable of more extensive hydrolysis of peptides than the jejunal brush-border membrane. It is therefore conceivable that as the luminal contents move along the intestine from the jejunum to the ileum, the rate of appearance of free amino acids in the lumen gradually increases, whereas the luminal concentration of dipeptides and tripeptides gradually decreases. Another contributing factor to this phenomenon is the duration of contact between the peptide substrates and the peptidases, which increases as the contents move from the jejunum to the ileum. Indeed, numerous studies have reported that the appearance of free amino acids is appreciably greater in the ileum than in the jejunum (16–19). There is little doubt that the parallelism between the absorptive capacities for amino acids, dipeptides, and tripeptides and the luminal concentrations of the corresponding substrates along the jejunoileal axis enhances the efficiency of the absorptive process. Although the role of the large intestine in the digestion of dietary carbohydrates has been well recognized (20), until fairly recently the common belief was that this part of the intestinal tract did not participate in the digestion and absorption of proteins to any significant extent. Numerous studies have now demonstrated that the colonic epithelial cells are capable of absorption of various amino acids (21–25), as well as peptides (26). These findings are surprising because due to the efficient digestion and absorption in the small intestine, only small amounts of proteins and protein digestion products enter into the colon under normal physiologic circumstances. It is, however, conceivable that the large intestine serves a useful function in special situations such as in the immediate postnatal period (23,24) or in patients with ileostomies (27). Furthermore, the large intestine contains appreciable quantities of bacterial proteins and their

degradation products. Amino acids, dipeptides, and tripeptides arising from these bacterial proteins may be absorbed in the colon, but the physiologic significance of colonic absorption of protein digestion products remains controversial.

GENERATION OF DRIVING FORCES FOR ACTIVE TRANSPORT SYSTEMS IN THE ENTEROCYTE Absorption of amino acids and peptides across the intestinal epithelium is mediated by a multitude of transport systems that are expressed differentially in the lumen-facing brush-border membrane and the blood-facing basolateral membrane. The transport processes that occur via these systems can be divided into two categories: active and passive. This classification is based on whether the transport process is dependent on metabolic energy. Active transport processes are energized by some form of driving force and are able to mediate uphill movement of their substrates against an electrochemical gradient. In contrast, passive transport processes do not require any type of driving force and are only capable of mediating the movement of their substrates down an electrochemical gradient. The driving force for the active transport systems in the intestinal brush-border and basolateral membranes comes from transmembrane ion gradients and membrane potential. Figure 65-2 describes the cellular mechanisms responsible for the generation of these driving forces across the two membranes in the enterocyte. The ultimate energy source for these processes is adenosine triphosphate (ATP), the cellular currency of energy. Na+-K+,ATPase, located exclusively in the basolateral membrane of enterocytes, uses ATP to mediate the uphill transport of Na+ from the cell into blood and uphill of transport of K+ from blood into the cell. This generates an inwardly directed Na+ gradient (∆pNa) and an outwardly directed K+ gradient (∆pK) across the basolateral membrane. Because the Na+:K+ stoichiometry for this transport process is 3:2, the transport system also generates an inside-negative membrane potential (∆ψ). The K+ channel located in the basolateral membrane mediates the efflux of K+ down its concentration gradient, a process that serves as an additional mechanism for the generation of the insidenegative membrane potential. The brush-border membrane expresses an Na+-H+ exchanger (NHE) that uses the transmembrane Na+ gradient as the driving force to facilitate the efflux of H+ from the cell into the intestinal lumen. This generates a transmembrane H+ gradient (∆pH) across the brush-border membrane. This active efflux of H+ is responsible for the formation of an acidic microclimate pH known to exist on the luminal surface of the brush-border membrane (28,29). This creates an approximately 10-fold concentration gradient for H+ (outside > inside) across this membrane. There is also a Cl− channel in the brush-border membrane that mediates the efflux of Cl− into the intestinal lumen down the electrochemical gradient. Together, these transport systems are responsible for the maintenance of lower concentrations of Na+ and Cl− and greater concentration of K+ inside the enterocyte compared with extracellular fluid. The luminal

PROTEIN DIGESTION AND ABSORPTION / 1671

1

ATP

∆Ψ

3

∆pCl

∆pNa

2

∆pH

∆pK

4

∆Ψ

+ + + − − − Na+ H+ NaCl from diet and Gl secretions

2

ATP 3Na+ 2K+

1

ADP + Pi Cl−

3

K+

4 BLOOD

LUMEN

+ + +

− − −

− − −

+ + + Na+

Na+ K+ K+ Cl− Basolateral membrane

Cl− H+ Brush border membrane

FIG. 65-2. Mechanisms for the generation of driving forces for active transport systems in the intestinal brush-border and basolateral membranes. (1) Na+-K+,ATPase; (2) Na+-H+ exchanger; (3) chloride channel; (4) potassium channel; ∆ψ, membrane potential (inside-negative); ∆pNa, transmembrane Na+ gradient; ∆pK, transmembrane K+ gradient; ∆pCl, transmembrane Cl− gradient; ∆pH, transmembrane H+ gradient. ADP, adenosine diphosphate; ATP, adenosine triphosphate; GI, gastrointestinal.

fluid contains substantial amounts of Na+ and Cl− arising from dietary sources, as well as from salivary and gastrointestinal secretions. Thus, there are five different driving forces, namely, an inwardly directed Na+ gradient, an inwardly directed H+ gradient, an inwardly directed Cl− gradient, an outwardly directed K+ gradient, and an inside-negative membrane potential, which provide energy to support the active transport processes mediated by various amino acid and peptide transport systems in the brush-border and basolateral membranes.

ENTRY OF PROTEIN DIGESTION PRODUCTS INTO THE ENTEROCYTE ACROSS THE BRUSH-BORDER MEMBRANE Amino Acid Transport The subject of amino acid transport across animal cell membranes is complex, primarily because of the existence of multiple transport systems with overlapping

substrate specificities. Christensen and his coworkers (30–32) have been largely responsible for classification and characterization of amino acid transport systems. Initial attempts to investigate and categorize amino-acid transport systems in the small intestine were based largely on the strategies developed by these investigators. However, it soon became apparent that the classical nomenclature of amino acid transport systems, which was deduced from information available for amino acid transport across the plasma membrane of nonpolarized cells, is not applicable to the brush-border membrane of the polarized intestinal epithelial cells. Unlike nonpolarized cells, the enterocyte should be equipped with transport systems at the two poles of its plasma membrane that exhibit differential characteristics, to enable the cell to perform vectorial transport. The brush-border membrane and the basolateral membrane are in contact with fluids of vastly different chemical composition, namely, the luminal fluid and the extracellular fluid, respectively. Thus, for the chemical environment, it is the basolateral membrane, not the brushborder membrane, that resembles the plasma membrane of nonpolarized cells. Not surprisingly, the amino acid transport systems in the intestinal basolateral membrane conform to the traditional nomenclature applicable to the plasma membrane of nonpolarized cells. In contrast, the intestinal brush-border membrane possesses many amino acid transport systems that have not been described in nonpolarized cells. In the past, there have been several reviews classifying the amino acid transport systems known to exist in the intestinal brush-border membrane (12,33–35). These classifications were primarily based on transport characteristics and substrate specificity of various transport systems identified in purified intestinal brush-border membrane vesicles at the functional level. In recent years, however, specific proteins responsible for several of these amino acid transport systems have been cloned and characterized (36–38). Based on this information, we now know the molecular identity of these transporter proteins. Table 65-1 classifies these transport systems and lists their substrate specificity and dependence on ion gradients. Most of these transport systems are active and able to mediate uphill transport of substrates. This characteristic is important especially for the transport systems in the intestinal brush-border membrane because the physiologic function of these transport systems is to effectively absorb amino acids from the intestinal lumen. If this process is mediated by energy-independent facilitative transport systems, the absorptive process will not be complete, resulting in significant loss of amino acids in the feces. Figure 65-3 describes how each of the amino acid transport systems that are expressed in the intestinal brush-border membrane is coupled to its driving forces, indicating the directionality of the movement of the amino acid substrates and the cotransported ions. System B0 The major transport system responsible for the transport of neutral amino acids across the intestinal brush-border membrane was originally identified as the neutral brush-border

1672 / CHAPTER 65 TABLE 65-1. Amino acid transport systems in the brush-border membrane of the small intestine Transport system

Molecular identity

Substrates

B0 B0,+

B0AT1 ATB0,+

IMINO β X −AG ASC PAT b0,+

SIT1/IMINO TAUT EAAT3 ASCT2/ATB0 PAT1 b0,+AT-rBAT

Neutral L-amino acids Neutral L-amino acids Cationic L-amino acids Certain neutral D-amino acids Imino acids Taurine, β-alanine Anionic amino acids Neutral L-amino acids Small neutral amino acids Neutral L-amino acids Cationic L-amino acids Cystine

Dependence on Na+

Involvement of other ions

Yes Yes

No Cl−

Yes Yes Yes Yes No No

Cl− Cl− K+, H+ No H+ No

ASCT2, second member of the ASC amino acid transporter family; ATB0,+, amino acid transporter B0,+; B0AT1, the first transport protein responsible for the amino acid transport system B0; b0,+AT, b0,+ amino acid transporter; EAAT3, excitatory amino acid transporter 3; rBAT, related to b0,+ amino acid transport; TAUT, taurine transporter; SIT1, sodium-coupled imino acid transporter 1.

(NBB) system (39) and subsequently renamed system B (40) to highlight its broad (“B” for broad) substrate specificity. This transport system is Na+-dependent and accepts all or nearly all neutral amino acids that possess the amino group in the α position as substrates (39). Imino acids and β amino acids, although neutral in terms of electrical charge, are excluded by the system. Cationic and anionic amino acids are also not substrates for this transport system. The rules for the classification of amino acid transport systems have been revised (41). According to this newer classification system, the Na+ gradient–dependent transport systems are identified by uppercase letters, whereas the Na+independent transport systems are identified by lowercase letters. Furthermore, the superscripts such as “0,” “+,” “−,” and “0,+” are added to describe the electrical nature of the BLOOD

amino acid substrates recognized by the transport systems. According to this new nomenclature, the amino acid transport system previously known as NBB or B is now called B0 because it has broad substrate specificity, is energized by an Na+ gradient, and recognizes only neutral amino acids (net charge on the substrate molecule is zero) as its substrates. Because the transport function of system B0 involves symport of Na+ and neutral amino acids, the transport process is electrogenic (40). Therefore, under physiologic conditions, an inwardly directed Na+ gradient and an inside-negative membrane potential provide the driving force for this system. There is evidence that this system is expressed in the small intestine even in early gestation (42). The molecular identity of the protein responsible for this transport system in mouse has been established by Broer and colleagues (43). LUMEN AA0 Na+

B0

AA0/AA+ 2 or 3 Na+ Cl−

B0,+ ∆pNa, ∆pCl, IMINO ∆ψ β

AA− K+

3 Na+ H+

AA0 Na+ AA0

AA+, cystine AA0 H+

X−AG

∆pNa, ∆ψ

∆pNa, ∆pH, ∆pK, ∆ψ

ASC b0,+

∆ψ

PAT

∆pH, ∆ψ

FIG. 65-3. Amino-acid transport systems in the intestinal brush-border membrane. Arrows indicate the direction of movement of amino acids/ions across the brush-border membrane in vivo. AA0, neutral amino acid; AA+, cationic amino acid; AA−, anionic amino acid.

PROTEIN DIGESTION AND ABSORPTION / 1673 The protein is identified as B0AT1, meaning “the first transport protein responsible for the amino acid transport system B0.” The human B0AT1 also has been cloned and functionally characterized (44,45). The human B0AT1 (also known as SLC6A19) consists of 634 amino acids and belongs to the solute-linked carrier (SLC) gene family SLC6. The gene coding for this protein is located on human chromosome 5p15.33. System B0,+ System B0,+ is similar to system B0, but accepts neutral and cationic amino acids as substrates (36–38). This unique substrate specificity is indicated by the superscript “0,+” in the name of the system. This transport system is dependent not only on a transmembrane Na+ gradient, but also on a transmembrane Cl− gradient. The transport process is also electrogenic. Thus, there are three different driving forces for this transport system, namely, a Na+ gradient, a Cl− gradient, and the membrane potential. The interaction of basic amino acids with this system is evident from the observations that the uptake of lysine into intestinal brush-border membrane vesicles is Na+ dependent (46,47). This system also is expressed in the brush-border membrane of the human colon carcinoma cell line Caco-2 (48). In this cell line, the transport of phenylalanine across the apical membrane is predominantly Na+ dependent, and this transport is significantly inhibited by lysine. The protein responsible for the activity of system B0,+ has been cloned (49,50). The protein is identified as ATB0,+ (amino acid transporter B0,+). The transport characteristics of the cloned protein are similar to those described for system B0,+. ATB0,+ transports neutral and cationic amino acids and the transport process is electrogenic and obligatorily dependent on Na+ and Cl−. The expression of ATB0,+ is more predominant in the large intestine than in the small intestine (51). A unique feature of this transport system is its ability to transport several amino acids in their D-isomeric form (51). Because colonic bacteria produce significant quantities of D-amino acids, it is possible that ATB0,+ expressed in the brush-border membrane of the colonocytes, plays a role in the absorption of these amino acids. Even though it is generally believed that D-amino acids do not participate in mammalian metabolism, it is becoming increasingly evident that this may not be true. For instance, D-serine has been identified as the endogenous activator of glutamate receptor in glutamatergic neurons, and an enzyme responsible for the synthesis of this D-amino acid has been cloned from the brain (52). Therefore, the ability of ATB0,+ to transport D-amino acids may have physiologic significance. This transport system, identified as SLC6A14, also belongs to SLC6 gene family, and the gene coding for the protein is located on human chromosome Xq23-q24. The unusually broad substrate selectivity of this transport system is receiving increasing attention because of the potential of this transport system for the delivery of amino acid–based drugs and prodrugs (53). ATB0,+ has the ability

to transport various nitric oxide synthase inhibitors (54) and the antiviral agents valacyclovir (55) and valganciclovir (56). System b0,+ A high-affinity, Na+-independent transport system for neutral and cationic amino acids has been described in the intestinal brush-border membrane (33,47,57). The lack of Na+ dependence is the primary characteristic that distinguishes system b0,+ from system B0,+. Interestingly, system b0,+ also is capable of transporting the disulfide amino acid cystine (Cys-S-S-Cys) (36–38). This is the primary transport system for the absorption of cystine in the intestine and in the kidney. Studies of the molecular aspects of this transport system have shown an unexpected feature. System b0,+ functions as a heterodimer, consisting of two different proteins (36–38,58,59). The heavy subunit of this transport system was first cloned in 1992 (60,61). This subunit is known as rBAT (i.e., related to b0,+ amino acid transport), which in itself has neither any transport function nor the membrane topology characteristic of an authentic transporter. The light chain, known as b0,+AT (i.e., b0,+ amino acid transporter), cloned and characterized at the functional level (62–64), is responsible for the transport function. The role of rBAT is to heterodimerize with b0,+AT during biogenesis via disulfide cross-linking and facilitate the trafficking of the heterodimer to the brush-border membrane. Even though rBAT does not have any transport function of its own, it can influence the kinetic parameters of the transport function of b0,+AT (65). The characteristics of the transport function of the rBATb0,+AT heterodimer are similar to those of system b0,+. The transport function is Na+ independent and is specific for cationic amino acids and cystine. Another interesting feature of this transport system is that it functions as an obligatory amino acid exchanger. Under physiologic conditions, it mediates the entry of cationic amino acids and cystine into enterocytes in exchange for neutral amino acids. Thus, the absorption of cationic amino acids and cystine via this transport system is coupled to the release of neutral amino acids into the intestinal lumen. When the system functions in the entry of cationic amino acids into the cell coupled to the efflux of neutral amino acids, the transport process becomes electrogenic. Under these conditions, the inside-negative membrane potential is expected to provide the driving force for the entry of cationic amino acids. rBAT, also known as SLC3A1, belongs to the SLC3 gene family, and the gene coding for this protein is located on human chromosome 2p16.3-p21. b0,+AT, also known as SLC7A9, belongs to the SLC7 family of amino acid transporters, and the gene coding for this protein is located on human chromosome 19q13.1. IMINO System The IMINO system of the intestinal brush-border membrane is one of the best-characterized amino acid transport systems in this tissue at the functional level. Detailed investigations have been made with a wide range of substrates (66,67).

1674 / CHAPTER 65 The system is exclusive for imino acids such as proline, hydroxyproline, and pipecolic acid. It is present in the jejunum, as well as in the ileum (68). The transport process is strictly Na+ dependent. In addition to Na+, Cl− also plays an obligatory role in the catalytic process (69). The stoichiometry for Na+:Cl−:proline is 2:1:1 (69,70), rendering the transport process electrogenic. The protein responsible for this transport activity has been identified at the molecular level recently (see note added in the proof). It is known as SIT1 (sodium-coupled imino acid transporter 1) or IMINO. It is also identified as SLCGA20. System β Among the amino acid transport systems in the intestinal brush-border membrane, system β occupies a unique position because, unlike other transport systems, it recognizes taurine, a nonprotein amino acid, as a high-affinity substrate. Studies with purified intestinal brush-border membrane vesicles have shown that this transport system interacts exclusively with β amino acids of small size (71–74). System β has no affinity for α amino acids. Anionic and cationic amino acids also are excluded by this system. Among the β amino acids, taurine shows the highest affinity. This transport system has an absolute requirement for Na+, as well as Cl−, being energized by transmembrane gradients for Na+ and Cl−. The Na+:Cl−:taurine stoichiometry is 2 or 3:1:1, which makes the transport process electrogenic. Thus, in the intact intestinal epithelium, three driving forces provide energy for active transport of taurine, namely, an inwardly directed Na+ gradient, an inwardly directed Cl− gradient, and an inside-negative membrane potential. This transport system also is expressed in HT-29 cells, which are of human colon carcinoma origin (75). This transport system is sensitive to inactivation by Ca2+. Preparation of the brush-border membrane vesicles in the presence of Ca2+ (73) or treatment of the brush-border membrane vesicles with Ca2+ (76) drastically reduces the activity of this transport system. The protein responsible for the transport function of system β has been identified at the molecular level (77,78). This transporter, known as TAUT (taurine transporter) and also identified as SLC6A6, belongs to the SLC6 gene family, and the gene coding for the transporter protein is located on human chromosome 3p26-p24. Munck and Munck (12,13,69) have described a transport system for β-alanine in the rabbit ileum. This system is also Na+ and Cl− dependent and is electrogenic with a Na+:Cl−: β-alanine stoichiometry of 3:1:1. But this system does not accept taurine as a substrate, and thus does not represent system β. The ileal transporter strongly interacts with neutral and cationic amino acids (12). The exact identity of this transport system is unknown. System X −AG − System X AG is defined as a transport system that transports the anionic amino acids aspartate and glutamate

exclusively and with high affinity. There is evidence for the existence of this transport system in the small intestine (79,80). − System X AG shows an absolute dependence on Na+, being energized by an inwardly directed Na+ gradient. The presence of an outwardly directed K+ gradient markedly stimulates the Na+-dependent activity of this system (80,81), implying that the movement of Na+ and the amino acid substrate from outside to inside is coupled to the movement of K+ from inside to outside. The transport process is electrogenic, resulting in the transfer of a positive charge across the membrane, and this characteristic is demonstrable in isolated brushborder membrane vesicles (82), as well as in intact tissue (83). Because aspartate and glutamate exist as monovalent anions under physiologic conditions, the electrogenic nature of the transport system suggests that multiple Na+ ions are involved in the catalytic process. The simplest stoichiometry of Na+:amino acid:K+ is 3:1:1. The activity of system X¯AG also is modulated by H+, and now it appears that H+ is indeed an additional cotransported ion. At the molecular level, the protein responsible for the transport activity of system X¯AG is known as EAAT3 (excitatory amino acid transporter 3); it was first cloned and characterized by Kanai and Hediger (84). Heterologous expression of EAAT3 in Xenopus laevis oocytes and detailed characterization of the kinetic aspects of its transport function have shown that the transport process involves entry of one anionic amino acid, three Na+ and one H+ in exchange for one K+ (85). Therefore, there are four different driving forces for the energization of EAAT3, namely, an inwardly directed Na+ gradient, an inwardly directed H+ gradient, an outwardly directed K+ gradient, and an inside-negative membrane potential. The transporter, designated as SLC1A1, represents the first member of the SLC1 gene family. The gene coding for the transporter is located on human chromosome 9q24. System ASC We cloned a transport system from rabbit intestine and the human colon carcinoma cell line Caco-2, the transport function of which was Na+ dependent and the substrate specificity was similar to that of system B0 (86,87). The protein is expressed in the brush-border membrane of the intestine (88). We believed that this transporter was responsible for the functional activity of system B0, and hence designated the transporter protein as ATB0 (amino acid transporter B0). However, later studies showed that the cloned transporter is an Na+-dependent obligatory amino acid exchanger (89,90). The transport function involves the entry of Na+ and a neutral amino acid into the cell coupled to the efflux of Na+ and a neutral amino acid out of the cell. This exchange phenomenon is not a feature of system B0 characterized in purified intestinal brush-border membrane vesicles. Therefore, it is now apparent that this protein is not responsible for the transport activity of system B0. The transport characteristics of ATB0 are similar to those of system ASC, a Na+-coupled transport system with preferential affinity for the amino acids alanine, serine, and cysteine. Consequently, the transporter

PROTEIN DIGESTION AND ABSORPTION / 1675 is now referred to as ASCT2, the second member of the ASC amino acid transporter family. ASCT2 (SLC1A5) belongs to the SLC1 gene family (85), and the gene coding for the protein is located on human chromosome 19q13.3.

acids and peptides along the small intestine, and dissimilarity between the absorptive processes for amino acids and peptides in their adaptational and developmental responses. Dependence of Peptide Transport on Metabolic Energy

System PAT A proton-coupled electrogenic transport system for shortchain amino acids (glycine, alanine, and proline) has been described in the brush-border membrane of the human colon carcinoma cell line Caco-2 (91,92). The protein (PAT1) responsible for this activity has been cloned (93,94). This protein is expressed exclusively in the intestinal brush-border membrane in humans (95) and mediates the H+-coupled electrogenic transport of amino acids such as glycine, alanine, and proline. Because there is an inwardly directed H+ gradient across the intestinal brush-border membrane, PAT1 functions as an active transport system in vivo, energized by an inwardly directed H+ gradient and also by the inside-negative membrane potential. Studies by Foltz and colleagues (96) have uncovered a novel feature of this transport system. In addition to transporting short-chain amino acids by an H+-coupled electrogenic mechanism, PAT1 also mediates the transport of short-chain fatty acids such as butyrate by an H+-coupled electroneutral mechanism. The affinity for short-chain fatty acids, however, is lower than for amino acids. These findings may be of physiologic significance because PAT1 is expressed in the colon (94) where high levels of short-chain fatty acids are generated by bacterial fermentation of unabsorbed carbohydrates and dietary fiber. It is possible that PAT1 plays a role in the entry of these short-chain fatty acids into colonocytes. Because these bacteria-derived fatty acids serve as the preferred metabolic fuel for colonocytes (97), PAT1 as a transporter for short-chain fatty acids may be relevant to the maintenance of colonic health and function. Peptide Transport Independence of Peptide Transport from Amino Acid Transport There is overwhelming evidence indicating that amino acids and peptides are absorbed in the small intestine by different mechanisms (98). The most convincing support for the presence of a distinct transport mechanism for small peptides is the observation that in the amino acid transport defects Hartnup disease and cystinuria, the affected amino acids are poorly absorbed when presented in the free form, but the absorption is normal when these amino acids are presented in the form of small peptides. Additional evidence for the independence of peptide transport includes lack of competition between peptides and amino acids during absorption, enhanced absorption in most studies of amino acids from peptide solutions compared with amino acid solutions of equivalent composition, differential sensitivities of amino acid and peptide transport processes to protease treatment, distinct regions of maximal absorptive capacity for amino

Hydrolysis of peptide substrates during transport studies by ectopeptidases associated with the intestinal brush-border membrane has been a significant problem in the interpretation of experimental data for involvement of metabolic energy in peptide transport. The use of hydrolysis-resistant peptides has, for the most part, overcome this problem. Three such peptides are Gly-Sar, Gly-Sar-Sar, and carnosine, and concentrative uptake into the enterocyte could be demonstrated with all these peptides (99–101). This is clear evidence that intestinal peptide transport is an active process. The dependence of the process on metabolic energy is indicated by the demonstration of inhibition of peptide transport by hypoxia, ATP depletion, and metabolic inhibitors such as cyanide and dinitrophenol (99–101). Driving Force for Peptide Transport Even though for many years it was widely believed that intestinal peptide transport was energized by the Na+ gradient, there was no convincing proof for this mechanism. The dependence of the transport process on Na+ is at best partial, because the inhibition in transport caused by Na+ replacement ranges only from 35% to 65% (102,103). A substantial portion of peptide transport still persists even after total replacement of Na+. Importantly, the Na+-independent component of the transport process is carrier mediated because it can be blocked by various dipeptides (103). Experiments with isolated intestinal brush-border membrane vesicles have provided strong evidence for Na+ independence of peptide transport (104). Surprisingly, despite this Na+ independence, peptide transport in intact intestinal epithelium (105–108), as well as in intestinal brush-border membrane vesicles (109–111), is electrogenic, resulting in transfer of positive charge across the membrane. This electrogenic nature is demonstrable both in the presence and absence of Na+. Because the charge transfer occurs even in cases of peptides that predominantly exist as zwitterions under experimental conditions, it is apparent that peptides are cotransported with an ion other than Na+. A clue to the identity of this ion first came from studies in which peptide transport was found to be stimulated by an inwardly directed H+ gradient (109,110). The H+ dependence of intestinal peptide transport has now been widely accepted (112–115). The acidic microclimate pH that normally exists on the luminal surface of the intestinal brush-border membrane (28,29) provides the driving force for the peptide transport system (Fig. 65-4). There exists a functional coupling between the peptide transport system and the NHE in the brush-border membrane (116,117). The isoform of NHE that is expressed in the intestinal brush-border membrane is NHE3 (118). This functional coupling may explain some of the previous studies that showed partial Na+ dependence of intestinal peptide transport.

1676 / CHAPTER 65 LUMEN

BLOOD

3

1

Dipeptides, tripeptides H+

2

Na+

Dipeptides, tripeptides H+

FIG. 65-4. Transport of small peptides across the enterocyte from the lumen into blood. (1) H+/peptide cotransporter (PEPT1) in the brush-border membrane; (2) Na+-H+ exchanger (NHE3) in the brush-border membrane; (3) peptide transport system(s) in the basolateral membrane.

Influence of Chain Length on Peptide Transport Because the intestinal luminal contents contain peptides of varying chain length, the question arises whether the peptide transport system in the brush-border membrane is capable of transporting all these peptides without regard to chain length. Most studies on peptide transport have used only dipeptides as substrates. There is, however, considerable evidence for the acceptance of tripeptides by the intestinal peptide transport system. Most of the evidence comes from competition experiments that have demonstrated inhibition of dipeptide transport by various tripeptides. There are also reports that directly describe the characteristics of tripeptide transport in the intestine using nonhydrolyzable peptide substrates (106,111,119). Thus, there is little doubt that dipeptides, as well as tripeptides, are excellent substrates for the intestinal peptide transport system. Transport of tetrapeptides occurs only to a small extent, if at all (120,121). Peptides with chain length greater than four amino acids do not appear to be absorbed via mediated pathways in the intestine. There is ample evidence for significant absorption of longer peptides, but the process may involve nonmediated mechanisms (122). Molecular Identity of the Intestinal Peptide Transport System The protein responsible for the intestinal peptide transport activity has been cloned and functionally characterized (123,124). When expressed in heterologous systems, the cloned transporter, known as PEPT1 (peptide transporter 1), mediates H+-coupled and electrogenic transport of dipeptides and tripeptides (125–127). This transporter, designated SLC15A1, belongs to the SLC15 gene family, and the gene coding for the protein is located on human chromosome 13q33-q34. The human PEPT1 consists of 708 amino acids, and immunolocalization studies have shown unequivocally the expression of this transporter in the intestinal brush-border

membrane (128). Detailed studies have been performed on the substrate selectivity of this transport system (126,127). The minimal structural features required for recognition by PEPT1 consist of the charged amino and carboxyl groups separated by a carbon backbone with a distance of 5.5 to 6.3 Å. This essential structural requirement accommodates dipeptides and tripeptides, but not free amino acids and peptides longer than tripeptides. It is therefore readily apparent why PEPT1 accepts only dipeptides and tripeptides as substrates. Interestingly, even though there are 400 different dipeptides and potentially 8000 different tripeptides present in the intestinal lumen as the result of digestion of dietary proteins, PEPT1 is solely responsible for handling this wide array of peptides. The affinity of PEPT1 for its peptide substrates is low, with the Michaelis constant in the submillimolar range. This makes sense for the physiologic function of this transporter. The concentration of dipeptides and tripeptides resulting from the digestion of proteins in the intestinal lumen can be as high as 100 mM. Therefore, the presence of a low-affinity and high-capacity transport system is highly suitable for the absorption of these peptides under these conditions. The broad-substrate selectivity of this transport system represents a unique feature that has received increasing attention because of the potential of such a system for oral delivery of drugs and prodrugs. PEPT1 does indeed possess the ability to transport a variety of drugs. This includes β-lactam antibiotics, angiotensin-converting enzyme inhibitors, and anticancer drugs, among others (129–131). Surprisingly, as long as the minimal structural requirements are met, even the peptide bond does not appear to be critical for recognition as a substrate by PEPT1. This is evident from the ability of this transporter to transport p-aminophenylacetic acid (132), ω amino fatty acids (133), δ-aminolevulinic acid (134), valacyclovir (135), and valganciclovir (136). Thus, PEPT1 has tremendous therapeutic relevance and potential as a delivery system for a variety of drugs and prodrugs. PEPT1 is expressed throughout the small intestine, and the capacity of this transporter for the absorption of dipeptides and tripeptides is high. Under normal conditions, little protein digestion end products leave the terminal small intestine. Accordingly, there is little or no expression of PEPT1 in the large intestine. However, the expression of the transporter in the large intestine appears to be induced under certain pathologic conditions. Colonic expression of PEPT1 has been noted in patients with short bowel syndrome (137), ulcerative colitis, and inflammatory bowel disease (138). These findings may have biological significance. In short bowel syndrome, significant quantities of protein digestion end products may pass through the shortened small intestine unabsorbed and enter the large intestine. The expression of PEPT1 in colon may serve to absorb the peptides as a compensatory mechanism. Similarly, in inflammatory bowel disease, the expression of PEPT1 in colon may be related to the ability of the transporter to transport the bacteria-derived chemotactic peptide N-formylMet-Leu-Phe (139,140). Abnormal expression of PEPT1 in the large intestine may expose the gut-associated immune

PROTEIN DIGESTION AND ABSORPTION / 1677 system to this chemotactic peptide and influence transepithelial neutrophil migration and initiation of inflammatory processes.

FATE OF ABSORBED AMINO ACIDS AND PEPTIDES IN THE ENTEROCYTE According to the scheme proposed in Figure 65-1 for intestinal protein digestion and absorption, the peptide transport system localized in the brush-border membrane of the enterocyte transports dipeptides and tripeptides into the cell, followed by intracellular hydrolysis of the transported peptides. Thus, intracellular peptidases in the enterocyte play a vital role in the terminal stages of protein assimilation. The enterocyte is one of the richest sources of peptidase activity against small peptides, and these enzymes act primarily on dipeptides and, to a lesser extent, on tripeptides (141). The specificity of these peptidases with respect to the amino acid chain length of the peptide substrates fits well with the corresponding specificity of the peptide transport system in the brushborder membrane. In addition to hydrolyzing the exogenous peptides arising from absorption from the lumen, the intracellular peptidases of the enterocyte also participate in the breakdown of endogenous proteins. Accordingly, the activity of these enzymes increases during starvation, a condition that leads to enhanced protein breakdown in the enterocyte (142). In contrast, if animals are put on a protein-deficient but isocaloric diet, a condition that leads to a decrease in the entry of exogenous peptides into the enterocyte, the activity of these enzymes decreases (143). Another interesting observation that is relevant to the role of intracellular peptidases in the hydrolysis of absorbed small peptides in the enterocyte is the regional variation in the activities of these enzymes along the length of the small intestine. The cytoplasmic peptidases show greatest activity in the proximal and/or middle segment of the small intestine, sites at which the absorptive capacity of the intestine for small peptides is high (15,144). Even though the presence of intracellular peptidases is not unique to the intestinal epithelium, because they are present in other tissues as well, the abundance of these enzymes in the intestine in comparison with other tissues supports a role for these enzymes in the hydrolysis of absorbed peptides. It is likely that some of the absorbed peptides that are resistant to hydrolysis by intracellular peptidases appear intact on the serosal side. There is evidence for the appearance of intact peptides containing proline and hydroxyproline in the blood after ingestion of gelatin (145,146), and these peptides are known to be relatively resistant to hydrolysis by cellular peptidases. The dipeptide carnosine also is absorbed intact to a significant extent (147,148). Gardner and colleagues (149) have reported that approximately 10% of the amino nitrogen entering the mesenteric blood during absorption of a partial hydrolysate of casein in vivo is in the form of peptides. Obviously, the extent of amino nitrogen absorption in the form of intact peptides across the enterocyte is influenced by the amino acid composition and sequence of the dietary

proteins, because these factors determine the susceptibility of the peptides arising from luminal digestion to hydrolysis by brush border and cytosolic peptidases of the enterocyte. Free amino acids arising either from hydrolysis of peptides within the enterocyte or from transport from the lumen into the enterocyte feed into several metabolic pathways, namely, degradation, conversion into other amino acids, incorporation into proteins, and transport into blood. The small intestine is a metabolically active tissue. It is a site of synthesis of mucins and apolipoproteins, and its cell renewal rate is rapid. These metabolic processes use amino acids that enter into the cell either from the lumen or from the blood (150,151). Glutamine, glutamate, and aspartate have been shown to be quantitatively the most important amino acids as respiratory fuel in the intestinal epithelium (152). Glutamine is readily deamidated to glutamate, and transamination initiates the metabolism of glutamate and aspartate. The metabolic products of these amino acids that appear in the intestinal venous blood include CO2, NH3, lactate, citrulline, and alanine (153). The capacity of the intestinal epithelium to use glutamine, glutamate, and aspartate is underscored by the observations that little glutamate and aspartate enter the blood even when the animals are fed a high concentration of these amino acids (154,155). Arginine also is extensively metabolized in the small intestine. A significant fraction of the absorbed amino acids is used in protein synthesis in the small intestine. Interestingly, amino acids available from the lumen are incorporated into proteins more readily than those available from the blood (156).

EXIT OF PROTEIN DIGESTION END PRODUCTS ACROSS THE BASOLATERAL MEMBRANE Amino Acid Transport Available information (33–35,157) suggests that there are at least six amino acid transport systems in this membrane, two of them being Na+-dependent (system A and GLYT1), three being Na+ independent (y+, L, and Asc), and one being Na+ independent or dependent depending on the substrate (y+L) (Table 65-2; Fig. 65-5). It has been suggested that the Na+-independent pathways are responsible for transport of amino acids from the cell into the blood, thus participating in the overall process of transcellular absorption of amino acids from the intestinal lumen, whereas the Na+-dependent pathways play a role in supplying the intestinal absorptive cells with amino acids for cellular nutrition during periods between meals (158). System A System A is Na+ dependent and accepts all neutral amino acids including imino acids as substrates (30–32). Ghishan and colleagues (159) have shown that one of the transport pathways for glutamine in the intestinal basolateral membrane

1678 / CHAPTER 65 TABLE 65-2. Amino acid transport systems in the basolateral membrane of the small intestine Transport system

Molecular identity

Substrates

Dependence on Na+

Involvement of other ions

A GLY y+ L y+L

ATA2/SNAT2 GLYT1 CAT1 LAT2-4F2hc y+LAT1-4F2hc

Neutral L-amino acids Glycine Cationic L-amino acids Neutral L-amino acids Neutral L-amino acids Cationic amino acids Neutral L-amino acids Cationic amino acids Small neutral L- and D-amino acids

Yes Yes No No Yes No Yes No No

No Cl− No No No No No No No

y+LAT2-4F2hc Asc

Asc1-4F2hc

ATA2, amino acid transporter A 2; CAT1, cationic amino acid transporter 1; GLYT1, glycine transporter 1; LAT1, L-amino acid transporter 1; SNAT2, sodium-coupled neutral amino acid transporter 2.

is Na+ dependent. This pathway, from its substrate specificity, appears most likely to be system A. This is an electrogenic transport system and derives its energy from an Na+ gradient, as well as from a membrane potential under physiologic conditions. The orientation of these driving forces in vivo is appropriate for active transport of amino acids from blood into the intestinal cells. Therefore, this system is unlikely to play any role in the exit of protein digestion end products from the enterocyte into portal circulation. Its physiologic role is to mediate the entry of amino acids such as glutamine into enterocytes from blood. This might be a physiologically important process to maintain the amino acid nutrition of the intestinal cells when entry of amino acids across the brushborder membrane is limited as may be the case between meals or during illness. The molecular identity of the protein responsible for system A activity remained unknown for a long time. Recently, three different proteins, named ATA1-3 (amino acid

transporter A 1-3), which exhibit transport characteristics similar to those of system A, have been cloned (160). Tissue expression pattern of these three transporter proteins suggests that ATA2 (currently known as SNAT2 [sodium-coupled neutral amino acid transporter 2]) is most likely the transporter responsible for system A activity expressed in the intestinal basolateral membrane. SNAT2 is expressed ubiquitously in mammalian tissues, including the small intestine. It mediates Na+-dependent electrogenic transport of shortchain neutral amino acids (161,162). Glutamine is an excellent substrate for this transporter, a finding relevant to glutamine uptake by enterocytes from blood. Immunolocalization studies have shown that SNAT2 is expressed exclusively in the basolateral membrane of the intestinal epithelium (Fig. 65-6). SNAT2 (SLC38A2) belongs to the SLC38 gene family, and the gene coding for the transporter is located on human chromosome 12q.

BLOOD ∆pNa, ∆ψ

A

∆pNa, ∆pCl, ∆ψ

Gly GLYT1 2Na+ Cl−

∆ψ

y+

L

y+L

Asc

LUMEN

AA0 Na+

AA+ AA0 AA0 AA0 Na+ AA0

AA+ AA0

FIG. 65-5. Amino-acid transport systems in the intestinal basolateral membrane. Arrows indicate the direction of movement of amino acids/ions across the basolateral membrane in vivo. AA0, neutral amino acid; AA+, cationic amino acid; GLYT1, glycine transporter 1.

PROTEIN DIGESTION AND ABSORPTION / 1679

A

B

FIG. 65-6. Immunolocalization of system A (ATA2/SNAT2 [sodium-coupled neutral amino acid transporter 2]) in rat intestinal basolateral membrane (blm). (A) Immunolocalization of ATA2/SNAT2 in rat intestine using fluorescein isothiocyanate–conjugated secondary antibody. (B) Hematoxylin and eosin staining. bbm, brush-border membrane. (See Color Plate 34.)

Glycine Transporter 1 Glycine is a constituent of glutathione, an antioxidant tripeptide found in high concentrations in intestinal epithelial cells. The availability of glycine has potential to control the cellular levels of glutathione in enterocytes. There are two different transporters for glycine in mammalian cells, namely, GLYT1 (glycine transporter 1) and GLYT2 (163). Of these two, GLYT1 has been shown to be expressed in the intestine. Immunohistochemical studies have established the preferential localization of the transporter in the basolateral membrane of enterocytes (164). The transport of glycine via GLYT1 is coupled to the movement of Na+ and Cl−, with a Na+:Cl−: glycine stoichiometry of 2:1:1. Thus, the transport process is electrogenic and is energized by multiple driving forces. The role of GLYT1 in vivo is to import glycine into enterocytes from blood for cellular utilization in metabolic pathways such as glutathione synthesis. Because of the magnitude and direction of the driving forces involved in this transport process, it is unlikely that the transporter plays any role in the efflux of glycine from the cells across the basolateral membrane, and hence in the absorption of diet-derived glycine. System y+ y+ is defined as a system that transports cationic amino acids (lysine, arginine, and ornithine) by an Na+-independent mechanism (30–32). It is present in the intestinal basolateral membrane (157). Because this transport system mediates the transport of positively charged amino acids, its transport function is electrogenic, and the process is energized by the insidenegative membrane potential. Therefore, it is unlikely that this system plays any role in the exit of cationic amino acids from the enterocyte into portal circulation. As is the case with system A, system y+ may function to maintain the amino

acid nutrition of intestinal cells during times of limited amino acid entry across the brush-border membrane. Three different proteins, known as CAT1-3 (cationic amino acid transporter 1-3), have been cloned that exhibit transport features similar those of system y+ (165,166). Among these three proteins, CAT1 is responsible for system y+ in the basolateral membrane of enterocytes. This transporter, identified as SLC7A1, belongs to the SLC7 gene family, and the gene coding for the protein is located on human chromosome 13q12-q14. System L System L is the major Na+-independent system in the intestinal basolateral membrane for transport of neutral amino acids (157). Imino acids, though zwitterionic in nature, are excluded by the system. Studies on the molecular aspects of this transport system have identified two different isoforms of system L (166). Both of them function as heterodimers, consisting of a heavy subunit, known as 4F2hc (heavy chain associated with the 4F2 antigen) or CD98, which is common for both isoforms, and a light subunit. The light subunit differs between the two isoforms, LAT1 (L-amino acid transporter 1) being specific for one and LAT2 being specific for the other. The 4F2hc-LAT1 and 4F2hc-LAT2 heterodimers behave as obligatory amino acid exchangers. Among these two isoforms, 4F2hc-LAT2 is the primary contributor to the activity of system L in the intestinal basolateral membrane. Because this transporter is an obligatory amino acid exchanger, its function will not result in a net influx or efflux of amino acids across the membrane. What it would do is mediate the efflux of neutral amino acids from the enterocyte coupled to the influx of neutral amino acids into the enterocyte. Which neutral amino acids participate in the efflux and which in the influx would depend on the relative concentrations of different

1680 / CHAPTER 65 neutral amino acids on the two sides of the basolateral membrane. As is the case with the rBAT-b0,+AT heterodimer, the role of 4F2hc in the 4F2hc-LAT2 complex is to facilitate the trafficking of the light chain to the basolateral membrane. 4F2hc does not have a membrane topology characteristic of a transporter, whereas LAT2 does. Thus, LAT2 is the actual transporter, and 4F2hc plays only an accessory role in bringing LAT2 to the membrane. 4F2hc (SLC3A2) has structural similarity to rBAT, and both proteins belong to the same gene family. LAT2, known as SLC7A8, belongs to SLC7 gene family. The gene coding for 4F2hc is located on human chromosome 11q13, and the gene coding for LAT2 is located on human chromosome 14q11.2. System y+L Transport of cationic amino acids across the basolateral membrane from the cell into blood during absorption has to occur against an electrical gradient, and this raises the question of how this process occurs in vivo. The answer to this question came from the finding that some neutral amino acids, especially leucine, stimulate absorption of cationic amino acids across the intestinal epithelium (167–170). Subsequent studies showed the presence of a separate transport system that transports neutral amino acids, as well as cationic amino acids (171). The substrate selectivity for neutral amino acids resembles that of system L, and the interaction with cationic amino acids is similar to system y+. Curiously, the transport of neutral amino acids is Na+ dependent, whereas the transport of cationic amino acids is Na+ independent. These characteristics led to naming this transport system y+L. Studies, however, have shown that y+L is also an obligatory amino acid exchanger (166). Under physiologic conditions, the transport system in the basolateral membrane mediates the entry of neutral amino acids from blood into enterocytes in an Na+-coupled manner in a process coupled to the efflux of cationic amino acids from the cells. This exchange process is electroneutral, and thus the exit of positively charged cationic amino acids from the enterocytes is facilitated without any interference by the inside-negative membrane potential. System y+L exists in two isoforms, each functioning as a heterodimer. As is the case with system L, 4F2hc is the common heavy subunit for both isoforms of y+L. The light subunit, which is the actual transporter, is specific for each isoform. The light subunits are known as y+LAT1 and y+LAT2. Both isoforms are expressed in the basolateral membrane of intestinal epithelial cells. Both y+LAT1 (SLC7A7) and y+LAT2 (SLC7A6) belong to the SLC7 gene family. The gene coding for y+LAT1 is located on human chromosome 14q11.2, and the gene coding for y+LAT2 is located on human chromosome 16q22.1-q22.2. System Asc Asc is defined as a transport system with substrate specificity that is similar to that of system ASC, but with transport function that is Na+ independent. It transports short-chain

amino acids such as glycine, alanine, serine, cysteine, and threonine. There are at least two isoforms of this transport system, Asc 1 and Asc 2. Both isoforms function as heterodimers, consisting of 4F2hc as the heavy subunit and an isoformspecific light subunit. Of these two isoforms, system Asc 1 is expressed in the intestinal basolateral membrane (166). The light subunit for this isoform is Asc 1. Thus, the transport function of system Asc in the intestinal basolateral membrane is mediated by the heterodimeric complex 4F2hc-Asc 1. This transporter functions as an amino acid exchanger (172,173). The uniqueness of this transport system is its ability to transport D-amino acids such as D-serine with high affinity. In mouse, Asc 1 is expressed abundantly in the small intestine, but not in the colon (172). The expression pattern in the human intestinal tract is unknown. Because this transport system is an amino acid exchanger, it is likely to participate in the efflux of amino acids from the enterocyte into blood across the basolateral membrane. The ability of the transport system to recognize D-amino acids as high-affinity substrates raises the possibility that it may function in the intestinal absorption of bacteria-derived D-amino acids. However, because the colon rather than the small intestine is the site of bacterial colonization, it would be of interest to determine if the transporter is expressed in human large intestine. If it is expressed in the colon, it might function in conjunction with ATB0,+ to facilitate the absorption of bacteria-derived D-serine and other D-amino acids. ATB0,+ would mediate the active entry of D-amino acids from the lumen into enterocytes across the brush-border membrane, and system Asc would facilitate the exit of these amino acids across the basolateral membrane.

Peptide Transport Although it is widely accepted that a small but significant amount of intact peptides enters the blood during protein assimilation, until recently the mechanisms involved in the transfer of these peptides across the intestinal basolateral membrane have remained unknown. A report by Dyer and colleagues (174) was the first to investigate peptide transport in the basolateral membrane. This study has provided evidence for the presence of a peptide transport system in this membrane. The transport system is relatively specific for small peptides. Even though the substrate selectivity of the basolateral membrane peptide transport system is similar to that of the brush-border membrane peptide transport system, the former is insensitive to transmembrane H+ gradient (175,176). Therefore, the exit of hydrolysisresistant dipeptides and tripeptides from the enterocyte into portal circulation occurs via a separate peptide transporter that does not involve cotransport of H+ (see Fig. 65-4). There have been some studies aimed at characterizing the basolateral membrane peptide transporter at the molecular level (177), but these attempts have not yet been successful in establishing the molecular identity of the protein responsible for this transport process.

PROTEIN DIGESTION AND ABSORPTION / 1681 TRANSPORT OF GLUTATHIONE IN THE SMALL INTESTINE The tripeptide glutathione (γ-Glu-Cys-Gly) is the most abundant soluble thiol in animal cells. There is a substantial amount of glutathione in the intestinal lumen, but it is not derived from dietary proteins. The luminal glutathione originates primarily from biliary secretions. Current interest in the intestinal transport of glutathione stems from the following observations (178–184): glutathione functions as an important antioxidant protecting cellular molecules, particularly the membrane lipids, from reactive electrophiles; glutathione is absorbed intact to an appreciable extent across the intestine; manipulation of glutathione levels in the intestinal lumen modulates plasma levels of this tripeptide, and thus may prove to be useful in the prevention of cellular damage involving free radicals and oxidative injury; glutathione is critical for normal intestinal function and for the prevention of absorption of lipid hydroperoxides; and glutathione levels are decreased in intestine under pathologic conditions such as ulcerative colitis and Crohn’s disease. The characteristics of the glutathione transport systems present in the intestinal brush-border and basolateral membranes have been reviewed (185). These transport systems are unique and differ from the systems available for small peptides originating from dietary proteins. Glutathione, although a tripeptide, contains an amide bond that is formed by the γ-carboxyl group of glutamate rather than by the α-carboxyl group. The glutathione transport systems present in both poles of the enterocyte do not recognize normal dipeptides and tripeptides as substrates. Similarly, the intestinal peptide transport system does not accept glutathione as a substrate. The transport system responsible for the transport of intact glutathione across the brush-border membrane is not energized by an Na+, K+, or H+ gradient, but is activated by Na+ and K+ (186,187). The activation process is catalytic, rather than energetic. The system does not interact with other γ-glutamyl compounds or with oxidized glutathione, but does accept S-substituted glutathione derivatives and glutathione ethyl ester as substrates in addition to glutathione. Therefore, it appears that the transport is highly specific for glutathione and its derivatives that possess the γ-glutamyl group and the tripeptide moiety of glutathione. However, in contrast with the results obtained by Vincenzini and her coworkers (186) with isolated brush-border membrane vesicles indicating the Na+ independence of the glutathione transport system in this membrane, Hagen and Jones (180) have found that the transepithelial transport of glutathione from the lumen into the blood is dependent on the presence of Na+ in the luminal fluid. The reasons for this discrepancy are unknown. The glutathione transport system in the intestinal basolateral membrane exhibits characteristics different from those of the brush-border membrane transport system (179,180). The basolateral transport system is active, Na+ dependent, electrogenic, and accepts various γ-glutamyl amino acids as substrates. It is likely that this transporter normally functions to transport glutathione from the blood into the enterocyte.

However, because the transporter is reversible (180), it may also participate in the transport of glutathione from the enterocyte into the blood under certain conditions.

GENETIC DISORDERS OF INTESTINAL AMINO ACID AND PEPTIDE TRANSPORT There are numerous genetic disorders of amino acid transport involving the small intestine, kidney, or both (188). Three of these disorders, Hartnup disease, cystinuria, and lysinuric protein intolerance, have been well characterized with respect to transport function in the small intestine. Investigations of intestinal function in these disorders have contributed immensely to the understanding of the mechanisms involved in the intestinal absorption of amino acids and peptides. These studies provided the first evidence for the independence of transport of free amino acids and intact peptides in the intestine. They also provided one of the earliest indications that the brush-border and basolateral membranes of the intestinal epithelium do not have the same transport mechanisms for amino acids.

Hartnup Disease Hartnup disease is a recessive genetic disorder in which intestinal and renal transport of neutral amino acids (alanine, serine, threonine, valine, leucine, isoleucine, histidine, glutamine, asparagines, phenylalanine, tyrosine, and tryptophan) is defective (189). The transport of cationic amino acids and anionic amino acids is normal. The biochemical feature of this disease is the hyperexcretion of neutral amino acids in urine. Obviously, this disease arises from a genetic defect in the function of a specific transport system that is involved in the intestinal and renal absorption of neutral amino acids. Despite the defect in the intestinal absorption of neutral amino acids, patients with Hartnup disease do not exhibit obvious symptoms of protein malabsorption. This observation was puzzling until it was discovered that the affected amino acids are absorbed normally in these patients when presented in the form of small peptides (190–192). Because protein digestion products are primarily absorbed in the form of small peptides, these patients obtain adequate amounts of the affected amino acids via the peptide transport mechanism. However, patients with Hartnup disease excrete increased levels of neutral amino acids in urine. Unlike the intestine in which amino acids are absorbed predominantly in the form of dipeptides and tripeptides, absorption of amino acids in the renal tubule occurs primarily in the form of free amino acids. This is because amino acids exist in plasma predominantly in free form, rather than in the form of peptides. Therefore, the transport defect in the kidney manifests in the form of hyperaminoaciduria specific for neutral amino acids. As can be predicted, the urinary levels of cationic and anionic amino acids are normal. In addition, most patients with this disease have increased urinary excretion of indolic

1682 / CHAPTER 65 compounds, primarily indican (indoxyl sulfate). These indolic compounds arise in the intestinal tract from the bacterial metabolism of unabsorbed tryptophan. Patients with Hartnup disease experience niacin deficiency (pellagra) with its associated symptoms: diarrhea, dermatitis, photosensitive skin rash, and neurologic symptoms such as cerebellar ataxia and psychosis. The severity of these clinical symptoms varies among patients depending on the status of dietary intake of proteins. The symptoms are mild in affluent nations where there is optimal intake of dietary proteins that compensates for the increased urinary loss of amino acids in patients with Hartnup disease. In contrast, the symptoms are more severe in underdeveloped countries because of the low levels of proteins in the normal diet. The decreased dietary protein nutrition coupled with increased excretion of amino acids in urine leads to decreased plasma levels of amino acids in patients with Hartnup disease in these countries. This precipitates the clinical symptoms. The pathologic basis of niacin deficiency in patients with Hartnup disease is that tryptophan, a neutral amino acid, is one of the amino acids of which intestinal and renal absorption is affected in this disease. About 50% of the niacin requirement in humans is met by endogenous synthesis of this vitamin from tryptophan. Patients with Hartnup disease are likely to have tryptophan deficiency caused by increased urinary excretion. This decreases the availability of this amino acid for the synthesis of niacin. Tryptophan is also a precursor for the synthesis of the neurotransmitter serotonin. Decreased synthesis of serotonin in patients with Hartnup disease may contribute to the pathogenesis of the neurologic symptoms associated with this disease. Genetic mapping studies have shown that the gene responsible for the disease is located on chromosome 5p15 (193). The transporter coded by the gene has been identified as B0AT1 (44,45). This transporter is responsible for the function of system B0 in the intestine and kidney. The protein is expressed exclusively in the brush-border membrane of the intestinal and renal epithelial cells. It is an Na+-coupled transporter for neutral amino acids, and defects in this transport system explain the hyperexcretion of neutral amino acids in urine in patients with Hartnup disease. Several mutations in this gene have been identified in patients with Hartnup disease, and all of these mutations lead to defective function of this transporter.

Cystinuria In cystinuria, there is a defect in the intestinal and renal absorption of cationic amino acids (arginine, lysine, and ornithine) and cystine (194). This is the most common primary inherited aminoaciduria (worldwide incidence, 1/7000). It is also a recessive disorder. Again, as with the patients with Hartnup disease, cystinurics do not show any evidence of malnutrition because the affected amino acids are absorbed adequately in the form of small peptides (195,196). However, cystinuria is a severe disease because of the inability of the kidney to reabsorb cystine from the glomerular filtrate.

The thiol-containing amino acid cysteine exists in plasma predominantly in the oxidized form cystine (Cys-S-S-Cys). Cystine is not freely soluble in water. When the levels of cystine in aqueous solutions increase beyond 300 mg/L, it crystallizes. The physiologic concentrations of cystine in plasma (10–20 mg/L) are well less than these levels; thus, cystine stays in solution. In the normal kidney, cystine is filtered at the glomerulus and is effectively reabsorbed by the tubular epithelium together with water. In patients with cystinuria, because of a defect in the transport system responsible for the absorption of cystine, the filtered cystine is not reabsorbed in the kidney. However, water is reabsorbed normally in these patients. This leads to a gradual increase in the concentration of cystine along the nephron. The tubular concentrations exceed the solubility limits, and cystine crystallizes and forms stones. Distinctive cystine crystals appear in urine, and radiopaque cystine stones develop in kidneys in affected individuals. Nephropathy caused by cystine stones is the primary clinical symptom associated with cystinuria. Notably, plasma levels of cystine are within normal limits in these patients. Because the transport defect lies in an amino acid transport system that handles not only cystine, but also cationic amino acids, patients with cystinuria excrete increased levels of arginine, lysine, as well as ornithine in urine, in addition to cystine. Urinary excretion of neutral amino acids such as tryptophan is within normal limits. The disease is classified as type I and non–type I based on the excretion pattern of cystine and cationic amino acids in heterozygotes. In type I, heterozygotes are silent with no evidence of hyperexcretion of cystine and cationic amino acids in urine. In contrast, in non–type I, heterozygotes exhibit moderately increased excretion of these amino acids in urine. Prevention of urolithiasis in patients with cystinuria requires increased water intake to enhance urinary output as a means of keeping cystine in solution in the tubular fluid. Oral bicarbonate solutions that alkalinize the urine and consequently enhance the solubility of cystine are useful. Oral sulfhydryl agents such as D-penicillamine also are useful. These agents react with cystine in blood and in tubular fluid to form mixed disulfides (e.g., D-penicillamine-S-S-Cys), which are more water soluble than cystine. The transport system involved in cystinuria is b0,+. Because this transport system functions as a heterodimer consisting of the heavy subunit rBAT and the light subunit b0,+AT, defects in either protein will lead to defective transport function. Analyses of mutations in patients with cystinuria have shown that this indeed is the case (194,197). Patients with type I cystinuria have mutations in the gene coding for rBAT (chromosomal location, 2p16-p21) and patients with non–type I cystinuria have mutations in the gene coding for b0,+AT (chromosomal location, 19q13.1).

Lysinuric Protein Intolerance Lysinuric protein intolerance is a genetic defect in the transport of cationic amino acids with no involvement of

PROTEIN DIGESTION AND ABSORPTION / 1683 cystine (198). This disease differs from cystinuria primarily in the manner in which the affected amino acids are handled in the intestine when presented in the form of peptides. In contrast with cystinuria, the cationic amino acids are absorbed poorly in patients with lysinuric protein intolerance regardless of whether these amino acids are presented in the free form or in the peptide form (199). Thus, there is no backup mechanism for absorption of the affected amino acids in these patients, which explains the symptoms of protein malnutrition associated with this disease. The defective absorption of peptide-bound cationic amino acids indicates that the site of the defect is the basolateral membrane. If the transport of cationic amino acids across the basolateral membrane were normal, the absorption of peptide-bound cationic amino acids (a process that involves transport of peptides across the brush-border membrane via the peptide transport system), generation of free amino acids inside the enterocyte by intracellular hydrolysis, and transport of the free amino acids across the basolateral membrane should remain unaffected. Obviously, this is not the case. The defect in this disease is not confined to the small intestine and kidney, but instead is a generalized phenomenon evident in other tissues such as hepatocytes (200). The characteristic protein intolerance seen in this disease is a consequence of postprandial hyperammonemia, which results from a deficiency of the urea cycle intermediate ornithine, one of the affected amino acids. Nausea and vomiting occur after meals, and therefore aversion to protein-rich meals develops in affected individuals. Because the intestinal and renal absorption of arginine (a semiessential amino acid) and lysine (an essential amino acid) is diminished in this disease, affected individuals experience protein malnutrition and fail to thrive. The disease also is associated with osteoporosis and variable degrees of mental retardation. Treatment consists of protein restriction and oral supplementation of citrulline. Citrulline is a neutral amino acid that is absorbed normally in these patients, and then is converted to ornithine in the liver for participation in the urea cycle. The gene responsible for this transport defect has been identified (197,198). The gene codes for y+LAT1, the light subunit of one of the isoforms of the transport system y+L. y+LAT1 functions as a heterodimer together with the heavy subunit 4F2hc. This transport system is expressed differentially in the basolateral membrane of intestinal and renal epithelial cells. Even though this transport system is a heterodimer, lysinuric protein intolerance is a monogenic disease, caused by mutations only in the gene coding for the light subunit. Currently, no mutations have been identified in the gene coding for the heavy subunit 4F2hc as causative of lysinuric protein intolerance. This is a notable distinction between lysinuric protein intolerance and cystinuria. Also important is that mutations in y+LAT2 have not been linked to lysinuric protein intolerance even though the y+LAT24F2hc complex is expressed in the intestinal and renal basolateral membrane and serves a transport function similar to that of the y+LAT1-4F2hc complex.

Genetic Defects in Intestinal Peptide Transport No genetic disorder has been reported in which the primary defect is in the intestinal peptide transporter PEPT1. However, structure-function studies have identified specific regions in the PEPT1 protein that are critical for transport function (125–127). Mutations in these regions may have marked influence on the ability of this transporter to absorb peptides. Polymorphisms, affecting the transport function, have been detected in the gene coding for PEPT1 in humans, but in vivo consequences of these mutations are not known (see note added in the proof).

NUTRITIONAL, CLINICAL, AND PHARMACOLOGIC RELEVANCE OF INTESTINAL PEPTIDE TRANSPORT It is now well recognized that protein digestion products are absorbed into enterocytes across the brush-border membrane largely as small peptides. Absorption in the form of peptides rather than free amino acids appears to have nutritional advantages (3,201). Human and animal studies have shown convincingly that mixtures of free amino acids are nutritionally inferior to mixtures of small peptides of comparable amino acid composition. The reasons for this include: (1) faster absorption of amino acids when given in the form of peptides than in the form of free amino acids; (2) more even appearance of amino acids in blood after absorption from peptide mixtures than from amino acid mixtures; (3) avoidance of competition during transport between amino acids when absorbed as peptides instead of free amino acids; (4) conservation of metabolic energy in transporting amino acids as oligomeric peptides, rather than as monomers; and (5) relative resistance of peptide transport compared with amino acid transport to numerous adverse conditions such as starvation, protein-calorie malnutrition, vitamin deficiency, and intestinal diseases. The nutritional advantage in the intestinal absorption of amino nitrogen in the form of peptides versus free amino acids has clinical implications. A wide variety of commercially available formulas are currently used for enteral nutritional support in hospitalized patients. A few of them are elemental formulas containing amino nitrogen in the elemental form, namely, free amino acids. These formulas originally were selected on the assumption that protein digestion products are primarily absorbed as free amino acids in the small intestine. Because it is now known that this assumption is incorrect, the possible clinical advantage and nutritional efficacy of enteral diets consisting of small peptides instead of free amino acids are being increasingly recognized in clinical practice. In addition to the physiologic advantages of amino acid absorption in peptide form, a peptide-based enteral diet has many other desirable features. Enteral diets based on amino acids in the free form are hyperosmolar, which may be at least one of the contributing factors in the commonly encountered diarrheal complications associated with feeding

1684 / CHAPTER 65 hyperosmolar formulas. The tonicity of these solutions can be considerably decreased by providing the amino acids in the peptide form. Furthermore, optimal enteral nutrition should be provided with a “complete” amino acid solution including essential and nonessential amino acids, but amino acid–based enteral solutions generally lack tyrosine, glutamine, and cysteine, because tyrosine is insoluble and glutamine and cysteine are unstable. These amino acids can be conveniently included in the form of dipeptides in peptidebased solutions because peptides containing these amino acids are highly soluble and stable. The essential role of glutamine in the maintenance of optimal nutrition in hospitalized patients, especially those with hypercatabolic conditions, has received much attention (202–205). Glutamine is used as a major metabolic fuel by enterocytes and colonocytes. Uptake and metabolism of glutamine in the intestine increase during stress. In a variety of animal models of intestinal atrophy, injury, and adaptation, glutamine-supplemented parenteral and enteral nutrition enhances intestinal mucosal growth, repair, and function; decreases the incidence and severity of sepsis; and improves nitrogen balance. Therefore, omission of this amino acid in most of the currently available liquid-based elemental diets poses a real problem that can be easily solved by adding glutamine in the form of a dipeptide. The current dry-powder elemental diets do, however, contain glutamine. Inclusion of glutamine in the form of small peptides has been recommended and has been used clinically not only in solutions meant for enteral nutrition, but also in those meant for parenteral nutrition, because these peptides are well tolerated and efficiently used by the body (206). In addition to these obvious advantages, a peptide-based enteral diet may be especially suitable for certain patients. Although intestinal diseases such as celiac sprue and tropical sprue are associated with defective amino acid and peptide absorption, the extent of impairment in peptide absorption is much less than the impairment in amino acid absorption. A peptide-based enteral diet would certainly be more beneficial than an amino acid–based diet in these patients. The intestinal peptide transport system has been shown to act as a carrier for absorption of orally active β-lactam antibiotics (129,130), which underscores the pharmacologic relevance of the peptide transport system. Aminocephalosporins and aminopenicillins possess structural features similar to those of tripeptides, and consequently are recognized by the peptide transport system as substrates. Although the normal peptide substrates are predominantly hydrolyzed inside the enterocyte soon after transport across the brushborder membrane, the β-lactam antibiotics remain resistant to the action of enterocyte peptidases and appear intact in the blood in their pharmacologically active form. The affinity of these antibiotics to the intestinal peptide transport system is therefore an important determinant of their therapeutic efficacy. In addition to orally active β-lactam antibiotics, other pharmacologically active drugs such as captopril, bestatin, and renin inhibitors also appear to be absorbed in the small intestine at least in part via the peptide transport system. Studies with valacyclovir and valganciclovir have shown that

the intestinal peptide transport system can be used as an effective delivery system for prodrugs (129,130). Acyclovir and ganciclovir are not substrates for the intestinal peptide transport system, but when these drugs are modified by the addition of valine in an ester linkage, the resultant valacyclovir and valganciclovir are recognized as substrates by the peptide transport system. These compounds do not even possess a peptide bond, and yet the peptide transport system is able to facilitate their intestinal absorption. Once transported into the enterocyte, cytoplasmic esterases hydrolyze these prodrugs and release the therapeutically active acyclovir and ganciclovir into the blood. Thus, the intestinal peptide transport system can be exploited as a delivery system for drugs and prodrugs to improve their oral bioavailability.

REGULATION OF INTESTINAL AMINO ACID AND PEPTIDE TRANSPORT The ability of the small intestine to absorb amino acids and peptides varies significantly in response to several factors. These variations are seen during development, pregnancy, and lactation, and also in response to diseases, intestinal resection, and the quantity as well as the quality of the diet. The underlying mechanisms for this process may be nonspecific (e.g., changes in the absorptive surface area, changes in the physical state of the membrane across which absorption occurs) or specific (e.g., changes in the density of particular transporters, changes in the function of transporters by covalent modification). Nonspecific mechanisms are involved in those instances where there are generalized alterations in the absorptive ability of the small intestine affecting all nutrients, whereas specific mechanisms are involved in those instances where there are changes in the absorption of an individual or a specific group of amino acids and peptides. The target transporters may be localized in the brush-border membrane, the basolateral membrane, or both. The chemical signals responsible for these effects may be exogenous or endogenous in origin and may elicit their actions either from the luminal side or from the serosal side.

Developmental Regulation The absorption rates for amino acids and peptides depend on the age of the organism. In several animal species including humans, amino acid and peptide transporters are present in the small intestine even before birth, even though the exact time of appearance may be different for each transporter. At birth, the gastrointestinal tract abruptly assumes the responsibility of supplying the organism with nutrients. Therefore, the transport mechanisms responsible for absorption of vital nutrients must already be in place at birth, and the amino acid and peptide transport mechanisms are no exception. It has been suggested that the prenatal appearance of these transport mechanisms in the small intestine may have physiologic relevance, enabling the newborn to survive in case of

PROTEIN DIGESTION AND ABSORPTION / 1685 premature birth and to absorb nutrients or factors that are essential for development from swallowed amniotic fluid (207). In fact, glutamine, which is the primary fuel for enterocytes and is essential for the maintenance of intestinal health, is present at high concentrations in amniotic fluid. The transport rates of most amino acids, when expressed per wet weight of the intestinal tissue, decline with age (208, 209). These rates are maximal at or near birth and decrease to a level 2.5 to 5 times lower in the adult. This decline, at least in the case of aromatic amino acids, is associated with an increase in transport affinity and a decrease in transport capacity (209). The age-dependent decrease in the transport of phenylalanine, one of the aromatic amino acids, is demonstrable in isolated brush-border membrane vesicles, showing that it is the transport mechanism in this membrane that is subject to the ontogenetic regulation (210). Similarly, the transport of taurine, studied in isolated enterocytes (211), as well as in isolated brush-border membrane vesicles (72), decreases considerably with age. An interesting aspect of this developmental regulation is that the extent of postnatal decline in intestinal transport is not the same for all amino acids. The decline is significantly greater for essential amino acids than for nonessential amino acids (207). The ability of the small intestine to transport peptides also has been investigated in developing animals (212). The capacity to absorb peptides is maximal at birth, and then decreases with age to reach adult levels. Furthermore, the postnatal decline is much greater for peptide transport than for amino acid transport. This decline, however, appears exclusively to reflect a decrease in the maximal transport capacity because there is no significant change in the affinity of the transport system. Studies have shown that these developmental changes in intestinal peptide transport function are accompanied by parallel changes in the levels of peptide transporter messenger RNA (mRNA) and protein (213–216).

Dietary Regulation The transport rates of most nutrients in the small intestine are regulated by substrate levels in the intestinal lumen, but the underlying mechanisms are complex (217). In general, the presence of high levels of protein or amino acids in the diet increases intestinal transport of amino acids. The pattern of this up-regulation, however, varies significantly for different amino acid transport systems. The increase in transport activity with respect to the nitrogen content of the diet is monotonic in the case of anionic amino acids and imino acids, but nonmonotonic in the case of cationic and neutral amino acids. Individual amino acids do not necessarily induce their own transport, but may increase the transport of unrelated amino acids. For example, the neutral amino acids glutamine and valine induce the transport of anionic amino acids, the anionic amino acid aspartate is the best inducer of cationic amino acid transport, and the cationic amino acid arginine, but not lysine, is a good inducer of anionic amino acid transport. In some cases, individual amino acids do induce their

own transport effectively. Examples of this include aspartate, valine, lysine, and glutamine. These findings suggest that there is no obvious pattern for this type of regulation. Restriction of dietary intake is also known to alter the intestinal ability to transport amino acids. In most studies, short-term fasting (semistarvation) increases amino acid transport (218–221), whereas long-term fasting (total starvation) reduces amino acid transport (221,222). Intestinal transport of peptides also is up-regulated by the presence of high levels of protein in the intestinal lumen (217). Long-term deprivation of protein in the diet, which decreases intestinal transport of amino acids, surprisingly does not affect peptide transport to any significant extent (223). Since the establishment of the molecular identity of the transporter protein (PEPT1) responsible for the intestinal peptide transport function, several studies have investigated the dietary regulation of peptide transport at the molecular level (224–228). Starvation enhances the intestinal expression of PEPT1, apparently in an attempt to maintain protein nutrition under conditions of limited availability of exogenous proteins (224,225). This up-regulation is not seen with other nutrient transporters under identical conditions. The protein content of the diet by itself is an important regulator of PEPT1 expression and activity in the intestine (226). High-protein content enhances PEPT1 expression. This effect can be simulated by certain amino acids and peptides. These amino acids and peptides exert their effects by interacting with a specific region of the promoter in this transporter gene and enhancing its transcription. This region is called the “amino acid– responsive element.” Similar studies have shown that dipeptides are able to up-regulate the expression and activity of PEPT1 in the human colon carcinoma cell line Caco-2 (227,228).

Hormonal Regulation Several hormones and second messengers have been shown to alter intestinal amino acid transport. Somatostatin decreases intestinal transport of glycine, leucine, and lysine (229,230). Vasoactive intestinal peptide also decreases leucine transport (230). In contrast, neurotensin, cholecystokinin, and secretin enhance leucine transport (230). Epidermal growth factor increases intestinal transport of glutamine and alanine, and this increase is demonstrable in brush-border membrane vesicles isolated from animals that have been pretreated with the hormone (231). Other hormones that are known to alter intestinal amino acid transport include adrenocorticotrophic hormone (232), prolactin (233), β-casomorphins and enkephalins (234,235), prostaglandin E1 (236), catecholamines (237), and thyroid hormones (238,239). Transport of taurine in the intestinal cell lines Caco-2 and HT-29 is inhibited by treatment with phorbol esters, which are activators of protein kinase C (240). Because many hormones are known to activate protein kinase C, it is likely that the intestinal transport of taurine also is regulated by certain hormones. Studies have shown that the intestinal peptide transporter PEPT1 is subject to regulation by several hormones (241,242).

1686 / CHAPTER 65 Insulin stimulates PEPT1 activity in Caco-2 cells (243,244). The stimulation is rapid and involves the insulin receptor. Because the insulin receptor is located on the basolateral membrane of the intestinal epithelial cells, it appears that the binding of the hormone to its receptor on the basolateral membrane causes changes in the expression of PEPT1 in the brush-border membrane. This effect occurs without any change in the steady-state levels of PEPT1 mRNA or translation rate. Insulin produces its stimulatory effect by facilitating the trafficking of the transporter protein from an intracellular pool to the brush-border membrane. Uncontrolled diabetes in streptozotocin-treated rats also enhances peptide transport activity in the intestine, but in this case, the increase in transport activity is associated with an increase in steadystate levels of the transporter mRNA (245). Short-term treatment with epidermal growth factor increases peptide transport activity in Caco-2 cells (244). This stimulation is not associated with any change in steady-state levels of PEPT1 mRNA or with any detectable change in transmembrane H+ gradient, which serves as the driving force for the peptide transporter. In contrast, long-term treatment of the cells to epidermal growth factor causes a decrease in the activity of PEPT1 with a parallel reduction in the steadystate levels of PEPT1 mRNA and protein (246). Leptin is another hormone that regulates PEPT1 activity in the intestine (247). By acting on the leptin receptor expressed on the brush-border membrane of the enterocytes, this hormone enhances the trafficking of PEPT1 protein from an intracellular pool to the brush-border membrane, and consequently increases the peptide transport activity. Mechanistically, the actions of insulin and leptin on PEPT1 are similar. Thyroid hormones inhibit intestinal peptide transport activity by interfering with gene expression in Caco-2 cells (248), as well as in rat intestine (249). Growth hormone has been shown to increase the intestinal peptide transport activity (250–252).

Diurnal Rhythm The expression and activity of intestinal peptide transport system are subject to diurnal rhythm (253–255). In rats maintained under a 12-hour lighting schedule (8:00 AM to 8:00 PM), the intestinal peptide transport activity is maximal at 8:00 PM and minimal at 8:00 AM (253). These changes are accompanied by parallel changes in peptide transporter mRNA and protein. However, this diurnal rhythm appears to be primarily caused by the feeding cycle in these animals, rather than the light cycle, as evidenced from the disappearance of the diurnal rhythm under fasting conditions (254,255). Na+-H+ Exchanger and Regulation of Intestinal Amino Acid and Peptide Transport The amino acid transporter PAT1 and the peptide transporter PEPT1 are energized by an inwardly directed H+ gradient.

Therefore, changes in the magnitude of this driving force can alter the transport function of these two transporters. The inwardly directed H+ gradient across the intestinal brushborder membrane is generated and maintained by NHE3. Under in vivo conditions, there is a functional coupling between NHE3 and PEPT1, as well as between NHE3 and PAT1. Theoretically, one would expect changes in NHE3 activity to influence PEPT1 and PAT1 activities in parallel. This indeed is the case (95,116,117). Inhibition of NHE3 activity by hormones or second messengers leads to a decrease in the activity of PEPT1 and PAT1, whereas activation of NHE3 by external Na+ leads to an increase in the activity of these two transporters. These findings may have clinical implications for alterations in amino acid and peptide nutrition under pathologic conditions such as cholera and infection with enterotoxigenic Escherichia coli, which are associated with decreased activity of NHE3.

CONCLUSIONS AND FUTURE PERSPECTIVES The most important aspect of protein digestion and absorption in the gastrointestinal tract is that the protein digestion products are absorbed into enterocytes predominantly in the form of dipeptides and tripeptides, rather than free amino acids. This absorptive process is mediated by a single transporter, known as PEPT1, expressed in the intestinal brushborder membrane. However, protein digestion end products enter the portal blood primarily in the form of free amino acids, after intracellular hydrolysis of absorbed peptides. Absorption of free amino acids does occur across the brushborder membrane, but only to a small extent. Different sets of amino acid transporters participate in the transfer of free amino acids across the brush-border and basolateral membranes in the enterocyte. Consequently, genetic defects in amino acid transporters that are expressed exclusively in the brush-border membrane do not lead to serious clinical complications, whereas genetic defects in amino acid transporters that are expressed predominantly in the basolateral membrane are associated with severe clinical symptoms. Because the absorption of small peptides via the intestinal peptide transporter is critical for maintenance of protein nutrition, what would be the consequences if there are genetic defects in this transporter protein? There are no reports in the literature on deleterious mutations in the gene coding for the transporter. Currently, no rodent models with disruption of the intestinal peptide transporter gene have been reported in the literature. However, based on the importance of the transporter in the supply of amino acids to the organism, we predict that disruption of the transporter function would have deleterious consequences. C. elegans is being increasingly used as an animal model to understand the functional aspects of membrane transport systems because of the relative simplicity with which specific genes can be knocked down in this organism. The nematode ortholog of the mammalian intestinal peptide transporter has been identified and

PROTEIN DIGESTION AND ABSORPTION / 1687 functionally characterized (256). There are deletion mutants of C. elegans that lack this transporter, and these mutants exhibit a severely retarded phenotype associated with reduced progeny and body size (257). Lack of the transporter also leads to disruption of nutrient sensing in this organism, suggesting that transport of peptides in the intestine from dietary proteins via the transporter is critical for normal growth and development (257). Absorption of free amino acids in the intestine apparently provides sufficient essential amino acids for survival, but this process is not able to compensate adequately for the lack of peptide absorption to support normal growth and development. Interestingly, these mutants have increased lifespan and reduced fat content (257–259). These effects are likely to be caused by a mechanism similar to caloric restriction. These studies with C. elegans provide strong evidence for the physiologic importance of the intestinal absorption of peptides. Rodent models lacking the intestinal peptide transporter are sorely needed to determine whether these findings from the nematode are reproducible in higher organisms. Such vertebrate animal models also would be useful to investigate the relevance of the transporter to inflammatory processes in the intestinal tract. Because the transporter is expressed in the large intestine under pathologic conditions such as ulcerative colitis, animal models with disruption of the transporter gene would be valuable to evaluate the role of the transporter in the large intestine in the initiation or development, or both, of these pathologic processes. Note added in proof: The molecular identity of the protein responsible for the amino acid transport system IMINO has been established recently (Takanaga H, Mackenzie B, Suzuki Y, Hediger MA. Identification of mammalian proline transporter SIT1 (SLC6A20) with characteristics of classical system imino. J Biol Chem 2005;280:8974–8984; Kowalczuk S, Broer A, Munzinger M, Teitze N, Klingel K, Broer S. Molecular cloning of the mouse IMINO system: an Na+- and Cl−-dependent proline transporter. Biochem J 2005;386:417–422.). Recent studies have also identified polymorphisms that significantly decrease the function of the transporter in the gene coding for PEPT1 in humans (Zhang EY, Fu DJ, Pak YA, Stewart T, Mukhopadhyay N, Wrighton SA, Hillgren KM. Genetic polymorphisms in human proton-dependent dipeptide transporter PEPT1: implications for the functional role of Pro586. J Pharmacol Exp Ther 2004;310:437–445; Anderle P, Nielsen CU, Pinsonneault J, Krog PL, Brodin B, Sadee W. Genetic variants of the human dipeptide transporter PEPT1. J Pharmacol Exp Ther 2005; in press.).

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1692 / CHAPTER 65 227. Walker D, Thwaites DT, Simmons NL, Gilbert HJ, Hirst BH. Substrate upregulation of the human small intestinal peptide transporter, hPepT1. J Physiol (Lond) 1998;507:697–706. 228. Thamotharan M, Bawani SZ, Zhou X, Adibi SA. Mechanism of dipeptide stimulation of its own transport in a human intestinal cell line. Proc Assoc Am Physicians 1998;110:361–368. 229. Krejs GJ, Browne R, Raskin P. Effect of intravenous somotostatin on jejunal absorption of glucose, amino acids, water, and electrolytes. Gastroenterology 1980;78:26–31. 230. Chen YF, Feng ZT, Wen SH, Lu GJ. Effect of vasoactive intestinal peptide, somatostatin, neurotensin, cholecystokinin octapeptide, and secretin on intestinal absorption of amino acid in rat. Dig Dis Sci 1987;32:1125–1129. 231. Salloum RM, Stevens BR, Schultz GS, Souba WW. Regulation of small intestinal glutamine transport by epidermal growth factor. Surgery 1993;113:552–559. 232. Mahmood A, Varma SD, Wagle DS. Effect of hormonal treatments in rats on the intestinal transport of methionine in vitro. Indian J Exp Biol 970;8:330–331. 233. Mainoya JR. Effect of prolactin on sugar and amino acid transport by the rat jejunum. J Exp Zool 1975;192:149–154. 234. Ermisch A, Brust P, Brandsch M. β-Casomorphins alter the intestinal accumulation of L-leucine. Biochim Biophys Acta 1989;982:79–84. 235. Meyer G, Botta G, Rossetti C, Cremaschi D. Enkephalin regulation of L-valine transport in rabbit ileum. Arch Int Physiol Biochem 1989;97: 65–69. 236. Nassar CF, Haddad ME, Habbal ZM. Prostaglandin E1 inhibition of lysine transport across the rat, rabbit and turtle small intestine. Comp Biochem Physiol 1982;72A:483–487. 237. Kinzie JL, Grimme NL, Alpers DH. Cyclic AMP-dependent amino acid uptake in intestine-the importance of β-adrenergic agonists. Biochem Pharmacol 1976;25:2727–2731. 238. London DR, Segal S. In vitro studies of the intestinal transport of amino acids and a sugar in hypothyroid rats. Endocrinology 1967;80:623–628. 239. Syme G, Levin RJ. The effects of hypothyroidism and fasting on electrogenic amino acid transfer: possible evidence for multiple neutral amino acid carrier systems in rat jejunum. Biochim Biophys Acta 1977;464:620–628. 240. Brandsch M, Miyamoto Y, Ganapathy V, Leibach FH. Regulation of taurine transport in human colon carcinoma cell lines (HT-29 and Caco-2) by protein kinase C. Am J Physiol 1993;264:G939–G946. 241. Adibi SA. Regulation of expression of the intestinal oligopeptide transporter (Pept-1) in health and disease. Am J Physiol 2003;285: G779–G788. 242. Nielsen CU, Brodin B. Di/tri-peptide transporters as drug delivery targets: regulation of transport under physiological and patho-physiological conditions. Curr Drug Targets 2003;4:373–388. 243. Thamotharan M, Bawani SZ, Zhou X, Adibi SA. Hormonal regulation of oligopeptide transporter Pept-1 in a human intestinal cell line. Am J Physiol 1999;276:C821–C826. 244. Nielsen CU, Amstrup J, Nielsen R, Steffansen B, Frokjaer S, Brodin B. Epidermal growth factor and insulin short-term increase

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Role of Membrane and Cytosolic Fatty Acid Binding Proteins in Lipid Processing by the Small Intestine Nada Abumrad and Judith Storch Cellular Fatty Acid Uptake, 1693 Fatty Acid Delivery to Cells, 1694 Facilitated Membrane Transfer of Fatty Acids, 1694 Fatty Acid Uptake by Enterocytes, 1695 Proteins Implicated in Fatty Acid Transport That Are Expressed in Enterocytes, 1696 Plasma Membrane Fatty Acid–Binding Protein, 1696 FATP4 and the Family of Fatty Acid Transport Proteins, 1696 CD36-Facilitated Fatty Acid Uptake: Role in Chylomicron Production and Secretion, 1698

Intracellular Fatty Acid Transport, 1699 Fatty Acid–Binding Proteins, 1700 Enterocyte Fatty Acid–Binding Protein Structure and Equilibrium Binding of Ligand, 1700 Regulation of Fatty Acid–Binding Protein Expression in the Intestine: Functional Implications, 1701 Functions of the Fatty Acid–Binding Proteins in the Proximal Intestine, 1702 Future Directions, 1704 References, 1704

After a meal, hydrolysis of triacylglycerols (TGs) in the intestinal lumen by pancreatic lipases generates fatty acids (FAs) and monoglycerides, which are absorbed by enterocytes. Inside the enterocyte, the FAs are incorporated into triglycerides, phospholipids, and cholesteryl esters; these lipids are packaged with apoproteins into chylomicrons, the intestinally produced triglyceride-rich particles. Chylomicrons are secreted into the mesenteric lymph for transport to peripheral tissues where their constituent triglycerides are hydrolyzed by lipoprotein lipase located at the surface of capillaries. The released FAs are taken up rapidly by tissues, where they are used for various cellular pathways. In general, there are two aspects of cellular FA uptake that are relevant to and can impact chylomicron metabolism. The first one relates to the ability of the enterocyte to uptake and

package dietary FA for secretion. The second, as discussed in later sections, relates to the capacity of peripheral tissues to take up FA, because this appears to be linked to efficient clearing of blood chylomicrons.

N. Abumrad: Departments of Medicine and Nutritional Sciences, Washington University, St. Louis, Missouri 63110. J. Storch: Department of Nutritional Sciences, Rutgers University, New Brunswick, New Jersey 08901. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

CELLULAR FATTY ACID UPTAKE To enter the cell, FAs have to cross the plasma membrane. Because of their lipophilic nature, it would appear that FAs can easily transfer across lipid bilayers. However, the membrane of mammalian cells is much more complex than a simple phospholipid bilayer, and there is evidence to indicate that it presents a significant barrier to diffusion of longchain FAs (1). Furthermore, it would appear to be important for cells to possess mechanisms by which to regulate FA uptake because these molecules have potent effects and can play a multitude of roles inside the cell. FAs are precursors to several pathways and regulators of the activity or localization of many proteins. FAs also serve as signaling mediators and as activating ligands of the peroxisome proliferator-activated receptors (PPARs), which are lipid-regulating nuclear factors (2–5). Thus, FAs modulate activity of many pathways and

1693

1694 / CHAPTER 66 gene expression and, as a result, can exert a profound influence on cell development and function. Based on this, a tight regulation of FA uptake and metabolism would be advantageous to avoid disturbances that could result in various cellular aberrations and dysfunctions. Transport of other major substrates such as glucose (6) or amino acids (7) is tightly regulated in line with cellular needs, and it is likely that FA uptake is regulated in a similar manner. Most tissues rely heavily on FAs and, except for liver, fat, and mammary gland, have limited or no capacity for FA synthesis. Thus, the needed FAs are taken up from the circulation. Consequently, cells need to tailor uptake to constant changes in substrate supply and demand as the organism navigates the transitions between feeding and fasting or rest and activity. For example, demand for FA-derived energy increases during fasting because it is essential to spare glucose to meet the needs of glucose-dependent tissues (8). In contrast with glycogen, which can be depleted in a short period, the amount of stored fat is substantial and can provide a more sustainable substrate. As a result, the ability of cells to regulate uptake and to accomplish substrate shifts, especially between glucose and FAs (9,10), is a physiologically important adaptation for surviving caloric restriction or oversupply.

with the cell membrane, or of the monomeric FA in solution after its dissociation from the micelle. The first possibility was discounted based on the findings that lipid constituents of micelles exhibited independent rates of cellular uptake. Based on several lines of evidence, it is now thought that enterocyte FA uptake occurs from the FA monomers in the aqueous phase, and this, in turn, drives the dissociation of more FA from the micelle. It is reasonable to consider the FA/albumin and FA/micelle delivery systems as similar and with parallel roles in cellular FA uptake. They act to solubilize millimolar (mM) amounts of FA and provide cells with a steady supply of monomeric FA or ubFA at nanomolar (nM) or low micromolar (µM) concentrations (22,23). If, as suggested, the FA monomer dissociated from micellar solutions is important for enterocyte uptake and because its concentration is likely to be extremely low in the acid environment of the intestinal lumen, the existence of membrane proteins that recruit the FA and direct it to metabolic sites would be advantageous and efficient for enterocytes. The following section highlights the evidence accumulated during the late 1990s and early 2000s that is related to membrane FA uptake. Most studies to date have used FA complexed to albumin and cells other than enterocytes; thus, these are described first before reviewing the work dealing with intestinal tissue or cells.

FATTY ACID DELIVERY TO CELLS In the circulation, FAs (0.2–2 mM) are carried quantitatively bound to albumin, which greatly increases the amount of FA solubilized and accessible to cells for uptake (11–13). Complexes of FA and albumin also are used in uptake assays in vitro (14). In the absence of albumin, because FAs are poorly soluble in water, the FA concentration that can be used is in the low micromolar (µM) range and will be depleted by cells instantaneously; thus, the linear portion of the uptake time course would be too short to measure. With mixtures of FA and albumin, uptake is a function of unbound fatty acid (ubFA), which is the monomeric form in equilibrium with albumin-bound FA. ubFA increases and so does uptake by cells as the molar ratio of FA to albumin is increased, promoting FA dissociation from the protein (5,15,16). Early estimates for ubFA levels in the circulation (12) have been revised and currently are thought to be in the range of 5 to 50 nM (13,17). In contrast with other cells, which derive FA from the FAalbumin complexes present in the circulation, the enterocyte on its apical membrane is exposed to another system of FA delivery, with FA being solubilized in the intestinal lumen by bile salt micelles. In the lumen, incorporation of FA into bile salt micelles, similar to albumin in the circulation, greatly increases the amount of FA that is available for uptake (18–20). Micelles will solubilize millimolar concentrations of FA, whereas the monomeric free FA that can be present in solution is extremely low. As formulated by the early studies of Westergaard and Dietschy (21), there are several ways by which uptake of micellar FA could occur: via uptake of the whole micelle, of the lipid after interaction of the micelle

FACILITATED MEMBRANE TRANSFER OF FATTY ACIDS Our current understanding of the mechanism of FA uptake is that it is facilitated, at least in part, by membrane proteins (15,16,24). This contradicts the previously held view that transfer occurs exclusively by passive diffusion through the membrane governed by extracellular FA concentration and intracellular metabolism. The molecular details of how proteins would facilitate FA transfer remain unknown. However, the protein-facilitated component appears to play the major role in vivo where it is postulated to coexist with a component of passive FA diffusion. Early studies of FA transport under conditions in which membrane FA permeation was the rate-limiting step documented saturation kinetics of the process as a function of ubFA (in equilibrium with albumin). This was not consistent with a process of passive diffusion, which would be expected to exhibit a linear dependence and no saturation. Based on the revised ubFA estimates, the transport Km obtained by numerous studies in mammalian cells is less than 10 nM, which is in the range of ubFA levels in the circulation, currently estimated to average 7.5 ± 2.5 nM. At physiologic ubFA levels, the saturable component of FA permeation would account for most (>90%) cellular uptake (24–26). The linear, diffusion-like component would be more significant at high FA/albumin ratios, although a study with adipocytes could not detect this component and suggested that all uptake in mammalian cells was saturable and protein facilitated (1).

ROLE OF MEMBRANE AND CYTOSOLIC FATTY ACID BINDING PROTEINS IN LIPID PROCESSING / 1695 Saturability of FA permeation was demonstrated in many cell types. These included adipocytes (27,28), muscle cells (29,30), and intestinal cells (29,145). Some features of the transport system appear similar across studies and cell types, and these relate to specificity for long-chain FA as opposed to short-chain FA and a transport Km in the low nanomolar range. There is disagreement on whether FA transport is active, adenosine triphosphate (ATP) dependent, or Na coupled as opposed to being facilitated down a concentration gradient. Active transport can lead to intracellular FA concentrations that exceed those present outside the cell, whereas facilitated diffusion would ultimately only equilibrate FA levels across the membrane. Initial studies with adipocytes suggested that transport was energy independent (27). Studies with hepatocytes (31) documented reduction of uptake by metabolic inhibitors and with substitution of medium Na+ by Li+, K+, or choline. One complication of the studies with hepatocytes is that effects of the manipulations on FA metabolism cannot be completely ruled out. The difficulty in determining whether FA uptake is passive or active reflects the inability to measure intracellular FA concentrations during uptake. For example, in the case of glucose transport by adipocytes under basal conditions, intracellular glucose can be measured and usually is found to be about one fourth of the level of extracellular glucose. This indicates that metabolic activity exceeds that of transport. Insulin addition increases transport capacity about 30-fold, eventually equilibrating glucose inside and outside the cell, in line with the process being that of facilitated diffusion. In the case of FAs, such information is difficult to obtain. Although the concentration of ubFA can be estimated for the extracellular medium, from the total amounts of FA and FA carrier and the related binding constants, similar measurements cannot be made easily for the intracellular milieu. The distribution of FA between cytosolic fatty acid–binding protein (FABPs) and intracellular membranes is largely unknown, making it difficult to estimate the level of unbound FA inside the cell. However, a study with adipocytes measured intracellular free FA (FFA(i)) by microinjecting cells with ADIFAB, a fluorescently labeled FABP that can be used to measure unbound FA (1). At steady state, FFA(i) was found to be, on average, twofold greater than FFA outside the cells, and this gradient was abolished by depleting cellular ATP. These interesting observations support energy coupling of FA uptake and provide a new approach to study this process. Further studies using ADIFAB and similar methods are needed to reexamine the energy dependence of FA transport.

FATTY ACID UPTAKE BY ENTEROCYTES Several studies have examined FA transport in the intestine using multiple experimental systems. These ranged from intestinal tissue, isolated enterocytes, model cell lines, or brush border membranes (32–39). Although some studies proposed a single diffusion mechanism, most reached the conclusion that uptake is likely to involve both protein-facilitated and

diffusion-like components. Using everted sacs, Chow and Hollander (37,38) found that the uptake of linoleic acid was saturable at low micellar concentration, whereas it showed diffusion-like behavior at high concentrations. The absence of information related to the monomeric FA concentration makes it difficult to interpret these data with respect to saturation kinetics. However, the observations remain intriguing with respect to the different kinetic behavior described for low versus high FA concentrations. Several studies with isolated intestinal cells using FA complexed to albumin have been reported and generally proposed the existence of a saturable protein-facilitated system for uptake of long-chain FAs. Stremmel and colleagues (34) reported that uptake of [3H]-oleate by isolated rat jejunal mucosal cells was saturable as a function of increasing unbound oleate with a km in the nanomolar range. Furthermore, it exhibited temperature dependence with an optimum at 37°C (40). A more recent study with isolated rat enterocytes documented saturability of initial uptake rates as a function of unbound FA and measured a Km in the nanomolar range. In addition, maximal capacity or Vmax averaged 6.9 nmol per 106 cells per minute and was reduced considerably by intraduodenal oleate infusion (41). Studies of FA uptake with immortalized cell line models of enterocytes such as Caco-2 (36) and intestinal epithelial cell 6 (IEC-6) (42) cells provided data that were in line with those obtained with isolated enterocytes with respect to the existence of saturable and nonsaturable FA uptake. They also highlighted additional features of FA transport in the intestine. In both cell lines, it was shown that FA uptake could be strongly inhibited by 2-monoacylglycerol (2-MG), another digestion product of triglyceride hydrolysis in the intestinal lumen. In the case of IEC-6 cells, triolein, glycerol, and monooctanoate also were tested, but had no effect. In Caco-2 cells, diacylglycerol was found to have no effect on FA uptake. In line with this, initial rates of MG uptake were sensitive to inhibition by long-chain FA (35,42). These findings suggest the interesting possibility that the membrane FA transporter(s) in enterocytes may coordinate the uptake of 2-monoglyceride and FA, the two substrates needed to re-form triglycerides inside the cell. Studies with both jejunal and ileal brush-border membranes, where uptake can be examined in the absence of metabolic activity, documented saturable kinetics for both jejunum- and ileum-derived membranes. Binding of oleate to the membranes was shown to be time and temperature dependent, inhibited by addition of excess cold oleate, and decreased by heat denaturation or trypsin digestion of the membranes (34). Furthermore, regulation of brush-border membrane transport by dietary lipid was demonstrated in another study in which a diet rich in unsaturated FA enhanced oleic acid uptake, whereas it was decreased by feeding an isocaloric diet containing only saturated FA (32). The studies cited earlier provided kinetic evidence in support of the existence of protein-facilitated FA uptake in enterocytes and documented susceptibility of the process to regulation by dietary FAs or by membrane-modifying agents.

1696 / CHAPTER 66 Other supportive but more indirect evidence can be derived from studies showing either genetically determined variability in FA uptake by intestinal segments (43) or a differential effect of dietary fat composition on long-chain FA uptake (44,45). Although these differences could theoretically be related to variations in the biophysical nature of the intestinal mucosa, the lack of major alterations in mucosal composition or morphology and the specificity of the observed effects suggest that they involved altered expression levels of mucosal membrane transporters. For example, using intestinal rings, Keelan and colleagues found significant differences in uptake of long-chain FA compared across the intestines of various strains of mice. The differences were not observed for cholesterol and could not be explained by variations in food uptake, body weight, or the weight of the intestine. These data suggested that genetic differences in FA uptake exist and may be related to variations in the expression of protein-mediated components of lipid uptake (43). Additional evidence can be derived from the extensive information available related to the ability of the intestine to adapt functionally in response to environmental challenges. For example, a threefold to fourfold increase in intestinal FA uptake is observed after bowel resection (46) and is important for improving nutritional outcome in response to the loss of a portion of the small intestine or with fasting and malnutrition. This intestinal adaptability involves differential changes in brush-border membrane transport (47). The specificity of these changes to some FAs, the modulation of the adaptive response by fat in the diet, and the lack of effects on other lipophilic nutrients make it likely that they involve up- or down-regulation of carrier-mediated transport.

PROTEINS IMPLICATED IN FATTY ACID TRANSPORT THAT ARE EXPRESSED IN ENTEROCYTES Several membrane proteins have been identified that enhance cellular uptake of FA, including the plasma membrane fatty acid–binding protein (FABPpm), fatty acid transport proteins (FATP1-6) (5), and fatty acid translocase (FAT) or CD36 (6). FABPpm, CD36, and FATP4 are expressed in the intestine.

component and could be inhibited by an antibody against FABPpm. Schoeller and colleagues (49), using rat and rabbit jejunal brush-border membrane vesicles, showed that intestinal uptake of oleic acid was reduced when brush-border membrane vesicles were incubated with a rat liver FABPpm antibody in the absence, but not in the presence, of opposing Na+-H+ gradients. These authors postulated that oleic acid uptake into enterocytes occurs both by diffusion and by a process involving activation of the Na+-H+ exchanger and FABPpm. Despite the accumulated evidence linking FABPpm to FA uptake, its function in the process is still debated for several reasons. The protein was shown to be a membrane-associated form of mitochondrial aspartate aminotransferase, mASpAT, an enzyme that functions in the malate-aspartate shuttle, which plays an important role in importing reducing equivalents into the mitochondria (50,51). This raised concerns that FABPpm recovery with plasma membranes reflected contamination of these fractions with mitochondrial proteins. Careful studies by Berk and Stump (48) effectively addressed these concerns. However, the factors that regulate membrane association of mASpAT and whether they operate in some cells and not others remain poorly understood. Studies using immunofluorescence and Western blot analysis documented FABPpm association with the plasma membrane in many tissues including liver, adipose tissue, cardiac muscle, intestine, and vascular endothelium (48). However, immunoelectron microscopy could only document membrane association in few of these tissues (50). Strong labeling of mitochondria was observed in all tissues examined, but there was no detectable cell-surface labeling in the heart or the liver where FABPpm is postulated to play an important role in FA uptake. In contrast, specific labeling was observed on the cell surface of kidney tubules, of arteriolar endothelial cells, and of lymphocytes (50). These findings may cast some doubt on the proposal that FABPpm directly functions in membrane FA transport in liver or muscle. However, they do not rule out involvement of the protein in FA uptake by these tissues. There is considerable evidence to link FABPpm levels and regulation to alterations in FA metabolism (52,53). For example, the reported changes in expression or localization of mASpAT may reflect an important interaction between activity of the malate-aspartate shuttle and cellular FA uptake, a possibility that has not yet been examined.

Plasma Membrane Fatty Acid–Binding Protein FATP4 and the Family of Fatty Acid Transport Proteins FABPpm was the first protein postulated to function in FA uptake, and it was isolated from rat liver plasma membranes and from jejunal microvilli (34,40,48). Using an oleateagarose affinity column, Stremmel and colleagues identified a 40-kDa FABP and named it FABPpm to highlight its association with plasma membranes. FA binding to purified FABPpm and inhibition of uptake by an antibody raised against the protein (48) were described later. Expression of FABPpm in Xenopus laevis oocytes and in 3T3 fibroblasts was associated with an increase in FA uptake rates. The increase in 3T3 cells reflected acquisition of a saturable, high-affinity

The first member of the FATP family was identified in 1994 by Schaffer and Lodish (54) using an expression cloning strategy. Screening of COS7 cells for uptake of a fluorescent FA after expression of complementary DNA (cDNA) from a 3T3-L1 adipocyte cDNA library identified a novel integral membrane protein (63 kDa), which was designated as FATP (later renamed FATP1). A second protein with high analogy to liver fatty acyl-coenzyme A synthetase (ACS) also was identified with the screen. FATP1-transfected cells showed a significant increase in uptake of long-chain FA versus

ROLE OF MEMBRANE AND CYTOSOLIC FATTY ACID BINDING PROTEINS IN LIPID PROCESSING / 1697 a small change in uptake of short-chain FAs. FATP1 is an integral membrane protein predicted to have at least one transmembrane domain (Fig. 66-1) and several membraneassociated sequences (55). An adenosine monophosphate– binding site is present in FATP, and in addition to mediating ATP binding, it appears essential for enhancement of FA uptake by the protein. The FATP protein family has 6 members, and they all share a conserved 311-amino-acid signature motif (15). The proteins have different tissue distribution. FATP1 is highly expressed in adipose tissue, heart muscle, and skeletal muscle, whereas FATP2 is in liver and kidney. FATP3 is found in liver pancreas and lung; FATP4 has high expression in the intestine, but also is expressed in fat, skin, and heart (56). Expression of FATP5 is specific to the liver and FATP6 is specific to the heart, where it is the most abundant FATP (57). All FATPs have FA-acyl-coenzyme A (CoA) synthase activity, but they differ in substrate specificity. FATP1 and FATP4 have broad specificity and can activate both longand very long-chain FAs (58,59). It could be argued that the synthase activity may mediate or be linked to the ability to enhance FA transport by preventing efflux of the FA from the cell. In line with this, the ATP-binding domain that is present in all FATPs appears essential for FA transport activity

because its mutation in FATP1 eliminated the ability of the protein to enhance transport (16). In contrast, studies of the various FATPs expressed in yeast strains compromised for long-chain FA metabolism (transport, activation, and synthesis of FA) indicated that the transport and enzymatic functions did not always correlate well (60). Findings in FATP1 null mice have been complex to interpret (61). Blood lipids levels and FA uptake in skeletal muscle were unaltered with FATP1 deficiency, which does not support a major contribution of FATP1 to FA uptake in vivo. However, when the null mice were fed a high-fat diet, there was protection from fat-induced insulin resistance of muscle glucose metabolism. This protective effect was associated with lower levels of intramuscular FA-acyl-CoA. Furthermore, intramuscular triglycerides, which were increased in wild-type mice fed the high-fat diet, remained unaltered in FATP1 null mice under the same conditions. These data were interpreted by the authors to document importance of FATP1 in metabolism of FA under conditions of high FA supply. They also linked FATP1 activity to accumulation of FA-acyl-CoA, which has been implicated in insulin resistance. FATP4 is the only FATP expressed in the small intestine and is localized to the apical surface of enterocytes (15).

Intestinal lumen

CD36 FA

FATP4

Enterocyte plasma membrane

FABP FA Intracellular compartment

FIG. 66-1. Diagram highlighting possible interactions between membrane and cytosolic proteins that cooperate to recruit and then target the fatty acid (FA) to metabolic sites. Diagram is speculative and based only on the concept that lipid uptake and processing in the enterocyte and other cell types likely involves a network of cooperating members. CD36, which binds FA with high affinity, is shown to function in recruiting the FAs to the membrane vicinity, where the FAs may be transferred to another membrane protein such as FATP4 (fatty acid transport protein 4), which possesses acylcoenzyme A synthase activity, and thus would help trap the FAs, preventing their efflux back into the lumen. Alternatively, FA transfer to FATP4 or other intracellular acylating enzymes may be mediated by cytosolic fatty acid–binding proteins (FABPs).

1698 / CHAPTER 66 In vitro studies with FATP4 overexpressing 293 cells documented increased FA uptake. In line with this, reduction of FATP4 expression in primary enterocytes by antisense oligonucleotides decreased FA uptake by 50%, supporting a role of FATP4 in FA uptake in the small intestine. FATP4 has been shown to have acyl-CoA synthase activity that is similar to that of FATP1 in having a broad specificity for FA of different chain length. The synthase activity may contribute or be essential to the function of FATP4 in uptake by trapping the FA intracellularly and preventing its efflux. In vivo, the role of FATP4 in intestinal FA uptake has been difficult to document because FATP4 deficiency was either embryonically or neonatally lethal. Gimeno and colleagues (62) showed that FATP4 deficiency caused embryonic lethality because mating of heterozygous FATP4 mice did not produce any homozygous offspring. These findings would support the concept that deletion of FATP4 is lethal by blocking fat absorption, but direct data to support this could not be obtained. Herrmann and colleagues (63) also generated FATP4 null mice. Although they did not observe embryonic lethality, the mice died soon after birth with features of restrictive dermopathy. Lipid analysis of intestine, liver, lung, brain, and dermis tissues showed no significant differences between null and wild-type mice. However, the FATP4-deficient epidermis had reduced content of phosphatidylcholine and altered FA composition of ceramides with decreases in the very long-chain FA C26:0. This implicated FATP4 in skin development. Similarly, a restrictive dermopathy-like phenotype also was described for a previously uncharacterized, spontaneous, autosomal recessive mouse mutation in FATP4 (64). Thus, in summary, more information is needed to understand what role FATP4 plays in FA uptake in enterocytes. It is possible that its function in transport involves, in addition to acyl-CoA synthase activity, interaction with other membrane proteins. For example, there may be association between FATP4 in enterocytes and other membrane proteins implicated in facilitating FA uptake (see Fig. 66-1). In cardiomyocytes, there is evidence for colocalization of FATP6 with CD36, possibly reflecting a functional interaction. CD36 is a scavenger receptor that has been shown to facilitate FA transport (see the next section) and might partner with a different FATP in different tissues. The possibility that CD36 partners with FATP4 in enterocytes is attractive. In addition to efficiently recruiting and trapping FAs, the association would help to salvage essential or very long-chain FAs that constitute a small fraction of dietary FAs. Because FATP4 is the only FATP present in the intestine, it would be predicted that its deficiency would result in defective uptake of very long-chain FAs.

CD36-Facilitated Fatty Acid Uptake: Role in Chylomicron Production and Secretion CD36 is an integral membrane glycoprotein, which has been identified on the surface of a variety of cells such as megakaryocytes, platelets, monocytes, dendritic cells,

adipocytes, myocytes, retinal and mammary epithelial cells, and endothelial cells of the microvasculature (65,66). CD36 is a member of a family of glycoproteins expressed both at the cell surface and within lysosomes. The CD36 family includes the high-density lipoprotein receptor SR-BI (scavenger receptor type B class I; also CLA-1), which is also highly expressed in the small intestine. CD36 and SR-BI share a hairpin membrane topology (see Fig. 66-1) with two transmembrane domains and with both termini in the cytoplasm (67). CD36 was identified as a facilitator of FA uptake by binding sulfosuccinimidyl oleate (SSO), a reactive oleic acid derivative, under conditions where SSO inhibited FA transport into rat adipocytes by 70% (68). The protein purified from adipose tissue and called FAT was later shown to bind native long-chain but not short-chain FAs in vitro (69). Distribution of CD36 is consistent with its role as a FA transporter, because it favors tissues with a high metabolic capacity for FAs (70). Expression is high in adipose tissue, where FA is stored as triglyceride, and in the heart, which relies on FA oxidation for energy. In skeletal muscle, CD36 expression favors muscles with a predominance of oxidative fibers. Deficiency of CD36 in mice results in impaired FA uptake by heart muscle, skeletal muscle, and adipose tissues (71,72). Fasting plasma TG and FA levels are increased. In contrast, muscle-targeted CD36 overexpression is associated with reduced levels of plasma FA and TG (73). In the spontaneously hypertensive rat, mutations in the CD36 gene were linked to the hypertriglyceridemia, high FA levels, and insulin resistance that are characteristic of this model (74), and these symptoms were improved by expression of the wild-type protein (75). In humans, CD36 deficiency is relatively common (2–7%) in persons of Asian and African descent (76,77), and it is associated with abnormalities of plasma lipids and insulin resistance (78–82). In the postprandial state, Kuwasako and colleagues (78) reported increased plasma levels of TG and apolipoprotein B-48. Plasma TG also was increased in fasting. Low-density lipoprotein–associated cholesterol was increased (81), whereas that associated with high-density lipoprotein was decreased (82). In white subjects, common polymorphisms (40–50% incidence) in the CD36 gene are associated with high blood FAs and an increased risk for diabetes-linked cardiovascular disease (83). The abnormal plasma lipids in humans with CD36 deficiency or with polymorphisms in the CD36 gene are likely to reflect abnormal peripheral clearance of plasma FA by muscle and adipose tissue. For example, defective FA uptake by the myocardium of CD36-deficient subjects has been well documented (84–86). Although the role of abnormal lipid processing by the small intestine has not been examined, it may be suggested by the postprandial lipemia and the high levels of apolipoprotein B-48 (78). The role of CD36 in intestinal lipid absorption has been assessed in rodents. CD36 is highly expressed in the small intestine and presents a proximal to distal gradient that would be consistent with its function in lipid transport. Poirier and

ROLE OF MEMBRANE AND CYTOSOLIC FATTY ACID BINDING PROTEINS IN LIPID PROCESSING / 1699 colleagues (87) have reported that FAT/CD36 messenger RNA (mRNA) is expressed predominantly in the proximal small intestine. Expression was localized to epithelial cells located in the upper two-thirds of villi, but was not detectable in crypt and submucosal cells. Protein expression was limited to the brush border of enterocytes and was not detectable in goblet cells. High-fat diets rich in long-chain FAs greatly increased FAT mRNA abundance, whereas diets rich in medium-chain FAs did not. Chen and colleagues (41) also showed that FAT/CD36 mRNA was differentially expressed along the gut axis, with the highest levels found in the jejunum and duodenum. CD36 mRNA also was detected in the gastric and colonic mucosa. In contrast with Poirier and colleagues (87), Chen and colleagues (41) found that intraduodenal administration of oleate down-regulated FAT/CD36 mRNA levels within 1 hour by about 80%, and this was associated with a decrease in uptake of FAs by enterocytes. These data documented opposite effects of acute versus chronic fat administration on CD36 expression levels in the intestine, and their impact on the processing of dietary lipid remains to be determined. To define the role of CD36 in the intestine, Goudriaan and colleagues (88) examined appearance of radioactivity in plasma after oral administration of 3H-labeled triolein and 14 C-labeled palmitic acid as an olive oil bolus to wild-type and CD36 null mice. No differences were observed. These observations were reproduced by Drover and colleagues (89), but a more comprehensive examination of the effects of acute and chronic fat feeding of wild-type and CD36 null mice on lipid uptake and secretion by the small intestine uncovered an important role of CD36 in intestinal lipid processing. CD36 null mice given a fat bolus by gavage or fed a high-fat diet accumulated neutral lipid in the proximal intestine indicating abnormal lipid processing. Lipoprotein secretion measured using the lymph fistula model was reduced by around 45%. The secretion defect appeared to reflect an impaired ability of enterocytes to efficiently synthesize TG from dietary FAs in the ER (89). These findings indicate that CD36 may play an important role in directing the FAs to the triacylglycerol pool destined for secretion, which is likely to involve interaction with various intracellular proteins such as the cytosolic FABPs (see the next section). An intriguing question that was suggested by these studies relates to why the role of membrane CD36 would differ between enterocyte and adipocyte or cardiomyocyte. CD36 is essential for net uptake of palmitate or oleate in heart or fat tissue, whereas it appears not to be essential for the process in the small intestine because CD36 null mice exhibit normal lipid absorption rates. However, it is possible that a defect in FA uptake exists in proximal segments of CD36 null mice and is compensated for by distal segments. Consistent with this interpretation is the observation that the small intestine is increased in length in CD36-deficient mice (Nada A. Abumrad, unpublished observations). Studies designed to examine different intestinal segments individually are needed and would be in line with the expression gradient of CD36 in this tissue. A role of CD36 in uptake of monoglycerides also should be

examined because they represent an important fraction of lipid uptake after intraluminal hydrolysis. Clearance of intestinally derived particles also was found to be defective in CD36 null mice (89). In plasma, the reduced intestinal lipid secretion was masked by slow clearance of chylomicrons. Whereas wild-type mice rapidly cleared plasma triglyceride after an oral olive oil load, CD36 null mice exhibited hypertriglyceridemia that persisted for several hours. This impaired clearance occurred despite normal lipoprotein lipase activity and likely reflected feedback inhibition of the lipase by FA as a result of their defective removal from the plasma. This indicated that cellular FA uptake could impact chylomicron clearance by modulating lipase activity. Thus, in summary, CD36 deficiency appears to result in hypertriglyceridemia both in the postprandial and fasting states, and in humans, it may constitute a risk factor for diet-induced type II diabetes and cardiovascular disease.

INTRACELLULAR FATTY ACID TRANSPORT After transfer across the enterocyte plasma membrane, the large load of diet-derived FAs must be transported to intracellular sites, particularly the ER, where FA activation by ACSs and re-formation of TG occur. The enterocyte contains two pathways for TG synthesis, the predominant monoacylglycerol acyltransferase (MGAT) pathway and the glycerol3-phosphate (G3P) pathway. Although both are localized in the ER, it has been reported that the MGAT pathway is associated largely with the smooth ER, whereas the G3P pathway is associated with the rough ER (90). FA taken up into the enterocyte may also be activated and incorporated into phospholipids, with the phospholipid biosynthetic pathways localized in the ER as well. Mitochondrial and peroxisomal oxidation of FAs in the enterocyte may also occur, although likely to a limited extent relative to esterification processes. Also notable is that FAs are taken up into the enterocyte across the basolateral plasma membrane as well, with subsequent metabolism occurring in various subcellular compartments. It is well appreciated that metabolic utilization of FAs almost always requires their activation to acyl-CoA esters; thus, the ACSs are presumably initial sites for FA trafficking. ACS5 is highly expressed in the small intestine (91), ACS3 is expressed at a low level (92), and ACS1, with widespread tissue distribution, is also found (93). Although surprisingly little is known about the enterocyte, distinct subcellular distributions and functional properties for different ACS forms in the liver have been reported (94). ACS5 is thought to be linked to phospholipid synthesis and FA β-oxidation, and ACS1 may be linked to TG synthesis (95). As noted earlier, plasma membrane–localized FATP4 also has ACS activity. It appears, therefore, that differential trafficking of FAs to several intracellular sites within the intestinal cell is likely to occur. Several mechanisms by which FA translocation could occur may be envisioned. Monomeric diffusion of unbound FA is one possibility. This would entail the spontaneous

1700 / CHAPTER 66 desorption of FA from the inner leaflet of the plasma membrane and subsequent redistribution to all subcellular membranes based on physical chemical properties of the FA and relative organelle membrane density. As discussed earlier, it is unlikely that the absorption and assimilation of this vital nutrient would occur in such an unregulated, untargeted manner. Another possible mechanism for movement of FA from the plasma membrane to the ER is via vesicular transport, because large amounts of FA can partition into membrane bilayers and at physiologic pH, in fact, it has been shown that FA themselves form bilayer structures (96). Although endocytosis occurs at both surfaces of the enterocyte, FA uptake via intercalation in the plasma membrane followed by bulk vesicular trafficking has not been described. It is of interest that, in other cell types, CD36/FAT has been found to be localized to caveolin-containing lipid rafts; however, studies indicate that FA uptake is not dependent on endocytosis of the caveolae (97). Another potential mechanism involves the binding of FA transferred across the plasma membrane by intracellular proteins, which would serve to increase the cytoplasmic FA concentration, thereby maintaining a transmembrane gradient, as well as to target the FAs to specific intracellular sites of utilization. It is thought that FABPs are involved in the intracellular transport of FAs and, in some cases, other hydrophobic ligands.

high I-FABP expression from the duodenum through the ileum, in contrast with a somewhat narrower distribution of L-FABP in duodenum and jejunum (105). FABPs generally are regulated at the transcriptional level; thus, it is likely that I-FABP and L-FABP protein concentrations in the intestinal tract closely reflect levels of the corresponding mRNA. In addition to the cephalocaudal gradient, FABP expression parallels dietary lipid assimilation along the crypt-to-villous axis as well. Expression of L-FABP begins at the crypt-villus junction and is highest in villous cells, with a small decline as cells reach the villous tip (106). At the subcellular level, it was reported that I-FABP and L-FABP have similar cytoplasmic distributions (107). Other immunohistochemical studies suggested that L-FABP levels are greater in basolateral relative to apical cytosol (108). Thus, the expression of L-FABP and I-FABP in the intestine follows the distribution of lipid uptake and intracellular lipoprotein formation, because both processes are maximal in the villous cells of the proximal jejunum (109–111). The somewhat shifted profiles of I-FABP and L-FABP expression, as well as other characteristics (see later), likely reflect differences in functional properties between the two proteins.

FATTY ACID–BINDING PROTEINS

Members of the FABP family are composed of 10 antiparallel β-strands, which form a barrel-like structure containing the ligand-binding cavity (see Fig. 66-1). The barrel is capped by two 8- to 9-residue α-helical segments (112). These helixes and the closely positioned β-turns are referred to as the portal region of the FABP, where it is thought that ligand entry into and exit from the binding cavity is likely to occur. All the FABPs with structures that have been currently solved exhibit a remarkably similar overall fold, despite low primary sequence similarities (may be as low as 25%). I-FABP and L-FABP thus have generally similar tertiary structures (113–116). Inspection of their holo-structures, however, demonstrates unique FA-binding characteristics. Biochemical and spectroscopic studies had suggested differential ligand-binding stoichiometries for I-FABP and L-FABP (117–119), as well as the potential for different FA orientations within the respective binding pockets (118,120). These studies were confirmed by X-ray crystallography, where cocrystallization of I-FABP with various FAs resulted in a 1:1 protein/ligand complex (121), whereas the L-FABP crystal structure showed a 2:1 ratio of oleic acid/L-FABP (113). For I-FABP, the carboxylate group of the FA ligand forms part of a five-member hydrogen-bonding network that includes the interior Arg106 residue. For LFABP, the carboxylate of one of its bound oleates is also part of an extensive hydrogenbonding network, including the interior Arg122, with the acyl chain largely shielded from the exterior of the protein. In contrast, the carboxylate of the other L-FABP–bound oleate has a more solvent-exposed position, interacting with

There are 12 members of the mammalian FABP family, each with specific tissue expression. FABPs typically are expressed at high levels of approximately 1% to 3% of total cytosolic protein (98). As their name suggests, the FABPs noncovalently bind long-chain FAs, with high affinity and a 1:1 molar stoichiometry in most cases. The intestinal absorptive cell exhibits robust expression of two types of FABPs. This contrasts with most other cell types, including those with a large flux of FAs such as the adipocyte or the skeletal muscle myocyte, which express a single predominant form. High levels of liver-type (L-FABP) and intestinal-type FABP (I-FABP) are expressed in rodent small intestinal cells, where the two FABPs together represent approximately 5% of mRNA (99,100). Estimations of protein concentrations reflect this high mRNA level and yield roughly equivalent concentrations for I-FABP and L-FABP of about 0.1 and 0.3 mM (99,101), respectively, in the rat. In the human intestine, however, a considerably greater level of L-FABP relative to I-FABP is found, although absolute I-FABP levels are still high (102). The pattern of I-FABP expression parallels that of dietary lipid absorption, indirectly indicating a role in FA assimilation. mRNA levels for L-FABP and I-FABP are greatest in the mouse jejunum, with L-FABP expression peaking in the proximal jejunum and that of I-FABP peaking in the distal jejunum (103,104). The range of expression along the duodenal to ileal axis is broader for I-FABP than L-FABP, with

Enterocyte Fatty Acid–Binding Protein Structure and Equilibrium Binding of Ligand

ROLE OF MEMBRANE AND CYTOSOLIC FATTY ACID BINDING PROTEINS IN LIPID PROCESSING / 1701 several residues near the portal region of the protein (113). Multidimensional nuclear magnetic resonance studies indicate that the two FA-binding sites of L-FABP become occupied in a sequential fashion (122). L-FABP and I-FABP differ in their ligand specificities, as well as their FA-binding capacities. Although both proteins bind long-chain length FAs and interact minimally or not at all with medium- and short-chain length FAs (
Regulation of Fatty Acid–Binding Protein Expression in the Intestine: Functional Implications The expression of I-FABP and L-FABP mRNA in rat small intestine is first detected during late gestation. A dramatic increase in expression occurs in the first postnatal day and is maintained during the suckling period. A decline in expression

of both FABPs at the suckling-weaning transition is presumed to occur secondary to the decrease in dietary fat content, although only L-FABP expression has been found to be clearly regulated by dietary fat in the adult proximal intestine (146). After the suckling-weaning transition, further increases in expression of both enterocyte FABPs are observed during adult growth (147). The developmental regulation of FABP expression, together with their aforementioned expression pattern along the proximal-to-distal axis, provides additional correlative evidence for a role in dietary lipid transport and metabolism. Nutritional regulation of FABP expression may nevertheless be limited to modification of genetically programmed patterns of expression, as proposed for intestinal lipid metabolic enzymes (148,149). Intestinal isograft implantation studies showed that the developmental and proximal-distal gradients of intestinal FABP expression are not dependent on the presence of luminal lipid or luminal contents per se (150). In contrast, the villous-to-crypt gradient of L-FABP mRNA expression was modified in neonatal intestinal isografts, with segments not exposed to luminal contents exhibiting a more homogeneous distribution throughout the villus relative to that found for normal intestine (151). Because this is similar to what has been found after fasting (152), it is possible that signals from the intestinal lumen, such as lipids or other nutrients, or alternatively, hormonal or nervous stimuli, are involved in regulating the levels of enterocyte FABP expression. It appears likely that nutrient-dependent regulation is involved, because the effects of lipids on FABP expression also were found in Caco-2 cells (153). The regulation of L-FABP and I-FABP levels by dietary lipid shows similarities, as well as differences. Long-term feeding of a diet containing 20% to 40% fat resulted in increased L-FABP expression in the proximal small intestine (123,154), but increased I-FABP levels only in the distal small intestine (155). Low-fat/high-carbohydrate diets led to decreased levels of both FABPs (123,155). When the lipid content of a mouse diet was changed from 3% to 6% by weight, 2- to 3-fold increases in proximal intestine L-FABP mRNA and protein levels were observed (153). In this study, no alterations were found in IFABP levels, perhaps because lower levels of lipid were used. Also notable is that the various studies have used different dietary fat sources, and in some cases, control groups have been fed chow diets that contain a different lipid composition than the experimental diets, rendering it uncertain whether lipid type or amount, or both, is responsible for the observed changes in gene expression. L-FABP is normally expressed at low levels in mouse ileum; however, direct ileal infusion of various saturated and unsaturated long-chain FAs led to the appearance of increased L-FABP message in that segment. Because a nonmetabolizable analogue of palmitic acid had the greatest modulatory effect on LFABP expression in this system, this suggested that unesterified FAs, rather than their metabolites, may be responsible for the effects of dietary lipid on expression (153).

1702 / CHAPTER 66 Various aspects of the regulation of enterocyte FABP expression further suggest that within the general context of lipid transport and metabolism, the specific roles of I-FABP and L-FABP are likely to be distinct. In the adult rat, L-FABP expression was induced by peroxisome proliferators such as clofibrate, which caused more than twofold increases in intestinal L-FABP mRNA and protein levels (156). The L-FABP promoter was later shown to contain a peroxisome proliferatorresponse element (PPRE) located between nucleotides −66 and −75 (157) similar to those found in a number of genes involved in peroxisomal FA oxidation, suggesting a role for LFABP in this process, perhaps related to substrate delivery. No PPRE has been described in the IFABP gene, and clofibrate treatment caused no change in IFABP mRNA levels. A small increase in IFABP protein levels was observed and could be a secondary effect because of alterations in other peroxisome proliferator-responsive genes (156). It is likely that the regulation of intestinal L-FABP expression in response to lipid may be mediated by nuclear PPAR transcription factors. The PPAR-α, PPAR-γ, and PPAR-δ subtypes have been identified in the intestine (153,158). In contrast, the negligible induction of I-FABP by clofibrate and dietary FAs may reflect the absence of a PPRE. Several copies of a 14-base pair (bp) sequence that binds the steroid hormone receptors hepatic nuclear factor-4 and apolipoprotein regulatory protein-1 are present in the I-FABP promoter region (159), but not that of L-FABP. The 5′ flanking regions of cellular retinol-binding protein II and apolipoprotein A-I, which are also highly expressed in the intestinal absorptive cell, also contain this 14-bp sequence, indicating the potential for coordinate regulation of several genes thought to be involved in the cellular processing of dietary lipid. It is possible that each of the enterocyte FABPs could be involved in modulating the others’ expression, although somewhat conflicting results have been obtained. In Caco-2 cells transfected with the human I-FABP gene, expression of L-FABP was found to decline (138). In contrast, mice null for the I-FABP gene showed normal levels of L-FABP expression (160), and mice null for L-FABP displayed normal I-FABP levels in the intestine (161). The functional implications of the differential regulation of L-FABP and I-FABP expression are unclear, because the unique roles of these proteins are still under active examination. It may be speculated that L-FABP, despite its relatively high level of constitutive expression, can be induced to accommodate increased lipid load caused by either dietary fat or lipid metabolic flux, which is consistent with a possible role as a cellular buffer for FAs. Perhaps I-FABP has a more specialized role in FA disposition in the enterocyte; its constitutive expression may already be maximal, and increased FA levels can be accommodated by its homologue, L-FABP. Taken together, the robust expression, ligand-binding properties, tissue localization, and regulation of expression of I-FABP and L-FABP point to a central role for these proteins in intestinal lipid absorption and processing. Their precise roles at the molecular and cellular levels are being investigated using in vitro, model cell line, and whole-animal approaches.

Functions of the Fatty Acid–Binding Proteins in the Proximal Intestine Since their discovery in the 1970s, the FABPs have been hypothesized to be intracellular FA transporters (155). Yet this family of proteins is referred to as binding proteins, because this equilibrium property is clear, whereas a true transport function is less definitive. If transport is defined simply to mean the property of ligand binding, with consequent effects on ligand partitioning to cell membranes or enzymes, which may, in turn, influence net uptake and cellular lipid metabolism, then the FABPs may be so considered. Typically, however, a transporter is deemed to be a protein that is involved in the movement of ligand from one site to another, as in the case of the transmembrane transporters discussed earlier. For an intracellular protein to function as a transporter, it must play a role in ligand delivery, participating in the movement of ligand from one site to another. Evidence has accumulated indicating that L-FABP and I-FABP are involved in the overall process of cellular FA transport; however, it is not yet definitively known whether they function as direct transporters or whether they act indirectly as cytoplasmic sites of FA partitioning. A series of studies were undertaken that used transfection of the L-FABP or I-FABP gene into L-cell fibroblasts or embryonic stem cells, followed by evaluation of different aspects of lipid transport and metabolism (162–168). Results have not provided a consistent picture of FABP effects. It was shown that the increase in fluorescence intensity was twofold greater when the fluorescent cis-parinaric FA analogue was added to cells expressing L-FABP compared with control cells or those expressing I-FABP. This was interpreted as evidence for an L-FABP–mediated transport function (165); however, the fluorescence quantum yield for cis-parinaric acid bound to L-FABP compared with I-FABP differs by approximately twofold as well, making this interpretation uncertain. When I-FABP was expressed at a twofold greater concentration in L-cells, it was found that cis-parinaric acid uptake decreased relative to the cells with lower I-FABP levels (167). Furthermore, transfection studies in embryonic stem cells did not demonstrate dose-dependent effects of I-FABP expression on FA uptake or metabolic incorporation, and effects observed were modest (168). Similarly, although some evidence for differential metabolic targeting of oleate by L-FABP relative to I-FABP was found, the changes were small and, in some cases, inconsistent (165,166). Caco-2 intestinal cells express low levels of I-FABP, but physiologic levels of L-FABP (169,170). Stable transfection of Caco-2 with I-FABP was found to increase FA incorporation into cellular TG and to decrease basolateral TG secretion (170). In seeming contrast, net FA incorporation was found to be decreased in Caco-2 cells transfected with approximately physiologic levels of I-FABP (171). A comparison of the two human I-FABP forms showed that when transfected with the Thr54 form of human I-FABP, Caco-2 cells exhibited a twofold increase in net FA uptake and secreted fivefold more TG into the basolateral medium relative to

ROLE OF MEMBRANE AND CYTOSOLIC FATTY ACID BINDING PROTEINS IN LIPID PROCESSING / 1703 Ala54-IFABP–transfected cells (138). Endogenous L-FABP levels remained several-fold greater than I-FABP levels; however, the absolute levels of L-FABP were decreased relative to untransfected and control cells (138). Overall, interpretation of the Caco-2 cell transfection data for I-FABP function is not straightforward. A comparative study of human fetal intestines from wild-type (Ala54/Ala54) and Thr54/Ala54 heterozygotes showed that the Thr-containing allele was associated with increased TG secretion and chylomicron output, clearly demonstrating the potential for clinically significant effects on cellular and perhaps whole-body lipid metabolism (172). An interesting series of experiments developed by Luxon and Weisiger (173–175) used fluorescence recovery after photobleaching (FRAP) to monitor the effective diffusion (Deff ) of the fluorescent FA NBD-stearate (NBDS) in LFABP–expressing hepatocytes or Hep G-2 cells. The results showed that the Deff for NBDS correlated closely with the cytosolic L-FABP levels and with the relative binding of the fluorescent FA by the L-FABP (173–175). Interpretation of these results was that by binding the FA, the cytoplasmic L-FABP decreased their partitioning to “immobile” membranes, hence increasing their rates of lateral diffusion in the cell. This interpretation was supported by so-called model cytoplasm FRAP experiments, where L-FABP caused a concentration-dependent increase in the rate of NBDS diffusion and decrease in membrane-bound NBDS. A similar experiment performed with embryonic stem cells transfected with I-FABP also showed a dose-dependent increase in NBDS Deff (168). These solution FRAP experiments provide an important demonstration that the enterocyte FABPs can perform a direct function in intracellular FA transport. Studies in model systems have added support for a role for I-FABP and L-FABP in enterocyte FA transport and utilization. For example, LFABP was found to increase the flux of oleic acid through a lipid–water interface by threefold (176) and to increase the extent and rate of movement of FAs between two compartments (177). In vitro experiments using a fluorescence resonance energy transfer assay provided a quantitative, mechanistic comparison of the FA transfer properties of L-FABP and I-FABP (178). The results showed that long-chain fluorescent FA transfer from I-FABP to membranes occurred approximately 10-fold faster than from L-FABP (178, 179). These studies also showed that L-FABP and I-FABP used different mechanisms to transfer FA to membranes. For L-FABP, the data indicated that the FA transfer rate was independent of the membrane acceptor, and transfer occurred by dissociation of the FA from L-FABP followed by aqueous diffusion (178,180). In marked contrast, the FA transfer rates from I-FABP were directly proportional to the concentration of acceptor vesicle phospholipid and showed a large degree of regulation by the phospholipid composition of the membranes (178). These results were interpreted as suggesting an interaction between I-FABP donor and membrane acceptor, implying the existence of an intermediate in which the protein and the membrane are in physical contact. Kinetic modeling and experimental analysis of protein-mediated FA transport to

model membranes supported these divergent transfer mechanisms for L-FABP and I-FABP (181). Both of the enterocyte FABPs are therefore hypothesized to function in the transport of FA, but appear to use distinct mechanisms and most likely have distinct cellular effects. To simulate the role of FABPs in FA transfer within a cell, Vork and colleagues (182) modeled the transfer of FA between two membranes in the absence and presence of a lipid-binding protein. They showed that only when collisional interaction of the FABP with the membranes was included in the model was a substantial impact on simulated net transfer between the two membrane surfaces observed. A direct interaction of I-FABP with membranes has been experimentally demonstrated. Preincubation of negatively charged phospholipids vesicles with I-FABP was shown to prevent the subsequent binding of the peripheral membrane protein, cytochrome c, indicating that I-FABP was bound to the vesicle surface (183). Furthermore, infrared spectroscopic studies indicated that monolayers of dimyristoylphosphatidic acid develop lipid domains on addition of I-FABP to the aqueous subphase (184). The fluorescent FA transfer rate was catalyzed to the greatest extent by anionic membrane phospholipids, suggesting that positively charged amino acid residues on the surface of I-FABPs were involved in the formation of a collisional complex with the membrane (178). It has been shown that neutralization of I-FABP surface lysine residues by chemical modification eliminated the effects of membrane surface charge on FA transfer rate, supporting this suggestion (185). Several lines of evidence, including studies with other members of the FABP family, indicate that the helix/portal domain could be the site of I-FABP–membrane interactions. A “helixless” construct of I-FABP was used to test this hypothesis (186). The results of fluorescent FA transfer experiments and cytochrome c competition studies clearly showed that the mechanism of FA transfer from the helix-less I-FABP did not involve a collisional interaction with the acceptor membrane, which was in marked contrast with results for the wild-type protein (183). In further studies, a pair of chimeric proteins with the “body” (ligand-binding domain) of I-FABP and the α-helical region of L-FABP (αLβIFABP), and the ligandbinding pocket of L-FABP and the helical domain of I-FABP (αIβLFABP), respectively, were constructed. The results showed that the mechanism of FA transfer was changed when the α-helical domains were exchanged, with transfer from aLbIFABP occurring by aqueous diffusion and transfer from aIbLFABP occurring via protein-membrane collisional interactions (187). Overall, these studies suggest strongly that each of the resident intestinal FABPs functions uniquely in the enterocyte and provide a structural basis underlying potential functional differences. Insight into the functions of the enterocyte FABPs also are beginning to emerge from studies of null mice. Mice lacking expression of I-FABPs showed no apparent compensation with either liver FABPs or other FABPs, further implying distinct functions for the individual enterocyte FABPs. The I-FABP knockout mice gained more weight and had greater

1704 / CHAPTER 66 levels of serum triglycerides (160), which could indicate an involvement of the protein in lipid absorption, metabolism, or secretion. The incorporation of radiolabeled oleic acid into TGs relative to phospholipids in jejunal mucosa was markedly increased in the I-FABP−/− mice, suggesting that I-FABP may normally be involved in the partitioning of FAs toward phospholipid synthesis, or alternatively, that the intracellular milieu was altered to promote TG synthesis (Storch, Zhou, and Wu, unpublished observations). The increased relative synthesis of TGs in the jejunal mucosa may be responsible for the observed hypertriglyceridemia. The I-FABP−/− mice also experienced development of hyperinsulinemia that appeared to be independent of body weight gain, which is an atypical dissociation (160). Interestingly, the effects on body weight and serum triglycerides were observed only in male mice (160), suggesting a previously unappreciated interaction between I-FABPs and sex hormones. The hyperinsulinemia was not sex specific, however, and the effects on metabolic partitioning of FAs also were found in both female and male knockout mice (Storch, Zhou, and Wu, unpublished observations). Several studies of mice null for the L-FABP gene have appeared (161,188–191). Although little work currently has focused on its enterocyte functions, it is notable that smallintestinal I-FABP expression was not altered in the L-FABP−/− mice (161). In the liver, where L-FABP also is highly expressed, several effects of the L-FABP knockout on FA disposition are consistently observed. The net uptake of FA by the liver is decreased, and liver TG content and incorporation of labeled FAs into TGs are decreased, particularly after a fast (161, 188,191). β-Oxidation of long-chain FA was little changed in the fed state, but was significantly decreased under ketogenic conditions such as starvation (161,190). In addition, despite indications that L-FABP may be mediating ligand delivery to the nuclear transcription factor PPAR-α (192), the L-FABP−/− mouse exhibited no alterations in PPAR-α target gene expression, or in PPAR-α expression itself (161, 190). Although additional work needs to be done, current studies point to defects within the liver (as opposed to changes in serum FA levels or initial rates of uptake) likely having to do with intrahepatic substrate delivery/availability. A decrease in the rate of fluorescent FA lateral diffusion (191) in hepatocytes isolated from L-FABP−/− provides a potential explanation for reduced substrate availability. Collectively, studies of proximal intestinal FABPs suggest the potential for one or both to act as targeting proteins, conveying their ligands to particular domains on organellar membranes, or perhaps to specific protein partners. Although a number of protein–protein interactions involving various members of the FABP family have been described, several may be particularly relevant to the intestine and suggest a regulated, vectorial transport of FAs within the cell, as well as the potential for functional actions of these proteins. In milk-fat globule membranes, direct interaction between hearttype FABPs and the cytoplasmic domain of CD36/FAT was demonstrated using gel filtration and coimmunoprecipitation (193), suggesting the possibility that this transmembrane FA

transporter is acting to direct incoming FAs to intracellular binding proteins (see Fig. 66-1). As coordinate regulation of the gene expression (194,195) of cytoplasmic FABPs and membrane FA transporters has been demonstrated, it is certainly possible that these proteins act in a concerted fashion to effectively carry out FA transport. An association between L-FABP and the lipid-activated transcription factor, PPAR-α, was shown using pull-down assays and coimmunoprecipitation (192), suggesting that L-FABP could directly traffic its FA ligand, perhaps obtained from an interaction with CD36/FAT, to the nucleus, and thus function in the regulation of gene expression. The physiologic significance of this interaction requires clarification, however, given the apparent absence of effect of L-FABP knockout on PPAR-α function (190).

FUTURE DIRECTIONS As discussed in the previous sections, cellular uptake of FAs is likely to play an important role in intestinal lipid metabolism and in the adaptability of the intestine to various challenges. Membrane transporters are the first step in FA processing by enterocytes and a probable site of regulation. What we know about these transporters in the intestine remains limited partly because of our insufficient knowledge related to how these proteins function in other cells. We have little information related to what determines the specificity of transport and to how transporters interact with intracellular proteins, either FABPs or others, to influence the metabolic fate of the FAs. A better understanding of how FA transport and binding proteins channel FA to intracellular targets appears to be crucial. It is likely that membrane or intracellular protein partners, or both, play crucial roles in determining transport specificity, direction, and regulation.

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165. Prows DR, Murphy EJ, Schroeder F. Intestinal and liver fatty acid binding proteins differentially affect fatty acid uptake and esterification in L-cells. Lipids 1995;30:907–910. 166. Murphy EJ, Prows DR, Jefferson JR, Schroeder F. Liver fatty acidbinding protein expression in transfected fibroblasts stimulates fatty acid uptake and metabolism. Biochim Biophys Acta 1996;1301:191–198. 167. Prows DR, Schroeder F. Metallothionein-IIA promoter induction alters rat intestinal fatty acid binding protein expression, fatty acid uptake, and lipid metabolism in transfected L-cells. Arch Biochem Biophys 1997;340:135–143. 168. Atshaves BP, Foxworth WB, Frolov A, Roths J, Kier A, Oetama B, Piedrahita J, Schroeder F. Cellular differentiation and I-FABP protein expression modulate fatty acid uptake and diffusion. Am J Physiol 1998; 274:C633–C644. 169. Le Beyec J, Delers F, Jourdant F, Schreider C, Chambaz J, Cardot P, Pincon-Raymond M. A complete epithelial organization of Caco-2 cells induces I-FABP and potentializes apolipoprotein gene expression. Exp Cell Res 1997;236:311–320. 170. Gedde-Dahl A, Kulseth MA, Ranheim T, Drevon CA, Rustan AC. Reduced secretion of triacylglycerol in CaCo-2 cells transfected with intestinal fatty acid-binding protein. Lipids 2002;37:61–68. 171. Darimont C, Gradoux N, Persohn E, Cumin F, De Pover A. Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells. J Lipid Res 2000;41:84–92. 172. Levy E, Menard D, Delvin E, Stan S, Mitchell G, Lambert M, Ziv E, Feoli-Fonseca JC, Seidman E. The polymorphism at codon 54 of the FABP2 gene increases fat absorption in human intestinal explants. J Biol Chem 2001;276:39679–39684. 173. Luxon BA, Weisiger RA. Sex differences in intracellular fatty acid transport: role of cytoplasmic binding proteins. Am J Physiol 1993; 265:G831–G841. 174. Luxon BA. Inhibition of binding to fatty acid binding protein reduces the intracellular transport of fatty acids. Am J Physiol 1996;271: G113–G1120. 175. Luxon BA, Milliano MT. Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein. Am J Physiol 1997;273: C859–C867. 176. Weisiger RA, Pond SM, Bass L. Albumin enhances unidirectional fluxes of fatty acid across a lipid-water interface: theory and experiments. Am J Physiol 1989;257:G904–G916. 177. Peeters RA, Veerkamp JH, Demel RA. Are fatty acid-binding proteins involved in fatty acid transfer? Biochim Biophys Acta 1989;1002:8–13. 178. Hsu KT, Storch J. Fatty acid transfer from liver and intestinal fatty acid binding-proteins to membranes occurs by different mechanisms. J Biol Chem 1996;271:13317–13323. 179. Storch J, Herr FM, Hsu KT, Kim HK, Liou HL, Smith ER. The role of membranes and intracellular binding proteins in cytoplasmic transport of hydrophobic molecules: fatty acid-binding proteins. Comp Biochem Physiol 1996;115B:333–339. 180. Kim HK, Storch J. Free fatty acid transfer from rat liver fatty acidbinding protein to phospholipid vesicles: effect of ligand and solution properties. J Biol Chem 1992;267:77–82. 181. Zucker SD. Kinetic model of protein-mediated ligand transport: influence of soluble binding proteins on the intermembrane diffusion of a fluorescent fatty acid. Biochemistry 2001;40:977–986. 182. Vork MM, Glatz JFC, Van Der Vusse GJ. Modeling intracellular fatty acid transport: possible mechanistic role of cytoplasmic fatty acidbinding protein. Prostaglandins Leukot Essent Fatty Acids 1997;57: 11–16. 183. Corsico B, Cistola DP, Frieden C, Storch J. The helical domain of intestinal fatty acid binding protein is critical for collisional transfer of fatty acids to phospholipid membranes. Proc Natl Acad Sci U S A 1998;95:12174–12178. 184. Wu F, Corsico B, Flach CR, Cistola DP, Storch J, Mendelsohn R. Deletion of the helical motif in the intestinal fatty acid-binding protein reduces its interactions with membrane monolayers: Brewster angle microscopy, IR reflection-absorption spectroscopy, and surface pressure studies. Biochemistry 2001;40:1976–1983. 185. Corsico B, Franchini GR, Hsu KT, Storch J. Fatty acid transfer from intestinal fatty acid binding protein to membranes: electrostatic and hydrophobic interactions. J Lipid Res 2005;46:1765–1772. 186. Kim K, Cistola DP, Frieden C. Intestinal fatty acid-binding protein: the structure and stability of a helix-less variant. Biochemistry 1996;35: 7553–7558. 187. Corsico B, Liou HL, Storch J. The alpha-helical domain of liver fatty acid binding protein is responsible for the diffusion-mediated transfer

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CHAPTER

67

Genetic Regulation of Intestinal Lipid Transport and Metabolism Zhouji Chen and Nicholas O. Davidson Overview, 1711 Intestinal Lipid Transport, 1711 Intestinal Lipoprotein Assembly, 1712 Major Pathways and Genes Involved in Intestinal Triglyceride-Rich Lipoprotein Assembly, 1712 Apolipoprotein B: Protein Structure and Functional Domains, 1712 MTTP: Structure and Functional Domains, 1714 General Model of Apolipoprotein B–Containing Lipoprotein Assembly, 1714 Genetic Defects in APOB and MTTP, 1717 Familial Hypobetalipoproteinemia, 1717 Abetalipoproteinemia, 1720 Apolipoprotein B Messenger RNA Editing: Overview, Molecular Mechanisms, and Functional Relevance, 1721 Apolipoprotein B Messenger RNA Editing: Molecular Machinery and Tissue-Specific Regulation, 1722

Functional Importance of Intestinal Apolipoprotein B Messenger RNA Editing, 1723 Other Genes Involved in Intestinal Lipoprotein Biogenesis: Apolipoproteins A-I and A-IV, 1723 Major Pathways and Genes Involved in Intestinal Sterol Transport, 1724 Sitosterolemia and ABCG5/G8, 1726 Identification of NPC1L1, the Putative Intestinal Cholesterol Transporter, 1727 Acyl-Coenzyme A Cholesterol Acyltransferase 2 and the Regulation of Intestinal Cholesterol Absorption, 1728 Other Genetic Defects of Intestinal Lipoprotein Assembly and Secretion and Potential New Pathways, 1728 Acknowledgments, 1729 References, 1729

OVERVIEW

biliary lipid secretion contributes ~50 g lipid per day in the form of phospholipid and cholesterol, which, together with small amounts of membrane-derived lipids contributed by sloughed villous epithelial cells, represents a further source of intestinal lipid. Accomplishing the task of absorbing and processing greater than 95% of the neutral lipid presented to the small intestinal enterocyte has led to the emergence of complex genetic adaptations, many of which are detailed in this review. Beyond the task of absorbing and processing large quantities of bulk lipid as an efficient energy source, the mammalian intestinal enterocyte has evolved important mechanisms to discriminate and adapt to the need for subtle distinctions in the types of lipid absorbed, including lipovitamins, xenobiotics, and lipid-soluble by-products of commercial food processing. A key example of this phenomenon is in the regulation of cholesterol absorption. The average U.S. diet contains approximately 300 to 500 mg cholesterol, which is presented to the intestinal enterocyte, together with approximately 2 g biliary cholesterol. As detailed in

Intestinal Lipid Transport Dietary lipid represents a major component of the daily caloric intake in most Western cultures. The adult U.S. diet contains upward of 120 g fat per day, derived largely in the form of long-chain (>14 carbon atoms) triglyceride, combined with smaller amounts (~10 g/day) of the phospholipid lecithin. In addition to this daily dietary lipid input,

Z. Chen: Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, St. Louis, Missouri 63110. N. O. Davidson: Division of Gastroenterology, Washington University School of Medicine, St. Louis, Missouri 63110. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1711

1712 / CHAPTER 67 this chapter, absorption of cholesterol is genetically and metabolically regulated, and important adaptations have emerged to discriminate and partition cholesterol absorption in relation to other sterols, in particular, the varying amounts of other plant sterols (β-sitosterol, campesterol, stigmasterol), as well as sterols derived from shellfish (brassicasterol) consumed in our diet. The late 1990s/early 2000s has witnessed great advances in understanding how the process of intestinal lipid transport is so efficiently coordinated. In particular, we now understand more completely the range of genes involved and the accompanying defects that underlie several syndromes of defective intestinal lipid transport and metabolism. These pathways and their regulation have focused research interest on the role of the small intestine as an active participant in systemic lipoprotein metabolism. In addition, the interaction of genetic susceptibility traits and dietary or other environmental factors has led to renewed interest in the role of dietary modulation of intestinal lipid metabolism.

Intestinal Lipoprotein Assembly Intestinal lipid transport is a multistep process that requires the coordinated regulation of a series of pathways that lead to the transport of lipolytic products and micellarized lipid across the brush-border membrane, through vectorial delivery through the apical cytoplasmic compartment to the endoplasmic reticulum (ER). From the ER, the site at which initiation of lipoprotein biogenesis is coordinated, the primordial particles are remodeled and undergo maturation through the Golgi profiles, which ultimately results in disgorgement of nascent intracellular chylomicrons into the pericellular spaces surrounding the basolateral membrane and lymphatic fenestrae. This chapter addresses developments in the carrier-mediated and bidirectional flux of sterols at the brush-border membrane and the emergence of new candidate cholesterol transporter proteins. We discuss the current understanding of intestinal lipoprotein biogenesis and the role of dominant genes in orchestrating triglyceride mobilization. Finally, we discuss developments in the molecular basis for several genetic diseases in which intestinal lipid metabolism is disrupted, because these provide important insight into the function of these pathways in health and under conditions of altered lipid intake.

MAJOR PATHWAYS AND GENES INVOLVED IN INTESTINAL TRIGLYCERIDE-RICH LIPOPROTEIN ASSEMBLY Triglyceride-rich lipoproteins, specifically chylomicrons and very low-density lipoproteins (VLDLs), play an indispensable role in the absorption of triglycerides and fatsoluble vitamins from enterocytes lining the small intestine. Triglyceride-rich lipoproteins are macromolecules composed of a large neutral lipid (triglyceride) core surrounded by

more polar components, including phospholipids, free cholesterol, and apolipoproteins (1). There are two dominant genetic requirements for intestinal and hepatic triglyceriderich lipoprotein assembly that must be met simultaneously for this process to proceed to completion. These include expression of a key structural protein, apolipoprotein B (ApoB) (2,3), which is the acceptor for the neutral lipid donor, microsomal triglyceride transfer protein (MTTP) (4). Details of their physical interaction and mechanisms for their obligate requirement in lipoprotein assembly are discussed in detail in this chapter (Fig. 67-1). Among the apolipoproteins, ApoB is a structurally unique, obligate integral component of the surface of lipoprotein particles. No triglyceride-rich lipoprotein can be assembled without the participation of ApoB. This is most clearly evidenced in the phenotype associated with familial hypobetalipoproteinemia (FHBL), in which structural defects in the ApoB protein preclude normal lipoprotein assembly. Triglyceride-rich lipoprotein assembly is regulated in a tissuespecific manner, reflecting, in part, the tissue-specific production of different forms of ApoB. Human liver produces VLDLs, each particle containing a molecule of the full-length form of ApoB (ApoB100) surrounding a particle of 30 to 80 nm in diameter. The intestine produces even larger particles (chylomicrons) with diameters as large as 75 to 1200 nm, as would be predicted from the need to accommodate the absorption of bulk dietary lipid. Chylomicrons contain ApoB48 (the N-terminal 48%), instead of the full-length ApoB100 molecule. ApoB48, a physiologically truncated form of ApoB, is formed as a result of posttranscriptional editing of the ApoB mRNA transcript in the intestine (5) that converts codon 2153 from a CAA (glutamine) to a UAA (in-frame stop codon; see later for a detailed discussion). As noted earlier for hepatic VLDL assembly, intestinal chylomicrons express a single copy of the ApoB molecule (6), but also contain multiple copies of the smaller, soluble apolipoproteins including apoA-I, apoA-IV, and apoC-III (1,2). However, unlike ApoB, none of the soluble apolipoproteins is absolutely required for the formation of VLDL or chylomicron particles, in either the liver or small intestine, respectively.

Apolipoprotein B: Protein Structure and Functional Domains The APOB gene is located on the short arm of human chromosome 2 (7,8) and in the syntenic locus of mouse chromosome 12 (9). The single-copy APOB gene spans 43 kb and contains 29 exons, of which exon 26, at 7572 base pair (bp), is the largest and encodes most of the full-length ApoB protein (7,8). Indeed, exon 26 of the APOB gene is one of the largest known mammalian exons. The liver and small intestine clearly represent the major sites for ApoB gene expression, although ApoB also is expressed at low levels by cardiomyocytes (10) and human placenta (11). ApoB100 is a large (Mr 550 kDa) and extremely hydrophobic polypeptide composed of 4536 amino acid residues (12). The predicted

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1713 Proteasomal degradation

ApoB synthesis ApoB mRNA

Rough ER

Smooth ER

Nascent apoB polypeptide chain

1

2

MTP

TG

Degradation Degradation by proteasomes or other mechanism

Primordial lipoprotein

MTP

1b 1a

TG

Secretion

ER Lumen

3 Fusion MTP or other cofactors?

TG

Secretory compartments

4

TG

MTP

TG

Lumenal lipid droplet

Mature lipoprotein

Budding from ER and fusion with Golgi

COPII proteins

FIG. 67-1. A two-step model of assembly of intestinal triglyceride-rich lipoproteins. The nascent apolipoprotein B (ApoB) polypeptide is translocated across the endoplasmic reticulum (ER) membrane cotranslationally. When lipid is available, the N-terminal domain of ApoB interacts with microsomal triglyceride transfer protein (MTP). This interaction facilitates neutral lipid transfer from the ER membrane to the nascent ApoB peptide, leading to the formation of a primordial lipoprotein particle (1a). This primordial particle is modified through the second step of lipoprotein assembly with bulk triglyceride (TG) addition and eventually is secreted. When lipid availability is limited or MTP function impaired, the nascent ApoB polypeptide becomes misfolded (1b), undergoes ubiquitination, and is degraded by the proteasomal pathway. This degradation step occurs either outside the ER or within the ER lumen. MTP also directs the assembly of ApoB-free, triglyceride-rich particles within the adjacent smooth ER (2). During the second step of lipoprotein assembly, the primordial lipoprotein particle fuses with these smooth ER TG-rich lipid droplets, resulting in prechylomicron formation (3). The role of MTP in mediating this fusion step is unknown. After acquiring vesicular transport proteins, including coat protein II complex (COPII) proteins, the prechylomicron particles are incorporated into a vesicular complex through ER membrane budding to fuse with membranes of the Golgi apparatus (4). Vectorial transport of these vesicular structures through the Golgi apparatus results in secretion of the nascent chylomicron particles into the pericellular spaces adjacent to lymphatic fenestrae.

structure indicates both hydrophobic lipid binding regions and hydrophilic regions that interact with the aqueous plasma environment. Segrest and colleagues (13,14) have postulated a pentapartite model for human ApoB100 on plasma low-density lipoprotein (LDL). According to this model, human ApoB100 is composed of three amphipathic α-helical domains alternating with two β-strand domains in an NH2-α1-β1-α2-β2-α3-COOH configuration (13,14). ApoB48 comprises the first two domains (α1-β1-) and a portion of the third domain (α2). The β1 and β2 domains may exist in the form of beltlike structures wrapped around the particle and may be irreversibly associated with the lipid core of the lipoprotein, whereas the α helices of the α domains, which are similar to those found in the “exchangeable” apolipoproteins such as ApoA-I and ApoE, are thought to confer reversible lipid-binding properties. The α1 domain is a highly

disulfide-bonded globular domain, containing seven of the eight paired disulfide bonds found in ApoB100 (13,14). ApoB100 also shares significant primary sequence homology with vitellogenin (15,16), an ancient lipid transport and storage protein that plays a critical role in delivery of nutrients to the egg yolk (16,17). The detailed structure of vitellogenin has been worked out, and it is predicted to contain a globular amino-terminal β-barrel, an extended α-helical structure, and a substantial C-terminal lipid-binding cavity (18). Based on its sequence homology to vitellogenins, it has been proposed that the α1 domain of ApoB can be further divided into two subdomains, an N-terminal β-barrel domain (amino acids 1–263) and a C-terminal α-helical domain (amino acids 294–592). Thus, the N-terminus of ApoB also is referred to as the βα1 domain (14). The βα1 domain is incapable of forming lipoprotein particles, but is

1714 / CHAPTER 67 required for lipoprotein synthesis, and its absence ablates the capability of ApoB to assemble lipoprotein particles (19). It is believed that this α1 domain of ApoB plays a critical role in the initiation of ApoB-lipoprotein assembly. However, the major lipid-binding domains of ApoB100 are localized primarily within the C-terminal 80% of the protein. The size of the ApoB C-terminal region is an important structural factor determining the capacity of a given ApoB variant to transport triglycerides. Most of the plasma ApoB-containing particles are metabolized via the low-density lipoprotein receptor (LDLR)– mediated pathway after they are secreted into the bloodstream. The LDLR recognition site of ApoB resides within the β2 domain of ApoB100 (20,21), and thus is not present in ApoB48. Catabolism of the ApoB48-containing particles is mediated by the interaction of ApoE with the LDLR and other lipoprotein receptors (22). ApoE is a high-affinity ligand for the LDLR, which is either cosecreted with the hepatic VLDL particles or acquired by the intestinal chylomicrons after they are remodeled into remnant lipoproteins in the plasma (22).

The importance of MTTP in ApoB-lipoprotein assembly and production is best illustrated by its ability to convert nonlipoprotein-secreting cell lines to ApoB-lipoprotein– secreting cells when the large MTTP subunit is coexpressed with plasmids encoding lipid-binding competent forms of ApoB (28). In contrast, inactivation of MTTP function using chemical MTTP inhibitors (4,29) or through genetic manipulation of the MTTP large subunit gene (Mttp) (30–32) selectively blocks ApoB-lipoprotein secretion in both liver and intestinal cells. Moreover, as discussed in detail later, truncation-specifying or missense mutations of the MTTP gene lead to abetalipoproteinemia (ABL) (23,24,29), an autosomally recessive genetic lipid disorder that is characterized by the absence of ApoB-containing lipoprotein production by the liver and intestine in homozygous individuals. These studies have established unequivocally the role of MTTP in the assembly and secretion of triglyceride-rich lipoproteins.

MTTP: Structure and Functional Domains

Assembly of VLDLs and chylomicrons is a complex process that brings together a large ApoB polypeptide and four different classes of lipids in a fixed temporal sequence, which requires coordinated functions of ApoB, MTTP, and lipid synthesis enzymes. The mechanism underlying the biogenesis of ApoB-lipoproteins has been a topic of intense investigation and major progress has been made. Although most of these studies have used human or rat hepatoma cell lines to study this process, hepatocytes and enterocytes may share many common features in their pathways for assembly and secretion of VLDLs and chylomicrons, respectively. In general, a twostep model has been proposed to explain the process involved in ApoB-lipoprotein synthesis and secretion (see Fig. 67-1). This model suggests that ApoB is first assembled into a lipidpoor primordial lipoprotein particle in the rough ER, which then acquires additional bulk triglycerides from the ApoBfree lipid particles within the smooth ER lumen through a “fusion” process, leading to enrichment of the core components and expansion of the lipoprotein particle. Based on this model, the particle size of the mature lipoproteins may depend on the size of the lipid droplet within the smooth ER, which may, in turn, be influenced by availability of neutral lipids. Depending on the status of lipid availability, ApoB-containing lipoprotein particles may be secreted as dense VLDLs, large triglyceride-rich VLDLs, or chylomicrons (33,34). Some of the primordial particles may escape the second step and are secreted directly as high-density lipoprotein (HDL)–like particles. This is especially true for ApoB48 when lipid is not sufficiently available (35,36).

Although ApoB serves as an indispensable backbone for VLDLs and chylomicrons, it is not capable of synthesizing buoyant lipoprotein particles without adequate lipidation, which requires the complementary lipid transfer function of MTTP (4,5). MTTP is a heterodimeric neutral lipid transfer protein complex found in the lumen of the ER of ApoBcontaining, lipoprotein-secreting cells. MTTP contains a smaller 55-kDa subunit and a larger 97-kDa subunit (4). The 55-kDa subunit is the ubiquitous ER resident enzyme protein disulfide isomerase (PDI), whereas the 97-kDa MTTP large subunit is a unique protein coexpressed with the ApoB gene mainly by liver and intestine (4). PDI is thought to serve as a resident ER chaperone for MTTP in delivering the functional transfer protein activity into proximity with the elongating ApoB protein as it inserts across the ER membrane. The large 97-kDa subunit of MTTP is a single polypeptide of 894 amino acids (23,24). This subunit alone is responsible for the lipid-binding and transfer activity of the MTTP complex. MTTP is a member of the large lipid transfer protein family including insect, nematode, and vertebrate MTTPs (25); the vitellogenins (17,26); and ApoB (27). Based on its sequence homology with vitellogenin, MTTP has been predicted to contain three domains: an N-terminal β-barrel, a central α-helical domain, and a C-terminal lipid-binding domain (26,27). The MTTP large subunit does not itself contain a signature KDEL ER retention sequence; rather, the MTTP heterodimer relies on the KDEL domain on PDI to be retained at the site of ApoB translocation within the ER (4). MTTP is capable of transferring all the lipid classes found in ApoB-lipoproteins including triglycerides, cholesteryl esters, free cholesterol, and phospholipids. However, the neutral core lipid components (triglycerides and cholesteryl esters) appear to be its preferred substrates (4).

General Model of Apolipoprotein B–Containing Lipoprotein Assembly

First Step of Lipoprotein Biogenesis The processes involved in the first step of ApoB-containing lipoprotein assembly may have an important role in governing the number of lipoprotein particles produced. These include

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1715 physiologic variation in the level of ApoB gene expression, the rates of ApoB synthesis, and the rates and efficiency of secretion of the newly synthesized ApoB protein. Numerous studies have shown that the rates of ApoB secretion are not regulated by modulating the rates of ApoB synthesis. The APOB gene is constitutively expressed, and the synthetic rates of ApoB protein remain unchanged under most, if not all, circumstances, both in vitro and in vivo (37,38). One of the best in vitro examples for this phenomenon is the finding that supplementation of oleic acid to the media of cultured human hepatoma cells (HepG2 cells) does not affect either ApoB mRNA levels or the synthetic rates of ApoB100, whereas it rapidly stimulates ApoB100 secretion (38–40). Similar findings have been reported in several other in vitro systems (40,41). The limited number of studies examining intestinal ApoB synthesis after dietary lipid challenge appears to confirm these findings as well (42). Dietary or hormonal conditions that stimulate hepatic or intestinal ApoB production in vivo also fail to influence ApoB gene expression or ApoB synthesis (43–45). Thus, the rate of secretion of ApoB-containing lipoproteins is believed to be regulated at a posttranslational level, rather than by altering de novo synthesis. In this model, secretion efficiency of ApoB is determined by the extent to which the nascent protein escapes presecretory degradation.

How is ApoB regulated in intestinal cells? In contrast with the above findings in hepatoma cell lines, studies in Caco-2 cells, an intestinal cell line that is commonly used for intestinal lipoprotein secretion studies, suggested that there was little, if any, intracellular ApoB degradation unless MTTP activity was blocked using specific MTTP inhibitors (58). These findings suggested that ApoB may not be subjected to presecretory degradation in intestinal enterocytes. However, a study by Xie and colleagues (59) has demonstrated that significant amounts of ApoB100 are degraded (range 70–90%) without being secreted in enterocytes isolated from Apobec-1−/− mice. Presecretory degradation of ApoB100 by these Apobec-1−/− enterocytes, however, was inhibited by providing supplemental lipids to the cells (59). Xie and colleagues (59) also demonstrated presecretory degradation of ApoB48 by the enterocytes of wild-type mice, but to a much lesser extent (~30%) than noted for ApoB100. Similarly, ApoB48 has been shown to be more efficiently secreted and appears less susceptible to presecretory degradation in cultured hepatoma cells (60) or rat hepatocytes (51), regardless of lipid availability. Thus, presecretory degradation is an important mechanism for regulating intestinal ApoB48 secretion, but appears quantitatively less important than in liver cells. Formation of Primordial Lipoprotein Particles

Presecretory Degradation of Apolipoprotein B In HepG2 cells, the majority of the newly synthesized ApoB100 is degraded (>70%) cotranslationally when lipid availability is low, for example, in the absence of oleic acid supplementation to the media (39,46). Studies reported by Ginsberg and coworkers (37,47–49) have demonstrated that the presecretory degradation of ApoB100 in HepG2 cells is mediated mainly by the proteasomal pathway. This ApoB100 degradative pathway involves ubiquitination of the ApoB100 nascent polypeptide when it is not properly translocated across the ER membrane or becomes misfolded in the ER because of insufficient lipidation (48,49). In one proposed pathway, the ubiquitinated ApoB100 is transported via retrograde transport back through the ER membrane (dislocation) and degraded rapidly by proteasomal proteases (48). Proteasomal degradation of ApoB100 also occurs in a rat hepatoma cell line, McA-RH7777 (37,50). Newly synthesized ApoB (both ApoB100 and ApoB48) also is subjected to presecretory degradation, as evidenced from studies in cultured rat or mouse hepatocytes (51–54). However, unlike the findings in hepatoma cells, there is little evidence that the proteasomal degradative pathway plays a significant role in ApoB degradation in primary cultures of murine liver cells (52,55,56). It appears in this situation that presecretory degradation of ApoB occurs mainly through a post-ER protein degradation mechanism including the LDLR pathway (52,54,56,57). Thus, ApoB degradation, which is a major mechanism for regulating the assembly and secretion of triglyceride-rich lipoproteins, is regulated in a cell-specific manner, as evidenced from numerous studies in liver cells.

The assembly of ApoB-containing lipoprotein particles begins with translocation of the nascent ApoB peptide across the ER membrane while the polypeptide is still attached to the polysome and before protein translation of the full-length protein is finished (37,61). During this process, the N-terminal region of the ApoB (within the first 700 residues) interacts directly with MTTP to facilitate folding of the ApoB polypeptide and the acquisition of neutral lipid (27,62,63). Mutations within the putative MTTP binding sites in the N-terminal region of ApoB impairs secretion of ApoB from cultured cell lines, as a result of fatal defects in the ability of the ApoB protein to fold correctly (27,63). Under physiologic circumstances, formation of primordial lipoprotein particles involves release of the nascent ApoB polypeptide together with lipids derived from the ER membrane, acquired through fusion with MTTP, as a protein-lipid complex into the ER lumen (see Fig. 67-1). This primordial lipoprotein particle is likely to contain ApoB, together with a phospholipid monolayer and a limited amount of neutral lipids (5,34). Manchekar and colleagues (64) showed that the first 1000 amino acid residues of ApoB1000 are capable of forming a phospholipid-rich lipid pocket that may be involved in the initiation of ApoB-lipoprotein assembly. This study also suggested that the ApoB sequence between residues 931 and 1000 is critical for the formation of this lipid pocket and the primordial lipoprotein particle (64). One of the major functions of forming a primordial particle is to render the ApoB molecule secretion competent. It is notable that a number of resident ER chaperone proteins have been demonstrated to play an important role in the folding of ApoB (65).

1716 / CHAPTER 67 Importantly, this step is likely to be conserved in both hepatocytes and enterocytes. The ApoB molecules that fail to achieve to this step are degraded intracellularly. It has been shown that ApoB48 undergoes translocation more efficiently than ApoB100 and requires less lipidation to become secretion competent (51,60,66). This may explain why less ApoB48 than ApoB100 undergoes presecretory degradation within enterocytes, which, in turn, leads to a more efficient secretion pattern with ApoB48 compared with ApoB100. Independent of ApoB synthesis, triglyceride-rich lipid particles are formed inside the lumen of smooth ER (see Fig. 67-1). It has been proposed that MTTP plays a critical role in extracting this membrane pool of triglycerides from the ER membrane (31,33,67,68). This process of membrane triglyceride mobilization also requires a constant supply of neutral lipids, especially triglyceride, which may be synthesized by the diacylglycerol acyltransferases within the ER membrane (69). Evidence for the synthesis of lipid droplets is largely derived from electron microscopic observations. It was originally showed by Alexander and colleagues (70) that the tubules and vesicles of a typical hepatocyte smooth ER did not contain ApoB, but did contain lipid droplets. Based on this observation, it was suggested that the triglyceriderich lipid droplets originate from the smooth ER and receive ApoB synthesized from the rough ER (70). Further evidence supporting this notion came from studies of intestinal ApoBdeficient mice (71). These intestinal ApoB-deficient mice were generated by crossing the ApoB-null mice with human ApoB-transgenic mice in which the transgene was expressed in the liver, but not in the intestine (3). In this compound knockout-transgenic mouse, the intestine is incapable of synthesizing any ApoB protein. Electron microscopy examination of the intestine of these intestine-specific ApoB null mice showed that even without ApoB synthesis, there were large lipid particles present in the ER lumen (71). These lipid droplets occasionally were even bigger than chylomicronsized particles seen within enterocyte of wild-type mice. This study established that the ability of the enterocyte to elaborate ApoB does not affect the formation of large lipid droplets in the ER lumen, provided MTTP is expressed at physiologic levels. Further insight into this process has emerged from the study of Raabe and colleagues (31) using hepatocytes from liver-specific MTTP-deficient mice. This study has provided strong evidence supporting a pivotal role for MTTP in the formation of the smooth ER–associated lipid particles (31). In these liver-specific MTTP-knockout mice, hepatic MTTP gene expression was reduced by more than 95%, and the hepatocytes of these mice accumulated large amounts of triglyceride in the cytosol, because of their inability to assemble VLDLs. Together with the lack of VLDL secretion, Raabe and colleagues (31) showed that the large, spherical, lipid-laden particles also were absent from the rough and smooth ER, and that the Golgi apparatus was empty in the MTTP-deficient hepatocytes compared with wild-type control animals. In a complementary in vitro system using

hepatoma cells, pharmacologic blockade of MTTP activity also leads to reduced accumulation of labeled triglycerides in the microsomal lumen, thus adding additional confirmation of the observations noted earlier (33,67,68). The exact mechanism for the formation of these lipid droplets is unknown, but the data suggest that the two simultaneous events occur, and both are considered part of the so-called first step of lipoprotein biogenesis and primordial lipoprotein biogenesis. These include: (1) fusion of ApoB with MTTP and its resultant cotranslational lipidation; and (2) the production of triglyceride droplets within the ER lumen. The first of these distinct processes requires both ApoB and MTTP, whereas the second process requires only MTTP. Second Step of Lipoprotein Biogenesis After assembly of the primordial particle, it is proposed that acquisition of additional core lipid by these small, dense primordial particles proceeds by fusion with the luminal lipid droplets. Although there is plenty of evidence demonstrating that the formation of primordial lipoprotein particles and lumenal lipid particles in smooth ER is a crucial first step in the biogenesis of VLDLs and chylomicrons, little is known about how the second step (i.e., the fusion of these two particles) occurs. Whether this second step requires the actions of MTTP remains a subject of debate. Based on the results from ultrastructural analysis of rat liver, Alexander and colleagues (70) proposed that the loading of a bulk of neutral lipid onto nascent lipoprotein particles could occur at the junction of the rough and smooth ERs in a quantum fashion. Studies in rat hepatoma McA-RH7777 cells (67) and rabbit enterocytes (33) have provided additional evidence supporting this notion. It is reasonable, therefore, to predict that during the postprandial state, the rapid supply of newly synthesized triglyceride may facilitate generation of larger luminal lipid droplets in the smooth ER lumen, which, in turn, leads to production of chylomicronsized particles. In this scenario, the size of the luminal triglyceride droplets may determine the size of the lipoprotein particle synthesized. The newly synthesized triglycerides appear to be preferentially incorporated into the chylomicron core (72). Under many pathophysiologic conditions, the rates of secretion of triglyceride-rich lipoproteins may be regulated at the step of converting the smaller, denser nascent particles into the larger, more buoyant particle; a full understanding of this step of the ApoB-lipoprotein assembly will provide important information on the control of VLDL and chylomicron production. Although there is a large body of evidence for a crucial role of MTTP in the formation and stability of the luminal lipid droplets, the role for MTTP in the fusion of the primordial lipoprotein particle and the luminal lipid droplet is unclear. It has even been suggested that MTTP may not be directly involved in this process (29,34), and it is unknown whether other cofactors are required for this step. This and other questions require additional study.

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1717 Transport of Prechylomicrons through the Secretory Pathway Nascent intracellular VLDL and prechylomicron particles leave the ER rapidly and are transported through the secretory pathway after budding from the membranous elements of this organelle. Developments have begun to shed light on the mechanisms by which nascent lipoprotein particles are delivered vectorially and transported through the distal elements of the secretory pathway. The coat protein II complex (COPII) machinery has been shown to have a critical role in transporting ER cargo destined for secretion through the Golgi apparatus (73). Emerging evidence now shows that the COPII machinery may also play a pivotal role transporting nascent triglyceride-rich lipoprotein particles from the ER to the Golgi apparatus (74–77). Studies of rat enterocytes by Siddiqi and coworkers (75) have shown that sequential interaction of distinct pairs of heterodimeric COPII proteins (Sec 23/24 and Sec 13/31) is required for transporting the nascent prechylomicrons from ER to the Golgi apparatus. Sar1 was demonstrated to play a pivotal role in the final assembly of a vesicular transport complex that was then capable of fusion with Golgi membranes. These investigators demonstrated in particular that COPII proteins are required for Golgi fusion, but not ER budding of the prechylomicron transport vesicle (75). In contrast, work from Gusarova and colleagues (76) in a rat hepatoma cell model demonstrated that a functional COPII complex was, in fact, required for ER budding of the nascent ApoB100-VLDL particle. The reasons for this apparent discrepancy in the role of COPII proteins in ER export of triglyceride-rich lipoproteins is unknown, but may reside in the different cell systems (rat intestine vs rat hepatoma cells) used. Obviously, more studies are needed to elucidate the mechanisms underlying the transport of large lipoprotein particles through the secretory pathway. Nonetheless, as discussed later, Sar1b, a member of the Sar1/Arf family of small GTPases, is an integral component of the COPII machinery (74). Mutations in the SARA2 gene, encoding Sar1b, have been shown to be associated with chylomicron retention disease (77), in which enterocytes are capable of assembling prechylomicrons in the ER, but fail to transport them through the secretory pathway, resulting in their accumulation in the membrane-bound compartments of the enterocytes (78,79). It is important to emphasize that this mutation does not affect VLDL secretion by the liver. This observation implies that there is either a tissue-specific defect or an effect mediated by the intrinsic difference between ApoB48 and ApoB100. Further studies on these topics are likely to provide new insights into the mechanism controlling intestinal lipid transport.

GENETIC DEFECTS IN APOB AND MTTP Familial Hypobetalipoproteinemia FHBL is an autosomal codominant disorder of lipoprotein metabolism characterized by low (<5th percentiles for age

and sex) plasma concentrations of total cholesterol, LDL cholesterol, and ApoB (80,81). The genetic cause(s) of most of the FHBL cases are unknown, and FHBL could be genetically heterogeneous. However, FHBL can be caused by nonsense and frameshift mutations in the APOB gene that lead to formation of C-terminally truncated ApoB (80,81). These subjects with such mutations represent the bestcharacterized FHBL cases to date. The truncated ApoB variants are designated according to a centile nomenclature (Fig. 67-2). Approximately 45 different premature stop codon–specifying mutations in the APOB gene have been reported. The predicted size for the genetic variants of APOB produced by these mutant APOB alleles ranges from ApoB0.5 to ApoB89 (see Fig. 67-2) (82–113). The frequency of APOB gene mutations causing truncated ApoBs and hypobetalipoproteinemia is unknown. Based on a study of 3873 total subjects participating in the Framingham Offspring Study, it was estimated that the frequency of FHBL attributable to truncation-specifying mutations in the APOB gene was 1 in 3000 (114). Heterozygous FHBL subjects often are symptomatic, but have plasma LDL-cholesterol and ApoB concentrations that are one fourth to one third of normal (see Fig. 67-2). Low plasma cholesterol levels appear to be well tolerated and do not appear to be associated with apparent defects in intestinal triglyceride absorption. The clinical and biochemical features of homozygous FHBL, in contrast, often are similar to that noted later for subjects with homozygous ABL. Patients with homozygous FHBL may have deficiencies in fat-soluble vitamins secondary to severe malabsorption of fat (82,87,88,94,101). Only a few cases have been reported of homozygous FHBL with ApoB mutations, including homozygotes for ApoB2 (82), ApoB25 (87), ApoB27.6 (88), ApoB38.7 (94), ApoB45.2 (98), ApoB49.6 (101), and ApoB87 (113). Most of these homozygous subjects have no detectable LDL-cholesterol. Mechanisms Underlying the Phenotype with Familial Hypobetalipoproteinemia Truncated ApoBs smaller than ApoB27.6 lack intrinsic lipid-binding properties and are usually absent from the plasma (82–87). It is unclear whether this is caused by their rapid clearance, low production rates, or both. Transfection of plasmids encoding truncated ApoBs into rat hepatoma cells shows that ApoB truncations as short as ApoB13 are secreted efficiently (115,116). Thus, it is unlikely that the liver and intestine are incapable of secreting the short ApoB variants. However, it is unknown whether or how the location of the premature termination codon (PTC) affects the stability of the mutant ApoB transcript in vivo. A PTC near the 5′ end of the transcript usually destabilizes the mutant transcript much more than a similar truncation at the 3′ end (117). Thus, it is possible that the small truncated ApoB variants, where PTCs would be located at the 5′ end of the gene, are synthesized at low rates as a result of intrinsic instability of the mutant ApoB mRNA. Nevertheless, it is

1718 / CHAPTER 67

FIG. 67-2. Genetic variants of apolipoprotein B (ApoB) in familial hypobetalipoproteinemia (FHBL) and their density distribution in plasma. (A) Domain structures of the two physiologic ApoB isoforms, ApoB100 and ApoB48. (B) The predicted truncated forms of ApoB associated with FHBL. Plasma low-density lipoprotein-cholesterol (LDL-Chol) levels in the subjects with heterozygous FHBL, density distribution of the truncated ApoBs, and a list of the corresponding references (Ref.) are provided in the three columns on the right. HDL, high-density lipoprotein; IDL, intermediate-density lipoprotein; NA, nonapplicable for LDL-Chol value as the corresponding subjects with FHBL are homozygous for ApoB mutations and have no detectable LDL in the plasma; ND, not determined; VLDL, very low-density lipoprotein.

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1719 possible that these short ApoB variants are not stable in the plasma or are rapidly cleared. Because of their absence from the plasma of subjects with FHBL, no information has been available on the potential pathway for the catabolism of these short ApoB truncations. Importantly, even when present in the plasma of subjects with FHBL, the truncated ApoBs are invariably found at low concentrations, usually 10% to 30% of ApoB100 encoded from the normal allele in FHBL heterozygotes (80–113). There is another important dilemma, and an unexpected feature of FHBL, namely, that plasma levels of ApoB100 in individuals with heterozygous FHBL are much less than 50%, the level predicted based on the preservation of one normal APOB100 allele. Lipoprotein kinetic studies of the human subjects with heterozygotes FHBL indicate that production rates of ApoB100 are reduced by 70% to 80%, instead of the expected 50% (118–120). Thus, the presence of a PTC-specifying mutation in one ApoB allele may somehow impair the synthesis or secretion, or both, of ApoB100, the gene product of the unaffected allele from the liver. A full understanding of the cellular mechanism underlying this phenomenon will require detailed mechanistic studies using appropriate animal models, as described later in this chapter. Accompanying the low production rates of ApoB100VLDL and the truncated ApoB-lipoproteins, rates of hepatic VLDL-triglyceride secretion also are reduced by 50% to 60% in the FHBL heterozygotes (120,121). Fatty livers are frequently associated with FHBL (81,86,94,103,113,121). By using magnetic resonance spectroscopy as a tool to assess liver fat content, Schonfeld and colleagues (121) showed that subjects with heterozygous FHBL with a range of ApoB truncations (ApoB4 to ApoB89) have a liver fat content threefold greater than control subjects. Presumably, the reduced hepatic triglyceride-exporting capacity plays a major role in the development of fatty livers in these subjects. The diameters of the truncated ApoB-containing lipoproteins particles vary directly with the length of the ApoB truncation (88–113) (see Fig. 67-2B). Thus, ApoB89-bearing lipoproteins have sizes and densities similar to normal VLDLs and LDLs (96), whereas ApoB27.6 and ApoB31 circulate in the plasma only as lipid-poor, HDL-sized particles (88,90). The lipoproteins containing the “intermediate”sized, truncated ApoB variants, such as ApoB45.2, ApoB46, ApoB52, or ApoB54.2, circulate in the plasma mainly as small LDL and VLDL particles (98–104). It is important to emphasize that because of ApoB mRNA editing, production of ApoB truncations larger than ApoB48 would not be likely to occur in the small intestine. Accordingly, the liver is responsible for the production of truncated ApoBs larger than ApoB48, whereas both the liver and the intestine contribute to the lipoproteins particles bearing truncated ApoB variants smaller than ApoB48. Stable isotope tracer methodology has been used to examine the in vivo kinetics of ApoB in subjects with FHBL (81). ApoB89 (122) and ApoB75 (109) are produced at slightly lower rates than ApoB100-containing lipoproteins and also are cleared more rapidly from the plasma. These “large”

truncated ApoB variants manifest increased affinities for the LDLR and are metabolized primarily by the liver (109,122). Particles containing “intermediate” or “small” variants of truncated ApoB such as ApoB70.5 (108), ApoB54.8 (104), ApoB38.9 (95), or ApoB31 (90) are produced at lower rates than the larger forms of truncated ApoB variants and, because they lack the LDLR-binding domain, are not intrinsically capable of binding to the LDLR (21). By analogy to ApoB48containing lipoproteins (22), however, it is predicted that the majority of these particles probably acquire ApoE by exchange from other plasma lipoproteins, and then undergo clearance by the liver via LDLRs and the LDLR-related protein, LRP. Notwithstanding this prediction, turnover studies in animals injected with lipoproteins containing truncated ApoB proteins isolated from plasma of subjects with FHBL have shown that a large proportion of the injected lipoproteins are catabolized in the kidney (21,123). Chen and colleagues (21) further demonstrated that megalin, a member of the LDLR gene family, may be responsible for the renal uptake and catabolism of these truncated ApoB variants. Most of the studies of FHBL have focused on derangements of plasma and hepatic lipid metabolism, and relatively little is known about the effect of ApoB-truncation mutants on intestinal lipid metabolism. However, the currently available data indicate that intestinal fat absorption and secretion may not be affected in the FHBL heterozygotes. Averna and colleagues (124) have reported findings from a dietary fat absorption study involving 10 patients with heterozygous FHBL with ApoB truncations larger than ApoB48 (ApoB89, ApoB75, ApoB54, and ApoB52), 6 patients with heterozygous FHBL with ApoB truncations smaller than ApoB48 (ApoB46, ApoB40, and ApoB31), and 16 control subjects matched for age, sex, body mass index characteristics, and dietary intake. These authors found no difference in response to a constant vitamin A oral fat loading test between the FHBL heterozygotes and the control subjects, indicating that one allele specifying the intestinal production of ApoB48 may be sufficient for normal fat absorption. Similarly, Ruotolo and colleagues (100) reported that postprandial lipid metabolism of an ApoB48.4 FHBL heterozygote responded normally to fat loading. Together, these studies strongly suggest that one normal APOB allele specifying intestinal production of ApoB48 may be sufficient for physiologic intestinal lipid absorption and secretion. Mouse Models of Familial Hypobetalipoproteinemia As mentioned earlier, subjects with heterozygous FHBL usually are asymptomatic. This poses ethical barriers to obtaining liver and intestinal biopsies for in-depth studies to understand the mechanisms underlying the full manifestations of a heterozygous FHBL phenotype, particularly whether there are subtle defects in lipoprotein assembly and secretion. Embryonic stem cell gene targeting technology has made it possible to generate mice lacking specific gene products and containing subtle mutations in any gene of interest. During the late 1990s/early 2000s, researchers have

1720 / CHAPTER 67 generated several lines of Apob gene–targeted mice, including Apob gene–null mice (125,126) and mice bearing ApoB70 (127), ApoB81 (128), ApoB39 (129), ApoB38.9 (130), or ApoB27.6 (131). These mice display a range of typical FHBL phenotypes, including low-plasma ApoB100and truncated ApoB-containing lipoproteins (125–131) and hepatic steatosis as well (130,131). Studies of these mice also have shown an essential role for ApoB in nutrient transport during embryonic development in rodents (125–131). Specifically, mice homozygous for gene-targeted Apob allele disruption usually die during midgestation because of the inability of the visceral yolk sac endoderm to produce sufficient triglyceride-rich lipoproteins to support critical functions, particularly nervous system and neural tube development (125–131). It should be emphasized that these lethal developmental defects represent an important departure in the phenotype from human FHBL, in which no such systemic effects have been noted. The ApoB38.9-bearing mouse (130) has proved particularly valuable in understanding features of the FHBL syndrome. This mouse was created using conventional homologous recombination in combination with the Cre-loxP system to avoid retention of the extraneous vector sequence in the Apob gene, eliminating the potential unwanted effect of the gene-targeting on the expression of the Apob gene or other genes nearby. The resultant ApoB38.9 mice carry only a single nucleotide deletion in their ApoB38.9-specifying allele, thus closely resembling the naturally occurring ApoB38.9specifying mutation in human subjects with FHBL (95). Using these mice, Chen and coworkers (130) demonstrated that an ApoB38.9-specifying mutation reduced steady-state mRNA levels of the affected Apob allele by 40% in the liver and intestine (130), providing evidence that reduced mRNA expression of the mutant Apob gene could be an important mechanism for the low production rates of truncated ApoB variants in individuals with FHBL. The availability of these mice also provides an opportunity to directly analyze the assembly and secretion of ApoB38.9-containing lipoproteins. ApoB38.9 is secreted efficiently by hepatocytes, but mainly as triglyceride-poor HDL particles, whereas ApoB48, the major physiologic ApoB isoform of mouse liver, is secreted mainly as VLDL and IDL particles. These results demonstrate that deleting the C-terminal 400-amino-acid residues for ApoB48 greatly reduces its ability to assemble into triglyceride-rich lipoproteins. Because of the inability of ApoB38.9 to transport triglycerides, the ApoB38.9 mice have low rates of hepatic triglyceride secretion and experience development of fatty livers (130). The ApoB27.6-bearing mouse was created by inserting a neomycin selection marker cassette gene as a gene-targeting vector into exon 25 of the endogenous murine Apob gene. Homologous recombination and mRNA splicing lead to formation of a premature stop codon specifying ApoB27.6 (131). ApoB27.6 is secreted by the hepatocytes exclusively as dense HDL particles, indicating its inability to assemble very low-density, triglyceride-rich particles. Furthermore, the ApoB27.6 heterozygotes had lower rates of hepatic

triglyceride secretion than the ApoB38.9 heterozygotes, and consequently experience development of more severe fatty livers (131). Thus, studies from these mice clearly demonstrate the importance of the C-terminal region of ApoB48 in determining its lipid transport capacity. To investigate the mechanism underlying the larger than expected decrease in ApoB100-VLDL production in the FHBL heterozygotes, researchers crossed the ApoB38.9 mice with ApoB100-only mice (132) or Apobec-1−/− mice (133), to eliminate ApoB48 formation in the liver and small intestine of the compound ApoB38.9/B100 heterozygotes (53). As a result of this intercross, the livers of the compound knockout mice produce only ApoB38.9 and ApoB100 (53). Direct measurement of in vivo secretion rates of ApoB100 demonstrates that the ApoB38.9-mutation causes a 70% to 80% reduction in ApoB100 secretion, as compared with the control mice in which both Apob alleles are used for ApoB100 formation. Ex vivo studies using hepatocytes isolated from these compound knockout mice showed that ApoB100 synthetic rates of the ApoB100/38.9 mice are reduced by 50%, compared with hepatocytes from ApoB100/100 mice. In addition, the newly synthesized ApoB100 of ApoB100/38.9 hepatocytes does not appear to be secreted as efficiently as that of the ApoB100/100 hepatocytes (53). These data suggest that the ApoB truncation-specifying mutation in FHBL heterozygotes may affect the secretion of ApoB100 of the normal Apob allele in a posttranslational manner. Little information is available on how the corresponding intestinal lipoprotein machinery responds to the reduced availability of ApoB48 in these FHBL mouse models. Nevertheless, based on the findings in murine hepatocytes, one would predict that malabsorption of lipid might be encountered in mice with FHBL.

Abetalipoproteinemia Mutations in the structural gene encoding MTTP cause ABL, a rare autosomal recessive disorder characterized by a selective absence of ApoB-containing lipoproteins in blood (134,135). The condition typically presents as failure to thrive in infancy because of severe fat malabsorption, or in older children as an atypical pigmented retinopathy associated with spinocerebellar degeneration (134–136). The clinical manifestations of homozygous ABL were initially described by Bassen and Kornzweig in 1950 (137). About 100 cases of ABL currently have been reported (see review by Berriot-Varoqueaux and colleagues [29]). In fasting patients, the plasma cholesterol levels are low (20–50 mg/dl) and triglyceride levels are typically less than 10 mg/dl (135). Even after a fat load, chylomicrons and ApoB48 do not appear in plasma. These findings in plasma are coupled with neutral lipid accumulation in both enterocytes and hepatocytes. Most obligate heterozygotes (~80%), in contrast, have normal plasma lipid levels, and some even have high plasma cholesterol levels (>240 mg/dl), whereas a small group of ABL heterozygotes also have been reported to have

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1721 relatively low cholesterol (130–165 mg/dl) (29,30). The mechanisms underlying the phenotypic heterogeneity among obligate heterozygotes are not fully understood. MTTP Mutations and Abetalipoproteinemia The clinical manifestation of ABL suggest that ABL is caused by a defect in the assembly of ApoB-containing lipoproteins. Thus, it was initially thought that this defect was linked to an absence of ApoB synthesis (138). However, subsequent biochemical (139,140) and genetic linkage studies (141,142) dismissed this possibility and established that the defect resided in a locus on chromosome 4q22-24, distinct from the APOB locus on chromosome 2. Wetterau and colleagues (143) first established a link between MTTP and ABL in 1992, demonstrating that both the large 97-kDa subunit of MTTP and triglyceride transfer activity were absent in intestinal biopsies from subjects with homozygous ABL. Subsequent work by two other groups (23,24) led to the cloning of MTTP and identification of the MTTP defects causing ABL. Currently, only a small number of patients with ABL (~20) have been studied at the molecular level (23,24,27,144–150; see also review by Berriot-Varoqueaux and colleagues [29]). Most of the MTTP mutations result in formation of PTCs and the production of C-terminally truncated forms of MTTP (144–150), indicating a critical functional role for this C-terminal domain in ApoB-lipoprotein assembly. Mouse Models of Abetalipoproteinemia Raabe and colleagues (30) attempted to generate an Mttp knockout mouse to study the role of MTTP in lipoprotein secretion and mouse embryonic development. Unfortunately, homozygous Mttp deletion in mice results in embryonic lethality, with the same phenotype observed in mice with truncating Apob mutations (see earlier) (30). This phenotype was not unexpected based on that ApoB function and lipoprotein secretion are required for normal murine embryonic central nervous system development (see earlier discussion). In contrast, the heterozygous Mttp mice showed a somewhat surprising phenotype. MTTP activity and mRNA levels were reduced by 50% in both the liver and intestine. Accompanying these changes, hepatic ApoB-containing lipoprotein secretion also was reduced by 20% to 30%. Thus, mice with one targeted allele for Mttp appeared to manifest an autosomal dominant trait, where elimination of one allele reduced the function of the gene by ~50%. Humans with heterozygous MTTP mutations, in contrast, have no such haploinsufficiency and manifest normal MTTP activity in the intestine, which is consistent with an autosomal recessive trait. These heterozygous Mttp targeted mice also displayed resistance to diet-induced hypercholesterolemia (30). However, although decreases in plasma cholesterol levels have been reported in some human subjects heterozygous for ABL, the majority of obligate heterozygotes do not show any abnormality in

plasma lipoprotein levels (see review by BerriotVaroqueaux and colleagues [29]). To develop an appropriate model to understand the role of MTTP in lipoprotein assembly, two independent groups (31,32) have produced two lines of liver-specific Mttp knockout mice. Effective hepatic MTTP ablation (>95%) was achieved in both lines after induction of Cre gene expression (31,32). Inactivation of MTTP in the liver dramatically reduced secretion of all classes of plasma lipoproteins in these mice. However, an important discrepancy was noted in these studies. In one study, near-absent hepatic secretion of both ApoB100- and ApoB48-containing lipoproteins was reported (32). In the other study, ApoB100 secretion was virtually eliminated, but ApoB48 secretion was relatively preserved, despite ablation of MTTP function (31). The reasons underlying this important discrepancy have yet to be adequately explained. The question of differential sensitivity to MTTP inhibition of ApoB100 versus ApoB48 has obvious implications for intestinal triglyceride-rich lipoprotein secretion. Attempts to understand the role of MTTP in lipoprotein secretion have taken advantage of chemical inhibitors of MTTP function, using cultured cell lines and primary cells. Although a number of in vitro studies have shown that ApoB48 secretion is less sensitive than ApoB100 to MTTP inhibitors in cell culture (68,151), the near-normal rates of hepatic ApoB48 secretion in the Mttp knockout mouse was unexpected. Liver-specific inactivation of MTTP also was accompanied by disappearance of VLDL-sized lipid particles within the ER and Golgi (31), providing the first in vivo evidence for a role of MTTP in promoting formation of ApoB-free lipid particles in the ER. Thus, although hepatic ApoB48 secretion was not completely blocked in Mttpdeficient mice, the second step of VLDL assembly was inhibited, leading to severe impairment in hepatic triglyceride export and fatty livers in these mice. It is not yet known whether intestinal enterocytes also are capable of secreting ApoB48 when intestinal Mttp is inactivated, but this question will have important implications for therapeutic drug development. The characterization of intestine-specific Mttp mice will have great appeal in this regard.

APOLIPOPROTEIN B MESSENGER RNA EDITING: OVERVIEW, MOLECULAR MECHANISMS, AND FUNCTIONAL RELEVANCE As noted earlier, ApoB is a requisite component for the assembly and secretion of triglyceride-rich lipoproteins by both enterocytes and hepatocytes. ApoB is expressed in a tissue-specific manner with ApoB48, the amino-terminal half of the protein, being expressed in human enterocytes, whereas ApoB100, the corresponding full-length isoform, is expressed virtually exclusively in the liver (2). A single-copy mammalian APOB gene is transcribed in both the liver and small intestine to a large (>14 kb) mRNA, which then undergoes tissue- and cell-specific substitutional editing of the

1722 / CHAPTER 67 nuclear transcript (152). A site-specific C to U deamination converts a CAA codon at position 2153 (specifying glutamine in the unedited, SpoB100 mRNA) to a UAA or translational stop codon in SpoB48 mRNA (153,154). This exquisitely site-specific RNA editing reaction thereby creates an inframe stop codon in the edited ApoB mRNA, which is then translationally terminated at codon 2152, causing ApoB48. Intestinal lipoprotein assembly and, in particular, chylomicron assembly and triglyceride secretion are critically dependent on ApoB48. ApoB mRNA editing takes place in mammalian enterocytes, as well as in the liver of rats, mice, and certain other species (155). As a result, hepatic lipoprotein assembly and secretion in these species uses both ApoB100- and ApoB48-specific pathways. The functional impact and presumed survival advantages of this adaptation are discussed later. Human liver, in contrast, contains only unedited ApoB mRNA (156), and hepatic lipoprotein assembly and secretion uses ApoB100-dependent pathways exclusively (2).

Apolipoprotein B Messenger RNA Editing: Molecular Machinery and Tissue-Specific Regulation Intestinal ApoB mRNA editing is mediated by a holoenzyme that contains a minimal core with two components: apobec-1, the catalytic deaminase, and an RNA-binding subunit, apobec-1 complementation factor (157–159). Apobec-1 mediates an exquisitely site-specific deamination of a single cytidine nucleotide in a nuclear ApoB mRNA of more than 14,000 bases. Although apobec-1 is essential for ApoB mRNA editing, it alone is not sufficient. ApoB mRNA editing requires the auxiliary cofactor apobec-1 complementation factor. The tissue-specific distribution of ApoB mRNA editing and its restriction in humans to intestinal ApoB reflects the tissue- and cell-specific distribution of apobec-1, which is expressed in enterocytes and subepithelial cells throughout the luminal gastrointestinal tract (160–162). As noted earlier, human liver expresses only ApoB100; this is reflected in the absence of apobec-1 mRNA (156). Expression of apobec-1 complementation factor mRNA and proteins is predominantly in the liver, kidney, and intestine, but low-level expression (using sensitive reverse transcriptase-polymerase chain reaction amplification of RNA) demonstrates the presence of apobec-1 complementation factor in almost all tissues in humans, rats, and mice (163–165). Site-specific C to U deamination of a single nucleotide in the nuclear ApoB mRNA requires the coordinated interaction of apobec-1, the catalytic deaminase, and apobec-1 complementation factor, the RNA binding subunit, in a critical stoichiometry that is likely governed by other protein–protein interactions (158,166,167). ApoB mRNA editing takes place on spliced, polyadenylated mRNA, and thus represents a posttranscriptional modification (168,169). A number of proteins (ABBP-1, ABBP-2, CUGBP2, GRY-RBP, hnRNP-C1) have been identified through their interactions with either ApoB RNA or apobec-1, or both (see review by Anant and colleagues [152]). These interactions, in turn, modulate the efficiency and

extent of ApoB mRNA editing, in either murine hepatoma cell lines and/or in vitro assays (152,170). However, the role, if any, of these proteins in modulating mammalian intestinal ApoB mRNA editing has yet to be demonstrated. Earlier studies suggested that mammalian intestinal ApoB mRNA editing is developmentally regulated; these studies in human intestine and Caco-2 cells demonstrated a general concordance with the developmentally regulated expression of apobec-1 mRNA (160). In contrast, the expression of apobec-1 complementation factor mRNA in human intestine appears to be unrelated to the extent of endogenous RNA editing (163,165). It bears emphasis that in both neonatal and adult rat and mouse liver, ApoB mRNA editing is regulated by a variety of nutritional, hormonal, and environmental stimuli and through mechanisms that involve transcriptional regulation of apobec-1 or apobec-1 complementation factor mRNA, or both (see review by Anant and colleagues [152]). Modulation of hepatic ApoB mRNA editing in these settings represents an important metabolic adaptation for the efficient secretion of hepatic triglyceride. In contrast, intestinal ApoB mRNA editing is a developmentally regulated event, with a progressive increase in the proportion of intestinal ApoB48 mRNA culminating in greater than 90% editing in postnatal mammalian enterocytes. There is no evidence for metabolic regulation of ApoB mRNA editing in the neonatal or adult mammalian intestine. Furthermore, gene expression of the core components of the intestinal ApoB mRNA editing enzyme, in adult animals, appears to be constitutive. This said, there is compelling evidence that apobec-1 and apobec-1 complementation factor proteins shuttle between nuclear and cytoplasmic compartments (170,171). The question of whether apobec-1 complementation factor and apobec-1 shuttle independently or as a complex has yet to be firmly resolved, but evidence from transfection studies using epitope-labeled proteins suggests that each protein has the intrinsic capacity to shuttle. This observation is consistent with the presence in each protein of both a nuclear localization sequence and a cytoplasmic retention sequence. This implies the possibility that the intracellular distribution of apobec-1 complementation factor and of apobec-1 may be modulated through protein–protein interactions that result in selective accumulation in a particular compartment. As a corollary, the functional consequences and possible range of RNA targets of each protein may also be subject to modulation. Evidence for alternate targets of apobec-1 include transcripts that contain the canonical binding site UUUN[A/U]U embedded within an A+U rich sequence (172). This cisacting element is present in the 3′ untranslated region of several mRNA species, including certain cytokines and protooncogenes. apobec-1 has been demonstrated to bind with high affinity to many of these candidate target mRNA, including c-myc and cox-2 mRNA, both key players in intestinal growth and malignant transformation (172,173). The possibility therefore exists that apobec-1 and apobec-1 complementation factor each have a range of targets distinct from ApoB mRNA, although the biological implications of this possibility have yet to be clearly delineated.

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1723 Functional Importance of Intestinal Apolipoprotein B Messenger RNA Editing ApoB mRNA editing is a recent evolutionary development, roughly coinciding with the appearance of a lymphatic vasculature after the divergence of birds and reptiles, phyla that express exclusively unedited ApoB in the intestinal tract (174,175). The chicken homolog of apobec-1 is a cytidine deaminase that is incapable of mediating C to U deamination of either the endogenous avian ApoB, or a synthetic mammalian ApoB RNA in an in vitro assay (174). In addition, there is sequence divergence of avian and frog ApoB mRNA in the region corresponding to the domain containing the edited base in mammalian ApoB, which renders the corresponding cytidine incapable of undergoing C to U editing in an in vitro assay using the mammalian machinery (175). As a result, birds and reptiles secrete ApoB100-containing lipoproteins from their intestine into the portal circulation in the form of large triglyceride-rich droplets referred to as portomicrons. ApoB mRNA editing generates a truncated protein that undergoes effective lipidation and directs intracellular chylomicron formation. The amino terminal 20% of ApoB contains almost all the N-glycosylation sites, as well as the domains that direct physical interaction with MTTP within the ER (176). The truncation of ApoB48 at residue 2152, however, eliminates two crucial domains that regulate important elements of systemic ApoB-containing lipoprotein metabolism. These include two stretches of basic residues in the region of residue 3500 that directs the physical interaction of ApoB with the LDLR. The other is a single unpaired cysteine residue at position 4326 that directs covalent association between ApoB100 in LDLs and the plasminogenrelated gene apolipoprotein(a) to form the atherogenic lipoprotein particle, Lp(a) (177,178). As a result of ApoB mRNA editing, ApoB48-containing chylomicron particles interact much less efficiently with the LDLR, and instead undergo preferential uptake by the related receptor, LRP, which is largely expressed in the liver. In addition, intestinal ApoB48-containing chylomicrons are incapable of forming chimeric Lp(a) particles. These metabolic consequences support one plausible explanation for the emergence of intestinal ApoB mRNA editing in mammals, namely, that the production of intestinal ApoB48 coincides with the need to establish dual pathways for cholesterol, lipovitamin, and triglyceride transport and targeted delivery of intestinal lipid to the liver. The corollary hypothesis is that ApoB100 serves a distinct role in lipoprotein metabolism by virtue of its targeted interaction with the LDLR, and thus the ability of mammals to regulate circulating plasma cholesterol levels in response to cellular demands. Examination of the hypothesis that intestinal lipid transport is intrinsically more efficient in an ApoB48 background was accomplished through gene targeting of Apobec-1, and the consequent elimination of ApoB mRNA editing (133,179,180). Apobec-1−/− mice are healthy and fertile and appear to eat and grow normally. However, when challenged

with a high-fat bolus, Apobec-1−/− mice absorb triglyceride over a longer time interval than wild-type (ApoB48) control animals (59,181). In addition, chylomicrons isolated from the media of cultured enterocytes from Apobec-1−/− mice are paradoxically larger than wild-type control animals (59,181). Accordingly, one suggestion emerging from these data is that the intrinsic capacity to assemble chylomicron-sized particles using either of the two natural isoforms of ApoB is a property of mammalian enterocytes. This suggestion is consistent with the observation that mammalian hepatocytes secrete triglyceride-rich lipoproteins in the size range of VLDLs with either ApoB100 or ApoB48, but not large particles in the size range of chylomicrons. The findings collectively suggest that intestinal chylomicron assembly and triglyceride transport in the background of ApoB100 results in the secretion of fewer, larger particles in response to a lipid bolus and less rapid intestinal triglyceride delivery into the systemic circulation than in wild-type (ApoB48) control animals. The production of fewer particles is consistent with the observation that posttranslational degradation of intestinal ApoB100 is much more extensive than that with intestinal ApoB48 (59). That is, less molecules of ApoB100 survive through posttranslational lipidation and primordial lipoprotein biogenesis to become nascent intracellular lipoprotein particles. ApoB100 availability thus becomes rate limiting, which, in turn, constrains lipoprotein assembly as a result of the requirement for one molecule of ApoB per particle. A further consequence of eliminating ApoB mRNA editing from these mice is that serum ApoB100 levels increase and the regulation of plasma cholesterol becomes critically dependent on the expression of hepatic and extrahepatic LDLRs for ApoB-containing lipoprotein particle uptake. When Apobec-1−/− mice were crossed into Ldlr−/− mice, the compound double-knockout animals represented a phenocopy of homozygous familial hypercholesterolemia, with plasma cholesterol levels of more than 400 mg/dl while consuming a low-fat chow diet (182). These animals experienced extensive atherosclerosis without dietary manipulation as a result of the augmented production and reduced clearance of ApoB100. Thus, a further consequence of ApoB mRNA editing is the production of lipoprotein particles that are intrinsically less atherogenic than those containing ApoB100. It bears emphasis, however, that the role of intestinal versus hepatic ApoB100 production in this setting has yet to be delineated.

OTHER GENES INVOLVED IN INTESTINAL LIPOPROTEIN BIOGENESIS: APOLIPOPROTEINS A-I AND A-IV ApoB is an obligate component for the assembly of triglyceride-rich lipoproteins in both the liver and the small intestine. ApoB is unique among the apolipoprotein family in that it does not exchange between lipoprotein particles; once assembled into a lipoprotein particle, ApoB remains physically associated with the lipid core during its intracellular

1724 / CHAPTER 67 maturation and secretion and throughout its journey within the plasma compartment and uptake at the cell membrane using either receptor-dependent or -independent pathways. Two other apolipoproteins are expressed as abundant species (>1% total mRNA) in the mammalian small intestine, namely, ApoA-I and ApoA-IV. The small intestine represents a major site of ApoA-I and ApoA-IV production in most mammals, whereas in humans, ApoA-IV is virtually only expressed in the small intestine. ApoA-I and ApoA-IV, in comparison with ApoB, are freely exchangeable between different lipoprotein classes, principally HDL and chylomicron particles. In the plasma compartment, ApoA-I is the major apoprotein in HDL. ApoA-IV also exists in the plasma compartment in both a lipoprotein-associated (chylomicron and HDL) and -unassociated (i.e., free) pool, in approximately equal proportions (183). Thus, beyond their role in the assembly and secretion of intestinal lipoproteins, ApoA-I and ApoA-IV have an important role in the regulation of systemic lipoprotein metabolism, particularly the regulation of HDL formation and catabolism. The functional role, if any, for ApoA-I in intestinal lipoprotein assembly is unknown. Studies have yet to be reported in ApoaI− /− mice to examine triglyceride absorption and normal production of chylomicrons. However, cholesterol absorption was increased in ApoaI− /− mice compared with wild-type control mice (184). ApoA-IV, in contrast, has been linked to triglyceride absorption through several indirect observations. These include the early findings that intestinal ApoA-IV mRNA and protein expression is increased, and that lymphatic secretion increases after triglyceride absorption (185,186). In addition, serum ApoA-IV levels have been reported to be decreased in subjects with ABL (183). In contrast, intestinal triglyceride absorption and chylomicron production were completely unimpaired in both ApoaIV− /− mice (187) and intestinal-specific transgenic ApoA-IV overexpressing mice (188), suggesting that the role of ApoA-IV may be permissive rather than required. In other studies, Lu and colleagues (189) have demonstrated that forced adeno-associated viral expression of ApoA-IV in a porcine intestinal cell line enhanced chylomicron formation and triglyceride secretion, independent of other genes involved in lipid transport (including MTTP and ApoB), suggesting that ApoA-IV may augment intestinal lipoprotein assembly and secretion under certain circumstances. Whether the findings in this porcine cell line are applicable to other species in vivo remains unknown. Studies by Gallagher and colleagues (190) have further illuminated this area by demonstrating a significant protein–protein interaction between ApoA-IV and a region in the amino terminus of ApoB, which perturbs the intracellular trafficking and secretion of ApoB. Among the important findings from this study were that ApoA-IV physically interacts with a domain between ApoB21 and ApoB25, and that this interaction is independent of MTTP (190). Expression of ApoA-IV with a carboxyl-terminus KDEL sequence (which results in retention within the ER) produced ~80% suppression of ApoB secretion from COS (African green monkey derived cell line transformed with SV40)

cells, suggesting that the interaction of ApoB with ApoA-IV delays transit of ApoB-containing lipoproteins through the distal elements of the secretory pathway, perhaps permitting greater opportunity for the nascent lipoproteins to acquire more core lipid, resulting in particle expansion. The findings are broadly consistent with a permissive role for ApoA-IV in modulating chylomicron formation and suggest some important opportunities for experimental validation. As alluded to earlier, ApoA-I has not been shown to play a requisite role in the assembly and secretion of intestinal triglyceride-rich lipoproteins. Nevertheless, ApoA-I plays a key role in cellular cholesterol mobilization, through the actions of efflux pump adenosine triphosphate (ATP)– binding cassette A1 (ABCA1). The prototype example of this function is in Tangier disease, which is a rare autosomal recessive disease associated with cholesterol accumulation in the intestine, spleen, and tonsils in association with virtually no detectable HDLs (191,192). The gene defect in Tangier disease is accounted for by mutations in ABCA1 (193–196), the result of which is defective delivery of membrane cholesterol and phospholipids to an ApoA-I acceptor particle. Under physiologic circumstances, cholesterol and phospholipids transferred to an ApoA-I acceptor would result in the formation of discoidal HDL (Fig. 67-3), followed by esterification of cholesterol through the actions of lecithin cholesterol acyltransferase, which generates spherical HDL containing a core of cholesterol ester. In contrast, as a result of the defective lipidation of ApoA-I and the absence of HDLs in Tangier disease, ApoA-I levels are extremely low because the free protein is cleared rapidly by renal filtration. Despite an important role of ABCA1 in cellular cholesterol mobilization into HDL particles, ABCA1 is not considered a major player in the regulation of cholesterol absorption (see detailed discussion in the following section).

MAJOR PATHWAYS AND GENES INVOLVED IN INTESTINAL STEROL TRANSPORT The small intestine represents a major portal of entry of cholesterol into the body and a barrier to the unrestricted absorption of cholesterol and other related sterols. Mammalian cholesterol homeostasis is tightly regulated, with input from absorption and de novo synthesis being balanced by daily losses through biliary excretion and bile salt synthesis. Although it has been recognized for decades that cholesterol absorption is a regulated process, details of the molecular mechanisms and pathways involved remained obscure until independent work in two distinct areas, namely, the genetics of sitosterolemia and the cloning of the transporter NPC1L1 (197), helped to resolve key pieces of this mystery. As illustrated in Figure 67-3, the sterol products of micellar lipid delivery traverse the microvillus membrane via the NPC1L1 transporter. These sterol products include cholesterol, as well as plant sterols (sitosterol, campesterol, sitostanol, and others) and shellfish sterols (e.g., brassicasterol), which are then selectively metabolized

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1725

FIG. 67-3. Pathways and transporter molecules regulating intestinal cholesterol (CHOL) transport and metabolism. Lumenal cholesterol and other sterols (including plant and shellfish sterols) are presented to the enterocyte brush-border membrane in the form of mixed micelles (M). Free sterol is transported across the brush border via NPC1L1, a 13-transmembrane protein with a sterol-sensing domain. After uptake, cholesterol is transported to the endoplasmic reticulum where it undergoes esterification to cholesterol ester (CE) through the actions of acyl coenzyme A cholesterol acyltransferase 2 (ACAT2). Sitosterol (SITOST) and other plant sterols are less effective substrates for ACAT2 and are transported back into the intestinal lumen through the paired half transporters ABCG5/G8. Intestinal cholesterol also is secreted back into the lumen through ABCG5/G8. Other apical transporters have been described, including scavenger receptor class B type I (SR-BI) and CD36. The significance of these two transporters in the absorption of cholesterol and other sterols, however, has yet to be established. Intestinal cholesterol arises through de novo synthesis, from uptake via lowdensity lipoprotein receptors (LDLR), high-density lipoprotein (HDL) receptors (including SR-BI), and through mobilization of membrane free cholesterol (FC). Intracellular free cholesterol arising through these sources may enter the metabolic pool from which ACAT2 derives its substrate. In addition, membrane free cholesterol may be mobilized together with phospholipid through the actions of ABCA1, resulting in transfer to extracellular ApoA-I and the formation of discoidal HDL particles that enter the lymphatic circulation. ABC, adenosine triphosphate–binding cassette; TG, triglyceride.

to their sterol esters through the actions of acyl-coenzyme A cholesterol acyltransferase 2 (ACAT2) (69). Sitosterol and other sterols that are ineffective substrates for ACAT2 (see Fig. 67-3) are directed to the efflux pump ABCG5/G8 for resecretion back into the lumen. This includes at least some of the excess intracellular free cholesterol (see Fig. 67-3, broken line). The bulk of newly absorbed cholesterol is delivered for esterification and incorporation into the core of nascent intracellular chylomicron particles. Additional intracellular free cholesterol may be mobilized for secretion through the actions of the basolateral efflux pump ABC1A1

(see Fig. 67-3), the coordinated actions of which allow the transfer of membrane cholesterol to an ApoA-I acceptor particle resulting in the production of HDL particles (193–196,198). ABCA1 was initially postulated to play a role in regulating intestinal cholesterol absorption, but this appears unlikely based on the results of targeted deletion of the murine ABCA1 gene, which demonstrated no change from the wild type (199). Another cholesterol transporter, the HDL receptor scavenger receptor class B type I (SR-BI), was considered a candidate gene in regulating intestinal cholesterol uptake by virtue of its known functions in selective

1726 / CHAPTER 67 sterol uptake in peripheral cells and also from its expression on both the apical and basolateral membrane of intestinal enterocytes (200,201). However, SR-BI−/− mice demonstrated no change in intestinal cholesterol absorption from their wild-type control mice (202). Another receptor, the multifunctional protein CD36, also is expressed on both the microvillous membrane and the basolateral membrane of mammalian enterocytes (203). CD36−/− mice, however, exhibited no alteration in intestinal cholesterol absorption compared with wild-type control mice. Although the cumulative evidence does not preclude a role for either SR-BI or CD36 in regulating cholesterol absorption, the evidence suggests that neither gene plays a major independent role in this regard. The current view (see summary in the next section) is that intestinal cholesterol absorption is regulated through dominant pathways that are regulated by ABCG5/G8 and by NPC1L1 (see Fig. 67-3).

Sitosterolemia and ABCG5/G8 As alluded to earlier, a breakthrough in the understanding of intestinal cholesterol absorption emerged from the cloning and characterization of two novel ABC transporter members, ABCG5/G8 (see Fig. 67-3). Two groups independently identified mutations in this coupled transporter as the genetic basis for the disease sitosterolemia, a condition in which there is indiscriminate hyperabsorption of plant sterols, as well as cholesterol (204,205). In one approach, Berge and colleagues (204) capitalized on the observation that treatment of mice with a chemical agonist of the orphan nuclear receptor liver X receptor (LXR) resulted in a striking decrease in cholesterol absorption, together with an increase in expression of another ABC-type cholesterol efflux/transporter, ABCA1 (see Fig. 67-3), which previously had been suspected of playing a role in regulating cholesterol absorption (206). Berge and colleagues (204) then used microarray analysis of intestinal RNA from LXR agonist–treated mice to identify other ABC-type transporters and discovered the paired halftransporter complex ABCG5/G8 and demonstrated that their mRNA was coordinately expressed in liver and intestine, the target organs suspected of orchestrating the metabolic response to altered cholesterol intake. In a separate approach, Patel and colleagues (207) independently narrowed the putative locus for sitosterolemia using linkage analysis to chromosome 2p21 and subsequently mapped a series of expressed sequence tags and candidate genes within the region of interest, one of which was expressed in the intestine and encoded ABCG5 (205). Both groups then identified a range of mutations in the human ABCG5/G8 genes of patients with sitosterolemia, establishing a molecular genetic basis for this disease. The genes for ABCG5/G8 are organized in a head-to-head (i.e., 3′ to 5′ and 5′ to 3′) configuration, resulting in transcription of genes in the opposite orientation that are coordinately regulated by a shared promoter (204). Each gene generates a half transporter with six-transmembrane–spanning domains.

Neither ABCG5 nor ABCG8 alone is capable of assembling a functional transporter. Homodimeric complexes composed of either ABCG5 or ABCG8 have not been demonstrated under normal physiologic circumstances (208), and presumably, if they exist at all, would be confined to the ER. Instead, the functional transporter is normally composed of an obligate 1:1 heterodimeric complex of ABCG5 and ABCG8, resulting in a 12-membrane–spanning duplex protein that is inserted into the brush-border membrane (208). This heterodimeric protein complex assembles in the ER and undergoes inefficient yet directed transport to the microvillous membrane of the enterocyte and also to the canalicular membrane of hepatocytes and the apical membrane of cells lining the gallbladder (209). Further study has shown that the majority of mutations in ABCG5 and ABCG8 result in a failure of the transporter to be transported to the apical surface of the cell; consequently, the mutant proteins are retained in the ER (208). Mutations have been demonstrated throughout the locus spanning ABCG5 and ABCG8, the presumption being that mutations in either half transporter cripple the ability of the mutant protein to form heterodimers with the other partner. Both ABCG5 and ABCG8 have potential N-linked glycosylation sites in their predicted extracellular domain (see Fig. 67-3), and these have been established to play an important role in the folding of the proteins, their interaction with chaperone proteins (calnexin), and their heterodimerization (208). Notwithstanding the demonstration that ABCG5/G8 is transported to the microvillous membrane of the enterocyte as a heterodimeric complex, it bears emphasis that a mouse ABCG8 knockout, generated by Klett and colleagues (209,210), demonstrated expression of ABCG5 at the enterocyte brush-border membrane in a distribution no different from that observed in wild-type mice. These findings raise the possibility that transport of the individual components of the duplex transporter to their eventual destination may occur. However, reconstitution of a functional sterol transporter likely requires expression of both half transporters in an appropriate stoichiometry. Role of ABCG5/G8 in Modulating Cholesterol Absorption The prevailing view is that, similar to other ABC-type transporters, ABCG5/G8 functions in the regulated efflux of sterols from enterocytes and other polarized epithelial cells in which this tandem gene duplex is expressed. In its role in regulating intestinal cholesterol absorption, the most plausible function for this transporter is the regulated efflux of sterol from the cell back into the intestinal lumen. Accordingly, one likely scenario is that, in response to intestinal uptake of dietary sterols or oxysterol derivatives, sterol signals are transmitted through the orphan nuclear receptors LXR α (206,211,212) and LRH-1 (213), resulting in transcriptional up-regulation of the ABCG5/G8 promoter and increased expression of the functional transporter at the enterocyte villous brush-border membrane. Increased expression of the transporter results in an increase in sterol efflux,

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1727 thereby effectively pumping increased amounts of sterol (preferentially plant sterols together with cholesterol) from the cell to the lumen (see Fig. 67-3). This model is consistent with the observations that ABCG5/G8 mRNA and protein expression are increased in mouse intestine after increased dietary cholesterol intake (204). Further confirmation of this general concept was established in experiments where the ABCG5/G8 gene was disrupted by homologous recombination, resulting in a 3-fold fractional increase in plant sterol absorption (cholestanol, campesterol, and sitosterol) coupled with a 30-fold increase in plasma sitosterol levels (214). Finally, variance in the relative expression levels of ABCG5 and ABCG8 appeared to correlate strongly with the fractional cholesterol absorption noted in different strains of inbred mice (215). Taken together, a strong case can be made that ABCG5/G8 is one of the central players in physiologic regulation of intestinal cholesterol and plant sterol absorption by modulating selective efflux. An alternative scenario has been proposed for understanding the mechanism of regulating cholesterol absorption and the role of ABCG5/G8 (216). A study examined the effects of stanol esters in modulating cholesterol absorption in hamsters. Stanol esters have been proposed to reduce cholesterol absorption by a variety of mechanisms, including competing with cholesterol for micellar solubilization, and by selective up-regulation of the efflux transporters in enterocytes. This work, however, demonstrated that intestinal expression of ABCG5/G8 transporter mRNA species decreased after stanol ester supplementation, in contrast with the expected increase in expression (216). These authors argue that decreased intestinal cholesterol flux (through mechanisms that were not identified) led to a decrease in expression of the ABCG5/G8 transporter mRNA. Whether such an adaptive response occurs in human intestine is currently unknown, although the question is certainly relevant because of the widespread use of plant stanol supplementation in commercial oils and fats consumed by Western populations. Regulation of plant sterol absorption has attracted considerable attention since the identification of the gene responsible for sitosterolemia and the knowledge that patients with this disease have accelerated atherosclerosis in association with increased serum levels of cholesterol and plant sterols (217). However, studies in ABCG5/G8 knockout mice crossed into hypercholesterolemic LDLR knockout mice showed no incremental increase in serum cholesterol levels in the compound knockout mice (i.e., LDLR−/−, ABCG5/G8−/−) compared with LDLR−/− mice and no increase in atherosclerosis despite 20-fold greater levels of plant sterols (217). These findings suggest that increased serum levels of plant sterols are not intrinsically more proatherogenic than cholesterol. In addition, there was no evidence in human subjects for an association of increased plant sterol levels with coronary artery calcium deposits, a surrogate marker of atherosclerosis development (217). This latter finding was somewhat unexpected given the information from subjects with sitosterolemia, where a strong correlation exists between increased plant sterol levels and premature coronary atherosclerosis.

Nevertheless, subjects with sitosterolemia are also hypercholesterolemic, suggesting that the augmented cholesterol absorption and increased plasma cholesterol levels, perhaps in combination with increased plant sterol levels, contributes to the increased propensity to vascular disease. Accordingly, these findings collectively suggest that plant sterol supplementation in the diet of healthy subjects is unlikely to add appreciable risk for atherosclerosis even if these sterols were absorbed in significant quantity.

Identification of NPC1L1, the Putative Intestinal Cholesterol Transporter A second major breakthrough in the understanding of intestinal cholesterol absorption emerged recently from a search for targets of a therapeutic agent (ezetimibe), a competitive inhibitor of cholesterol absorption that is used in the drug treatment of patients with hypercholesterolemia. Ezetimibe, an azetidinone derivative, was demonstrated to inhibit intestinal cholesterol uptake in both experimental animals and humans, and its presumed mechanism of action is via binding to a brush-border membrane cholesterol transporter (197,218,219). Using a bioinformatics approach coupled with analysis of expressed sequence tags from intestinal cDNA libraries, a single candidate gene was identified that fulfilled the criteria of a putative intestinal cholesterol transporter (197). The candidate gene, NPC1L1, has ~50% amino acid homology to the Niemann-Pick type C (NPC) transporter (197). The protein has 13 predicted transmembrane domains (see Fig. 67-3) and a sterol-sensing domain similar to domains present in other sterol-regulated gene products including sterol regulatory element-binding protein/cleavageactivating protein and Niemann-Pick type C (197). The sterol-sensing domain facilitates structural and conformational alterations in response to alterations in the ambient sterol concentration, and thereby mediates metabolic signaling. This particular characteristic, namely, the ability to respond to alterations in sterol flux, would be anticipated from a membrane cholesterol transporter in the small intestine and likely increased the stringency of the authors’ interrogation of the databases in yielding NPC1L1. NPC1L1 Function in Intestinal Cholesterol Absorption Endogenous NPC1L1 mRNA is expressed in murine small intestine and at much lower levels in the liver. Within the small intestine, NPC1L1 mRNA is expressed predominantly in the jejunum and is confined to the enterocyte compartment (197). In contrast, human NPC1L1 mRNA is expressed at equivalent levels in the small intestine and liver, as well as in the stomach, ovary, lung, and muscle, a distribution not immediately accountable for by its presumed function in sterol uptake (197). The function of this transporter in these extrahepatic and extraintestinal sites requires further investigation. Further evidence supporting a central role of NPC1L1 in regulating intestinal cholesterol uptake is the finding that

1728 / CHAPTER 67 NPC1L1−/− mice demonstrated a 69% reduction in cholesterol uptake (197), similar to the reduction observed after treatment of wild-type mice with ezetimibe. However, the target of ezetimibe has yet to be determined definitively. It would be interesting to examine the binding of ezetimibe to microvillous membrane extracts prepared from wild-type and NPC1L1−/− mice to compare binding affinity in the absence of at least one putative cholesterol transporter. In further studies, Davis and colleagues (220) demonstrated a similar decrease in both sitosterol and cholesterol absorption in NPC1L1−/− mice, suggesting that initial sterol uptake by this transporter does not discriminate between closely related sterols. It will be of interest to examine the range of sterols transported by NPC1L1, but the current data suggest that NPC1L1 represents a common portal of entry for most dietary sterols. Intestinal triglyceride absorption, in contrast, was completely normal in NPC1L1−/− mice, suggesting that this transporter has no function in the uptake of dietary long-chain fatty acids. These findings are consistent with the observations on patients treated with ezetimibe, in whom there has been no evidence of intestinal triglyceride malabsorption. In response to augmented intestinal cholesterol uptake, after 7 days of dietary manipulation with increased cholesterol and cholic acid, wild-type mice responded with a 75% decrease in the abundance of intestinal NPC1L1 mRNA (220), suggesting that expression of this cholesterol transporter is subject to negative feedback regulation in response to cholesterol uptake. In contrast, NPC1L1−/− mice demonstrated an increase in the mRNA encoding 3-hydroxy-3-methylglutaryl coenzyme A synthase, one of the rate-limiting enzymes in cholesterol synthesis, suggesting that the reduced intestinal cholesterol absorption in NPC1L1−/− mice in turn stimulated a compensatory increase in endogenous cholesterol synthesis (220). Taken together, the available evidence strongly suggests that NPC1L1 functions as a sterol transporter in the intestine, with activity toward cholesterol and other sterols. After uptake into the enterocyte, cholesterol enters a metabolic pool that is regulated by ACAT2, whereas sitosterol and other plant sterols that are inefficient substrates for ACAT2 undergo efflux through ABCG5/G8.

Acyl-Coenzyme A Cholesterol Acyltransferase 2 and the Regulation of Intestinal Cholesterol Absorption Cellular cholesterol esterification is accomplished by two enzymes: ACAT1, which is widely distributed, but expressed at low levels in the liver and intestine; and ACAT2, which is the enzyme responsible for cholesterol esterification in these tissues (69). To examine the role of ACAT2 in regulating intestinal cholesterol absorption, Repa and colleagues (221) generated ACAT2−/− mice and demonstrated reduced (but not eliminated) cholesterol absorption, particularly in the setting of increased dietary cholesterol intake. In addition, the reduction in NPC1L1 mRNA seen in wild-type animals after cholesterol feeding was amplified in ACAT2−/− mice, suggesting that the accumulation of free intracellular

intestinal cholesterol further down-regulated NPC1L1 expression beyond the effects noted in wild-type mice. In addition, these workers demonstrated an increase in ABCG5/G8 mRNA abundance in chow-fed ACAT2−/− mice, but no further increase after cholesterol supplementation, suggesting that the regulation of ABCG5/G8 expression in response to sterol supplementation involves ACAT2. A striking increase was noted in the expression of the basolateral efflux transporter ABCA1 in chow-fed ACAT2−/− mice, and a further increase in ABCA1 mRNA abundance was noted in ACAT2−/− mice fed a high-cholesterol diet. These data suggest that free cholesterol accumulation in enterocytes in response to ACAT2 deficiency leads to up-regulation of basolateral cholesterol efflux mechanisms, although the form of the lipoprotein particles associated with this augmented cholesterol secretion have yet to be identified. However, based on current information, the most likely candidate would be HDL particles formed by the actions of ABCA1 and ApoA-I (see Fig. 67-3).

OTHER GENETIC DEFECTS OF INTESTINAL LIPOPROTEIN ASSEMBLY AND SECRETION AND POTENTIAL NEW PATHWAYS Transport of chylomicrons through the distal elements of the ER to the Golgi compartment is a regulated step that requires ER budding and the formation of transport vesicles containing COPII proteins (75). Insight into the mechanisms regulating this pathway emerged from studies of patients with defective intestinal chylomicron secretion associated with three related syndromes, namely, Anderson’s disease, chylomicron retention disease, and chylomicron retention disease associated with Marinesco–Sjögren syndrome. This group of diseases is characterized by shared features including presentation in infancy as failure to thrive, which is associated with fat malabsorption, low-serum cholesterol, and absence of chylomicrons and ApoB48. Importantly, and in contrast with the phenotype encountered in subjects with ABL, subjects with Anderson’s disease/chylomicron retention disease have normal plasma levels of ApoB100 and no hepatic steatosis. Jones and colleagues (77) studied 12 patients from 8 families, narrowing a locus to chromosome 5q31.1, within which all of the probands were found to have mutations in the SARA2 gene, encoding the Sar1b protein. Humans express two isoforms of Sar1 (Sar1 and Sar1b) that differ by 20 residues. The process of ER budding is dependent on the activation of Sar1 through the presence of guanosine triphosphate (GTP) in the binding pocket, which results in a conformational change that facilitates membrane binding and interaction with the COPII heterodimer Sec23/24. Binding of the Sec23/24 heterodimer subsequently recruits additional COPII proteins including Sec13/31, which associate with the prechylomicron transport vesicles in the budding complex (74). All the mutations described in the Anderson’s disease/chylomicron retention disease probands are predicted to disrupt the GTP binding pocket of Sar1b,

GENETIC REGULATION OF INTESTINAL LIPID TRANSPORT AND METABOLISM / 1729 thus leading to a failure of ER budding and retention of prechylomicrons within profiles of the ER membrane. These predictions are consistent with the phenotype observed in these patients, where large triglyceride droplets are demonstrated within the ER, with no lipoprotein particles visible in the Golgi (77). Another pathway for intestinal lipoprotein assembly and secretion appears to involve phosphatidylinositol transfer proteins (PITPs), of which evidence has implicated one, PITPα, in intestinal chylomicron secretion. Studying mice with targeted deletion of the PITPα gene, Alb and colleagues (222) demonstrated early neonatal death in 40% and failure to thrive with intestinal and hepatic steatosis in the remainder. The intestinal steatosis was accompanied by accumulation of large multilobed lipid droplets within the ER, together with decreased plasma postprandial triglyceride levels (222). These data are reminiscent of the intestinal phenotype observed with Anderson’s disease/chylomicron retention disease, although it is important to emphasize that hepatic steatosis is not a feature of these latter patients. The data therefore implicate PITPα in the transport of chylomicrons from the ER, perhaps at the stage of budding of the ER into transport vesicles. Further study is needed to discern the mechanism involved and to understand the accompanying defect in hepatocytes.

ACKNOWLEDGMENTS This work was supported by grants HL-38180, DK-56260, DK-52574 (N.O.D.), and HL-73939 (Z.C.).

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CHAPTER

68

Digestion and Intestinal Absorption of Dietary Carotenoids and Vitamin A Alexandrine During and Earl H. Harrison Carotenoid and Vitamin A Metabolism: Overview, 1735 Dietary Sources and Forms, 1737 Solubilization of Carotenoids and Retinoids, 1738 Conversion of Provitamin A Carotenoids to Retinoids, 1739 Carotene Cleavage Enzymes, 1739 Regulation by Nutritional Factors, 1740 Digestion of Retinyl Esters, 1740 Pancreatic Enzymes, 1740 Intestinal Enzymes, 1741 Intestinal Absorption of Carotenoids, 1741 Kinetics of β-Carotene Transport through Intestinal Cells, 1742 Selective Uptake of All-Trans β-Carotene versus Its Cis Isomers by Intestinal Cells, 1743

Differential Intestinal Transport of Individual Carotenoids, 1743 Carotenoid Interactions during Intestinal Absorption, 1744 Intestinal Absorption of Vitamin A, 1744 Mechanisms of Cellular Uptake and Efflux of Unesterified Retinol in Enterocytes: Portal Secretion, 1744 Intracellular Transport by Cellular Retinol Binding Protein (II), 1745 Reesterification and Incorporation into Chylomicrons: Lymphatic Secretion, 1746 Summary, 1747 References, 1748

Vitamin A deficiency affects more than 100 million children throughout the world (1,2). Thus, knowledge about the mechanisms of absorption of vitamin A can lead to better approaches for enhancing its absorption and could be helpful in ameliorating some of the deficiencies. The major sources of vitamin A in human diet are the provitamin A carotenoids in fruits and vegetables and retinyl esters found in foods of animal origin. In humans, carotenoids are either cleaved to generate retinol or absorbed intact. In contrast, retinyl esters are completely hydrolyzed in the intestinal lumen, and free retinol is taken up by enterocytes. The intestinal absorption

and metabolism of dietary carotenoids has been the subject of several reviews (3–5), as have the biochemical and molecular mechanisms involved in the digestion and absorption of vitamin A (6).

A. During: Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205. E. H. Harrison: Phytonutrients Laboratory, United States Department of Agriculture Human Nutrition Research Center, Beltsville, Maryland 20705. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

CAROTENOID AND VITAMIN A METABOLISM: OVERVIEW Carotenoids are synthesized in plants and in certain microorganisms such as some bacteria, algae, and fungi. They are a group of pigments that are widespread in nature and are responsible for the yellow/orange/red/purple colors of many fruits, flowers, birds, insects, and marine animals. More than 600 carotenoids have been isolated from natural sources, but only ~60 of them are detected in the human diet (7) and ~20 of them in human blood and tissues. β-Carotene, α-carotene, lycopene, lutein, and β-cryptoxanthin are the five most prominent carotenoids present in the human body. All carotenoids are derived from the basic linear polyisoprenoid structure of lycopene that contains 40 carbon atoms

1735

1736 / CHAPTER 68 and an extended system of 13 conjugated double bonds. Carotenoids derive from this parent structure by cyclization (i.e., formation of β- or ε-ionone rings) at one (i.e., γ-carotene) or two ends (i.e., β-carotene and α-carotene) of the polyene chain and by dehydrogenation, oxidation, or both. Figure 68-1 shows the structures of several major carotenoids. The carotenoid group is divided into the carotenes, hydrocarbon carotenoids with unsubstituted rings, and the xanthophylls, which are carotenoids with at least one oxygen atom. They exist mostly in the all-trans configuration, but they can be subject to a cis isomerization at any double bond of their polyene chain, resulting in a large number of mono- and poly-cis isomers (8). Capability for the de novo synthesis of compounds with vitamin A activity is limited to plants and microorganisms (9,10). Thus, higher animals must obtain vitamin A from the diet, either as the preformed vitamin or as a provitamin carotenoid such as β-carotene. In the intestinal mucosa, β-carotene is converted to retinal by β-carotene 15,15′ oxygenase (BCO), and the retinal is then reduced to retinol by a retinal reductase (11). In the human intestine, about half the dietary provitamin A carotenoids are converted to retinol and about half are absorbed intact (12). The major dietary forms of preformed vitamin A are long-chain fatty acid esters of retinol (13). These esters must be hydrolyzed before intestinal absorption. Hydrolysis of the esters is catalyzed both by enzymes secreted by the pancreas into the intestinal lumen and by those associated directly with intestinal cells.

After the hydrolysis of dietary retinyl esters, the free retinol is then taken up by the mucosal cell (14). The free retinol, resulting either from hydrolysis of dietary retinyl esters or conversion of dietary provitamin A carotenoids, is reesterified with long-chain, mainly saturated, fatty acids by the enzyme lecithin:retinol acyltransferase (LRAT), which is membrane bound (15,16). The resulting retinyl esters are incorporated with other neutral lipid esters (i.e., triacylglycerols and cholesteryl esters) and intact carotenoids into chylomicrons (CMs) and absorbed via the lymphatics (17,18). In the vascular compartment, much of the CM triacylglycerol is hydrolyzed by lipoprotein lipase in extrahepatic tissues, resulting in the production of a “chylomicron remnant” that contains most of the newly absorbed retinyl esters (19,20). In the rat, the CM remnants are rapidly and almost quantitatively taken up by the liver, and there is evidence that the retinyl esters are rapidly hydrolyzed and reesterified during this process (21–23). Under conditions of adequate vitamin A nutriture, the liver is the main site of vitamin A storage, with more than 95% of the total neutral retinoid being present as retinyl esters, predominately retinyl palmitate and stearate (24–27). Although CM remnants (and the retinyl esters they contain) are initially taken up exclusively by the hepatocytes in the liver, the retinyl esters are then transferred largely to the perisinusoidal stellate cells (21,28). In vitamin A–adequate rats, the stellate cells account for approximately 80% of the total retinyl ester store, with the remainder being in

Lycopene OH

HO

all-trans β-Carotene

Zeaxanthin OH

HO

α-Carotene

Lutein

O

OH

O

β-Cryptoxanthin

Canthaxanthin 15

15 15′

15′

9′

13-cis β-Carotene 9-cis β-Carotene

γ-Carotene

FIG. 68-1. Structures of various carotenoids.

13′

DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1737 hepatocytes (28–30). In both cell types, the retinyl esters are stored in cytoplasmic lipid droplets together with other neutral lipids. Before mobilization from the liver, the retinyl esters are hydrolyzed, and free retinol is complexed to serum retinol-binding protein for secretion from the liver (31). Unesterified retinol is delivered to peripheral tissues where it is converted to biologically active metabolites such as 11-cisretinal, the chromophore for the visual pigment rhodopsin, or to retinoic acids (RAs), the ligands for two families of hormone-dependent transcription factors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Thus, vitamin A is essential for vision and for cell differentiation and development in vertebrates including humans. Figure 68-2 shows the structures of the major dietary retinoids and their metabolites and outlines their functions. Several epidemiologic studies have reported that consumption of carotenoid-rich foods is associated with a reduced risk for certain cancers, cardiovascular disease, and agerelated macular degeneration (32–35). These preventive effects of carotenoids could be related to their major function as vitamin A precursors and/or their actions as antioxidants, modulators of the immune response, and inducers of gap-junction communications (36). All carotenoids do not have a similar protective effect against a specific disease.

DIETARY SOURCES AND FORMS As mentioned earlier, vitamin A activity in the diet derives from two sources: preformed vitamin A as retinyl esters in foods of animal origin and provitamin A carotenoids, such as β-carotene, α-carotene, and β-cryptoxanthin, found in

plant-derived foods. Stoichiometric conversion of 1 mol β-carotene (with two β-ionone rings) would yield 2 mol retinol (via retinal), whereas conversion of 1 mol of either β-cryptoxanthin or α-carotene (each with only a single β-ionone ring) would yield a single mole of retinol. β-Carotene is the most potent vitamin A precursor of all provitamin A carotenoids. The carotenoid molecule must have at least one unsubstituted β-ionone ring and the correct number and position of methyl groups in the polyene chain to exhibit a provitamin A activity (37). In practice, α-carotene, β-cryptoxanthin, and γ-carotene show 30% to 50% of provitamin A activity (38,39) and 9-cis and 13-cis isomers of β-carotene less than 10% (40) compared with all-trans β-carotene. In the human diet, plant food sources are the major contributors of carotenoids: carrots, squash, and dark green leafy vegetables for β-carotene; carrots for α-carotene; tomatoes and watermelon for lycopene; kale, peas, spinach, and broccoli for lutein; and sweet red peppers, oranges, and papaya for β-cryptoxanthin. Foods in the U.S. diet with the greatest concentrations of preformed vitamin A are avian and mammalian livers (4–20 mg retinol/100 g), instant powdered breakfast drinks (3–6 mg/100 g), ready-to-eat cereals (0.7–1.5 mg/100g), and margarines (about 0.8 mg/100 g) (41). Except for liver, these sources derive their high retinyl ester contents from fortification. The greatest concentrations of vitamin A as provitamin A carotenoids are found in carrots, sweet potatoes, pumpkin, kale, spinach, collards, and squash (roughly 5–10 mg retinol activity equivalents per 100 g) (41). A retinol activity equivalent (RAE) is equal to 1 µg retinol, 12 µg β-carotene, or 24 µg of α-carotene or β-cryptoxanthin (42). Analysis of

Functions of retinoids Nervous system Epithelia Bone

Others??

Immune system Skin

Differentiation

Vision

Retinoids

Reproduction

Chemical structures of the most common retinoids O OH Retinol

OH

O Retinal

Retinoic acid

O O O Retinyl palmitate

11-cis -retinal

O

OH

9-cis -retinoic acid

FIG. 68-2. Structures of the major retinoids and physiologic functions of the retinoids.

1738 / CHAPTER 68 the NHANES 2000 data for food consumption in the United States shows that the majors contributors to the intake of preformed vitamin A are milk, margarine, eggs, beef liver, and ready-to-eat cereals, whereas the major sources of provitamin A carotenoids are carrots, cantaloupes, sweet potatoes, and spinach. Analysis of NHANES data (43), for both sexes and all age groups, showed that the mean intake of vitamin A in the United States was about 600 µg RAE/day from food, and that 70% to 75% of this was as preformed vitamin A (retinol). The provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin were ingested in amounts of approximately 1750, 350, and 150 µg/day, respectively. The intakes of the nonprovitamin A lycopene and the xanthophylls (zeaxanthin + lutein) were about 6000 and 1300 µg/day, respectively.

SOLUBILIZATION OF CAROTENOIDS AND RETINOIDS Carotenoids are hydrophobic molecules, and thus are located in lipophilic sites of cells, such as bilayer membranes. Their hydrophobic character is decreased with an increased number of polar substituents (mainly hydroxyl groups free or esterified with glycosides), thus affecting the positioning of the carotenoid molecule in biological membranes. For example, the dihydroxy carotenoids such as lutein and zeaxanthin orient themselves perpendicular to the membrane surface as a “molecular rivet” to expose their hydroxyl groups to a more polar environment. In contrast, the carotenes such as β-carotene and lycopene could position themselves parallel to the membrane surface to remain in a more lipophilic environment in the inner core of the bilayer membranes (8,44). Thus, carotenoid molecules can have substantial effects on the thickness, strength, and fluidity of membranes, and thus affect many of their functions. To move through an aqueous environment, carotenoids can form complexes with proteins. For example, the ketocarotenoids (i.e., canthaxanthin and astaxanthin) interact with proteins by formation of Schiff’s bases between their keto groups and specific lysine residues of the proteins, whereas the other carotenoids (e.g., the carotenes) form mostly hydrophobic interactions in amphipathic areas of the proteins or with the lipid components of lipoproteins. Specific carotenoid–protein complexes have been reported mainly in plants and in invertebrates (e.g., cyanobacteria, crustaceans, and silkworm) (45–47). In vertebrates, data on the existence of carotenoproteins are limited. Although no intracellular β-carotene–binding protein was found in bovine liver and intestine (48), a cellular carotenoid-binding protein with a high specificity for the carotenes was reported in ferret liver (49) and a specific xanthophyll-binding protein was reported in the human retina and macula (50). As an alternative mechanism for their water solubilization, carotenoids could use small cytosolic carrier vesicles (48). In nature, carotenoids can be also present in fine physical dispersions (or crystalline aggregates) in aqueous media;

oranges, tomatoes, and carrots are well-known examples of sources that contain such aggregates (51). These different physicochemical characteristics—that is, chemical structure, positioning in biological membranes, and interaction with proteins—may account for the differences observed among individual carotenoids in their absorption and metabolism, as well as their biological activities. Retinoids are also lipophilic molecules that require solubilization. Unesterified retinol exerts detergent-like properties on cellular membranes and usually is sequestered in cells by a variety of retinol-binding proteins (see later for a more detailed discussion). The very nonpolar retinyl esters usually are found in cells in stabilized lipid droplets and emulsions. The digestion of retinyl esters and the conversion of carotenoids to retinoids require catalysis by enzymes that use these water-insoluble substrates. There is the involvement of a lipid–water interface in the catalytic process and the presence of heterogeneous phases: an aqueous phase with the water-soluble enzyme (e.g., BCO or various retinyl ester hydrolases [REHs]) and a lipid phase containing the water-insoluble substrates. In some cases, the enzymes that metabolize retinoids or carotenoids are themselves hydrophobic and membrane bound. The presence of heterogeneous phases per se, and the fact that they change in composition during the course of the enzyme reaction, make the interpretation of enzyme kinetic data much more complicated than for homogeneous catalysis. A full discussion of such “interfacial” kinetics is beyond the scope of this chapter; however, Brockman (52) edited an excellent monograph on this topic. A few points of particular relevance to the study of the metabolism of highly apolar lipids such as retinyl esters and carotenoids should be made. The composition and packing of nonsubstrate molecules at the interface (the “quality” of the interface) plays a large role in the binding of the enzyme and the rates of substrate conversion observed. Obviously, the availability of substrate molecules is also important. The point is that the observed kinetic “preference” for one substrate over another may reflect more its interactions with other lipids that allow it to achieve a high concentration at the interface than preferential binding to the enzyme itself. That is, the apparent specificity of the enzyme may reflect mostly the physical availability of the substrate at the interface. The physiologic relevance of this is that the detailed composition of luminal lipids has a major impact on the digestion and absorption of dietary vitamin A and carotenoids. Studies of the enzyme catalyzed reactions of retinyl esters and carotenoids generally have been conducted under conditions where the interfacial concentration of substrate (and other lipids or detergents) is undefined. This does not preclude making certain operational comparisons of the rates of reaction of different potential substrates. It does, however, mean that it is problematic to conclude that the higher rates of reaction of one substrate over another tells much about the specificity of the enzyme. This is especially true when one considers that even fairly well-characterized substrate forms (i.e., micelles, liposomes, or monolayers of

DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1739 CONVERSION OF PROVITAMIN A CAROTENOIDS TO RETINOIDS

defined composition) probably do not closely resemble the physical forms adopted by dietary retinyl esters and carotenoids in vivo (e.g., the complex emulsions, micelles, and vesicles in the intestinal lumen and protein-bound substrates in the mucosal cell). It is also notable that there is almost no detailed information on the physical forms or “phases” that retinyl esters and carotenoids adopt in the intestinal lumen. Much more detailed information on these issues is available for other major dietary lipids such as triglycerides, phospholipids, and cholesterol (53,54). Nonetheless, it is clear from studies both in experimental animals and humans that the coingestion of dietary fat markedly enhances the intestinal absorption of dietary vitamin A and carotenoids (55,56). The presence of dietary fat in the intestine can stimulate retinyl ester digestion and provitamin A conversion by: (1) stimulating pancreatic enzyme secretion; (2) stimulating the secretion of bile salts, which serve to form mixed micelles of lipids; and (3) providing products of lipid digestion (i.e., lysophospholipids, monoglycerides, and free fatty acids), which themselves can serves as components of micelles. Finally, fat ingestion promotes vitamin A and carotenoid absorption by providing the lipid components for intestinal CM assembly (see later for a more detailed discussion).

15′

β-Carotene

Carotene Cleavage Enzymes Two pathways have been described for the cleavage of β-carotene to retinoids (vitamin A): central and eccentric (Fig. 68-3). The major pathway is the central cleavage catalyzed by a cytosolic enzyme, BCO, which cleaves β-carotene at its central double bond (15,15′) to yield retinal, a direct precursor of retinol and retinoic acid (RA). Two mechanisms for the enzymatic central cleavage of β-carotene have been proposed. The first is a dioxygenase reaction that requires molecular oxygen and yields an unstable dioxetane intermediate that is rapidly converted into retinal (57). More recently, a monooxygenase reaction mechanism that requires two oxygen atoms from two different sources (molecular oxygen and water) and yields an epoxide as intermediate has been proposed (58). Regardless of the mechanism, the final product of the central cleavage of β-carotene is retinal. Using intestinal preparations, investigators clearly showed that the stoichiometry of this reaction was 2 mol retinal formed per 1 mol β-carotene cleaved (59,60). One of the major recent advances has been the characterization of BCO on a molecular level in Drosophila melanogaster (61), chicken (62),

14′

12′

10′

8′

15′

Eccentric cleavage

14′

15

12′

10′

8′

CHO

15′

β-Apo-8′-carotenal 14′

15

12′

10′

12′

CHO

15′

Central cleavage

β-Apo-10′-carotenal 14′

15 15′

β-Apo-12′-carotenal

15

CHO

Retinal

15COOH

CH2OH

15

Retinol

Retinoic acid

FIG. 68-3. Products of the central and eccentric cleavages of β-carotene.

CHO

β-Apo-8′carotenoic acid

β-Apo-10′carotenoic acid β-Apo-12′carotenoic acid

1740 / CHAPTER 68 mouse (11,63,64), and human (65). In these different species, the identified complementary DNA sequence encoded a protein with the ability to catalyze the cleavage of β-carotene into retinal and consisting of ~550 amino acids (with a predicted molecular weight of ~65 kDa). This sequence is well conserved among the different species and showed a high sequence homology with RPE65, a retinal pigment epithelium protein that may have an important role in vitamin A metabolism of the vertebrate eye. Although the purification of the recombinant human protein showing a specific activity of 10 nmol retinal/min/mg protein has been successfully achieved (66), that of the native protein remains problematic (64). The second pathway of β-carotene metabolism is the eccentric cleavage, which occurs at double bonds other than the central 15,15′-double bond of the polyene chain of β-carotene to produce β-apocarotenals with different chain lengths. Given that only trace amounts of apocarotenals are detected in vivo from tissues of animals fed β-carotene (67), and that they can be formed nonenzymatically from β-carotene autooxidation (68), the existence of this pathway has been the subject of debate. However, the identification and characterization of an eccentric cleavage enzyme from mouse that acts specifically at the 9′, 10′-double bond of β-carotene to produce β-apo-10′-carotenal and β-ionone (69) provides evidence for the occurrence of some eccentric cleavage in animals. Based on in vitro observations (70), it was suggested that eccentric cleavage could occur preferentially under oxidative conditions (when antioxidants are insufficient) such as smoking and diseases involving an oxidative stress or in the presence of high β-carotene levels, or both (5). In contrast, under normal physiologic conditions (when antioxidants are adequate), central cleavage would be the predominant pathway. The two major sites of β-carotene conversion in humans are the intestine and the liver. By direct determination of BCO activity in human small intestine and liver samples, it was estimated that, in a human adult, the maximum capacity for β-carotene cleavage by the two organs combined was 12 mg β-carotene/day (71); this amount is much greater than the observed average daily intake of 1.5 mg β-carotene/day in the United States or even the greater daily intake of 6 mg β-carotene/day suggested by some authors as necessary to meet the goal of 90% of vitamin A intake (72). Human liver was shown to have a four times greater capacity for metabolizing β-carotene than the small intestine (71). This is consistent with the earlier prediction, using a multicompartmental model, that, in humans, β-carotene cleavage takes place in the liver to a greater extent than in the intestine (73). In rats, the highest activities of BCO also were found in the small intestine and liver, followed by the brain, lung, and kidney (74). In agreement with the tissue distribution of the BCO activity, high levels of human BCO mRNA were reported in the jejunum, liver, and kidney, whereas lower levels were present in the prostate, testis, ovary, and skeletal muscle (66). In contrast with humans, rats convert most β-carotene dietary in the intestine and only small amounts of β-carotene reach the liver.

Regulation by Nutritional Factors In rats, BCO activity was enhanced by vitamin A deficiency (75), dietary polyunsaturated fats (76), copper depletion, and fructose feeding (77) and was inhibited by protein deficiency (78). However, the mechanisms by which these nutritional factors affect β-carotene conversion remain to be clarified. The cloning of BCO offers the possibility of answering some of these questions. A direct repeat of retinoic acid response elements (RARE) was identified in the promoter of the mouse BCO gene, suggesting that RA and 9-cis RA could down-regulate the intestinal enzyme in rat and chicken at the transcriptional level via their interaction with RARs, RXRs, or both (79). This mechanism of regulation could be generalized to all pro-RA retinoids and carotenoids and could explain why vitamin A deficiency status enhanced the intestinal enzyme activity. Next, a direct repeat of the peroxisome proliferator-response element (PPRE) was identified in the promoter of the mouse BCO gene (80). The authors demonstrated that peroxisome proliferatoractivated receptors (PPARs; most likely PPAR-γ) bind specifically the PPRE site in the promoter of BCO in TC7 and PF11 cells, and that binding probably required the heterodimerization of PPAR-γ with RXR-α. Polyunsaturated fats, known as activators of PPARs (81), could thus regulate BCO at the transcriptional level by this mechanism, which also occurs for the cellular retinol-binding protein II (CRBP[II]), another gene in carotenoid/retinoid metabolism that contains the PPRE site (82). Finally, it should be indicated that these mechanisms of regulation of BCO appear to be tissue specific (79).

DIGESTION OF RETINYL ESTERS Pancreatic Enzymes In 1968, Erlanson and Borgström (83), using a gel filtration column, reported the partial separation of two different pancreatic REH activities in the rat. These two activities hydrolyzed different physical forms of the retinyl palmitate substrate; the early peak mainly hydrolyzed retinyl palmitate, which was dispersed in millimolar concentrations of taurodeoxycholate (a condition known to stimulate carboxylester lipase [CEL] and to inhibit pancreatic triglyceride lipase [PTL]) (84), whereas the subsequent peak was more effective in hydrolyzing dispersed retinyl palmitate in the absence of bile salt. These two different REH elution patterns were consistent with CEL and PTL, respectively. Pancreatic CEL catalyzes the hydrolysis of cholesteryl esters, triglycerides, and lysophospholipids. It was thought to hydrolyze retinyl esters also in the intestine. CEL knockout (CEL KO) mice were generated to study the functions of CEL (85,86). Although neither CEL KO nor wild-type (WT) mice absorbed nonhydrolyzable cholesteryl ether, CEL KO mice absorbed about half the amount of cholesterol provided as cholesteryl ester compared with WT mice. These data

DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1741 indicated that hydrolysis of cholesteryl esters is necessary before absorption, and that CEL plays an important role in cholesterol absorption. In contrast with the results for cholesteryl ester, CEL KO mice absorbed the same amount of retinol, when provided as retinyl ester, as did WT mice. In contrast, neither mouse absorbed the nonhydrolyzable retinyl hexadecyl ether. These data suggested that retinyl ester hydrolysis was required for absorption, and that CEL was not the responsible enzyme (at least under conditions where the amount of dietary retinyl ester was in the microgram range, as used in this study). Triglyceride absorption also was comparable between CEL KO and WT mice, indicating that absence of CEL does not affect triglyceride hydrolysis. Therefore, if intestinal retinyl ester absorption is unaffected in CEL KO mice, one or more other retinyl ester hydrolytic enzymes must be present in the gut lumen or on the enterocyte surface. Studies then were conducted to identify the non-CEL pancreatic REH activity that appeared to be present in CEL KO mice, as well as to investigate this activity in WT mice and in rats. Several lines of evidence suggest that this activity is caused by PTL (87). First, the dependence of pancreatic REH on different types of bile salt was investigated in pancreatic homogenates of WT mice and rats. When assayed using different bile salt conditions, cholesteryl ester hydrolase activity was detected only in the presence of trihydroxy bile salts, which is consistent with previous results (88,89). Pancreatic REH activity, however, was not absolutely dependent on trihydroxy bile salts. Retinyl ester hydrolysis was detected not only in the presence of trihydroxy bile salts, but also in the presence of dihydroxy bile salts and CHAPS, a bile salt analog, as well as in the absence of bile salts. The finding that REH activity was supported by dihydroxy bile salts is consistent with PTL-mediated hydrolysis. Second, when total pancreatic homogenates obtained from rat and WT and CEL KO mice are used to assay REH activity, a considerable stimulation of the REH activity by colipase was observed, indicating that PTL was at least partially contributing to the pancreatic bile salt–dependent REH activity. Third, when pancreatic homogenates from rats and WT and CEL KO mice were applied to diethyl aminoethyl (DEAE)-chromatography, the majority of REH activity coeluted with PTL activity in the unbound fraction. In contrast, a minor peak eluted with CEL activity during the KCl gradient elution in both species. This further supports the notion of PTL-mediated retinyl ester hydrolysis. Fourth, because of the possibility of multiple proteins being involved in pancreatic retinyl ester hydrolysis, the enzymatic characteristics of purified human PTL, using either triolein or retinyl palmitate as a substrate, was studied. Both REH and TGH activities of the enzyme were completely dependent on the presence of colipase. In addition, identical patterns of bile salt inhibition were observed using either triolein or retinyl palmitate as a substrate. Although these data strongly suggest that PTL may be a major REH in rat and mouse intestinal lumen, they do not provide final proof for this concept. In addition, other enzymes synthesized and secreted by pancreas may also play a role in the lumenal hydrolysis of retinyl esters. For example, some

triglyceride hydrolysis was observed in the absence of colipase in pancreatic homogenates from both rats and mice, which may point to the presence of other related enzyme activities, such as pancreatic lipase–related protein 2 (PLRP2). It is possible that the triglyceride hydrolase activity observed in the absence of colipase may be caused by endogenous colipase present in the pancreatic homogenates. Nonetheless, PLRP2 is 65% identical to PTL, hydrolyzes phospholipids, and shows activity toward triglycerides in the classical PTL assay (90). Currently, the percentage contribution of PLRP2 to pancreatic bile salt–dependent REH activity is unknown. Also, yet another pancreatic lipase–related protein (PLRP1) has been cloned that is 68% homologous to PTL, but for which the substrate remains unknown (91). Thus, more than one enzyme may be responsible for the complete hydrolysis of retinyl esters in the intestinal lumen.

Intestinal Enzymes In addition to pancreatic bile salt–dependent REH activities, an REH activity intrinsically located in the brush-border membrane of the absorptive enterocytes was shown in rat and human intestines (92,93). This enzyme activity was suggested to be caused by an intestinal phospholipase B (PLB) (94). These authors showed that the brush-border membrane, isolated from rats in which the common pancreatic duct had been ligated for 2 days (thus prohibiting contamination of brush-border membrane with any enzymes secreted by pancreas such as CEL or PTL), had a greatly decreased hydrolytic activity against short-chain retinyl esters (in the presence of trihydroxy bile salts) and a smaller (30%) decrease in the activity against long-chain retinyl esters (such as retinyl palmitate) compared with sham-operated rats. Therefore, they suggested that short-chain REH was mainly caused by enzymes of pancreatic origin (and could be caused by CEL), whereas the majority (70%) of long-chain REH was intrinsic to the brush border. The remaining 30% of REH activity, however, could be caused by PTL, because this REH activity was detected in the presence of both trihydroxy and dihydroxy bile salts. It is important to note, however, that the relative activities observed in vitro may not reflect the relative contributions of the various enzymes in vivo. It is likely that both PTL and PLB contribute to retinyl ester digestion. It will become essential to perform retinyl ester absorption experiments in the appropriate KO mice strains and mice deficient in more than one enzyme to determine their relative roles in intestinal retinyl ester digestion and absorption.

INTESTINAL ABSORPTION OF CAROTENOIDS Knowledge about human carotenoid absorption is mostly derived from studies conducted with β-carotene (5,95–98). Rodents, because of their high efficiency of cleaving provitamin A carotenoids in intestine, are not a good

1742 / CHAPTER 68 animal model for studying human carotenoid absorption. Ferrets, preruminant calves, and gerbils have been used as alternatives (99–101). However, none of these animal models completely mimics carotenoid metabolism in humans (102). There are different methods to quantify the intestinal absorption of carotenoids in humans, such as the intake-excretion “balance” approach and the total plasma “carotenoid response” approach. Both of these methods give only a rough estimate of intestinal absorption per se. Approaches using stable isotopes, coupled with mass spectral analysis of the carotenoid and its newly synthesized metabolites isolated from the postprandial triglyceride-rich lipoprotein plasma fraction, are the most promising methods for accurate measurement of carotenoid absorption. However, such studies are costly and complex, and the data generated currently are limited and difficult to compare because of the use of different experimental designs (73,103–105). Although such methods have a great promise in assessing carotenoid bioavailability and bioefficacy from different food sources in humans (106–108), they do not provide mechanistic information about the carotenoid absorption process itself. The in vivo intestinal absorption of carotenoids involves several crucial steps: (1) release of carotenoids from the food matrix; (2) solubilization of carotenoids into mixed lipid micelles in the lumen; (3) cellular uptake of carotenoids by intestinal mucosal cells; (4) incorporation of carotenoids into CMs; and (5) secretion of carotenoids and their metabolites associated with CMs into the lymph. In this overall process, several basic aspects still remain to be clarified such as the absolute absorption efficiencies of the different carotenoids, the nature of luminal and intracellular factors regulating the process of absorption, the mechanisms of intracellular transport of carotenoids and of their incorporation into CMs, and the nature of interactions among carotenoids occurring during their intestinal absorption. Given the limitations of using human subjects for these kinds of investigations, much of our knowledge of the mechanisms of intestinal carotenoid absorption on the molecular level has come from studies of in vitro intestinal cell-culture model systems mimicking the in vivo intestinal absorption of carotenoids (i.e., the third through the fifth steps mentioned above) (109). Under normal cell-culture conditions, human intestinal Caco-2 cells are unable to form CMs. However, when supplemented with oleic acid (OA) and taurocholate (TC) (110), highly differentiated parent Caco-2 cells (without BCO activity) and the derived TC7 cells (with BCO activity) cultured on membranes were able to form and secrete CM. The high OA concentration is necessary to induce intracellular triglyceride synthesis, and thus CM assembly. Because Caco-2 cells were more efficient than TC7 cells for both CM formation and β-carotene transport, and because β-carotene cleavage might complicate studies on provitamin A carotenoid absorption per se, the parent Caco-2 cell line has been used in most studies. CM secreted by Caco-2 cells were characterized as particles rich in (newly synthesized) triglycerides (~90% of total secreted) containing apolipoprotein B (ApoB;

~30% of total secreted) and phospholipids (~20% of total secreted) and with an average diameter of ~60 nm (determined by laser light scattering) (109). These characteristics are similar to those of CMs secreted in vivo by the enterocytes. Thus, this in vitro model provides the possibility of dissociating experimentally two important processes of the intestinal carotenoid absorption: cellular uptake and secretion. Under conditions mimicking the postprandial state (TC and OA supplementation), differentiated Caco-2 cells were able (1) to take up carotenoids at the apical side and incorporate them into CMs and (2) to secrete them at the basolateral side, associated with CM fractions. In this model, no attempt currently has been made to reproduce the in vivo physiochemical conditions occurring in the intestinal lumen, such as carotenoid release from the food matrix and solubilization into mixed lipid micelles. Carotenoids were delivered to Caco-2 cells in aqueous suspension with Tween 40 (109). Using this cell-culture system in conjunction with an in vitro digestion procedure (see the first two steps mentioned earlier in this section) (111), in which carotenoids are transferred from the food to bile salt micelles, could be useful to assess the bioavailability of carotenoids from different types of food matrices in vitro. These first two steps of carotenoid absorption have been mimicked using Caco-2 cells cultured on plastic (112). Kinetics of β-Carotene Transport through Intestinal Cells Only a few studies have been done on the kinetics of carotenoid absorption. Based on earlier rat studies (113,114), the intestinal absorption of carotenoids was thought to be a passive diffusion process determined by the concentration gradient of the carotenoid across the intestinal membrane. The kinetics of β-carotene transport through Caco-2 cell monolayers, characterized for both steps (cellular uptake and secretion in CMs), showed curvilinear, time-dependent and saturable, concentration-dependent (apparent Km 7–10 µM) processes (109). Thus, these data suggest that the intestinal transport of carotenoids might be facilitated by the participation of a specific epithelial transporter, a hypothesis that contrasts with previous investigations (113,114). The contrast between studies by During and colleagues (109) and El-Gorab and colleagues (113) and Hollander and Ruble (114) could be because of the use of different models, human cells and rats, respectively, and possibly because of the use of different β-carotene concentration ranges, 0.5 to 23 µM (109) and 0.5 to 11 µM (114) or 6 to 60 µM (113), respectively. The saturation of β-carotene transport through Caco-2 cell monolayers occurred at β-carotene concentrations of 15 to 20 µM, which is equivalent to a daily β-carotene intake of 100 mg or more. It was estimated that the β-carotene concentration of 1 µM at the apical side of cells (or 400 pmol β-carotene/cm2 of Caco-2 cell monolayer) was close to the physiologic level of β-carotene found in the gut (200 pmol

DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1743 β-carotene/cm2 of surface of absorption) after ingestion of a daily β-carotene dose of 5 mg (109). Under linear concentration conditions (for a β-carotene concentration range of 0.12–6 µM) at 16-hour incubation and under cell-culture conditions mimicking the in vivo postprandial state, the extent of absorption of all-trans β-carotene through Caco-2 cell monolayers was 11%, a value similar to that reported from different human studies. In humans, the bioavailability of a single dose of β-carotene (in oil or in capsule) was 9% to 17% using the lymph-cannulation approach (115), 11% using carotenoid and retinyl ester response in the triglyceride-rich lipoprotein plasma fraction approach (116), and 3% to 22% using most recent isotopic tracer approaches (73,104). That the extent of β-carotene absorption obtained with Caco-2 cells falls within the range observed in vivo adds confidence in the in vitro model for studying human intestinal absorption of carotenoids. Finally, of the total β-carotene secreted by Caco-2 cells, 80% was associated with CMs, 10% with very low-density lipoproteins (VLDLs), and 10% with the nonlipoprotein fraction (109), pointing to the importance of CM assembly for β-carotene secretion into the lymph in vivo. Selective Uptake of All-Trans β-Carotene versus Its Cis Isomers by Intestinal Cells Human studies (117–121) have consistently reported a preferential accumulation of all-trans β-carotene in total plasma and in the postprandial triglyceride-rich lipoprotein plasma fraction compared with its 9-cis isomer. These differences in plasma response between the two geometric isomers suggested either a selective intestinal transport of all-trans β-carotene versus its 9-cis isomer or an intestinal cis-trans isomerization of 9-cis β-carotene into all-trans β-carotene. This later possibility was proposed by a study (121) showing a significant accumulation of [13C]-all-trans β-carotene in plasma of subjects who ingested only [13C]-9-cis β-carotene. Starting with an initial concentration (1 µM) for the three geometric isomers of β-carotene applied separately to the in vitro system described earlier, it was demonstrated that both 9-cis and 13-cis β-carotene were taken up by Caco-2 cells to only one-fifth the extent of all-trans β-carotene (109). The extents of absorption of the two cis isomers through Caco-2 cell monolayers were less than 3.5% (compared with 11% for all-trans β-carotene), indicating that the discrimination between β-carotene isomers occurred at the cellular uptake level of the intestinal absorption process. The β-carotene isomer selectivity was tissue specific; a preferential uptake of the all-trans isomer was shown in hepatic stellate HSC-T6 cells and in a cell-free system from rat liver microsomes, but not in endothelial EAHY cells or U937 monocyte/macrophages (109). When Caco-2 cells were incubated with only 9-cis β-carotene, all-trans β-carotene did not increase in cells or in the basolateral medium, indicating that there is no cis-trans isomerization occurring in intestinal cells. Thus, the isomerization of 9-cis β-carotene observed

in vivo (121) could take place in the gastrointestinal lumen before the cellular uptake, probably under the action of enzymes related to gut microflora because the spontaneous isomerization of 9-cis β-carotene to all-trans β-carotene is not favored thermodynamically (122). The data on the selective uptake of β-carotene isomers by Caco-2 cells also support the idea of a specific transport involved in the intestinal absorption process of carotenoids.

Differential Intestinal Transport of Individual Carotenoids Data on the intestinal absorption of carotenoids other than β-carotene are limited. It appears that the more polar carotenoids (xanthophylls) are absorbed better than the carotenes. Supporting this idea, it was reported that the plasma response for lutein was twice as high as it was for β-carotene when single doses of those carotenoids were given in oil (123), and that both lutein and zeaxanthin versus β-carotene were preferentially increased in CMs after ingestion of a carotenoid mixture, “Betatene” (124). In addition, the relative bioavailability of lutein from vegetables was reported to be five times greater than that of β-carotene (125), but in the same study, the plasma response of lutein was substantially smaller than that of β-carotene after simultaneous ingestion of pure lutein and β-carotene dissolved in oil. In fact, in these different human studies, the difference in plasma response between carotenoids may not reflect a difference in true absorption. There are several factors for which each carotenoid appears to follow a different pattern; for example, (1) the differential transfer of carotenoids from food matrices to the lipid micelles: lycopene (from tomato puree) was reported to be less efficiently transferred to the micellar phase of the duodenum than β-carotene (from carrot puree) and lutein (from chopped spinach) in vivo (126); (2) the differential stability of carotenoids: lycopene and β-carotene decomposed more rapidly than lutein and zeaxanthin on exposure to various prooxidants in vitro (127); (3) the differential metabolism of carotenoids: at least 35% (up to 75%) of the absorbed β-carotene is converted to retinyl esters in intestinal cells (115,116,128), whereas xanthophylls are nonprovitamin A carotenoids; and finally, (4) the differential clearance rate of carotenoids from the plasma once absorbed. These many factors, which make it difficult to compare the actual absorption of the different carotenoids in vivo, can be avoided by using in vitro cell-culture systems. A differential transport of carotenoids through Caco-2 cell monolayers was shown as follows: all-trans β-carotene (11%) ≈ α-carotene (10%) > lutein (7%) > lycopene (2.5%). These in vitro data (109) and several studies with animals (129,130) and humans (128,131) converge to indicate that lycopene is poorly absorbed compared with other carotenoids. In addition, these data (109) agreed with a human study (128) that showed that β-carotene is preferentially absorbed compared with lutein. In contrast, it was reported that plasma β-carotene response was less than plasma lutein response when the two carotenoids

1744 / CHAPTER 68 were ingested separately (123–125). However, in these studies, the retinyl ester fraction formed during the intestinal absorption of β-carotene was not analyzed, a fact that could contribute to an underestimated β-carotene absorption compared with lutein absorption. Interestingly, for the four individual carotenoids tested (β-carotene, α-carotene, lycopene, and lutein), the extents of secretion varied over a wider range (2.5–11%) than the extents of cellular uptake (15–18%), indicating that the carotenoid structure might be a major determinant in their ability to be incorporated into CMs.

Carotenoid Interactions during Intestinal Absorption Carotenoids compete for their absorption and metabolism, but data are conflicting, as indicated in a review (132). In humans, β-carotene reduced the apparent lutein absorption (123,133,134), whereas lutein had either no effect (123) or reduced the apparent β-carotene absorption (133,134). This inhibitory effect of lutein on plasma β-carotene response observed in vivo could be attributed, at least in part, to that lutein inhibits β-carotene cleavage enzyme as suggested in rats (39), but not confirmed in humans (133,134). Furthermore, β-carotene was shown to improve the apparent lycopene absorption (131), whereas lycopene had no effect on that of β-carotene in humans (131,134). When carotenoids were provided in their natural vegetable matrices, it was reported that adding a second carotenoid to a meal that contained another carotenoid diminished the CM response of the first carotenoid (135). However, in this postprandial study (135), it was difficult to define clearly specific interaction between two carotenoids because some of the meals contained more than two carotenoids. In addition, “pharmacologic” doses of carotenoids are commonly used in these interaction studies; these are doses at which the efficiency of carotenoid absorption appears to decrease probably in relation to the limited capacity of micellar incorporation of carotenoids in the lumen (36,108,132). Thus, it is difficult to interpret the results for interaction at the cellular level. Using in vitro cell-culture systems and a range of physiologic concentrations (1–5 µM), neither lutein nor β-carotene significantly affected the transport of each other through Caco-2 cell monolayers, whereas the main carotenoid interactions were observed between nonpolar carotenoids (β-carotene/α-carotene and β-carotene/lycopene). The discrepancy between these in vitro (109) and in vivo data (123,131–135) might be because plasma carotenoid response measured in in vivo studies does not reflect only intestinal absorption, as mentioned earlier. Thus, the specific interactions observed in the in vitro study (109) indicate that two carotenoids exhibiting similar structural characteristics could follow a similar pathway in intestinal cells, and thus compete for their cellular uptake or their incorporation into CMs, or both. For instance, in CM particles, carotenoids may organize themselves differently on the basis of their structural properties; the more polar carotenoids (xanthophylls) may remain at the surface with the less polar carotenoids (carotenes) in

the core of CMs. Finally, these mutual interactions also are consistent with the idea of a facilitated uptake process. In summary, the concentration dependence (saturation) of β-carotene uptake and secretion in CMs, the discrimination between β-carotene isomers for their cellular uptake, and the differential absorption of different carotenoids and their interactions observed during transport through Caco-2 cells all suggest that the intestinal transport of carotenoids might be facilitated by the participation of a specific epithelial transporter. This hypothesis is supported by the recent identification of a scavenger receptor with a high sequence homology to the mammalian class B scavenger receptors (SR-BI and CD36) mediating the cellular uptake of carotenoids in Drosophila (136). In Drosophila, which only require vitamin A for vision, two mutants, ninaB and ninaD, lack the visual chromophore of the fly, 3-hydroxyretinal, when raised on a media with carotenoids as the sole source of vitamin A. Analysis of the ninaB mutant demonstrated that the gene encoded a carotenoid cleavage enzyme. It then was demonstrated that mutation of the ninaD gene resulted in defects in the cellular uptake and distribution of carotenoids in the fly, and that this gene encoded a scavenger receptor with significant sequence identity to the mammalian receptors. Thus, carotenoid uptake by intestinal cells may involve epithelial transporter(s), which need to be identified in mammals. Lipid transporters in intestinal epithelial cells probably also are involved in the uptake or efflux, or both, of unesterified retinol (see later).

INTESTINAL ABSORPTION OF VITAMIN A Mechanisms of Cellular Uptake and Efflux of Unesterified Retinol in Enterocytes: Portal Secretion Studies concerning the uptake of retinol by the human intestinal cell line, Caco-2, indicated that retinol at physiologic and pharmacologic concentrations was taken up by saturable, carrier-mediated processes and nonsaturable, diffusion-dependent processes, respectively (137). The retinol taken up by these cells was esterified and the retinyl esters mainly contained palmitic acid and OA (137,138). Our studies showed that retinol uptake is a rapid process with a half-life of minutes (139). This uptake was not affected by the presence of high concentrations of free fatty acids. Experiments in our laboratory have further suggested the role of transporters in retinol flux in Caco-2 cells (140). When cells were incubated with 3 µM retinol for varying times up to 24 hours, cellular retinol plateaued within 2 hours, whereas there was continuous formation of retinyl esters. Both retinol and retinyl esters secreted in basolateral medium increased linearly with time (up to 20 hours). Retinyl esters were associated with CMs, and retinol with the nonlipoprotein fraction. After incubation with retinol concentrations of 0.5 to 130 µM, cellular uptake of retinol was directly proportional to initial retinol concentration. However, the kinetics of efflux of retinol into basolateral

DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1745 medium revealed two processes. Retinol secretion showed saturation at concentrations less than 10 µM, implying a mediated transport out of the cell, and linearity with greater concentrations, implying passive diffusion. One interpretation of these data is that free retinol enters into intestinal cells by simple diffusion, whereas its secretion may require a facilitated transport at physiologic doses. Other experiments in which glyburide, a known inhibitor of the adenosine triphosphate–binding cassette A1 (ABCA1) transporter, caused marked inhibition of the efflux of free retinol into basolateral medium (but not cell uptake) support this notion. Of course, it is also possible that cellular uptake at the apical membrane is facilitated as well, but that the rapid esterification of retinol after uptake makes it difficult to demonstrate kinetically that the transport is rate limiting. Early studies using intestinal segments also suggested that the unesterified retinol was taken up by protein-mediated facilitated diffusion and passive diffusion mechanisms at physiologic (<150 nM) and pharmacologic concentrations (450–2700 nM), respectively (141,142). Other evidence for protein-mediated uptake of retinol has been presented using intestinal segments (14). However, no protein currently has been identified and characterized that might be involved in the uptake of retinol. There is an enormous amount of current interest in the role of membrane-bound lipid transporters in the cellular uptake and efflux of fat-soluble molecules. For example, three different membrane-bound proteins—CD36, membrane-bound fatty acid binding protein, and a fatty acid transport protein— that might be involved in fatty acid uptake have been identified (see Abumrad [143] and Glatz and colleagues [144] for review). In the case of cholesterol, SR-BI, CD36, NPC1L1, and a variety of ABC transporters have been implicated in its uptake or efflux, or both, from various cells (145–150). It is possible that these or other proteins may play a role in the transport of retinol across cell membranes. Defining the exact mechanisms of cellular uptake of retinol (or other lipids) is complicated by the fact that, as indicated earlier, multiple mechanisms (both facilitated and passive) may exist in a single cell. An additional problem is that much of the work in this area relies on the use of membrane transporter inhibitors, and there is increasing evidence that some of these compounds inhibit multiple transporter types (151). A final complication is that some of the transport inhibitors also affect the expression of transporter genes, and thus exert effects on transporter number and function simultaneously. The general perception that retinol is efficiently absorbed and quantitatively transported on CMs may need reevaluation (see Blomhoff and colleagues [152,153] for review). First, it should be noted that the recovery of ingested retinol into lymph varies between 20% and 60% in various studies (18,115,153). Second, Hollander (154) showed that approximately 60% and 30% of the absorbed retinol is secreted into lymph and portal circulation, respectively. Furthermore, he showed that secretion of retinol into lymph was modulated by the presence of different concentrations of TC and different

fatty acids (154). Third, oral supplementation of retinol into patients with abetalipoproteinemia, who do not assemble and secrete CMs, results in partial recovery from symptoms of retinol deficiency (155). Fourth, cell-culture studies showed that free retinol or its metabolized products are transported across the cells independent of the assembly and secretion of lipoproteins (139). Thus, much of the absorbed retinol is secreted into lymph in esterified form. However, a significant amount also is secreted into portal circulation probably as free retinol. The transport of free retinol to the portal circulation is expected to be physiologically significant in pathologic conditions that affect the secretion of CMs. Thus, the transport of free retinol may be an essential backup mechanism for the homoeostasis of vitamin A under some conditions.

Intracellular Transport by Cellular Retinol Binding Protein (II) After cellular uptake, free retinol is probably immediately sequestered by cellular retinol-binding proteins (CRBPs). Two CRBPs, CRBP(I) and CRBP(II), have been purified and characterized extensively. They have considerable sequence identity and belong to a family of fatty acid–binding proteins. Both of the proteins are highly conserved during evolution, indicating their physiologic importance. These proteins share considerable structural, genetic, and biochemical properties. However, the cellular expression pattern of these proteins is different. The CRBP, a 14.6-kDa polypeptide, is expressed in many tissues, whereas CRBP(II), a 16-kDa polypeptide, is expressed primarily in the absorptive cells of the small intestine. The CRBP(II) is one of the most abundant proteins and accounts for approximately 1% of the total soluble proteins recovered from the jejunal mucosa. The tissue distribution and abundance indicate that it must be uniquely suited for retinol absorption by the intestine (see reviews by Li and Norris [156], Newcomer and colleagues [157], and Ong [158,159]). In vitro studies indicated that CRBP(II) can play several functions in the trafficking of retinol. It has been speculated that it can bind to specific transporters on the brush-border membrane and permit facilitated diffusion. It can serve as a reservoir to keep the concentrations of free retinoids low and protect cells from their detergent-like properties. More important, it may present retinoids to different enzymes and direct their metabolism (158,159). In vivo studies showed that CRBP(II) mRNA levels are increased in the small intestine of the retinoid-deficient rats (160) and rats fed with long-chain fatty acids (161). In Caco-2 cells, CRBP(II) mRNA was shown to be increased (twofold to threefold) after treatment of the differentiated cells with RA (162). More importantly, this resulted in increased absorption and intracellular esterification of radiolabeled retinol. Furthermore, absorption and esterification also was increased after the overexpression of CRBP(II) in these cells (163). These studies indicate that changes in CRBP(II) expression results in the modulation of retinol metabolism (162,164,165). However, it is unknown whether

1746 / CHAPTER 68 the increased expression of CRBPII results in increased secretion of retinyl esters in CMs. Characterization of the CRBPII KO mouse has clarified that CRBPII plays an important, but not absolutely essential, role in the intestinal absorption of vitamin A (166). Using the methodology originally described by Dew and Ong (14), Xueping and coworkers (166) showed that the saturable component of retinol uptake by jejunal segments was reduced to about half that of tissue from WT littermates. The CRBPII KO mice, when maintained on a vitamin A–enriched diet, also had reduced (about 40%) hepatic stores of vitamin A, suggesting that the incomplete impairment of retinol processing in the intestine nonetheless affects whole-body vitamin A status.

Reesterification and Incorporation into Chylomicrons: Lymphatic Secretion Early studies in intact rats and humans clearly demonstrated that after uptake of newly absorbed retinol, the retinol was largely reesterified with long-chain fatty acids (mostly palmitate) and secreted into the lymphatics together with other dietary lipids in CMs. Caco-2 cells have been used to study mechanisms of vitamin A absorption that are difficult to study in intact animals. The differentiated Caco-2 cells were shown to express CRBPII, acyl-coenzyme A (CoA):retinol acyltransferase (ARAT), lecithin:retinol acyltransferase (LRAT), and retinal reductase (137,138). Studies on retinol secretion showed that Caco-2 cells supplemented with no fatty acids secreted only free retinol. However, cells incubated with OA were shown to secrete retinyl esters in addition to free retinol (138). Based on these observations, it has been suggested that lipoprotein particles secreted by these cells may contain retinol and retinyl esters (138). However, no data about the secretion of either free or esterified retinol as part of different lipoprotein particles were reported in these studies. In the cells, the free retinol can remain associated with its CRBPs. However, much of the retinol is usually esterified and stored. In enterocytes, two enzymes, LRAT and acylCoA:retinol acyltransferase, have been identified that can catalyze the esterification of free retinol in vitro (15,16). It has been suggested, but not shown, that retinyl esters formed by LRAT and acyl-CoA:retinol acyltransferase may be targeted for secretion with CM and storage, respectively (152,153). The characterization of the LRAT KO mouse has largely resolved the question of whether enzymes other than LRAT play any physiologic role in retinol esterification. Given that the LRAT KO mouse has no detectable tissue retinyl esters (167), it is unlikely that other enzymes such as acyl-CoA: retinol acyltransferase are physiologically involved in retinol esterification in intestine. Obviously, this issue warrants further investigation. It is possible, and even likely, that other acyltransferases can catalyze the acyl-CoA–dependent esterification of retinol in vitro. It is also possible that other

enzymes may be involved in the esterification of retinol if given to animals in large quantities. It is generally believed that retinol is mainly secreted into the lymph as retinyl palmitate. During metabolic studies, analysis of the plasma showed that most of the retinyl esters are present in small CMs (168). Significant amounts of retinyl esters also are found in large CMs, followed by smaller amounts in VLDLs (168). In contrast with triglycerides, cholesterol esters, and other lipids, retinyl esters are not present in other lipoproteins such as intermediate-, low-, or high-density lipoproteins. These studies indicate that retinyl esters are mainly present in large and small CMs and behave differently from other neutral lipids, such as triglycerides and cholesterol esters. Studies were conducted in which differentiated Caco-2 cells were supplemented with radiolabeled retinol under conditions that support (postprandial) or do not support (fasting) CM secretion (139) to understand the mechanism of secretion of retinyl ester by the intestine during the fasting and postprandial states. These cells assimilated vitamin A by a rapid uptake mechanism under both conditions. After uptake, the cells stored retinol in both esterified and nonesterified forms. Under fasting conditions, the cells mainly secreted variable amounts of free retinol unassociated with lipoproteins. However, under postprandial conditions, these cells secreted significant amounts of retinyl esters mainly with CMs. The secretion of retinyl ester with CMs was independent of the rate of uptake of retinol, intracellular free and esterified retinol levels, and was dependent on the assembly and secretion of CMs. The secretion of retinyl ester was correlated with the secretion of CMs and not with the secretion of total ApoB. Inhibition of CM secretion by Pluronic L81 decreased the secretion of retinyl ester and did not result in their increased secretion with smaller lipoproteins. These data strongly suggest that retinyl ester secretion by the intestinal cells is a highly specific and regulated process that is dependent on the assembly and secretion of CMs. These data also indicate that the incorporation of retinyl esters into CMs is not a passive process, but rather is an exquisitely orchestrated event. Retinyl ester secretion does not always occur. It is induced when cells can assemble and secrete CMs. Thus, it appears that intestinal cells may have a specific mechanism for the targeting of retinyl esters to nascent CMs. As discussed earlier, Caco-2 cells do not secrete retinyl esters under conditions simulating fasting state. In our work on the kinetics of retinol uptake by Caco-2 cells, we conducted a 5-day “washout” experiment in which cells were incubated with retinol for 16 hours to accumulate cellular retinol and retinyl esters, and then incubated with retinoid-free medium (containing fatty acids, and thus mimicking the “fed” state) that was changed every 24 hours (140). This resulted in the release of the free retinol, but not the accumulated retinyl esters. This implies that only newly synthesized retinyl is incorporated into CMs, and that preformed retinyl esters cannot be used for CM assembly.

DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1747 Thus, the synthesis of retinyl esters and their incorporation into CMs appear to be concerted processes. Because of the specificity of the secretion of retinyl esters, it was proposed that retinyl esters can be used as signposts to study CM assembly (169,170). Two different mechanisms have been proposed for the assembly and secretion of CMs (see Hussain and colleagues [20,169,170] for review). First, in the independent pathway, VLDLs and CMs have been proposed to be synthesized by two independent mechanisms (171,172). If CM assembly occurs by an independent pathway that is induced during the postprandial state, retinyl esters may be used to differentiate between CM and VLDL assembly pathways. Second, the sequential assembly mechanism suggests that the assembly of CMs and VLDLs involves a common first event resulting in the synthesis of “primordial lipoproteins.” Another important event hypothesized for the assembly of CMs and other lipoproteins is the formation of

Lumen

different-sized lipid droplets. Subsequently, the larger lipid droplets are proposed to fuse with primordial lipoproteins resulting in the “core expansion” of primordial lipoproteins (20,169). If this is true, retinyl esters may be incorporated at the terminal stages of lipoprotein assembly. Both of these mechanisms can be distinguished by studying the presence of retinyl esters in different intracellular lipoproteins. In any case and as stated earlier, it is clear that retinyl ester synthesis and their incorporation and secretion in CMs are concerted processes.

SUMMARY Figure 68-4 gives an overview of the mechanisms involved in the intestinal metabolism of vitamin A and carotenoids. It emphasizes the many proteins that participate

Enterocyte

Dietary RE

PLB

Lymph/Blood

ROH

PTL

CEL

RT? LRAT

CRBP2 ROH apoB

RT?

RE, car

BCO ROH

β-Carotene CT? Carotenoid

Carotenoid

FIG. 68-4. Overview of intestinal absorption of dietary vitamin A and carotenoids. Dietary retinyl esters (REs) are hydrolyzed in the lumen by the pancreatic enzyme, pancreatic triglyceride lipase (PTL), and intestinal brush-border enzyme, phospholipase B (PLB). Studies of the carboxylester lipase (CEL) knockout mouse suggest that CEL is not involved in dietary RE digestion. Unesterified retinol (ROH) is taken up by the enterocyte, perhaps facilitated by a yet unidentified retinol transporter (RT). Once in the cell, retinol is complexed with cellular retinol–binding protein type 2 (CRBP2), and the complex serves as a substrate for reesterification of the retinol by the enzyme lecithin:retinol acyltransferase (LRAT). The REs then are incorporated into chylomicrons, intestinal lipoproteins containing other dietary lipids such as triglyceride, phospholipids, cholesterol, and cholesteryl esters and apolipoprotein B (ApoB). The incorporation of some of these lipids is dependent on the activity of microsomal triglyceride transfer protein. Dietary provitamin A carotenoids (e.g., β-carotene) and other carotenoids are taken up by intestinal cells perhaps by facilitated processes involving carotenoid transporters (CT). β-Carotene is partially converted to retinol by β-carotene 15,15′ oxygenase (BCO) and retinal reductase. The formed retinol then is metabolized similar to that originating from preformed vitamin A. The intact carotenoids are incorporated into nascent chylomicrons. Chylomicrons containing newly absorbed retinyl esters and carotenoids (RE,car) then are secreted into the lymph. Unesterified retinol also is absorbed into the portal circulation, and its efflux from the basolateral cell membrane may also be facilitated by retinol transporter (RT) proteins.

1748 / CHAPTER 68 TABLE 68-1. Proteins involved in the digestion, absorption, and transport of dietary carotenoids and vitamin A 1. Carotenoid cleavage β-Carotene oxygenases (BCO1 and BCO2(?)) 2. Carotenoid uptake and transfer Carotenoid transporters (CD36, SR-BI, NPC1L1, ABCA1, etc.) (?) Carotenoid-binding proteins? 3. Retinyl ester hydrolysis a. Lumen Pancreatic triglyceride lipase Pancreatic lipase–related protein 1 (?) Pancreatic lipase–related protein 2 (?) Carboxyl ester lipase (probably not involved) b. Brush-border membrane Phospholipase B 4. Retinol uptake, transfer, and efflux Retinol transporters (CD36, SR-BI, NPC1L1, ABCA1, etc.) (?) Cellular retinol–binding protein I (?) Cellular retinol–binding protein II 5. Retinyl ester synthesis Lecithin:retinol acyltransferase Acyl-coenzyme A:retinol acyltransferase (probably not involved) 6. Chylomicron assembly Apolipoprotein B Microsomal triglyceride transfer protein

in these processes; Table 68-1 also summarizes these proteins. Note added in proofs. Recent evidence has demonstrated that the intestinal absorption of β-carotene and lutein is at least partially facilitated by the scavenger receptor class B type I (Reboul and colleagues [173], van Bennekum and colleagues [174], During and colleagues [175]. For β-carotene other lipid transporters, that are the target of the drug Ezetimibe (e.g., the Nieman-Pick type C1 Like 1 protein), are also likely involved in facilitating its intestinal absorption (During and colleagues [175]).

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DIGESTION AND INTESTINAL ABSORPTION OF DIETARY CAROTENOIDS AND VITAMIN A / 1751 123. Kostic D, White WS, Olson JA. Intestinal absorption, serum clearance, and interactions between lutein and beta-carotene when administered to human adults in separate or combined oral doses. Am J Clin Nutr 1995;62:604–610. 124. Gartner C, Stahl W, Sies H. Preferential increase in chylomicron levels of the xanthophylls lutein and zeaxanthin compared to betacarotene in the human. Int J Vitam Nutr Res 1996;66:119–125. 125. van het Hof KH, Brouwer IA, West CE, Haddeman E, SteegersTheunissen RPM, van Dusseldorp M, Weststrate JA, Eskes TKAB, Hautvast JGAJ. Bioavailability of lutein from vegetables is 5 times higher than that of beta-carotene. Am J Clin Nutr 1999;70:261–268. 126. Tyssandier V, Reboul E, Dumas JF, Bouteloup-Demange C, Armand M, Marcand J, Sallas M, Borel P. Processing of vegetable-borne carotenoids in the human stomach and duodenum. Am J Physiol Gastrointest Liver Physiol 2003;284:G913–G923. 127. Siems WG, Sommerburg O, van Kuijk FJ. Lycopene and beta-carotene decompose more rapidly than lutein and zeaxanthin upon exposure to various pro-oxidants in vitro. Biofactors 1999;10:105–113. 128. O’Neill ME, Thurnham DI. Intestinal absorption of beta-carotene, lycopene and lutein in men and women following a standard meal: response curves in the triacylglycerol-rich lipoprotein fraction. Br J Nutr 1998;79:149–159. 129. Clark RM, Yao L, She L, Furr HC. A comparison of lycopene and canthaxanthin absorption: using the rat to study the absorption of non-provitamin A carotenoids. Lipids 1998;33:159–163. 130. Bierer TL, Merchen NR, Erdman JW. Comparative absorption and transport of five common carotenoids in preruminant calves. J Nutr 1995;125:1569–1577. 131. Johnson EJ, Qin J, Krinsky NI, Russell RM. Ingestion by men of a combined dose of beta-carotene and lycopene does not affect the absorption of beta-carotene but improves that of lycopene. J Nutr 1997;127:1833–1837. 132. van den Berg H. Carotenoid interactions. Nutr Rev 1999;57:1–10. 133. van den Berg H. Effect of lutein on beta-carotene absorption and cleavage. Int J Vitam Nutr Res 1998;68:360–365. 134. van den Berg H, van Vliet T. Effect of simultaneous, single oral doses of beta-carotene with lutein or lycopene on the beta-carotene and retinyl ester responses in the triacylglycerol-rich lipoprotein fraction of men. Am J Clin Nutr 1998;68:82–89. 135. Tyssandier V, Cardinault N, Caris-Veyrat C, Amiot M-J, Grolier P, Bouteloup C, Azais-Braesco V, Borel P. Vegetable-borne lutein, lycopene, and beta-carotene compete for incorporation into chylomicrons, with no adverse effect on the medium-term (3-wk) plasma status of carotenoids in humans. Am J Clin Nutr 2002;75: 526–534. 136. Kiefer C, Sumser E, Wernet MF, von Lintig J. A class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila. Proc Natl Acad Sci U S A 2002;16:10581–10586. 137. Quick TC, Ong DE. Vitamin A metabolism in the human intestinal Caco-2 cell line. Biochemistry 1990;29:11116–11123. 138. Levin MS. Cellular retinol-binding proteins are determinants of retinol uptake and metabolism in stably transfected Caco-2 cells. J Biol Chem 1993;268:8267–8276. 139. Nayak N, Harrison EH, Hussain MM. Retinyl ester secretion by the intestinal cells is a specific and regulated process that is dependent on the assembly and secretion of chylomicrons. J Lipid Res 2001;42: 272–280. 140. During A, Harrison EH. Kinetics of retinol uptake and secretion by Caco-2 cells: mechanistic implications. FASEB J 2003;17:A314. 141. Hollander D. Intestinal absorption of vitamins A, E, D, and K. J Lab Clin Med 1981;97:449–462. 142. Hollander D, Muralidhara KS. Vitamin A1 intestinal absorption in vivo: influence of luminal factors on transport. Am J Physiol 1977; 232:E471–E477. 143. Abumrad N, Harmon C, Ibrahimi A. Membrane transport of longchain fatty acids: evidence for a facilitated process. J Lipid Res 1998; 39:2309–2318. 144. Glatz JFC, van Nieuwenhoven FA, Luiken JJFP, Schaap FG, van der Vusse GJ. Role of membrane-associated and cytoplasmic fatty acidbinding proteins in cellular fatty acid metabolism. Prostaglandins Leukot Essent Fatty Acids. 1997;57:373–378. 145. Altmann SW, Davis HR Jr, Zhu LJ, Yao X, Hoos LM, et al. NiemannPick C1 like 1 protein is critical for intestinal cholesterol absorption. Science 2004;303:1201–1204. 146. Chen HC. Molecular mechanisms of sterol absorption. J Nutr 2001; 131:2603–2605.

147. Davis HR Jr, Zhu LJ, Hoos LM, Tetzloff G, Maguire M, et al. Niemann-Pick C1 like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Biol Chem 2004;279:33586–33592. 148. Turley SD, Dietschy LM. Sterol absorption by the small intestine. Curr Opin Lipidol 2003;14:233–240. 149. van Heek M, Farley C, Compton DS, Hoos L, Davis HR. Ezetimibe selectively inhibits intestinal cholesterol absorption in rodents in the presence and absence of exocrine pancreatic function. Br J Pharmacol 2001;134:409–417. 150. Wang DQ. New concepts of mechanisms of intestinal cholesterol absorption. Ann Hepatol 2003;2:113–121. 151. Nieland TJ, Chroni A, Fitzgerald ML, Maliga Z, Zannis VI, et al. Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide. J Lipid Res 2004; 45:1256–1265. 152. Blomhoff R, Green MH, Berg T, Norum KR. Transport and storage of vitamin A. Science 1990;250:399–404. 153. Blomhoff R, Green MH, Green JB, Berg T, Norum KR. Vitamin A metabolism: new perspectives on absorption, transport, and storage. Physiol Rev 1991;71:951–990. 154. Hollander D. Retinol lymphatic and portal transport: influence of pH, bile, and fatty acids. Am J Physiol 1980;239:G210–G214. 155. Kane JP, Havel RJ. Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic and molecular bases of inherited disorders. New York: McGraw-Hill, 1995;1853–1885. 156. Li E, Norris AW. Structure/function of cytoplasmic vitamin A-binding proteins. Annu Rev Nutr 1996;16:205–234. 157. Newcomer ME, Jamison RS, Ong DE. Structure and function of retinoid-binding proteins. Subcell Biochem 1998;30:53–80. 158. Ong DE. Vitamin A-binding proteins. Nutr Rev 1985;43:225–232. 159. Ong DE. Cellular transport and metabolism of vitamin A: roles of the cellular retinoid-binding proteins. Nutr Rev 1994;52:S24–S31. 160. Rajan N, Kidd GL, Talmage DA, Blaner WS, Suhara A, Goodman DS. Cellular retinoic acid-binding protein messenger RNA: levels in rat tissues and localization in rat testis. J Lipid Res 1991;32:1195–1204. 161. Suruga K, Suzuki R, Goda T, Takase S. Unsaturated fatty acids regulate gene expression of cellular retinol-binding protein, type II in rat jejunum. J Nutr 1995;125:2039–2044. 162. Levin MS, Davis AE. Retinoic acid increases cellular retinol binding protein II mRNA and retinol uptake in the human intestinal Caco-2 cell line. J Nutr 1997;127:13–17. 163. Lissoos TW, Davis AE, Levin MS. Vitamin A trafficking in Caco-2 cells stably transfected with cellular retinol binding proteins. Am J Physiol 1995;268:G224–G231. 164. Suruga K, Mochizuki K, Suzuki R, Goda T, Takase S. Regulation of cellular retinol-binding protein type II gene expression by arachidonic acid analogue and 9-cis retinoic acid in caco-2 cells. Eur J Biochem 1999;262:70–78. 165. Takase S, Tanaka K, Suruga K, Kitagawa M, Igarashi M, Goda T. Dietary fatty acids are possible key determinants of cellular retinol-binding protein II gene expression. Am J Physiol 1998;274: G626–G632. 166. Xueping E, Zhang L, Lu J, Tso P, Blaner WS, et al. Increased neonatal mortality in mice lacking cellular retinol-binding protein II. J Biol Chem 2002;277:36617–36623. 167. Batten ML, Imanishi Y, Maeda T, Tu DC, Moise AR, et al. Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. J Biol Chem 2004; 279:10422–10432. 168. Lemieux S, Fontani R, Uffelman KD, Lewis GF, Steiner G. Apolipoprotein B-48 and retinyl palmitate are not equivalent markers of postprandial intestinal lipoproteins. J Lipid Res 1998;39: 1964–1971. 169. Hussain MM. A proposed model for the assembly of chylomicrons. Atherosclerosis 2000;148:1–15. 170. Hussain MM, Kedees MH, Singh K, Athar H, Jamali NZ. Signposts in the assembly of chylomicrons. Front Biosci 2001;6: D320–D331. 171. Tso P, Balint JA. Formation and transport of chylomicrons by enterocytes to the lymphatics. Am J Physiol 1986;250:G715–G726. 172. Tso P, Drake DS, Black DD, Sabesin SM. Evidence for separate pathways of chylomicron and very low-density lipoprotein assembly and transport by rat small intestine. Am J Physiol 1984;247: G599–G610.

1752 / CHAPTER 68 173. Reboul E, Abou L, Mikal C, Ghiringhelli O, Andre M, Portugal H, Jourdheuil-Rahmani D, Amiot MJ, Lairon D, Borel P. Lutein transport by Caco-2 TC-7 cells occurs partly by a facilitated process involving the scavenger receptor class B type I (SR-BI). Biochem J 2005; 387:455–46. 174. van Bennekum A, Werder M, Thuahnai ST, Han C-H, Duong P, Williams DL, Wettstein P, Schulthess G, Phillips MC, Hauser H.

Class B scavenger receptor-mediated intestinal absorption of dietary β-carotene and cholesterol. Biochemistry 2005;44:4517–4525. 175. During A, Dawson HD, Harrison EH. Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1 and ABCA1 is down-regulated in CACO-2 cells treated with ezetimibe. J Nutr 2005;135:2305–2312.

CHAPTER

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Vitamin D3: Synthesis, Actions, and Mechanisms in the Intestine and Colon J. Wesley Pike, Makoto Watanuki, and Nirupama K. Shevde Overview of Vitamin D Production and Physiology, 1754 Discovery of Vitamin D, 1754 Synthesis, Activation, and Degradation of Vitamin D, 1754 Regulation of Calcium and Phosphorus Homeostasis by 1,25-Dihydroxyvitamin D, 1755 Molecular Mechanism of Action of 1,25-Dihydroxyvitamin D, 1756 Discovery, Properties, and Cloning of the Vitamin D Receptor, 1756 Vitamin D Receptor Is a Member of the Steroid Receptor Gene Family, 1756 Vitamin D Receptor Functions as a Heterodimer with Retinoid X Receptor, 1757 Interaction of the Vitamin D Receptor with Target Genes, 1758 Process of Transcriptional Modulation, 1758 Dynamics of Vitamin D Receptor/Retinoid X Receptor Colocalization and Cofactor Recruitment to Target Genes in Intact Cells, 1759 Role of 1,25-Dihydroxyvitamin D in Activation of Gene Expression through Vitamin D Receptor, 1760 Proof That the Vitamin D Receptor Mediates 1,25-Dihydroxyvitamin D Action, 1760

Transport of Calcium across the Intestinal Epithelium, 1761 Calcium Transport Process, 1761 Calcium Transporters, 1761 Transient Receptor Potential Vanilloid Types 5 and 6 Are the Gatekeepers of the Calcium Absorption Process, 1762 Genetic Deletion of the Components of Transepithelial Calcium Transport, 1762 Regulation of Calcium Transporter Expression by 1,25-Dihydroxyvitamin D, 1763 Physiology of 1,25-Dihydroxyvitamin D Regulation, 1763 Regulation of the Expression of Calcium Transporting Genes, 1763 Mechanisms of 1,25-Dihydroxyvitamin D Regulation of the Calbindins D9K and D28K and of Transient Receptor Potential Vanilloid Types 5 and 6, 1763 Vitamin D Therapeutics and the Calcemic Side Effects, 1765 Vitamin D Actions in the Colon: Anticarcinogenic Actions and a New Vitamin D Receptor Ligand, 1765 Summary, 1766 References, 1767

Studies earlier in this century by a number of investigators including the groups of Mellanby (1), McCollum (2), Steenbock (3), and Windaus (4) led to the discovery and characterization of an important new bioactive substance termed vitamin D. Aside from the critical role of this vitamin in maintaining calcium and phosphorus homeostasis in

vertebrates, perhaps its most important feature was its ability to cure rickets in children and osteomalacia in adults (5). Although vitamin D was found to be produced in the skin after exposure to sunlight, more recent studies have shown that vitamin D undergoes sequential hydroxylations initially in the liver to 25-hydroxyvitamin D (25OHD) (6), and then in the kidney to 1,25-dihydroxyvitamin D (1,25(OH)2D3) (7), the final biologically active form of the original vitamin. The discovery that the renal synthesis of 1,25(OH)2D3 is tightly regulated by numerous factors that monitor the mineral status of the organism led to the initial suggestion that 1,25(OH)2D3 might be a steroid hormone, rather than a true vitamin (8). This insight proved to be correct as a result

J. W. Pike, M. Watanuki, and N. K. Shevde: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1753

1754 / CHAPTER 69 of a series of subsequent mechanistic studies that were performed over the past several decades. These studies have demonstrated that 1,25(OH)2D3 operates in target tissues including the intestine, kidney, and bone through its capacity to activate a nuclear receptor/transcription factor termed the vitamin D receptor (VDR) (9). This regulatory protein functions, in turn, to modulate the expression of specific genes with protein products that are responsible for maintaining calcium and phosphorus homeostasis. Regulation is enabled through direct interaction of the 1,25(OH)2D3-bound VDR with specific DNA sequence elements located within the gene promoters (10). The localization of the VDR at these sites provides a nucleation center for the recruitment of sets of coregulatory complexes that are essential for modifying gene transcription (11). Although variable, common features of this mechanism are used by all the steroid hormones, including the sex and adrenal steroids, thyroid hormone, the retinoids, and a variety of additional ligands that are produced in cells and that serve to modulate intracellular activity in either an autocrine or paracrine fashion, or both (12). 1,25(OH)2D3 functions to integrate the calcium and phosphorus regulating actions of the intestine, kidney, and bone, such that blood calcium and, to a lesser extent, blood phosphorus levels are maintained within tight limits (13). An increase in circulating 1,25(OH)2D3 levels accelerates the absorption of calcium and phosphorus by the intestine, accelerates the reabsorption of filtered calcium at the kidney, and may induce the mobilization of calcium and phosphorus from the skeleton. 1,25(OH)2D3 is produced in response to parathyroid hormone (PTH), an 84-amino-acid peptide, the elaboration of which from the parathyroid gland is sensitive to blood calcium levels (14). Thus, decreasing blood calcium triggers increased production of PTH by the parathyroid gland, which causes increased production of 1,25(OH)2D3 and enhanced actions on the three tissues responsible for calcium and phosphorus homeostasis. Calcitonin may provide a reciprocal regulation during conditions of high blood calcium (15) and fibroblast growth factor 23 (FGF23), a recently discovered phosphatonin-like factor, may also play a direct and essential role in phosphorus homeostasis (16). The general mechanism whereby calcium is absorbed across the intestinal epithelium is known to be complex and to involve several sequential steps (17). Early studies identified several components that were essential to the absorption process, including the cytosolic calbindin D9K and D28K calcium-binding proteins (18), basolateral calcium ATPase PMCA1b (plasma membrane calcium ATPase 1b) (19), and the sodium/calcium exchanger NCX1 (20). The expression and relative levels of these proteins are known to be regulated by the vitamin D hormone. Components essential to the uptake of calcium at the apical surface of the intestinal cell have been identified only recently, however, and include the two ion channel genes TRPV5 and TRPV6 (transient receptor potential vanilloid types 5 and 6) (21,22). These channel genes represent the “gatekeepers” of calcium uptake and are likewise dependent on 1,25(OH)2D3, as well as calcium, for their expression (23). Studies are now focused on the details

by which 1,25(OH)2D3 regulates the expression of these genes at the molecular level. This chapter provides an historical overview of the vitamin D endocrine system, as well as a contemporary view of the mechanisms through which the vitamin D hormone functions to modulate gene expression in the intestine, kidney, and bone. We then focus on the process of intestinal calcium transport across the intestinal epithelium and the molecular mechanisms whereby 1,25(OH)2D3 regulates the expression of the transport genes including the paramount “gatekeeper” TRPV6. The final section discusses the presence of the VDR in the colon, its activation by lithocholic acid (LCA), and the unexpected role of the VDR as an inducer of enzymes responsible for the detoxification of fecal bile acids (24).

OVERVIEW OF VITAMIN D PRODUCTION AND PHYSIOLOGY Discovery of Vitamin D Sir Edward Mellanby was the first to discover that cod-liver oil could cure rickets in experimental animals, prompting the suggestion that this substance might contain a fat-soluble, antirachitic substance (1). Although the activity initially was attributed to vitamin A, McCollum and coworkers (2) subsequently demonstrated that the substance was novel, and they named it vitamin D. It was demonstrated that this antirachitic vitamin could be produced through the exposure of skin or food substances to ultraviolet light (3), thereby providing a scientific explanation why in children rickets could be cured with sunlight or artificially produced ultraviolet light (5). The isolation and chemical identification of the two forms of the vitamin, vitamin D2 and vitamin D3, were eventually achieved by Askew and colleagues (25) and Windaus, Schenck, and von Werder (4) in 1936. Although this brought the initial discovery phase of research on vitamin D to a close, it was just the beginning in a long and continuing effort to elucidate the physiologic and molecular details of an intricate endocrine system essential to the maintenance of calcium and phosphorus homeostasis.

Synthesis, Activation, and Degradation of Vitamin D Extensive work beginning more than half a century ago has now demonstrated the biochemical processes whereby vitamin D is produced, activated, and degraded. As indicated earlier, vitamin D is produced in the skin after exposure to sunlight through a process that involves initial photolysis of cutaneous 7-dehydrocholesterol (provitamin D) to previtamin D, followed by rapid isomerization to authentic vitamin D (26). Vitamin D was discovered to undergo further metabolism, however; first in the liver to 25OHD (6), and then in the kidney to 1,25(OH)2D3 (7). The enzymes responsible for these conversions are cytochrome P450–containing, mixed-function oxidases (27). Several hydroxylases have been identified that

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1755 can carry out 25-hydroxylation of vitamin D, including mitochondrial CYP27A1 and microsomal CYP2D11, CYP2D25, CYP2J2/3, Cyp3A4, and most recently, CYP2R1 (27). CYP2R1, a 25-hydroxylase (25OHase) discovered in the liver by Cheng and colleagues (28) demonstrates high affinity for vitamin D and is perhaps the most compelling of the 25OHases. By far the most important hydroxylation of vitamin D occurs in the kidney through the actions of a mitochondrial CYP27B1, which results in 1,25(OH)2D3 (7). First discovered by Fraser and Kodicek in 1970 (29), this enzyme was cloned by St Arnaud and coworkers (30) and was shown to be responsible for the synthesis of 1,25(OH)2D3. Interestingly, genetic mutations in both CYP2R1 and CYP27B1 are associated with human disease, the former with a unique and perhaps rare form of hereditary rickets (31), and the latter with the syndrome of vitamin D–resistant rickets type 1 (VDDR-1) (32). The biochemical and skeletal phenotype of this latter syndrome has been recapitulated using homologous recombination through genetic deletion of the Cyp27b1 gene in mice (33,34). The activity of renal CYP27B1 is critical to the production and maintenance of physiologic levels of circulating 1,25(OH)2D3. Consequently, the synthesis and activity of CYP27B1 is tightly regulated by factors that are elaborated in response to changes in blood calcium. Most notable is PTH, which is produced in the parathyroid glands in response to hypocalcemia and which acts directly on the kidney to stimulate CYP27B1 gene expression (14,35). As serum levels of calcium increase to greater than acceptable limits, thyroid C-cell production of calcitonin suppresses CYP27B1 response; the mode of calcitonin action is not well characterized, however (15). CYP27B1 also is modulated independently by both calcium and phosphate signals, although the mechanisms through which regulation is carried out currently are undefined (36). Studies also have suggested a key role for FGF23 in the modulation of CYP27B1 gene expression. FGF23 appears to be the long sought after hormone phosphatonin (or a member thereof), which is thought to play a central role in phosphorus homeostasis (16). Linkage of FGF23 to the regulation of phosphate currently is derived from the phenotypes associated with tumorinduced osteomalacia (37), autosomal dominant hypophosphatemic rickets (38), and X-linked hypophosphatemic (39) syndromes, as well as from studies in which the FGF23 gene has been either deleted in mice (40) or overexpressed (41). The molecular mechanisms and signaling pathways whereby PTH, calcitonin, and FGF23 modulate the expression of renal CYP27B1 currently are under investigation. Importantly, completion of the vitamin D endocrine circuit involves a potent feedback mechanism through which 1,25(OH)2D3 acts to suppress CYP27B1 gene expression in the kidney and to down-regulate PTH expression and production by the parathyroid gland (16,42). A number of additional factors also regulate CYP27B1 including the sex hormones and adrenal hormones, prolactin and growth hormone (27). 1,25(OH)2D3 is degraded through the primary actions of CYP24A1, a microsomal enzyme that is found in virtually

all vitamin D target tissues and the expression of which is strongly induced by 1,25(OH)2D3 itself (43). The degradative pathway involves a third hydroxylation of 1,25(OH)2D3 at carbon 24 to 1,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3), followed by multistep destruction of the vitamin D side chain to calcitroic acid (44). 25OHD also is hydroxylated by CYP24A1, leading to high circulating levels of 24,25dihydroxyvitamin D3 (24,25(OH)2D3) (27). Although biological roles for 24,25(OH)2D3 have been hypothesized for many years, little in vivo evidence exists to support these views. Indeed, although deletion of the Cyp24a1 gene in the mouse by St Arnaud and coworkers (45) resulted in a phenotype of hypercalcemia, hypercalciuria, renal calcification, and skeletal abnormalities, all of these effects could be attributed to the toxic circulating levels of 1,25(OH)2D3, which coexist as a result of the deletion of Cyp24a1 (46). As indicated earlier, one of the fundamental actions of 1,25(OH)2D3 in all target cells is to stimulate the expression of CYP24A1, thus initiating the means of its own selfdestruction. The mechanism through which 1,25(OH)2D3 induces CYP24A1 transcription at the molecular level is now well understood (47,48) and has provided a useful paradigm for the study of transcriptional regulation by the vitamin D hormone.

Regulation of Calcium and Phosphorus Homeostasis by 1,25-Dihydroxyvitamin D Calcium and phosphorus homeostasis is maintained through the actions of the intestine, kidney, and bone (Fig. 69-1). These tissues serve to acquire mineral from the diet, to conserve mineral from glomerular filtrate, and to provide an immediately available source of skeletal mineral when the diet is deficient in calcium, phosphorus, or both. Integrating the actions of these tissues so that serum calcium and phosphorus levels are maintained within tight limits and at supersaturating concentrations relative to mineralized bone is the central function of 1,25(OH)2D3 (49). The actions of 1,25(OH)2D3 in the intestine are therefore focused on stimulating the production of proteins essential to the processes of dietary calcium and phosphorus absorption. 1,25(OH)2D3 also is able to promote the mobilization of calcium and phosphorus from the skeleton, a process that involves both stimulation of bone resorbing osteoclast activity and induction of new osteoclast formation from cellular precursors. This mechanism has a particularly profound consequence when dietary levels of calcium are insufficient. Accordingly, a physiologic attempt is made to maintain serum calcium and phosphorus levels at the expense of bone, which leads to bone demineralization, a weakening of the structure of the skeleton and increased risk for bone fracture. Depletion of serum levels of calcium and phosphorus may also lead to a failure of bone to mineralize, resulting in rickets or osteomalacia. 1,25(OH)2D3 also acts on the kidney to increase the resorption of calcium. Although this action is modest, the actual amount of calcium recovered through reabsorption can be highly significant because

1756 / CHAPTER 69 MOLECULAR MECHANISM OF ACTION OF 1,25-DIHYDROXYVITAMIN D

Bone

Discovery, Properties, and Cloning of the Vitamin D Receptor (−) 1,25(OH)2D3

PTG

Intestine

1(OH)ase

Ca/P

(+)

PTH (+) Kidney

(−) Npt2a

FGF23 Pi

Bone

FIG. 69-1. Regulation of calcium and phosphorous homeostasis in higher organisms. FGF23, fibroblast growth factor 23; PTG, parathyroid gland; PTH, parathyroid hormone.

of the large daily load of calcium that is filtered by the kidney. Central to the orchestration of intestinal, kidney, and bone actions by 1,25(OH)2D3 is the system that monitors the content of blood calcium and phosphorus. In the case of calcium, the level of this ion is monitored continually by the calcium-sensing receptor in the parathyroid gland, which responds when calcium levels decrease by increasing parathyroid gland secretion of PTH (50,51). As noted earlier, increased PTH levels enhance the synthesis of 1,25(OH)2D3, stimulating, in turn, both calcium uptake at the intestine and calcium reabsorption by the kidney (14,35). The actions of 1,25(OH)2D3 in conjunction with increased PTH on bone serve to promote bone mobilization, which also leads to an increase in blood levels of mineral. Although 1,25(OH)2D3 also acts to increase phosphate levels in the blood via actions on the intestine (49), both PTH and the newly identified phosphatonin FGF23 act collectively on the kidney to modulate phosphate reabsorption (40,41). Both the source of FGF23 and the sensing mechanism by which FGF23 secretion is accomplished during changes in phosphate levels remain unknown. These and additional actions on both the kidney and bone restore calcium and phosphorus concentrations to appropriate levels in the blood. Feedback mechanisms described earlier then act to prevent calcium and phosphorus levels from increasing beyond acceptable limits, thus maintaining mineral levels within narrow boundaries. The molecular mechanisms whereby 1,25(OH)2D3 induces the uptake of calcium and phosphorus across the intestinal epithelium, as well as its molecular actions in kidney and bone, are considered later in this chapter.

Evidence that vitamin D might function at the level of gene expression and could operate in a fashion similar to that of a steroid hormone prompted an early search for a binding protein or receptor capable of such action. After an initial indication of the existence of such an entity via in vivo studies (52), a binding protein eventually designated VDR was discovered first in the chick intestine (53,54), and then in other tissues such as kidney, bone, and parathyroid glands (55). Subsequent studies have shown the presence of the VDR in almost all vertebrate tissues, many of which do not play a role in calcium and phosphorus homeostasis (56). The VDR is a protein of 50 to 60 kDa that resides largely, although perhaps not exclusively, in the nucleus. The protein is able to bind 1,25(OH)2D3 with high affinity and selectivity, and it undergoes a significant biochemical change on ligand association. The VDR was subsequently discovered to bind directly to DNA through a domain separate from that which binds ligand (57,58). These functional properties of the VDR provided further support for the hypothesis that it might mediate the actions of 1,25(OH)2D3 at the level of transcription. The DNA-binding capacity of the VDR enabled investigators to purify the protein from chick intestine and to generate both polyclonal and monoclonal antibodies useful in the characterization of the protein (59,60). Immunologic and other techniques using these reagents demonstrated the rather extensive distribution of the VDR in tissues other than the intestine, kidney, and bone, suggesting additional roles for 1,25(OH)2D3 (61,62). A role for 1,25(OH)2D3 in the regulation of cellular growth and differentiation was concurrently demonstrated (63,64). Perhaps most importantly, a chicken VDR cDNA was cloned in 1987 (65), with the human (66) and rat (67) homologues closely following. This latter achievement opened the door to a myriad of studies that have shown not only the molecular structure of the protein, but precise details of its function as well.

Vitamin D Receptor Is a Member of the Steroid Receptor Gene Family The cloning of the VDR provided the final evidence that 1,25(OH)2D3 operated in a manner mechanistically similar to that of other steroid receptors. Indeed, the overall structural similarities of the VDR with that of several other steroid receptors that were cloned during that period were striking (Fig. 69-2) (68). The most salient of these structures was the presence of a highly conserved DNA-binding domain located in the central or amino-terminal region of the protein. This region is found in virtually all the 48 human steroid receptors

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1757 DNA binding

H12

C N 1

24

89

242

427

Hinge region

Ligand binding

A N-term H1 H9

H10 H8

H4 Peptide

H5 H7

H3 H12 C-term

H2 H6 H11 G218

M159

B FIG. 69-2. Organization of the vitamin D receptor (VDR). (A) The domain structure of the human VDR. (B) Ribbon diagram of the crystal structure of the ligand-binding domain of the rat VDR in association with both 1,25-dihydroxyvitamin D and an LxxLL peptide derived from Med220 (111).

currently known to exist (69). The steroid receptor gene family is perhaps the largest of the known transcription factor families of regulatory proteins and is composed of receptors for the bone fide steroidal ligands estrogen, androgens, progestins, glucocorticoids, mineralocorticoids, and thyroid hormone, as well as retinoids and 1,25(OH)2D3 (8). This steroid receptor gene family also contained a number of receptors with unknown ligands (70). Many of the ligands for these receptors have now been identified including several fatty acid derivatives, prostaglandins, oxysterols, cholesterolderived bile acids, and mevalonate pathway derivatives, as well as a variety of xenobiotics and drugs (71). Clearly, this family of transcription factors is involved in modulating an incredibly wide range of biological processes including those that impact growth, development, differentiation, and adult homeostasis. Surprisingly, members of this family of transcription factors comprise a substantial percentage of the genome of Caenorhabditis elegans (69).

Vitamin D Receptor Functions as a Heterodimer with Retinoid X Receptor Early studies using recombinant VDR indicated that the ability of the VDR to bind to DNA was dependent on an unknown nuclear factor (72,73). The identity of this protein was subsequently revealed when it was discovered that specific members of the steroid receptor family termed retinoid X receptors (RXRs) were capable of forming heterodimeric complexes with the VDR (74,75). Importantly, one of the roles of 1,25(OH)2D3 on ligand activation of the VDR is to increase the affinity of this receptor for an RXR partner such that cooperative, high-affinity DNA binding can be accomplished (76,77). Three different genes encode the RXRs, including RXRα, RXRβ, and RXRγ (78). Although all interact with the VDR in vitro, the precise hierarchy of interaction with these related proteins that may occur in vivo is unknown. The RXRs also represent heterodimeric binding

1758 / CHAPTER 69 partners for many other steroid receptors, including those for retinoic acid, thyroid hormone, and peroxisome proliferators (79). Interestingly, RXR can be activated independently by its own ligands, which include 9 cis-retinoid acid, as well as other fatty acids (80,81). This feature of the RXRs both as an independent transactivator and as a common interacting component of heterologous steroid receptors potentially links specific ligand-activated signaling systems together in cells where they are coexpressed. The role of RXR in VDR action appears to be largely one of facilitating and enhancing specific DNA binding to the promoter regions of target genes. Evidence has accumulated, however, to suggest that RXR also participates in the transcriptional activating process as well (82–84). The partnership between VDR and RXR was largely identified in vitro through biochemical and molecular techniques that involve overexpression of the two proteins, an approach with inherent artifacts. However, the association of RXR with the VDR on target genes and the dependency of RXR DNA binding on VDR activation has been determined in living cells using chromatin immunoprecipitation (ChIP) techniques (85). Additional studies have shown that depletion of RXRα but not RXRβ, in bone cells using small interfering RNA techniques reduces the capacity of 1,25(OH)2D3 to stimulate Cyp24a1 gene expression (86). Future studies are likely to clarify the role of the individual isoforms of RXR in 1,25(OH)2D3 action.

Interaction of the Vitamin D Receptor with Target Genes 1,25(OH)2D3 is known to regulate the expression of a number of genes, not only in the intestine, kidney, and bone, but in other tissues as well. Perhaps its most ubiquitous regulatory target is CYP24A1, which as indicated earlier functions negatively to down-regulate cellular levels of 1,25(OH)2D3 (27). In the intestine, 1,25(OH)2D3 regulates the transcriptional output of the calcium-binding proteins (calbindin D9K and D28K) (87,88), basolateral calcium-stimulated ATPases such as PMCA1b (89), and TRPV6 (which is considered in more detail later in this chapter). Proteins with analogous functions also are regulated by 1,25(OH)2D3 in the kidney. In bone, however, 1,25(OH)2D3 regulates a variety of additional genes, including the matrix proteins such as osteocalcin, osteopontin, and bone sialoprotein, that are involved in bone formation (90,91). 1,25(OH)2D3 also regulates RANKL (92) and OPG (93), factors that participate in the activation and induction of osteoclasts central to bone calcium and phosphorous mobilization (94). Investigation of each of these transcriptional targets at the molecular level has shown the presence of specific DNA sequences or vitamin D response elements (VDREs) that are located within the promoter regions of each gene and that represent high-affinity binding sites for the VDR/RXR heterodimer (Fig. 69-3) (36). VDREs are composed largely, but not exclusively, of two hexanucleotide half-sites with the consensus sequence AGGTCA that are separated by three base pairs of DNA. The details of individual VDRE sequences,

Canonical VDRE

5' -AGGTCA 3' -TCCAGT 5'Half-site

XXX XXX Spacer

AGGTCA-3' TCCAGT-5' 3'Half-site

FIG. 69-3. Structural organization of a canonical vitamin D response element (VDRE).

their number and promoter location within target genes, have been described extensively (36). The response elements that mediate the actions of 1,25(OH)2D3 in cis are similar, but not identical, to those for other nuclear receptors that use RXR as a partner (78). Most striking is that although the arrangement of the half-sites and their sequences are retained, the degree of nucleotide spacing that exists between the two half-sites is different. Thus, the diagnostic for a potential VDRE is not the sequence of the half-sites, but rather their separation by three base pairs (94). Interestingly, it is known from both molecular and structural studies that VDR and RXR bind in a fixed configuration, with VDR binding to the upstream 5′ half-site, whereas RXR binds to the downstream 3′ half-site (95,96). This orientation is determined by specific domains within the two proteins that enable the formation of a dimer during their association with DNA (97). 1,25(OH)2D3 also is known to suppress the expression of genes, among others those that are targets of negative feedback regulation such as the PTH gene (98) and CYP27B1 (99). Although the molecular basis for the suppression of CYP27B1 currently is not understood, the cis element that mediates PTH downregulation has been identified (98). Small differences in the sequence of one or both of the half-sites are currently suspected of dictating suppression, suggesting that DNA can allosterically regulate VDR activity.

Process of Transcriptional Modulation Like that of all members of the steroid receptor family, the association of the VDR/RXR heterodimer with specific DNA sequences initiates the processes essential to the modulation of transcriptional output of target genes. Although early studies suggested that the interaction of gene regulators such as the VDR with basal transcriptional machinery was sufficient to modulate gene output, it is now known that the process of transcriptional modulation is exceedingly complex and may involve a number of comodulatory complexes each with a unique function (Fig. 69-4) (100,101). Overcoming or restoring the inherent repressive state of chromatin requires the presence of regulatory machinery able to shift or displace nucleosomes (chromatin remodeling), or both; alter the condensation state, and therefore the architecture of chromatin (histone modification); and facilitate the entry of RNA polymerase II (RNA pol II). At least three complexes

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1759 Mediator complex 80

SWI/SNF (Chromatin remodeling)

150 205

130

230

Med6

100 Med7

BAF180 BAF155 BAF53

Histone acetylation BAF 60

BRG/hBRM CBP P300

SRC1 p160 PCAF

HAT RXR-VDR

ATP

CTD TFIIH

TFIIE

TFIIA TFIIB

VDRE Nucleosomes

RNA poll TFIID TFIIF

General transcription complex

FIG. 69-4. Model for the components and complexes essential to the activation of gene expression by 1,25-dihydroxyvitamin D. ATP, adenosine triphosphate; HAT, hitone acetyltransferase; RXR, retinoid X receptor; SRC-1, steroid receptor coactivator 1; SWI/SNF, switching defective/sucrose nonfermenting complex; VDR, vitamin D receptor; VDRE, vitamin D response element.

are believed to accomplish these specific actions. The first are the vertebrate ATPase-containing homologs of the yeast switching defective/sucrose nonfermenting (SWI/SNF) complex that uses the energy of ATP to remodel and reposition nucleosomes (102). This action increases the availability of binding sites for additional transactivators and facilitates the changes necessary for transcription to proceed. The second are complexes that contain either acetyltransferases or methyltransferases, deacetyltransferases (histone deacetylase complex [HDAC]), or demethylases, each of which function to modify the lysine- or arginine-containing tails of histone 3, histone 4, or both (100). These include the p160 family of coactivators steroid receptor coactivator 1 (SRC-1), SRC-2, and SRC-3 (103), as well as cyclic AMP response element binding protein (CREB) binding protein (CBP)/ p300 (104), the corepressors SMRT (silencing mediator of retinoid and thyroid hormone receptors) and nuclear receptor corepressor (NCoR), and one of several HDACs (105,106). These modifications alter the local condensation state of the target gene, thereby permitting or limiting the entry of additional factors essential to transcriptional modulation. The third complex is mediator, believed to facilitate the entry of RNA pol II and perhaps to play a role in transcriptional reinitiation (107,108). In addition to these three complexes, there are a number of additional complexes that are known to interact with nuclear receptors, although their precise roles in transcriptional modulation remain to be elucidated (109). Importantly, separate from its capacity to facilitate the

formation of VDR/RXR heterodimers and DNA binding is the role of 1,25(OH)2D3 to promote the formation of VDR/RXR-comodulator complexes. Because complex formation is mediated through a common domain within the VDR (and likely RXR) (110,111), two unanswered questions emerge: (1) Is there a temporal order to comodulator recruitment?; and (2) Are the coregulatory complexes recruited by receptors to every gene promoter in an identical fashion?

Dynamics of Vitamin D Receptor/Retinoid X Receptor Colocalization and Cofactor Recruitment to Target Genes in Intact Cells The bulk of the aforementioned studies examined the capacity of 1,25(OH)2D3 to stimulate VDR interactions with both RXR and coactivators using molecular techniques that involve overexpression, either in broken cell preparations or after transfection into living cells. More recent studies, however, have explored the ability of 1,25(OH)2D3 to induce VDR and RXR localization on natural target gene promoters using chromatin immunoprecipitation (ChIP) techniques in both intact intestinal and bone cells (85). These studies also examined the capacity of 1,25(OH)2D3 to stimulate coactivator recruitment and to induce covalent modifications to chromatin in the vicinity of these genes. In bone cells, 1,25(OH)2D3 induced the binding of both VDR and RXR to the promoter regions of Cyp24a1 and osteopontin (Opn). The binding of the VDR was

1760 / CHAPTER 69 dynamic in its initial phases and reached a maximum at approximately 3 hours. Coactivators responsible for acetylation such as the p160 family members, CBP and p300, also were recruited in response to 1,25(OH)2D3, as was the comodulator Med220, the protein that links activated VDR to Mediator (112). The recruitment of members of the acetyltransferase complex occurred earlier and correlated with lysine acetylation at histone 4. Med220 recruitment was slightly delayed and correlated directly with both VDR binding and the entry of RNA pol II. These studies provide strong “in vivo” evidence not only for the concept that VDR functions as an RXR heterodimer, but also for the idea that coregulators are recruited by the receptor heterodimer and are essential for the transcriptional activation of Cyp24a1, as well as Opn. A similar examination of these actions in intestinal cells is described later in this chapter.

Role of 1,25-Dihydroxyvitamin D in Activation of Gene Expression through Vitamin D Receptor The previous sections have demonstrated a role for 1,25(OH)2D3 in promoting both RXR heterodimer formation and the recruitment of cofactors both in vitro and in intact cells “in vivo.” It is now clear from molecular and structural studies that the potentiation of these interactions by 1,25(OH)2D3 is because of the ability of the ligand to induce changes in VDR conformation such that its affinity for both RXR and coregulators is increased. The evidence for 1,25(OH)2D3-induced conformational changes in the VDR has been derived from early, as well as more recent, in vitro experiments that demonstrate increased VDR resistance to proteolysis in the presence of ligand (58). Conformational changes in the VDR are likely induced on entry of 1,25(OH)2D3 into the carboxy-terminal ligand-binding pocket, where it is stabilized by direct contacts with specific VDR amino acid (111,113). Unfortunately, although the crystal structures of both the human and rat VDR ligand-binding domains (LBDs) provide detailed molecular support for receptor structure in the presence of 1,25(OH)2D3 (see Fig. 69-2) (111,113,114), relevant crystal structures for VDR in the absence of ligand have not been obtained. Conclusions drawn from the crystal structures of the unliganded LBDs of other nuclear receptor appear to be relevant, however, because the overall organization in nuclear receptor LBDs exhibits a significant degree of similarity (115). The conformational changes that are induced by 1,25(OH)2D3 result in a rearrangement of several of the 12 α helices that create both a contact heterodimer surface for RXR and a binding site for the coregulators. The coregulator binding site in the VDR is composed of an interaction among helices 3, 4, and 12. A molecular view of this relation has been acquired through cocrystallization of the rat VDR LBD with a peptide sequence that mimics the coactivator interacting domain of Med220 (see Fig. 69-2) (111). This sequence conforms to the leucine-charged receptor interacting region of most coregulators and is composed of a motif with a central core that contains the amino-acid

sequence LXXLL. A corepressor domain with the motif LXXXIXXX I/L binds to the same region of the VDR (116). These studies detail in exquisite terms the consequence of 1,25(OH)2D3 binding to VDR structure. Activation of the VDR by 1,25(OH)2D3 also leads to several additional events. The first event is the induction of a rapid phosphorylation of the VDR, which on the human protein occurs at VDR serine 208 (117,118), and perhaps at several additional serine residues (119,120). The role of this phosphorylation event(s) is unclear, although it may influence the ability of the receptor to interact with coactivators, and therefore to stimulate transcription (121). It is also possible that this event triggers the eventual degradation of the VDR and the termination of transcriptional activation. Future research is necessary to identify phosphorylation sites in VDRs from other species and to establish with certainty the role of these modifications in receptor function. Second, activation of the VDR by 1,25(OH)2D3 serves to up-regulate the VDR both at the VDR messenger RNA and protein levels (122). In intestinal and bone cells, up-regulation of the VDR protein is rather dramatic, resulting in at least a 10-fold increase (85). The mechanisms and consequences of both VDR phosphorylation and up-regulation remain to be determined.

Proof That the Vitamin D Receptor Mediates 1,25-Dihydroxyvitamin D Action Despite delineation of the mechanism of action described earlier, unequivocal evidence for the central role of the VDR as the mediator of the actions of 1,25(OH)2D3 is derived from experiments of nature. Early clinical studies defined a rare human syndrome termed vitamin D dependency rickets type 11, which was characterized by hypocalcemia, hypophosphatemia, severe skeletal and dental deformities, and in a portion of the cases, alopecia (123,124). Additional features include reduced circulating levels of 24,25(OH)2D3, and perhaps most striking, extremely high blood levels of 1,25(OH)2D3. This recessive syndrome results from consanguineous marriages and is manifested at a young age, suggesting a hereditary ideology. Patients generally are unresponsive to treatment with 1,25(OH)2D3. Significantly, treatment with 1,25(OH)2D3 was usually, although not always, ineffective. Interestingly, however, infusion with calcium and phosphorus in at least one case led to a normalization of serum calcium and phosphorus levels and improved the skeletal physiology (125). Importantly, as in the patients, fibroblastic cells derived from these affected individuals appeared to be fully resistant to the transcriptional activating abilities of 1,25(OH)2D3 (126). This syndrome exhibits all the features of a hormone resistance syndrome and was renamed hereditary 1,25-dihydroxyvitamin D resistant rickets type 11 (HVDRR type 11). It was subsequently shown that this syndrome derives from genetic mutations in the VDR gene, first in the exons encoding the DNA-binding domain that prevent association with target gene VDREs (127,128),

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1761 and then later in exons that encode domains that affect stability, RXR heterodimerization, and coregulator complex formation (129). Many of the features of HVDRR type 11 including those at the skeleton were recapitulated when Li and colleagues (130) and Yoshizawa and colleagues (131) successfully prepared mice nullizygous for the VDR alleles. These genetic strains of mice have proved useful in clarifying the skeletal actions of 1,25(OH)2D3, as well as identifying new physiologic actions not previously recognized.

TRANSPORT OF CALCIUM ACROSS THE INTESTINAL EPITHELIUM Calcium Transport Process As indicated earlier, the maintenance of extracellular calcium and phosphorus is vital to the proper functioning of virtually all cell types in vertebrates and other organisms (13). Although both the kidney and bone participate in mineral homeostasis, it is the intestine that ultimately is charged with making both minerals available from external sources. Thus, this section focuses on the events that occur in the intestine, although the process of conserving minerals at the kidney is highly related. Not surprisingly, the essentiality of calcium absorption, coupled to the need for this process to respond dynamically to the changing needs of the organism, places this process under the regulatory control of a wide variety of developmental, physiologic, homeostatic, and endocrine regulators, including 1,25(OH)2D3. Because of the complexity of the subject, we focus entirely on the regulation of calcium. More information is available in extensive reviews of the regulation of phosphorus metabolisms by 1,25(OH)2D3 in the intestine, as well as kidney and bone (132), and in other chapters in this textbook, particularly Chapter 77.

Calcium absorption occurs across the intestinal enterocyte. Although both paracellular and transcellular routes are operable, it is the transcellular route that facilitates the net movement of calcium from the gut lumen into the circulation (17). Transepithelial calcium transport is a complex, multistep process that entails the movement of the cation through the glycocalyx region and across the brush-border membrane into the cell interior, the translocation of the cation through the cytosol to the basolateral membrane, and finally its extrusion into the extracellular fluid of the lamina propria, where it is absorbed into the circulation (Fig. 69-5) (17). A number of physicochemical and thermodynamic parameters pertain to this uptake process, such that energy is required for the process to occur despite the presence of a steep downhill lumen to blood gradient (133,134).

Calcium Transporters Calcium Entry Although a number of transport mechanisms have been postulated to account for the uptake of calcium into the intestinal epithelial cell, the identity of the key components and the mechanisms of their action only recently have come together. It is now believed that the two ion channels TRPV5 and TRPV6, the latter perhaps more important, play paramount roles in the entry of calcium into the intestinal cell (see Fig. 69-5) (21,22,133,134). TRPV5 and TRPV6 were identified via functional expression cloning techniques by Hoenderop and colleagues (21) and Peng and colleagues (22), respectively, and were shown to retain an appropriate level of calcium-selective gating activity. TRPV5 and TRPV6 are members of a large superfamily of genes with products that are nonvoltage-gated ion channels that function as receptors

Ca2+

1,25(OH)2D3 Basolateral

Apical Ca2+ 3Na+ Intestine

TRPV6

Transcription

Ca2+ NCX1

ATP

Kidney

TRPV5

Entry

ADP

Calbindin-D

Translocation

Ca2+

PMCA1b

Extrusion

(Modified: Nijenhuis et al. [135])

FIG. 69-5. Model for the mechanism of transepithelial calcium transport by the intestine and kidney. 1,25(OH)2D3, 1,25-dihydroxyvitamin D; ADP, adenosine diphosphate; ATP, adenosine triphosphate; NCX, sodium/calcium exchanger; PMCA, plasma membrane calcium ATPase; TRPV, transient receptor potential vanilloid.

1762 / CHAPTER 69 for a diverse set of external stimuli and facilitate a wide range of biological processes (134). Perhaps most importantly, the tissue expression profiles, as well as the calciumtransferring capabilities of TRPV5 and TRPV6, support the idea that these ion channels represented the anticipated proteins responsible for the transcellular transport of calcium across both kidney and intestinal epithelia (134,135). Extensive work since the cloning of these genes has demonstrated important biophysical properties of the channels, their pore-forming properties, and the mechanisms by which they manifest calcium selectivity and inward-rectifying and gating capability (134). The mechanisms through which the protein channel activities are modulated and the components essential to this modulation are beginning to emerge. The discovery of these channels and the subsequent characterization of their mode of action in the intestine, as well as in the kidney, fill a crucial gap in our understanding of transepithelial calcium transport that has existed for many decades, and must therefore be considered a significant scientific advance in gastrointestinal biology. Calcium Diffusion The influx of calcium representing the first step in transepithelial transport must be buffered such that it does not perturb low intracellular calcium levels and transported through the cytoplasm to the basolateral membranes where it can be extruded into the circulation (17). Several hypotheses have been developed to account for these two events, and although the actual mechanisms are not yet defined, it is clear that the calcium-binding proteins calbindin D9K and D28K play essential roles in these processes (see Fig. 69-5). The calbindins are highly conserved cytosolic calcium-binding proteins that are rather ubiquitously expressed (87,88). They were proposed early on to act as cytosolic buffers capable of maintaining low intracellular calcium levels during transcellular calcium transport and to serve as shuttles for the diffusion of calcium. Physical interactions between the calbindins and both TRPV5 and TRPV6, as well as basolateral membrane calcium ATPase, have been postulated based on the coexpression of calbindin D9K and TRPV channels in tissues (135,136) and through the ability of calbindin D28K to enhance the calcium ATPase activity (137). Solid demonstration of these interactions would bolster the concept of a role for the calbindins in calcium transfer across the enterocyte. As discussed later, however, calcium transport does not correlate directly with the calcium-binding proteins (138,139) and can occur in the absence of these calciumbinding proteins. It is currently unclear whether the calbindins are inessential components of the calcium transfer process or whether they and many of their related family members simply play redundant roles. Calcium Extrusion at the Basolateral Surface Calcium is extruded from the enterocyte against a significant electrochemical gradient (17). Thus, this process

requires the energy of ATP. Members of both the sodium/ calcium exchangers (NCXs) and the PMCAs are expressed in the intestine (19,20), although it appears that the former functions primarily in the reabsorption of calcium by the kidney (136,140,141). These proteins likely mediate the extrusion of calcium from the enterocyte (see Fig. 69-5). PMCAs are expressed ubiquitously and are composed of at least four isoforms, two of which are believed to function in the general maintenance of cellular calcium homeostasis (142). PMCA1b, the isoform that is expressed predominantly in the intestine, is likely the calcium-regulated pump that functions to extrude calcium from the enterocyte and into the circulation (89,134).

Transient Receptor Potential Vanilloid Types 5 and 6 Are the Gatekeepers of the Calcium Absorption Process Calcium influx at the apical membrane in renal epithelium correlates directly with apical to basolateral calcium movement, suggesting that the rate of calcium influx represents the rate-limiting step in the net reabsorption of calcium (143). This finding, together with additional evidence, has prompted Hoenderop and colleagues (144) and others to characterize TRPV5 and TRPV6 as “gatekeepers” of transepithelial calcium reabsorption. If this concept proves to be true for the intestine, as well as the kidney, the roles of TRPV5 and TRPV6 in maintaining calcium homeostasis take on enormous significance. Perhaps as importantly, therefore, are the systemic and cellular factors that serve to regulate the expression of these genes or their activities. It is perhaps no coincidence that one of the primary regulators of TRPV gene expression in the intestine is 1,25(OH)2D3, the master regulator of calcium homeostasis.

Genetic Deletion of the Components of Transepithelial Calcium Transport The previous sections have described the component of the calcium absorption process in the intestine. They include apical TRPV6 (and TRPV5), cytosolic calbindin D9K (and 28K), and basolateral PMCA1b. Importantly, several of these genes have been deleted in mice through homologous recombination techniques, thereby providing additional in vivo support for the essentiality of their roles in the calcium transport process. Perhaps most illuminating were the phenotypes of the TRPV5 (145) and TRPV6 (146) knockout mice. In the former case, most significant was the observation that TRPV5 null mice exhibited a dramatic increase in the secretion of urinary calcium from the kidney, supporting the predicted role for TRPV5 in the kidney. The mechanism underlying this enhanced secretion was pinpointed in detail. Interestingly, normal serum calcium levels were maintained in the TRPV5 null mice primarily through an increased rate of absorption of calcium via the intestine. This increase in

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1763 absorption was likely caused by enhanced levels of serum 1,25(OH)2D3 evident in the TRPV5 null mice and increased levels of expression of both TRPV6 and calbindin D9K. Thus, the phenotype of this mouse supports both the role of TRPV5 in renal calcium reabsorption, as well as the relation between TRPV6 and calbindin D9K expression and calcium absorption by the intestine. It also hints at the central role of 1,25(OH)2D3 in the regulation of the components necessary for calcium homeostasis. Genetic deletion of TRPV6 in the mouse is likewise informative (146). TRPV6 null mice when placed on a calciumdeficient diet exhibited a significantly reduced capacity to absorb calcium. Although the phenotype of this animal will require additional evaluation, it is clear from current work in both the TRPV5 and TRPV6 null mice that each of these genes are critical to the normal maintenance of calcium homeostasis. Interestingly, the phenotype of a homozygous mutation in the calbindin D28K gene also showed an increase in calcium excretion coexistent with normal serum calcium levels (147,148). This finding suggests the possibility that the intestine is able to compensate via increased absorption of calcium through a mechanism at least related to that seen in the TRPV5 null mouse, and it supports a role for calbindin D28K in renal calcium reabsorption. The calbindin D9K gene has not been deleted and as such provides no information. As indicated earlier, one complication to the study of these phenotypes is the possibility that the functions of calbindin D9K and D28K and the myriad of other calciumbinding proteins are redundant, thus alleviating a potential phenotype. Unfortunately, deletion of PMCA1b (149), as well as NCX1 (150), results in embryonic lethality, preventing any conclusion with respect to the contribution of this transporter to the transepithelial calcium transport process.

REGULATION OF CALCIUM TRANSPORTER EXPRESSION BY 1,25-DIHYDROXYVITAMIN D Physiology of 1,25-Dihydroxyvitamin D Regulation As described earlier, vitamin D deficiency leads to a dysregulation of calcium homeostasis that results in hypocalcemia and hypophosphatemia, and eventually to rickets, osteomalacia, or both (13). Although this effect has long been known to be caused by a depletion of circulating 1,25(OH)2D3, the critical role of the hormone in this action is highlighted most effectively in the skeletal phenotype of the human syndrome of HVDRR-1 wherein mutations in the CYP27B1 gene prevent the production of 1,25(OH)2D3 (27). This phenotype also has been reproduced in mice nullizygous for the Cyp27b1 alleles (33,34). Importantly, in both the human syndrome and the animal model, the skeletal defects are fully normalized after treatment with physiologic levels of 1,25(OH)2D3 (151,152). These results provide unequivocal support for the central role of the vitamin D hormone in the regulation of calcium homeostasis.

Regulation of the Expression of Calcium Transporting Genes 1,25(OH)2D3 is known to regulate each of the components that mediate transepithelial calcium transport, including PMCA1b, the calbindins D9K and D28K, and both TRPV5 and TRPV6. The degree of regulation of PMCA1b is modest at best, however, and it currently remains unclear whether this is a direct effect of 1,25(OH)2D3 on PMCA1b transcription or an indirect effect mediated via a calcium flux that is capable of independently enhancing gene expression (153–155). The calbindins D9K and D28K, in contrast, are well-recognized targets of vitamin D action, having been discovered originally as a result of the physiologic activities of vitamin D (87,88). Accordingly, the expression levels of these proteins are reduced as a result of vitamin D deficiency (87,88,130,131), as well as genetic deletion of Cyp27b1 alleles in more recent studies (33,34). Importantly, a single dose of 1,25(OH)2D3 induces an up-regulation of calbindin D9K in the intestine and a modest up-regulation of calbindin D28K in the kidney in both systems (151,152). Perhaps most profound, however, is the capacity of 1,25(OH)2D3 to regulate the expression of renal TRPV5 and intestinal TRPV6 (156). Although numerous experiments have been conducted, the most compelling are those performed in both Cyp27b1 and VDR null mice (157–161). Both mouse strains show a dramatic depletion in the expression of TRPV5 and TRPV6. As with the calbindins, however, the reduced expression of TRPV5 and TRPV6 can be rescued directly in the Cyp27b1 null mouse by treatment with 1,25(OH)2D3 (162). The genetic deletion of the VDR prevents 1,25(OH)2D3 action, and therefore fully abrogates a similar rescue by 1,25(OH)2D3. Additional support for the regulatory role of 1,25(OH)2D3 in TRPV5 and TRPV6 expression derives from studies by Song and colleagues (163), who demonstrated that 1,25(OH)2D3 was able to up-regulate the expression of the two genes, as well as the calbindins, as a function of both dose and time. The Caco-2 colon tumor cell-culture model also supports such regulation in vitro (164,165). These and other studies provide unequivocal support for the capacity of 1,25(OH)2D3 to regulate the expression of genes with products that are essential for the transport process in both the intestine and the kidney.

Mechanisms of 1,25-Dihydroxyvitamin D Regulation of the Calbindins D9K and D28K and of Transient Receptor Potential Vanilloid Types 5 and 6 Although the mechanism by which 1,25(OH)2D3 induces the PMCA1b gene has not been explored, considerable effort has focused on understanding the molecular details associated with the regulation of both calbindin D9K and D28K by 1,25(OH)2D3. In addition to the calcium homeostatic organs of the intestine, kidney, and bone, mammalian calbindins also are expressed in a variety of other tissues (87,88). Their expression is regulated by a number of hormones and

1764 / CHAPTER 69 evaluation of the promoter regions of the genes for TRPV5 and TRPV6 using in silico techniques suggested the presence of potential canonical VDREs (160). This approach, however, is not definitive for numerous reasons. First, single base pairs can alter what may appear to be attractive VDRbinding sites. Second, the availability of these sites to the VDR/RXR heterodimer is uncertain in intact cells in the context of chromatin, and therefore must be assessed through functional means. Third, evidence has accumulated that the capacity of a transcription factor to bind an element does not always correlate with the ability of the factor to modulate the gene to which it is bound. Experimental investigations of both TRPV5 and TRPV6 promoter regions are nevertheless being conducted. With respect to the human TRPV6 gene, some progress has been made in defining both the locations of the VDREs and the identity of the coactivators that participate in the activation process. Interestingly, none of the VDREs located in the proximal promoter region of the TRPV6 gene and originally proposed to mediate 1,25(OH)2D3 action have emerged as functional. Figure 69-6 depicts at least five VDREs identified through a more recent in silico analysis of the human TRPV6 gene. These elements are located considerably upstream of the proximal promoter region of the TRPV6 gene. Using ChIP techniques, Watanuki and colleagues (174) scanned the first 5 kb of the TRPV6 promoter using human Caco-2 cells and demonstrated that despite substantial sequence identity with canonical VDREs, only those VDRE sequences located at −2.1 and −4.3 kb, and perhaps at −1.2 kb, actually represented binding sites for the VDR/RXR heterodimer. The −2.1- and −4.3-kb elements, but not the sequences at −1.2 or −3.5 kb, were subsequently shown to facilitate the primary transcriptional responses to 1,25(OH)2D3 when linked directly to a viral promoter and introduced via transfection into host cells. Subsequent studies using ChIP showed that the 1,25(OH)2D3-induced response was accompanied by a time-dependent recruitment of several coactivators to this region, including SRC-2 and SRC-3, as well as extensive acetylation to histone 4. These studies use a new approach to identify and characterize the molecular mechanisms involved in the regulation of TRPV6 gene expression in intact cells by 1,25(OH)2D3. Future studies are likely to focus on similar details of TRPV5 regulation.

factors, as well as by 1,25(OH)2D3 (166). Early studies of these genes suggested that 1,25(OH)2D3 might regulate their expression by both transcriptional and posttranslation mechanisms. Initial efforts focused on the molecular mechanisms whereby 1,25(OH)2D3 regulated calbindin D28K gene expression in the intestine of chickens (167). Surprisingly, to date, these efforts have failed to show regulatory elements or VDREs within the chicken promoter capable of significantly modulating the transcriptional output of this gene. Efforts focused on the mammalian calbindins D9K and D28K have been met with greater success. Indeed, similar approaches using both the rat calbindin D9K (168) and the mouse calbindin D28K (169) gene promoters showed the presence of VDREs located between −445 and −489 nucleotides in the rat gene and between −170 and −200 nucleotides in the mouse gene. These elements exhibited some similarity to the VDREs identified previously in the osteocalcin, Opn, and Cyp24a1 genes (36). There is considerable divergence, however, both in half-site sequence and in the spacing from the canonical VDREs found in the above-mentioned genes, suggesting the potential for additional complexity, perhaps related to the participation of calcium directly in the activation of calbindin gene regulation. Interestingly, neither of these VDREs manifest a strong capacity to drive transcriptional output in transfection studies, providing additional support for the hypothesis that the primary mechanism of calbindin D regulation by 1,25(OH)2D3 occurs at a posttranslational level (170,171). It is also notable that a calbindin D9K promoter gene construct containing the regulatory VDRE described earlier was introduced as a transgene into mice in vivo. This gene failed to demonstrate a response to 1,25(OH)2D3, although the inclusion of additional upstream sequence of the promoter restored that response (172,173). Once again, it appears as though other regulatory elements in the calbindin D9K gene may be required for synergistic modulation by 1,25(OH)2D3. Regardless, these efforts have advanced our understanding of the molecular mechanisms by which 1,25(OH)2D3 induces the expression of calbindin D gene targets. Studies focused on the molecular regulation of TRPV5 and TRPV6 by 1,25(OH)2D3 are less advanced, largely because the discovery of these genes has been more recent. An early

TRPV6 gene promoter TATAA 5

4

3

2

1

AGGTCAagaGGTCTA

AGGTCAtttAGTTCA

GGGGTAgtgAGGTCA

−7

−6

ATG

−5

GGGGCAgagAGGTCA AGGTCTtggGGTTCA

−4

−3

−2

−1

Kilobases

FIG. 69-6. Structural organization and location of vitamin D response elements (VDREs) in the human transient receptor potential vanilloid type 6 (TRPV6) promoter.

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1765 VITAMIN D THERAPEUTICS AND THE CALCEMIC SIDE EFFECTS In view of its role in calcium regulation, vitamin D and many of its more active metabolites including 1,25(OH)2D3 have been used for many years in the treatment of skeletal diseases such as rickets, osteomalacia, renal osteodystrophy, and various types of osteoporosis. Subsequent studies, however, showed that VDR was expressed in a wide variety of tissue types, including skin and both immune and tumor cells, and that 1,25(OH)2D3 could also function to modulate growth, cellular differentiation, activity, and survival (175). These discoveries led to the recognition that the hormone might also be useful therapeutically in the treatment of both proliferative disorders and immune system function (49,56,176,177). Ironically, the normal calcium homeostatic actions of 1,25(OH)2D3 in the intestine, kidney, and bone largely preclude the use of the hormone, because treatment results in increased uptake of calcium uptake from the gut, the mobilization of calcium from bone, and consequent hypercalcemia. These responses are detrimental to both softtissue calcium content and the skeleton, and they can prove to be lethal. This complication of what is viewed as a rather normal 1,25(OH)2D3 action in calcium homeostasis prompted the development of new vitamin D analogs, the altered biochemical and perhaps pharmacokinetic properties of which might make them more therapeutically useful in situations where high levels of the drug are required for efficacy. Many of these analogs failed to provide efficacy and most, similar to 1,25(OH)2D3, also stimulated the calcium homeostasis response. Interestingly, however, a series of vitamin D agonists was discovered that displayed significant potency and efficacy in suppressing tumor cell growth, yet no longer exhibited the ability to stimulate intestinal calcium uptake or bone calcium mobilization (178–181). It remains too early to conclude whether these compounds will indeed demonstrate significant clinical efficacy, particularly for such indications as psoriasis, autoimmune diseases, and cancer. However, the results of a number of preliminary trials have not been entirely encouraging. The vitamin D analogs such as those mentioned earlier display blends of biological potency, efficacy, and selectivity in vivo that differ from that seen for 1,25(OH)2D3. Despite considerable efforts, the underlying mechanisms that lead to these unusual effects remain unclear. Studies have suggested that ligand potency is largely caused by altered interaction with carrier proteins such as vitamin D–binding protein in the blood and both systemic and local Cyp24a1-mediated 1,25(OH)2D3 inactivation (182). In contrast, the biological selectivity of vitamin D ligands, as evidenced by the compounds that exhibit a reduced capacity to induce hypercalcemia and more recently by compounds that favor anabolic bone activity, remains unknown. Experiments using proteolytic digestion techniques have shown that a number of vitamin D ligands are able to alter the conformation of the VDR in ways that appear to differ from that induced by

1,25(OH)2D3 (183–185). Although not shown to be the direct result of conformational change, the activation of the VDR by these analogs in vitro results in VDRs with differing capacities to interact with RXR, members of the p160 family of coactivators, or Med220 (186–189). These altered interactions have been proposed to account for the tissue selectivity observed in vivo. Although an attractive hypothesis, none of these altered interactions between the VDR and either its heterodimer partner or the coregulators observed in vitro have been linked directly to a tissue or gene-selective action. Even more puzzling, the crystal structures of the VDR LBD in complex with several of these selective vitamin D analogs do not support a VDR conformation that is different from that with 1,25(OH)2D3 (111,114). These results suggest an alternative explanation that the tissueselective actions of many vitamin D analogs that exhibit a reduced ability to stimulate hypercalcemia arise from the pharmacokinetic features of the compound in vivo, rather than from the conformational differences observed in the VDR in vitro. It must be stated, however, that altered, ligandspecific VDR conformations that are capable of producing changes in VDR/coactivator interaction and novel transcriptional responses remain a formal possibility. At least one vitamin D analog that antagonizes 1,25(OH)2D3-induced Cyp24a1 induction fails to stimulate both VDR/RXR binding to that promoter and recruitment of p160 coactivators (85). This compound appears to induce a unique VDR conformation (115). Further studies are needed to determine fully why certain vitamin D analogs do not manifest calcium homeostatic capabilities.

VITAMIN D ACTIONS IN THE COLON: ANTICARCINOGENIC ACTIONS AND A NEW VITAMIN D RECEPTOR LIGAND Epidemiologic observations have implicated vitamin D in the prevention of colon and prostate cancer, as well as a reduction in autoimmune disease (190–192). This relation is believed to be caused by sunlight, with the higher latitudes resulting in a lower ultraviolet exposure that leads to greater incidences of these diseases (193). Importantly, the involvement of vitamin D in colonic health is supported by direct experimental evidence that has accumulated to support these relations such that supplementation of vitamin D reduces tumor development in a rat model of fat-induced carcinogenesis (194,195). At least a portion of these effects is likely to be caused by the growth suppressing effects of 1,25(OH)2D3 on colonic cells (196), similar to that described earlier. Some studies have shown that 1,25(OH)2D3 is able to induce detoxifying enzymes such as human CYP3A4 and perhaps other cytochrome P450 metabolizing enzymes (197,198), as well as the mouse homolog Cyp3a11 (199), in both the liver and intestine. These enzymes serve to detoxify not only a wide variety of xenobiotic agents such as drugs, carcinogens, and toxins, but also secondary bile acids such as LCA (200,201).

1766 / CHAPTER 69 The induction of human CYP3A4, as well as mouse Cyp3a11, is mediated by the VDR/RXR heterodimer through specific VDREs that are located within the promoter regions of these genes (Fig. 69-7) (198,202). Interestingly, these DNA sequence targets also are activated by two additional members of the steroid receptor gene family, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) (203–205). Once orphan receptors, these two receptors currently are known to be activated by a number of drugs including phenobarbital, rifampicin, clotrimazole, and high levels of dexamethasone. Interestingly, the capacity of VDR to bind to DNA sequences that represent PXR response elements now appears to be reflected in a perfect crossover; an observation suggests that ligand-activated PXR is able to induce Cyp24a1, the quintessential target gene for 1,25(OH)2D3 (206). Significant to Cyp3A4 action from a carcinogenic aspect is the capacity of the enzyme to detoxify LCA (see Fig. 69-7). LCA, as well as additional secondary bile acids, arises from bacterial activity and concentrates within cells where it is capable of inhibiting cellular enzymes and causing direct damage to DNA (207–209). Thus, the conversion of LCA to more polar hydroxylated or sulfonated by-products that can be eliminated is essential. The ability of 1,25(OH)2D3 to induce Cyp3A4 and other detoxifying enzymes of this class therefore likely contributes to reduced LCA levels, which, together with its growth-inhibiting properties, reduces the risk for colon cancer. Interestingly, cells of the colon represent one of several extrarenal sites in which the CYP27B1 gene is expressed and 1,25(OH)2D3 is produced from its 25OHD3 substrate (210). In a surprising and exciting finding, Makishima and colleagues (199) have shown that LCA can bind directly to the VDR and induce the binding of the receptor to VDREs located in several Cyp genes including CYP3A4 and Cyp3a11. Further studies in the PXR null mouse demonstrate that, similar to vitamin D compounds, LCA can also

A

1,25(OH)2D3 LCA(3-KetoLCA) RXR

VDR

hCYP3A4 −7722/−7719

−169/−152

+1

B hCYP3A4 LCA

Sulfolithocholic acid 60H Lithocholic acid

Excretion

FIG. 69-7. (A) Mechanism of induction of CYP3A4 by 1,25dihydroxyvitamin D (1,25(OH)2D3) and lithocholic acid (LCA). (B) Steps in LCA degradation by CYP3A4. RXR, retinoid X receptor; VDR, vitamin D receptor.

stimulate the expression of Cyp3a11 in both the liver and the intestine in vivo. These observations suggests that LCA induces the mechanism of its own detoxification in a manner virtually identical to that through which 1,25(OH)2D3 also induces it degradation. This has led to the proposed concept that the VDR represents a bile acid sensor. Regardless of whether the VDR is activated by 1,25(OH)2D3 or LCA, or both, these findings suggest a novel action for the VDR in the colon that contributes to the maintenance of bile acid homeostasis and perhaps a reduction in the risk for colon cancer.

SUMMARY This chapter has summarized the physiologic and molecular actions of vitamin D in the intestinal tract. Vitamin D is metabolized to 1,25(OH)2D3, the active form that functions through actions in the intestine, kidney, and bone to regulate calcium and phosphorus metabolism. 1,25(OH)2D3 operates as a steroid hormone in these tissues through its capacity to activate the VDR and to modulate the expression of genes. The mechanism of this modulation involves a primary association of the VDR and its heterodimeric partner RXR with DNA sequences located in the promoter regions of the target gene. Coregulators are subsequently recruited to this site, where they act to mediate the events required for transcriptional modulation. The vitamin D hormone is now known to regulate the expression of a number of genes that are essential to the process of calcium and phosphorous absorption by the intestine, as well as the reabsorption of mineral by the kidney and resorption of mineral from bone. Key genes with regard to intestinal calcium absorption include the apical ion channels TRPV5 and TRPV6, the soluble calcium-binding proteins calbindin D9K and D28K, and the basolateral ATPase pump PMCA1b. The molecular mechanisms whereby these genes are regulated by 1,25(OH)2D3 are currently being investigated. An understanding of the details by which 1,25(OH)2D3 regulates these genes is essential to our basic understanding of the process of calcium absorption. Vitamin D also plays a role in events unrelated to calcium homeostasis that involve the regulation of cellular growth, proliferation, and survival. These events suggest that vitamin D and certain analogs may be useful in the therapeutic treatment of hyperproliferative disorders such as psoriasis, autoimmune disease, and cancers. Because 1,25(OH)2D3 and many of its synthetic analogs hyperactivate calcium homeostatic mechanisms inherent to the intestine, kidney, and bone, this potential is unlikely to be realized until a fuller understanding of these basic mechanisms is appreciated. A novel class of vitamin D analogs has been discovered with a reduced tendency to activate the calcium absorption mechanism in the intestine and to mobilize bone calcium. The mechanism of this selective action remains to be identified. In another vein, studies have highlighted the ability of 1,25(OH)2D3 through its receptor to activate cytochrome P450–containing enzymes responsible for drug detoxification

VITAMIN D3: SYNTHESIS, ACTIONS, AND MECHANISMS IN THE INTESTINE AND COLON / 1767 in the colon. These enzymes also play a role in the detoxification of secondary bile acids such as LCA, which are known colonic carcinogens. The striking finding that LCA can also bind directly to the VDR and induce the enzymes responsible for the detoxification of LCA suggests that the VDR may play a homeostatic role in bile acid metabolism. Thus, the well-recognized impact of vitamin D in the prevention of colon cancer may be caused by a complex dual mechanism whereby both the cell growth–regulating and detoxification properties of the vitamin D hormone act in concert to prevent tumor development. The capacity of LCA to promote its own detoxification via the VDR (and PXR) indicates that this transcription factor may indeed represent a bile-acid sensor and adds an additional level of protection in the colon to a known carcinogenic agent.

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CHAPTER

70

Vitamin E and Vitamin K Metabolism Ronald J. Sokol, Richard S. Bruno, and Maret G. Traber Vitamin E, 1773 Structure and Biochemistry, 1773 Intestinal Absorption of Vitamin E, 1774 Hepatic α-Tocopherol Transfer Protein and Regulation of Serum Levels, 1775 Vitamin E Oxidation and Metabolism, 1776 Vitamin E Function, 1777 Physiologic Role of Vitamin E, 1778 Causes and Symptoms of Vitamin E Malabsorption and Deficiency, 1778 Assessment of Vitamin E Status, 1779 Treatment of Vitamin E Deficiency, 1779 Toxicity of Vitamin E, 1780 Vitamin K, 1781 Structure and Biochemistry, 1781 Dietary Sources and Human Requirements, 1781

Intestinal Absorption of Vitamin K, 1782 Vitamin K Metabolism, 1782 Vitamin K Function, 1782 Physiologic Role of Vitamin K, 1783 Causes of Vitamin K Malabsorption and Deficiency, 1784 Assessment of Vitamin K Status, 1784 Treatment of Vitamin K Deficiency, 1785 Toxicity of Vitamin K, 1785 Acknowledgments, 1785 References, 1785

VITAMIN E

group substitutions on the chromanol ring and in their bioactivity (1). All tocopherols occurring naturally in foods have the RRR stereochemistry in the side chain. Most vitamin supplements and fortified foods contain synthetic forms of vitamin E. Because α-tocopherol has three asymmetric carbon atoms, it has eight possible stereoisomers, seven of which are found only in synthetic preparations. Synthetic vitamin E (all-rac-α-tocopherol, incorrectly labeled dl-αtocopherol historically) contains all eight stereoisomers in equal amounts. Although RRR-α-tocopherol is the most biologically active, the other 2R-sterosiomers generally have higher activity than the 2S-stereoisomers, but considerably less than the RRR-isomer (2). The naturally occurring stereoisomer is RRR-α-tocopherol (incorrectly labeled d-αtocopherol historically), which is labeled natural source vitamin E when marketed. Esterification of the labile hydroxyl group on the chromanol ring helps to prevent its oxidation, and thus will prolong the shelf life for products containing these esters. For this reason, vitamin E supplements typically are synthesized as acetate, succinate, or nicotinate esters of tocopherol. These esters require hydrolysis before intestinal absorption, but this apparently occurs efficiently in the small intestine of healthy humans.

Structure and Biochemistry The term vitamin E refers to a group of eight naturally occurring compounds, including the four tocopherols and the four tocotrienols, which consist of substituted hydroxylated chromanol ring systems linked to isoprenoid side chain (Fig. 70-1). Tocotrienols differ from tocopherols in that they have an unsaturated side chain rather than a saturated one. Within each class, the four major forms of vitamin E (α, β, γ, and δ) differ by the number and position of the methyl

R. J. Sokol: Department of Pediatrics, Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Pediatric General Clinical Research Center, University of Colorado School of Medicine, The Children’s Hospital, Denver, Colorado 80218. R. S. Bruno: Department of Nutritional Sciences, University of Connecticut, Storrs, Connecticut 06269. M. G. Traber: Department of Nutrition and Exercise Sciences, Linus Pauling Institute, Oregon State University, Corvallis, Oregon 97331. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1773

1774 / CHAPTER 70 R1

Tocopherols

HO α β γ δ

CH3 R2

O R3

CH3

H

H

CH3

H

CH3

CH3

R1

R1 CH3 CH3 H H

R2 CH3 H CH3 H

R3 CH3 CH3 CH3 CH3

Tocotrienols

HO

CH3 O

R2 R3

CH3

CH3

CH3

CH3

FIG. 70-1. Chemical structures of the four tocopherols and four tocotrienols.

to assess activity. More recently, it has been proposed that less bioactivity be assigned to the synthetic forms because they are not maintained in serum (1). For each form of vitamin E, there is a different conversion factor from milligrams to International Units (1).

Because the various forms of vitamin E are metabolized and secreted in different manners in humans, they have different bioactivities. α-Tocopherol (which has three methyl groups on the chromanol ring) has the highest biologic activity in humans and is the predominant form in foodstuffs, together with γ-tocopherol (soy and corn oils). Tocotrienols are the predominant form of vitamin E in palm and rice bran oils. The common dietary sources of vitamin E are oilcontaining grains, plants, and vegetables. The Recommended Dietary Allowance (RDA) for α-tocopherol is 15 mg/day among individuals 14 to 70 years old, 6 to 11 mg/day for those between 1 and 13 years old, and 19 mg/day in lactating women (1). None of the other seven forms of vitamin E has a dietary requirement because they are poorly recognized by the α-tocopherol transfer protein (α-TTP) and they cannot be converted to α-tocopherol (1). The interconversion of milligrams to International Units (IU) or USP (United States Pharmacopeia) units is 1 mg dl-α-tocopheryl acetate (all racemic-α-tocopherol) = 1 IU or USP unit. One milligram of RRR-α-tocopherol was calculated to be equivalent to 1.49 IU (USP) vitamin E using the rat fetal absorption assay

Intestinal Absorption of Vitamin E All vitamin E compounds are highly lipophilic based on their chemical structure. Therefore, after oral ingestion in foods or in a vitamin supplement, vitamin E requires solubilization by bile acids secreted by the liver into mixed micelles, so that the highly hydrophobic vitamin E can traverse the aqueous environment in the intestinal lumen to reach the surface of the absorptive enterocyte, where 20% to 40% of the vitamin is absorbed (3) (Fig. 70-2). Before intestinal absorption, most esters of vitamin E are hydrolyzed by esterases either secreted by the pancreas or located in the intestinal mucosa (4). Intraluminal bile acid concentrations greater than the critical micellar concentration are critically important in promoting Hepatic uptake

Vitamin E

Circulating lipoproteins

Vitamin E & fatty acids to tissues

Vitamin E Lipoprotein lipase

Circulating plasma

Pancreas

Liver

Vitamin E enriched chylomicrons Bile & pancreatic secretions

Dietary vitamin E

Mesenteric iymphatics Vitamin E

Small intestine

Mixed micelles

FIG. 70-2. Intestinal absorption of vitamin E. Absorption is dependent on proper functioning of the liver, pancreas, enterocyte, and mesenteric lymphatics.

VITAMIN E AND VITAMIN K METABOLISM / 1775 adequate solubilization of tocopherols that is essential for their absorption (5). All forms of vitamin E are absorbed by a nonsaturable, noncarrier-mediated passive diffusion process, dependent on an adequate surface area of intestinal mucosa (3). Once inside the enterocyte, α- and γ-tocopherol are incorporated with other products of dietary lipid digestion and with apolipoproteins into newly synthesized chylomicrons and very low-density lipoproteins (VLDLs), which are secreted into the intercellular space and transported via mesenteric lymphatics and the thoracic duct into the systemic circulation. All forms of vitamin E studied have shown similar efficiencies of absorption and subsequent secretion into chylomicrons (6,7). During chylomicrons catabolism by lipoprotein lipase, some vitamin E is redistributed to all of the circulating lipoproteins and to the tissues (8). Impairment of any of the processes involved in vitamin E solubilization, digestion of dietary lipids, absorption, or transport leads to malabsorption and eventual deficiency of vitamin E if prolonged (3,4). Chylomicron remnants that contain newly absorbed vitamin E are taken up by the liver by receptor-mediated uptake. This pool of vitamin E is secreted by the liver in VLDLs, with preferential secretion of only one form of vitamin E, RRR-α-tocopherol (Fig. 70-3) (6). Thus, it is the liver, and not the intestine, that is responsible for the preferential enrichment in plasma of α-tocopherol to the expense of other forms of vitamin E. Current evidence points to the hepatic α-TTP as being responsible for this discrimination (9). Almost all circulating vitamin E is found in lipoproteins, and red blood cell membranes may also be enriched. Tissues acquire vitamin E through LPL lipolysis of circulating chylomicrons and VLDLs, during high-density lipoprotein (HDL)–mediated delivery, during low-density lipoprotein (LDL) uptake via the LDL receptor, and probably by nonspecific transfer between lipoproteins and tissues (1). Vitamin E also rapidly transfers among various circulating

α-T enriched VLDL α- and γ-CEHC P450-mediated metabolism

α-TTP mediated

Lipolysis HDL

Biliary excretion

Liver

α-T

α-T Vitamin E

Tissues

LDL

Chylomicron remnants

FIG. 70-3. Hepatic vitamin E secretion, metabolism, and excretion. α-CEHC, 2,5,7,8-tetramethyl-2-(2′-carboxyethyl)6-hydroxychroman; γ-CEHC, 2,7,8-trimethyl-2-(2′carboxyethyl)6-hydroxychroman; HDL, high-density lipoprotein; LDL, low-density lipoprotein; α-TTP, α-tocopherol transfer protein; VLDL, very low-density lipoprotein.

lipoproteins and between lipoproteins and membranes (10). Vitamin E is primarily stored in adipose tissue, which can be released slowly during times of dietary deprivation. Hepatic α-Tocopherol Transfer Protein and Regulation of Serum Levels α-TTP was first identified in 1977 (11) and was shown to transfer α-tocopherol between liposomes and microsomes (12). It is now believed that α-TTP is the hepatic protein that recognizes RRR-α-tocopherol from incoming chylomicrons and preferentially regulates its resecretion into hepatic-derived VLDLs (13). Hepatic α-TTP has been isolated, and its complementary DNA sequences have been reported from a variety of species including human, mouse, rat, dog, and cow. The human protein, which encodes 238 amino acids, has 94% homology to the rat protein and some homology to the retinaldehyde binding protein in the retina and to sec14, a phospholipid transfer protein (14). The human gene has been sequenced and localized to 8q13. 1-13.3 of chromosome 8 (14,15). α-TTP was crystallized by two different groups (16,17). The structure includes a α-tocopherol–binding pocket, which has a hinge and cover that closes entrapping α-tocopherol. Although expression of α-TTP was first reported to be limited to hepatocytes (18), α-TTP messenger RNA (mRNA) also has been detected in rat brain, spleen, lung, and kidney (19), and α-TTP protein has been detected in human brain (20). Furthermore, α-TTP is present in pregnant mouse uterus and human placenta (21,22), suggesting that it functions to ensure adequate α-tocopherol transfer to the fetus during pregnancy. In fact, placental α-TTP mRNA expression was second only to that in the liver (23). It was also reported that α-TTP was localized not only in the cytosol, but was predominantly located in nuclei of the trophoblast and in the endothelium of the fetal capillaries. The cellular mechanism by which α-TTP facilitates preferential secretion of α-tocopherol into plasma by the liver has not been clarified. Triglyceride-rich chylomicrons and VLDLs and LDLs carrying vitamin E are taken up by the liver via receptor-mediated endocytosis. Horiguchi and colleagues (24) suggest that α-TTP translocates from the cytosol to endosomes to acquire α-tocopherol, and then the α-TTP/αtocopherol complex moves to the plasma membrane where α-tocopherol is released to the membrane to be acquired by circulating lipoproteins, especially VLDL. Zha and colleagues (25) have reported that the adenosine triphosphate (ATP)– binding cassette protein A1 (ABCA1) in endosomes also plays a role in endocytosis by acting as a flippase to translocate phosphatidyl serine to the outer membrane and potentiate membrane budding. Because ABCA1 can also transfer α-tocopherol (26), ABCA1 could enrich the outer membrane of the endocytic vesicles with α-tocopherol; then α-TTP could preferentially remove RRR-α-tocopherol from the outer leaflet of the endosomal membrane for transfer to the plasma membrane. It remains to be clarified whether ABCA1

1776 / CHAPTER 70 participates in α-tocopherol transfer directly to and from α-TTP, as was suggested by Horiguchi and colleagues (24), or if some other proteins also are involved in α-tocopherol trafficking. It now appears that the affinity of α-TTP for vitamin E analogs is one of the critical determinants for plasma concentrations of the various forms of vitamin E (27). α-TTP has highest affinity for RRR-α-tocopherol (100%), followed by β-tocopherol (38%), γ-tocopherol (9%), δ-tocopherol (2%), α-tocopherol acetate (2%), α-tocopherol quinone (2%), SRR-α-tocopherol (11%), α-tocotrienol (12%), and trolox (9%) (27). Proof of the importance of this protein in regulating plasma levels is derived from knockout mice and humans with a genetic deficiency of this protein. In α-TTP knockout mice, plasma and tissue α-tocopherol concentrations are 2% to 20% of control mice (28,29), and the mice lack the ability to discriminate between the naturally occurring RRR-αtocopherol and synthetic, all-rac-α-tocopherol (28). Since the mid-1980s, several dozen patients with neurologic findings consistent with vitamin E deficiency have been found to have low plasma levels, but no evidence of fat malabsorption (30,31). These patients with “ataxia with vitamin E deficiency” (AVED) were unable to maintain normal plasma α-tocopherol concentrations without supplementation with large oral doses of vitamin E. Although they had normal intestinal absorption of vitamin E, they were unable to secrete α-tocopherol from the liver in VLDLs (9) and were unable to discriminate between forms of vitamin E (32). AVED subsequently was shown to result from homozygous mutations in the gene coding for α-TTP (31,33). Thus, genetic mutations of α-TTP in humans and gene manipulation in mice conclusively demonstrate the importance of α-TTP in regulating normal serum concentrations of vitamin E. An additional cytosolic protein that regulates tissue α-tocopherol concentrations has been identified in bovine liver as the 46-kDa tocopherol-associated protein (TAP) (34). Subsequently, the human homologue, hTAP, was cloned (35). hTAP is expressed the most in the liver, brain, and prostate (35). It has been found that TAP is identical to supernatant protein factor (SPF) (35,36), which enhances cholesterol synthesis by stimulating squalene conversion to lanosterol (36). Of interest was the finding that human TAP/SPF complexes with RRR-α-tocopheryl quinone, the oxidation product of α-tocopherol (37), suggesting a potential role in regulating tocopherol catabolism. The physiologic function of TAP/SPF remains under investigation.

Vitamin E Oxidation and Metabolism α-Tocopherol can be oxidized to the α-tocopheroxyl radical (Fig. 70-4) by free radical attack and may be reduced back to the unoxidized α-tocopherol by reducing agents, such as vitamin C, glutathione, and coenzyme Q (38). Further oxidation of the tocopheroxyl radical generates tocopheryl quinone, which is not converted back to tocopherol. Other oxidation products, dimers, and trimers occur in vitro; however, their role in vivo is unknown.

• O

R

H3 C

Tocopheroxyl Radical

O

CH3

CH3 CH3

CH3

Tocopherol oxidation R

CH3

CH3

e-

H3 Tocopherol

HO

CH3 O

H3 C CH3

CH3

CH3

Cytochrome P450 mediated

CH3

CH3

Tocopherol metabolism

R HO COOH

H3 C

O

CH3

CEHC

CH3

FIG. 70-4. α-Tocopherol oxidation and metabolism.

All forms of dietary vitamin E are absorbed and delivered to the liver, but only α-tocopherol is preferentially recognized by hepatic α-TTP. Unlike other fat-soluble vitamins, vitamin E does not accumulate in the liver to toxic levels, implying that excretion and metabolism of vitamin E are important physiologic processes. The two major vitamin E metabolites, α-CEHC (2,5,7,8-tetramethyl-2[2′-carboxyethyl]-6-hydroxychroman) and γ-CEHC (2,7,8trimethyl-2-[2′carboxyethyl]-6-hydroxychroman), result from degradation of the phytyl tail without changes of the chromanol ring (Fig. 70-5). These substances have a shortened tail with a carboxyl group. CEHCs are derived from both tocopherols and tocotrienols (39). These metabolic, not oxidative, products result from a cytochrome P450 (CYP)–initiated process. Hepatic CYP4F2 is involved in ω-oxidation of α- and γ-tocopherols (40), but CYP3A may also be involved (41–44). It is likely that liver synthesizes CEHCs (42,45), but other tissues involved in vitamin E metabolism have not yet been identified. Vitamin E metabolism is similar to that of xenobiotics. Initial ω-oxidization by CYPs is followed by removal of two carbon units through β-oxidation (40–42), followed by glucuronidate conjugation or sulfation (46–48), and finally by excretion in urine (49) or bile (50). Xenobiotic transporters are likely candidates for mediating hepatic CEHC excretion. In addition, α-tocopherol is excreted into bile via multidrug resistance gene 2 (MDR2; p-glycoprotein) (51), an ABC transporter that facilitates biliary phospholipid excretion. γ-Tocopherol and γ-tocotrienol are more actively metabolized to CEHCs than are α-forms (39,48,52). High α-tocopherol intakes, for example, most vitamin E supplements, lead to plasma increases of α-tocopherol, decreases of γ-tocopherol (53), and increases in excretion of both α-CEHC (54) and γ-CEHC (39). Studies in patients with end-stage renal disease demonstrated that vitamin E

VITAMIN E AND VITAMIN K METABOLISM / 1777 CH3 HO

HO COOH O

H3C CH3

CH3

α-CEHC

COOH H3C

O CH3

CH3

γ-CEHC

FIG. 70-5. Major metabolites of vitamin E. α- and γ-CEHC (carboxyethyl-hydroxychroman) are the final excretory metabolites of α- and γ-tocopherol, respectively.

supplementation was accompanied by a concomitant decrease (∼1 µM) in circulating γ-tocopherol and a ∼1-µM increase in circulating γ-CEHC (54), suggesting that α-tocopherol decreases γ-tocopherol concentrations by increasing γ-tocopherol metabolism. Thus, metabolism appears to be a key regulator of plasma γ-tocopherol concentrations and may be dependent on α-tocopherol intakes (55,56). Because non–RRR-α-tocopherols such as γ-tocopherol or all-rac-α-tocopherol are preferentially metabolized (52), it appears that forms of vitamin E that are not actively transported from liver to the plasma are expendable. However, it is unclear how certain forms of vitamin E are diverted within the hepatocyte to be metabolized. One possibility is that there is a specific vitamin E pool destined to be metabolized, such as the vitamin E that is delivered to the liver by HDL, analogous to reverse cholesterol transport (57). HDL could deliver tocopherols for excretion into bile via the SR-BI (scavenger receptor class B type I) pathway (58). In support of this hypothesis, Mardones and colleagues (59) showed in SR-BI knockout mice that SR-BI–mediated hepatic α-tocopherol uptake was coupled to its biliary excretion. Moreover, vitamin E–deficient rats had increased liver SR-BI protein levels, suggesting that SR-BI is regulated by α-tocopherol (60). Increased SR-BI would serve to increase vitamin E delivery via HDL to the liver, potentially providing a metabolizable pool of vitamin E. HDL γ-tocopherol could be preferentially metabolized or excreted from this pool. Clearly, this is a pathway for tocopherol excretion into bile; identification of a CEHC excretion pathway remains to be described.

Vitamin E Function Vitamin E functions as a chain-breaking antioxidant that prevents propagation of free radical reactions. It functions as a peroxyl radical and singlet oxygen scavenger, and prevents peroxidation of polyunsaturated fatty acid (PUFA) residues within membrane phospholipids and in circulating lipoproteins. The phenolic hydroxyl group on the chromanol ring reacts with the peroxyl radical, yielding the tocopheroxyl radical and a stable organic hydroperoxide. In this manner, vitamin E is the major lipid-soluble antioxidant preventing lipid peroxidation of cellular membranes. In addition, γ-tocopherol, because of its unsubstituted position on the chromanol head, has reactive nitrogen oxide scavenging ability that results in the formation of 5-nitro-γ-tocopherol. Thus, it may

protect other biomolecules from peroxynitrite-mediated damage (61). Several other functions of vitamin E have been identified. α-Tocopherol inhibits protein kinase C (PKC) in smooth muscle cells, human platelets, and monocytes (62). PKC is involved in cell proliferation and differentiation. Vitamin E also has been shown to regulate expression of a number of genes, including down-regulation of intercellular adhesion molecule-1, and up-regulation of cytosolic phospholipase A2 and cyclooxygenase-1 (63). This latter effect may explain the ability of tocopherol to enhance release of prostacyclin, a vasodilator and platelet aggregation inhibitor. Most recently, vitamin E has been found to interact with the pregnane X nuclear receptor (PXR) (64). Nuclear receptors are a superfamily of ligand-activated transcription factors, which stimulate expression of genes by binding to short DNA sequence motifs (response elements) located in the regulatory regions of target genes. PXR is a broad-specificity nuclear receptor that recognizes xenobiotics (65,66). On ligand binding, PXR, as a heterodimer with the retinoid X receptor (RXR), binds to the promoter region of genes and induces oxidation systems (phase I), conjugation systems (phase II), and transporters (phase III) that effectively clear xenobiotics from the liver (67). PXR-regulated genes include those that encode some CYPs, aldehyde dehydrogenases, uridine diphosphate glucuronosyltransferases (UGTs), sulfonyl transferases, glutathione S-transferases (GSTs), and transporters such as MDR1 and organic anion transporter peptide 2 (68). Phase II systems conjugate various substrates, for example, UGT glucuronidate and sulfotransferase sulfate substrates. PXR regulates some UGTs (Ugt1a1 and Ugt1a9) that are involved in glucuronidation of bilirubin and phenolics (69) and also regulates dehydroepiandrosterone sulfotransferase (70). PXR regulation of phase III systems has not been studied extensively. Although PXR appears to regulate multidrug-associated protein 2 (MRP2) that secretes conjugated xenobiotics into the bile (71), it does not regulate MRP1 that secretes conjugates into plasma (72). In an in vitro PXR-reporter assay, γ-tocotrienol, the form of vitamin E with the most PXR agonist activity, stimulated both a PXR-reporter construct and CYP3A expression by hepatocytes (64) as strongly as rifampicin, a known stimulator of both PXR and CYP3A. In addition, hepatocyte incubation with rifampicin and vitamin E resulted in production of increased amounts of vitamin E metabolites (41). Moreover, in hepatocytes, tocotrienols activate SXR (human

1778 / CHAPTER 70 form of PXR) and induce CYP3A4 expression, but have no effect on UGT1A1 (phase II) and MDR1 (phase III) expression, whereas in intestinal cells, tocotrienols induce UGT1A1 and MDR1 gene expression, but have no effect on CYP3A4 expression (73). Thus, tocotrienols provide the first example of a novel class of SXR ligands, which may influence metabolism of xenobiotics and that of vitamin E. Moreover, interactions with other nuclear receptors may facilitate effects of vitamin E on regulation of other genes.

Physiologic Role of Vitamin E The physiologic role of vitamin E in humans can be inferred by the effects of clinical deficiency. Symptoms of vitamin E deficiency suggest that its antioxidant properties play a major role in protecting tissues from oxidative stress. Clinical deficiency of vitamin E in humans causes hemolysis of varying degrees depending on age and status of other antioxidants, prooxidants, and PUFAs. Thus, preterm infants on diets high in PUFAs and receiving iron supplements experienced development of hemolysis and peripheral edema that was associated with vitamin E deficiency (74). Patients with cystic fibrosis (CF) and vitamin E deficiency caused by fat malabsorption experienced a mild degree of hemolysis with mild anemia (75,76). The primary and most serious clinical manifestation of human vitamin E deficiency is a peripheral neuropathy with degeneration of large-caliber axons in sensory neurons (5,77–79). Involvement also includes the spinocerebellar tracts, cranial nerve nuclei 3 and 4, posterior columns of the spinal cord, gracillus and cuneatus nuclei in the brainstem, skeletal muscle, and ocular retina (79–82). The development of a progressive ataxia appears more rapidly in infants and young children with malabsorption from birth than in adults, suggesting that the developing nervous system is dependent on adequate vitamin E for normal structural and physiologic development (83). Vitamin E also appears to be essential in preserving immunologic function in elderly individuals (84,85), especially concerning the immune synapse among CD4+ T lymphocytes that is important for T-cell signaling and immune function (86). Both animal (87,88) and human studies (85) suggest that relative vitamin E deficiency enhances and vitamin E supplementation prevents aging-associated reductions in immune function. However, studies suggest that vitamin E supplementation in the elderly may not be as protective against lower respiratory tract infectious illness as previously postulated (89).

Causes and Symptoms of Vitamin E Malabsorption and Deficiency Malabsorption is the most common cause of serious vitamin E deficiency because tocopherols are ubiquitous in the Western diet. Thus, clinically significant vitamin E deficiency

almost never occurs on a dietary basis. Rather, conditions in which there is impaired gastrointestinal, pancreatic, lymphatic, or hepatic function can interfere with the digestion or absorption of dietary lipid and vitamin E and can lead to clinical deficiency (4,90). Indeed, low concentrations of vitamin E have been observed in patients with a variety of steatorrheic disorders, including abetalipoproteinemia and other disorders of lipoprotein secretion, chronic cholestatic hepatobiliary disease in children and in adults, CF and other causes of chronic pancreatic insufficiency, short bowel syndrome, radiation enteritis, congenital intestinal lymphangiectasia, intestinal pseudoobstruction, Crohn’s disease, and others (91–93). The major exception is the isolated vitamin E deficiency syndrome (also called AVED syndrome), an autosomal recessive genetic cause of α-TTP deficiency, which causes symptomatic vitamin E deficiency despite normal intestinal absorption of lipid and vitamin E (15,30). Early suspicion and recognition of vitamin E deficiency is important because it is possible to prevent, reverse, or stabilize neurologic deterioration by correcting the deficiency. Characteristic clinical signs include loss of deep-tendon reflexes, truncal and limb ataxia, impaired vibratory and position sensations, ophthalmoplegia, muscle weakness, a widebased gait, and, if severe, dystonic facial appearance, pes cavus, visual loss, and scoliosis (5,77–82). In advanced cases, the child or adult may be confined to a wheelchair and require assistance for basic needs. These clinical findings are corroborated by abnormalities on electrophysiologic tests, including decreased action potential of sensory nerves, mild degeneration of muscles, delayed conduction through the posterior columns of the spinal cord, abnormal visual-evoked responses, and abnormal electroretinograms (77,80,81). Sural nerve and skeletal muscle biopsies show characteristic lesions. In sural nerves the loss of large caliber myelinated axons (94–96) corresponds to the diminished vibratory and position sensation that characterize the peripheral neuropathy symptoms. In skeletal muscle, there is accumulation of multiple autofluorescent inclusions that show strong acid phosphatase and esterase reactivity (80). By electron microscopy, the inclusions laying between myofibrils were membrane-bound dense bodies having characteristics of both lysosomes and lipopigment material. This material was similar to that observed in vitamin E–deficient animals and was believed to be formed in response to disordered intracellular lipid peroxidation. A more rapid course of neurologic degeneration has been observed in children with cholestatic liver diseases arising in infancy (e.g., Alagille syndrome, biliary atresia, and progressive familial intrahepatic cholestasis), suggesting the presence of other unidentified deficiencies or the presence of an additional oxidative stress. Thus, hyporeflexia and ataxia may appear between 18 and 24 months of age with disabling ataxia developing by age 10 years if the vitamin E deficiency is left untreated (77–79,83). In children with abetalipoproteinemia, neurologic symptoms appear at a later time during childhood (82). Adults with profound vitamin E malabsorption are less likely to experience clinical signs of deficiency either because it may take some time before stores of

VITAMIN E AND VITAMIN K METABOLISM / 1779 vitamin E become depleted or the mature neuromuscular system is relatively resistant to the effects of vitamin E deficiency (97,98). Isolated vitamin E deficiency syndrome presents during childhood and shows progression into adulthood if not diagnosed and treated appropriately. Significant cognitive and behavioral abnormalities also have been described in association with prolonged vitamin E deficiency (99,100). Assessment of Vitamin E Status Evaluating vitamin E status is primarily conducted through measurement of serum levels of α-tocopherol by high-performance liquid chromatography and relating these to a measure of serum lipids (101–103). Because vitamin E circulates entirely in lipoproteins, serum vitamin E levels are directly related to total serum lipid concentrations and, to a lesser extent, to serum cholesterol levels. Thus, in individuals with either increased or diminished total lipid concentrations, serum vitamin E levels may be more or less than normal, despite normal nutritional status (102). For this reason, a serum vitamin E/total serum lipid concentrations ratio (expressed as milligrams tocopherol per grams of total lipids) has been adopted to compensate for high or low serum lipid concentrations (101–103). A serum vitamin E/lipid ratio greater than 0.8 mg/gm for individuals older than 1 year and greater than 0.6 mg/gm for infants 1 year or younger indicates vitamin E sufficiency (102,103). Others have recommended using serum vitamin E/total cholesterol ratios to monitor patients with abnormal lipids (104); however, this was not as accurate as the serum E/total lipids ratio in children with cholestasis (101). In individuals with normal serum lipid concentrations, it is not necessary to calculate a vitamin E/lipid ratio, but rather to assess vitamin E status based on serum tocopherol levels alone. Functional tests have been developed to determine the relative protection of vitamin E against an oxidative stress. The best known of these is the peroxide hemolysis test in which red blood cells are exposed to hydrogen peroxide and the degree of hemolysis is quantitated (105). This correlates with functional vitamin E sufficiency assuming that other antioxidants are present at normal levels. A different version of this test measures production of malondialdehyde, a lipid peroxide product, instead of hemolysis (106). Because vitamin E is primarily stored in adipose tissue, quantitation of adipose tissue levels has been proposed as a means to assess body stores and overall nutritional adequacy (107). This has been particularly useful in abetalipoproteinemia and related disorders of β-lipoprotein synthesis, diseases in which circulating vitamin E levels are so low (because of markedly diminished VLDL and LDL levels) that they do not reflect vitamin E status. Treatment of Vitamin E Deficiency Detection, treatment, and prevention of vitamin E deficiency are customized to the cause of the deficiency state.

In cholestatic liver diseases, the reduction in bile flow leads to diminished concentrations of intraluminal bile acids in the intestine, causing failure of micellar solubilization and impairment of absorption of vitamin E together with other fat-soluble vitamins (5). In infants with chronic cholestasis, early findings of neurologic injury caused by vitamin E deficiency (hyporeflexia and wide-based gait) can be detected on physical examination by 18 months old (77–79,83,95). In cholestasis, it is essential to assess vitamin E status using the vitamin E/total lipid ratio because the hyperlipidemia associated with cholestasis may cause serum vitamin E levels to increase into the normal range despite the presence of deficiency (101). Thus, patients have been described with clear signs of clinical vitamin E deficiency reflected by subnormal vitamin E/total lipids ratios, yet with normal serum vitamin E levels. Most infants and children with significant chronic cholestasis (serum direct bilirubin > 2 mg/dl), and at least 20% of adults with similar cholestasis, experience development of biochemical evidence of vitamin E deficiency if not receiving supplements. In children, treatment of vitamin E deficiency (defined by serum E/total lipid ratio) should be initiated with 25 to 50 IU/kg/day RRR-αtocopherol, the most bioactive form; and in adults, 800 to 1200 IU/day should be started (108). Serum E/total lipid ratio should be monitored each 2 to 4 weeks until the ratio is normalized. Doses of vitamin E may need to be increased further if not absorbed. Because of the severe reduction in absorption of dietary lipids, most patients do not absorb large doses of standard free and esterified forms of α-tocopherol (109). Alternatively, a truly water-soluble form of vitamin E, d-α-tocopheryl polyethylene glycol-1000 (TPGS), which has been shown to form its own micelles and be absorbed in the virtual absence of bile flow, can be administered at doses of 10 to 25 IU/kg/day (109–111). This form of vitamin E has been demonstrated to correct low serum E/lipid ratios and produce improved neurologic function, particularly if started within the first few years of life. There is little reported toxicity of TPGS; however, because a small percentage of free polyethylene glycol is absorbed (109), caution should be entertained in using TPGS in patients with significant renal insufficiency. Intramuscular preparations of vitamin E are available in some countries and can be administered to correct vitamin E deficiency (112–114). In abetalipoproteinemia and related disorders, the failure to assemble normal chylomicrons leads to an inability to transport vitamin E from the enterocyte to mesenteric lymphatics, and failure to synthesize VLDL limits the ability of the liver to secrete newly absorbed vitamin E into the systemic circulation. These two perturbations of vitamin E absorption and transport lead to profoundly depressed serum and adipose tissue levels (91). The resulting vitamin E deficiency leads to progressive ataxia and pigmented retinopathy (115). Long-term treatment trials with massive supplements of vitamin E have conclusively demonstrated that neurologic function can be preserved if vitamin E deficiency is corrected at a young age (82,115). Because serum vitamin E levels do not reflect vitamin E status in these

1780 / CHAPTER 70 disorders, adipose tissue levels have been measured as an index of vitamin E stores (107). All patients with abetalipoproteinemia should be assumed to be vitamin E deficient and must receive 100 to 200 mg/kg/day of RRR-α-tocopherol in divided doses to achieve enough vitamin E absorption to prevent deficiency (82,90,115). Vitamin E status can be monitored by serial adipose tissue analysis of vitamin E, neurologic examinations, or serial electrophysiologic studies (somatosensory-evoked potentials or visual-evoked response). Vitamin A treatment also is recommended in abetalipoproteinemia by most authorities (116). In CF and other causes of pancreatic insufficiency, serum vitamin E levels and vitamin E/lipid ratios accurately reflect vitamin E status. Most patients with CF will remain vitamin E deficient despite the provision of oral pancreatic enzyme supplements and standard multiple vitamins (117). Patients with CF without significant liver involvement require treatment with an additional 5 to 10 IU/kg/day of vitamin E administered with pancreatic enzymes and meals (90). If there is concomitant cholestatic liver disease, treatment should proceed as for cholestasis. A follow-up study of infants randomized to newborn screening for CF showed an association between the duration of vitamin E deficiency in infancy and neurocognitive outcome at 7 to 17 years old (118), emphasizing the importance of preventing vitamin E deficiency and monitoring vitamin E status in this disease. Patient with other fat malabsorption conditions can be monitored with serum vitamin E levels or vitamin E/total lipid ratios (if serum lipids are greater than or less than normal values) and supplemented with large oral doses of vitamin E if found to be deficient. Patients have been described with neurologic findings of vitamin E deficiency secondary to virtually any cause of fat malabsorption, including celiac disease, short gut syndrome, radiation enteritis, intestinal lymphangiectasia, Crohn’s disease, mesenteric vascular thrombosis, and others. Therefore, it is essential that vitamin E status be monitored periodically in all patients with any degree of significant fat malabsorption. Isolated vitamin E deficiency syndrome, or AVED, is an autosomal recessive disorder with a clinical phenotype resembling Friedreich’s ataxia and is caused by an isolated deficiency of vitamin E in the absence of fat malabsorption (30–32). The disorder appears to be more common in certain ethnic groups, with a relatively high prevalence reported in Tunisia (119) and Morocco (120). The neurologic symptoms include ataxia, dysarthria, hyporeflexia, head titubation, and decreased vibration sense (121). Neuropathologic examination shows involvement of the retina, the central axons of dorsal root ganglion cells, the pyramidal tract, and peripheral sensory nerves to a lesser extent. In these patients, all other causes of secondary vitamin E deficiency and fat malabsorption are absent. Oral absorption tests, using either large oral doses of standard forms of α-tocopherol or physiologic doses of stable isotopes (deuterated) of tocopherols, demonstrate normal intestinal absorption of α-tocopherol, but impaired hepatic secretion of α-tocopherol into VLDLs (9,30,31). Molecular evaluation of the human α-TTP gene

shows point mutations or deletions at several locations (121), which code for proteins in which the amino-acid sequence is changed significantly, impairing the function of the protein. Vitamin E status is assessed by serum vitamin E or the vitamin E/total lipid ratio. Patients show biochemical and clinical response to 800 to 1200 IU per day of RRR-αtocopherol, with normalization of serum vitamin E (30,119). Apparently, tocopherol is transferred from chylomicrons directly to other circulating LDLs and HDLs, thus bypassing the defective function of the hepatic α-TTP. If vitamin therapy is initiated at a young age, neurologic symptoms may gradually remit; however, when initiated when symptoms are advanced, therapy will only prevent further deterioration of neurologic function (30,119). Asymptomatic first-degree relatives should be screened and treated if low serum vitamin E levels are present. Serum vitamin E levels and neurologic examinations should be monitored serially and doses adjusted accordingly.

Toxicity of Vitamin E Substantiated toxic effects of large doses of vitamin E in humans are rare. Documented side effects include an increased risk for the development of sepsis and necrotizing enterocolitis in small preterm infants treated with high intravenous doses of vitamin E (122). The cause of this potentially severe side effect of parenteral vitamin E therapy in neonates appears to be the inhibition of the oxidative burst in neutrophils that is essential for the killing of organisms. Another form of parenteral vitamin E used in preterm infants in the 1980s (which has since been removed from the market) contained polysorbate, a detergent that produced a fatal hepatic venoocclusive disease and renal failure (123,124). Doses of vitamin E greater than 1000 IU/day also have been associated with excessive prolongation of the prothrombin time (PT) in adults receiving Coumadin (warfarin) for anticoagulation prophylaxis for thrombosis (125). Thus, it is recommended that vitamin K status be managed in patients with fat malabsorption who are receiving high-dose therapy of vitamin E, and that patients receiving anticoagulant medications that interfere with normal vitamin K metabolism be monitored closely if receiving high doses of vitamin E. Other reports of adverse effects of vitamin E supplements in humans had been extremely rare, leading the Food and Nutrition Board to set the tolerable upper intake level for α-tocopherol at 1000 IU/day (1). Findings from several clinical trials have suggested adverse vitamin E effects in humans under special circumstances. One study was a 3-year, double-blind trial of mixed antioxidants (vitamins E and C, β-carotene, and selenium) in 160 subjects with hyperlipidemia receiving simvastatin-niacin or placebo therapy (126). In subjects taking antioxidants, there were lower HDL cholesterol levels than was expected (127), and there was an increase in the clinical end points of arteriographic evidence of coronary stenosis, or the occurrence of a first cardiovascular event (death, myocardial infarction,

VITAMIN E AND VITAMIN K METABOLISM / 1781 stroke, or revascularization) (126). Another study, the Women’s Angiographic Vitamin and Estrogen (WAVE) Trial, was a randomized, double-blind trial of 423 postmenopausal women who at baseline had at least one coronary artery stenosis identified by coronary angiography (128). In postmenopausal women receiving hormone replacement therapy, all-cause mortality was increased in women assigned to antioxidant vitamins compared with placebo (hazard ratio, 2.8; 95% confidence interval [CI], 1.1–7.2; p = 0.047). To help explain these surprising findings, it is notable that both simvastatin (129) and estrogen (130) undergo hepatic metabolism by CYP3A4. Tocopherols are ligands for PXR, a regulator of transcription of CYP3A4. Thus, high doses of tocopherol may indirectly alter metabolism of both estrogens and lipid-altering medications, perhaps explaining these reported adverse effects of vitamin E. The long-term results of a placebo-controlled vitamin E supplementation trial were reported by the Health Outcomes Prevention Evaluation (HOPE) and the HOPE-The Ongoing Outcomes (HOPE-TOO) studies (131). In these studies, a mean of 4.5 years and then 7.2 years of a daily dose of 400 IU RRR-α-tocopherol given to patients at least 55 years old with vascular disease or diabetes mellitus resulted in no reduction in cancer incidence or cancer deaths, or in major cardiovascular events. However, vitamin E supplementation was associated with a greater risk for heart failure (relative risk [RR], 1.13; 95% CI, 1.1–1.26; p = 0.03) and hospitalization for heart failure (RR 1.21; 95% CI, 1.00–1.47; p = 0.045). In addition, a meta-analysis of 19 studies involving 135,967 subjects found that there was a small but significant dose-dependent increase (RR, 1.04; p = 0.04) in all-cause mortality of vitamin E when taken at a dose of 400 IU or more daily (132). Thus, there is sufficient reason to recommend precaution in recommending high-dose vitamin E supplementation of adults until therapeutic advantage for specific disorders is documented. Despite these studies demonstrating no benefit of tocopherol supplementation, there are several specific disorders in which vitamin E may be of benefit. In patients with advanced renal failure undergoing hemodialysis, vitamin E may reduce the risk for myocardial infarction (133). In age-related macular regeneration of moderate or greater severity, an antioxidant combination plus zinc and copper in patients older than 55 years may reduce the risk for progression to visual loss (134). Trials are currently underway to determine whether vitamin E and selenium affect the incidence of prostate cancer in men, as suggested by smaller studies (135). The mechanisms explaining these possible toxicities of vitamin E currently are speculative, but could involve vitamin E–induced alterations in metabolism of lipoproteins or medications used to treat patients with coronary artery disease. The mediator of this effect may be the interaction of tocopherols with nuclear receptors that regulate gene expression. For example, tocopherols bind to the nuclear receptor PXR, which regulates P450 metabolism of medications and other endogenous and exogenous substrates (66). An additional interaction is the potential effect of

A

O

O 3

B

O

O

n

FIG. 70-6. Chemical structure of the vitamin K compounds, phylloquinone or vitamin K1 (A) and menaquinones or vitamin K2 (B). n = 6 to 13 isoprenyl subunits for menaquinone.

antioxidants on the enzymatically determined cellular concentrations of oxysterols, known activators of RXR/PXR that regulate genes affecting lipoprotein metabolism (136). Clearly, basic science advances in coming years will clarify these new important nonantioxidant actions of vitamin E and other “antioxidants.”

VITAMIN K Structure and Biochemistry Vitamin K functions as a coenzyme for the synthesis of several biologically active proteins involved in blood coagulation and bone metabolism (137). Vitamin K compounds are 3-substituted 2-methyl-1,4 naphthoquinones that can be classified into two groups: phylloquinone (vitamin K1), the major form in plants that contain a phytyl group, and the menaquinones (vitamin K2), the form that is synthesized by bacteria in the colon and distal small bowel and contains a polyisoprenyl side chain, with 6 to 13 isoprenyl subunits at the 3-position (Fig. 70-6). One specific menaquinone, MK-4, is not produced by bacteria, but can be formed by alkylation of menadione, and from phylloquinone as well (138). Vitamin K3, menadione, is a synthetic form of the vitamin with other metabolic activities not shared by vitamins K1 and K2 (139).

Dietary Sources and Human Requirements The major dietary sources of phylloquinone include green leafy vegetables and some vegetable oils, whereas menaquinones can be found in dairy products including cheeses and fermented soybean products (137). Specifically, spinach,

1782 / CHAPTER 70 collards, broccoli, and iceberg lettuce are the major contributors of vitamin K to the diet of U.S. children and adults. The breast-fed infant receives vitamin K through ingestion of mother’s milk. The adequate intakes for vitamin K have been calculated by the Food and Nutrition Board of the Institute of Medicine (137). The adequate intake for infants 6 months or younger is based on the reported average intake of milk of 780 ml/day and an average phylloquinone concentration in human milk of 2.5 mg/L, yielding the adequate intakes of 2.0 µg/day. This assumes that the infant received prophylactic vitamin K at birth as recommended by the American Academy of Pediatrics. At age 7 through 12 months, the adequate intake is 2.5 µg/day; however, this does not take into account the contribution to daily intake of vitamin K in weaning foods for which accurate vitamin K content analysis is not available. If the adequate intake of adults is extrapolated down to these infants, the adequate intake would be 23 µg/day. It should be noted that vitamin K content of cow milk (5 µg /L) and infant formulas (50–100 µg/L) is greater than that of human milk. Although significant amounts of menaquinone-4 have been detected in cow milk, its physiologic function is unknown. There is also no information about the bioavailability of vitamin K in infant formulas because vitamin K reversibly binds to a protein in cow’s milk. For older children, the vitamin K adequate intake is based on the greatest median intake as reported by the Third National Health and Nutrition Examination Survey (NHANES III) conducted from 1988 to 1994 in the United States. Thus, there is a significant increase in adequate intake compared with infancy because of the inclusion of vitamin K–rich fruits and vegetables as the child’s diet becomes more diversified. The adequate intake is 30 µg/day for children 1 to 3 years old, 55 µg/day for children 4 to 8 years old, 60 µg/day for children 9 to 13 years old, and 75 µg/day for teenagers 14 to 18 years old. Similarly, the adequate intake for adults is based on the medium intake data form NHANES III. For men 19 to older than 70 years, the adequate intake is 120 µg/day; for women 19 to older than 70 years, the adequate intake is 90 µg/day. This does not change significantly during pregnancy or lactation.

Intestinal Absorption of Vitamin K Phylloquinone is absorbed in the jejunum and ileum and is dependent on adequate flow of bile and pancreatic secretions being enhanced by coadministration of dietary fat (140). Absorption of vitamin K1 is by a saturable process requiring metabolic energy, whereas vitamin K2 absorption occurs by passive diffusion. Under normal circumstances, the absorption of dietary vitamin K is only 5% to 15%, partly because the vitamin is bound to vegetable substrates. In conditions causing pancreatic insufficiency, cholestasis, loss of normal absorptive surface area, or obstruction of mesenteric lymphatics, poor absorption of vitamin K occurs. Absorbed phylloquinone is incorporated into newly synthesized

chylomicrons by the enterocyte and is secreted into the intercellular space, absorbed into mesenteric lymphatics, and finally transported into the systemic circulation (137). Circulating phylloquinone is found primarily in VLDLs and chylomicrons. Phylloquinone is taken up by the liver in chylomicron remnants where it is presumably resecreted into VLDLs to be transported to other tissues. Menaquinones are generated by intestinal and colonic bacteria and play an important role in vitamin K nutrition, especially in the breast-fed infant who receives little vitamin K from mother’s milk. Absorption of these lipophilic compounds resulting from bacterial production has been difficult to demonstrate; however, it is generally accepted that dietary menaquinones are well absorbed. That vitamin K deficiency can occur with dietary restriction of phylloquinones suggests that the intestinally derived vitamin K compounds are not as bioavailable (137). One specific menaquinone, MK-4, is formed in animal tissues from phylloquinone, as well as from menadione (138). This compound is present in greater concentrations than phylloquinone in pancreas, salivary gland, brain, and sternum. Vitamin K stores in the liver are rapidly depleted (within a few weeks to a month) when dietary vitamin K is restricted or malabsorbed (141). MK-4 appears to have a unique, but unidentified, role in human nutrition.

Vitamin K Metabolism Vitamin K is rapidly catabolized in the liver and excreted mainly in bile. It has been thought that vitamin K proceeds through oxidative degradation of the phytyl side chain to K acid 1 and K acid 2, followed by glucuronide conjugation and urinary excretion. Comparison of the metabolites of vitamins E and K show striking similarities, which has led Landes and colleagues (142) to propose that they are metabolized through the same P450 pathway, and that vitamin K, like tocopherols, was capable of inducing PXR-controlled gene expression. Using a cationic amino acid transporter (CAT) reporter gene, they showed that vitamin K induced PXR-regulated CAT activity, with menaquinone inducing 8.4-fold activation and phylloquinone 2.8-fold activation, compared with 2.5-fold activation by α-tocopherol. The induction by these compounds was similar to that observed with rifampicin. Thus, the initial ω hydroxylation of large doses of vitamin K and E compounds by CYPs appears to be regulated by PXR, as if the liver were disposing of a potentially harmful xenobiotic.

Vitamin K Function Vitamin K plays an essential role in the posttranslational conversion of glutamyl residues to γ-carboxylglutamyl residues (Gla) in a limited number of proteins (143). These proteins include the clotting factors, plasma prothrombin (factor II), VII, IX, and X. Because γ-carboxylation is essential for full coagulant activity of these proteins, the classical

VITAMIN E AND VITAMIN K METABOLISM / 1783 Physiologic Role of Vitamin K

Vitamin-dependent carboxylase γ-carboxy glutamate

O2+CO2+glutamate

Reduced vitamin K (Hydroquinone)

2,3 Vitamin K epoxide

Oxidized thiol Vitamin K epoxide reductase Reduced thiol NAD(P)+ Vitamin K reductase

Vitamin K quinone

Reduced thiol Vit. K epoxide reductase Oxidized thiol

NAD(P)H

FIG. 70-7. The vitamin K cycle, which catalyzes the γ-carboxylation of glutamic acid residues on specific proteins by the vitamin K–dependent carboxylase and regenerates reduced vitamin K (the hydroquinone).

sign of vitamin K deficiency is a vitamin K–dependent increase in the PT, which can lead to a sometimes lifethreatening hemorrhagic event. The Gla residues bind calcium, and calcium binding by the fully γ-carboxylated clotting factors is essential for optimal activation or activity of these factors in blood. The enzyme catalyzing this process is the vitamin K–dependent carboxylase, a 94-kDa protein composed of a single polypeptide chain of 758 amino acids located in the endoplasmic reticulum and the Golgi apparatus (144). The γ-carboxylation reaction requires molecular oxygen, carbon dioxide, and the fully reduced form of vitamin K, the vitamin K hydroquinone generated by hepatic enzymes (Fig. 70-7). γ-Carboxylation of each Gla residue is coupled stoichiometrically to the formation of a single molecule of vitamin K 2,3 epoxide, which is reduced back to the hydroquinone by the hepatic enzymes, vitamin K epoxide reductase and dehydrogenases that use NADH as a cofactor. This cycle establishes the vitamin K redox cycle. Vitamin K epoxide reductase is the target for the anticoagulant drug, warfarin, which irreversibly prevents the production of the reduced vitamin KH2. Because large doses of vitamin K can reverse this effect of warfarin, the second dehydrogenase pathway is believed to be responsible for regeneration of vitamin K under these circumstances. There are also three vitamin K–dependent proteins in bones, osteocalcin, matrix Gla protein, and protein S (145). Osteocalcin is exclusively synthesized by osteoblasts, whereas matrix Gla protein and protein S are synthesized in chondrocytes, vascular smooth muscle cells, and epithelium as well. Osteocalcin appears to be a negative regulator of bone formation. Matrix Gla protein is a powerful inhibitor of tissue calcification.

The two major physiologic roles of vitamin K involve the clotting system and bone metabolism. Vitamin K is necessary to ensure normal production of clotting factors II, VII, IX, and X, without which spontaneous or stimulated hemorrhage can occur at any age. This is most dramatic in the young infant, who is born with low vitamin K stores and who receives little additional vitamin K in mother’s milk. Hemorrhagic disease of the newborn develops during the first week of life and includes bleeding in the skin or from mucosal surfaces, circumcision, or venipuncture sites. Internal bleeding, including intracranial hemorrhage and retroperitoneal hematomas, may rarely be the primary site of hemorrhage, sometimes being fatal or leaving the infant with devastating long-term neurologic consequences (146). These instances provide the rationale for the use of vitamin K prophylaxis in neonates (147). This disorder is more common in infants receiving human milk in contrast with cow’s milk formulas, primarily because of the comparative vitamin K content (146). Administering parenteral vitamin K can quickly correct this deficiency state; however, infusions of fresh-frozen plasma, to more rapidly correct the clotting factor deficiencies, are required if clinically significant bleeding is evident. Prophylactic vitamin K has been used at the time of birth for about a half century to prevent this potentially life-threatening disorder. Vitamin K (500 µg to 1 mg) is given intramuscularly to the newborn immediately after birth in most countries, some choosing to administer oral vitamin K supplements after birth as an alternative. At these doses, vitamin K administration has not been shown to cause significant morbidity and is extremely effective in preventing hemorrhagic disease of the newborn. Vitamin K deficiency–induced hemorrhage still rarely occurs between 1 and 11 weeks of age, with the majority of cases occurring in infants with fat malabsorption caused by undiagnosed cholestatic liver disease (148). The first possibility that vitamin K may play a role in bone health was suggested in the mid-1970s when bone malformations were reported in children born to women who had received vitamin K agonists during the first trimester of their pregnancy (149). Later, lower circulating phylloquinone concentrations were reported in patients with osteoporosis who had fractures of the femur or vertebrae (150). This led to examining vitamin K levels in patients with decreased bone mineral density; most reports showed an inverse correlation between the two (151). Population studies have demonstrated that low bone mineral density and increased risk for bone fractures are associated with low dietary or circulating levels of vitamin K or high levels of undercarboxylated osteocalcin (see review by Bugel [145]). Osteocalcin is a substrate for the carboxylation steps catalyzed by vitamin K; thus, increased levels of osteocalcin have been taken by some to indicate vitamin K deficiency. Notably, multiple techniques have been used to assay osteocalcin, and the results of all studies have not been consistent. The strongest data linking vitamin K intake with bone health

1784 / CHAPTER 70 was the Nurses’ Health Study, in which investigators reported an inverse relation between vitamin K intake and the risk for hip fractures in a 10-year follow-up period (152). A food frequency questionnaire was used to estimate vitamin K intake in 71,327 women between 38 and 63 years old. Women in the lowest quintile with intakes less than 109 µg/day had a greater risk for hip fracture. Vitamin K supplementation trials have shown that vitamin K reduces the undercarboxylated osteocalcin levels, reduces urinary calcium excretion, and improves indices of bone turnover in short-term studies. Administering 46 mg/day menaquinone to patients with osteoporosis for 6 months led to an increase in metacarpal bone density, increased total osteocalcin, and reduced urinary calcium excretion (153). However, there was no effect on lumbar bone mineral density. In another study, supplementation with menaquinone plus vitamin D led to increased bone mineral density over a 2-year period (154). In another study, supplementation with phylloquinone for 2 years prevented bone loss (155). It would be expected that patients receiving long-term warfarin therapy would be in a constant state of relative vitamin K deficiency, and it has been proposed that these patients would be at risk for impaired bone health if vitamin K played an important role. A meta-analysis of nine studies examined the effect of long-term exposure to oral anticoagulants in relation to bone mineral density and found that there was not a significant effect on most important bones (156). Thus, although some studies are promising, the results of larger long-term studies that are in progress will be needed to determine the clinically significant effect of different dosing strategies for vitamin K therapy to improve bone health.

Causes of Vitamin K Malabsorption and Deficiency The typical Western diet contains generally adequate amounts of vitamin K to prevent clinically significant coagulation problems. Most cases of human vitamin K deficiency are either in patient groups with inadequate stores (newborns), those receiving medications that impair function of the vitamin K cycle (warfarin), patients with fat malabsorption, or patients receiving broad-spectrum antibiotics for long periods that alter intestinal flora. Patients with gastrointestinal diseases that impair intraluminal digestive processes (e.g., bacterial overgrowth of the small bowel, use of bile acid–binding resins such as cholestyramine, gastric hypersecretory states), cause decreased bile flow (e.g., biliary atresia, α1-antitrypsin deficiency, Alagille syndrome, primary biliary cirrhosis, cholestatic hepatitis), cause pancreatic insufficiency (e.g., CF, chronic pancreatitis, alcoholic pancreatitis), cause a loss of absorptive surface area (e.g., celiac disease, tropical sprue) or shortened intestinal length (e.g., congenital intestinal atresia, intestinal resections, Crohn’s disease), trigger defective synthesis of chylomicrons (e.g., abetalipoproteinemia, hypobetalipoproteinemia), or cause lymphatic obstruction (e.g., intestinal lymphangiectasia, lymphatic obstruction by malignancy,

lymphoma) are at risk for vitamin K deficiency (157,158), particularly if these patients receive broad-spectrum antibiotics. Because vitamin K stores are limited, the time to depletion may be only several weeks in older children and adults and days in young infants.

Assessment of Vitamin K Status The most commonly used indicator for vitamin K status is the PT. The PT is dependent on the proper functioning of the vitamin K–dependent clotting factors and is considered a functional test of vitamin K adequacy, particularly if it corrects after parenteral administration of vitamin K (159). The International Normalized Ratio (INR) has been used to standardize the PT for differences in techniques of performance of the PT assay in different laboratories and in different countries. However, it is notable that the PT is not a sensitive indicator of vitamin K for several reasons. First, the PT must be decreased by about 50% before it is considered outside of the normal range (159). PT also does not respond to a change in dietary vitamin K in healthy subjects. Finally, PT also is affected by loss of hepatic synthetic function, regardless of vitamin K status. Despite these shortcomings, the PT is associated with adverse clinical consequences (hemorrhage, bruising, etc.), which is not the case for the other proposed indices of vitamin K status. Some have proposed plasma factor VII levels as a means to monitor vitamin K status, with its half-life of 6 hours. Although some studies have indicated changes with dietary deprivation, others have shown that, in the absence of oral antibiotic therapy, it is not a sensitive indicator of vitamin K status (160). Factor VII levels also decrease dramatically during acute liver failure because of hepatic synthetic failure unrelated to vitamin K status. Plasma levels of phylloquinone and menaquinone have been used to assess vitamin K status in the research setting. Phylloquinone levels reflect recent dietary intake and will change in response to dietary intake within 24 hours (161). Consequently, this measurement does not bear on vitamin K stores and status, but rather is more of an indicator of recent intake. These levels also correlate with serum lipid levels, because phylloquinone is carried in lipoproteins (162). Thus, older individuals have higher plasma levels than younger people. Other than in a research setting, these levels are not helpful in the clinical assessment of a patient. Undercarboxylated Prothrombin Vitamin K deficiency in humans results in the secretion into plasma of biologically inactive, under-γ-carboxylated forms of the vitamin K–dependent clotting factors. These proteins are referred to as proteins induced by vitamin K absence (PIVKA), and when referring to prothrombin, are known as PIVKA-II. Commercial assays currently are available to measure these proteins. PIVKA-II levels vary little with age, yet increase in response to dietary limitation of

VITAMIN E AND VITAMIN K METABOLISM / 1785 vitamin K intake (163). An increased PIVKA-II may be observed in a patient with a normal PT and no evidence of bleeding. Thus, the test is more sensitive for the detection of vitamin K deficiency than is the PT; however, it may not correlate with clinical findings or outcomes. In addition, circulating decarboxylated prothrombin also is increased in patients with hepatocellular carcinoma (HCC) (164), and some consider it a better marker than serum α-fetoprotein in the diagnosis of HCC. PIVKA-II is a useful clinical test to determine if an increased PT is indicative of vitamin K deficiency, particularly in patients with acute or chronic liver disease in the absence of HCC. Under-γ-Carboxylated Osteocalcin Similar to PVKA-II, undercarboxylated osteocalcin circulates in the blood and has been considered a possible marker of suboptimal vitamin K status. A variety of assays and techniques for its measurement have led to confusion in the literature as to the role of this compound as an indicator of vitamin K status (137). A monoclonal antibody specific for this form of osteocalcin may help to standardize testing that should lead to better interpretation of study results. Currently, measuring this compound is not helpful in clinical practice. Treatment of Vitamin K Deficiency If vitamin K deficiency is associated with significant clinical bleeding, fresh-frozen plasma is administered immediately to replete vitamin K–dependent clotting factors in the short term. In addition, intravenous, intramuscular, or subcutaneous injections of vitamin K are given that should lead to increased clotting factors within 12 to 24 hours. Typically, 10 to 20 mg vitamin K is given in an adult and 2.5 to 5 mg in a child. Large doses of vitamin K, about 100 mg, are needed to overcome bleeding caused by warfarin. In patients with chronic fat malabsorption, daily doses of 2.5 to 5 mg vitamin K may be given to prevent deficiency. If vitamin K malabsorption is severe, such as in severe cholestatic liver disease, monthly parenteral administration of vitamin K may be necessary. Toxicity of Vitamin K Of all the fat-soluble vitamins, vitamin K has the lowest toxicity profile. Vitamin K1 has not been shown to be toxic even if administered in large doses (165). Vitamin K3, a synthetic form of vitamin K that is well absorbed, has been shown to induce hepatotoxicity and may induce hemolysis in glucose6-phosphatase dehydrogenase–deficient infants (166).

ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (grants RO1 DK38846, U54 RR019455, UO1DK062453, RO1 DK59576, and 5MO1 RR00069), the

Environmental Health Sciences Center at Oregon State University (National Institute of Environmental Health Sciences grant P30 ES002100), the Madigan Foundation, and the Abby Bennett Liver Research Fund.

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osteoporosis: a double-blind multicenter comparative study with 1-ahydroxyvitain D3. J Bone Miner Res 1992;7:S122. Ushiroyama T, Ikeda A, Sakai M, Higashiyama T, Ueki M. Effects of the combined use of calcitonin and 1 alpha-hydroxycholecalciferol on vertebral bone loss and bone turnover in women with postmenopausal osteopenia and osteoporosis: a prospective study of long-term and continuous administration with low dose calcitonin. Maturitas 2001;40:229–238. Braam LA, Knapen MH, Geusens P, Brouns F, Hamulyak K, Gerichhausen MJ, Vermeer C. Vitamin K1 supplementation retards bone loss in postmenopausal women between 50 and 60 years of age. Calcif Tissue Int 2003;73:21–26. Caraballo PJ, Gabriel SE, Castro MR, Atkinson EJ, Melton LJ 3rd. Changes in bone density after exposure to oral anticoagulants: a metaanalysis. Osteoporos Int 1999;9:441–448. Rashid M, Durie P, Andrew M, Kalnins D, Shin J, Corey M, Tullis E, Pencharz PB. Prevalence of vitamin K deficiency in cystic fibrosis. Am J Clin Nutr 1999;70:378–382. Argao EA, Heubi JE. Fat-soluble vitamin deficiency in infants and children. Curr Opin Pediatr 1993;5:562–566. Suttie JW. Vitamin K and human nutrition. J Am Diet Assoc 1992;92: 585–590.

160. Bach AU, Anderson SA, Foley AL, Williams EC, Suttie JW. Assessment of vitamin K status in human subjects administered “minidose” warfarin. Am J Clin Nutr 1996;64:894–902. 161. Sokoll LJ, Booth SL, O’Brien ME, Davidson KW, Tsaioun KI, Sadowski JA. Changes in serum osteocalcin, plasma phylloquinone, and urinary gamma-carboxyglutamic acid in response to altered intakes of dietary phylloquinone in human subjects. Am J Clin Nutr 1997;65:779–784. 162. Kohlmeier M, Saupe J, Drossel HJ, Shearer MJ. Variation of phylloquinone (vitamin K1) concentrations in hemodialysis patients. Thromb Haemost 1995;74:1252–1254. 163. Booth SL, O’Brien-Morse ME, Dallal GE, Davidson KW, Gundberg CM. Response of vitamin K status to different intakes and sources of phylloquinone-rich foods: comparison of younger and older adults. Am J Clin Nutr 1999;70:368–377. 164. Yuen MF, Lai CL. Serological markers of liver cancer. Best Pract Res Clin Gastroenterol 2005;19:91–99. 165. Lane PA, Hathaway WE. Vitamin K in infancy. J Pediatr 1985;106: 351–359. 166. Zinkham WH. Peripheral blood and bilirubin values in normal full-term primaquine-sensitive Negro infants: effect of vitamin K. Pediatrics 1963;31:983–995.

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CHAPTER

71

Intestinal Absorption of Water-Soluble Vitamins Hamid M. Said and Bellur Seetharam Folate, 1792 Mechanism of Intestinal Folate Transport, 1792 Molecular Identity of the Intestinal Folate Uptake System, 1795 Regulation of Intestinal Folate Uptake Process, 1796 Cell Biology of the Human Reduced Folate Carrier Protein in Intestinal Epithelia, 1797 Thiamin (Vitamin B1), 1797 Mechanisms of Intestinal Thiamin Absorption, 1798 Molecular Identity of the Intestinal Thiamin Uptake Systems, 1798 Regulation of the Intestinal Thiamin Uptake Process, 1800 Cell Biology of the Intestinal Thiamin Transporters, 1801 Biotin (Vitamin H), 1802 Mechanism of Intestinal Biotin Transport, 1802 Molecular Identity of the Intestinal Biotin Uptake System, 1804 Regulation of the Intestinal Biotin Uptake Process, 1805 Vitamin C (Ascorbic and Dehydroascorbic Acids), 1807 Mechanism of Intestinal Vitamin C Transport, 1807 Molecular Identity of the Intestinal Ascorbic Acid and Dehydro-L-Ascorbic Acid Uptake Systems, 1807

Regulation of the Intestinal Ascorbic Acid Uptake Process, 1808 Cell Biology of the Intestinal Ascorbic Acid Transporters, 1808 Vitamin B6, 1809 Mechanism of Intestinal Vitamin B6 Transport, 1810 Regulation of the Intestinal Vitamin B6 Uptake Process, 1810 Riboflavin (Vitamin B2), 1811 Mechanism of Intestinal Riboflavin Transport, 1811 Regulation of the Intestinal Riboflavin Uptake Process, 1811 Niacin (Nicotinic Acid; Vitamin B3), 1812 Pantothenic Acid, 1812 Cobalamin (Vitamin B12), 1813 Gastrointestinal Absorption of Cobalamin, 1813 Acquired Causes of Cobalamin Malabsorption, 1815 Inherited Causes of Cobalamin Malabsorption, 1815 Structure and Function of Cobalamin Transport Proteins, Intrinsic Factor, and Transcobalamin, 1816 Structure and Function of Cubilin, 1817 Cellular Synthesis and Regulation of Expression of Intrinsic Factor and Transcobalamin, 1818 Cellular Synthesis and Expression of Cubilin, 1819 Acknowledgments, 1819 References, 1819

H. M. Said: Departments of Medicine and Physiology/ Biophysics, University of California, Irvine, California, and VA Medical Center, Long Beach, California 90822. B. Seetharam: Departments of Medicine and Biochemistry, Medical College of Wisconsin and Clement Zablocki VA Medical Center, Milwaukee, Wisconsin 53295. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

The water-soluble vitamins represent a group of structurally and functionally unrelated compounds that share the common feature of being essential for normal cellular functions, growth, and development. With the exception of some endogenous production of niacin, human cells cannot synthesize water-soluble vitamins, and thus must obtain these micronutrients from exogenous sources via intestinal absorption. The intestine therefore plays a critical role in maintaining and regulating normal body homeostasis of these micronutrients, and interference with its normal ability to process and/or absorb these compounds could lead to conditions of deficiency and

1791

1792 / CHAPTER 71 suboptimal levels. Such interferences occur in a variety of conditions including congenital defects in the intestinal digestion phase of the dietary forms of the vitamins or in the involved uptake system, in intestinal diseases, in drug interactions, with chronic alcohol use, and after intestinal resection. Our understanding of the mechanisms involved in the intestinal absorption of water-soluble vitamins and their regulation has progressed significantly during the last few decades. It is now known that absorption of physiologic concentrations of all these micronutrients occur via specialized uptake mechanisms. In many cases, the driving force involved in the uptake event and the molecular identity of the gene product involved in the uptake system has been identified. Information also has been forthcoming regarding regulation of the intestinal vitamin uptake events by intracellular and extracellular factors/conditions. In addition, information regarding the structure of the genes of some of the involved transporters and their basal and 5′-regulatory regions (promoters) has been emerging. Furthermore, the mechanisms involved in intracellular trafficking and membrane targeting of the uptake systems of some of the water-soluble vitamins in intestinal epithelial cells has been described. Progress also has been made in our understanding of the ability of the large intestine to absorb some of the bacterially synthesized vitamins. Efficient and specialized carriermediated uptake systems have been identified for a number of the water-soluble vitamins produced by the normal microflora of the large intestine. Although the contribution of this source of vitamins toward overall normal body homeostasis is unclear and in need of further study, it is believed that this source of vitamins contributes significantly toward the cellular vitamin homeostasis in the localized colonic epithelial cells. This chapter aims to describe our current knowledge and understanding of the mechanisms involved in the intestinal absorption process of dietary and bacterially synthesized water-soluble vitamins and their regulation with special emphasis on the progress that has been made during the late 1990s/early 2000s.

FOLATE The term folate (Fig. 71-1) refers to a group of one-carbon derivatives of the vitamin folic acid that are required for the synthesis of pyrimidine and purine nucleotides, precursors of DNA and RNA, respectively. Folate also is involved in the metabolism of several amino acids including homocysteine. At the cellular level, deficiency of folate leads to derangement of one-carbon metabolism, defect(s) in DNA synthesis and methylation, misincorporation of uracil into DNA, and disturbance in the metabolism of several amino acids. Clinically, folate deficiency is associated with a variety of abnormalities that include megaloblastic anemia and growth retardation. In contrast with the deleterious effects of folate deficiency, increasing folate intake and optimizing folate body homeostasis can lead to a great reduction in the incidence of neural tube defects (the most common birth defect in humans), and

may also provide protection against certain types of cancers (e.g., colorectal cancer). Folate deficiency is a highly prevalent vitamin deficiency throughout the world and occurs because of a variety of causes including impairment in intestinal absorption of the vitamin. A variety of conditions have been reported to affect/interfere with the normal intestinal folate absorption process. These include reported defects in the intestinal folate uptake in (1), intestinal diseases (e.g., celiac disease, tropical sprue), drug interactions (e.g., sulfasalazine, trimethoprim, pyrimethamine, diphenylhydantoin), and chronic alcohol consumption.

Mechanism of Intestinal Folate Transport Two sources of folate are available to the gut: a dietary source that is mainly processed in the small intestine, and a bacterial source where the vitamin is generated by the normal microflora of the large intestine (2) and is absorbed in that part of the gut. Dietary folate exists as a mixture of free folate (i.e., folate monoglutamate) and as folate polyglutamates (see Fig. 71-1), with the latter form being the predominant form in the average diet. The conjugated polyglutamate forms of folate cannot be absorbed because of their size and multiple negative charges; thus, they must undergo an obligatory process of progressive hydrolysis to the folate monoglutamate form before uptake. This hydrolysis step is performed by the specific action of the enzyme folate hydrolase (also known as folylpoly-γ-glutamate carboxypeptidase). Two forms of this enzyme have been identified in intestinal epithelial cells. One form is expressed at the apical brushborder membrane (BBM) domain, and the other form is expressed intracellularly (most probably in the lysosomes) (3–6). The BBM form of the enzyme appears to be expressed mainly in the proximal part of the small intestine, whereas the intracellular form appears to be expressed uniformly along the entire length of the small intestine (7,8). The intestinal BBM form of the folate hydrolase has been cloned (8,9) and has been shown to be up-regulated in the condition of dietary folate deficiency (10). Impairment in the activity of the BBM form of the enzyme occurs in disease conditions that affect the intestinal mucosa such as celiac disease and tropical sprue (11–13). In addition, activity of the BBM enzyme also has been shown to be impaired after chronic alcohol administration and use of the pharmacologic agent sulfasalazine (14–17). The mechanism of uptake of dietary folate monoglutamates by the small intestine (which also applies to the folate that enters the small intestine via enterohepatic cycling of the vitamin) (18,19) has been studied extensively using a variety of intestinal preparations from a number of species including humans (reviewed by Rose [20]). These studies have established that dietary folate monoglutamates are absorbed mainly in the proximal part of the small intestine, and that the process involves a specialized carrier-mediated system. Animal studies have shown that folate absorption in the ileum is limited and occurs via simple diffusion (21). The latter

A

N

N

H2N

COOH CH2

N N

CO

NH

NH

COOH

CH2

CH

CH2

CO n

NH

CH

CH2

COOH

CH2

OH

B

Cl O N

N

S

N N

Thiamine

C H

O H N HN 1

O

S H

H

3 NH

N H

O

HOOC

CH2 CH2 CH2 CH2 C

S

Biotin

NH NH

CH2 CH2 CH2 CH2

CH O

C

Biocytine OH

O

H N

HOOC

H OH

O

CH3

O H3C

S

S

Pantothenic acid Lipoate

D

O

O O

O

O

O

O

O

O

O O

O

Ascorbic acid

E

Dehydroascorbic acid

H

O

X

CH2 NH2 Cl

(b)

CH2

HO

R CH3

N H3C

O O

N

N

R′ R H3C

Pyridoxamine

Pyridoxine

N

H

H3C

F N N

H3C

N OH

CO

O O

O

NH O

P

CH2

N

O

N O

(e)

N

CH2

G

O

CH3

(f) CH2

N

Pyridoxal

CH3 R′

CH2OH H3C

CH3

N

N

R

H3C

Co

H3C

CHO HO

(d)

CH3 R′

CH2OH

O

O O

CH

O H3C

O O

Riboflavin

N

Niacin

Cobalmin

FIG. 71-1 Chemical structure of water-soluble vitamins. (A) Folate polyglutamate. (B) Thiamin. (C) Biotin, biocytin, pantothenic acid, and lipoate. (D) Ascorbic acid and dehydroascorbic acid. (E) Pyridoxine, pyridoxal, and pyridoxamine. (F) Riboflavin. (G) Niacin. (H) Structure of cobalamin. The variable axial ligand is indicated by X.

1794 / CHAPTER 71 4

PteGlu uptake (pmole/mg protein/IO sec)

0.6

0.25 µM PteGlu

1/

V

2

0.4

0.5 µM PteGlu −2.2

0.2

4

5

6 7 Incubation buffer pH

2 4 [5-CH3H4 PteGlu]

8

FIG. 71-2. Effect of incubation buffer pH on initial rate of folic acid uptake by human jejunal brush-border membrane vesicles. (Modified from Said and colleagues [22], by permission.)

8

6

0.25 µM PteGlu

1/

V

4 0.5 µM PteGlu

situation, however, changes after resection of the proximal part of the small intestine, where a carrier-mediated uptake system is induced in the remaining ileal segment (21). The small-intestinal carrier-mediated folate uptake system is highly dependent on acidic extracellular pH with a markedly greater uptake at pH 5.0 to 5.5 compared with neutral or alkaline pH (Fig. 71-2). Studies with purified intestinal brush-border membrane vesicles (BBMVs) also have shown that the folate uptake system at the apical BBM is capable of transporting the negatively charged substrate (folate exists as an anion at physiologic pH values; pKa values for its α- and γ-carboxyl groups are 3.5 and 4.8, respectively) against a concentration gradient, and that the transport event is electroneutral in nature (22–25). In addition, the transport event was shown to be sensitive to the inhibitory effect of the anion transport inhibitors 4,4′-diisothiocyano-2-2-disulfonic acid stilbene (DIDS) and acetamidoisothiocyanostilbene-2,2′ disulfonic acid (SITS) (22,23). The effect of extracellular acidic pH on intestinal folate uptake was found to be mediated in part via a transmembrane pH gradient (suggesting the involvement of a folate−:OH− exchanger or a folate−:H+ cotransport mechanism, or both) and in part via the direct effect of the acidic extracellular pH on the activity of the folate carrier (22,23). Other studies have shown that the intestinal folate uptake process has similar affinity for reduced (e.g., 5-methyltetrahydrofolate [5-MTHF] and 5-formyltetrahydrofolate), oxidized (e.g., folic acid), and substituted (e.g., methotrexate) folate derivatives (22–25) (Fig. 71-3). The latter two observations—that is, the preference for acidic extracellular pH and the similar affinity for reduced oxidized and reduced folate derivatives—are unique to the intestinal folate uptake process and are different from the widely studied folate uptake process of the mouse leukemia cells. These differences exist despite that both processes are mediated via the same carrier system (see later). The folate uptake system in cells such as the mouse leukemia cells functions optimally at alkaline/neutral extracellular pH and has a greater preference for reduced overoxidized folate

−1.4

2 [MTX]

4

1/

V

4

6

0.25 µM PteGlu

2 0.5 µM PteGlu

−1.4

2 4 [5-CHOH4 PteGlu]

6

FIG. 71-3. Dixon plot of the effect of reduced (5-methyltetrahydrofolate [5-CH3H4PtcGlu]; 5-formyltetrahydrofolate [5-CHOPteGlu]) and substituted (methotrexate [MTX]) folate derivatives on the initial rate of 3H-folic acid (PtcGlu) uptake by human jejunal brush-border membrane vesicles. (Modified from Said and colleagues [22], by permission.)

derivatives; hence, it is referred to as the reduced folate carrier (RFC) (26–28). As to the mechanism of exit of folate out of the enterocyte, that is, transport across the basolateral membrane (BLM), studies using purified intestinal basolateral membrane vesicles (BLMVs) have shown the involvement of a specialized carrier-mediated system in this event. This system again was found to transport the substrate via an electroneutral process, to have similar affinity for oxidized, reduced, and substituted folate derivatives, and to be sensitive to the inhibitory effect of the anion transport inhibitor DIDS (29). As to the bacterial source of the vitamin, previous studies have shown that a substantial portion of this folate exists in the absorbable monoglutamate form, and that the large intestine is indeed capable of absorbing some of this vitamin (30). The mechanism of folate uptake in the large intestine has been investigated using purified colonic apical membrane vesicles (AMVs) isolated from the colon of human organ

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1795 donors and using the human-derived, nontransformed, colonic epithelial NCM460 cells (31,32). These studies have shown that folate uptake in the colon is efficient and occurs via a specialized, carrier-mediated system. This system was found to be similar to that of the small intestine in being highly dependent on extracellular acidic pH, electroneutral, and DIDS sensitive (31,32). These results have suggested that the same folate uptake system is functional in both regions of the intestinal tract. The identification of an efficient carriermediated system for folate uptake in the human large intestine lends further support to the belief that this source of folate may contribute to the host’s normal folate homeostasis, especially that of the localized colonic epithelial cells, and may also help explain the cause(s) of the localized folate deficiency observed in colonic epithelial cells believed to be associated with premalignant changes in the colonic mucosa (33,34).

Molecular Identity of the Intestinal Folate Uptake System Two specialized transport processes have been identified for folate uptake by mammalian cells. The first process involves the RFC, which is a classical cell membrane transport protein. The second process involves the folate receptor (FR), a protein that is anchored to the exterior surface of the cell membrane via glycosylphosphatidylinositol linkage and transports folate via a receptor-mediated endocytic process. Functional, molecular, and immunologic studies have indicated the FR is not functional/not expressed in the normal intestine (35–37); rather, intestinal folate uptake occurs via the carrier-mediated mechanism. The molecular identity of the intestinal folate carrier-mediated system has been determined by means of cloning the system from mouse, rat, and human intestine (38,39) (GenBank accession number U38180). The sequence of the open reading frame of the cloned human intestinal folate carrier was found to be the same as that of the folate carrier cloned from a number of other human tissues, that is, being that of the human RFC (hRFC) (40,41). Differences, however, were found in the 5′ untranslated region of the hRFC cloned from the different tissues. These differences were caused by alternate splicing of RNA transcripts from the same human SLC19A1 gene and result from the use of multiple promoters (28). Five hRFC splice variants have been identified in the different human tissues, with variant I being the predominant intestinal form (28). The open reading frame of the hRFC showed a high degree of sequence homology with RFCs of other mammals at both the nucleotide and the amino-acid levels. The hRFC is predicted to encode a protein of 591 amino acids with 12 predicted transmembrane domains, 3 putative protein kinase C (PKC) phosphorylation sites, and 1 potential N-glycosylation site. The RFC polypeptide is predicted to carry a net positive charge at physiologic pH, which may be important for its interaction with the negatively charged substrate (38,39). Functional identity of the cloned intestinal hRFC has been verified by expression in Xenopus oocytes, which showed a

significant and specific increase in 5-MTHF uptake (38,39). The induced folate uptake was found to be DIDS sensitive and saturable as a function of folate concentration and exhibited an apparent Km similar to that of native intestine. Differences, however, were found in the pH profile, as well as in the affinity of the expressed system toward oxidized and reduced folate derivatives, when compared with that of native intestine. Although folate uptake in the native intestine is markedly greater at acidic extracellular pH compared with neutral and alkaline pH (22,24), no such pH dependence was seen in the expressed system in Xenopus oocytes (38,39). In addition, the induced folate uptake in Xenopus oocytes showed preferential inhibition by reduced folate compared with the oxidized derivatives, which is in contrast with the situation in the native intestine where affinity for oxidized and reduced folate derivatives is similar (22,24). Further investigations into the cause(s) of these differences have indicated that the RFC system displays characteristics that are dependent on the type of cell that it is expressed in, and that these differences are probably because of disparities in membrane composition, cell-specific posttranslational modification(s) of the expressed protein, and/or involvement of cell-specific auxiliary protein(s) that modulate activity of the expressed RFC system (42). A study has reported that the RFC system may also transport the monophosphate derivative of thiamin in murine leukemia cells (43). This scenario, however, is unlikely to occur in the intestine because of the efficient dephosphorylation of phosphorylated thiamin derivatives in the lumen by the abundant intestinal phosphatases. The distribution of the RFC message, together the vertical axis of the intestine, has been examined by Northern blot analysis and in situ hybridization. Expression was found to be significantly higher in the mature and differentiated epithelial cells of the villous tip compared with the immature and undifferentiated epithelial cells of the crypt (38,39). These findings suggest that expression of intestinal RFC system is differentiation dependent. However, further studies are needed to address this issue. Distribution of the RFC message in the small and large intestines also has been examined, and the message was found in both regions, but at different levels (38,39). These findings further support the earlier observations that the functional folate uptake systems in the small and large intestines are identical. In addition, immunoblotting studies have shown that hRFC protein is expressed in both surface membrane domains of polarized enterocytes with a significant enrichment in the apical membrane (31). The bipolar expression may be important to ensure efficient extraction of folate from the intestinal lumen and its vectorial transport to the blood. Knowledge about structural-functional activity of the RFC system also has been forthcoming. Early studies have used group-specific reagents and have shown the possible involvement of histidine residues in the function of the RFC protein (44). These histidine residues were believed to be located at or near the folate-binding sites of RFC. More recent studies using a mutational approach have reported the importance of a number of positively charged amino-acid residues

1796 / CHAPTER 71 within the RFC transmembrane domains in the function of the carrier system (45). In addition, preliminary evidence also has been reported that assigned functional importance to other specific amino-acid residues in the function of the hRFC protein (46). Furthermore, studies have reported a functional role for the intracellular loop between transmembranes 6 and 7 of the RFC polypeptide (47), although no such role was found for the N- or C-terminal tails of the RFC protein (48).

Regulation of Intestinal Folate Uptake Process

Uptake (pmol/mg proteins/ 5 min)

The intestinal folate uptake process appears to be under the regulation of intracellular and extracellular factors/ conditions. There are reports suggesting that the intestinal folate uptake process is under the regulation of an intracellular protein tyrosine kinase (PTK)–mediated pathway (32,35,36). Pretreatment of the human-derived colonic epithelial NCM460 cells and the rat-derived intestinal epithelial IEC-6 cells with specific inhibitors of the PTK-mediated pathway was found to lead to a significant inhibition in folate uptake. The inhibition was found to be mediated via a decrease in the Vmax (but not the apparent Km) of the folate uptake process (Fig. 71-4), suggesting that the effect on the decrease in activity is caused by a decrease in the activity (and/or number) of the functional folate carriers without changes in their affinity. Similarly, an intracellular cyclic adenosine monophosphate (cAMP)–mediated pathway was reported to play a role in regulating intestinal folate uptake, but this regulation appears to be independent of the protein kinase A (PKA) regulatory pathway (32,35). The intestinal folate uptake process also was found to be regulated by extracellular folate levels. Dietary folate deficiency in rats was found to lead to a specific and significant up-regulation in intestinal carrier-mediated folate uptake (10).

The up-regulation was mediated via an increase in the Vmax (with no changes in the apparent Km) of the folate uptake process and was associated with a parallel increase in intestinal RFC protein and RNA levels. These findings suggest the possible involvement of a transcriptional regulatory mechanism(s) in this adaptive up-regulation in the intestinal folate uptake process. These observations have been confirmed in studies with the human-derived intestinal epithelial Caco-2 cells (49). In the latter studies, maintaining Caco-2 cells in a folate-deficient growth medium led to an up-regulation in folate uptake that was associated with an induction in hRFC protein and RNA levels. In addition, activity of the hRFC promoter B (the promoter that drives the expression of variant I of hRFC, i.e., the predominant intestinal variant) (28) fused to the Firefly luciferase reporter gene and transfected into Caco-2 cells was found to be significantly higher in cells maintained in folate-deficient medium compared with those maintained in control growth medium (Fig. 71-5). The most responsive sequence of the hRFC promoter B to folate deficiency was found to be encoded in a sequence between −2016 and −1431 (using the A of the initiation ATG sequence as position 1), which is outside the minimal region required for basal activity of this promoter in Caco-2 cells (the latter is encoded in a sequence between −1088 and −1043) (49) (see Fig. 71-5). The intestinal folate uptake process also was found to be regulated ontogenically (50). Transepithelial transport of folate in rat intestine was found to undergo clear changes during early stages of life (50). This was mediated via a progressive decrease in the Vmax (with no changes in the apparent Km) of the folate uptake process with maturation (i.e., from suckling to weanling to adult). These ontogenic changes were found to involve the entry step of folate across the apical BBM of the polarized enterocytes (51). In addition, the changes were found to be associated with a parallel decrease in the level of the RFC protein and messenger RNA (mRNA),

3

2

1

1 2 Folic acid concentration (µM)

3

FIG. 71-4. Effect of genistein on initial rate of folic acid uptake by the rat-derived intestinal epithelial IEC-6 cells. (Modified from Said and colleagues [35], by permission.)

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1797

600

400

−1043bp

−1431bp

0

−1671bp

200

−2016bp

Relative expression over pGL3 basic (x fold)

800

FIG. 71-5. Effect of folate deficiency on the activity of hRFC promoter B in the human-derived intestinal epithelial Caco-2 cells. (Modified from Subramanian and colleagues [49], by permission.)

membrane have begun to emerge (54,55). Confocal imaging studies using live human-derived intestinal epithelial cells and a series of truncated fusion proteins of hRFC with the enhanced green fluorescent protein (i.e., hRFC-EGFP) to image the targeting and trafficking dynamics of the hRFC polypeptide have shown that the molecular determinants that dictate the targeting of the hRFC protein to the cell membrane reside within the hydrophobic backbone of the polypeptide and not within its N- or C-terminal domains (55). These studies also have shown that the integrity of the hRFC backbone is critical for exporting the polypeptide from the endoplasmic reticulum to the cell surface. Numerous trafficking vesicles that contain the hRFC-EGFP fusion protein also have been identified and appear to be involved in the intracellular movement of the protein (55) (Fig. 71-6; real-time movies can be viewed online at: http://www.jbc.org/cgi/ content/full/277/36/33325/DC1). This intracellular trafficking process was found to be critically dependent on the existence of an intact microtubule network because its disruption led to a severe inhibition in the motility of the hRFC-containing trafficking vesicles, as well as in the final expression of the hRFC protein at the cell membrane (55).

THIAMIN (VITAMIN B1) as well as a decrease in the transcription rate of the RFC gene, as indicated by results of Western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and nuclear run-on assays, respectively (51). These findings suggest that the ontogenic regulation of the intestinal folate uptake process occurs via a transcriptional regulatory mechanism(s). Additional studies are needed to determine the details of this mechanism. As stated earlier, the intestinal folate uptake process is adaptively regulated in response to dietary folate deficiency and during ontogeny (10,49–51). In both of these cases, the regulation appears to involve transcriptional regulatory mechanisms; thus, knowledge about the 5′-regulatory regions of the hRFC gene is important to understand the exact mechanisms involved. Such knowledge has been emerging after the cloning and characterization of the 5′-regulatory region of the hRFC gene (reviewed by Sirotnak and Tolner [28]). Three TATA-less promoters have been identified that are capable of driving the transcription of the multiple variants of the hRFC gene in the different human tissues (28). A number of nuclear factors that interact with the hRFC promoters also have been identified (28,52). The hRFC promoter B appears to be responsible for driving the transcription of variant I, the predominant hRFC splice variant expressed in the human intestine (28,53).

Cell Biology of the Human Reduced Folate Carrier Protein in Intestinal Epithelia Studies on the mechanisms involved in membrane targeting and intracellular trafficking of the hRFC protein to cell

Thiamin (see Fig. 71-1) in its coenzyme form, that is, thiamin pyrophosphate, is involved in a variety of metabolic reactions that include the decarboxylation of pyruvic and α-ketoglutamic acids and the use of pentose in the hexose monophosphate shunt. Thiamin deficiency in humans leads to a variety of clinical abnormalities including neurologic (neuropathy, Wernicke–Korsakoff syndrome, or both) and

10 µM

FIG. 71-6. Involvement of trafficking vesicles in the intracellular movement of the human reduced folate carrier fused to the enhanced green fluorescent protein in the human-derived intestinal epithelial HuTu-80 cells. (Modified from Marchant and colleagues [55], by permission.)

1798 / CHAPTER 71 cardiovascular (peripheral vasodilatation, biventricular myocardial failure, edema, potentially acute fulminant cardiovascular collapse) disorders. In contrast, optimization of thiamin levels appears to have potential in preventing diabetic retinopathy and blocking hyperglycemic tissue damage caused by uncontrolled diabetes (56). Thiamin deficiency and suboptimal levels occur in a high percentage of alcoholic and diabetic patients (57–60), in patients with coeliac and renal diseases (61,62), and in subjects receiving long-term use of the diuretic furosemide (63). A tissuespecific deficiency of thiamin also has been reported in thiamin-responsive megaloblastic anemia (TRMA), an autosomal recessive disorder characterized by manifestations of sensorineural deafness and diabetes mellitus (64,65). A genetic defect in the human thiamin transporter-1 (the product of the SLC19A2 gene) is believed to be the cause of TRMA (66–69).

Mechanisms of Intestinal Thiamin Absorption The intestinal tract encounters two sources of thiamin: a dietary source and a bacterial source (that is generated by the normal microflora of the large intestine (2). Dietary thiamin exists mainly as the pyrophosphate forms and is hydrolyzed by the abundant intestinal phosphatases to free thiamin before absorption (70). Absorption of the liberated free thiamin then takes place mainly in the proximal part of the small intestine. The mechanism of absorption of thiamin in the small intestine has been the subject of intense investigation using a variety of intestinal preparations from a number of species including humans (71–74). Collectively, these studies have shown the involvement of a specialized carriermediated mechanism in the uptake of the cationic thiamin. This mechanism is inhibited by thiamin structural analogues, but not by unrelated organic cations (71–75). Studies with purified BBMVs isolated from human and animal small intestine have shown that the thiamin uptake process across the apical membrane domain of the polarized enterocyte occurs via a pH-dependent (but not Na+-dependent), electroneutral, and amiloride-sensitive carrier-mediated mechanism (72,73). When an outwardly directed H+ gradient (pH in < pH out) was imposed across the membrane of BBMVs, the involved thiamin uptake system(s) transported the substrate against a concentration gradient in the intravesicular space. The mechanism involved in the exit process of thiamin out of the intestinal absorptive cells, that is, transport across the BLM, also has been studied using purified intestinal BLMV isolated from human and rat intestine (76,77). In both cases, evidence was obtained to show the involvement of a specialized, pH-dependent, electroneutral, carrier-mediated mechanism. With regard to potential metabolic alterations in the thiamin molecule during absorption in the guts, studies have shown that some of the absorbed thiamin is converted into the phosphorylated forms of the vitamin (mainly to thiamin pyrophosphate); however, only free thiamin was found to exit the intestinal absorptive cells across the BLM into the serosal side (71).

Absorption of the bacterially synthesized thiamin in the large intestine, a second source of thiamin, has been studied using the human-derived colonic epithelial NCM460 cells as a model to human colonocytes. The results showed the existence of an efficient thiamin uptake mechanism that is similar to that of the small intestine in being specific, carrier-mediated, and able to transport the vitamin via a thiamin+-H+ exchange mechanism (78). The existence of an efficient carrier-mediated system for thiamin uptake in the large intestine supports the belief that the normal microflora may contribute to the host’s normal thiamin homeostasis, and especially that of the localized colonocytes.

Molecular Identity of the Intestinal Thiamin Uptake Systems The molecular identity of the intestinal thiamin uptake systems has been elucidated after the cloning of two thiamin transporters, the thiamin transporter-1 (for humans, it is referred to as hTHTR-1; for mice, it is referred to as mTHTR-1) and the thiamin transporter-2 (for human, hTHTR-2; for mouse, mTHTR-2), from a number of human and mouse tissues (66–69,79–81). These transporters are the products of the human SLC19A2 (Slc19a2 for mouse) and the human SLC19A3 (Slc19a3 for mouse) genes, respectively. The hTHTR-1 encodes a protein of 497 amino acids, and the hTHTR-2 encodes a protein of 496 amino acids. Both transporters have 12 putative membrane-spanning domains, and both have the N- and the C-terminal tails extending into the cell interior. The hTHTR-1 and hTHTR-2 share 48% identity and 64% similarity with one another; they also share 40% and 39% identity at the amino acid level, respectively, with the hRFC (67,80,81). However, the hTHTR-1 and the hTHTR-2 transport folate and the RFC do not transport free thiamin (79). One study, however, has reported that the RFC system is capable of transporting thiamin monophosphate in L1210 murine leukemia cells (43). Although this may represent an alternative mechanism for thiamin import into nonintestinal cells, it is unlikely that this route contributes to normal intestinal absorption of dietary thiamin because of the efficient dephosphorylation of phosphorylated dietary thiamin derivatives in the intestinal lumen before absorption by the abundant intestinal phosphatases. Studies on the expression of hTHTR-1 and the hTHTR-2 in the intestinal tract have shown that both of these transporters are expressed at the RNA and the protein levels (82,83). The hTHTR-1 message was found to be expressed along the intestinal tract with levels in the following order: duodenum > jejunum > colon > cecum > rectum > ileum (82); the hTHTR-2 message was found to be expressed with levels in the following order: duodenum > jejunum > ileocecum > colon > ileum > rectum > cecum (83). The membrane domain of the polarized human enterocyte at which the hTHTR-1 and hTHTR-2 proteins are expressed also has been investigated using an immunoblotting approach (using specific polyclonal antibodies and native human jejunal

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1799 BBM and BLM preparations) and a confocal imaging approach (using fusion proteins of hTHTR-1-EGFP and hTHTR-2-EGFP expressed in living human intestinal epithelial Caco-2 cells) (Fig. 71-7) (83). Results of both approaches have shown that although the hTHTR-1 protein is expressed at both the apical and the BLM domains of the polarized human enterocytes (Figs. 71-7B and D), expression of the hTHTR-2 protein appears to be restricted only to the apical membrane domain of the intestinal absorptive epithelial cells (Figs. 71-7A and C) (83). These findings may have physiologic implications for the role of each of these transporters in regulating the intestinal thiamin absorption process and body homeostasis of the vitamin in health and disease. Indeed, studies from our laboratory have shown that these two thiamin transporters respond differently to altered nutritional conditions (see later).

Because the hTHTR-1 and the hTHTR-2 are both expressed in intestinal epithelial cells, it was important to understand their relative contribution toward total carriermediated intestinal thiamin uptake. This was achieved in studies by Said and colleagues (83) using the approach of selective silencing of the hTHTR-1 and the hTHTR-2, either individually or together, using gene-specific small interfering RNA (siRNA), followed by determination of thiamin uptake via the carrier-mediated process. The results showed that both of the thiamin transporters play a significant role in the normal intestinal thiamin uptake process, and that together they account for total carrier-mediated thiamin uptake by human intestinal epithelial cells (Fig. 71-8). It is of interest that patients with TRMA (SLC19A2 defective) and knockout mice with targeted disruption of the Slc19a2 gene both have normal thiamin plasma levels (66,69,84).

C

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FIG. 71-7. Distribution of human thiamin transporter-1 (hTHTR-1 [B, D]) and hTHTR-2 (A, C) fused to the enhanced green fluorescent protein in the human-derived intestinal epithelial Caco-2 cells grown on filters. (See Color Plate 35.) (Modified from Said and colleagues [83], by permission.)

1800 / CHAPTER 71 Regulation of the Intestinal Thiamin Uptake Process

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FIG. 71-8. Thiamin uptake by control Caco-2 cells and those treated with gene-specific small interfering RNA (siRNA) for human thiamin transporter-1 (hTHTR-1) and hTHTR-2. (Modified from Said and colleagues [83], by permission.)

This finding points to the involvement of another intestinal thiamin uptake mechanism that is capable of absorbing this vitamin, thus providing indirect support for the earlier findings that both THTR-1 and THTR-2 are involved in intestinal thiamin uptake. Knowledge about the structure-function relation of the hTHTR-1 also has been forthcoming from both clinical and experimental findings. Clinical findings have reported the existence of 15 distinct mutations in the hTHTR-1 in patients with TRMA (85,86). Four of these mutations were found to be missense in nature, whereas the rest were nonsense mutations resulting in an early truncation of the carrier protein. The inhibitory effect of three of the missense mutations on functional activity of the hTHTR-1 has been demonstrated experimentally by means of site-directed mutagenesis followed by expression of the mutant constructs in HeLa cells (87). Other studies have shown that amino acid 138 of the hTHTR-1 polypeptide (which is the only conserved anionic amino acid in any of the predicted transmembrane domains of the protein) is critical for the normal function of the carrier protein in transporting the cationic thiamin (88). In addition, both of the potential N-glycosylation sites predicted in the hTHTR-1 polypeptide (i.e., the one at position 63 and the one at position 314) were indeed found to be glycosylated in the native protein. However, neither of these glycosylation sites was found to be necessary for the function of the hTHTR-1 or for the expression of the carrier protein at the plasma membrane (87). Regarding the structure-function relation of the hTHTR-2, nothing currently is known on the subject.

The intestinal thiamin uptake process appears to be under the regulation of intracellular and extracellular factors/ conditions. Evidence from studies with the human-derived intestinal epithelial Caco-2 cells and the human-derived colonic epithelial NCM460 cells has suggested that the intestinal thiamin uptake process is under the regulation of an intracellular Ca2+/calmodulin-mediated pathway (75,78). This pathway appears to act through decreasing the Vmax, but not the apparent Km, of the intestinal thiamin uptake process, suggesting that the effect is most likely mediated via changes in the activity (and/or number), but not affinity, of the thiamin uptake carriers, respectively. The intestinal thiamin uptake process also was found to be adaptively regulated by extracellular thiamin levels (74,88). Thiamin deficiency in a human subject has been reported to lead to an increase in thiamin uptake compared with uptake in control subjects. This effect was reported to be mediated via changes in the Vmax and the apparent Km of the intestinal thiamin uptake process (74). The finding of adaptive regulation in the intestinal thiamin uptake process during thiamin deficiency has been confirmed in studies with mice (88). In the latter studies, dietary thiamin deficiency was found to lead to an induction in intestinal carrier-mediated thiamin uptake. This increase in thiamin uptake was associated with a significant increase in the mRNA and protein levels of mTHTR-2, but not that of mTHTR-1. In addition, activity of the luciferase reporter gene in transgenic mice expressing the human SLC19A3 promoter-luciferase construct was found to be significantly greater in the intestine of thiamin-deficient mice compared with the intestine of their pair-fed counterparts (88). In contrast, no changes in the activity of the luciferase reporter gene in transgenic mice expressing the human SLC19A2 promoter-luciferase construct were observed during thiamin deficiency (88). These data provide evidence that the adaptive up-regulation during intestinal thiamin uptake process in thiamin deficiency is mediated via an induction in the level of expression of the THTR-2, but not that of the THTR-1, and that this induction involves transcriptional regulatory mechanism(s). Studies have shown that the intestinal thiamin uptake process also is regulated during ontogeny (89). A decrease in thiamin uptake by mouse intestinal BBMV was observed with maturation (suckling to weanling to adult). The decrease noted in thiamin uptake was associated with a decrease in the level of mRNA of both the mTHTR-1 and the mTHTR-2 (89). These studies also have shown that in transgenic mice expressing the human SLC19A2 promoterluciferase construct and those expressing the human SLC19A3 promoter-luciferase construct, the activity of the luciferase reporter gene in the gut decreases with maturation (89). These data provide strong evidence that ontogenic regulation in the intestinal thiamin uptake process is mediated via a decrease in the expression of both the THTR-1 and the THTR-2, and that the decrease is mediated via transcriptional regulatory mechanisms.

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1801 The intestinal thiamin uptake process is adaptively regulated in response to dietary thiamin deficiency, as well as during ontogeny (74,88,89), with both appearing to involve transcriptional regulatory mechanisms. The 5′-regulatory regions of the SLC19A2 and the SLC19A3 genes have been cloned (82,90,91). The 5′-regulatory region of the SLC19A2 gene has been characterized and its functionality confirmed by expression of the cloned promoter fused to the Firefly luciferase reporter gene in human intestinal epithelial Caco-2 cells (82,90). In addition, the minimal promoter region required for basal activity of the SLC19A2 promoter has been determined and found to be mainly encoded in a sequence between −356 and −36 (using the A of the initiation ATG sequence as position 1), and also has been shown to include multiple putative cis-regulatory elements (82,90) (Fig. 71-9). A number of these cis-elements (namely, gutenriched Krupple-like factor [GKLF], nuclear factor-1 [NF-1], and stimulating protein-1 [SP1]) were found to play an important role in regulating the activity of the SLC19A2 promoter as shown by studies involving mutational analysis, oligonucleotide competition assays, and electromobility shift and supershift assays (90). Studies also have confirmed the functionality and established the physiologic relevance of the full-length and the minimal SLC19A2 promoterluciferase constructs in vivo in transgenic mice (90). The pattern of tissue distribution of the luciferase activity in the transgenic mice expressing these human promoter constructs was found to be similar to the expression of the hTHTR-1 mRNA in different human tissues. Other investigations also have determined the transcription initiation site(s) of the SLC19A2 gene in intestinal epithelial Caco-2 cells using 5′-rapid amplification of complementary DNA (cDNA) ends (5′-RACE). Three such sites have been identified at positions −183, −192, and −220. In separate studies, the human thiamin transporter SLC19A2 was shown to be a target for activation by the p53 tumor-suppressor transcription factor in the murine erythroleukemia cells (92), but it is unclear whether similar activation occurs in intestinal epithelial cells. The 5′-regulatory region of the human SLC19A3 gene also has been cloned, characterized, and its functionality confirmed in the human intestinal epithelial Caco-2 cells

in vitro and in transgenic mice in vivo (91). In addition, the minimal promoter region required for basal activity of this promoter has been determined and found to be mainly encoded in a sequence between −77 and +59 (using the start of transcription initiation as position 1) (91). This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for an SP1/guanylyl cyclase (GC)–box binding site (at −48/−45 base pair [bp]) being established using mutational analysis, electromobility shift, and supershift assays, and after expression in Drosophila SL2 cells. The transcription initiation site for the SLC19A3 gene in Caco-2 cells also was determined and found to be at position −88.

Cell Biology of the Intestinal Thiamin Transporters Studies on the intracellular events involved in the intracellular trafficking and membrane targeting of the human thiamin transporters in human intestinal epithelial cells have begun to emerge (93). Confocal imaging studies using live human-derived intestinal epithelial HuTu-80 cells and Caco-2 cells (83,93) and a series of truncated hTHTR-1-EGFP fusion proteins have shown that the full-length fusion protein is expressed at the cell membrane of Hutu-80 and Caco-2 cells. The expression was observed at both the apical and BLM domains of these cells (83,93). Analysis of the expression pattern of truncated mutants of the hTHTR-1 have shown that although the C-terminal region of the polypeptide has no role in directing the protein to cell membrane, an essential role was found for both the N-terminal tail and the integrity of the backbone of the polypeptide in the targeting event (Fig. 71-10). These studies also have shown that truncation of the hTHTR-1 polypeptide within a region where several TMRA truncations are clustered results in intracellular retention of the mutant protein. Regarding intracellular trafficking of the hTHTR-1 protein, the protein was found to be inside numerous intracellular trafficking vesicles with the movement of these vesicles being temperature dependent and interrupted by treating the cells with microtubule (but not microfilament) destabilizing agents (93;

FIG. 71-9. Diagrammatic representation of the minimal region required for basal activity of the SLC19A2 promoter.

1802 / CHAPTER 71 A

D

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FIG. 71-10. Distribution of truncated human Na+-dependent biotin transporter fused to the enhanced green fluorescence protein (hTHTR1-EGFP) constructs in the human-derived intestinal epithelial HuTu-80 cells. (See Color Plate 36.) (Modified from Subramanian and colleagues [93], by permission.)

real-time movies can be viewed online at: http://www.jcb. org/cgi/content/full/278/6/3976/DC1).

BIOTIN (VITAMIN H) Biotin (see Fig. 71-1) acts as a coenzyme for five carboxylases in mammalian cells. These carboxylases are propionyl-coenzyme A (CoA) carboxylase, methylcrotonylCoA carboxylase, pyruvate carboxylase, and the two isoforms of acetyl-CoA carboxylase. The biotin-dependent carboxylases are involved in a number of important metabolic reactions that include fatty acid biosynthesis, catabolism of certain amino acids and fatty acids, and gluconeogenesis. Biotin also appears to play a role in the regulation of oncogene expression (94) and in regulating the intracellular level of cGMP (95–97). Thus, it is not surprising that deficiency of this micronutrient leads to serious clinical abnormalities, including growth retardation, neurologic disorders, and dermatologic disorders. Furthermore, animal studies have shown that biotin deficiency during pregnancy leads to embryonic growth retardation, congenital malformation, and death

(98–101). An increase in the incidence of biotin deficiency and suboptimal levels has been reported. Biotin deficiency occurs in patients with inborn errors of biotin metabolism (102), in patients receiving long-term therapy with anticonvulsant agents (103–106), and in patients receiving long-term parenteral nutrition (107). Suboptimal levels of biotin have been reported during pregnancy (108), in substantial numbers of alcoholics (109), and in patients with inflammatory bowel disease (110).

Mechanism of Intestinal Biotin Transport Two source of biotin are available to the host: (1) a dietary source that is mainly processed in the small intestine; and (2) a bacterial source where the vitamin is synthesized by the normal bacterial flora of the large intestine (2), which is processed in that segment of the gut. Dietary biotin exists in the free and protein-bound forms. Ingested protein-bound forms of biotin are first broken down by gastrointestinal proteases and peptidases to biocytin (biotinyl-L-lysine; see Fig. 71-1) and biotin-short peptide conjugates (111).

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1803 Free biotin then is liberated from biocytin and biotin-short peptides in the intestinal lumen before absorption. This is accomplished by the specific action of biotinidase, an enzyme that is believed to be of pancreatic origin (111). Clinical and experimental evidence have demonstrated the importance of this hydrolysis step for efficient absorption and optimal bioavailability of dietary protein-bound biotin (111,112). Biotinidase has been cloned, and its gene structure has been determined (113). Several clinical mutations in this enzyme have been identified that lead to the autosomal recessive metabolic disorder of “biotinidase deficiency” (114). In this disorder, the affected individuals cannot recycle/digest biocytin (and biotin-short peptides) to free biotin, and thus their cellular uptake of biotin (including their ability to absorb dietary protein-bound biotin) is diminished (111). Supplementation of free biotin to these patients can prevent (and in some cases reverse) the clinical symptoms of biotinidase deficiency, which include seizure, vision problems, alopecia, developmental delay, and hearing loss (111,114). The mechanism of uptake of free biotin in the small intestine that originates from the diet has been the subject of intense investigation using a variety of intestinal preparations (reviewed by Said and colleagues [115,116]). Collectively, these studies have shown the involvement of a Na+-dependent, carrier-mediated mechanism in the intestinal biotin absorption process. Functional studies using purified BBMVs and BLMVs isolated from the small intestine of a number of species (including humans) have shown that the Na+dependent, carrier-mediated mechanism is operational only at the apical BBM domain of the polarized intestinal epithelial cells (117–122). This has been confirmed by means of Western blotting using specific polyclonal antibodies against the Na+-dependent biotin carrier (i.e., against the sodiumdependent multivitamin transporter [SMVT]; see later) and BBM and BLM proteins isolated from the jejunum of human organ donors (Fig. 71-11). The findings also have been confirmed by confocal imaging of living intestinal BBM

BLM

FIG. 71-11. Expression of the human sodium-dependent multivitamin transporter (hSMVT) protein in native human jejunal brush-border membrane (BBM) and basolateral membrane (BLM). Western blot analysis was run using 150 µg protein of native human jejunal BBM and BLM isolated by an established procedure. The blot was probed with specific polyclonal antibodies directed against a specific peptide of the hSMVT protein and detected using the enhanced chemiluminescence system.

epithelial cells expressing the human Na+-dependent biotin transporter fused to the enhanced green fluorescence protein (i.e., hSMVT-EGFP) (Fig. 71-12). In both of these approaches, expression of the Na+-dependent biotin transporter was found to be only at the apical BBM domain, and not at the BLM domain of the polarized enterocytes. The apical Na+dependent carrier system was shown to be capable of transporting biotin against a concentration gradient and represents the rate-limiting step in the overall absorption process of the vitamin across the intestinal epithelia (117–119). The importance of Na+ in driving biotin movement across the intestinal BBM appears to be mediated via its inwardly directed gradient, and not through the mere presence of the cation in the extracellular compartment (117). Other studies have shown biotin uptake to be higher in the proximal compared with the distal part of the small intestine (120). Regarding the mechanism of exit of the negatively charged biotin out of the intestinal absorptive epithelial cells across the BLM, this process was again found to occur via a specialized, carriermediated mechanism (121,122). The latter mechanism, however, was found to be Na+ independent and electrogenic in nature being significantly stimulated on induction of a positive transmembrane electrical potential (121,122). An interesting observation regarding the intestinal transport system of biotin was the subsequent recognition that this system also is used by two other functionally unrelated nutrients, namely, pantothenic acid and lipoate (see Fig. 71-12) (123,124). Pantothenic acid, a water-soluble vitamin, is essential for the synthesis of coenzyme A and acyl carrier proteins in mammalian cells, and thus is important in the metabolism of carbohydrate, fat, and protein. Lipoate is a potent intracellular and extracellular antioxidant and is needed in the redox cycling of other antioxidants such as vitamins C and E, as well as in the up-regulation of intracellular glutathione level. The utilization of the three functionally unrelated micronutrients (i.e., biotin, pantothenic acid, and lipoate) for the same transport system is not unique to the intestine, but also occurs in other cellular systems that include the brain, the heart, and the placenta (125–127). These observations have increased the physiologic and nutritional importance of the intestinal biotin uptake system, which is now called the SMVT system. The intestinal SMVT system also appears to interact with certain antiepileptic drugs (128,129). Studies have shown that carbamazepine and primidone competitively inhibit biotin uptake in human jejunal BBMVs (128,129). These latter findings add pharmacologic and clinical relevance to the SMVT transport system. Regarding the bacterial source of biotin, previous studies have shown that a substantial portion of this biotin exists in the large-intestinal lumen in the form of free unbound biotin, and thus is available for absorption (2). Furthermore, in vivo studies in humans, rats, and minipigs have shown that the colon is capable of absorbing significant amounts of luminally introduced biotin (130–132). The mechanism involved in the uptake of biotin in the large intestine also has been examined using the human-derived colonic epithelial NCM460 cells as a model for human colonocytes (123). The results showed

1804 / CHAPTER 71 A

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FIG. 71-12. Distribution of human sodium-dependent multivitamin transporter (hSMVT) fused to the enhanced green fluorescence protein (EGFP) in the human-derived intestinal epithelial Caco-2 cells grown on filters. (A) Lateral confocal image (x-y) showing Caco-2 cell expressing human hSMVTEGFP and dsRed (a cytoplasmic dye) imaged 48 hours after transient transfection. (B) Fluorescence distribution in a Caco-2 cell transfected with EGFP alone. (C, D) Axial confocal sections (x-z) of the same Caco-2 cells shown in A and B, expressing hSMVT-EGFP (C) or EGFP alone (D). Notice the exclusive expression of hSMVT at the apical membrane of Caco-2 cells. (See Color Plate 37.)

the existence of an efficient and specialized Na+-dependent, carrier-mediated mechanism for biotin uptake by these cells that is similar to that seen for the vitamin transport in the small intestine. The argument that the bacterial source of biotin contributes to the host’s normal biotin homeostasis, and especially that of the localized colonic epithelial cells, is supported by the identification of an efficient carrier-mediated system for biotin uptake in the large intestine.

Molecular Identity of the Intestinal Biotin Uptake System The cloning from the intestine of a number of species (including the rat, rabbit, human, and more recently, mice; GenBank accession number AY572835) (133,134) has allowed the molecular identity of the intestinal biotin uptake system to be determined. High degree of sequence homology was found between the SMVT clones of the different species at both the nucleotide and the amino-acid levels. The SMVT protein was predicted to have 12 membrane-spanning domains with both the N- and C-terminal tails on the cytoplasmic side of cell membrane. In addition, a number of potential posttranslational modification sites that include phosphorylation and glycosylation sites were identified. Functional identity of the cloned SMVTs has been established by expression of the cloned cDNA in a number of heterologous systems that showed the protein to transport biotin and pantothenate in an Na+-dependent manner with kinetics similar to that observed

in the native intestine. Molecular studies in the rat also have shown the existence of significant heterogeneity in the 5′ untranslated region of the rat SMVT, with four distinct variants (I, II, III, and IV) being identified (133). Variant II was found to be the predominant form that is expressed in the rat small and large intestines (133). Distribution of the SMVT mRNA along the vertical and longitudinal axes of the intestine also has been delineated. A 2.6-fold greater level of expression of the SMVT message was found in the mature differentiated cells of the villous tip compared with the immature and undifferentiated cells of the crypt. Carrier-mediated biotin uptake paralleled this finding with a significantly greater uptake in intestinal villi compared with crypt epithelial cells (133). Distribution of the SMVT message along the longitudinal axis of the gut was found to be similar in the different regions of the gut (133,134). The latter finding is in contrast with the greater level of biotin uptake observed in the proximal part of the small intestine compared with the ileum and colon (120,135). These findings suggest the possible involvement of specific posttranslational modifications that ultimately may affect the functionality of the biotin uptake system in the different regions of the gut. Two functional studies on biotin uptake in nonintestinal cells (the human peripheral blood mononuclear cells and the human keratinocytes) have reported the potential existence of an additional biotin uptake system that operates at the nanomolar range (apparent Km of this putative system was estimated at 2.6 nM) (136,137). The putative system also was reported to be specific for biotin and does not transport

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1805 pantothenic acid (136,137). To determine whether the human intestine also expresses this high-affinity biotin transporter, Balamurugan and colleagues (138) used physiologic and molecular biology approaches to address the issue. In the physiologic approach, the characteristics of biotin uptake by human-derived intestinal epithelial Caco-2 cells at the nanomolar concentration range were investigated. In the molecular biology approach, the endogenous SMVT system of Caco-2 cells was selectively silenced with the use of hSMVT gene–specific siRNA, followed by examination of carrier-mediated biotin uptake in siRNA treated and control cells. Their findings showed the initial rate of biotin uptake as a function of concentration over the range of 0.1 to 50 nM to be linear. In addition, the presence of 100 nM unlabeled biotin, desthiobiotin, or pantothenic acid in the incubation medium did not affect the initial rate of uptake of 2.6 nM 3 H-biotin. Pretreatment of Caco-2 cells with SMVT-specific siRNA, which led to a substantial reduction in SMVT mRNA and protein levels, caused a severe inhibition in initial rate of carrier-mediated biotin (2.6 nM) uptake (Fig. 71-13). These findings indicated that the putative human high-affinity biotin uptake system does not function in human intestinal epithelial cells, and that the SMVT system is the major (if not the only) biotin uptake carrier that operates in these cells. Little currently is known about the structure-function relation of the SMVT system. Using group-specific reagents, Said and Mohammadkhani (139) have shown the involvement of histidine residues and sulfhydryl groups in the function of the biotin transporter in intestinal BBMVs. The histidine residues appear to be located at or near the substrate-binding site(s) as addition of biotin to the BBMV suspension before treatment with the histidine-modifying reagent (diethyl pyrocarbonate) led to a significant protection in biotin uptake against the effect of the inhibitor (139). Further studies using site-directed mutagenesis and deletion experiments are needed to delineate the structure-function relation of the intestinal SMVT system.

Carrier-mediated [3H]Biotin uptake (fmol/3min/mg protein)

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FIG. 71-13. Initial rate of carrier-mediated biotin uptake by small interfering RNA (siRNA) pretreated and untreated Caco-2 cells. (Modified from Balamurugan and colleagues [138], by permission.)

Regulation of the Intestinal Biotin Uptake Process Both intracellular and extracellular factors/conditions appear to regulate the intestinal biotin uptake process. Evidence now exists to suggest that the intestinal biotin uptake process is under the regulation of an intracellular PKCmediated pathway (132,140). Activation of PKC was found to lead to a significant inhibition in biotin uptake, whereas PKC inhibition was found to lead to a slight (but significant) increase in the substrate uptake by Caco-2 cells and by the human-derived colonic epithelial NCM460 cells (132,140). The PKC-mediated regulatory effect was found to occur via a decrease in the Vmax (but not the apparent Km) of the biotin uptake process, suggesting a possible decrease in the activity (and/or the number), but not affinity, of the SMVT system. Interestingly, two putative PKC phosphorylation sites do exist in the SMVT sequences (133,134), but their involvement in the PKC-mediated effect on biotin uptake is unclear. Another intracellular regulatory pathway, namely, the Ca2+/calmodulinmediated pathway, also appears to be involved in the regulation of the intestinal biotin uptake process (124). Specific inhibitors of this pathway were found to cause a significant inhibition in initial rate of carrier-mediated biotin uptake (124). This inhibition again appears to be mediated via a decrease in the Vmax (but not the apparent Km) of the intestinal biotin uptake process. The latter findings suggest that the effect is probably mediated via changes in the activity (and/or the number) of the SMVT system with no changes in its affinity. The Ca2+/calmodulin-mediated pathway appears to exert its regulatory effect on intestinal biotin uptake via a different mechanism than that of the PKC-mediated pathway. This conclusion is based on the observations that simultaneous activation of the PKC pathway and inhibition of the Ca2+/ calmodulin pathway leads to an additive increase in the degree of inhibition in biotin uptake (124). The intestinal biotin uptake process also was found to be regulated by extracellular biotin levels (140). Biotin deficiency in rats leads to a specific and significant up-regulation in intestinal carrier-mediated biotin uptake. In contrast, oversupplementation of rats with biotin was shown to lead to a specific and significant down-regulation in intestinal biotin uptake. These adaptive changes in intestinal biotin uptake were found to be mediated mainly via changes in the Vmax of the substrate uptake process with no significant changes in its apparent Km. The latter findings suggest that changes in the number (and/or activity) of the biotin transporters mediates the regulation of the intestinal biotin uptake by substrate levels, and not changes in their affinity. The findings with rats described above have been confirmed in studies with the human-derived intestinal epithelial Caco-2 cells and with the young adult mouse colonic (YAMC) epithelial cells (J. Reidling and H. M. Said, unpublished observations). In the latter studies, biotin uptake by cells maintained in biotindeficient growth medium was found to be associated with a significant increase in intestinal SMVT protein and mRNA levels. No change, however, in the stability of the intestinal SMVT RNA in biotin deficiency was found (Reidling J,

1806 / CHAPTER 71 Nabokina S, Said HM; unpublished observations). These findings clearly suggest the involvement of transcriptional regulatory mechanism(s) in the adaptive regulation of intestinal biotin uptake during biotin deficiency. Further studies are required to identify and characterize these regulatory elements. The intestinal biotin uptake process also is regulated ontogenically (141,142). Transepithelial transport of biotin in rat intestine was found to undergo clear changes during early stages of life (141). A change in the preferential site of absorption was observed through maturation with the site changing from the ileum to the jejunum (141). The observed changes in the jejunum were found to be mediated via a progressive increase in the Vmax and the apparent Km of the biotin carrier-mediated uptake process with maturation (i.e., from suckling to weanling to adult rats). The latter changes were found in studies to involve the entry step of biotin across the apical BBM of the polarized enterocytes (142). In addition, the changes were found to be associated with a parallel increase in SMVT protein and mRNA levels in the jejunum and in the transcription rate of the SMVT gene, as indicated by results of Western blotting, RT-PCR, and nuclear run-on assays, respectively (142). These findings suggest that the ontogenic regulation of the intestinal biotin uptake process involves transcriptional regulatory mechanism(s). Additional studies, however, are needed to determine the exact nature of this mechanism(s). It is notable that although clear ontogenic changes occur in the intestinal biotin uptake process, no such changes take place in the renal biotin uptake process or in the level of expression of the SMVT in the renal epithelia during development (142). These observations point out that differences exist in the regulation of the biotin uptake process in the intestine and kidney. As stated earlier, expression and function of the SMVT in rat intestine in vivo is dependent on the differentiation state of the intestinal epithelial cells (133). New findings using the human-derived intestinal epithelial Caco-2 cells as a model (these cells differentiate spontaneously in culture on reaching confluence) (143) have supported this finding and showed the level of expression of the hSMVT mRNA and protein, as well as that of carrier-mediated biotin uptake, to be markedly greater in postconfluent and differentiated Caco-2 cells (4 days after confluence) compared with preconfluent and undifferentiated cells (1 day after seeding) (J. Reidling and H. M. Said, unpublished observations). These findings also suggest the possible involvement of transcriptional regulatory mechanism(s) in the differentiation-dependent regulation of intestinal biotin uptake and hSMVT expression. The latter possibility has been confirmed in studies involving hSMVT promoters fused to the Firefly reporter gene and transfected into preconfluent and postconfluent Caco-2 cells, which showed a significantly greater promoter activity in the former compared with the latter cell type (unpublished observations). The observation of a differentiation-dependent regulation of hSMVT expression is not unique to this transporter, but also has been observed in intestinal epithelial cells with a number of other transporters including that of the

water-soluble vitamin C (144) (see also the Vitamin C (Ascorbic and Dehydroascorbic Acids) section later in this chapter). As mentioned earlier, the intestinal biotin SMVT system appears to be expressed at different levels in differentiated and undifferentiated intestinal epithelial cells, is adaptively regulated in response to biotin levels, and is under ontogenic regulation (133,140–142). In all these conditions, the regulation appears to involve transcriptional regulatory mechanisms; thus, knowledge about the 5′-regulatory region of the SMVT gene is required to better understand the mechanisms involved in these different types of regulation. Such knowledge has become available with the cloning and characterization of the 5′-regulatory region of the rat and human SMVT genes (145,146). As discussed earlier, four distinct transcript variants have been identified for the rat SMVT. These variants arise from heterogeneity at the 5′-untranslated region of the SMVT gene (133), and their existence suggests the possible involvement of multiple promoters in the regulation of transcription of the SMVT gene. Genome-walking studies have shown that this indeed is the case (145). Three distinct putative promoters (1, 2, and 3) that are separated by exons of the four reported variants have been identified. Promoter 1 was found to contain multiple putative cis-regulatory elements that include GATA-1, activator protein-1 (AP-1), AP-2, and C/EBP, as well as several repeats of purine-rich regions and two TATA-like elements. The other two promoters were found to be GC rich and also contained many putative cis-regulatory elements. Activity of the identified promoters has been demonstrated by fusing the promoter constructs to the Firefly luciferase reporter gene, transfecting the fusion constructs into the rat-derived intestinal epithelial IEC-6 cells, and assaying for luciferase activity. Promoter 1 was found to be the most active SMVT promoter in intestinal epithelial cells. Other investigations also have determined the minimal regions required for basal activity of each of the rat SMVT promoters in IEC-6 cells (145). These regions were found to be encoded in a sequence between −5311 and −5209 for promoter 1, between −4548 and −4439 for promoter 2, and between −4441 and −4319 for promoter 3. With regard to the 5′ regulatory region of the human SMVT gene, this region also has been cloned and two promoters (1 and 2) have been identified (146; and Reidling J and Said HM; unpublished observations). Both promoter sequences were found to be TATA-less and CAAT-less, to contain high GC-rich regions, and to have multiple putative cis-regulatory elements. Activity of these human promoters has been confirmed using the Firefly luciferase reporter gene assay after transient transfection into the humanderived intestinal epithelial HuTu-8o and Caco-2 cells. The minimal region required for basal activity of each promoter also was determined in these cells and found to be encoded by a sequence between −4830 and −4603 for promoter 1 and by a sequence between −4417 and −4303 for promoter 2 (Fig. 71-14). Furthermore, computational analysis (Mat Inspector) showed these minimal regions to contain a number of putative cis-regulatory elements, which include SP1, AP-2,

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1807 P1 -4830 CP2 NFkB/CRE L AP2 GK LF/SP-1 AP4/AP-2 TACCTCGGGTAGCGCCA GAGCCCTTTC CACGCCCA GAGCGAGGAGCAGGG CGGCAGCCCA GG GK LF AP-2/GK LF -4728 GCACCAGGG TCGTAAGACTACC CGGG CCGTGAGGCGCCATTTTCCGTTCCCTTGGTGCTCCGC CpG Island TGCTCGCGCGACCCGGCCCCGCGGCCCCGCCCCGCAGCGCGTCAGGCCCTCTTCCCCGGGCGT Exon 1a GGCCTAAGCGGCCCGGTCCAGTCGCCCTGGGGCTGCTTGGGGGCT -4603 P2 -4417 SP-1 CGGCTTCGCAGAAACTCGGGCCCCTCCATCCGCCCTCAGTAAACATGGCGGCACGG CGAGCG SP-1 SP-1 SP-1 AP-2 Exon1b GGG CGGGCAAGGGG CGGGCAGGGGG CGGGCAGGGGGGCCGGGGCCCCC GCGGGCTCCCGGC -4303

FIG. 71-14. Diagrammatic representation of the minimum regions required for basal activity of the hSMVT (human sodium-dependent multivitamin transporter) promoters 1 and 2 (J. Reidling and H. M. Said, unpublished observations).

AP-4, NF-κB, CP2, and GKLF (see Fig. 71-14). Further studies are required to examine the role, if any, of these putative cis-elements in the regulation of activity of the hSMVT promoters and the type of nuclear factors that interact with them.

VITAMIN C (ASCORBIC AND DEHYDROASCORBIC ACIDS) Vitamin C exists in two physiologic forms: the reduced ascorbic acid (AA) form and the oxidized dehydro-L-ascorbic acid (DHAA) form (see Fig. 71-1). The reduced form of vitamin C acts as a cofactor in a myriad of metabolic reactions in which it maintains metal ions such as iron and copper in their reduced forms. It also acts as a free radical scavenger and as a cofactor in reactions involving the synthesis of collagen, carnitine, and catecholamine, as well as in peptide amidation and tyrosine metabolism. Studies also have suggested that AA acts as a biological regulator of cystic fibrosis transmembrane conductance regulator–mediated chloride secretion in epithelial cells (147). Vitamin C deficiency leads to a variety of clinical abnormalities including scurvy, poor wound healing, vasomotor instability, and connective tissue disorders. With regard to DHAA, this compound is structurally different from AA and is similar to glucose. Its toxicity is similar to that of alloxan, which has long been used by investigators to induce diabetes in experimental animals.

Unlike a number of other water-soluble vitamins where two sources are available to the gut (a dietary source and a bacterial source in the large intestine), only the dietary source is available in the case of vitamin C because no net synthesis of the vitamin occurs by the normal microflora of the large intestine (2). The mechanism of uptake of dietary AA has been investigated using a variety of intestinal tissue preparations from a number of species including humans (see review by Rose [20]). These investigations have concluded that intestinal AA uptake occurs via a concentrative, carriermediated, and Na+-dependent mechanism. These findings were confirmed in studies with purified intestinal BBMV preparations (148,149). The exit process of AA from the enterocyte occurs by a carrier-mediated system that is not dependent on Na+ (148). Little metabolic alterations occur in the absorbed AA during transport in the enterocytes. Regarding the absorption of DHAA, the enterocyte takes up this form of vitamin C and metabolizes it to the reduced form by the action of DHAA reductase (148,150,151). It is through this mechanism that the intracellular level of DHAA is maintained at low nontoxic levels. Studies on the cellular uptake of DHAA have shown that the enterocyte takes up this compound across the BBM by an Na+-independent process (148). Because of its structural similarity with glucose, uptake of DHAA was found to be competitively inhibited by sugars (152). Substantial uptake of DHAA from the serosal surface of the intestinal epithelial cells, that is, across the BLM, also has been reported and is believed to occur via an exchange with the reduced form of the vitamin (153).

Mechanism of Intestinal Vitamin C Transport Most mammals generate vitamin C from D-glucose through gulonic acid in the liver. Humans, other primates, and guinea pigs cannot synthesize this vitamin because they lack the enzyme L-gulonolactone oxidase. Thus, they must obtain the vitamin from exogenous sources via intestinal absorption.

Molecular Identity of the Intestinal Ascorbic Acid and Dehydro-L-Ascorbic Acid Uptake Systems Two AA transport systems from different human and animal tissues have been cloned (154–159). These systems are the sodium-dependent vitamin C transporter-1 (SVCT-1, the

1808 / CHAPTER 71 product of the SLC23A1 gene) and the SVCT-2 (the product of the SLC23A2 gene) (154–159); in humans, these isoforms are referred to as hSVCT-1 and hSVCT-2. SLC23A1 message appears to be expressed mainly in epithelial cells including those of the small intestine, whereas SLC23A2 message appears to be expressed in most other tissues (except the lung and skeletal muscles) including the small intestine. In addition, the intestine appears to express a splice variant of SVCT-1, which leads to four additional amino acids being inserted yielding a nonfunctional version of SVCT-1 (158). Expression of SVCT-1 in the intestine is higher than that of SVCT-2. The amino-acid sequence of the SVCT-1 and SVCT-2 are 66% identical and also is conserved among different species. In addition, both isoforms encode proteins with 12 predicted transmembrane domains that have multiple potential N-glycosylation sites and a number of putative protein kinase phosphorylation sites. The hSVCT-1 encodes a protein of 598 amino acids with a predicted molecular mass of 65.2 kDa and with both the N- and the C-terminal tails being directed toward the cell interior. The hSVCT-2 encodes a protein of 650 amino acids with a predicted molecular mass of 70.4 kDa and with both the N- and C-termini being directed toward the cell interior. Functionality of both of the cloned SVCT-1 and SVCT-2 cDNA has been confirmed by expression in Xenopus oocytes, COS-7 cells, and other cellular systems, and in all cases, they were shown to transport the negatively charged AA via a specific, electrogenic, Na+dependent process with an AA:Na+ stoichiometric ratio of 1:2. Affinity of the SVCT-2 system for AA, however, appears to be higher than that of SVCT-1 (156), but both transporters have greater selectivity for L-ascorbic acid compared with D-isoascorbic acid and neither transport DHAA. An interesting feature of SVCT-1 and SVCT-2 systems was the recognition that both are able to also act, in the absence of AA, as Na+ uniporters allowing Na+ to leak into the cell (154). Other studies have reported that flavonoids (like quercetin) are reversible and noncompetitive inhibitors of AA transport by the SVCT-1 system, although they themselves are not transported by this transporter (160). Regarding the molecular identity of the system(s) involved in the intestinal absorption of DHAA, studies have shown that the glucose transporters GLUT1, GLUT3, and GLUT4 (but not GLUT2, GLUT5, or SGLT-1) are able to transport this form of vitamin C (see Liang and colleagues [156] for review). These transporters, however, do not transport the reduced form of vitamin C, that is, AA (156).

version of the hSVCT-2 and results from the deletion of 345 bp that corresponds to a sequence in transmembrane domains 4, 5, and 6. Although this deletion does not result in a frameshift, the resulting product is believed to act as a potential regulator of AA transport by interacting with the function of both the SVCT-1 and the SVCT-2 systems in a dominant-negative manner (161). Although expression of the short SVCT-2 was found in all tissues examined, expression in the intestine has not been tested (161). Other studies have shown that functionality of the hSVCT-1 and the hSVCT-2 is regulated by an intracellular PKC-mediated pathway, but via different mechanisms (162). Although the PKC-mediated pathway appears to affect hSVCT-1 via redistribution of the carrier from cell surface to intracellular membranes, the effect on hSVCT-2 appears to be mediated via reduction of the catalytic activity of the carrier protein (162). The latter studies (162) have been performed in COS-1 cells; thus, it is unclear whether the same mode of regulation of AA transport also occurs in intestinal epithelial cells. Other studies have used the human-derived intestinal epithelial Caco-2 cells (which differentiate spontaneously in culture on reaching confluence to become enterocyte-like cells) (144) to show that the intestinal AA uptake process also is regulated during differentiation (163). This differentiationdependent up-regulation of AA uptake was found to be associated with a significant increase in the level of expression of the hSVCT-1 (but not the hSVCT-2) message (163). Whether a similar situation exists for up-regulation of AA uptake in vivo currently is unknown. The intestinal AA uptake process was found to be adaptively regulated by extracellular substrate levels. Studies have shown that supplementation of guinea pigs with AA leads to a downregulation in intestinal AA absorption (164,165). This observation has been confirmed in studies with the human intestinal epithelial Caco-2 cells in culture (166). In the latter studies, the decrease in AA uptake after supplementation with AA also was found to be associated with a decrease in the level of expression of the hSVCT-1 message. Currently, nothing is known about the molecular mechanism involved in this adaptive regulation in AA uptake. Little also is known about the 5′ regulatory region of the SLC23A1 and SLC23A2 genes and their activity in intestinal epithelial cells. One study, however, has reported the identification of the transcription start site of the SLC23A1 gene and has shown it to be at position −47 relative to the ATG initiation site (167).

Regulation of the Intestinal Ascorbic Acid Uptake Process

Cell Biology of the Intestinal Ascorbic Acid Transporters

The intestinal AA uptake process appears to be regulated by intracellular and extracellular factors/conditions. Studies have reported the existence of an intracellular dominantnegative inhibitor of AA transport (161). This inhibitor originates from alternative splicing of the hSVCT-2 transcript (161). The transcript of this inhibitor encodes a shorter

Studies on the mechanisms involved in the membrane targeting and intracellular trafficking of the AA transporters in intestinal epithelial cells has begun to emerge (168). Confocal imaging studies and video rate measurements using live human-derived intestinal epithelial Caco-2 cells grown on filters and fusion proteins of hSVCT-1 with the

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1809 A

B

FIG. 71-15. Distribution of human sodium-dependent vitamin C transporter-1 (hSVET-1) fused to the yellow fluorescent protein (YFP) in Caco-2 cells grown on filters. (A) Axial (xz) section of Caco-2 cells expressing hSVCT1-YFP. (B) Axial (xz) section of Caco-2 cells expressing YFP alone. (See Color Plate 38.)

yellow fluorescent protein (hSVCT1-YFP) have demonstrated that the hSVCT-1 protein is expressed at the apical membrane domain of these cells and also resides in a heterogeneous population of intracellular structures with discrete dynamic properties (Fig. 71-15) (168). Three types of these structures have been identified: small vesicles, tubular-like structures, and large, hollow vesicular structures (these structures can be viewed online at: http://www.jbc.org/cgi/ content/full/M400876200/DC1). Mobility of these structures was found to be temperature and microtubule dependent in nature. The targeting signal that dictates apical membrane expression of the hSVCT-1 also was determined by means of progressive truncations of the hSVCT-1 polypeptide and was found to be embedded in the 10-amino-acid sequence PICPVFKGFS (amino acids 563–572) of the cytoplasmic C-terminal tail (Fig. 71-16) (168). Interestingly, this

sequence was found to share significant homology with apical targeting motifs in the cytoplasmic tail of two other members of the sodium-dependent transporters, namely, the ileal bile acid transporter (169) and the glutamate transporter (170). These findings suggest that such conservation may be reflected topologically through the adoption of a β-turn in the cytoplasmic C-terminal tail of each of these transporters.

VITAMIN B6 Vitamin B6 represents a group of three structurally related compounds: pyridoxine, pyridoxal, and pyridoxamine (see Fig. 71-1). These compounds exist in varying proportions in the diet in both the phosphorylated and the nonphosphorylated forms. Pyridoxal 5′-phosphate represents the most biologically

Extracellular

COOH-terminal NH2-terminal

Cytoplasm

FIG. 71-16. Sorting sequence that dictates targeting of the human sodium-dependent vitamin C transporter-1 (hSVCT-1) protein to the apical membrane of polarized Caco-2 cells grown on culture inserts.

1810 / CHAPTER 71 active form of the vitamin and is a cofactor for enzymes that catalyze transaminase, decarboxylase, and synthetase reactions in pathways that include carbohydrate, protein, and lipid metabolism. Vitamin B6 deficiency in humans leads to a variety of clinical abnormalities that include sideroblastic anemia, weakness, insomnia, and neurologic disorders. Deficiency of vitamin B6 occurs in conditions such as alcoholism and diabetes, in patients with celiac and renal diseases, and after long-term use of hydrazines (e.g., isoniazid) and penicillamine. Patients with vitamin B6–dependent seizure, an autosomal recessive disorder believed to be caused by an abnormality in pyridoxine transport into cells, also show low levels of vitamin B6 in their blood (171).

Mechanism of Intestinal Vitamin B6 Transport As with a number of other water-soluble vitamins, two sources of vitamin B6 are available to the intestine: a dietary source and a bacterial source (where the vitamin is produced by the normal microflora that resides in the large intestine) (2). As mentioned earlier, dietary vitamin B6 exists in the phosphorylated and nonphosphorylated forms. The phosphorylated forms are first hydrolyzed to the free forms in the intestinal lumen before absorption (172–174). A variety of intestinal preparations have been used to delineate the mechanisms(s) involved in the absorption of vitamin B6 in the gut. Some studies have reported the inability of the intestine to accumulate vitamin B6 (175), whereas others have reported the lack of a saturable process in the vitamin uptake process. Based on these findings, it has been concluded (172,176) that the intestinal vitamin B6 uptake process occurs via a nonspecific simple diffusion process. This conclusion, however, has been challenged by the findings of Said and colleagues (177), who used the human-derived intestinal epithelial Caco-2 cells

to show the existence of an efficient, specialized, carriermediated system for pyridoxine uptake. The latter studies also have shown that the intestinal uptake process of pyridoxine is Na+-independent in nature, but is highly dependent on acidic buffer pH. In addition, amiloride was found to competitively inhibit pyridoxine uptake by Caco-2 cells with an apparent inhibition constant (Ki) of 0.39 mM. These findings on pyridoxine uptake by Caco-2 cells are similar (except for the effect of buffer pH) to those reported using renal epithelial opossum kidney (OK) cells (178). Currently, little is known about the structure of the intestinal pyridoxine carrier in any species or about its regulation. However, the structure of the first vitamin B6 transporter in Saccharomyces cerevisiae has been determined (179). This system was predicted to have 12 transmembrane domains and appears to be regulated by substrate availability in the surrounding environment (179).

Regulation of the Intestinal Vitamin B6 Uptake Process The intestinal vitamin B6 uptake process appears to be under the regulation of an intracellular PKA-mediated pathway (177). This conclusion is based on the observation that pretreatment of Caco-2 cells with compounds that lead to an increase in intracellular cAMP levels, and thus activate PKA, cause a significant inhibition in the initial rate of pyridoxine uptake. This inhibition was found to be mediated via a decrease in the activity (and/or the number), but not the affinity, of the pyridoxine uptake carriers (177) (Fig. 71-17). The latter conclusion is based on the observations that pretreatment of Caco-2 cells with dibutyryl cAMP leads to a significant decrease in the Vmax, but not the apparent Km, of the pyridoxine uptake process, respectively. The molecular mechanism through which cAMP exerts its effect on the intestinal pyridoxine uptake is unclear and requires further study.

Pyridoxine uptake (pmol/mg protein/3 min)

50

40 Control 30

dBcAMP 1mM

20

10

0 0

5

10 15 Pyridoxine conc (µM)

20

FIG. 71-17. Effect of pretreatment of Caco-2 cells with dibutyryl cyclic adenosine monophosphate (DBcAMP; 1 mM; solid circles) on pyridoxine uptake as a function of concentration. Open circles indicate controls. (Modified from Said and colleagues [177], by permission.)

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1811 RIBOFLAVIN (VITAMIN B2) Riboflavin (RF; see Fig. 71-1), in its coenzyme forms (riboflavin-5-phosphate [FMN] and flavin adenosine dinucleotide [FAD]), plays key metabolic roles as an intermediary in the transfer of electrons in biological oxidation-reduction reactions. These reactions include carbohydrate, amino acid, and lipid metabolism and conversion of folic acid and vitamin B6 compounds into their active coenzyme forms. Thus, it is not surprising that deficiency of RF (which occurs in humans under a variety of conditions that include inflammatory bowel disease and chronic alcohol consumption) leads to a variety of clinical abnormalities that include degenerative changes in the nervous system, endocrine dysfunction, skin disorders, and anemia.

Mechanism of Intestinal Riboflavin Transport Host’s intestine encounters two sources of RF: a dietary source that is processed and absorbed in the small intestine, and a bacterial source that is generated by the normal bacterial flora in the large intestine (2) and is absorbed there. Dietary RF exists mainly in the form of FMN and FAD that is bound noncovalently to proteins. These coenzymes are first released from proteins via the combined action of gastric acid and hydrolases; they then get hydrolyzed via the action of alkaline phosphatases to free RF before absorption (180). The mechanism of intestinal absorption of free RF in the small intestine has been the subject of extensive investigations using a variety of human and animal intestinal preparations that include intact intestinal tissue preparations, isolated, purified membrane vesicles, and cultured intestinal epithelial cell lines (181–188). Collectively, these studies have shown that transport of dietary RF occurs mainly in the proximal part of the small intestine and involves an efficient, specialized, Na+-independent, carrier-mediated system. This system was inhibited by structural analogues of RF (including lumiflavin) and by the Na+-H+ exchange inhibitor amiloride in a competitive manner (the inhibition constant, Ki, for amiloride was reported at 0.48 mM) (182). In addition, the intestinal RF uptake process was found to be sensitive to the effect of the tricyclic phenothiazine drug chlorpromazine, a compound that shares structural similarity with RF (183). Some degree of RF metabolism (phosphorylation) also has been reported after transport of the substrate into the cell (189). Regarding exit of RF out of the polarized intestinal epithelial cells across the BLM, this issue also has been addressed using purified intestinal BLMV and has been shown to involve a specialized, electroneutral, carrier-mediated mechanism (184). With regard to the bacterially synthesized RF, the amount of the vitamin produced by the normal microflora of the large intestine was found to depend on the type of the ingested diet and is greater after ingestion of a vegetable-based compared with a meat-based diet (190). Also, considerable amounts of the bacterially produced RF were found to exist in the large-intestinal lumen in the form of free RF (190,191),

and thus are available for absorption. Other investigations have shown that the large intestine is indeed capable of absorbing luminally introduced free RF (131,192), but little was known about the mechanism involved in the uptake process. Using the human-derived colonic epithelial NCM460 cells as a model for human colonocytes, Said and colleagues (193) have shown RF uptake by this cellular system to be via an efficient, specialized carrier-mediated mechanism that is similar to that described in the small intestine (193). The findings described above on the existence of a carrier-mediated system for RF uptake in cultured human colonic epithelial cells has been confirmed in studies with native rat colon tissue preparations (194). With the report of an efficient carrier-mediated system for RF uptake in the large intestine, the belief that this source of RF may contribute to the overall host normal RF homeostasis and especially to the cellular homeostasis of the vitamin in the localized colonic epithelial cells is supported. Currently, nothing is known about the molecular identity of the intestinal RF uptake system of any species. Currently, little is known about the structure-function relation of the intestinal RF uptake system. Studies using specific sulfhydryl group reagents have suggested the possible involvement of sulfhydryl groups in the function of the intestinal RF uptake system (182). Pretreatment of the humanderived intestinal epithelial Caco-2 cells with the sulfhydryl group reagents, p-chloromercuribenzene sulfonate and eosin maleimide, was found to lead to significant inhibition in RF uptake (182). The inhibition was reversed by treating Caco-2 cells with reducing agents dithiothreitol or mercaptoethanol. Because the sulfhydryl group reagents used in that study are membrane impermeable, it is reasonable to assume that the inhibited sulfhydryl groups in the RF carriers are located at the exofacial side of cell membrane.

Regulation of the Intestinal Riboflavin Uptake Process The intestinal RF uptake process appears to be under the regulation of intracellular and extracellular factors/conditions. Evidence has been presented to suggest that the intestinal RF uptake process is under the regulation of an intracellular PKA-mediated pathway (186). Activation of this regulatory pathway leads to a significant inhibition in RF uptake by the enterocyte-like Caco-2 cells. This inhibition was found to be reversible and appears to be mediated via a significant decrease in the Vmax of the RF uptake process with no changes in its apparent Km. The latter findings suggest that the PKAmediated pathway acts through decreasing the activity (and/or the number) of the RF uptake carriers, but not their affinity. Other findings (using cycloheximide, actinomycin D, colchicine, and cytochalasin D) have suggested that the PKA-mediated inhibition in RF uptake by Caco-2 cells is not mediated via inhibition in the synthesis of the RF carrier protein(s) or through inhibition in the recruitment of preexisting carrier protein(s) into the plasma membrane (186). The intestinal RF uptake process also appears to be under

1812 / CHAPTER 71 the regulation of another intracellular regulatory pathway, the Ca2+/calmodulin-mediated pathway (186). Regulation by this pathway appears to be mediated via changes in both the Vmax and the apparent Km of the RF uptake process, suggesting an influence on both the activity (and/or number) and the affinity of the involved RF uptake carriers. The intestinal RF uptake process also was found to be regulated by extracellular substrate levels (182,195). This has been shown in vitro in studies with cultured intestinal epithelial cells and in rats supplemented in vivo with different levels of RF. In the in vitro studies, maintaining Caco-2 cells in an RF-deficient growth medium was found to lead to a significant up-regulation in RF uptake, whereas maintaining these cells in a medium supplemented with high pharmacologic concentrations of the vitamin leads to down-regulation in RF uptake (182). These adaptive changes were found to be mediated via changes in the Vmax (but not the apparent Km) of the RF uptake process, suggesting that the changes are mediated via an increase in the number (and/or activity), but not the affinity, of the RF carriers (182). It is of interest that similar adaptive changes in RF uptake by Caco-2 cells were observed when lumiflavin was used in place of RF in such experiments. Lumiflavin is an RF structural analogue that uses the same RF uptake system for its own internalization, yet it cannot be phosphorylated or used as an RF-like vitamin by cells (182). These findings suggest that the availability of a transportable form of the substrate in the growth medium that is recognizable by the RF uptake system is what triggers the adaptive response in intestinal RF uptake. In studies with the human colonic epithelial NCM460 cells (where similar adaptive changes in RF uptake occur in response to RF extracellular levels) (193), the induction in RF uptake caused by maintaining the cells in an RF-deficient medium was inhibited significantly by the addition of the transcription inhibitor actinomycin D. This finding suggests the possible involvement of a transcriptional regulatory mechanism in the adaptive response of the intestinal RF uptake process during substrate deficiency. Further studies are needed, however, to confirm this suggestion and to determine the exact mechanism involved. The findings described earlier on the adaptive regulation of RF uptake in cultured intestinal epithelial cells in vitro has been confirmed in rats fed different levels of RF (195). In these studies, induction of RF deficiency in rats was found to lead to a significant and specific up-regulation in intestinal RF uptake, whereas oversupplementation with RF leads to a significant and specific down-regulation in intestinal RF uptake. The intestinal RF uptake process also was found to be regulated ontogenically (196). Transepithelial transport of RF in rat intestine was found to undergo a clear decline with maturation (196). This was mediated via a decrease in the Vmax and an increase in the apparent Km of the carrier-mediated RF uptake carriers with maturation (i.e., from suckling to weanling to adult rats). These findings suggest that ontogeny is associated with a decrease in the number (and/or activity) of the RF uptake carriers and a decrease in their affinity. Again, the molecular mechanism(s) involved in this type of

regulation in the intestinal RF uptake process is unknown and in need of further study.

NIACIN (NICOTINIC ACID; VITAMIN B3) The main function of niacin (see Fig. 71-1) is as a precursor for the synthesis of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). Most of the NAD- and NADP-linked enzymes are involved in reactions that maintain the redox state of cells such as glycolysis and the pentose phosphate shunt. Niacin also appears to have lipid-reducing effects and is in use clinically for that purpose. Severe niacin deficiency in humans leads to pellagra, a disease characterized by dementia, dermatitis, and diarrhea. Deficiency and suboptimal levels of niacin occur in alcoholic patients and in those with Hartnup’s disease. Patients with the latter disorder have mutations in the gene that encodes the transporter of tryptophan, which is a precursor for the endogenous production of niacin. Humans have access to niacin from endogenous and exogenous sources. The endogenous source of niacin is provided via the metabolic conversion of the amino-acid tryptophan to niacin. The exogenous source is provided through the diet by absorption in the intestine (the normal microflora of the large intestine synthesize some niacin, but it is believed that this niacin is fixed inside the microorganisms, and thus is not available to the host) (2). The mechanism involved in intestinal niacin absorption is not well understood. Previous investigations into the subject using purified intestinal BBMV preparations isolated from rat and rabbit intestine have suggested that the process occurs via a carrier-mediated mechanism (197,198). The apparent Km of the system involved, however, was reported to be between 3.52 and 17.0 mM, which is rather high considering that the luminal concentration of the vitamin is in the micromolar range (199). This raises a concern regarding the physiologic relevance of the described system. Some studies, however, have used the human-derived intestinal epithelial Caco-2 cells as a model and found evidence for the involvement of a specialized carrier-mediated system for uptake of physiologic concentrations of niacin (S. Nabokina and H. M. Said, unpublished observations). This system has been found to function in the micromolar range, is highly dependent on extracellular acidic buffer pH, and is Na+ independent in nature. The structure of the intestinal niacin uptake system and its regulation is unknown. The mechanism by which niacin exits the intestinal absorptive cells via the BLM also is unknown. Some degree of intracellular metabolism has been observed where the vitamin is converted into intermediates of NAD biosynthesis.

PANTOTHENIC ACID This B vitamin (see Fig. 71-1) is required for the biosynthesis of coenzyme A and acyl carrier protein in mammalian

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1813 tissues, and thus is involved in the metabolism of carbohydrate, fat, and to a lesser extent, protein. Spontaneous pantothenic acid deficiency has not been reported in humans, most likely because of the ubiquitous nature of the vitamin. The intestinal tract is exposed to two sources of pantothenic acid, one being dietary and the other bacterial (i.e., the vitamin is produced by the normal microflora of the large intestine) (2). Dietary pantothenate exists mainly in the form of coenzyme A, which is hydrolyzed to free pantothenic acid in the intestinal lumen before uptake (200). Free pantothenic acid then is transported into the absorptive cells via the SMVT, which also transports biotin and lipoate (123,124,133,134) (see also the Biotin (Vitamin H) section earlier in this chapter). Absorption of the bacterially synthesized pantothenic acid in the large intestine also appears to use the SMVT system of that region (123). Little is known about regulation of the intestinal pantothenic uptake process, but it may be reasonable to assume that factors/conditions that regulate the intestinal biotin uptake process may also be involved in the regulation of the uptake of this vitamin because they share the same intestinal uptake system.

COBALAMIN (VITAMIN B12) Cobalamin (Cbl; vitamin B12) is a water-soluble vitamin of 1357 molecular weight, and it is present in meat, eggs, and dairy products. Its structure (see Fig. 71-1H) consists of four reduced pyrrole rings that are linked together and are known as “corrins” because it forms the core of the molecule. All compounds containing this ring are called corrinoids. The prefix cob designates the presence of a cobalt atom. The corrinoids active in human or mammalian metabolism are called Cbl, whereas those active in microorganisms are referred to as vitamin B12. Above the plane of the corrin ring, axial ligands (designated as X) are present coordinating to the central cobalt. Biologically active forms of Cbl in all higher mammals contain either methyl (CH3) or 5′-deoxyadenosyl groups as the axial ligand and are therefore known as methyl-Cbl and adenosyl-Cbl, respectively. Other axial ligands present include OH (hydroxo-Cbl) and H2O (aquacobalamin). CN (cyanocobalamin, CN-Cbl) is an artifact of isolation, but it is stable and primarily used for research and medical purposes. The tetrapyrrole ring is substituted with acetamide (CH2CONH2) and propionamide (CH2CH2CONH2) residues at positions R and R′, respectively. Below the corrin ring, 1-α-D-ribofuranosyl-5,6-dimethylbenzimidazole-3-phosphate is linked to the rest of the molecule at two points: (1) through a coordination linkage to the central cobalt via one of its nitrogen atoms; and (2) via a phosphodiester linkage to a 1-amino-2 propanol substituent on the propionamide f. Intracellular Cbl is converted to coenzyme forms, methylCbl and 5′-deoxyadenosyl-Cbl, and is used for the enzymatic remethylation of homocysteine to methionine and for the conversion of methylmalonyl CoA to succinyl CoA, respectively (201). These are the only two known cellular metabolic

reactions needing Cbl, and currently, it is not fully understood how aberrations in these two reactions noted in Cbl deficiency cause two major diseases, megaloblastic anemia and neurologic disorders (202). Cbl deficiency also is known to affect the differentiation, proliferation, and metabolic status of rapidly proliferating cells, and this observation may help to explain the development of gastrointestinal (epithelial) and immunologic (lymphocytes) disorders noted in Cbl deficiency.

Gastrointestinal Absorption of Cobalamin Because of its highly polar nature, physiologic amounts of Cbl that are needed on a daily basis (2–5 µg) cannot traverse the lipid membranes by diffusion, an inefficient process. A large amount of gastric intrinsic factor is secreted from the stomach and binds to dietary Cbl before it is absorbed. Nearly 8 to 10 µg Cbl representing both dietary and biliary sources is presented to the enterocytes on a daily basis. However, the capacity for intrinsic factor–mediated Cbl absorption by the gut is limited to about 1 to 1.5 µg/day. This limited capacity of intrinsic factor–mediated Cbl transport in the intestine is because of confined expression of intrinsic factor-Cbl receptor to the terminal ileum (203), low number of intrinsic factor-Cbl binding sites at this location (204), and the slow turnover of the receptor (205). However, because the daily loss of Cbl from the human body stores (5 mg) is estimated, based on animal studies (206), to be about 0.02% (1 µg/day), it is likely that the limited capacity of ileal Cbl transport in humans may be adequate to compensate for the amount of Cbl lost on a daily basis. Three distinct steps (Fig. 71-18) are required to process and present Cbl to the ileal receptor before it is taken up via receptor-mediated endocytosis. First, in the stomach, combined action of acid/pepsin results in the release of most of food protein-bound Cbl. Second, haptocorrin (HC), derived from saliva and gastric secretions because of its higher affinity for Cbl than intrinsic factor at gastric pH, sequesters Cbl released from the food proteins (207). Third, although intrinsic factor is stable in the intestinal lumen for the action by pancreatic proteases, such as trypsin, chymotrypsin, and others, HC or HC-Cbl complex is not. Thus, Cbl released from HC in the intestinal lumen binds to intrinsic factor (208). Additional details of these earlier events can be found in previous review articles (209,210). After the binding of intrinsic factor-Cbl to its receptor, the complex is endocytosed from the luminal side and there is delay of about 4 hours before Cbl enters the circulation bound to another Cbl binding protein, transcobalamin (Fig. 71-19). A number of earlier studies have shown that intrinsic factorCbl is processed via the classical endosomal-lysosomal pathway (210, and reference citations therein). At some stage during the endosomal-lysosomal processing of intrinsic factor-Cbl complex, intrinsic factor is degraded most likely by the enzyme cathepsin L, (211) and Cbl liberated is transported out of the lysosomes. Although not identified directly, it has been speculated that Cbl enters a secretory vesicle that

1814 / CHAPTER 71 Pathophysiology of acquired disorders of Cbl absorption

The three phases of Cbl-absorption r eta Di bl yC

IF

Stomach

Bile

ases ote r P

HCCb l

Atrophic gastritis, partial or total gastrectomy-Partial or total loss of IF Cbl HC

HC Cbl IF

Pancreatic insufficiencyImpaired transfer of Cbl from HC to IF

HC ed rad eg +Cbl IF

Pancreas Infestation and bacterial overgrowth-Competition for dietary and biliary Cbl. Ileal disease-resection-Loss of cubilin

D

IF-Cbl

IF-Cbl

Terminal Ileum

FIG. 71-18. Three phases involved in the gastrointestinal absorption of cobalamin (Cbl). The pathophysiology of acquired disorders of Cbl absorption is shown on the right. HC, haptocorrin; IF, intrinsic factor.

Lysosomal Cbltransporter Lysosomes

Endosomes

Degraded

X3

X

2

Secretory vesicle X 2

Megalin

Cbl X 1 IF

TC Cubilin

X4 ER

FIG. 71-19. Transcellular transport of cobalamin (Cbl) bound to intrinsic factor (IF) in polarized epithelial cells. IF-Cbl is processed via endosomal-lysosomal pathway, and Cbl liberated from within the lysosomes binds to transcobalamin (TC) and enters circulation. The four types of inherited disorders that cause blocks in the transcellular transport of Cbl are indicated (X): type 1: lack of formation of IF-Cbl caused by lack of IF synthesis or synthesis of dysfunctional IF; type 2: lack of IF-Cbl binding to cubilin caused by defects in the cubilin molecule by itself or in both cubilin and its accessory protein (Amn); type 3: intracellular retention of Cbl in the lysosomes because of possible defects in the transporter; and type 4: lack of formation of TC-Cbl complex caused by lack of TC synthesis. ER, endoplasmic reticulum.

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1815 contains transcobalamin as a cargo. Transcobalamin-Cbl then is secreted via the BLM to enter circulation. The importance of lysosomes during the transcellular transport of Cbl is supported by the evidence that lysosomal inhibitors such as chloroquine or leupeptin prevented intrinsic factor degradation resulting in a failure to liberate Cbl bound to it. This resulted in a failure of Cbl transfer to transcobalamin, a necessary step before the exit of Cbl into circulation. Thus, a failure to degrade intrinsic factor will result in inhibition of Cbl transcytosis (212,213). Receptor for intrinsic factor-Cbl, now known as cubilin (see later) (214), is a large (460-kDa) (215) protein located in the ileal apical BBM (216); at this location it is bound to megalin, another large endocytic receptor of 600 kDa molecular mass. The interaction between these two proteins has been suggested to be important for the endocytosis of intrinsic factor-Cbl (217). Although cubilin and megalin are bound to each other during endocytosis of intrinsic factor-Cbl, it is unknown whether these two membrane proteins recycle back to the apical membranes still bound to each other or whether they recycle separately. Thus, the essential components important for normal absorption of Cbl include normal healthy ileal cells, secretions from the stomach and exocrine pancreas, and functional expression of the three gene products, intrinsic factor, cubilin, and transcobalamin. Some of the known details of transcellular transport of Cbl across the ileal cell are shown in Figure 71-19. Because of the complexity of the absorption process, there are many acquired and inherited causes that lead to Cbl malabsorption.

Acquired Causes of Cobalamin Malabsorption The most common cause, other than total lack of dietary intake (as in vegans and strict vegetarians), for the development of Cbl deficiency is its malabsorption. Many details of this complex process have been learned from patients who were not able to absorb dietary Cbl for one reason or another. Malabsorption of Cbl through acquired causes occurs due to diseases of multiple organs, including stomach, pancreas, terminal ileum, and thyroid diseases (209,210). Malabsorption of Cbl because of diseases of the stomach, such as atrophic gastritis (218,219) and partial or total gastrectomy (220,221), is because of the lack or absence of intrinsic factor resulting from either injury or loss of parietal cells that produce intrinsic factor. In addition, because of the lack of acid and pepsin, there may be inadequate release of Cbl bound to food proteins. Patients with gastric tumors (Zollinger–Ellison syndrome) (222) may experience slow development of Cbl deficiency because of a slow transfer of Cbl from HC to intrinsic factor. However, these patients can absorb crystalline Cbl better than food protein-bound Cbl. Exocrine pancreatic insufficiency (208) or intestinal diseases such as bacterial overgrowth and infestation (223,224), tropical sprue (225,226), acquired immune deficiency syndrome (227), ileal disease, and Crohn’s disease also are known to cause Cbl malabsorption (228,229).

Cbl malabsorption in patients with pancreatic insufficiency is correctable with pancreatic supplements (208), and in patients with intestinal infestation and bacterial overgrowth, it can be corrected after elimination of these causes. Patients with permanent loss of intrinsic factor because of total gastric surgery or cubilin due to terminal ileal resection will require Cbl supplementation. Cbl deficiency in some of these patients can be treated with oral Cbl, if there is sufficient stomach remaining to produce intrinsic factor or sufficient gut remaining to absorb intrinsic factor-Cbl. Usually, a large dose of Cbl is required and 1-mg Cbl tablets are available for this purpose, which are being used widely in treating Cbl deficiency in Europe. In instances where the oral Cbl therapy does not correct Cbl deficiency, regular intramuscular injections of Cbl often is an alternate option. The mechanism of Cbl malabsorption noted occasionally in patients with hypothyroidism (230) is unknown and appears to depend on the severity of the disease. Based on animal studies, Cbl malabsorption in such cases appears to involve deficit of the intrinsic factor-Cbl receptor (231). Several reviews in the literature provide more details on Cbl malabsorption due to these acquired causes (209,210,232). The pathophysiology of Cbl malabsorption caused by acquired disorders is summarized in Figure 71-18.

Inherited Causes of Cobalamin Malabsorption Cbl malabsorption is known to occur rarely in children because of a variety of rare inherited disorders. There are at least four types of genetic disorders identified in children with selective malabsorption of Cbl. In the first type, because of a lack of intrinsic factor synthesis or synthesis of dysfunctional intrinsic factor (201, 202,233), patients do not form an intrinsic factor-Cbl complex. Although the molecular basis for the expression of dysfunctional intrinsic factor is unknown, lack of intrinsic factor synthesis causing megaloblastic anemia has been shown to occur despite the presence of a full-length gene (234). The mutation has been detected in the coding region and includes a small (four-nucleotide) deletion (235) or a single base change at codon 5 (236). How these specific mutations cause intrinsic factor deficit is unknown. Patients with the second type have normal production of functional intrinsic factor, but they exhibit selective malabsorption of Cbl. This disorder originally was known as Imerslund–Grasbeck syndrome and included all causes of malabsorption of Cbl in patients who synthesized functional intrinsic factor and had a normal mucosal architecture (237). More recent studies have shown that Cbl malabsorption noted in patients across the world is caused by mutations (238–241) in cubilin molecule or in its escort protein, amnionless (AMN) gene product, a 45-kDa protein that is thought to play a role in vectorial targeting of cubilin to the cell surface (242), or in both. The Scandinavian patients with mutations predominantly in the cubilin molecule had founder effects, whereas the Mediterranean patients had defects in both genes.

1816 / CHAPTER 71 The overall effects of these mutations include decreased binding of intrinsic factor-Cbl and lack of cubilin delivery to the cell surface. Studies from a pedigree of dogs with selective Cbl malabsorption have shown a deficit of cubilin at ileal cell surface (243), and this phenotype appears to be the homologue of the human disease (244). In patients with the third type, Cbl is retained in the intracellular acidic vesicles (245) after its uptake by cells. This disorder also is known as Cbl F and is caused by a failure of Cbl transport out of the acidic vesicles (endosomes or lysosomes). It is unknown whether retention of Cbl in the lysosomes (246) is caused by a lack of expression or expression of a dysfunctional Cbl transporter, identified in hepatic lysosomes (247). In patients with Cbl F, Cbl is not transported across the enterocytes (248). Fourth, lack of transcobalamin synthesis is the most common form of transcobalamin deficiency noted in children. It results in Cbl malabsorption caused by a failure to mediate exit of Cbl into the circulation, after its uptake bound to intrinsic factor. Failure to synthesize transcobalamin is caused by private mutations unique to each family and also has been shown to be caused by a lack of transcobalamin mRNA. Lack of transcobalamin mRNA appears to be due to nonsensemediated decay of the transcript (249,250) or to errors in mRNA editing (251). Figure 71-19 presents the four types of inherited disorders of Cbl absorption and the step affected. It is important to diagnose Cbl deficiency in affected children as early as possible and to treat them with Cbl to prevent devastating complications that can affect their postnatal development and cause neurologic deficit.

Structure and Function of Cobalamin Transport Proteins, Intrinsic Factor, and Transcobalamin Molecular cloning and gene structure analysis of human Cbl transporters intrinsic factor and transcobalamin have strongly suggested that both of these proteins, despite their different chromosome localization, are derived by gene duplication from a common ancestral gene. Although intrinsic factor is localized to chromosome 11 (234), transcobalamin is localized to chromosome 22 (252). Phylogenetic analyses (253) have demonstrated that transcobalamin evolved earlier than intrinsic factor and HC (another chromosome 11 resident gene) (254). These observations have suggested that transcobalamin may have evolved when symbiosis existed between prokaryotes and eukaryotes. It is possible that transcobalamin mediated transport of Cbl synthesized by the prokaryotes to the eukaryotes. However, somewhere during evolution when symbiosis ceased, intrinsic factor may have evolved with the development of a rudimentary digestive system. Further studies are required to validate this hypothesis. Sequence alignment of both cDNA (255,256) and genomic levels (252) have provided some novel insights into the structure of intrinsic factor and transcobalamin. Although the overall homology between intrinsic factor and transcobalamin is only about 20% to 30%, there are several regions in their

sequence containing 10 to 15 residues where the homology is much higher with nearly 60% to 80% identity. In addition, both of these proteins contain conserved cysteine residues at six identical positions. Intrinsic factor and transcobalamin genes are approximately 20 kb and contain an identical number of exons of approximately the same size, and many of the highly conserved regions of these two proteins are localized to different exons. In addition, four of eight intronexon boundaries are at exactly identical locations, whereas the other four are located in close proximity (252). Based on these comparative studies, it is likely that the Cbl-binding regions in these proteins have evolved earlier and their different receptor binding regions evolved later. Studies (253) have shown that a highly conserved 15-residue region (174SVDTAAMAGLAFTCL188) represents Cbl-binding signature of transcobalamin and perhaps intrinsic factor (Fig. 71-20, lower box). Within this region, two polar and two alanine residues appear to be important to facilitate the initial event, that is, entry of the nucleotide moiety into the Cblbinding pocket. Earlier work has shown that Co-N coordination bonds (257) and the ribazole fragment of the nucleotide (258) appear to be essential for positioning the nucleotide at a critical distance from the corrin ring and for promoting conformational changes for corrinoid binding. Moreover, a deletion of 12% of the residues from the carboxyl terminus of human intrinsic factor resulted in loss of Cbl binding (259). Taken together, these studies strongly suggest that the 15-residue region and the residues identified therein play a crucial role in Cbl binding. However, the role of other structural elements of Cbl and other regions of transcobalamin in further stabilizing bound Cbl and influencing the affinity for binding cannot be ruled out. The importance of the overall topology of intrinsic factor and transcobalamin in Cbl binding is borne out by the observation that reducing agents that disrupt one or more of the three disulfide bonds of bovine transcobalamin (260) resulted in the liberation of bound Cbl. More recent studies (261) using in vitro mutagenesis of the six conserved cysteine residues of human transcobalamin have shown that not all six conserved cysteine residues that are involved in the formation of three disulfide bonds are equivalent in Cbl binding. Although disruption of two disulfide bonds, C98-C291 and C147-C187, resulted in loss of Cbl binding and in vivo stability, disruption of the disulfide bond C3-C249 had no effect. Because both intrinsic factor and transcobalamin have six conserved cysteine residues, it is likely that Cbl binding by intrinsic factor also is affected by the disruption of disulfide bonds formed by the cysteine residues located at or near the same positions as in transcobalamin. The location of the six conserved and the two nonconserved cysteine residues in intrinsic factor and transcobalamin from various species and the disulfide bonding pattern is shown in the top of Figure 71-20. In contrast with Cbl binding, a common property of intrinsic factor and transcobalamin, their binding to specific cell-surface receptors may involve different structural elements. The initial receptor-binding region of intrinsic

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1817 3

65 78

98

147

187

249

291 Location of cysteine residues in human TC Disulfide bonding pattern

N-terminus

100

200

300

SVDTAAMAGLAFTCL

Human

SVDTMAMAGMAFSCL

Bovine

SVDTEAMAGLALTCL

Mouse

SVDTEAMAGLAFTCL

Rat

TC

IF

SVDTGAVATLALTCM

Rat

FIG. 71-20. Disulfide bonding pattern and cobalamin (Cbl) binding. Linear diagram of transcobalamin (TC) indicating the positions of the conserved (closed circles) and nonconserved (open circles) cysteine residues, disulfide bonding pattern, position of the highly conserved 15-residue region, and important residues therein (downward arrowheads). IF, intrinsic factor.

factor is localized to residues 25 to 62 at the N-terminal region (259), where there is little homology of intrinsic factor with other Cbl-binding proteins. However, other contact points of intrinsic factor with its receptor may develop after this initial binding (262). This is not too surprising because cubilin is a polyvalent protein with multiple structural domains. The receptor recognition region of transcobalamin has not been identified yet, but preliminary studies from the authors’ laboratory indicate that the binding site is localized to the interior of the molecule. It is obvious from these studies that much needs to be learned about the structure-function relation of Cbl transport proteins.

Structure and Function of Cubilin The isolation (214) of an 11.2-kb gene that encoded a protein of 460 kDa molecular mass and bound intrinsic

factor-Cbl with high affinity was a major breakthrough that has helped in elucidating the structure and function of this receptor. Its linear structure (Fig. 71-21) consists of three structural domains, a 113-residue N-terminal followed by 8 epidermal growth factor–like repeats and a tandemly arranged cluster of 27 CUB domains. CUB is an acronym for domains consisting of about 110 amino acids that are found frequently in developmentally regulated proteins such as complement Clr/Cls, Uegf, and bone morphogenic protein-1 (263–265). Cubilin is polyvalent in nature and has the ability to bind to a structurally diverse set of ligands that include receptorassociated protein (215), albumin (266), apolipoprotein A-I/high-density lipoprotein (267,268), transferrin (269), immunoglobin light chains (270), hemoglobin (271), and vitamin D–binding protein (272). These proteins that bind to cubilin are largely undetected in the urine, but their levels are increased in the urine of dogs and humans with familial selective malabsorption of Cbl. Thus, these studies have 27-CUB domains

113-residue N-

C

N

1 2 3 4 5 6 7

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

8-EGF-Like

FIG. 71-21. The three distinct domains of cubilin (Cbl) and their function. The N terminus binds to megalin and the lipid bilayer. The epidermal growth factor (EGF)–like domains have no known function. The 27 CUB domains representing nearly 85% of the total mass of cubilin are polyvalent and bind to many ligands, including intrinsic factor (IF)-Cbl (CUB domains 6–8).

1818 / CHAPTER 71 clearly established that in addition to uptake of intrinsic factor-Cbl in the ileal mucosa, cubilin also functions in the tubular reabsorption of many serum proteins. Two regions of cubilin, the N-terminal 113-residue region and CUB domains 6 to 8 have been shown to bind intrinsic factor-Cbl and albumin (214,266,273). However, these two ligands do not compete for binding at both regions, indicating that the two ligands bind to sites that are spatially related yet distinct from each other (273). It is possible that similar spatial differences also exist for the binding of other ligands, such as vitamin D–binding protein to this region. Some patients who malabsorb Cbl because of a missense mutation, P1297 L in CUB domain 8 that decreased affinity for intrinsic factor-Cbl binding (239), do not excrete vitamin D–binding protein or 25(OH)-D3 in their urine (272). However, there was an increase in the urinary excretion of vitamin D–binding protein and 25(OH)-D3 in patients who had an intronic mutation that resulted in the introduction of premature termination codon and truncation of the cubilin molecule (238). One interesting and important feature of cubilin is that it functions in the endocytosis of so many ligands, but it has no cytoplasmic and transmembrane domains (214). In addition to binding intrinsic factor-Cbl and albumin, the N-terminal 113-residue region is thought to have other functions, such as aiding in the trimerization of the cubilin molecule (274) and interacting with megalin (231), both of which could be important for cubilin in making peripheral contact with the lipid bilayer. The structural elements of the N-terminal region of cubilin that makes contact with the lipid bilayer are unknown and might involve palmitoylation in this region because cubilin from OK cells has been shown to be palmitoylated (205). Based on the in vitro binding of cubilin to megalin, it is suggested (214) that both endocytosis of many ligands that bind to cubilin and its own membrane recycling are assisted by megalin. In addition, megalin is thought to be a chaperone for the plasma membrane delivery of cubilin (268). In vivo evidence for the importance of megalin–cubilin interaction has been obtained in whole-body irradiated rats that experienced development of severe albuminuria caused by loss of albumin and megalin, but not intrinsic factor-Cbl binding to cubilin (275). These observations have supported the distinct nature of albumin and intrinsic factor-Cbl binding sites of cubilin and may help to explain why some patients with Imerslund–Grasbeck syndrome exhibit proteinuria, whereas others do not. Although structural details of cubilinmegalin interaction are unknown, the N-terminal region of cubilin, including the eight epidermal growth factor domains, is implicated as one of the regions important for cubilin binding to megalin (231). Loss of this region because of increased proteolytic conversion of the 460-kDa form of cubilin in the intestine of thyrodectomized rats resulted in decreased association of cubilin with megalin and absorption and transport of orally administered Cbl (231). These studies have underscored the importance of cubilin–megalin interactions in the apical brush borders of kidney cortex and intestine, but additional studies are required to identify other regions of cubilin that

may interact with megalin and other proteins. Such studies will help in the better understanding of how cubilin is delivered to the apical brush border and functions at this site in the endocytosis of many ligands, including intrinsic factor-Cbl.

Cellular Synthesis and Regulation of Expression of Intrinsic Factor and Transcobalamin Intrinsic factor is a glycoprotein, and its expression is limited to cells derived from tissues originating from the foregut anlage and its secretion is regulated by a variety of factors (209,210; see also reference citations therein). Cellular/tissue sites of intrinsic factor synthesis include gastric parietal (human), chief (rat), and pancreatic duct (dog) cells. Transgenic mice experiments (276) using 1.1 kb of the 5′-flanking region of mouse intrinsic factor gene fused with the human growth hormone showed transgene expression in the parietal cells, but not the chief cells, the site of intrinsic factor expression in the mouse. These studies have underscored the complexity of cell-specific intrinsic factor/gene expression, and further studies are required to identify how various cis/trans interactions affect intrinsic factor gene activation or suppression in any given gastric cell type. Cortisone (277) and growth hormone (278) are known to modulate intrinsic factor mRNA levels in rats, but it is not known whether these hormones directly affect transcription of the intrinsic factor gene or exert their effects by altering the stability of the transcript. Unlike intrinsic factor, transcobalamin is not a glycoprotein and is secreted in a constitutive manner from many human cells in culture, and in vivo endothelial cells appear to be the primary source of plasma transcobalamin (279). Transcobalamin mRNA is expressed in many human (280) and rat (281) tissues, but at different levels. Transcobalamin mRNA is developmentally regulated in the rat intestine and kidney and by cortisone (281). Transcobalamin expression in vitro by cultured intestinal epithelial cells (282) and its synthesis in vitro by ileal villous cells (283) are important for the transcellular transport of Cbl across the mucosal cell barrier after the release of Cbl bound to gastric intrinsic factor. Transcobalamin is a product of a typical housekeeping gene in that its 5′-flanking region lacks a discernible TATA box, contains multiple start sites, and is GC rich (252). Deletion and mutagenesis of the 5′-flanking region and transfection in human cells have shown that transcobalamin promoter is weak and is restricted to a region −29 to −163. The weak activity of transcobalamin promoter appears to be because of transcriptional suppression caused by the binding of transcription factor Sp3 to a proximally located GC/GT box (−179 to −165), and the basal activity of transcobalamin promoter is caused by the binding of transcription factor Sp1 to a positively acting GC box (−568 to −559) (284). Thus, the differential levels of transcobalamin mRNA noted in tissues/ cells (280,281) may be related to relative ratios of Sp1 and Sp3, both of which bind to the GC/GT box (284). Further in vitro transfection studies (285) have shown that the physical

INTESTINAL ABSORPTION OF WATER-SOLUBLE VITAMINS / 1819 interactions between Sp1 that bind to GC-box and USF1 and USF2, transcriptional factors of the basic helix-loop-helix family that bind to E-box (−523 to −528), may play a role in high bidirectional transcriptional activity of the transcobalamin promoter. If the transcriptional activation of transcobalamin promoter by E-box–binding proteins occurred in vivo, it may help to explain increased plasma transcobalamin levels noted in patients with various forms of cancer. Other transcription factors of the basic helix-loop-helix family, such as Myc/Max, Mad/Max, and Max/Max, by binding to the E-box might transactivate the transcobalamin gene. Further studies are required to address these issues. In ECV304 cells, which have some properties similar to human umbilical vein endothelial cells, transcobalamin expression appears to depend on cell density and binding of an undefined transcription factor to a hexameric sequence TGGTCC localized 121 bp upstream of the transcription site (286).

Cellular Synthesis and Expression of Cubilin Cubilin is an epithelial cell product and its expression occurs in the intestine and kidney (204,243,287) of many species. In addition, cubilin also is expressed in the rat yolk sac (288). In all three tissues, cubilin activity as measured by binding to intrinsic factor-Cbl, is developmentally regulated (210); in the yolk sac, cubilin has been identified to be the same protein (gp280) (289) that was earlier identified as a teratogenic antigen present in the kidney brush borders (290). De novo synthesis of cubilin has been studied using OK cells (291) and rat kidney cortical slices (292), and these studies have shown that cubilin is retained to a large extent in an intracellular pool, that only about 10% to 25% is delivered to the apical brush border, and that this delivery requires an intact microtubule network. Although cubilin has been shown to bind to megalin in vitro (214) and to form a complex in the apical brush border (231), the functional significance of this interaction for the apical delivery or endocytosis of many ligands is unknown. In addition to megalin, cubilin also binds to AMN gene product, and transfection studies using a partial cubilin fragment have shown that its apical delivery requires the presence of AMN (242). Moreover, mutations in the AMN gene also result in Cbl malabsorption, suggesting strongly that apical membrane expression of cubilin requires the AMN gene product. In addition, more recent studies (293) have shown that adult AMN ← +/+ chimeras excrete large amounts of cubilin ligands such as albumin and transferrin. Further structural studies are required to understand the molecular and cellular basis of these interactions in the anterograde and retrograde pathways of cubilin in an epithelial cell. In the intestine, it is not known whether cubilin has any function other than Cbl absorption. However, because of its ability to bind to galectin-3 (294), intestinal cubilin may also play a role in regulating gastrointestinal cancers (295) and in inflammatory response (296). Cubilin generally is found in the perforin-containing granules of the uterine natural killer

cells and is endocytosed via galectin-3 (294). Thus, cubilin/ galectin-3 interactions may be important in uterine natural killer cell function and in the development of the fetus. Further investigations into the structure-function relation of cubilin are required to understand how by binding to different types of ligands, cubilin may affect intestinal and renal function and regulate fetal development. In the yolk sac, cubilin could play an important role in the endocytic mechanisms that are important in nutrient transport at the maternal–fetal interfaces.

ACKNOWLEDGMENTS This work was supported by grants from the Department of Veterans Affairs and the National Institutes of Health (DK 56061 and DK58057 to H.M.S.; DK 50052 to B.S.).

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72

Water Transport in the Gastrointestinal Tract Jay R. Thiagarajah and A. S. Verkman Epithelial Fluid-Transporting Mechanisms, 1828 “Standing-Gradient” Model, 1828 Transcellular versus Paracellular Fluid Transport, 1830 Solute Recirculation, 1830 Water “Pumps,” 1830 Countercurrent Multiplication, 1831 Luminal Hypotonicity Driven by Acidification, 1832 Summary, 1832 Aquaporins, 1832 Physiologic Role of Aquaporins in Nongastrointestinal Tissues, 1833 General Paradigms about Physiologic Functions of Aquaporins, 1835 Aquaporin Expression in the Gastrointestinal Tract, 1835

Fluid Transport Mechanisms and Aquaporins in Gastrointestinal Organs, 1835 Salivary Gland, 1835 Stomach, 1836 Liver, 1837 Gallbladder, 1838 Small Intestine, 1839 Colon, 1840 Summary and Perspective, 1841 References, 1841

Large quantities of fluid are transported across epithelial barriers in the gastrointestinal (GI) tract for secretion of saliva, gastric juice, bile, and pancreatic fluid, and for fluid absorption in the intestine. The quantity of fluid transported in the GI tract is second only to kidney, where ~180 L fluid per day are filtered by the glomerulus in humans and processed by various nephron segments. In the human GI tract, the salivary glands produce ~1.5 L fluid per day, the stomach secretes 2.5 L gastric juice, the liver produces 0.5 L bile, the pancreas produces 1.5 L enzyme and bicarbonate-rich fluid, the small intestine absorbs 7.5 L fluid, and the colon absorbs 1.3 L fluid against significant osmotic gradients (Fig. 72-1). The fluids transported across epithelial and endothelial barriers contain salts (~150 mM) and water (~55,000 mM). As discussed elsewhere in this textbook, there is a considerable body of data on the molecular identities of the major salt transporters in epithelial cells of the GI tract and their role in transcellular

ion transport. The molecular pathways for water transport in the GI tract have been identified relatively recently, remaining an evolving and, in some cases, controversial subject. As in other organ systems, the general paradigm in the GI tract is that water movement occurs secondary to osmotic driving forces created by active salt transport and to hydrostatic pressure differences. Based on a substantial body of evidence in the kidney and other epithelia carrying out active nearisosmolar fluid secretion or absorption, greater cell membrane water permeability produces greater net fluid movement (see the Epithelial Fluid-Transporting Mechanisms section later in this chapter for a description of current concepts in fluid-transporting mechanisms). Aquaporin (AQP) water channels provide the molecular pathway for cell membrane water transport in many cell types. The AQPs are a family of small, integral membrane proteins that transport water and, in some cases, water and small solutes. AQPs are expressed widely in cell plasma membranes in epithelial, endothelial, and other cell types in the GI tract and elsewhere. There is compelling evidence for the physiologic importance of some AQPs in some tissues based on studies in humans with AQP deficiency/mutations and phenotype analysis of transgenic mouse models of AQP deletion. However, the role of AQPs in the GI tract remains

J. R. Thiagarajah and A. S. Verkman: Departments of Medicine and Physiology, University of California San Francisco, San Francisco, California 94143. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1827

1828 / CHAPTER 72 +2 L

+1.5 L

Salivary gland

+0.5 L

Stomach +2.5 L

Liver Gallbladder +1.5 L Pancreas

−1.3 L Colon

Small intestine

−6.5 L 0.2 L

FIG. 72-1. Schematic of the gastrointestinal tract showing daily fluid secretion and absorption in humans.

largely uncertain, despite a considerable body of data on the expression pattern and cellular processing of AQPs in various GI cell types. The available data are reviewed in this chapter, and major unresolved questions are identified.

than that on membrane B, fluid will be transferred from compartments I to III in the absence of an osmotic pressure difference between compartments I and III.

“Standing-Gradient” Model

EPITHELIAL FLUID-TRANSPORTING MECHANISMS The mechanism of how water is absorbed and secreted across epithelia has puzzled physiologists for decades. Although large volumes of water cross the various epithelia of the GI tract, as diagrammed in Figure 72-1, there remains a lack of consensus as to the mechanisms by which water is transported across epithelia both generally and in specific regions of the GI tract. However, the general paradigm of water transport following active solute movement has remained the basis of most models of fluid transport. The original observation that fluid can be transported in the apparent absence of an osmotic gradient was made in a series of elegant studies by a few pioneering physiologists in the late nineteenth century (e.g., Hedenhain and also Reid [1,2]). It was demonstrated that fluid could be transported across an epithelial sheet such as rabbit ileum in the absence of an external osmotic pressure difference, and that this transport occurred only as long as the tissue remained viable, implying that “active” metabolism within the tissue is required. In the middle of the twentieth century, Ussing (3,4) demonstrated that sodium is actively transported across epithelia, providing the basis for a model of fluid transport. Curran (5) was the first to show that water transport was related linearly to Na+ transport, and he and Macintosh (6) thereafter proposed a “three-compartment model” (Fig. 72-2A) of fluid transport that accounted for the ability of epithelia to transport water in the absence of or against (“uphill”) an osmotic gradient. The model requires active solute transport from compartments I to II across membrane barrier A. Compartment II is then hypertonic to compartment I. If the reflection coefficient for the actively transported solute in membrane A is greater

After the anatomic observations of Diamond (7) and Whitlock and Wheeler (8), Diamond and Bossert (9,10) extended the Curran–Macintosh model by delineating the biological structures responsible for the different compartments and membranes within the epithelium, resulting in the “standing-gradient” model (see Fig. 72-2B). The model suggested that the lateral intercellular space (LIS) between adjacent epithelial cells was the central hypertonic compartment in the Curran–Macintosh model. The basic feature of the model, as illustrated in Figure 72-2B, is that the driving solute (Na+ in most cases) is actively transported from the lumen into the LIS, resulting in a steady-state (standing) osmotic gradient decreasing from the tight junction to the basolateral membrane. The osmotic gradient drives water flux through the cells into the LIS and across the basolateral membrane, resulting in an isosmolar or near-isosmolar absorbate. As originally conceived, the model predicted a number of important characteristics for an epithelium to absorb water in the absence of an osmotic gradient between the luminal and basolateral solutions, which include a water-impermeable tight junction between cells, relatively low water permeability for membranes facing the LIS, and clustering of Na+/K+ATPase pumps near the tight junction. However, subsequent experimental data indicated that many of the original requirements of the model were incorrect, leading to revision of the original model and development of alternative models. Hydraulic conductivity (Lp) measurements of various intestinal membranes generally have shown relatively high water permeabilities for both apical and basolateral membranes (11–18). This observation, together with a series of optical studies of water and solute dynamics in the LIS, led to a revised model in which the osmotic gradient localized in

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1829 I

II

OSMOTIC

Isotonic

Water & Na+

III

HYDROSTATIC

Hypertonic A

Isotonic B

Basement membrane

A

Capillary

B

Transcellular

Paracellular

Na+

Na+

Tight junction Na+

Na+

Aquaporins

C

Isotonic absorbate

D Na+

Water

Glucose

Hypertonic villus tip Water

SGLT-1

Na+

Countercurrent exchange Epithelium

E

F

FIG. 72-2. Mechanisms of water transport in epithelia. (A) Curran–Macintosh three-compartmental model of fluid transport. Isotonic transport of fluid is achieved from compartment I to III via hypertonic compartment II across semipermeable membranes A and B as a result of osmotic and hydrostatic pressures developed as a result of active ion transport from compartment I to II. (B) “Standing-gradient” model of fluid transport. Na+ is actively transported into the lateral intercellular spaces (LISs) between cells, resulting in transcellular movement of water into the LIS and isotonic transfer of fluid into the capillary circulation. (C) Routes of water flux across epithelia: transcellular across the lipid plasma membrane, paracellular across tight junctions between cells, transcellular via aquaporin channel proteins in the plasma membrane. (D) “Sodium recirculation” model of fluid transport. Na+ is actively transported into the LIS, resulting in hypertonic transfer of fluid across the basement membrane and paracellular water flux. Na+ is transported back into the LIS via basolateral transporters, resulting in net isotonic fluid absorption. (E) Water cotransport via the sodium/glucose cotransporter SGLT-1. Water is transported across the lipid bilayer, together with Na+ and glucose, as a result of conformational changes during the normal transport cycle of SGLT-1. (F) “Countercurrent” multiplier in small-intestinal villi. Blood flow through the villous capillary network results in exchange of small solutes in the villous interstitium. Active Na+ absorption via the epithelium, together with countercurrent exchange, causes an osmolarity gradient from villus tip to base. High villus tip osmolarity drives water transport across the epithelium into the central villus lacteals.

1830 / CHAPTER 72 the LIS is predicted to be so small (<15 mOsm) as to be essentially immeasurable (19–21). Another tenet of the revised model is that there is a much greater magnitude of water transport through the cell than through tight junctions. The validity of the revised standing-gradient model has remained uncertain, with an increasing number of theoretic inconsistencies and problems related to the difficulty in measuring many of the key parameters in physiologically relevant epithelial cells in vitro or in vivo. Reported water permeabilities of apical and basolateral membranes have differed over orders of magnitude, introducing considerable uncertainty about osmotic gradients required to drive fluid absorption (11,17,22). Also, the methodologies used to measure membrane water permeability have been criticized, as has the physiologic relevance of the model systems (18). The high water permeabilities measured by Spring and coworkers (12,23,24) in Necturus gallbladder and other systems form the basis for predicting small osmotic gradients in leaky epithelia. Earlier reported values of low water permeability have been criticized because of neglect of unstirred layer effects, although the validity of this criticism also has been questioned (17,25). Also, the ultrastructural techniques used to identify the putative Curran–Macintosh hypertonic middle compartment as the LIS have been criticized (26–28). Notwithstanding these criticisms, the revised standinggradient model of fluid movement by local osmotic gradients still remains the most widely accepted description of water transport across intestinal epithelia.

Transcellular versus Paracellular Fluid Transport Debate on the mechanism of solute-driven water transport in near-isotonic fluid transport in leaky epithelia (small intestine and gallbladder in vertebrate GI tract) has largely focused on whether water passes through cells (transcellular) or between cells (paracellular). The revised standinggradient model assumes that water movement is predominantly transcellular (19). Other models represent variations on the basic Curran–Macintosh model in which fluid transfer is largely paracellular, such as the solute recirculation model of Larsen and colleagues (29) or due to completely different mechanisms such as the water cotransport model proposed by Loo and coworkers (30,31). Central to the debate on the pathway of water transfer across the GI tract is the role of AQPs in fluid absorption and secretion. As discussed later, studies of AQP knockout mice in certain organs systems, such kidney proximal tubule, have clarified the extent of the transcellular pathway. In systems such as the small and large intestines, where no role has yet been found for AQPs in fluid movement, there is ongoing debate about the mechanism(s) responsible for fluid absorption and secretion (see Fig. 72-2C).

Solute Recirculation The idea of “solute recirculation” was proposed initially in 1989 by Ussing and Eskesen (32) and further explored in experiments in toad intestine (33). Subsequent studies confirmed and extended the initial experimental data, leading to a mathematical model of fluid absorption in which sodium recirculation accounts for absorbate isotonicity (29). In the proposed scheme (see Fig. 72-2D), sodium entering into the LIS from pumping through cells or across the tight junction results in predominantly paracellular water movement. A hypertonic fluid then is extruded across the basolateral membrane of the LIS, providing the vectorial water movement component for fluid absorption. Isotonicity of the resulting absorbate is created by reuptake of sodium, putatively by the Na+-K+-2Cl− cotransporter, into cells and consequent “recirculation” back into the LIS. The compartmental model of Larsen and colleagues (29) used their data from toad intestine, together with other experimental data, to model transepithelial solute fluxes, initially for electroneutral absorption, and subsequently for electrogenic absorption (34). Their conclusions of mainly paracellular water flux and basolateral Na+ recirculation were dependent on experiments using cesium as a paracellular probe to estimate the magnitude of solvent drag in the LIS and the movement of solute from the basolateral compartment. However, the use of cesium as a paracellular tracer has been criticized (35); thus, definitive testing of the recirculation hypothesis remains to be done. The conflicting evidence from experimental studies and modeling (24,34) about whether solute movement in the LIS represents true solvent drag resulting from water flux across the tight junction, or pseudo-solvent drag resulting from solute “swept” away by water flux through the cell, emphasizes the need for further testing of solute/solvent dynamics across epithelia.

Water “Pumps” An interesting hypothesis by Wright, Zeuthen, and coworkers (30,36–39) proposes that, in the small intestine (and gallbladder), water transport is mediated by “active” transport by cotransporters such as the sodium/glucose cotransporter (SGLT-1) present at the brush-border membrane of enterocytes. The theory proposed that water is cotransported stoichiometrically together with glucose and sodium as a consequence of conformational changes that occur during the normal transport cycle of SGLT-1 (see Fig. 72-2E). The principal evidence for active water transport comes from studies in Xenopus oocytes expressing SGLT-1, in which sodium current generated by the cotransporter is measured by microelectrodes, and cell swelling (a proxy for water transport across the membrane) is measured optically (30,38). Oocytes overexpressing SGLT-1 swell rapidly and manifest a parallel inward sodium current when exposed to an isotonic glucosecontaining solution, which is inhibited by the specific blocker

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1831 phlorizin. The initial fast rate of cell swelling was interpreted as the result of direct coupling of sodium and glucose transport to water transport (36). Additional evidence in support of this interpretation was the relatively slow rate of swelling produced by equivalent inward sodium currents generated by ion channels or ionophores (36–38,40,41). Also, it was shown that rapid changes in Na+ transport driven by applied voltage changes induced concomitant changes in the rate of cell swelling (36). SGLT-1 has an intrinsic passive water permeability, and in the steady state, sodium-glucose cotransport is expected to induce osmotic water flow by changing intracellular osmolarity. It was proposed that in the steady state, direct (nonosmotic) water cotransport contributes ~35% of the total water transport across the membrane (37). Follow-up work by Zeuthen and coworkers (42) has implicated active water transport by a variety of cotransporters, including the Na+-glutamate cotransporter in the nervous system, H+-lactate cotransporter in retinal pigment epithelium (43), and Na+dicarboxylate cotransporter in the kidney (44). The evidence on which the SGLT-1 water cotransport hypothesis is based has been criticized from a number of standpoints. The physiologic relevance of SGLT-1 water cotransport has been questioned based on studies of water transport in the kidney. Water permeability and fluid absorption in kidney proximal tubule are largely dependent on AQP1 as shown from knockout mouse studies (45,46) (see later). Also, according to the proposed stoichiometry of coupling of water to sodium and glucose (210:2:1; human SGLT-1 [hSGLT-1]), the measured quantities of sodium and glucose reabsorbed by the kidney cannot account quantitatively for the quantity of water reabsorbed (47,48). Although it appears likely that cotransporter-mediated active water transport is not important in the kidney, this is far from clear in the small intestine. AQP-dependent water transport in the small intestine currently has not been demonstrated (see later); thus, in the small intestine at least, the physiologic relevance of water cotransport remains an open question. Studies by Lapointe and coworkers (49–51) have called into question the interpretation of the experimental data and, in some cases, the accuracy of the data itself in studies that aimed to replicate the experimental models used by Wright, Zeuthen, and coworkers. They did not find good agreement between membrane voltage-induced changes in sodium current and cell swelling, concluding negligible water cotransport in the steady state. They proposed that the phenomena seen in the oocyte experiments can be accounted for quantitatively by the generation of local osmotic gradients close to the membrane (in effect, an unstirred layer), and they concluded that the existing model of passive water flow across membranes produced by compartmentalized osmotic gradients can explain water transport in intestinal epithelia. Thus, there remains significant debate on many of the details of the experimental evidence supporting the hypothesis of water cotransport. Although water cotransport may yet emerge as a water transport mechanism in some epithelia under some conditions, currently it is neither sufficiently

proven nor accepted to replace the established paradigm of water transport secondary to local osmotic gradients.

Countercurrent Multiplication An intriguing model of fluid absorption, involving countercurrent exchange in the small intestinal villus, was proposed by Kampp and coworkers during the 1960s (52). The idea that a countercurrent system operates in the villus was based initially on observations on the vascular morphology of mammalian villi (53–55). The villus contains a central arterial vessel surrounded by a dense capillary network and neighboring venous vessels. The hairpin arrangement of the arterial and venous vessels, resembling the renal microvasculature, could produce a “countercurrent” flow system in which blood flow occurs in opposite directions. The evidence that this countercurrent flow could operate as an exchange system came from measurements of relative absorption rates of the inert gases 133Xe and 85Kr from the intestinal lumen into the villus (56,57). These experiments, together with measurements of villous hemodynamics, suggested the presence of a villous countercurrent exchanger in several species including humans (58). These observations led to the proposal that the putative villous countercurrent exchanger could function as an osmotic “multiplier” as in the renal medulla (59). The evidence to support a multiplier function comes mainly from measurements of villous osmolality using cryoscopic techniques and sodium-sensitive microelectrodes (60–63). The villus was reported to have a large gradient of osmolality from tip to base with tip osmolality as high as 700 to 900 mOsm. From these measurements it was proposed that the villous interstitium was the hypertonic compartment described in the Curran–Macintosh model. Water absorption from the intestinal lumen could be driven by the large osmotic gradient/pressure head developed in the villous tip by the countercurrent multiplier system (see Fig. 72-2F). The theoretic basis of countercurrent exchange and its presence in intestinal villi has been criticized for a variety of reasons (see Jodal and Lundgren [64] for more theoretic details). The accuracy and validity of the techniques used to measure inert gas absorption to show countercurrent exchange have been criticized, and alternative explanations for the relative gas absorption rates have been proposed (65,66). Conflicting data also have emerged on the existence of countercurrent exchange in certain mammalian species, but not others, based on differences in villous anatomy (67). The in vitro techniques used to show high villous tip osmolality are thought to produce highly variable results, which may be an artifact of the tissue preparation. It has been noted that the microvascular anatomy in small-intestinal villi is quite different from that of the renal medulla, and in particular, there is no equivalent pathway in villi to the ascending limb of Henle in kidney (66). Also, it has been argued that the existence of a hypertonic compartment in the villous tip

1832 / CHAPTER 72 does not necessarily require a countercurrent mechanism, but does require a relatively water-impermeable epithelium. Thus, convincing in vivo evidence for a hypertonic villous compartment is lacking. Modern optical and fluorescence techniques may be able to elucidate the solute/solvent dynamics within the villus, although in situ experiments on the small intestine remain a significant technical challenge because of its complex geometry. In addition, the efficiency of the putative countercurrent exchange system is critically dependent on the water permeability of the microvasculature, suggesting that endothelial AQP1 might play an important role in villous absorption.

Luminal Hypotonicity Driven by Acidification An interesting model for fluid absorption in the small intestine in which the driving force for water movement across the small-intestinal mucosa is located at the brush-border membrane of enterocytes was proposed by Lucas (68). This model postulates that fluid transport is driven by the neutralization of bicarbonate ions contained within an unstirred layer at the luminal membrane of enterocytes. Sodium-proton exchange at the brush-border membrane produces local luminal acidification resulting from proton transport out of the cell. Bicarbonate ions diffusing from the bulk solution neutralize the protons, resulting in the production of water and carbon dioxide. The combination of two osmotically active ions to form a single osmotically active carbon dioxide molecule produces a hypotonic compartment adjacent to the luminal membrane that, together with an increase in intracellular osmolarity from sodium entry and carbon dioxide diffusion into the cell, drives water across the membrane. Evidence for this model comes from observations of increased fluid absorption for bicarbonate-containing balanced electrolyte solutions in the upper small intestine where luminal acidification is high, together with data from bacterial diarrheas (69–75). In Salmonella typhimurium infection, it was shown that the ileum, which normally exhibits robust electrogenic Na+/glucose absorption, exhibits jejenum-like characteristics with increased luminal acidification and increased plasma pCO2. This was proposed as evidence of increased Na+-H+ exchange leading to fluid absorption. Although the model suggests that the brush-border membrane is the key site of solute–solvent coupling, it does not ultimately contradict the original Curran–Macintosh idea that a basolateral or abluminal hypertonic compartment drives fluid absorption. In the case of electrogenic sodium absorption by sodium channels or sodium/glucose cotransport, or absorption in the absence of luminal acidification, sodium movement across the epithelium into a putative hypertonic compartment remains the primary driving force for fluid movement. In addition, the model does not sufficiently explain how an osmotic gradient across the apical membrane alone results in overall fluid transport from lumen to blood, and studies of bacterially induced fluid transport argue against a significant role (76). Notwithstanding these

caveats, the concept of a luminal hypotonic compartment potentially describes a number of unexplained observations in the upper small intestine. It remains unlikely, however, that it can describe fluid transport in other parts of the GI tract where luminal acidification does not normally occur.

Summary In summary, although the general paradigm of local osmotic and hydrostatic pressure differences driving fluid transport across epithelia has been sufficient to describe measured water flows in the GI tract, a number of variations of the basic theory have been proposed, as well as some completely new theories. The mechanisms involved in fluid absorption have, in general, received much more attention than the mechanisms of fluid secretion, particularly in complex epithelia such as the small intestine. Secretion of saliva, bile, and pancreatic fluid probably can be explained simply and adequately by water movement into a closed/slowly moving compartment driven by osmotic gradients produced by active salt pumping. However, in the small and large intestines and in the gallbladder, significant debate remains regarding the basic water/fluid-transporting mechanisms.

AQUAPORINS An extensive body of information exists on the tissue localization, regulation, structure, and function of the more than 10 currently identified mammalian AQPs. Structural studies of AQP1 indicate a homotetrameric assembly in membranes in which each monomer contains six tilted helical segments that form a barrel surrounding a water pore (reviewed by Fujiyoshi and colleagues [77]). Functional measurements indicate that AQP1, AQP2, AQP4, AQP5, and AQP8 are primarily water selective, whereas AQP3, AQP7, AQP9, and AQP10 (“aquaglyceroporins”) also transport glycerol and possibly other small solutes. Structural analysis and molecular modeling suggest that AQP water selectivity is a consequence of steric and electrostatic factors in which water moves through a narrow pore in a tortuous and single-file manner that does not permit continuous hydrogen bonding (78). The AQPs are expressed in many cell types involved in rapid fluid transport such as kidney tubules, exocrine gland epithelia, microvascular endothelia, choroid plexus, and ciliary epithelium. Physiologic roles of AQPs have been proposed based on their tissue expression pattern and, in some cases, the existence of human mutations, such as nephrogenic diabetes insipidus caused by AQP2 mutation (79–81); however, the current unavailability of AQP inhibitors suitable for use in vivo has precluded direct investigation of their function. As described later, phenotype analysis of AQP knockout mice has elucidated specific physiologic roles for many of the AQPs and has established general paradigms for their physiologic roles.

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1833 Physiologic Role of Aquaporins in Nongastrointestinal Tissues Aquaporins in the Kidney Tubules and the Urinary Concentrating Mechanism The role of AQPs in kidney function has been studied extensively. AQP1 is expressed in apical and basolateral plasma membranes in proximal tubule and thin descending limb of Henle and in microvascular endothelia of outer medullary descending vasa recta. AQP2 is expressed in collecting duct principal cells and undergoes antidiuretic hormone (vasopressin)-regulated trafficking between an intracellular vesicular compartment and the cell apical plasma membrane. AQP3 and AQP4 are coexpressed at the basolateral membrane of collecting duct epithelial cells, with relatively more AQP3 in proximal segments of collecting duct and more AQP4 in inner medullary collecting duct. Each of these AQPs plays a role in the formation of concentrated urine. Given free access to fluid, AQP1 and AQP3 null mice were remarkably polyuric, consuming 2 to 3 (AQP1) to 7 to 10 (AQP3) times more fluid than wild-type mice, whereas the AQP4 null mice were not polyuric, but manifested only a mild reduction in maximal urinary concentrating ability (82,83). After 36-hour water deprivation, urine osmolality in AQP1 null mice did not change, whereas that in AQP3 null mice increased to a submaximal level. Mutant AQP2 mice, in which a mutation causing nephrogenic diabetes insipidus in human subjects was introduced into the mouse genome, were severely polyuric and died within the first 10 days after birth (84). Mechanistic studies indicated that the urinary concentrating defect in AQP1 null mice results from defective proximal tubule fluid absorption and defective countercurrent multiplication, producing a relatively hypoosmolar medullary interstitium (45,85,86). Humans with AQP1 deficiency manifest a qualitatively similar urinary concentrating defect (87), although maximum urine osmolality in humans (~1200 mOsm) is much less than that in mice (>3000 mOsm). Defective urinary concentrating ability in AQP2, AQP3, and AQP4 deficiency results from reduced collecting duct water permeability and consequent impaired osmotic equilibration of tubular fluid with the hypertonic medullary interstitium. Aquaporins in Near-Isosmolar Fluid Absorption and Secretion As mentioned earlier, impaired fluid absorption in kidney proximal tubule in AQP1 deficiency indicates the need for high transepithelial water permeability for rapid, near-isosmolar fluid transport (45). Indeed, micropuncture studies showed remarkable luminal hypertonicity in late proximal tubule in AQP1 null mice resulting from the retrieval of a hypertonic absorbate (46). An important role for AQPs also was found for near-isosmolar fluid secretion in salivary (88) and airway submucosal (89) gland in AQP5 deficiency, and for AQP1-dependent secretion of cerebrospinal fluid by

choroid plexus (90) and aqueous fluid by the ciliary epithelium (91). For example, saliva secretion was reduced by more than twofold in AQP5-deficient mice, and secreted saliva was hypertonic as a consequence of active salt secretion in the acinar lumen of the salivary gland without adequate osmotic equilibration because of reduced epithelial water permeability. High transepithelial water permeability permits rapid water transport in response to active transepithelial salt transport. However, active fluid transport in many tissues does not appear to be AQP dependent, such as alveolar fluid absorption in AQP1/AQP5 deficiency (92) (93) and sweat secretion in AQP5 deficiency (94). The rate of active fluid absorption/secretion per unit epithelial surface area in alveolus and sweat gland is much less than that in kidney proximal tubule or salivary gland, suggesting that high AQP-dependent water permeability is not required to sustain relatively slow fluid absorption/secretion. Aquaporins in Brain Swelling and Neural Signal Transduction AQP4 has an interesting role in the central nervous system, where it is expressed strongly throughout the brain and spinal cord, especially in astroglial cells lining ependyma and pial surfaces in contact with cerebrospinal fluid and the blood–brain barrier. Although AQP4 null mice show no overt neurologic abnormalities, they had remarkably reduced brain swelling after cytotoxic (cellular) edema produced by acute water intoxication and ischemic stroke (95,96). However, brain swelling, intracranial pressure, and clinical outcome were worse in AQP4 null mice in models of vasogenic (leaky vessel) edema including intraparenchymal fluid infusion, cortical freeze injury, and brain tumor (97). Because AQP4 facilitates bidirectional water transport, its deletion in mice can thus reduce brain water accumulation in cytotoxic edema, resulting in improved clinical outcome, and reduce clearance of excess brain water in vasogenic edema, worsening clinical outcome. Interestingly, AQP4 also is expressed in the central nervous system, inner ear, and retina in supportive cells that are in close proximity to non-AQP–expressing electrically excitable cells—in astroglia supporting neurons, in retinal Müller cells supporting bipolar cells, and in cochlear Clausius and Hensen cells supporting hair cells. Mice lacking AQP4 showed reduced seizure susceptibility (98) and reduced evoked potential responses to light (99) and acoustic (100) stimuli. Also, mice lacking AQP4 showed remarkable retinal neuroprotection after ischemia-reperfusion challenge (101). Based on AQP4 and K+ channel Kir4.1 colocalization, it has been proposed that AQP4 in supportive cells may facilitate K+ recycling during electrical signal transduction in the brain, retina, and inner ear (102). In support of this hypothesis, we found using an in vivo fluorescence photobleaching method that the extracellular space was expanded in cerebral cortex in AQP4-deficient mice (98). Quantitative studies relating extracellular space volume and ionic concentration to neural stimuli are needed to establish the mechanism of altered neural signal transduction in AQP4 deficiency.

1834 / CHAPTER 72 Aquaporins in Corneal Edema and Transparency Studies of corneal swelling suggest yet another interesting role for AQPs. Maintenance of corneal transparency requires precise regulation of stromal water content. AQP1 is expressed in corneal endothelial cells and AQP5 in epithelial cells. Corneal thickness, water permeability, and response to experimental swelling were compared in wild-type mice and mice lacking AQP1 or AQP5 (103). Compared with wildtype mice with corneal thickness of 123 µm, corneal thickness was reduced in AQP1 null mice (101 µm) and increased in AQP5 null mice (144 µm). After exposure of the external corneal surface to hypotonic saline (100 mOsm), the rate of corneal swelling (5 µm/min) was reduced about twofold by AQP5 deletion. After exposure of the corneal endothelial surface to hypotonic saline by anterior chamber perfusion, the rate of corneal swelling (7 µm/min) was reduced about fourfold by AQP1 deletion. Although baseline corneal transparency was not impaired by AQP1 deletion, the recovery of corneal transparency and thickness after hypotonic swelling (10-minute exposure of corneal surface to distilled water) was remarkably delayed in AQP1 null mice, with ~75% recovery at 7 minutes in wild-type mice compared with 5% recovery in AQP1 null mice. The impaired recovery of corneal transparency in AQP1 null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium. A Nonwater-Transporting Role of Aquaporins: Aquaporin 3 in Skin Hydration and Aquaporin 7 in Fat Cells The water/glycerol-transporting protein AQP3 is expressed strongly in epidermal keratinocytes, which form the interface between the dermis/vasculature and the stratum corneum. The hypothesis that AQP3 is an important determinant of skin hydration and barrier/mechanical properties was tested by comparative measurements in wild-type and AQP3 null mice generated in a hairless genetic background (104). High-frequency skin conductance, a direct measure of stratum corneum hydration, was remarkably (about twofold) reduced in the AQP3 null mice, and this difference persisted even when mice were placed in a humidified atmosphere or when the skin was covered by an occlusive dressing. Transepidermal water loss measurements after tape-stripping showed significantly slowed recovery of barrier function in the AQP3 null mice. Skin elasticity, measured from the kinetics of skin displacement in response to rapidly applied suction, was remarkably reduced in the AQP3 null mice because of abnormal stratum corneum mechanical properties; skin elasticity of wild-type and AQP3 null mice became similar after removal of the stratum corneum by tapestripping. AQP3 thus appears to play an important role in skin hydration and barrier/mechanical properties. A detailed comparison of skin morphology and the composition of the stratum corneum and epidermis showed reduced glycerol content as the main abnormality in AQP3 deficiency (105). The various abnormalities in stratum corneum hydration, barrier recovery, and skin elasticity could be related to

reduced glycerol content based on the humectant (waterretaining) and biosynthetic functions of AQP3. In addition, topical or systemic glycerol replacement corrected the skin abnormalities in the AQP3 null mice (106), supporting the conclusion that the glycerol-transporting function of AQP3 is important for skin hydration and biosynthetic functions. AQP7, which is expressed strongly in adipocyte plasma membranes, also functions as water/glycerol-transporting protein. We found remarkable age-dependent hypertrophy in adipocytes in AQP7-deficient mice and attributed the phenotype to defective AQP7-dependent glycerol transport in adipocytes (107). Wild-type and AQP7 null mice had similar growth at 0 to 16 weeks as assessed by body weight, though by 16 weeks, AQP7 null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7 null mice 16 weeks old were greatly enlarged (39 µm in diameter) compared with wild-type mice (118 µm). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 vs 86 nmol/mg protein) and triglycerides (3.4 vs 1.7 µmol/mg protein). In contrast, at 4 weeks old, adipocyte volume and body fat mass were comparable in wild-type and AQP7 null mice. To investigate the mechanism(s) responsible for the progressive adipocyte hypertrophy, glycerol permeability and fat metabolism were studied in adipocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by 14 C-glycerol uptake was reduced threefold in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by 14C-glucose incorporation into triglycerides, were not affected by AQP7 deletion. The results supported the conclusion that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides, suggesting the possibility of pharmacologic up-regulation of adipocyte AQP7 expression/function to reduce adipocyte size and fat mass in obesity. Aquaporins in Angiogenesis and Cell Migration We have discovered a new function of AQPs in facilitating cell migration (108). The original motivation for studying the role of AQPs in angiogenesis was the observation of strong AQP1 expression in tumor microvessels. Remarkably, impaired tumor growth was found in AQP1 null mice after subcutaneous or intracranial tumor cell implantation, with reduced tumor vascularity and extensive necrosis. Reduced microvessel growth also was found in implanted pellets of Matrigel-containing vascular endothelial growth factor. A novel mechanism for the impaired angiogenesis was established from cell-culture studies. Although adhesion and proliferation were similar in primary cultures of aortic endothelia from wild-type versus AQP1 null mice, cell migration was greatly impaired in AQP1-deficient cells, with abnormal vessel formation in vitro. Stable transfection of nonendothelial cells with AQP1, or a structurally different water-selective transporter (AQP4), accelerated cell migration and in vitro wound healing. Interestingly, the motile AQP1-expressing cells had prominent membrane ruffles

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1835 at the leading edge with polarization of AQP1 protein to lamellipodia, where rapid water fluxes occur. As a possible mechanism for the dependence of cell migration on AQPs, it has been proposed that cell membrane protrusions are formed as a consequence of actin cleavage and ion uptake at the tip of a lamellipodium, creating local osmotic gradients that drive the influx of water through the cell membrane (109,110). Water entry is thought then to increase local hydrostatic pressure to cause cell membrane protrusion. AQPs may thus provide an important pathway for water entry into lamellipodia in some cell types and also play a role in a wide variety of processes such as tumor angiogenesis and invasion/metastasis, wound healing, and organ regeneration.

General Paradigms about Physiologic Functions of Aquaporins In summary, studies of the extra-GI phenotype of AQPdeficient mice suggest that AQP-facilitated water permeability is important in the following ways: (1) when water movement is driven across a barrier by a continuous osmotic gradient (as in kidney collecting duct); (2) for active, near-isosmolar fluid absorption/secretion (as in kidney proximal tubule and salivary gland); (3) for neural signal transduction (as in brain and inner ear); and (4) for cell migration (as in tumor vessel angiogenesis). Glycerol transport by the aquaglyceroporins is involved in skin hydration (AQP3) and adipocyte metabolism (AQP7). Another conclusion from the phenotype studies is that the tissue expression of an AQP does not ensure its physiologic significance; therefore, evaluation of tissue AQP function must be done on a case-by-case basis. For example, despite the expression of AQP4 in skeletal muscle, no phenotype abnormalities in AQP4 null mice were found (111), as was the case for several AQPs in airways and lung (112). From these general paradigms, a variety of diverse roles of AQPs in the GI tract are possible.

Aquaporin Expression in the Gastrointestinal Tract The expression of specific AQPs in the GI tract provides clues to possible functional roles. Several well-documented expression patterns provide indirect evidence for a possible role of AQPs in these tissues: AQP1 in intrahepatic bile duct epithelium (bile formation), intestinal lacteals (fat absorption), and microvascular endothelia throughout the GI tract (fluid absorption/secretion); AQP4 in basolateral membrane of parietal cells in the stomach (acid/fluid secretion) and of colon surface epithelium (fluid absorption); AQP5 in apical membrane of acinar cells in salivary glands (saliva secretion); AQP8 in salivary gland, liver, pancreas, and intestine; and AQP9 in hepatocytes. Figure 72-3 shows examples of AQP protein localization in the GI tract, with AQP1 in central lacteals of the small intestine (see Fig. 72-3A), AQP4 at the basolateral membrane of surface epithelial cells in the colon (see Fig. 72-3B), AQP8 in crypt epithelium in the ascending colon (see Fig. 72-3C), and AQP4 at the basolat-

eral membrane of gastric parietal cells (see Fig. 72-3D). Additional information about the localization of these and other AQPs is discussed later in this chapter, together with information about organ-specific, fluid-transporting mechanisms and available data from AQP knockout mice.

FLUID TRANSPORT MECHANISMS AND AQUAPORINS IN GASTROINTESTINAL ORGANS Salivary Gland Saliva secreted by the salivary gland is the first fluid with which ingested food comes into contact. The interstitial-toluminal transport of sodium and chloride across the acinar epithelium is the driving force for osmotic water movement (113). The salivary duct epithelium is believed to be relatively water impermeable, in which sodium and chloride are absorbed and potassium and bicarbonate secreted to produce hypotonic saliva. On stimulation, the salivary gland can secrete saliva at high rates (up to 50 ml/min per 100 g tissue in humans), which relies on rapid water movement from serosa-to-mucosa across the capillary endothelium and acinar cells. The possible involvement of AQPs in this process has been proposed based on the expression of AQP1 in microvascular endothelial cells of the salivary gland (114), AQP5 in apical membrane of acinar cells (115), and AQP8 in acinar cells (116,117). A more recent study of expression of AQP1, AQP3, AQP4, and AQP5 in the human salivary gland reported AQP1 in microvessels and AQP5 at the apical membrane of acinar cells, as found in the rodent salivary gland, as well as AQP3 at the basolateral membrane of both serous and mucous acinar cells (118). Our laboratory generated AQP5 knockout mice to investigate the role of AQP5 in saliva secretion (88). Pilocarpinestimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mOsm) and dramatically more viscous (Fig. 72-4). Amylase and protein secretion, which are functions of salivary mucous cells, were not affected by AQP5 deletion. Saliva secretion was not impaired in AQP1 or AQP4 knockout mice. A subsequent report confirmed the defect in saliva secretion in AQP5 null mice and reported reduced water permeability, as expected, in acinar cells isolated from the null mice (119). We measured saliva secretion in AQP8 knockout mice (120), but found no impairment, nor was there a difference in saliva secretion in AQP8/AQP5 doubleknockout mice compared with AQP5 knockout mice. One study has suggested that AQP5 has an abnormal intracellular distribution in patients with Sjögren syndrome (121), but another study reported the opposite (122). A immunolocalization study in rats showed that AQP5 in salivary gland acinar cells is primary at the apical plasma membrane under both basal and stimulated conditions (123). Although there may be a subset of patients with Sjögren syndrome with cellular mislocalization of AQP5, it is more likely that AQP5

1836 / CHAPTER 72

A

B

PC

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PF

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FIG. 72-3. Immunocytochemistry of aquaporin (AQP) expression in the gastrointestinal tract. (A) Immunofluorescence of mouse small-intestinal villi showing AQP1 protein in central lacteals. (B) AQP4 labeling of basolateral membrane colonic surface epithelium. (C) AQP8 labeling of colon crypt epithelium. (D) AQP4 labeling of basolateral membrane in gastric parietal cells (Pc). (Modified from Yang and colleagues [120], Ma and colleagues [143], and Wang and colleagues [205], by permission.)

mislocalization, if it occurs, is a consequence of the chronic immune damage to the salivary gland.

Stomach The large quantity of gastric fluid produced by the mammalian stomach is thought to be secreted mainly by fundic glands in the mucosa of the stomach body. These glands contain mucous

cells, chief cells, and parietal cells that secrete mucus, pepsinogen, and hydrochloric acid, respectively. During agonist-stimulated acid secretion, gastric juice is transported from the mucosal interstitium into the human gastric lumen at a rate of ~0.7 ml/min per 100 g tissue (124). Little information exists about the relative contributions of different cell types involved in gastric fluid secretion. Currently, AQP4 is the only AQP identified in the stomach. Rat AQP4 was immunolocalized to the basolateral membrane of

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1837 +/+ AQP5 −/−

* 0

100

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Osmolality (mOsm)

+/+ AQP5 −/− −/−

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+/− AQP5

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FIG. 72-4. Defective salivary gland secretion in (AQP5) null mice. (A) Photograph of saliva collected over 5 minutes from mice of indicated genotype. Salivation was stimulated by pilocarpine. (B) Averaged (± standard error of the mean) osmolality and sodium concentration of saliva collected from mice of indicated genotype. (Modified from Ma and colleagues [88], by permission.)

parietal cells (125). A human AQP4 homolog was subsequently cloned from the stomach and immunolocalized to both parietal and chief cells (126). A more recent evaluation of AQP4 expression in gastric glands showed AQP4 protein localization only in parietal cells in mid and deep regions of gastric glands (127). One interesting study addressed the possible importance of AQP4 assembly in orthogonal arrays of proteins (OAPs), which are lattice-like particle arrangements seen by freeze-fracture electron microscopy. AQP4 was shown to be the main component of OAPs from studies in AQP4-expressing CHO cells (128) and from the absence of OAPs in multiple tissues in AQP4-deficient mice (129). Carmosino and colleagues (130) reported that OAPs in an AQP4 transfected human gastric cell line was reduced by ~50% after histamine stimulation, suggesting that shortterm regulation of AQP4 assembly and water permeability might be possible. The physiologic significance of this observation remains unclear. It has been postulated that AQP4 is involved in gastric acid and pepsinogen secretion or cell volume regulation, or both. One study investigated the role of AQP4 in gastric acid and fluid secretion using AQP4 null mice (131). Gastric acid secretion was measured in anesthetized mice in which the stomach was perfused lumenally at 0.3 ml/min with normal saline containing 14C-PEG as a volume marker. Collected effluent was assayed for titratable acid content and 14C-PEG radioactivity. After a 45-minute baseline perfusion, acid secretion was stimulated by intravenous pentagastrin for 1 hour or by intravenous histamine plus intralumenal carbachol. Baseline gastric acid secretion was 0.06 ± 0.03 µEq/ 15 minutes in wild-type mice versus 0.03 ± 0.02 µEq/ 15 minutes in AQP4 null mice. Pentagastrin-stimulated acid

secretion was 0.59 ± 0.14 µEq/15 minutes in wild-type mice versus 0.70 ± 0.15 µEq/15 minutes in AQP4 null mice. Histamine/carbachol-stimulated acid secretion was 7.0 ± 1.9 µEq/15 minutes in wild-type versus 8.0 ± 1.8 µEq/ 15 minutes in AQP4 null mice. In addition, there was no effect of AQP4 deletion on gastric fluid secretion, gastric pH, or fasting serum gastrin concentrations. Thus, currently, there is no direct evidence to support a physiologic role of AQPs in the stomach.

Liver Several AQPs (1, 3, 8, and 9) have been reported to be expressed in liver and proposed to play a role in bile secretion. Early immunocytochemistry showed AQP1 expression on the apical and basolateral membrane domains of cholangiocytes (132). A series of studies by the LaRusso group have provided indirect evidence for a possible role of several AQPs in bile secretion. Semiquantitative water permeability measurements in isolated cholangiocytes and intrahepatic bile ducts suggested AQP-mediated water transport based on weak temperaturedependent water transport and inhibition by HgCl2 (133). Membrane fractionation studies suggested that secretin caused a redistribution of AQP1 from intracellular vesicles to the cell plasma membrane, and that the redistribution was blocked by colchicine and low temperature (134,135). If correct, these observations would indicate that unlike other cell types where AQP1 is expressed constitutively at the plasma membrane, the cholangiocyte possesses a unique regulatory mechanism for AQP1. However, the cell biology of regulated AQP1 trafficking requires elucidation, because AQP1

1838 / CHAPTER 72 (unlike AQP2 [136]) does not contain a consensus phosphorylation site at its C terminus. Immunocytochemistry in rat (137,138) and mouse (139) showed AQP8 protein expression in intracellular vesicles in hepatocytes. Further measurements of water permeability in isolated hepatocytes from rat showed moderate water permeability with Pf of 66 × 10−4 cm/sec at 37°C (140); however, follow-up studies from the same group reported a much lower Pf of <10−4 cm/sec (141). They also reported a cyclic adenosine monophosphate (cAMP) agonist-induced approximately twofold increase in rat hepatocyte water permeability, relocalization of intracellular AQP8 to the plasma membrane (138), and a sixfold increase in water permeability of canalicular plasma membrane domains in cAMP-treated rat hepatocytes (142). Work from our group showed AQP8 immunolocalization in the plasma membranes of hepatocytes in mice with weak intracellular labeling (120). AQP8 knockout mice served as control mice for immunolocalization analysis. Osmotic water permeability measured in freshly isolated hepatocytes from wild-type mice was low (Pf 6 × 10−4 cm/sec) and did not increase after cAMP agonists or was reduced in AQP8 deficiency, providing evidence against constitutive or cAMP-regulated AQP8 water permeability in hepatocytes in mice. As mentioned for AQP1, rat and mouse AQP8 do not contain consensus sequences for phosphorylation by protein kinases A or C. Studies in AQP1 and AQP8 knockout mice do not support a significant role of AQPs in bile secretion, at least in mice. Dietary fat misprocessing was observed in young, but not adult, AQP1 knockout mice (143). Whereas young wild-type mice gained 36% ± 5% body weight in 8 days, the AQP1 null mice lost 2% ± 1% body weight and developed steatorrhea. The weights became similar 6 days after return to a 6% fat diet. Despite a 50% decrease in ad libitum food intake in the knockout mice, averaged serum triglyceride concentrations were 137 mg/dl in wild-type versus 66 mg/dl in knockout mice on the high-fat diet. Semiquantitative analysis of stool fat content by a lipid extraction method showed increased stool fat in the knockout mice on the high-fat diet. Inclusion of pancreatic enzymes (lipase, amylase, and protease) in the high-fat diet partially corrected the weight loss and the increased stool fat content in the AQP1 knockout mice. Fluid collections done in older mice (which are less sensitive to a high-fat diet) by ductal cannulation showed threefold increased pancreatic fluid flow in response to secretin/cholecystokinin, but volumes, pH, and amylase activities were affected little by AQP1 deletion. Bile flow rates and bile salt concentrations were not affected by AQP1 deletion. The data established a dietary fat misprocessing defect in young AQP1 null mice, the cause of which is not fully resolved, although bile secretion is not impaired in adult AQP1-deficient mice. Also, direct measurements of water permeability and fluid secretion in cultures of mouse cholangiocytes do not support a significant role of AQP1 in bile secretion (144). Several studies have previously reported AQP1 expression in pancreatic ducts or pancreatic microvasculature, or both (145–149); however, the unimpaired pancreatic fluid secretion in AQP1 null mice raises

doubt about the physiologic significance of this observation, at least in mice. AQP5 and AQP8 expression in the pancreas also were found in several of these descriptive expression studies. As done for AQP1 null mice, the AQP8 null mice were stressed by a high-fat diet to expose potentially subtle defects in hepatobiliary function. Weight gain in wild-type and AQP8 null mice on a high-fat diet was similar, and the AQP8 null mice did not experience development of steatorrhea or abnormalities in serum lipid profile, liver function tests, or pancreatic enzymes. It was concluded that AQP8 does not have an essential role in hepatobiliary/pancreatic function, although a more definitive conclusion will require direct assessment of the secretion rate and composition of bile and pancreatic fluid. The only significant difference between wild-type and AQP8 null mice on a high-fat diet was mildly increased plasma triglyceride and cholesterol concentrations in the AQP8 null mice, the cause and significance of which are unclear. Thus, a physiologically significant role of AQPs in hepatobiliary and pancreatic secretory function remains unproven.

Gallbladder The gallbladder epithelium is perhaps the most intensely investigated area of the GI tract in relation to water transport, in part because it is relatively easy to study, consisting of a simple, relatively flat epithelium with large cuboidal cells that are homogenous in size and functional properties. Also, the gallbladder is a blind sac allowing assessment of water transport by relatively simple gravimetric techniques. Studies on rabbit gallbladder formed the original experimental basis for the standing-gradient model of fluid absorption in leaky epithelia (7,150–157). Follow-up studies in mammalian and Necturus gallbladder investigated the osmotic water permeability and morphology of the apical (lumen-facing) membrane and the epithelium as a whole in an effort to measure the primary parameters to differentiate among the various fluid transport models for transepithelial fluid transport (158–168). The gallbladder is primarily an absorptive epithelium that functions to concentrate sodium salts of bile acids by nearisotonic fluid absorption from the gallbladder lumen. Transcellular water flux across highly water-permeable apical and basolateral cell membranes is the prevailing view on the mechanism of fluid absorption across the gallbladder epithelium (160). However, this view has been questioned based on both methodologic and theoretic considerations (see the Epithelial Fluid Transport Mechanisms section earlier in this chapter). AQP1 is the only AQP localized in gallbladder so far, with initial immunohistochemistry suggesting expression in the basolateral membrane of epithelial cells (169). Currently, there are no direct studies of gallbladder function or water transport in AQP1 knockout mice, but no significant abnormalities were found in bile salt concentration (143), suggesting that AQP1 has little, if any, role in mediating fluid absorption in this epithelium.

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1839 Small Intestine The normally functioning small intestine carries out substantial net absorption of fluid (~6–7 L/day in humans), which represents a combination of absorptive and secretory fluid fluxes. In response to various stimuli and in disease states such as bacterial/viral infection, net fluid absorption switches to fluid secretion that can result in diarrhea (170). The small intestine is a geometrically complex epithelium with many villi protruding into the intestinal lumen, thereby increasing the effective surface area of the epithelium for solute and water absorption. Interspersed between the villi are the glandlike crypts of Lieberkuhn that form the progenitor cells for the villus tips. It has been postulated that fluid secretion occurs predominantly within the crypt cells and fluid absorption at the villus tip. This view has begun to be revised with both absorptive and secretory functions being ascribed to cells in both locations (171–184). The magnitude of fluid movement varies along the length of the small intestine with the bulk of fluid absorption occurring in the jejunum and ileum. As discussed in detail earlier in the Epithelial Fluid-Transporting Mechanisms section, the mechanism and pathway of water transport across the small intestinal epithelium remains unresolved. Expression of at least seven AQPs in the small intestine has been reported (137,185): AQP1 in endothelia and lacteals of the small intestine and microvascular endothelia throughout the intestine; AQP3 at the basolateral membrane of the epithelial cells lining the villus tip of the small intestine in rat; AQP4 at the basolateral membrane of crypt epithelium; AQP5 in the apical membrane of secretory cells in duodenal glands; AQP8 at apical membrane of epithelia in rat small intestine; and AQP9 in goblet cells in duodenum, jejunum, ileum. A new mercurial-sensitive water channel (AQP10) has been cloned (186,187), with transcript expression in human duodenum and jejunum and protein expression demonstrated in the apical membrane of the villus epithelium in ileum (188). Duodenum A primary role of the duodenum is the protection of the epithelium and underlying tissue from damage by gastric acid and proteases produced in the stomach. The duodenum achieves this primarily by the secretion of mucus and bicarbonate into the lumen from the epithelium and submucosal glands (Brunner’s glands), forming a protective mucousbicarbonate barrier (189–191). The mucous barrier is formed when mucins and water combine to form a bicarbonate-rich viscoelastic gel that creates a physiochemical barrier to acid and enzymes (192). Secretion of mucous fluid from Brunner’s glands and the epithelium clearly has an important role in normal duodenal function. However, the pathway for the water component of this secretion currently is undetermined. AQP5 has been found at the apical membrane of acinar cells in Brunner’s glands (193,194) suggesting a possible role in fluid secretion from glands. Currently, no functional evidence exists for facilitated water transport in duodenum.

Jejenum and Ileum Water movement in the jejenum and ileum in the healthy intestine is primarily absorptive (195). Bacterial toxins such as cholera toxin and Escherichia coli STA toxin act on enterocytes, resulting in massive secretion of NaCl and a consequent reversal of net water flux from absorptive to secretory, producing diarrhea (196). The role of AQPs in fluid absorption in the jejunum and ileum currently is unclear. Osmotic water permeability of the apical membrane of smallintestinal enterocytes, when measured minimizing confounding effects of unstirred layers, generally is found to be fairly high (25,197). Early perfusion studies suggested that proximal segments of the small intestine have greater osmotic permeability than distal segments (195). The proximal jejunum has been proposed to be highly water permeable to permit rapid osmotic equilibration of intestinal contents (198–200). It has therefore been proposed that (see Standing-Gradient Model section earlier in this chapter) the transcellular water permeability is sufficiently high to account for the majority of water transport across the epithelium. Whether AQPs are involved in this process is unclear. One possibility is that the specialized lipid composition of enterocyte plasma membranes and their convoluted microvillar ultrastructure confer high transcellular water permeability in the absence of AQPs. Another widely accepted model states that water moves across the epithelium primarily by a paracellular route across the tight junctions between neighboring cells. Because the small-intestinal epithelium has low electrical resistance, low reflection coefficients for small solutes (201), and high paracellular solute flux, it has been proposed that the tight junctions are also permeable to water. Smallintestinal tight junctions have been thought to be cation selective (202) and permeable to large polar nonelectrolytes. This led to the suggestion that osmotic gradients created by active transport of solutes, together with the passive ion permeability through the tight junction, results in “solvent drag” of water through the paracellular pathway (see also Solute Recirculation section earlier in this chapter). Several experimental and theoretic studies (33,203,204) based on the dynamics of various paracellular probes have supported the paracellular model for water movement; however, the interpretation of the data remains subject to considerable debate. An alternative pathway that has been proposed is that transport occurs via the SGLT (see Water “Pumps” section earlier in this chapter). Experiments on AQP knockout mice have found little difference in fluid absorption or secretion in the small intestine compared with wild-type mice, which leaves the role of AQPs in the small intestine (and colon) an open question. At least four AQPs have been reported in the epithelial cells: AQP4 in the basolateral membrane of enterocytes at the base of crypts in the ileum (137), AQP3 in the basolateral membrane of villus enterocytes (137,185), AQP8 in the apical membrane of both crypt and enterocytes (120), and AQP10 at the apical membrane of enterocytes in ileum (188). AQP4 is expressed only at the base of crypts in undifferentiated

1840 / CHAPTER 72 enterocytes, and thus is thought not to be responsible for fluid transport in the intestine. Also, it is unclear whether AQP3 is actually expressed in small-intestinal enterocytes with equivocal immunostaining reported in tissues (137,185). AQP8 may be the main AQP expressed in small-intestinal crypt cells (120,185). However, analysis of AQP8 null mice showed little phenotypic differences compared with wild-type mice (120). Osmotically-driven water secretion, isosmolar fluid absorption, and cholera toxin–induced active fluid secretion were compared in wild-type versus AQP8 null mice. Osmotically-driven water secretion was measured from luminal fluid osmolalities measured at 5 and 15 minutes after infusing a hyperosmolar solution (phosphate-buffered saline containing 300 mM mannitol) into closed jejunal loops in vivo. Osmotic equilibration did not differ significantly in wild-type versus AQP8 null mice. Apparent transepithelial osmotic water permeability coefficients, computed assuming the jejunum as a smooth cylinder, were 0.012 and 0.014 cm/sec for wild-type and AQP8 null mice, respectively. Isosmolar fluid absorption was measured from the increase in concentration of a volume marker (bluedextran) at 20 minutes after infusion of jejunal and descending colonic loops with an isosmolar saline solution, and it showed comparable rates of isosmolar fluid absorption in wild-type versus AQP8 null mice. Computed fluid absorption rates in jejunum were 2.0 ± 0.4 and 1.7 ± 0.4 µl/min/cm2 luminal surface for wild-type and AQP8 null mice, respectively. Cholera toxin–induced fluid secretion also was measured, and no significant difference between wild-type and AQP8 null mice was found. Thus, the current experimental data suggest that AQPs do not constitute a major pathway for water movement across the small-intestinal epithelium.

Colon Water transport in the colon is primarily absorptive and occurs against a significant osmotic and hydraulic resistance imposed by the luminal contents. The primary functions of the colon are the salvage of the remaining fluid and electrolytes entering from the small intestine and the dehydration and storage of feces. The colon is a tight epithelium with substantially greater electrical resistance than the small intestine and much lower paracellular permeability. The colonic epithelium consists of a flat surface epithelium and numerous deep crypts containing absorptive and secretory columnar epithelial cells. In addition, colonic structure and function vary along its length, with fecal dehydration becoming more important distally. Early studies showed that in contrast with the small intestine, the colon is able to transport fluid hypertonically, with absorbate osmolalities of 500 to 1000 mOsm (200). As in the small intestine, the route of water movement in the colon has been the subject of ongoing debate. At least two AQPs are expressed in the colon: AQP4 in surface epithelial cells and AQP8 in crypt epithelial cells.

To date, studies in AQP knockout mice have not supported a role for AQPs in colonic fluid absorption. AQP4 is localized at the basolateral membrane of surface colonic epithelial cells with the strongest expression in the proximal colon. Analysis of stool water content indicated no differences in cecal stool from wild-type versus AQP4 knockout mice, although a small but significantly greater water content was present in defecated stool from the knockout mice (205). The transepithelial osmotic water permeability coefficient (Pf) of the in vivo perfused colon was measured using 14C-polyethylene glycol as a volume marker. Pf of wild-type mice was 0.016 ± 0.002 cm/sec and was independent of osmotic gradient magnitude and direction, as well as the solute used to induce osmosis. Pf was significantly reduced in the AQP4 knockout mice in the proximal but not distal colon. However, despite the differences in water permeability with AQP4 deletion, theophylline-induced secretion was not impaired (50 ± 9 vs 51 ± 8 µl/min/g). Fluid absorption and secretion have been investigated in AQP8 knockout mice (120). Isosmolar fluid absorption measured in closed loops of descending colon was similar in wild-type versus AQP8 null mice (1.8 ± 0.1 vs 2.0 ± 0.4 µl/min/cm2, respectively). Also, stool water content did not differ significantly (60% ± 3% vs 59% ± 1%). The results from AQP knockout mice suggest that AQPs play, at most, a minor role in water absorption in the colon, suggesting that other mechanisms are involved. A significant proportion of water absorption occurs at the surface epithelium of both the proximal and the distal colon (180), although the exact route of water flux is unclear. The process of fecal dehydration occurs against substantial osmotic and hydraulic resistance imposed by feces as their water content is reduced from 95% to 70%. The mechanism by which the colon carries out fecal dehydration has not been resolved. Several studies have suggested that colonic crypts are the major site of both fluid absorption and agonist-stimulated fluid secretion (174,183,206). Studies by Naftalin and colleagues (180) using agarose gel cylinders to mimic the hydraulic resistances imposed by feces, as well as optical studies using fluorescent dextrans, indicated that colonic crypts play an important role in colonic fluid absorption. These studies led to a model of fecal dehydration based on the original Curran–Macintosh model, in which solute absorption by colonic crypts produces a hypertonic pericryptal compartment outside of the crypt lumen (Fig. 72-5). Distal colonic crypts are surrounded by a fenestrated membrane or “sheath” consisting of myofibroblast cells and extracellular matrix components, creating a diffusion barrier to sodium and providing mechanical rigidity to resist collapse of the crypt under negative pressure (175,179). Experiments using a fluorescent sodium indicator showed high sodium concentrations in the pericryptal space (176). The resultant osmotic gradient is proposed to drive water transport from the crypt lumen across a relatively water-impermeable barrier, allowing the mouth of the crypt to act as a suction device to absorb water out of fecal material within the colon lumen. Initial evidence for this mechanism was the demonstration

WATER TRANSPORT IN THE GASTROINTESTINAL TRACT / 1841

by photobleaching experiments of convective fluid flow within crypts (177). Additional evidence for a role for the “pericryptal sheath” in modulating fluid absorption comes from studies showing that proabsorptive stimuli, such as high plasma rennin-angiotensin II-aldosterone, correlate with a trophic effect on the pericryptal sheath and increased pericryptal sodium concentrations (207). However, although the suction hypothesis is currently the only mechanism proposed to account for fecal dehydration, direct measurements of crypt suction and water permeability are needed to examine this interesting mechanism further.

Phenotypic studies in AQP knockout mice suggest that AQPs can have physiologic importance for rapid water transport in response to osmotic gradients created by continuous flow (as in kidney) or active salt pumping (as in saliva secretion). AQPs also appear to be involved in electrically excitable tissues, perhaps by dissipating osmotic gradients created during rapid potassium recycling. The aquaglyceroporins (AQP3 and AQP7), which transport glycerol as well as water, appear to have physiologic importance in mechanisms that require high cell membrane glycerol permeability such as epidermal hydration and adipocyte triglyceride biosynthesis. However, phenotypic studies have shown that the functioning of many tissues expressing AQPs, such as lung, airways, various microvascular beds, and skeletal muscle, is not impaired by AQP deletion. The reasons for the expression of AQPs in many tissues without apparent functional significance remain unclear. The finding of a role for AQP1 in angiogenesis and cell migration might provide a clue about previously unrecognized roles for AQPs, which might explain the wide distribution of AQPs to cell types that do not carry out rapid water or glycerol transport. Many questions remain to be addressed regarding watertransporting mechanisms in GI physiology. Although fluid secretion by GI organs is now reasonably well understood, the basic mechanisms of how fluid is absorbed in small intestine and colon are unresolved. The controversial role of sodiumcoupled solute transporters in fluid absorption in the small intestine needs to be resolved, as does the crypt suction mechanism for fecal dehydration in colon. The finding of numerous AQPs in multiple organs of the GI tract without apparent function remains perplexing. Whereas AQP deletion has produced many interesting phenotypes in the kidney, central nervous system, eye, skin, and fat, few phenotypes have been found in GI organs. The role of AQP10 in fluid transport by the human intestine requires investigation, as does the expression of other possibly novel water-transporting proteins. Finally, possible roles of AQPs in diseases of the GI tract and the development of novel therapies based on AQP function warrant evaluation. For example, is AQP expression altered in biliary and pancreatic disease and in various diarrheas, and can pharmacologic modulation of AQP expression/function by novel agents or gene delivery alter the course of diarrheas, inflammatory bowel disease, hepatic fibrosis, Sjögren syndrome, and other GI disorders? The investigation of molecular water-transporting mechanisms in the GI tract has been an understudied area with considerable potential for significant advances.

SUMMARY AND PERSPECTIVE

REFERENCES

All membranes, including those lacking AQPs, are moderately water permeable such that “highly” water-permeable membranes are no more than ~50-fold more waterpermeable than water-impermeable membranes. Slow water transport occurs across plasma membranes in all cells for housekeeping functions such as volume regulation. AQPs probably have little importance in such processes.

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Colon lumen Fluid flow

Surface

Suction tension

high [Na+] Myofibroblast cells

Na+ H2O Pericryptal sheath

Epithelium

Colon crypt

FIG. 72-5. Model of colonic crypt fluid absorption. Na+ is transported across the crypt epithelium into the surrounding interstitium. The pericryptal sheath, consisting of myofibroblast cells and extracellular matrix elements, provides a barrier to Na+ movement, creating a hypertonic compartment. Water is transported across the crypt, creating a suction tension at the crypt opening. This tension causes fluid influx from the lumen, allowing dehydration of the luminal fecal contents.

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Na+-H+ Exchange in Mammalian Digestive Tract Pawel R. Kiela and Fayez K. Ghishan Overview, 1847 Mammalian Na+-H+ Exchanger Gene Family, 1848 Membrane Topology, 1851 Functional Domains, 1852 Transport Characteristics and Pharmacology, 1853 Gastrointestinal Na+-H+ Exchangers, 1855 NHE1, 1855 NHE2, 1856 NHE3, 1858 NHE4, 1863 NHE5, 1863 NHE6, 1863 NHE7, 1864

NHE8, 1864 NHE9, 1865 Physiologic Roles of Na+-H+ Exchange in the Digestive Tract, 1866 Na+-H+ Exchange and Salivary Gland Physiology, 1866 Na+-H+ Exchange and Liver Physiology, 1867 Na+-H+ Exchange and Pancreatic Physiology, 1868 Na+-H+ Exchange and Gastric Physiology, 1869 Na+-H+ Exchange and Intestinal Water and Sodium Absorption, 1870 Acknowledgments, 1873 References, 1873

OVERVIEW

posttranscriptional levels. Consequences of gene-targeted mutation of individual NHE isoforms (NHE1-4) are discussed in the context of the physiology of digestive organs. Where available, we also provide a review of pathophysiologic states related to aberrant expression or activity, or both, of NHEs within the confines of the digestive system. The first physiologic observations suggesting the existence of an Na+-H+ exchange mechanism in mammalian membranes were made by Mitchell and Moyle (1) and Brierley and colleagues (2) in rat liver and cow heart mitochondria, respectively. These observations were soon followed by similar findings in the prokaryotic plasma membrane by Harold and Papineau in Streptococcus faecalis (3) and by West and Mitchell in Escherichia coli (4). Since then, the field of Na+-H+ exchange has grown steadily. The annual number of Medline-indexed publications referring to this mechanism consistently rose from 2 in 1975 to a record 380 in 2002. In 1987, Goldberg and colleagues (5) cloned the first Na+-H+ antiporter from E. coli, later termed nhaA. Its mammalian counterpart was cloned shortly after by Sardet and colleagues and reported in 1988 (6) and 1989 (7). Cloning of this NHE, initially described as growth factor–activatable Na+-H+ antiporter, and later termed NHE-1, initiated the explosion of knowledge about function, structure, and regulation of what

The Slc9a family of nine Na+-H+ exchangers (NHEs) plays a critical role in neutral sodium absorption in the mammalian intestine, as well as in other absorptive and secretory epithelia of digestive organs. These transport proteins mediate the electroneutral exchange of Na+ and H+ and are crucial in a variety of physiologic processes, including the fine-tuning of intracellular pH (pHi), cell volume control and systemic electrolyte, and acid–base and fluid volume homeostasis. This chapter reviews the role of the Na+-H+ exchange mechanism as it relates to the physiology of organs and cells involved in nutrient intake and absorption and describes physiologic and molecular aspects of individual isoforms, including their structure, tissue, and subcellular distribution, as well as their regulation by physiologic stimuli at the transcriptional and

P. R. Kiela and F. K. Ghishan: Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1847

1848 / CHAPTER 73 was soon determined to be a family of proteins involved in membrane Na+-H+ antiport mechanism. Families of NHEs were identified in bacteria, yeast, plants, and animals. Although the basic principle of Na+-H+ exchange is consistent among species, there are also some considerable differences. In vertebrate animals, the ubiquitous plasma membrane Na+,K+-ATPase mediates the efflux of 3Na+ and the influx of 2K+, a process coupled to the hydrolysis of adenosine triphosphate (ATP). This electrogenic Na+-K+ exchange establishes an Na+ gradient across the plasma membrane that is used by the cell for a number of physiologic functions. These processes are largely dependent on Na+-H+ exchange, a mechanism critical in the regulation of intracellular and systemic pH, cell volume, absorption, and reabsorption of sodium in the intestinal tract and kidney, respectively. This physiologic need dictates the polarity of the exchange, with extracellular sodium being exchanged for intracellular proton. In contrast, in lower organisms such as bacteria, plants, and yeast, the ability to withstand extreme hypersaline or hyperalkaline conditions results from developed Na+-H+ antiport mechanisms leading to the net uptake of protons and net loss of sodium. In addition to differences in Na+-H+ exchange polarity, its stoichiometry also varies. For example, although all the described mammalian NHEs are electroneutral, E. coli bacteria exhibit electrogenic Na+-H+ exchange, with a stoichiometry of 1Na+/2H+ and 2Na+/3H+ for NhaA and NhaB isoforms, respectively (Fig. 73-1). In mammalian cells, tight pHi regulation is crucial for cell function and survival, because its subtle changes may significantly affect cellular physiology. Variations in pHi mediated by NHE mechanism have been associated with basic biological processes such as proliferation, cell adhesion and migration, cell volume regulation, and transepithelial electrolyte and water transport. This chapter focuses primarily on the structure, function, and regulation of NHEs expressed in the mammalian gastrointestinal (GI) tract. Several reports in the literature provide an overview on plants (8,9), Caenorhabditis elegans (10,11), and procaryotic Na+-H+ antiport (12,13). Comprehensive reviews on mammalian Na+-H+ exchange were published by Orlowski and Grinstein (14) and Zachos and colleagues (15), with particular emphasis on intestinal Na+-H+ exchange. Phylogenetic analysis of evolutionary relations among NHE sequences from all phyla, including sequences well characterized, as well as those electronically annotated based on sequence similarity, has been published by Brett, Donowitz, and Rao (16).

MAMMALIAN NA+-H+ EXCHANGER GENE FAMILY After the cloning of NHE1 (6,7), which was later considered a prototypical mammalian NHE, eight other isoforms from diverse species were described by various methodologic approaches (Table 73-1). The nine proteins forming the family of mammalian NHEs demonstrate considerable variation in their amino-acid sequence, ranging from less than

3H+

2H+ + −

NhaA

+ −

+ −

+ −

+ −

+ −

Na+

+ −

+ −

+ −

NhaB

2Na+

ADP+Pi

ATP

H+-ATPase H+ H+

E. Coli

Na+ + + + − − −

+ −

+ −

+ −

NHE

+ −

+ + + − − −

H+ 2K+

ADP+Pi ATP

α β

Na+ 3Na+

Na+/K+-ATPase

Mammals FIG. 73-1. The major driving forces for Na+-H+ exchangers (NHEs) in a prokaryotic (Escherichia coli) cell and in a mammalian cell. ADP, adenosine diphosphate; ATP, adenosine triphosphate. (Modified from Orlowski and Grinstein [14], by permission.)

12% (hNHE1 vs hNHE9) to more than 70% identity (hNHE6 vs hNHE7) (Table 73-2). Eight of the nine NHE isoforms cloned to date have been detected in the GI tract (Fig. 73-2) with segmental differences, crypt-villous gradients of expression, and different cellular localizations, all of which determine their proven or alleged functions. An additional isoform of chloride-dependent NHE has been cloned from the rat colon (17). This new purported isoform shares 100% homology of the 375 N-terminal amino acids with NHE1, and a divergent 63-amino-acid sequence in the C-terminal portion of the protein, which is suggestive of an alternative splice variant of the NHE1 gene. Surprisingly, although the rNHE1 gene is located on chromosome 5q36, the complementary DNA (cDNA) coding for the novel C-terminal 63 amino acids aligns fully with chromosome 17p14. Unless this cDNA represents a product of transsplicing of pre-mRNA (a phenomenon with little empirical evidence in mammalian cells), this novel sequence may represent a cloning artifact and will require independent verification.

NM_177353a AF482993 (213)

NM_004594 (206)

NM_006359 (209)

NM_032591 (51)

NM_015266a

NM_173653 (215)

Slc9A5

Slc9A6

Slc9A7

Slc9A8

Slc9A9

XM_230865a

XM_228416a

NM_138858 (295) XM_538181a

XM_545197a

AF260664a

AF124218a

AF123280a

NM_001007103 (292) AF184171a

AH010079a XM_531775a

Pig

Dog

aPartial

Numbers in parentheses next to the accession numbers refer to cloning articles in the References. and/or unpublished works or sequences predicted by computational analysis. NHE, Na+-H+ exchanger.

NM_172780a

NM_177084

CR627411a

Slc9A4 NM_173098 (132)

NM_012654 (132)

AF139194a

NM_004174 (133)

Slc9A3

NM_012653 (93)

XM_129721a

AF073299 (97)

Slc9A2

Rat NM_012652 (132)

NM_003047 (6)

Slc9A1

Mouse NM_016981

Human

NHE

M87007 (131)

L13733 (95)

Rabbit

AJ131764a

NM_174833a

Cow

TABLE 73-1. GenBank and RefSeq accession numbers for cloned members of mammalian SLC9 gene family

X68970 (293)

Hamster

CLU75970a

AF012894a

Prairie dog

100% 33.40% 56.50% 60.80% 30.90% 16.40% 16.70% 18.10% 16.40% hNHE-2 SLC9A2 NM_003048 2q11.2

100% 25.70% 15.80% 20.80% 21.80% 16.20% 12.40% 12.80% 22.90% 11.80% hNHE-1 SLC9A1 NM_003047

1p36.1-p35

5p15.3

100% 28.90% 30.10% 51.30% 12.30% 12.90% 13.00% 12.90% hNHE-3 SLC9A3 NM_004174 2q11-2q12

100% 83.50% 27.30% 13.80% 13.10% 17.20% 13.40% hNHE4 SLC9A4 CR627411 9q21

100% 29.00% 14.80% 13.60% 17.30% 13.90% rNHE-4 SLC9A5 NM_173098 16q22.1

100% 11.80% 11.00% 12.80% 13.60% hNHE-5 SLC9A6 NM_004594 Xq26.3

100% 71.10% 22.20% 61.20% hNHE-6 SLC9A7 NM_006359

Xp11.3p11.23

100% 21.20% 58.40% hNHE-7 SLC9A8 NM_032591

20q13.13

100% 23.80% hNHE-8 SLC9A9 NM_015266

3q24

NM_173653

100% hNHE9

Because human NHE4 has not been formally cloned, the sequence assumed to be hNHE4 based on computational analysis (Accession number CR627411) is presented next to its rat orthologue for illustrative purposes. hNHE, human Na+-H+ exchanger; rNHE, rat Na+-H+ exchanger.

hNHE-1 hNHE-2 hNHE-3 hNHE4 rNHE-4 hNHE-5 hNHE-6 hNHE-7 hNHE-8 hNHE9 Gene Symbol RefSeq/ GenBank Gene locus

TABLE 73-2. Protein homology and chromosomal location of the nine members of the Slc9a family of human Na+/H+ exchangers

1850 / CHAPTER 73

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1851 20%

40%

60%

80%

100% NHE

Cellular localization

Salivary gland

Stomach

Small intestine

hNHE1

PM (BL)

+

+

+

Colon Pancreas +

+

Liver Gallbladder +

Ref.

+

[2 8 9]

+

[93; 95; 103; 219; 241]

hNHE1

PM (AP)

+

+

+

+

+

+

hNHE4

PM (BL)

+

+

+

+

+

+

hNHE3

PM (AP), RE

+

+

+

+

+

+

+

[103; 133; 137; 138]

hNHE5

PM, RE

−

−

−

−

−

−

−

[206; 290]

hNHE6

RE, PM

+

+

[209; 212]

hNHE7

TGN

+

+

[5 1]

hNHE9

RE

+

[48; 215]

hNHE8

PM (AP), G

+

+

+

+

+

+

+

−

+

+

+

+

[132; 200; 202]

+

[49; 50; 213]

FIG. 73-2. Homology tree, tissue-specific expression, and cellular localization of the nine cloned mammalian Na+-H+ exchangers (NHEs) in the gastrointestinal tract. AP, apical membrane; BL, basolateral membrane; G, Golgi; PM, plasma membrane; RE, recycling endosomes; TGN, trans-Golgi network. (Modified from Zachos and colleagues [15], by permission.)

Membrane Topology Although the nine-member family of NHEs has not been studied systematically for secondary structure and membrane topology, modeling algorithms predicting hydrophobic and hydrophilic regions of the proteins suggest the same general arrangement. According to the modeling, approximately 60% of the amino terminus of the protein is amphipathic and contains 10- to 12-membrane-spanning α helices, which are relatively conserved among different isoforms. A much more hydrophilic and less conserved carboxyl terminus faces the cytoplasm (Fig. 73-3). This region of NHE proteins, as determined empirically and through prediction sequence analyses, contains multiple phosphorylation sites and sites responsible for interaction with accessory proteins, all of which are believed to serve regulatory functions. Of the nine NHEs cloned to date, two-dimensional structure of NHE1 and NHE3 has been most extensively studied using a variety of experimental approaches including cysteine substitution and accessibility (18), C-terminal truncation (19,20), identification of glycosylation sites (21,22), proteolytic cleavage (23), and epitope immunolocalization (24–27). Not all of these studies confirm this general model of NHE membrane topology, particularly with regard to the C-terminal region. The notion of an entirely cytoplasmic location of this domain has been contended by Khan (25) and Biemersderfer and colleagues (24) both in NHE1 and NHE3, respectively. Both studies argued that truncations or amino-acid substitution may alter the natural structure and folding of the protein, and they suggested that epitope immunolocalization of native proteins indicates at least some extracellular epitopes located with the C-terminal tail. This observation appears to be supported by more recent observations in Nhx1, a yeast homologue of mammalian NHEs, which has been shown to

be glycosylated on two asparagine residues located within the C terminus (28). However, considering that the C-terminal domain of NHE1 or NHE3 does not appear to have hydrophobic regions of sufficient length to form a membrane-spanning domain (15–20 amino acids), and that functional analyses of regulatory domains within this region suggest multiple interactions with cytoplasmic and cytoskeletal factors, it remains unclear whether the controversy over the topology of NHE C terminus is a result of methodologic differences or whether it reflects actual variations in membrane orientation. Because of the highly variable amino-acid sequence among various NHE isoforms, the N-terminal has been believed to contain a cleavable signal peptide. In NHE1, for example, the cleavage would occur before the experimentally confirmed glycosylation site in the first extracellular loop (see Fig. 73-3). If this hypothetical scenario were true, the predicted topology of NHE1 and NHE3 would change to an 11-membrane-spanning domain configuration. Experimental evidence from studies on NHE1 protein using cysteine substitution suggests, however, that the N-terminus is retained in the mature protein and remains in the cytosol (18). In contrast, experiments with in vitro translated NHE3 point to cleavage of the N-terminal signal peptide during processing in microsomes (29). Based on the results of these studies, membrane topology predictions should not be generalized to all members of NHE family, and secondary structures of each of the remaining NHE proteins need to be addressed experimentally. NHE1 and NHE3 have been demonstrated to form homodimers in the cytoplasmic membrane (30). The formation of dimers appears to depend on protein–protein interaction within the amphipathic membrane-spanning region of the proteins, but individual subunits were found to function independently within the complex. Kinetic analyses of renal brush-border membrane Na+-H+ exchange also suggested

1852 / CHAPTER 73 NHE1 Extracellular

EL 1

EL5

Cleavage? EL3

EL2

EL4

Na+

EL6 + + + + + + + + +

1

2

3

4

5

6

7

8

9

10 11 12 − − − − − − − − −− − − −

IL1

NH2−

IL2

IL3

H+

IL4

IL5

Cytoplasmic

−COOH

NHE3 Extracellular

EL 1

EL5

Cleavage? EL3

EL2

EL6

Na+

EL4 + + + + + + + + +

1

2

3

4

5

6

7

8

9

10 11

12 − − − − − − − − −− − − −

IL1 NH2−

IL2

IL3

H+ IL4

Cytoplasmic

IL5 −COOH

FIG. 73-3. Current models of Na+-H+ exchanger 1 isoform (NHE1) and NHE3 membrane topology. EL, extracellular loop; IL, intracellular loop.

the presence of dimers and tetramers dependent on the outside Na+ concentration (31).

Functional Domains Based on the secondary structure of NHE proteins and their membrane topology, two major domains can be distinguished: an N-terminal amphipathic region including all 10- to 12-membrane–spanning domains, and a C-terminal hydrophilic tail. The latter segment is believed to be intracellular and confers regulation of NHE activity by direct or indirect interactions with kinase, cytoskeletal, and other proteins. Within the amphipathic region, transmembrane domain 4 (TM4) is crucial for NHE1 function, with the residues Phe161, Phe162, Leu163, and Gly173 affecting affinity for Na+ or its resistance to inhibitors, or both (32–34). Slepkov and colleagues showed that within this transmembrane helix, Pro167 and Pro168 are critical for NHE1 activity, expression, and membrane targeting (35). Within TM7, Glu262

and Asp267 are indispensable for NHE1 activity, with their charge and acidity being the most critical (36). TM9 contains a sequence conferring sensitivity to antagonists. A hybrid NHE1, in which this transmembrane helix has been replaced with an analogous segment of the amiloride-resistant NHE3, was resistant to amiloride, ethylisopropylamiloride (EIPA), HOE-694, and cimetidine (37). Within this TM, His349 may be one of the critical moieties bestowing sensitivity to amiloride compounds, as described by Wang and colleagues (38). Mutation of amino acids Tyr454 and Arg458 within the TM11 has shown that both amino acids are essential in targeting NHE1 to the cell surface (39). TM11 and its neighboring intracellular loop 5 also have been implicated in pH sensing. Mutation of Gly455 or Gly456, although it did not affect the affinity of the protein for Na+ or H+ ions, resulted in an alkaline shift in NHE1 pH dependence. In contrast, mutation of Arg440 in intracellular loop 5 had the opposite effect. Based on these data, it has been postulated that both Arg440 in intracellular loop 5 and glycine residues in the conserved segment of TM11 constitute the putative pHi

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1853 sensor in NHE1. Other NHE isoforms have not been studied in such detail, although general location and functional consequences of the described functional domains are likely to be consistent among the Slc9a family. The long, hydrophilic, cytosolic domain of NHE1 (amino acids 500–815), which regulates the activity of the amphipathic N-terminal domain, is a target for phosphorylation by protein kinases and participates in binding of regulatory proteins. Several protein kinases are thought to phosphorylate NHE1, including extracellular signal–regulated kinases 1 and 2(ERK1/2), p90rsk, p160ROCK, p38, and an Nckinteracting kinase. By changing NHE1 pH dependence, phosphorylation increases its activity in a more alkaline pH. A number of regulatory proteins have been demonstrated to bind to the cytosolic tail of NHE1, including calmodulin, calcineurin homologous protein (CHP), tescalcin, and carbonic anhydrase II. (See published articles by Putney and colleagues [40], Slepkov and Fliegel [41], Baumgartner and colleagues [42], and the references therein for more detailed descriptions of modes of NHE1 activity regulation.) The role of the cytoplasmic C-terminal domain of NHE3 is described later in this chapter in the section titled Posttranscriptional Regulation p. 1860.

Transport Characteristics and Pharmacology Substrate Specificity The steady-state velocities of most NHE isoforms show a saturating, first-order dependence on the outside Na+o concentration (with KNa values 3–50 mM), consistent with simple, saturating, Michaelis–Menten kinetics, which is indicative of a single binding site (43–45). The kinetics of NHE4 isoform appears to be more complicated. Under unstimulated isotonic conditions, NHE4 appears inactive; however, under hypertonic stress, the kinetics of NHE4mediated Na uptake follow a sigmoidal rather than hyperbolic curve with increasing concentrations of extracellular Na+, a phenomenon characteristic of allosteric regulation (46). Most plasmalemmal NHE isoforms are specifically transporting Na+ in exchange for H+, with much lower efficiency toward Li+ or NH4+ and essentially no affinity for K+. NHE4 is an exception to this rule, because it shows similar exchange rates for Na+ and Li+, and an even greater rate for K+ (47), suggesting that this isoform functions as a nonspecific cationH+ exchanger. Similarly, NHE8, which is described as both an intracellular (48) and a plasmalemmal isoform (49,50), is capable of K+-H+ exchange when reconstituted in artificial liposomes (48). NHE7, an exclusively organellar isoform localized in the trans-Golgi, network, also functions primarily as a K+-H+ exchanger (51), a feature likely shared by other organellar NHE isoforms. Contrary to a typical first-order dependence of exchange kinetics for Na+, hydrogen concentration dependence does not follow a simple Michaelis–Menten equation that assumes no cooperativity, but rather it displays characteristics typical

of allosteric effect, with more than one binding site for H+. The kinetic analysis of Na+-H+ exchange appears to be in agreement with structural data suggesting presence of an H+i sensor in the TM11 and an element regulating pH set point located in the intracellular loop 5 (see the Functional Domains section earlier in this chapter). The allosteric regulation of Na+-H+ exchange by intracellular protons may not be a universal feature of all NHEs. For example, NHE5 displays a simple first-order dependence on the H+i concentration, when expressed in fibroblasts, suggesting no cooperativity. Adenosine Triphosphate Dependency All well-characterized mammalian NHEs have been classified as electrochemical potential-driven transporters, and they have been categorized into the monovalent cation proton antiporter family (CPA1; 2.A.36), according to classification developed by Busch and Saier (52) and endorsed by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). This classification is based on the fact that cation fluxes via the NHE mechanism are driven exclusively by the transmembrane gradients of substrates and are only secondarily dependent on ATP. Although NHE proteins do not bind or consume ATP directly, cellular ATP depletion results in marked inhibition of NHE1, NHE2, and NHE3 activities, despite a maintained transmembrane H+ gradient (53). ATP depletion affects NHE1 and NHE2 by reducing their sensitivity to intracellular pH (H+i), whereas NHE3 is characterized by impaired H+i sensing and reduced maximal velocity of transport (Vmax). Although the precise mechanism of these phenomena is unclear, a role for plasmalemmal phosphatidylinositol 4,5bisphosphate (PIP2) has been postulated. Because binding of PIP2 to the C terminus of NHE1 is critical in maintaining NHE1 activity and ATP depletion results in a decline in plasma membrane PIP2 content, this could represent a potential mechanism for ATP dependence of NHE1. It remains to be determined whether ATP depletion impairs NHE2 and NHE3 via similar mechanisms. Chloride Dependency Chloride-dependent Na+-H+ exchange has been functionally identified in the epithelium of the rat distal colon (17, 54–56). This phenomenon was postulated to be the result of functional coupling of the chloride channel to a novel NHE isoform, later cloned as a putative, but perhaps artifactual (see earlier discussion) Cl−-dependent NHE (17). Two other studies in the mouse, however, demonstrated no Cl−-dependent NHE in the colonic crypts (57,58). One explanation for this discrepancy is a possibility that not all Cl− replacements are equally inert, but this is not likely because two different anion substitutions with different permeabilities (nitrate or gluconate) produced the same results. Other explanations include the possibilities that the volume changes caused by Cl− depletion affect different cell types differently, or that coupled Cl−-anion exchange may be necessary for dissipation

1854 / CHAPTER 73 of the intracellular gradient in some cells. These discrepancies may also be because of species differences (rat vs mouse), site of analysis (crypt base or midcrypt region), or simply the use of different methodologic approaches. It also has been demonstrated that NHE1-3 isoforms can be Cl dependent to a certain extent (59). This phenomenon is incompletely understood, but may involve impaired H+i sensing with depletion of intracellular Cl−. Overall, the described inconsistencies in descriptions of Na+-H+ exchange Cl− dependence suggest that it may not be a ubiquitous function of colonic crypt epithelia. Inhibitors Over the years, a number of Na+-H+ exchange inhibitors have been developed, primarily as an effort to inhibit NHE1 in cardiac ischemia-reperfusion injury. The pharmacology of NHE inhibitors was reviewed by Masereel and colleagues (60). Amiloride, a K+-sparing diuretic, was the first described NHE inhibitor (61), which could also inhibit an electrogenic Na+ channels and the Na+-Ca2+ exchanger. NHE1 and NHE2 isoforms are the most sensitive to amiloride

inhibition, whereas NHE3 and especially NHE4 are amiloride-resistant isoforms (Table 73-3). The sensitivity of NHE8 and NHE9, two of the most recently cloned NHE isoforms, to currently known inhibitors has not been evaluated, and only limited information on sensitivity of NHE4 and NHE7 is available. Development of several pyrazine or phenyl derivatives of amiloride increased their potency toward NHEs, particularly NHE1, and more importantly increased their selectivity by eliminating the inhibitory potency toward the Na+ channel and Na+-Ca2+ exchangers. Of these molecules, dimethylamiloride (DMA), EIPA, HOE-694, and HOE-642 are the most frequently used in experimental settings. More recently, several NHE inhibitors based on a bicyclic template have been introduced, such as zoniporide, SM-20550, BMS-284640, T-162559, or TY-12533. Other compounds not related to amiloride also have proved useful, especially S-3226, as the first NHE3-specific inhibitor (62). Cimetidine, clonidine, and harmaline, although not frequently used, also have been reported to act as weak and nonspecific inhibitors of Na+-H+ exchange (63). A majority of the developed Na+-H+ exchange inhibitors and their affinities to different NHE isoforms are depicted in Table 73-3.

TABLE 73-3. Inhibitory potency of Na+-H+ exchanger inhibitors toward the different isoforms Inhibitory potency (Ki or IC50 [µM]) Inhibitor

NHE1

Amiloride and derivatives Amiloride 1–1.6; 5.3

Pyrazine EIPA DMA HMA Phenyl HOE-694 HOE-642 (cariporide) EMD-85131 (eniporide)

0.01–0.02; 25.1 0.023 0.013

NHE3

NHE4

NHE5

1.0

>100

813

21

0.08–0.5 0.25

2.4; 3.3 14 2.4

>10

0.42

0.085

640

9.1

4.3–62

1 to >100

>30

0.005–0.38

2–17

100–460

>30

1800 12

>30 >500

3.36

0.43 0.31

11 >30

2.3 80 330 42 330

0.02 6200; >1000 620 1000

0.003 3.6 26; 51 210 140

NHE6

NHE7

NHE8

NHE9

>2000

0.37

0.03–3.4

Bicyclic inhibitors BMS-284640 0.009 Zoniporide 0.059 TY-12533 0.017 SM 20550 0.01 T-162559(S) 0.001 T-162559(R) 35 Other inhibitors SL-591227 S-3226 Cimetidine Clonidine Harmaline

NHE2

230; >1000 Not active 940

DMA, dimethylamiloride; EIPA, ethylisopropylamiloride; NHE, Na+-H+ exchanger. Modified from Masereel B, Pochet L, Laeckmann D. An overview of inhibitors of Na(+)/H(+) exchanger. Eur J Med Chem 2003;38:547–554, by permission.

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1855 GASTROINTESTINAL NA+-H+ EXCHANGERS NHE1 NHE1, the first cloned mammalian NHE (6,7), is the most extensively studied NHE isoform, although the preponderance of information on NHE1 expression, activity, and regulation comes from systems other than the GI tract, and as such may or may not be applicable to GI physiology and pathophysiology. The large body of knowledge about this isoform precludes us from including it in this chapter; however, several detailed reviews of the biology of Na+-H+ exchange as mediated by NHE1 have been published, including reviews by Fliegel (64), Slepkov and Fliegel (41) and Putney and colleagues (40), and a more general review of mammalian NHEs was written by Orlowski and Grinstein (14). Only a rudimentary overview of this isoform is presented here, with particular emphasis on its role in the physiology of the digestive tract. Mammalian NHE1 is an 813- to 822-amino-acid protein with a calculated molecular mass of ~91 kDa. NHE1 contains consensus sequences for both N- and O-linked glycosylation, and there is evidence that Asn-75 in the first extracellular loop of NHE1 is glycosylated, explaining the appearance of the mature 110-kDa form of NHE1 in Western blotting (21). Its membrane topology has been studied extensively and is represented schematically in Figure 73-3.

this chapter. NHE1 also appears to regulate cell differentiation, because deletion or inhibition of NHE1 has been shown to impair differentiation pathways (70). A role for NHE1 in apoptosis regulation also has been postulated, because high NHE1 activity confers resistance to proapoptotic stimuli (40). In addition, NHE1 function is important in cytoskeletal organization and cell migration. The cytoplasmic tail of NHE1 acts as an anchor for actin filaments via binding of ezrin, radixin, and moesin (ERM) proteins, and disruption of these interactions or inhibition of NHE1 activity results in inhibition of cell migration and formation of focal adhesions (40). NHE1 knockout mice are viable, although they have stunted growth and reduced survival rates (71,72). They also exhibit severe neurologic defects (slow-wave epilepsy, ataxia, and neuronal degradation) and present with abnormalities in gastric histology (see the Na+-H+ Exchange and Gastric Physiology section later in this chapter) (71). It is unclear whether involvement of NHE1 in these mechanisms is equally critical in the cells of the GI tract. No intestinal defect was demonstrated in otherwise rapidly renewing intestinal epithelium in NHE1−/− mice, suggesting that the role of NHE1 in the intestinal crypt cell proliferation is minor. Also, the described role of NHE1 in cellular differentiation may not represent a ubiquitous mechanism because its expression along the crypt-villus axis did not correlate with the differentiation status of enterocytes (65). Transcriptional Regulation

Tissue Distribution and Cellular Localization NHE1 is expressed ubiquitously in almost all mammalian cell types where it resides exclusively on the plasma membrane. Depending on the cell type, NHE1 tends to accumulate in distinct membrane domains. In polarized epithelial cells, NHE1 is expressed on the basolateral membrane (65); in cardiac myocytes, it is concentrated around the intercalated disks and t-tubules (66); whereas in fibroblasts, it is found along the border of lamellipodia (67). In the rat smallintestinal epithelium, no detectable difference in segmental expression of NHE1 mRNA has been described, with only a minor decrease in expression along the crypt-villous axis in the jejunum (65). Similarly, no longitudinal differences in NHE1 expression have been detected in the human intestine (68). This relatively uniform expression of NHE1 is consistent with its perceived role as a “housekeeping isoform” participating in the regulation of pHi and volume. Physiologic Role NHE1 primarily serves to regulate pHi, and its activation is associated with a number of downstream cellular events. The transient increase in pHi induced by NHE1 participates in cell proliferation and promotes transit through the G2-M checkpoint of the cell cycle (69). This finding may be related to the role NHE1 plays in proliferative responses of hepatocytes and hepatic stellate cells (HSCs), as described later in

Regulated expression of NHE1 mRNA has been described in various systems, but especially in myocardium (41). Human, mouse, rabbit, and pig NHE1 gene promoter have been cloned and characterized to a various extent (73–76), with mouse NHE1 promoter analyzed in more detail than other species. The activity of this promoter is largely dependent on activator protein-2 (AP-2)–like transcription factors (74), as well as a poly (dA:dT) region of the promoter interacting with yet unidentified nuclear protein (77). Serum and growth factors have been shown to stimulate promoter activity in cardiomyocytes and fibroblasts through more distal elements of the promoter (0.8–1.1 kb) interacting with chicken ovalbumin upstream promoter (COUP) transcription factors (78,79); however, these in vitro findings do not correlate with data obtained from transgenic mice bearing NHE1 gene promoter reporter construct. In the latter studies, crossing these mice with AP-2α or COUP-TFI knockout mice did not change the reporter gene expression in embryonic mouse tissue (80). Regulation of NHE1 gene expression in GI tissues has not been studied extensively, although a limited amount of available data suggests that, consistent with its housekeeping role, NHE1 is not regulated at the mRNA level in situations where other NHE isoforms are regulated. Examples of such circumstances are metabolic acidosis (81), microvillous inclusion disease (82), small-bowel resection (83), glucocorticoid administration (84), or postnatal development (85).

1856 / CHAPTER 73 Posttranscriptional Regulation The cytoplasmic C-terminal regulatory domain is associated with a number of functionally distinct signaling molecules, including PIP2, CHP1, and actin-binding proteins of the ERM family. In the distal C-terminal region, NHE1 contains a number of serine residues phosphorylated by p90RSK and Ste20-like, Nck-interacting kinase on activation of growth factor receptors, and Rho kinase 1 on activation of integrin receptors and G protein–coupled receptors for thrombin and lysophosphatidic acid (LPA). Phosphorylation of C-terminal serine results in increased NHE1 activity, whereas phosphorylation of Ser703 by p90RSK promotes direct binding of the multifunctional adaptor protein 14-3-3, which conceivably serves as a focal point for the assembly of other signaling molecules. Additional proteins such as calmodulin, heat shock protein 70 (HSP70), and carbonic anhydrase II also have been shown to bind to this regulatory domain of NHE1. The latter interaction is particularly intriguing, because it may explain the ultimate changes in NHE1 activity observed on phosphorylation. It has been postulated that serum-induced phosphorylation within the last 178 amino acids of the C terminus facilitates binding of carbonic anhydrase II, which through catalysis of CO2 hydration causes local acidification and an increase in NHE1 activity (86). Baumgarter and colleagues (42) have provided an exhaustive overview of the signaling molecule scaffolding at the C terminus (see this article and the references therein for more details). It is unknown whether all the described mechanisms of posttranslational modifications of NHE1 activity are ubiquitous to all cell types, and whether they have functional consequences in the digestive tissues. Na+-H+ activity at the basolateral membrane of enterocytes increases with age (85,87), despite unchanged expression of NHE1 mRNA. This may represent age-dependent changes in NHE1 activity mediated by one or more of the mechanisms described earlier, especially because another potential basolateral isoform, NHE4, has not been detected in the small-intestinal epithelium (85,88). Pathophysiology Two studies implicated NHE1 in the pathophysiology of inflammatory bowel disease (IBD). In a rat model of acetic acid or trinitrobenzenesulfonic acid–induced colitis, Khan and colleagues (89) described an induction of NHE1 mRNA in the colonic mucosa. Also in vitro, in Caco-2 and HT-29 human intestinal epithelial cells, inhibition of Na+-H+ exchange with amiloride and other unrelated NHE inhibitors has been shown to reduce interleukin (IL)-1β–, tumor necrosis factor-α–, and lipopolysaccharide-stimulated IL-8 production; IL-1β–induced nuclear factor-κB (NF-κB) activation; and phosphorylation of ERK (90). In the latter study, amiloride administered in vivo to dextran sodium sulfate (DSS)–treated rats resulted in attenuated symptoms of colitis and decreased neutrophilic infiltration in the colonic mucosa. The interpretation of these results is complicated by that IC50 for inhibition of IL-8 production by amiloride was ~30-fold greater than

the IC50 required to inhibit NHE1 and NHE2, the two isoforms likely to be expressed in the selected cells used under culture conditions. Plasma concentration of amiloride in DSS-treated rats was not evaluated. It is possible, therefore, that the observed effects may represent nonspecific effects of the selected inhibitors. Moreover, analysis of NHE1 gene expression in human IBD contrasts with the data obtained from rodent models of colitis. Khan and colleagues (91) demonstrated decreased mRNA expression in colonic biopsies from patients with Crohn’s disease and ulcerative colitis compared with healthy colon samples (91). Therefore, the involvement of NHE1 in the pathogenesis of IBD is still unclear. In a rat model of necrotizing enterocolitis (NEC), the observed decrease in expression and activity of NHE1 was ascribed to cellular acidification, which was postulated to participate in the failure of the epithelial barrier, and consequently in the pathogenesis of NEC (92). The plausibility of NHE1 involvement in liver cirrhosis through activation of stellate cells, as well as in hepatic tumorigenicity, is discussed in the Na+-H+ Exchange and Liver Physiology section later in this chapter.

NHE2 NHE2 was first cloned from rat and rabbit intestinal cDNA libraries by Collins and colleagues, Wang and coworkers (93,94), and Tse and colleagues (95), respectively. Human NHE2 was cloned by Ghishan and coworkers (96), and later corrected by Malakooti and colleagues (97). Among the members of the human Scl9a family of NHEs, NHE2 protein is most similar with NHE4, especially within the cytoplasmic C terminus (see Table 73-2). Interestingly, in the human, rat, and mouse, the Slc9a2 and Slc9a4 genes cosegregate on chromosomes 2, 9, and 1, respectively (98,99). The adjacent chromosomal location of the two NHEs in all three species strongly suggests that they arose by gene duplication early in the evolution. The predicted molecular weights of NHE2 protein in rat, rabbit, and human are ~91 kDa, although its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels does not confirm these calculations. Mature rabbit NHE2, when expressed in PS120 fibroblasts, was shown to be an O-linked sialoglycoprotein (22). In these studies, neuraminidase shifted the mobility of NHE2 protein from 85 to 81 kDa, and O-glycanase further shifted the mobility of the 81-kDa protein to 75 kDa. Incubation of PS120/NHE2 cells with an O-glycosylation inhibitor benzyl N-acetyl-α-D-galactosaminide reduced the size of the 85-kDa protein to 81 kDa, although this was without consequence for the initial rate of Na+-H+ exchange in these cells (22). Tissue Distribution and Cellular Localization NHE2 is expressed in the epithelia of all digestive organs, with particularly high expression in the proximal colon (100–104). Outside of the GI tract, NHE2 activity or expression, or both, have been described in the kidney (cortical

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1857 thick ascending limb of the nephron, macula densa, distal convoluted tubules, and connecting tubules) (105,106), endometrium and placenta (107,108), chondrocytes (109), inner ear (110,111), heart, testes, and adrenal glands (96). Expression of NHE2 in the individual digestive organs is described later in this chapter (see the Physiologic Roles of Na+-H+ Exchange in the Digestive Tract section). With the exception of gastric epithelium, NHE2 was unambiguously demonstrated on the apical membrane of polarized epithelial cells. Because NHE2 has an exclusive ability to be activated by increased extracellular pH (pHo), it has been speculated that NHE2 may be the NHE isoform that mediates Na+-H+ exchange activation by an increase in interstitial HCO3− concentration during acid secretion in gastric epithelium (100,112), a hypothesis that assumes basolateral localization of this isoform. Immunohistochemical evidence for this assumption currently is lacking. In the intestinal epithelium, expression of NHE2 along the crypt-villous axis shows some species-dependent differences. In rabbits, NHE2 is present in the brush border of the entire villus of the small intestine, in colonic surface cells, and in the apical membrane of the upper half of the crypt (104). In the mouse colon, however, NHE2 is expressed predominantly in the crypt cells (57,58), suggesting a role for this isoform in cryptal pHi and volume homeostasis. Physiologic Role Despite a relatively wide expression of NHE2, its physiologic role remains elusive. NHE2 stably transfected in NHEdeficient Chinese hamster ovary (CHO) cells (AP-1) showed a relatively high affinity for amiloride and its analogues (see Table 73-3), with potencies in decreasing order of EIPA (IC50 = 79 nM) > DMA (IC50 = 250 nM) > amiloride (IC50 = 1.4 µM) > benzamil (IC50 = 320 µM). Nonamiloride compounds also inhibited NHE2 with the following order of potency: clonidine (IC50 = 42 µM) > harmaline and cimetidine (IC50 = 330 µM for both). Kinetic analyses showed that NHE2 Na+o dependence followed simple, saturating Michaelis–Menten kinetics with an apparent affinity constant for Na+ (KNa) of ~50 mM. Intracellular H+ activated NHE2 by a positive cooperative mechanism with an apparent half-maximal activation value of pK 6.90. Li+ and H+ acted as competitive inhibitors of NHE-mediated Na+ influx, whereas extracellular K+ had no effect on NHE2 activity (45). The information provided by analysis of NHE2−/− mice suggests a role in muscarinic stimulation of salivary secretion, as well as in gastric physiology (see later discussions). The involvement of NHE2 in gastric parietal cell homeostasis appears to be particularly significant because NHE2 gene ablation leads to a reduced number of parietal and chief cells, loss of net acid secretion (112), and progressive inflammation in the form of diffuse corporal gastritis (113). Other roles for NHE2 in the physiology of digestive organs, presumed from the expression and/or functional studies, are discussed in more detail later in this chapter (see the Physiologic Roles of Na+-H+ Exchange in the Digestive Tract section). Overall, the results of the available reports suggest that NHE2 plays a

negligible role in net Na+ or fluid absorption in the mouse digestive tract. The disparity between these results and the functional studies demonstrating contribution of NHE2 to various cellular functions (especially intestinal Na+ absorption) remains unresolved; however, unidentified compensatory mechanisms may help explain the significance of this gene in the physiology of intestinal and renal epithelia. Transcriptional Regulation Rat and human NHE2 promoters have been cloned and characterized (114,115). Both proximal promoters lack canonical TATA and CAAT boxes, are highly GC rich, and share about 59% homology with a number of conserved, predicted, regulatory elements. Only rudimentary analysis of the human NHE2 promoter has been performed, with prediction analyses indicating putative binding sites for the following trans-acting factors: Sp1, AP-2, Egr-1, p300, NF-κB, Oct-1, zinc finger protein-1, MyoD, two caudal-related homeobox (Cdx) family members, CdxA and Cdx-2, glucocorticoid receptor (GRE), thyroid hormone receptor, a CACCC site, and several polyoma viral enhancer 3 sites (114). Of all these sites, only Sp1, AP-2, CACCC, NF-κB, and Oct-1 were conserved in human and rat NHE2 promoters. A minimal promoter of the rat NHE2 was identified and found to be regulated by Sp transcription factors, with Sp1 acting as an activator and Sp3 and Sp4 playing inhibitory roles (116). NHE2, similar to NHE1, can be activated by serum (95) and by epidermal growth factor (EGF) (117). There also appears to be a transcriptional component to the mechanism by which NHE2 is activated by EGF. In suckling rats, parenterally administered EGF increased expression of NHE2 mRNA in the small-intestinal epithelium, but not in the kidney (118). This finding was confirmed in EGFtreated rat intestinal epithelial cells, which also showed activation of the rat NHE2 promoter in transient transfection experiments (118). In adult mice, however, neither exogenous EGF nor salivarectomy affected NHE2 mRNA expression in the small intestine (119), suggesting that this regulation may be species or age dependent, or both. Similar bimodal regulation of NHE2 by osmolarity has been described. In PS120 cells, hyperosmolarity inhibited NHE2 activity (120), but other reports showed activation of NHE2 in mouse inner medullary collecting duct (mIMCD-3) cells (121), AP-1 cells (53), and colonic crypt cells (57). In the case of renal mIMCD cells, mRNA expression also was induced by hyperosmotic stress (121,122). A TonE-like element and a novel cis-element, termed OsmoE, were identified in the rat NHE2 promoter as being responsible for the increased transcription of the NHE2 gene induced by hyperosmolarity, with both elements acting in concert to provide maximal transcriptional induction (122). The transcription factors interacting with these elements have not been identified, and currently, it is also unknown whether the same mechanism is present in the colonic crypts. During postnatal development, expression and activity of NHE2 in the rat small-intestinal epithelium dramatically

1858 / CHAPTER 73 increase around the time of weaning (123). This increase is caused by transcriptional activation of the NHE2 gene, as shown by nuclear run-on assay (123) and by reporter gene analysis in transgenic mice bearing −2.4 kb of the rat NHE2 promoter (P. R. Kiela and colleagues, unpublished observations). Interestingly, NHE2 expression in the rat kidney follows a reciprocal pattern, with highest expression in the suckling period and a decline toward adulthood (124), implying tissue-specific mechanisms regulating postnatal changes in NHE2 expression. Posttranscriptional Regulation NHE2 protein has a relatively short half-life (~3 hours) compared with other NHE isoforms (e.g., 24 hours for NHE1 and 14 hours for NHE3) and is subject to lysosomal degradation, as determined in PS120 fibroblasts and Caco-2 cells (125). This suggests that changes at the level of gene transcription or translation may be more critical for NHE2 regulation than for other isoforms with long half-lives. NHE2 is a residual plasma membrane protein, and unlike NHE3, it does not undergo endosomal recycling (125). Glycosylation of NHE2 may affect its cellular localization, because unglycosylated 75-kDa rabbit NHE2 was found predominantly intracellularly (126), although it is unclear whether this represents a regulatory mechanism or is simply related to the maturational stage of NHE2 protein synthesis. Of the two well-characterized apically expressed NHE isoforms, NHE2 and NHE3, NHE2 activity is considered relatively stable and is not regulated by many factors. Extracellular alkalinization activates NHE2, which is believed to propel increased proton extrusion in gastric parietal cells during secretagoguestimulated acid secretion (see the Na+-H+ Exchange and Gastric Physiology section later in this chapter). The maximal rate of exchange (Vmax) mediated by NHE2 was shown to be stimulated by serum, fibroblast growth factor (FGF), and protein kinase C (PKC) activator phorbol myristyl acetate in PS120 fibroblasts (127). Intracellular ATP depletion reduced the NHE2 activity by a dramatic decrease in H+ affinity, as well as Vmax, with virtual elimination of the allosteric effect of H+ (127). ATP depletion also eliminated the stimulatory effect of serum, suggesting that growth factor–stimulated NHE2 activity is mediated via its pH-sensing mechanism. Thrombin increased NHE2 Vmax without altering the Hill coefficient (127), although it is unclear whether this could be attributed to increased intracellular Ca2+ ascribed to thrombintreated fibroblasts. In the same study, thrombin also increased NHE3 activity, whereas it was shown later that increase of intracellular Ca2+ by thapsigargin in Caco-2/bbe cells inhibited NHE3 (128). Pathophysiology NHE2 has a particularly well-documented role in the gastric epithelium, although alterations in NHE2 expression or activity in gastric disorders have not been documented. Down-regulation of NHE2 activity and gene expression has

been documented in rats and Caco-2/bbe cells treated with IFN-γ (interferon-γ), implicating a role for NHE2 in inflammation-associated diarrhea (129). The lack of absorptive defect in the intestine of NHE2−/− mice, however, suggests that cytokine-mediated changes in NHE2 function may not be critical for electrolyte absorption in the inflamed intestinal mucosa. Surprisingly, enteropathogenic E. coli invasion of intestinal epithelial cells increased NHE2 activity by ~300%, whereas it inhibited activities of NHE3 and Cl−-OH− exchange (130). The authors speculated that NHE2 activity might represent a potential compensatory response to increased luminal fluid resulting from inhibition of NHE3 activity, disruption of tight junctions, inflammatory response, or alterations in anion exchanger activity.

NHE3 Rabbit and rat NHE3 were first cloned by Tse and colleagues (131) and Orlowski and coworkers (132), respectively. The second report also included partial cloning of a human ortholog, later fully cloned by Brant and colleagues (133), and mapped to chromosome 5p15.3 (134). The open reading frame of NHE3 mRNA codes for an 831- to 834amino-acid protein with a calculated molecular weight of ~93 kDa, with highest homology to NHE5 (51.3%) and NHE2 (33.4%; see Table 73-2). Based on the presence of potential N-glycosylation sites in the NHE3 protein of all three species, it was initially believed to be a glycoprotein. It appears, however, that glycosylation of NHE3 may be species specific. Rabbit and pig renal NHE3 was shown to be glycosylated and sensitive to glycopeptidase F and the general N-linked glycosylation inhibitor tunicamycin (135,136), whereas glycosylation of rat or canine NHE3 was not detected (21,136). The functional consequences of glycosylation are unclear. In vivo inhibition of N-glycosylation in tunicamycin-treated LLC-PK cells significantly decreased NHE3 activity, as measured by pH-dependent 22Na uptake and by Na-dependent pHi recovery from an acid load (135). This decrease in NHE3 function in tunicamycin-treated cells was accompanied by an intracellular accumulation of seemingly unglycosylated forms of the protein and a conceivably compensatory threefold increase in NHE3 mRNA. Based on these studies, it has been postulated that glycosylation of porcine NHE3 plays a role in membrane trafficking, and ultimately in NHE3 activity. In contrast, deglycosylation of rabbit renal brush-border protein did not impact acid-stimulated, amiloride-sensitive 22Na influx into the vesicles (136). Analogous experiments in vivo with NHE1 with mutated N-glycosylation sites, and with in vivo inhibition of O-glycosylation in NHE2, did not translate into detectable functional changes of the respective isoform (21,22). Therefore, the physiologic significance of NHE3 glycosylation remains unclear. The secondary structure of NHE3 follows the general model for all members of the Slc9a family and is discussed in more detail earlier in this chapter (see the Membrane Topology section).

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1859 Tissue Distribution and Cellular Localization The range of NHE3 gene expression in various tissues has been found to differ among species. Rabbit and rat NHE3 is consistently expressed at high levels in the absorptive epithelia of kidney cortex, colon, and small intestine, with lower levels detected in the stomach, brain, and heart (131,132), whereas human NHE3 also is expressed in relatively high levels in testes, ovaries, prostate, and thymus (133). Rat NHE3 is expressed in both acinar and ductal cells of the salivary glands (101), although its role in salivary secretions appears to be negligible (see the Na+-H+ Exchange and Salivary Gland Physiology section later in this chapter). Expression of NHE3 in cholangiocytes and gallbladder epithelium implies a role for this isoform in bile formation, and possibly in the pathogenesis of gallstones (137–139). Expression, cellular localization, and functional relevance of NHE3 in the gastric epithelium are somewhat controversial and are described in more detail later in this chapter (see the Na+-H+ Exchange and Gastric Physiology section). Expression of NHE3 appears to be higher in the ileum than in other intestinal segments in both rabbits and humans (68,104). In humans, NHE3 mRNA levels are greater in the jejunum and the colon, where NHE3 is present at approximately the same levels in both the ascending and the descending segments (68). In the small-intestinal and colonic epithelium, NHE3 may be considered a marker for the absorptive epithelial cells, because it is expressed only in the villous or surface epithelium, and not in the crypts (65,104). Atypical expression of NHE3 has been demonstrated in the colonic crypts of NHE2−/− mice, where it is believed to play a compensatory role in regulation of crypt cell volume and pHi (57). In polarized intestinal epithelial cells, the majority of NHE3 protein is localized to the apical membrane, where it can be found both on the microvilli and in the intervillous clefts (104). In Caco-2 cells, about 20% of total NHE3 protein is localized to a diffuse subapical pool, and recycling between plasma membrane and this endosomal compartment represents a mode of regulation of NHE3 by endocytosis/ exocytosis (140,141). Similar observations have been made in the renal proximal tubule epithelium (142). When expressed in AP-1 fibroblasts, ~90% of NHE3 protein was found in the juxtanuclear endomembrane vesicles, a pool further increased by inhibition of phosphatidylinositol 3-kinase (PI3K) (26). It has been suggested that the constitutive activity of PI3K is important in the maintenance of the steady-state level of NHE3 on the plasma membrane. The extent to which endosomal recycling participates in NHE3 regulation in native cells, although largely unknown, is discussed in more detail later in this section (see the Posttranscriptional Regulation subsection). In the brush-border membrane of rabbit ileal enterocytes, NHE3 is equally split between detergent-soluble and -insoluble fractions, and a part of the latter fraction is present in cholesterol-enriched lipid microdomains (lipid rafts) (143). Li and colleagues (143) demonstrated that the lipid raft

pool and its association with actin cytoskeleton play an important role in regulation of NHE3 activity through endocytosis. Physiologic Role Kinetics and regulation of NHE3 expressed in fibroblasts and intestinal epithelial cells were described by Levine and colleagues (127) and McSwine and colleagues (144). According to these two reports, NHE3 mediated Na+-H+ exchange, with evident cooperativity by intracellular H+ and with simple Michaelis–Menten kinetics for extracellular Na+ (Km ~17 mM), and was inhibited by phorbol esters and ATP depletion. NHE3 is relatively insensitive to amiloride and its analogues, with the highest affinity to the newly developed S3226 inhibitor (IC50 = 0.02 µM; see Table 73-3). In addition to its well-defined role in the absorptive epithelium of renal proximal tubules (see reviews by Burckhardt and coworkers [145] and Moe [146]), NHE3 plays a prominent role in epithelial sodium absorption in the small and large intestines, as evidenced by studies in mice with targeted disruption of the Slc9a3 gene (147,148). The intestinal absorptive defect observed in these mice is described in more detail later in this chapter (see the Na+-H+ Exchange and Intestinal Water and Sodium Absorption section). Transcriptional Regulation Rat NHE3 gene promoter was cloned at about the same time by Kandasamy and Orlowski (149) and Cano (150). A discrepancy in the transcriptional start site in those two reports was later resolved by Kiela and colleagues (151), who showed that the atypical TATA box located at −26/−31 bp (numbers according to the major transcription start site mapped by Kandasamy and Orlowski [149]) was not necessary and even detrimental for promoter activity, and that a −20/+8-bp fragment represents a functional, albeit atypical, initiator. Within the −81-bp upstream region, 3 Sp transcription factor binding sites were critical because their mutation drastically reduced promoter activity. The roles of Sp1 and Sp3 were further demonstrated by electromobility shift assay and by transactivation of the NHE3 promoter in SL2 cells by forced expression of Sp1 and Sp3. Both of these transcription factors were found to act synergistically with GATA-5 bound to a GATA box in exon 1 (+20/+23 bp). These studies demonstrated that the rat NHE3 promoter is initiator driven and controlled mainly by Sp1 and Sp3, which functionally interact with GATA-5. This interaction may represent a regulatory mechanism participating in a gradient of intestinal gene expression along the crypt-villous axis (151). Cloning of the human NHE3 5′-regulatory region (152) defined a maximal promoter activity in the −95/+5 nt region, a sequence with high homology with the proximal promoter of the rat NHE3, with overlapping and functional regulatory elements for Sp and AP-2 transcription factors (152). The following discussion of examples of long-term regulation of NHE3 involves reports describing changes in NHE3 mRNA expression that

1860 / CHAPTER 73 in only some instances have been confirmed to represent transcriptional regulation. Glucocorticoids The response of NHE3 to glucocorticoid hormones is biphasic, involving nontranscriptional activation of the protein mediated by serum- and glucocorticoid-induced protein kinase (SGK1), which is discussed later in this section, as well as transcriptional activation of NHE3 gene. Changes in NHE3 mRNA with glucocorticoid treatment were shown in the rabbit small intestine and colon by Yun and colleagues (153). In adult rats, dexamethasone increased NHE3 mRNA expression in the ileum and proximal colon, but not in the jejunum or distal colon (84); conversely, adrenalectomy reduced NHE3 expression in the rat ileum and proximal colon, but not in the jejunum. This regulation was not only segment specific, but also age dependent. Glucocorticoid responsiveness in the proximal small intestine was greatest in suckling rats and decreased with age to no detectable change in adults, whereas ileal NHE3 was induced by methylprednisolone only in adults (154). This age- and segment-specific responsiveness to glucocorticoid treatment correlated with the expression and ligand-binding capacity of the GRE in the enterocytes (154). Transcriptional regulation of the NHE3 gene by glucocorticoids was demonstrated by Cano (150) and Kandasamy and Orlowski (149) in cells transiently transfected with rat NHE3 promoter reporter constructs. Short-Chain Fatty Acids SCFAs are potent stimuli of sodium and water absorption in the colon (155–159), with butyrate being the most effective (157). It has been speculated that the SCFA-mediated increase in Na+ absorption is caused by the coupling of two exchange mechanisms, Na+-H+ and SCFA−-Cl− (see the Colonic Na+-H+ Exchange section later in this chapter). The use of amylase-resistant starch as an additive to oral rehydration solution proved effective in reducing diarrheal stool output in patients with cholera (160), thus showing that SCFAs can be potent antidiarrheal agents. Part of the NHE regulation by SCFA appears to be mediated by transcriptional induction of NHE3 gene expression. In rats fed 5% pectin-supplemented diet for 2 days, NHE3 mRNA, protein, and activity increased in the colonic epithelial cells (161). Similar results were obtained with Caco-2/bbe cells treated with SCFAs in vitro (161). Rat NHE3 promoter reporter construct, when transiently transfected into Caco-2 cells, also was significantly induced by SCFAs, especially butyrate (162). The mechanism of this induction, although largely undetermined, involves Ser/Thr kinase activity with a likely permissive role for PKA, because the activation of the promoter by butyrate was abrogated by H-7, Rp-cAMPS, and H-89 inhibitors, as well as by overexpression of a dominant-negative mutant form of the regulatory subunit of PKA (162).

Metabolic Acidosis Chronic metabolic perturbations in systemic acid–base balance can affect Na+ absorptive functions of the gut (163). Metabolic acidosis induced in rats by 5% NH4+Cl− in drinking water induced ileal expression of NHE2 and NHE3 mRNA and protein, as well as their activities (81). Transcriptional regulation of NHE3 was confirmed in opossum kidney cells (OKPs) transfected with the NHE3 promoter construct and subjected to prolonged (24-hour) exposure to acidified media (150). The precise mechanism of this induction has not been described. Intestinal Resection As an adaptive response to enhance the intestinal absorptive capacity, rat small-intestinal Na+-H+ activity was shown to increase, primarily in the segment distal from the resection (164). It was later shown that this increase was associated with ~3-fold increase of NHE3 mRNA and protein expression after a 50% massive proximal small-bowel resection in rats (83). The increase was again observed only in the ileal segment distal from the anastomosis site, suggesting that dietary rather than humoral factors might be responsible. Similar results were obtained in enterectomized mice (119). Intestinal Inflammation Diarrhea observed commonly in IBDs is a direct result of perturbations in colonic absorptive and secretory processes (165). A proinflammatory mediator, IFN-γ, down-regulates NHE3 mRNA and protein expression both in vivo and in vivo (129). In IL-2 knockout mice with a disease resembling human ulcerative colitis (166), a drastic reduction in colonic transepithelial net Na+ flux was paralleled by a reduction in electroneutral NaCl absorption and decreased NHE3 mRNA and protein expression in the proximal colon, and by abrogated aldosterone-stimulated electrogenic Na+ transport with decreased electrogenic sodium channel expression in the distal colon (167). This strongly suggests an involvement of NHE3 in the pathogenesis of diarrhea in ulcerative colitis. In contrast, in a rat model of NEC, ileal expression of NHE3 was not affected (92), implying differential involvement of inflammatory mediators in regulation of NHE3 expression and activity. Posttranscriptional Regulation Most knowledge on acute regulation of NHE3 activity comes from either heterologous cell expression systems or renal epithelial cells, although the described mechanisms are likely ubiquitous and will, perhaps with certain exceptions, apply to the epithelial cells of the digestive tract. These mechanisms have been reviewed in more detail by Zachos and colleagues (15), Moe (146), Weinman and colleagues (168), and Yun (169).

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1861 Phosphorylation The C-terminal domain of NHE3 contains numerous putative phosphorylation sites for various kinases (132). Deletion of this domain renders NHE3 activity constitutive, with partially preserved transport activity. Domain swapping experiments have shown that regulatory characteristics of one NHE isoform can be transferred to another by the cytoplasmic domain of the first. For example, replacement of the C-terminal cytoplasmic tail of NHE1, an isoform that is largely cyclic adenosine monophosphate (cAMP) insensitive, with an analogous domain of NHE3, transfers cAMPmediated inhibition to the hybrid molecule. The experiments strongly suggested the existence of functionally relevant phosphorylation sites located within the cytoplasmic Cterminal tail of NHE3. In response to increased intracellular cAMP, protein kinase A (PKA) phosphorylates NHE3 on multiple sites in the intact cell. Ser605 and Ser634 of rat NHE-3 are crucial for regulation of NHE3 by PKA, although of the two serines, only Ser605 has been phosphorylated in vivo (170). In this study, phosphorylation of Ser552 also was shown to participate in the NHE3 response to cAMP, although in another study by Kurashima and colleagues [171], Ser552 was not functionally important. The functional

A

consequence of PKA-mediated NHE3 phosphorylation is its reduced Vmax, decrease in the surface amount, presumably caused by increased endocytosis and decreased exocytosis. The recruitment of PKA to the C terminus of NHE3 involves a multiprotein complex including NHE regulatory factors (NHERF1 and NHERF2) and a scaffolding protein ezrin. NHERF1 and NHERF2 proteins contain two 80- to 90-aminoacid PDZ domains mediating physical interaction with short peptide sequences located at the C terminus of interacting proteins. Both NHERF1 (initially cloned and described as NHE3 kinase A regulatory protein, E3KARP [172]) and NHERF2 reconstitute PKA-dependent NHE3 inhibition when expressed in NHERF-deficient cells (172). NHERF1 and NHERF2 interact though their C-terminal 29 amino acids with cytoskeleton-associated ezrin (173,174), which functions as A kinase–anchoring protein (AKAP). Phosphorylation of NHE3 by PKA is therefore facilitated by bringing the catalytic subunit of PKA to the vicinity of the NHE3 cytoplasmic tail by a protein complex containing either of the two NHERF factors and the cytoskeletonassociated AKAP protein ezrin (Fig. 73-4A). A somewhat similar scaffolding mechanism lies behind glucocorticoid-stimulated NHE3 activity. In this case, however, the mediating kinase (SGK1) interacts directly and

B NHE3

NHE3

P P −COOH

−COOH

NHERF1/2 cAMP

NHERF2 Glucocorticoids

F-actin

FIG. 73-4. Putative model for protein–protein interactions in the vicinity of Na+-H+ exchanger 3 isoform (NHE3) carboxy terminus as it relates to cyclic adenosine monophosphate (cAMP)–mediated inhibition (A) and glucocorticoid-mediated stimulation (B) of NHE3 activity. PKA, protein kinase A. NHERF, NHE regulatory factor; SGK1, serum- and glucocorticoid-induced protein kinase 1.

1862 / CHAPTER 73 specifically with NHERF2, acting as a bridge between the kinase and NHE3, to stimulate activity of the latter (169). It is the second PDZ domain of NHERF2 that binds both NHE3 (amino acids 585 and 660) and SGK1. A model facilitating this assembly was proposed (175) in which NHERF2 dimerizes, as depicted in Figure 73-4B. It also has been postulated that the mechanism of posttranscriptional regulation of NHE3 by glucocorticoids is biphasic, with an initial phase involving phosphorylation of the preexisting membrane NHE3, and a later phase in which SGK1 and NHERF2 facilitate translocation of the newly synthesized NHE3 to the cytoplasmic membrane (169). The exact sites within the C terminus of NHE3 phosphorylated by SGK1 currently are unknown. Regulation of NHE3 does not require phosphorylation in all cases. Stimulation of NHE3 activity by FGF or PKCactivating phorbol esters did not coincide with detectable changes on the phosphorylation status of NHE3 (176). An indirect mechanism of action has been suggested, mediated via phosphorylation of associated regulatory factors indirectly affecting NHE3 activity. Association with Cytoskeleton The NHERF-mediated link with ezrin suggests the association of NHE3 with cytoskeleton as a likely mechanism controlling NHE3 activity. Consistent with this notion, NHE3 was found to cosediment with F-actin, and pharmacologic disruption of cytoskeleton induced a profound inhibition of NHE3 activity (177). Inhibition of two kinases controlling cytoskeletal assembly, Ras homolog A (RhoA) and Rho associated kinase (ROK), also inhibited NHE3 activity in CHO cells stably transfected with dominantnegative mutants of a respective kinase without altering NHE3 abundance in the cytoplasmic membrane (178). This mechanism may, at least in part, account for the inhibitory effect of cAMP on NHE3 activity. Increased PKA activity inhibits RhoA, resulting in altered cell morphology with disruption of the microfilament actin network (179). By analogy, expression of constitutively active forms of RhoA and ROK kinases attenuates PKA-mediated NHE3 inhibition by stabilizing actin filaments (180). Similarly, disruption of actin cytoskeleton by hyperosmotic stress may be responsible for the associated decrease in NHE3 activity (181). Endocytosis/Exocytosis As mentioned earlier, in addition to being present at the cell surface, NHE3 is detectable in intracellular vesicles of the juxtanuclear compartment consistent with recycling endosomes (182–184). NHE3 remains in a state of dynamic equilibrium between the cell surface and the intracellular compartment: It undergoes internalization via clathrincoated vesicles (182) and is exocytosed back to the cytoplasmic membrane in a PI3K-dependent manner (185). PI3K inhibition leads to decreased NHE3 activity correlating with depletion of the plasma membrane pool of NHE3 protein (26), whereas constitutively active PI3K or AKT transfected into NHE3-expressing PS120 cells stimulates the exchanger

and increases the percentage of NHE3 present on the plasma membrane (185). EGF and FGF have been shown to stimulate NHE3 activity by increasing the surface protein pool in a PI3K-dependent manner (186,187). Other factors increasing the apical pool of NHE3 include LPA (188) and endothelin-1 (189). Conversely, decreased NHE3 surface expression has been associated with inhibition of the transporter by PKC (141), by parathyroid hormone (190), and by dopamine (191). Collectively, these studies strongly suggest that redistribution of NHE3 between subcellular compartments is an effective means of transport regulation. In the cell, NHE3 exists in large multiprotein complexes that range from 400 kDa in the intracellular pool to ~1000 kDa at the plasma membrane. These complexes are dynamic and are influenced by physiologic stimuli participating in acute NHE3 regulation. This dynamic assembly, association with cytoskeletal proteins, endosomal recycling, and protein phosphorylation events all act in concert to provide highly regulated turnover and activity of NHE3 protein (15). Pathophysiology Holmberg and Perheentupa (192) and Booth and coworkers (193) described a form of congenital secretory diarrhea (CSD) caused by defective sodium/hydrogen exchange (OMIM %270420). Based on a close phenotypic resemblance between this rare disease and symptoms displayed by NHE3−/− mice, NHE3 became a likely candidate for linkage. Homozygosity mapping and multipoint linkage analysis studies in four candidate regions known to contain NHE1, NHE2, NHE3, and NHE5 genes have shown that CSD is an autosomal recessive disorder, but is not related to mutations in the NHE1, NHE2, NHE3, and NHE5 genes (194). Because location of the human NHE2 gene is most likely adjacent to NHE4, that latter gene can also be excluded as a candidate for CSD. It would appear, therefore, that another NHE isoform or a regulatory factor may be directly responsible for the loss of Na+-H+ exchange in this disease. Altered expression and activity of NHE3 in primary and diabetes-related hypertension have implied a potential role for this isoform in the pathogenesis of high blood pressure. Spontaneously hypertensive rats have increased NHE3 activity in the ileal brush-border membranes (195) and in renal proximal tubules (196), suggesting that increased intestinal sodium absorption and renal reabsorption may contribute to systemic sodium retention and the pathogenesis of hypertension. In both streptozotocin-induced diabetes and BB/W autoimmune diabetic rats, renal cortex brush-border Na+-H+ exchange (presumably mediated by NHE3) was significantly induced, likely because of acidosis and not hypoinsulinemia (197). The effect of diabetes on intestinal Na+-H+ exchange is not well described. In streptozotocin-induced diabetes in rats (used as a model for secondary vitamin D deficiency), ileal NHE3 mRNA was induced twofold (198). It is likely, however, that the observed difference was caused by vitamin D deficiency, because repletion of diabetic mice with 1,25(OH)2D3 brought NHE3 mRNA expression down to control levels.

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1863 NHE4 NHE4 was first cloned from rat stomach cDNA library by Orlowski and colleagues (132). Although human NHE4 has not been formally characterized, a cDNA sequence identified by the German cDNA Consortium with GenBank locus CR627411 appears to represent the actual human Slc9a4 isoform based on more than 83% homology to rat NHE4 (see Table 73-2 and Fig. 73-4) and on chromosomal mapping described by Szpirer and coworkers (99). The predicted primary structure of rat NHE4 is 717 amino acids with a calculated molecular weight of ~81.5 kDa. Among the nine members of the NHE gene family, NHE4 protein shares the greatest homology with NHE2 (56.5% hNHE2 vs hNHE4). Western blotting with polyclonal antibodies against fusion protein–containing amino acids 393 to 625 of rat NHE4 protein detected a band of ~100 kDa, suggesting posttranslational modifications, conceivably glycosylation, in stably transfected PS120 fibroblasts (199). In contrast, monoclonal antibodies raised against a similar fragment (565–675 amino acids) of rat NHE4 reacted with a predominant band of ~65 to 70 kDa and 2 minor bands at 45 to 50 kDa, and ~75 kDa (200). These described discrepancies in NHE4 molecular weight currently remain unresolved. Hydropathy plot analyses have indicated membrane topology similar to the general model for NHEs (132), although this issue was not addressed experimentally. Tissue Distribution and Cellular Localization NHE4 exhibits fairly limited tissue distribution, with the highest expression in the stomach (200) and lower levels in the kidney (199), pancreas (201,202), salivary glands (101), hippocampus (46), and endometrium (107). Its expression in the small and large intestines is uncertain. An initial cloning report described detectable NHE4 transcript in these tissues (132), although later studies with more specific cDNA probes found no expression of this isoform in the rat jejunum or colon (85,88). In all reported cases, NHE4 protein was localized on the basolateral membrane, with the exception of pancreatic acinar cells, where NHE4 was also detected on the zymogen granule membrane (202).

Physiologic Role Unlike other plasma membrane NHEs, NHE4 lacks sodium specificity and acts also as a K+-H+ and a Li+-H+ exchanger (47). Under basal conditions, NHE4 exhibits low cation-H+ exchange activity, which is further stimulated by hyperosmolarity (199) or treatment with stilbene derivative, DIDS (disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate) (47). Another distinguishing feature of NHE4 is its relative insensitivity to amiloride and EIPA inhibition. The inhibition constant for NHE4 in stably transfected LAP(−) cells was more than 800 µM for amiloride (5.3 and 309 µM for NHE1 and NHE3, respectively), with even larger differences observed for

EIPA (47). HOE-642 inhibition studies later showed that NHE1 and NHE2 are 11,000- and 180-fold more sensitive to inhibition by the compound than is NHE4, which exhibits sensitivity to HOE-642 similar to that of NHE3 (203,204). It has been hypothesized that in the kidney, where NHE4 is predominantly expressed on the basolateral membrane of the thick ascending limb epithelial cells, this isoform may be specifically involved in ammonium transport (203). Its role in the GI tract may be most significant in the stomach, because targeted disruption of the NHE4 gene in mice results in profound phenotypic changes (205). The role of NHE4 in gastric morphology and acid secretion is discussed later in this chapter (see the Na+-H+ Exchange and Gastric Physiology section). Little is known about regulation of NHE4 gene expression and protein activity. The mechanisms of the described stimulation of NHE4 activity by hyperosmolarity or DIDS have not been elucidated.

NHE5 Klanke and colleagues (206) identified a cosmid clone for NHE5 (SLC9A5) by low-stringency hybridization with a rat NHE2 cDNA probe. Two exons of SLC9A5 were sequenced and compared with the other family members. The sequence most closely matched that of NHE3. Northern blots showed that SLC9A5 is expressed in the brain, testes, spleen, and skeletal muscle. No expression in the organs of the GI tract has been described to date. Human NHE5 resides in the EKD2 region of chromosome 16, which is linked to the pathogenesis of familial paroxysmal kinesigenic dyskinesia. Although no mutations in the coding region, intron/exon boundaries, or the 5′ and 3′ untranslated regions of the gene were identified, possible sequence variations in introns or the regulatory region could not be ruled out (207).

NHE6 By sequencing random cDNA corresponding to relatively long transcripts from the human myeloid cell line KG-1, Nagase and colleagues (208) identified a cDNA, which they called KIAA0267, that encoded an incomplete sequence that was later identified by Numata and coworkers (209) as NHE6 (SLC9A6). Human NHE6 protein shares the greatest homology with NHE7 and NHE9 (71% and 61%, respectively; see Table 73-2) and has 669 amino acids. Its predicted membrane topology is similar to that reported for other NHEs, with 12 putative membrane-spanning domains, followed by a hydrophilic C terminus. Tissue Distribution and Cellular Localization Northern blot analysis detected an approximately 5.5-kb SLC9A6 transcript with the most abundant expression in mitochondrion-rich tissues such as brain, skeletal muscle,

1864 / CHAPTER 73 and heart, and to a lesser extent, in the liver and pancreas. Expression of NHE6 has not been confirmed in other GI organs, although it is conceivable that its ubiquitous pattern of expression includes a majority of cell types and organs. NHE6 has a putative mitochondrial inner membrane targeting signal at its N terminus. This fact and initial fluorescence microscopy studies with green fluorescent protein (GFP)– tagged NHE6 expressed in HeLa cells costained with mitochondrial stain MitoTracker Red (209), as well as organelle fractionation (51), initially suggested that NHE6 represents the putative mitochondrial NHE regulating intramitochondrial Na+ and H+ levels as suggested by Garlid (210). Later studies, however, provided evidence that the positivecharge–rich segment is not a mitochondrial-targeting sequence, and that NHE6 accumulates not in the mitochondria, but in the recycling compartment of the endoplasmic reticulum when overexpressed in mammalian cells (211,212). NHE6 also has been shown to transiently appear on the cytoplasmic membrane (211), as hemagglutinin tag introduced into the predicted extracellular loop 1 of NHE6 was detected on the surface of live cells at a temperature nonpermissive to endocytosis (4°C). The physiologic relevance of this phenomenon remains unclear. Physiologic Role NHE6 and its transport properties currently have not been evaluated. Its physiologic function can only be extrapolated from its cellular localization and homology to other members of the NHE gene family. Its high homology to NHE7 suggests that it too may represent a nonselective cation-H+ exchanger and may participate in organellar pH and volume regulation. It has been speculated that NHE6 may participate in vesicle fusion and lysosomal biogenesis, and by regulating pH in the early and recycling endosomes, NHE6 may participate in regulation of surface receptor recycling (211). Regulation of NHE6 expression, cellular trafficking, or activity by physiologic or pathophysiologic stimuli has not yet been described.

NHE7 The NHE7 isoform was cloned by Numata and Orlowski (51) through a combination of computational and molecular biology approaches. NHE7 is a 725-amino-acid, ~80-kDa transmembrane protein with more than 70% homology to NHE6 and ~58% homology with NHE9 (see Table 73-2). Hydropathy analysis predicts membrane topology similar to that of other members of the NHE family, with 12 α-helical TMs, followed by a hydrophilic cytoplasmic C terminus. Tissue Distribution and Cellular Localization Human NHE7 gene is expressed ubiquitously, but most prominently in the putamen and occipital lobe of the brain, in skeletal muscle, and in a number of secretory tissues such

as prostate, stomach, pancreas, pituitary gland, adrenal gland, thyroid gland, salivary gland, and mammary gland (51). It also is expressed in the liver, small intestine, and colon, and this broad pattern of expression implies that NHE7 serves a “housekeeping” function. Inducible expression of hemagglutinin-tagged NHE7 in CHO cells followed by Western blot analysis identified two diffuse bands of ~180 and 80 kDa, suggesting formation of moderately stable homodimers and a possibility of glycosylation (51). Dual-labeling experiments showed that NHE7 accumulates predominantly in a juxtanuclear compartment partially overlapping mannosidase II–positive medial and trans-cisternae of the Golgi apparatus, but has been further localized primarily to the trans-Golgi network (51). Physiologic Role Compared with control cells, NHE7-overexpressing CHO cells demonstrated ~75% greater rates of 22Na+ influx into intact intracellular membrane compartments after permeabilization of the plasma membrane with saponin. This NHE7mediated 22Na+ uptake was pH gradient sensitive, as H+-specific ionophore carbonyl cyanide m-chlorophenylhydrazone, rapidly dissipating the organellar transmembrane H+ gradient, significantly reduced NHE7-mediated 22Na+ influx. Alkalinization of endomembrane compartments by sustained exposure to NH4Cl, led to decreased 22Na+ influx, whereas rapid acidification of the intracellular compartment by pretreatment and rapid removal of NH4Cl increased 22Na+ uptake in both control and NHE7-overexpressing cells about threefold (51). Taken together, these data indicate the existence of an endogenous organellar Na+ influx pathway that depends on the transmembrane H+ gradient and that is up-regulated in NHE7HA-overexpressing cells. NHE7 also has been determined to be a relatively nonselective monovalent cation-H+ exchanger, which is able to transport Na+, K+, Li+, and Rb+ (51). Numata and Orlowski (51) postulated that because K+ is the main intracellular cation, NHE7 serves primarily as a K+-H+ exchanger, and its roles in the homeostasis of the trans-Golgi include providing a pathway for H+ efflux and participating in controlling the volume of the organelle through transmembrane K+ flux. Regulation of NHE7 expression, cellular trafficking, or activity by physiologic or pathophysiologic stimuli currently has not been described.

NHE8 The NHE8 isoform was cloned from a mouse kidney cDNA library by Goyal and colleagues (213). The characterized sequence encodes a 576-amino-acid protein, sharing 96% identity with its likely human ortholog (NM_015266) (48). Human NHE8 protein shares less than 24% homology with other known NHE isoforms (see Table 73-2). Hydropathy analysis predicts membrane topology similar to the general models for all NHEs with 10 to 12 TMs in the N-terminal

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1865 portion of the protein, followed by a relatively short (~100 amino acids) hydrophilic C-terminal tail (213). The molecular mass of NHE8 detected by Western blotting is ~85 kDa, which is significantly greater than the 64 kDa predicted from the length of open reading frame. Consistent with the prediction of four N-glycosylation sites, inhibition of glycosylation with tunicamycin reduced the size of detected protein, confirming the posttranslational modification of NHE8 (213). Tissue Distribution and Cellular Localization Initial expression analysis indicated ubiquitous expression of NHE8 in mouse tissues with predominant expression in the liver, skeletal muscle, kidney, and testes (213). In the kidney, NHE8 mRNA was localized by in situ hybridization primarily to the proximal tubules of the outer stripe of the outer medulla and, to a lesser extent, to the renal cortex, whereas NHE8 protein copurified with brush-border membranes. Further immunolocalization studies determined that renal NHE8 is expressed on both microvillar surface membranes and the coated pit regions in the epithelial cells of proximal tubules (49). The colocalization of NHE8 with megalin in the intermicrovillar coated pits and subapical tubules suggests that, similar to NHE3, NHE8 may be regulated by endocytic retrieval and recycling. Plasma membrane localization of NHE8 has not been confirmed in the heterologous expression system of human NHE8 in COS-7 cells, where NHE8 was convincingly shown to be expressed in the mid- to trans-Golgi compartments (48), and not on the plasma membrane. Although human NHE8 is expressed in the liver, small intestine, and colon (48), its role in the GI tract is unknown. Xu and colleagues (50) described cloning of rat NHE8 cDNA. Polyclonal antibodies raised against N- and C-terminal peptides detected NHE8 in the apical membrane of jejunal enterocytes both by Western blotting and by immunohistochemistry (50). The apparent discrepancies between subcellular localization of NHE8 in vitro and in vivo are unresolved. They may represent true variation in protein trafficking, or it may stem from methodologic differences or differences in protein distribution in polarized epithelial cells and nonpolarized fibroblast cell lines. Physiologic Role Human NHE8 protein was overexpressed, purified from S. cerevisiae where it is primarily associated with endoplasmic reticulum, and reconstituted in artificial liposomes for transport studies. Under outwardly directed pH gradient, proteolysosomes showed progressive intravesicular alkalinization, as well as 22Na uptake, which is consistent with the action of NHE8 as a sodium-proton exchanger (48). Interestingly, K+ also stimulated alkalinization of proteolysosomes, suggesting that NHE8 acts as a nonspecific cation-H+ exchanger, similar to other organellar NHEs, for example, NHE7. Consistent with these observations, overexpression of NHE8 dissipated the acidic pH of the Golgi complex and increased the pH by

~6.5, from pH ~0.78 to 7.28, indicating an active role of NHE8 in mediating intraorganellar pH (48). Analysis of NHE8 gene expression and brush-border protein abundance in preweaning and adult rats showed an age-dependent decrease in NHE8 expression in the smallintestinal epithelium (50). This trend is opposite to that shown for the other two major epithelial NHEs, NHE2 and NHE3, with which expression and activity increase around weaning (123,214) and may point to a possible role for NHE8 in smallintestinal Na+ absorption during early stages of postnatal development. No other data describing regulation of this transporter and its potential implications in the physiology and pathophysiology of the GI tract are currently available.

NHE9 The NHE9 isoform was only recently identified by de Silva and colleagues (215) as one of two genes affected by a pericentric inversion of chromosome 3, 46N inv(3)(p14:q21), associated with attention deficit hyperactivity disorder (ADHD). Computational analysis indicated homology with mammalian NHEs, with strongest similarity to human NHE6 (61.2% at the protein level; see Table 73-2). Phylogenetic analysis indicates that NHE9 belongs to the same clade as other organellar isoforms NHE6 and NHE7 (see Fig. 73-4). The SLC9A9 gene spans approximately 470 kb genomic DNA and has 16 exons, and its cDNA is approximately 3.4 kb in length. The predicted SLC9A9 protein has 645 amino acids and a molecular weight of 72.6 kDa, and its sequence analysis suggests a membrane protein with 10 (215) or 12 (48) TMs. Tissue Distribution and Cellular Localization Northern blot analyses indicate that NHE9 expression is fairly widespread, with the highest expression in the heart, skeletal muscle, and brain. In the GI tract, NHE9 was detected in the liver and in smaller quantities in the small intestine. No detectable levels were observed in the colon (215). The homology of NHE6 and NHE9 proteins suggested that both of these isoforms are located in the intracellular compartments. Coimmunolocalization studies in COS-7 cells combined with rhodamine-labeled transferrin chase imply that NHE9 is primarily expressed in late recycling endosomes, with a smaller proportion of NHE9 localizing to EEA1-positive early endosomes (48). Physiologic Role Although initial identification of NHE9 was purely computational, current study appears to confirm its identity as a NHE. In COS-7 cells, overexpression of NHE9 resulted in luminal alkalinization to near-cytosolic pH in the compartments in which NHE9 resided (from pH 6.73 to 7.14, as indicated by pH-sensitive GFP mutant enhanced yellow green flourescent protein [EYFP] fusion with NHE9) (48). Because overexpression systems (in this case,

1866 / CHAPTER 73 expression close to 100-fold greater than that of endogenous NHE9) may tend to overestimate the physiologic role of NHE9 in regulating intraorganellar pH, more data from genetargeting or knockdown experiments are needed to validate these results. The NHE9 gene has only recently been cloned, and there are not yet any available data pertinent to transcriptional and posttranscriptional modes of regulation of its expression and activity. Pathophysiology The role of NHE9 in the physiology of the GI tract has not been elucidated. A genome-wide scan of families affected with ADHD identified marker D3S1569 (logarithm of odds [lod] 1.37; p = 0.0060) located in intron 5 of the SLC9A9 gene (216). Although the lod score did not exceed the QTL threshold for significance, its location within the NHE9 gene suggests that it could play a role in the development of the ADHD phenotype. The expression of NHE9 in neuronal tissues (primarily medulla and spinal cord) appears to supports this hypothesis.

PHYSIOLOGIC ROLES OF NA+-H+ EXCHANGE IN THE DIGESTIVE TRACT Na+-H+ Exchange and Salivary Gland Physiology Saliva provides hydration and protection from mechanical and chemical insults for oral mucosa, oropharynx, and esophagus. Its more specialized functions include the initiation of digestion and antimicrobial defense. It has been postulated that saliva formation involves a two-stage secretory/absorptive process. In the first stage, acinar cells secrete an isotonic fluid, which is generated through the coordinated activities of membrane transport proteins driving net transepithelial Cl− movement and HCO3− efflux. During the second stage of secretion, ductal cells modify acinar secretions primarily by reabsorbing NaCl. Because the apical surfaces of salivary ducts are relatively impermeant to water, the resulting saliva is generally hypotonic. In the first stage of saliva formation, the action of basolateral Na+,K+-ATPase in the acinar cells produces a 10- to 15-fold inwardly directed Na+ gradient. Various transporters use this gradient to increase intracellular Cl− levels. Cl− uptake across the basolateral membrane of acinar cells is thought to be primarily mediated via the electroneutral Na+-K+-2Cl− cotransporter (Nkcc1). Most acinar cells also possess a second Cl− uptake pathway mediate via coupled basolateral Cl−-HCO3− and Na+-H+ exchange. These Na+-dependent Cl− uptake mechanisms result in intracellular Cl− concentrations greater than five times more than its electrochemical gradient, a requirement for Cl− exit via Cl− channels. Activation of Cl− channels coincides with activation of K+ channels, a mechanism necessary to maintain the electrochemical driving force for Cl− efflux. Rapid loss of intracellular Cl− and K+ produces a transepithelial potential difference that results in paracellular passive movement of cations, creating a

transepithelial osmotic gradient driving the movement of water. Apical Cl− efflux is accompanied by HCO3− efflux, primarily mediated by Cl− channels. Basolateral and apical HCO3− fluxes require intracellular HCO3− provided by the activity of carbonic anhydrases, and they produce an intracellular acid load that has to be rapidly buffered by an increase in Na+-H+ exchange activity (217). Several reports suggest that NHE1 is most likely the NHE isoform involved in this process (218). Indeed, in vivo salivary secretion in Nhe1−/− mice was reduced by 30% to 40% compared with their wild-type littermates, despite a compensatory increase in expression of Nkcc1 on the basolateral membrane (219). It also greatly reduced pH recovery from an acid load in resting and stimulated acinar cells (220). Ablation of the NHE2 gene, normally expressed on the apical membrane of acinar and ductal cells (101), did not alter pH recovery in acid-loaded acinar cells (220), but did blunt in vivo pilocarpine-stimulated saliva secretion by mouse parotid glands (219). It has been speculated that NHE2 is stimulated by alkaline secretions in the acinar lumen, thus providing support for NHE1 in regulating the pHi and participating in fine-tuning of the activity of the Ca2+-stimulated cystic fibrosis transmembrane conductance regulator (CFTR) channel during muscarinic receptor– induced fluid secretion (219). Targeted disruption of the NHE3 gene, normally expressed on the apical membrane of mouse, but not rat, salivary glands, did not result in significant alteration of the volume of stimulated salivary secretion (219), suggesting a minor or negligible role for NHE3 in the physiology of salivary acinar cells. Although NHE4 is expressed in the acinar cells, pH recovery studies suggest that it plays no active role in regulating pHi under normal physiologic conditions (101) (Fig. 73-5). The second stage of secretion is characterized by conservation of NaCl by ductal epithelial reabsorptive processes to generate hypotonic saliva. It is believed that reabsorption of Na+ by the ductal cells is mediated by at least two Na+ uptake mechanisms: Na+-H+ exchange located in the luminal membrane and amiloride-sensitive Na+ channel, possibly electrogenic sodium channels. Ductal epithelial cells express the same four NHE isoforms as acinar cells, with NHE1 and NHE4 located on the basolateral membrane and NHE2 and NHE3 located apically (101,221,222). With NHE2 and NHE3 as likely candidates for Na+ sparing in the ductal cells, it was surprising to find that concentrations of Na+, K+, Cl−, and osmolarity in saliva of NHE2−/− and NHE3−/− null mice were comparable with secretions from wild-type littermates (219), suggesting that these NHEs do not play a major role in NaCl reabsorption in salivary gland ducts. Expression of electrogenic sodium channel subunits α, β, and γ was increased in NHE2−/− and NHE3−/− mice, pointing to a possibility that sodium channels not only serve a primary role in ductal sodium reabsorption, but also that electrogenic sodium channel expression, and presumably its activity, compensate for the loss of the two apical NHE isoforms (219). The osmolality and Na+, K+, and Cl− content increased significantly in saliva from Nhe1−/− mice (219). This observation was somewhat puzzling, because sodium reabsorption in the salivary

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1867 Interstitium H+

Na+/K+-ATPase 3Na+

H+ ?

K+

HCO3−

ATP Na+

2K+

Na+

ADP+Pi

Na+ 2Cl−

Ca++

CO2

Cl−

K+

H2O

Carbonic anhydrase HCO3−

H+

Na+

Ca++

Na+

H+ Na+

H+ Cl−

HCO3−

?

H2O Na+

Lumen FIG. 73-5. Major membrane transport proteins involved in salivary secretion from the acinar cells of the parotid gland. Entry of Cl− across the basolateral membrane is mediated by a Na+-K+-2Cl− cotransporter (Nkkc1) and coupled Na+-H+ and Cl−-HCO3− exchange. Carbonic anhydrase generates HCO3− by catalyzing the reversible reaction of water and CO2 to form HCO3− and H+, providing driving forces for basolateral and apical Na+-H+ exchangers (NHEs) and for basolateral entry of Cl− via anion exchange. Cl− exit is across the apical membrane via Ca2+-stimulated cystic fibrosis transmembrane conductance regulator (CFTR) channel. Created electrochemical gradient provides a driving force for passive paracellular transport of sodium into the acinar lumen. ADP, adenosine diphosphate; ATP, adenosine triphosphate.

ducts is flow-rate dependent (223); therefore, one would expect that with a lower flow rate of saliva in NHE1−/− mice, osmolality, and ion content should decrease. This finding, however, points to a critical role of this exchanger in both acinar and ductal epithelial cells of the salivary glands, although the precise mechanism of NHE1 contribution to the biology of ductal epithelial cells is unclear. The roles of the two other NHE isoforms found to be expressed in the salivary glands (NHE7 and NHE8) remain to be elucidated.

Na+-H+ Exchange and Liver Physiology Relatively little is known about the role of NHEs in hepatic functions, although all NHEs except NHE5 have

been detected in this organ. In most cases, the cellular and subcellular distribution of NHEs in the liver has not been described. Na+-H+ exchange has been demonstrated by Arias and Forgac (224) in the sinusoidal but not canalicular plasma membrane of rat hepatocytes and suggested to be the major mechanism controlling pHi. Even earlier, Koch and Leffert (225) provided evidence for involvement of Na+-H+ antiport in hepatocyte proliferation in response to trophic stimuli. Increased hepatic plasma membrane NHE activity was demonstrated in hepatectomized (226) and neonatal rats (227), implicating Na+-H+ exchange in hepatic regeneration and growth. Stimulation by growth factors of DNA synthesis in hepatocytes has been shown to coincide with Na+-H+ exchange activation (228,229). More recently, the activity of basolateral Na+-H+ exchange in rat hepatocytes has been

1868 / CHAPTER 73 implicated not only as permissive in the stimulation of DNA synthesis, but perhaps also in tumor-promoting actions of known carcinogens (230). Na+/H+ exchange also has been described in the stellate cells, where it is the main pHi regulator. An increase in pHi mediated by Na+-H+ antiport (presumably by NHE1) was associated with activation and proliferative response of HSCs to platelet-derived growth factor (PDGF) (231). Activation, proliferation, and differentiation of HSCs into collagen-producing myofibroblasts reflect a wound-healing response of the liver, which when unbalanced, may lead to hepatic fibrosis. The effect of oxidative stress on fibrosis has been partially attributed to NHE activation in the HSCs, and the amiloride-mediated reduction in cell proliferation and collagen synthesis was postulated as a novel therapeutic option in liver fibrosis (232). Indeed, Benedetti and colleagues (233) showed that amiloride analogue EIPA prevented type I collagen accumulation and proliferation of HSCs in vitro, when induced with oxidative stress or PDGF. The same study also demonstrated that in vivo, administration of amiloride considerably reduced fibrosis in rats that underwent bile duct ligation or were administered dimethylnitrosamine. The interpretation of the in vivo studies was questioned by Häussinger (234), because the resulting plasma concentration of amiloride was much less than that expected to inhibit Na+-H+ exchange. Similar protective effects of NHE inhibition were, however, reported in a follow-up study with an unrelated and more specific NHE inhibitor: cariporide (235). These findings strongly suggest that Na+-H+ exchange inhibition may be beneficial in chronic liver diseases leading to fibrosis and cirrhosis, and perhaps in the treatment of an already established disease. In addition, by analogy to the role of NHE1 in the cardiac ischemia-reperfusion injury, results from inhibition of Na+-H+ exchange by EIPA in a rat model of partial hepatic ischemia are suggestive of a potential role of NHE1 in this tissue damage and imply inhibition of NHE as a novel strategy in reducing or preventing ischemic injury to the liver (236). Both hepatocytes and biliary epithelial cells (cholangiocytes) participate in biliary secretion. Biology of cholangiocyte secretion has been reviewed in more detail elsewhere (237, 238). Relatively little is known, however, about absorptive processes in the biliary epithelium. NHE3 has been identified on the apical membranes of mouse cholangiocytes, and targeted disruption of the NHE3 gene resulted in inhibition of fluid reabsorption from isolated bile duct units that followed forskolin-stimulated secretion (137). These findings warrant further investigation into the potential role of apical cholangiocyte Na+-H+ exchange in both physiologic states and in the pathogenesis of cholestatic liver disease. NHE3 also has been detected in human (239), calf (240), and prairie dog gallbladder epithelium (241). Na+-H+ exchange has been best characterized in the latter species, believed to represent a good animal model of human gallstone formation. Prairie dog gallbladder epithelial cells in primary culture demonstrated H+ gradient-dependent 22Na uptake, mediated by NHE1 (6%), NHE2 (66%), and NHE3 (~28% of total uptake),

as determined by DMA and HOE-694 inhibition (102). The significant contribution of Na+/H+ exchange to epithelial Na+ absorption, combined with data suggesting increased gallbladder Na+ and fluid absorption in the early stages of gallstone formation (242,243), makes the hypothesis about involvement of the apical NHEs in the pathogenesis of gallstones quite attractive, although further experimental evidence is required.

Na+-H+ Exchange and Pancreatic Physiology Secretion by the exocrine pancreas is carried out by two morphologically and functionally distinct epithelia: the acini and the ducts. The physiologic role of the pancreatic acinar cells is to synthesize, package in granules, and secrete zymogens in response to stimulation. In addition, pancreatic acinar cells also secrete a NaCl-rich primary fluid. This primary secretion is modified by the duct cells to generate the HCO3−rich pancreatic juice. Many aspects of the electrolyte fluxes participating in acinar and ductal secretion of pancreatic juice are analogous to those observed in the salivary glands. Acinar cells secrete a near-neutral primary fluid by Na+coupled secondary active Cl− transport. Basolateral Na+-H+ exchange, driven by the transmembrane Na+ gradient generated by Na+,K+-ATPase, increases pHi and promotes formation of HCO3− by hydration of CO2, catalyzed by carbonic anhydrase. The basolaterally expressed pNBC1 variant of an electrogenic sodium-bicarbonate cotransporter (244) provides an additional pathway for intracellular HCO3− accumulation. The HCO3− formed thus exits to the interstitium via a Cl−-HCO3− exchanger in the basolateral membrane, resulting in cytosolic Cl− accumulation greater than its electrochemical equilibrium concentration. Cl− exits the cell on secretagogue stimulation via apical Ca2+-sensitive Cl− channels, a flux accompanied by paracellular movement of Na+ intended to preserve electroneutrality. The role of Na+-H+ exchange in controlling pHi of the acinar cells is of particular interest because acidification could potentially impact Ca2+ signaling machinery, leading to abnormalities in secretagogue-stimulated secretion. Although their cellular distribution and subcellular localization has not been systematically studied, most of the characterized NHE isoforms have been shown to be expressed in the pancreas, except for NHE5 and NHE9 (expression of the latter was not evaluated in pancreatic tissues; see Fig. 73-4). NHE1 is expressed on the basolateral membrane of pancreatic acinar cells (201) and on the membranes of the zymogen granules (202). Basolateral NHE1 accounts for a majority of Na+-H+ exchange activity in mouse pancreatic acinar cells, because pHi of the acinar cells isolated from NHE1−/− mice could not recover from an acid load (245). The significance of this finding is further accentuated by that these cells were unable to recover from the acid challenge in the absence or presence of extracellular HCO3−, suggesting that basolateral Na+-HCO3− cotransport could not compensate for the absence of NHE1 in buffering pHi. It also indicates that other isoforms, such as NHE4, previously localized to the basolateral

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1869 membrane of pancreatic acinar cells (201), do not participate in pHi regulation. Although NHE4 knockout mice have been characterized (205), the role of this isoform in the pancreas has not been investigated. The expression of NHE2 and NHE3 in the acinar cells has not been verified, but their roles appear to be negligible because acini isolated from both NHE3−/− and NHE2−/− mice recover from an intracellular acidification with kinetics similar to that seen in wildtype animals (245). A possibility of intracellular localization of these two isoforms has been speculated, although earlier observations showed the absence of NHE2 and NHE3 proteins on the zymogen granule membranes (202). NHE1 activity also has been shown to be activated by muscarinic stimulation, suggesting an important role for NHE1 in secretagogue-stimulated fluid secretion, although otherwise pHsensitive Ca2+ signaling appeared unaltered in acinar cells isolated from NHE1−/− mice (245). The role of NHE1 and NHE4 expressed on the zymogen granules has not been investigated. It has been postulated, however, that they may participate in the fusion and exocytosis of the pancreatic enzymes. Indeed, cholecystokinin-mediated amylase release was inhibited with 50 µM EIPA and a high extracellular concentration of Na+ (~130 mM), but stimulated with a lower extracellular Na+ concentration (30 mM) (246). The postulated scenario is that secretagogue stimulation of pancreatic acinar cells increases activity of NHE1 on the basolateral membrane, which results in a transient increase in the intracellular Na concentration from resting 10 mM up to 5060 mM. This, in turn, may provide the Na+ gradient necessary for organellar NHEs to alkalinize intragranular space in the mature zymogen granules (247), thus facilitating fusion and secretion of the stored enzymes (248). Pancreatic ducts secrete the bulk of secretagogue-induced bicarbonate ions by moving HCO3− from the blood into the luminal pancreatic fluid through duct cells. The molecular mechanisms of bicarbonate secretion in the pancreatic ducts have been reviewed in detail by Steward and colleagues (249). In the classical model, CO2 diffuses into the duct cell across the basolateral membrane and is hydrated by intracellular carbonic anhydrase. The produced H+ ions are extruded by the basolateral Na+-H+ exchange mechanism driven by the Na+ gradient that is maintained by Na+,K+-ATPase. The HCO3− ions leave the cell at the apical membrane through an HCO3−/Cl− exchanger driven by the luminal Cl− concentration determined by secretin-regulated Cl− channels. Na+ follows passively via the paracellular pathway, because of a transepithelial potential difference, followed by osmotically driven water secretion. This model has been more recently challenged by demonstrating an additional pathway for HCO3− entry into pancreatic duct cells, involving HCO3− uptake at the basolateral plasma membrane via an electrogenic Na+HCO3− cotransporter, pNBC1 (250). Although NHE1 and NHE4 are present at the basolateral membrane of the duct cells and likely participate in the regulation of pHi, the contribution of the NHE mechanism to HCO3− secretion is now less certain. In the guinea pig pancreas, measurements of the decline of pHi in isolated guinea pig ducts after the

application of amiloride and H2DIDS (NBC1 inhibitor) indicated approximately equal contributions of the two transport systems to unstimulated secretion and pHi regulation (251). After secretin stimulation, however, the activity of pNBC1 increases and participates in ~75% of the basolateral HCO3− uptake. Bicarbonate secretion from the duct cells also is attenuated predominantly by pNCB1 inhibition and, to a significantly lesser extent, by NHE inhibition with amiloride analogue (by ~56% and 18%, respectively) (252). Juice collected from resting pancreatic ducts is relatively acidic, Cl− rich, and has relatively high pCO2, suggesting active acidification of the ductal lumen. Apical Na+-H+ exchange has been implicated in this phenomenon in mouse (253) and bovine pancreas (254) and has been thought to be mediated by apically expressed NHE2 and NHE3 isoforms (103). NHE2−/− mice, however, have normal luminal Na+dependent H+ efflux in ducts. In contrast, pancreatic duct cells isolated from NHE3−/− mice displayed 45% reduced luminal Na+-dependent H+ efflux (103). It also has been postulated that NHE3 plays an important role in HCO3− salvage under resting, low-flow conditions, especially in larger pancreatic ducts. This hypothesis is supported by the finding that CFTR enhances the cAMP-induced inhibition of NHE3, a phenomenon likely dependent on the physical interaction of the two transport proteins mediated through a scaffolding protein, ezrin-radixin-moesin–binding phosphoprotein 50 (EBP50; NHERF1) (255).

Na+-H+ Exchange and Gastric Physiology pHi regulation in gastric epithelial cells and the role of Na+-H+ exchange in this process are of considerable interest because of periodically low pH of the gastric lumen. Although there is some disagreement about pHi stability in resting and stimulated isolated parietal cells (256,257), their ability to maintain or increase pHi despite large H+ fluxes is quite remarkable. Na+-H+ exchange, first described in gastric mucosa by Paradiso and colleagues (258,259), has since been implicated in gastric mucosal pHi homeostasis (260), cell volume regulation associated with secretory stimulation (261), and gastric epithelial restitution after injury (262,263). Many of the currently identified NHE isoforms (except NHE5, NHE6, and NHE9; see Fig. 73-4) are expressed in the gastric epithelium, with NHE1-4 being best described. NHE1 is expressed on the basolateral membranes of surface and neck mucous cells, chief cells, and to a lesser extent, in parietal cells (100,264). Morphometric evaluations of gastric histology in NHE1−/− mice showed thinning of the glandular epithelium and considerable widening of the interstitial space between gastric glands (71). These morphologic changes were not attributable to inflammation, and they did not appear to translate into detectable perturbations in systemic acid–base homeostasis, with no difference in whole-blood pH, pCO2, pO2, or bicarbonate concentration between NHE1−/− mice and their wild-type littermates (71). It is not known whether stunted growth observed in 2-week-old and older

1870 / CHAPTER 73 NHE1−/− mice can be attributed to changes in gastric epithelial morphology. NHE2 has a tissue distribution similar to that of NHE1 in the gastric mucosa (100). Although lacking immunohistochemical support, NHE2 is believed to be located on the basolateral membrane. Unusually high sensitivity of NHE2 to extracellular pH led investigators to speculate that basolateral alkalinization, during stimulated acid secretion by parietal cells, results in increased basolateral NHE activity, likely mediated by NHE2. It also was speculated that increased NHE2 activity at the basolateral membrane in response to interstitial alkalinization could permit this isoform to participate in acid secretion, viability of parietal cells, and mucosal protection. Indeed, NHE2 homozygous mutants exhibited marked alterations in gastric mucosal histology and function. NHE2−/− mice have a sharply reduced number of parietal and chief cells, resulting in a loss of net acid secretion (112). The reduction of the number of zymogenic cells may be secondary to decreased viability of parietal cells, which develop normally, but undergo necrosis prematurely (112). This process is accompanied by progressive inflammation in the form of diffuse corporal gastritis that ranges from transmural neutrophilic infiltration to a profound atrophy consistent with chronic achlorhydria, dependent on age and stage of inflammation (113). Expression of the NHE3 isoform in the stomach is a somewhat controversial subject. Although documented in the rat (132), human, and guinea pig (265), it has not been detected in rabbit gastric mucosa (100,131). According to Kulaksiz and colleagues (265), NHE3 protein expression was confined to the basolateral membrane of surface mucous cells in both human and guinea pig specimens. This surprising subcellular distribution is likely to be true, because the same antibodies properly stained the brush-border membrane of the duodenal enterocytes (265). These findings were later challenged by functional studies in perfused, isolated rat gastric glands, which demonstrated NHE3-like activity in the parietal cell apical membrane (266). No descriptions of gastric abnormalities were reported in studies on NHE3-deficient mice (148); therefore, the expression and physiologic role of NHE3 in the gastric epithelium remain obscure. NHE4 is abundantly expressed in the gastric gland epithelium, with protein located on the basolateral membrane of parietal and chief cells and, to a lesser extent, in mucous cells (100,200). Similar to the putative function of NHE2 in the basolateral membrane of parietal cells, high expression of NHE4 was suggestive of an important role this NHE plays in maintaining pHi homeostasis and acid secretion. Normal gastric acidity in young NHE2−/− mice (112) was suggestive of another basolateral NHE isoform. Because NHE1 has relatively low expression in parietal cells, NHE4 was a likely candidate gene participating in parietal cell physiology. Indeed, data from gene-targeting studies showed that NHE4−/− mice had hypochlorhydria (205). In many ways, the phenotypic picture resembled that observed in NHE2−/− or AE2−/− mice, with a reduced number of parietal cells because of apoptosis and necrosis and with concomitant loss of mature

chief cells. One of the distinguishing differences between the two NHE-targeted mutations was that the achlorhydria in NHE4−/− mice was detected early in postnatal life and was not progressing with age. It has been speculated that NHE4 is normally coupled with the basolateral Cl−/HCO3− exchanger (AE2), and that it plays a critical role in regulating gastric acid secretion and foveolar epithelial cell differentiation.

Na+-H+ Exchange and Intestinal Water and Sodium Absorption The average daily luminal load of water and sodium in the GI tract of an adult human amounts to ~9 L water and 800 mEq Na+. Less than 20% of this comes from ingestion, and more than 80% represents secretory fluids (saliva, gastric and pancreatic juices, bile, and enteric secretions). Healthy gut is capable of absorbing more than 98% of this load, resulting in ~200 g daily stool output (subject to dietary influences). Absorption of water in the GI tract is closely coupled with solute transport in the intestinal epithelium. Traditionally, water movement across the intestinal epithelium has been attributed to the influence of hydrostatic and osmotic pressures and passive paracellular diffusion. More recently, an alternative mode of water transport has been proposed, in which an active apical Na+-glucose cotransporter, SGLT1, serves as a water channel with stoichiometry of 220 to 400 molecules water per 1 molecule glucose and 2 molecules sodium (267). The latter model remains a subject of scientific debate, as recently reviewed by Lapointe and colleagues (268). Transport of water across the intestinal epithelium is beyond the scope of this chapter and is only referred to as a secondary phenomenon related to NHE-mediated sodium absorption. Small-Intestinal Na+-H+ Exchange Sodium enters the enterocyte primarily by means of cotransport with glucose and amino acids, through apically expressed NHEs and through electrogenic Na+ channels. NHE2 and NHE3 are the two apically expressed NHE isoforms believed to participate in vectorial transport of Na+ across the intestinal epithelium. Apical expression of NHE8 has been described in rat jejunum, and participation of NHE8 in the small-intestinal Na+/H+ exchange in suckling animals was postulated (50). The exact contribution of this isoform to intestinal electroneutral Na+ transport remains to be determined. Although NHE2 appears to be the major functional isoform in the chicken ileum and colon (269), it is NHE3, and not NHE2, that mediates the majority of both basal and meal-stimulated Na+ absorption in the dog ileum (270–272). In rabbit ileum, NHE2 and NHE3 contribute equally to the Na+-H+ exchange, with the NHE3 contribution increasing to ~68% after glucocorticoid treatment (273). In developing rats, the contribution of NHE3 to jejunal Na+-H+ exchange dramatically increases with age from 59% in 2- to -3week-old to ~92% in 6-week-old animals (214). Therefore, it

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1871 has been postulated that NHE2 plays a greater role in smallintestinal Na+ absorption in the preweaning period than in adulthood. In light of findings of NHE8 in rat jejunum and its reciprocal pattern of postnatal expression (compared with NHE2 or NHE3) (50,123,214), the contribution of NHE2 to intestinal Na+ absorption in the early postnatal period may need to be reevaluated. Although not assessed in the suckling period, the relevance of NHE2 to intestinal Na+/H+ exchange in mice appears to be negligible, because NHE2null mice have no apparent intestinal absorptive defect (112) and no compensatory mechanism, for example, increased expression of NHE3 (147). Contrasting with these results and emphasizing the physiologic role of NHE3 in Na+ absorption, NHE3−/− mice display a number of phenotypic changes: moderate diarrhea, distention and accumulation of alkaline fluid in all intestinal segments, mild metabolic acidosis, low blood pressure, decreased body fat, and increased mortality when deprived of Na+ intake (147,148). Hypertrophy of the small intestine and colon (see discussion in the following section) likely represents a compensatory mechanism. NHE2 expression was not altered in the jejunal epithelium of NHE3−/− mice (147). Although a residual, EIPA-sensitive component of Na+ absorption remained in NHE3−/− mice, this probably did not represent NHE2 activity, because it could be inhibited by cAMP (147). Also, in double-knockout mice (NHE2−/−/NHE3−/−), the additional loss of NHE2 in NHE3-deficient mice caused no reduction in viability, no further impairment of systemic acid–base balance, and no apparent worsening of the diarrhea (274). The observed residual activity in NHE3−/− and in double-knockout mice may be mediated by NHE8, although kinetics of this isoform and its sensitivity to inhibitors and cAMP have not been evaluated. Increased local intestinal expression of IFN-γ, as well as a fivefold increase in serum levels, was found in the intestine of NHE3−/− mice (275). This was accompanied by increased expression of a number of IFN-γ–inducible genes identified by microarray analysis (275). There was no evidence of inflammation in the intestine of NHE3−/− mice, and it has been suggested that increased IFN-γ might participate in compensation for the defective Na+ absorption mechanisms by its antisecretory effects, such as decreased expression of CFTR (275). Colonic Na+-H+ Exchange In healthy individuals, daily ileocecal flow is approximately 2 L electrolyte-rich fluid. Of this amount, 1.5 to 1.9 L is absorbed in the colon, although the maximal capacity of the human large intestine to absorb fluids may be as high as 5 to 6 L/day (276). Therefore, there is a large margin within which a healthy colon can compensate for increased ileocecal flow ensuing from defective small-intestinal absorption. Exceeding the maximal capacity will result in diarrhea. In contrast, in colonic disease, relatively small changes in water and electrolyte absorption will produce a significant increase in stool water output, emphasizing the relevance of fine-tuning of colonic transport processes. The bulk colonic

electrolyte absorption occurs via electroneutral NaCl transport and takes place in both crypts and surface epithelia of the proximal and distal colon. This electroneutral absorption is mediated by coupled luminal Na+-H+ and Cl−-HCO3− exchange. The remaining electrolyte absorption is mediated by electrogenic sodium channels and by transcellular or paracellular absorption of Cl−, or both. For a more comprehensive review of colonic electrolyte absorption, see articles by Kunzelmann and Mall (165) and Geibel (277). Electroneutral NaCl absorption generally is believed to represent coupled apical membrane Na+-H+ and Cl−-HCO3− exchanges. This HCO3−-dependent component of electroneutral NaCl absorption is enhanced by adrenergic agonists and inhibited by cAMP, aldosterone, and increases in intracellular Ca2+ (165). Colonic NaCl absorption also is enhanced by SCFAs and especially by butyrate. Several components have been proposed in this interaction. SCFA absorption may stimulate electroneutral Na+ uptake by acidification of colonocytes and activation of apical NHEs (278). Cl− absorption is mediated via the apical Cl−/HCO3− exchanger, stimulated, in turn, by increased HCO3− production during SCFA metabolism (Fig. 73-6). Another model has been proposed, in which butyrate is taken up via nonionic diffusion or SCFA-HCO3−– mediated exchange, likely by MCT1 (monocarboxylate transporter 1) (279). Subsequently electroneutral NaCl absorption is activated by parallel Cl−-butyrate and Na+-H+ exchange (280) (see Fig. 73-6). SCFA absorption activates basolateral volume-sensitive Cl− channels, whereas basal and cAMP-activated, CFTR-mediated Cl− secretion is inhibited (281,282). Activation of Na+-H+ exchange and inhibition of chloride secretion are most likely the basis of the antidiarrheal effects of butyrate (165,283,284). In vitro functional studies with the rat distal colon indicated that butyratedependent Na+ absorption was mediated by both NHE2 and NHE3 (285). In C2bbe clone of Caco-2 cells, however, only NHE3 activity, and not NHE2 activity, was stimulated by butyrate (161). The increase in NHE3 activity was likely caused by transcriptional activation of NHE3, because both protein and mRNA were induced by SCFAs in vitro and in the colonic epithelium of rats fed 5% pectin-supplemented diet (161). This hypothesis is further supported by strong induction of rat NHE3 promoter by sodium butyrate in transient transfections (162). There are segmental differences with respect to the predominant mode of Na+ absorption present in the proximal and distal colon of various species. Overall, Na+ transport in the proximal colon is thought to be mediated primarily by Na+-H+ exchange, whereas in the distal colon, it is mixed (rat and mouse) or dominated by electrogenic absorption mediated by electrogenic sodium channels (rabbit, human, and guinea pig). Of the nine cloned NHEs, expression of NHE1-4, NHE7, and NHE8 has been described in the colon (see Fig. 73-2). The ubiquitous NHE1 is not regulated by dietary Na+ depletion (286) and is expressed in the basolateral membrane of all epithelial cells of the colon with no detectable segmental differences (68). NHE2 and NHE3 are both expressed on the apical side of colonic epithelial cells (104). Although NHE3

1872 / CHAPTER 73 SCFA

Lumen

Passive diffusion

Cl−

− Na+ Cl

SCFA−

Na+

H+ HCO3−

Ca++

H+

HCO3−

NaB

Metabolism H+

SCFA−

NHE3 mRNA

2K+

ADP+Pi

H+

ATP K+

Na+ Cl−

3Na+ Na+/K+-ATPase

Interstitium FIG. 73-6. Relation between short-chain fatty acid (SCFA) absorption and metabolism and electrolyte fluxes in the colonic epithelium. Both proabsorptive and antisecretory events have been depicted. Na+-H+ exchanger 2 isoform (NHE2) and NHE3 have been depicted in this diagram together for illustrative purposes only. It is assumed that NHE2 plays a more prominent role in this mechanism in colonic crypts, and NHE3 in surface epithelial cells. ADP, adenosine diphosphate; ATP, adenosine triphosphate; CFTR, cystic fibrosis transmembrane conductance regulator; MCT1, monocarboxylate transporter 1.

has been unambiguously described in the surface epithelium (65,68), expression of NHE2 has been somewhat controversial. Studies with human colonic biopsies showed uniform distribution on NHE2 mRNA along the vertical axis of the colonic crypts (68); however, later studies in mice demonstrated that, in the distal colon, NHE2 functions mainly in the crypts cells (57,58), which added to the body of evidence about absorptive properties of colonic crypts (e.g., see Singh and colleagues [287]). Although the Cl−-dependent Na+-H+ exchange mechanism has been described in the crypts of rat distal colon (17,56,288), others have failed to demonstrate such a mechanism in mouse colonic crypts (57,58). The reasons for this discrepancy remain unknown, as indicated in the earlier discussion on Cl− dependency. Despite the high expression of NHE2 in the colon and its established participation in pHi and cell volume regulation (57), NHE2−/− mice display no obvious intestinal absorptive defect (112,147). It has been speculated that ectopic expression of NHE3 in

colonic crypts of NHE2−/− mice may compensate for the lack of the latter isoform (57). In bird colon, NHE2 mediates most (~85%) Na+-H+ activity both under basal conditions and under Na+ depletion (269). In mice, in contrast, based on defective Na+ absorption in NHE3-deficient animals, NHE3 appears to be the major Na+ transporter in the mouse proximal colon (148), estimated to contribute ~70% of basal net Na+ absorption (167). Consistent with this observation, and as described earlier, NHE3−/− mice deprived of dietary Na+ exhibit moderate diarrhea with colonic distention and fluid accumulation, mild metabolic acidosis, lower blood pressure, and a high mortality (147,148). Colonic compensatory mechanisms counteracting the loss of NHE3 include hypertrophy and increased expression and activity of the apical electrogenic sodium channels in the distal colon and a dramatic induction of H+,K+-ATPase (cHKA) mRNA, with the latter presumably representing a K+-sparing mechanism in a state of increased electrogenic Na+ absorption (148).

Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1873 These compensatory increases in electrogenic sodium channels and cHKA were paralleled and likely mediated by an approximately fivefold increase in circulating aldosterone (148).

ACKNOWLEDGMENTS We are thankful to Carol Levine for her editorial assistance in the preparation of this chapter. This work was supported by the National Institutes of Health (grant DK041274).

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Na+-H+ EXCHANGE IN MAMMALIAN DIGESTIVE TRACT / 1879 258. Paradiso AM, Negulescu PA, Machen TE. Na+-H+ and Cl(−)-OH(HCO3−) exchange in gastric glands. Am J Physiol 1986;250: G524–G534. 259. Paradiso AM, Tsien RY, Machen TE. Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator. Proc Natl Acad Sci U S A 1984;81:7436–7440. 260. Kaneko K, Guth PH, Kaunitz JD. Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo. Pflugers Arch 1992; 421:322–328. 261. Sonnentag T, Siegel WK, Bachmann O, Rossmann H, Mack A, Wagner HJ, Gregor M, Seidler U. Agonist-induced cytoplasmic volume changes in cultured rabbit parietal cells. Am J Physiol Gastrointest Liver Physiol 2000;279:G40–G48. 262. Joutsi T, Paimela H, Bhowmik A, Kiviluoto T, Kivilaakso E. Role of Na(+)-H(+)-antiport in restitution of isolated guinea pig gastric epithelium after superficial injury. Dig Dis Sci 1996;41: 2187–2194. 263. Yanaka A, Suzuki H, Shibahara T, Matsui H, Nakahara A, Tanaka N. EGF promotes gastric mucosal restitution by activating Na(+)/H(+) exchange of epithelial cells. Am J Physiol Gastrointest Liver Physiol 2002;282;G866–G876. 264. Stuart-Tilley A, Sardet C, Pouyssegur J, Schwartz MA, Brown D, Alper SL. Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa. Am J Physiol 1994;266:C559–C568. 265. Kulaksiz H, Bektas H, Cetin Y. Expression and cell-specific and membrane-specific localization of NHE-3 in the human and guinea pig upper gastrointestinal tract. Cell Tissue Res 2001;303:337–343. 266. Kirchhoff P, Wagner CA, Gaetzschmann F, Radebold K, Geibel JP. Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands. Am J Physiol Gastrointest Liver Physiol 2003;285:G1242–G1248. 267. Zeuthen T, Meinild AK, Loo DD, Wright EM, Klaerke DA. Isotonic transport by the Na+-glucose cotransporter SGLT1 from humans and rabbit. J Physiol 2001;531:631–644. 268. Lapointe JY, Gagnon MP, Gagnon DG, Bissonnette P. Controversy regarding the secondary active water transport hypothesis. Biochem Cell Biol 2002;80:525–533. 269. Donowitz M, De La Horra C, Calonge ML, Wood IS, Dyer J, Gribble SM, De Medina FS, Tse CM, Shirazi-Beechey SP, Ilundain AA. In birds, NHE2 is major brush-border Na+/H+ exchanger in colon and is increased by a low-NaCl diet. Am J Physiol 1998;274:R1659–R1669. 270. Maher MM, Gontarek JD, Bess RS, Donowitz M, Yeo CJ. The Na+/H+ exchange isoform NHE3 regulates basal canine ileal Na+ absorption in vivo. Gastroenterology 1997;112:174–183. 271. Maher MM, Gontarek JD, Jimenez RE, Donowitz M, Yeo CJ. Role of brush border Na+/H+ exchange in canine ileal absorption. Dig Dis Sci 1996;41:651–659. 272. Yeo CJ, Barry K, Gontarek JD, Donowitz M. Na+/H+ exchange mediates meal-stimulated ileal absorption. Surgery 1994;116:388–395. 273. Wormmeester L, Sanchez de Medina F, Kokke F, Tse CM, Khurana S, Bowser J, Cohen ME, Donowitz M. Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange. Am J Physiol 1998;274:C1261–C1272. 274. Ledoussal C, Woo AL, Miller ML, Shull GE. Loss of the NHE2 Na(+)/H(+) exchanger has no apparent effect on diarrheal state of NHE3-deficient mice. Am J Physiol Gastrointest Liver Physiol 2001;281:G1385–G1396. 275. Woo AL, Gildea LA, Tack LM, Miller ML, Spicer Z, Millhorn DE, Finkelman FD, Hassett DJ, Shull GE. In vivo evidence for interferongamma-mediated homeostatic mechanisms in small intestine of the

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CHAPTER

74

Intestinal Anion Absorption Pradeep K. Dudeja and K. Ramaswamy Mechanisms of Intestinal Sulfate Absorption, 1881 Mechanisms of Intestinal Cl− Absorption, 1882 Electroneutral NaCl Absorption, 1882 Cl−-HCO3− (OH−) Exchange, 1883 Mechanisms of Short-Chain Fatty Acid Absorption, 1901 Diffusion versus SCFA−/HCO3− Exchange, 1901

Molecular Identity of Short-Chain Fatty Acid Transporters: Role of Monocarboxylate Transporters, 1904 Regulation of Short-Chain Fatty Acid Absorption in the Intestine, 1906 Conclusion, 1907 Acknowledgments, 1908 References, 1908

This chapter aims at providing an update about the current understanding of the molecular mechanisms of intestinal absorption of chloride and short-chain fatty acids (SCFAs), two of the most abundant anions in the mammalian small and large intestines, respectively. Since the previous edition of the book was published, the knowledge of the molecular identity of the electrolyte transporters has advanced rapidly. This has warranted the need for separate treatments of the molecular mechanisms of Na+, Cl−, and SCFAs to provide a detailed analysis of the absorption of these molecular mechanisms. In addition to Cl− and SCFAs, this chapter discusses, albeit briefly, the mechanisms of absorption of another anion, sulfate. However, mechanisms of absorption of other less abundant anions, for example, phosphate, are not included here, but are dealt with elsewhere in this textbook (see Chapter 77). Attempts are made to define the general transport mechanisms for Cl− and SCFA− absorption, the identity of the apical and basolateral molecular species of the transporters implicated in their absorption, as well as the mechanisms of their regulation. Because the general mechanisms of the intestinal electrolyte transport and the physiology of their integration are addressed in Chapter 76, this chapter

focuses, therefore, on the molecular identity and regulation of the transporters involved in Cl− and SCFA− absorption.

P. K. Dudeja: Department of Medicine, University of Illinois at Chicago College of Medicine, Research and Development, Jesse Brown VAMC, Chicago, Illinois 60612. K. Ramaswamy: Department of Medicine, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

MECHANISMS OF INTESTINAL SULFATE ABSORPTION Inorganic sulfate is an essential anion required by all organisms to sustain life. Sulfate has been shown to be involved in many physiologic processes including cell growth, development, biosynthesis, and detoxification via sulfation of many endogenous (e.g., steroids, cerebrosides, and catecholamines) or exogenous (e.g., ibuprofen, salicylates, and acetaminophen) compounds (1,2). Sulfate homeostasis in the body depends on the intestinal absorption of dietary sulfate, metabolism of sulfur-containing molecules, and renal reabsorption of inorganic sulfate. Because sulfate is a highly dissociated hydrophilic divalent anion, it cannot simply diffuse through the lipid bilayer of plasma membranes of the cells. Therefore, sulfate movements across the plasma membranes require specialized transporter proteins. This chapter addresses only the intestinal sulfate transporters expressed mainly in mammalian systems. The bulk of the sulfate transport occurs in the jejunum and ileum regions of the intestine. A number of studies in various animal species and in humans have been performed using purified brush-border (BBMVs) or basolateral membrane vesicles (BLMVs) or cells in culture to characterize the mechanisms of sulfate transport across the apical or basolateral domains of enterocytes. Sulfate transport across the apical membranes of the small-intestinal epithelial cells has been shown to be either via a sodium-dependent

1881

1882 / CHAPTER 74 sulfate cotransport or via a sulfate-anion exchange mechanism (3,4). In general, the sulfate transport characteristics of the intestinal brush-border membrane (BBM) and basolateral membrane (BLM) appeared similar to their counterparts in the renal BBM and BLM. For example, sodium-dependent sulfate transporter similar to the renal sulfate transporter was demonstrated to be present in pig jejunal (3) and rat (5) and rabbit (6) ileal BBMs. A sulfate-hydroxyl exchange process stimulated by outwardly directed OH− gradient and inhibited by typical anion exchange inhibitors 4-acetamido-4′isothiocyanatostilbene-2,2′-disulfonic acid (disodium salt) (SITS) and disodium 4,4′-diisothiocyanatostilbene-2,2′disulfonate (DIDS) also was shown to be present in pig jejunal BBM (3) and rabbit ileal BBM (7). Studies performed with differentiated human Caco-2 cells also demonstrated the presence of a sulfate-hydroxyl anion exchange process in apical membranes of these polarized cells, which have been used extensively to characterize electrolyte and nutrient transport characteristics of the human intestine (8). This SO42−-OH− exchanger was shown to be capable of transporting oxalate and chloride, but not nitrate or SCFAs, and appeared to be distinct from the Cl−/OH−, HCO3− exchanger residing in these membranes. In contrast with Cl−/OH−, HCO3− exchanger in apical membranes of these cells exhibiting relative resistance to inhibition by anion exchange inhibitors SITS and DIDS, this SO42−-OH− exchanger was highly sensitive to inhibition by these anion exchange inhibitors (8). Studies directly performed with purified apical membrane vesicles (AMVs) from the proximal colon of the organ donors also demonstrated the existence of a SO42−-OH− exchanger in the luminal membranes of the human colonic AMVs (9). Similar to the above studies using Caco-2 cells, this transporter also was shown to exhibit capacity to transport both oxalate and chloride, but was distinct from the luminal membrane Cl−/OH−, HCO3− exchanger. A sodium-dependent sulfate cotransporter could not be detected in these colonic AMVs (9). A carrier-mediated Cl−-SO42− exchange system similar to renal BLMV has been shown to exist in the BLMs of the rat jejunum (10) and rabbit ileum (11). The ileal BBM, sodiumdependent, sulfate cotransporter system is encoded by NaSi-I (12) and is identical in amino-acid sequence to renal NaSi-I (12). Studies suggest that NaSi-I is electrogenic with potential stoichiometry of 3Na:1SO42 (13). Based on studies in various species, a potential model of sulfate absorption in the mammalian intestine is shown in Figure 74-1. It appears that small-intestinal BBM expresses both the Na-SO42− cotransport and a SO42−-OH− exchanger. Relative roles of these two apical transporters, as well as their regulation, are not well defined. BLM sulfate exit appears to involve the Cl−-SO42− exchange system. Also, the molecular nature of some of these intestinal transporters (e.g., apical SO42−-OH− and basolateral Cl−-SO42− exchanger) has not been fully established. A gene family of sulfate transporters (SLC26 gene family members) has been identified (14). More detailed characteristics of these transporters and their potential role in sulfate and chloride transport are discussed in the following section.

Na+

SO42−

SO42−

OH−

Cl− (HCO3−)

SO42−

FIG. 74-1. Model of sulfate absorption in the mammalian intestine.

MECHANISMS OF INTESTINAL Cl− ABSORPTION Chloride is one of the most important anions in the mammalian intestinal lumen, and it is avidly absorbed throughout the intestinal tract. Its efficient absorption is critical to maintain the optimal levels of this key electrolyte in the body. Chloride has been shown to be absorbed along the length of the intestine via region-specific pathways. In general, three major chloride-absorptive pathways have been proposed: (1) throughout the intestine via a potential difference– dependent pathway; (2) via an electroneutral coupled NaCl absorptive pathway involving parallel functioning of the Na+-H+ exchanger (NHE) and Cl−-HCO3− exchanger mainly present in ileal and colonic regions (proximal colon > distal colon); and (3) via an HCO3− -dependent Cl− absorption pathway mainly in the ileum and colon via a Cl−-HCO3− exchanger, which is not coupled to a parallel NHE. This chapter focuses mainly on electroneutral NaCl absorption specifically involving functioning of the Cl−-OH−, HCO3− exchanger. Electroneutral NaCl Absorption Ample evidence suggests the involvement of electroneutral NaCl absorption in the mammalian ileum and colon as one of the major routes of Na+ and Cl− absorption. This electroneutral absorption could occur via either: (1) an electroneutral NaCl cotransporter; or (2) the coupled functioning of the NHE and Cl−-HCO3− exchangers. The evidence for a functional NaCl cotransporter is almost absent in the luminal membranes of the mammalian intestinal epithelial cells (15–22). This thiazide-sensitive transporter has been shown to be present in the kidney medulla (23) and in the intestine of winter flounder (24). Most of the evidence present in the literature supports the model involving coupled functioning of the Na+ and Cl− transporters (25) rather than a single cotransporter.

INTESTINAL ANION ABSORPTION / 1883 In this model, coupling of the NHE and Cl−-HCO3− exchangers appears to occur via changes in intracellular pH (pHi) and results in electroneutral NaCl absorption. The efflux of H+ via NHE makes the cytoplasm alkaline, which activates the Cl−-HCO3− exchanger functioning. The intracellular substrates, H+ and HCO3− , for these transporters are produced by the action of carbonic anhydrase. The net result is movement of Na+ and Cl− into the cell in exchange for the exit of H+ and HCO3− . The chloride at the BLM can exit either via a KCl cotransporter (26) or via the functioning of another anion exchanger (AE), for example, the Cl−-HCO3− exchanger (27,28). The identity and function of the transporters involved in basolateral Cl− efflux are not yet fully established. The clinching evidence for the coupling of the dual ion exchangers in NaCl absorption was derived from genetic disorders. Two different genetic disorders have been reported that are known to selectively abolish sodium or chloride absorption (29–32). For example, congenital sodium diarrhea and congenital chloride diarrhea (CLD) disorders involve selective impairment of intestinal sodium or chloride absorption, respectively, and are considered to be caused by the aberrant enterocyte luminal membrane ion transporters (29,30,33). Because both of these disorders are known to be autosomal recessive, this bodes well in support of the existence of two distinct transporters (one each for Na+ and Cl−) involved in NaCl absorption. Cl−-HCO3− (OH−) Exchange Cl−-HCO3− exchangers are key intestinal transporters known to play important roles in transepithelial chloride absorption, bicarbonate secretion, and maintenance of pHi and chloride concentrations. Numerous studies using purified AMVs or BLMVs from different regions of the intestine to measure pH gradient–driven 36Cl− uptake or using cell-culture models, as well as intact tissues, have shown the presence of these exchangers in the intestine. These exchangers have been shown to function in parallel with the NHE for electroneutral NaCl absorption both in colonocytes and ileal cells. Various studies have reported significant regional, surface-crypt axis and species differences with respect to the expression of these transporters and their regulation (18,19,21,34–38). A model of the expression of Cl−-HCO3− (OH−) and NHEs in luminal membranes of surface cells comparing their roles in various regions of the intestine (ileum, proximal, and distal colon) in rat, rabbit, and human is shown in Figure 74-2. Studies in rat distal colon using purified AMVs have shown the presence of two distinct exchangers (i.e., a Cl−-HCO3− and a Cl−-OH− exchanger) expressed on these membranes (39). In these studies, HCO3− gradient–driven uptake of Cl (Cl−-HCO3− exchange) into the vesicles was further stimulated by imposition of an outwardly directed OH− gradient. In contrast, OH− gradient–driven Cl uptake (Cl−-OH− exchange) was not stimulated in the presence of additional outward HCO3− gradient. Based on these observations, together with further evidence of distinct kinetic parameters and characteristics of inhibition

by the anion exchange inhibitor DIDS, the existence of two distinct chloride-transporting AEs on the luminal membranes of these cells was proposed (see proposed model in Fig. 742). It also was shown that although Cl−-HCO3− exchange was expressed mainly on apical membranes of surface cells, Cl−OH− exchange was shown to be present throughout the cryptsurface cell axis, but with much greater activity in surface cell membranes (40). The existence of two distinct chloride transporters in rat colonic AMVs was further supported by studies demonstrating differential regulation of the two transporters by aldosterone secondary to dietary salt deprivation in rats (40). Another earlier study in rat jejunal and ileal luminal membranes had also suggested the existence of two separate AEs in chloride transport (41). Although the physiologic significance of the two distinct electroneutral AEs for chloride transport in rat colon is not yet known, it is suggested that Cl−-HCO3− exchange may be the predominant chlorideabsorbing transporter, whereas Cl−-OH− exchange process may be important for other cellular functions, for example, maintenance of pHi (39,40). Studies with purified AMVs or in vitro studies with intact tissues from rabbit ileal (15), distal colon (34), human ileal (21), and colonic regions (17,20) also have shown the presence of an electroneutral Cl−-HCO3− (OH−) exchange process in luminal membranes of epithelial cells. Analysis of the possible existence of distinct Cl−-OH− versus Cl−-HCO3− exchange similar to the rat colon, did not support the existence of two distinct transporters in the human colonic membrane vesicles (17,20). In rabbit ileum, Cl−-HCO3− exchange activity was shown to be present in both the crypt and villus cells (42). Because the predominant sodiumabsorptive Na+-H+ exchanger isoform NHE3 is mainly expressed in apical membranes of surface cells, it is conceivable that Cl−-HCO3− (OH−) exchange in the surface cells is involved in electroneutral NaCl absorption, whereas the presence of Cl−-HCO3− or Cl−-OH− exchange in crypt cell membranes may have roles other than electroneutral NaCl absorption. The evidence for the existence of a Cl−-HCO3− exchange process in the human ileal and colonic luminal membranes also is derived from the congenital CLD disorder (30,33). In this disease, the large volumes of fecal fluids are known to contain high chloride concentrations, and metabolic alkalosis is observed. In vivo perfusion studies in the ileum and colon of these patients indicated gross impairment of chloride absorption parallel to an absence of bicarbonate secretion (30). These findings could be easily explained by an aberrant or missing Cl−-HCO3− exchange process in luminal membranes of the ileal and colonic epithelial cells (30,33). Furthermore, it appears that the presence of two distinct luminal chloride transporters, that is, Cl−-OH− and Cl−-HCO3− exchangers, is not a generalized phenomenon, because except for the rat intestine, no evidence for this effect could be seen in the human and rabbit intestinal luminal membranes. In the human intestine, a single transporter appears to function as a Cl−-OH− or Cl− -HCO3− exchanger (17,20). It should be clear from this

1884 / CHAPTER 74 Na+ NHE

H+

HCO3−

Na+

Na+

Proximal colon

Na+

Cl−

H+

HCO3−

HCO3−

Cl−

Na+

NHE

Distal colon

Cl−

NHE

Cl−

Cl−

HCO3−

Na+

Cl−

H+

H+

H+

HCO3−

NHE

NHE

Cl−

NHE

NHE H+

Ileum

Na+

Cl−

Na+

Cl−

NHE

H+

HCO3−

OH−

Rat

HCO3−

Rabbit

H+

HCO3−

Human

FIG. 74-2. Expression of the Cl−-HCO3− and Cl--OH− exchangers in luminal membranes of surface cells in the rat, rabbit, and human. NHE, Na+-H+ exchanger.

discussion that it is not appropriate to make a generalized statement with respect to the identity and mechanism of chloride transport without taking into account the species and regional differences. Studies performed using either purified BLMVs, cells in culture, or intact epithelial tissues mounted in perfusion chambers also have provided evidence for the existence of distinct BLM Cl−-HCO3− exchange processes in rat jejunal (43) and colonic (27) and in guinea pig (44) and human colonic epithelial cells (45). In contrast, a basolateral Cl−-HCO3− exchange process was shown to be absent in rabbit ileal crypt and villus epithelial cells (42). The role of basolateral transporters in transepithelial chloride transport is not entirely clear and may depend on ion gradients. These exchangers may play important roles in cell pH maintenance or other functions that currently remain to be defined. Regulation of Intestinal Cl−-HCO3− Exchange This section reviews studies about the physiologic regulation of mammalian (more importantly, human) intestinal Cl− -HCO3− exchange as the molecular identity of these

transporters has not yet been fully established. Figure 74-3 provides a diagrammatic summary of some of these regulatory pathways. Effects of Short-Chain Fatty Acids SCFAs, the most abundant anions in the colonic lumen, have long been known to stimulate NaCl absorption and inhibit Cl− secretion in the rabbit and rat distal colon. However, little information is available regarding the molecular mechanisms of the effects of SCFAs on Cl− uptake in the intestinal epithelial cells. With regard to stimulation of Cl− absorption by SCFA in the rat distal colon, three potential mechanisms were suggested to be responsible: (1) the generation of intracellular HCO3− during the oxidation of SCFAs as providing substrate for the apical Cl−-HCO3− exchanger; (2) the activation of volume-sensitive basolateral Cl− channels by SCFAs (46); and (3) the functioning of a unique unidirectional SCFA-Cl exchanger in rat colonic AMVs (for a more detailed discussion, see the Mechanisms of Short-Chain Fatty Acid Absorption section later in this chapter). Further studies are necessary to fully elucidate the molecular

INTESTINAL ANION ABSORPTION / 1885

−

− −

FIG. 74-3. Mechanism(s) of regulation of the human intestinal luminal membrane Cl--OH− (HCO3−) exchange. 5-HT, 5-hydroxytryptamine; cGMP, cyclic guanosine monophosphate; EPEC, enteropathogenic Escherichia coli; NO, nitric oxide; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PKG, protein kinase G; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; sGC, soluble guanylate cyclase. (Modified from Gill and colleagues [96], by permission.)

mechanisms of the effects of SCFAs on the luminal Cl− uptake in the human intestine. Role of Bile Acids Bile acid malabsorption has been shown to induce diarrhea (47) in various conditions. The underlying mechanisms of induction of diarrhea by bile acids are not fully known (47). In in vitro studies with short-circuited rabbit colon, addition of mucosal taurochenodeoxycholic acid (TCDC) was found to decrease net sodium flux by about 50%, and the net chloride flux was almost completely abolished (48). In that study, the addition of TCDC to the serosal compartment also abolished net sodium flux and stimulated electrogenic Cl− secretion (48). Previous studies also showed decreased net Na+ absorption and increased net Cl− secretion across rabbit jejunal mucosa (49) in response to TCDC. These results indicated that increased serum bile acid levels seen in chronic liver diseases could also modulate the colonic electrolyte and water transport. Perfusion studies performed in the human colon showed induction of water and electrolyte secretion by dihydroxy bile acids (50). The mechanisms of the effects of bile acids on the intestinal epithelial cells have been shown to involve multiple signal transduction molecules including intracellular cyclic adenosine monophosphate (cAMP), Ca+, and protein kinase C (PKC) (51–54). In other studies, the involvement of certain inflammatory mediators in this process also has been suggested (55). The effects of bile acids on Cl− uptake and the elucidation of the underlying mechanisms in the human intestine were examined using the Caco2 cell line as an experimental model (56). Caco-2 cells have been used extensively as an excellent experimental model to

investigate the mechanisms and regulation of a wide variety of nutrients and electrolytes in the human intestine (56–62). In one study, taurodeoxycholic and glycochenodeoxycholic acids were shown to reduce the apical Cl−-OH− exchange in Caco-2 cells via Ca+, phosphatidylinositol 3-kinase (PI3K), and PKC-dependent pathways (56) (see Fig. 74-3). Role of Inflammation In villus cells of the rabbit ileum, chronic inflammation has been shown to inhibit luminal membrane Cl−-HCO3− exchange activity (63). This inhibition was shown to be caused by changes in the affinity (Km) of the transporter for its substrate chloride, rather than changes in maximal velocity of the transporter. Glucocorticoid treatment, which has been shown to stimulate Cl−-HCO3− exchange in normal intestine by increasing the maximal velocity of this transporter, also was found to alleviate the inhibitory effects of inflammation on this transporter via restoring the changes in its affinity. Furthermore, a number of inflammatory mediators, which are increased in inflammatory disorders, such as substance P, interleukin-1β (IL-1β), and nitric oxide (NO), also have been shown to be involved in modulation of electrolyte absorption in enterocytes and colonocytes. However, no direct effects of these modulators, except NO (see later discussion), on intestinal Cl−-HCO3− exchange activity have been studied in detail. Role of Nitric Oxide NO has been shown to inhibit NaCl absorption in the intestine (64,65). Under normal physiologic conditions, NO

1886 / CHAPTER 74 has been suggested to be proabsorptive, whereas under pathophysiologic conditions, it has been shown to stimulate net secretion (66). Constitutive nitric oxide (cNO) was shown to have differential effects on crypt and villus Cl−HCO3− exchange activity (67). For example, cNO was shown to have no effect on the villus cell Cl−-HCO3− exchange activity, but was suggested to stimulate crypt cell Cl−-HCO3− exchange activity (67). NO was shown to inhibit the apical membrane Cl−-OH− exchange activity in Caco-2 cells (58). The inhibition of Cl−-OH− exchange activity was shown to be mediated via both cyclic guanosine monophosphate/PKG and PKC signaling pathways (see Fig. 74-3). In parallel studies, apical NHE3 activity also was shown to be inhibited by NO in Caco-2 cells (57). Because inhibition of NHE activity by NO was not mediated via PKC signaling, the NO-induced decrease in Cl−-OH− exchange activity appears to be a specific phenomenon, rather than being secondary to inhibition of NHE. The molecular studies pinpointing the nature of the AE regulated by NO have not been performed and would be of interest. Regulation by Hormones and Neuropeptides A number of studies have shown previously that several hormones and neuropeptides modulate intestinal electrolyte and water absorption in various species including rat, rabbit, and dog (68). For example, an increase in NaCl absorption has been shown to occur in response to catecholamines (69–72) glucocorticoids (73,74), thyroxine (75), and the neuropeptide YY (76,77), whereas serotonin (78–80) was shown to inhibit electroneutral NaCl absorption. A number of other molecules, for example, neurotensin, vasoactive intestinal peptide, acetylcholine, guanylin, prostaglandins, leukotrienes, histamine, bradykinin, adenosine, and platelet-activating factor, also have been shown to be prosecretory and antiabsorptive in nature (68). However, no direct studies evaluating the effects of these molecules on Cl−-HCO3− exchange activity in the mammalian intestine are available. Aldosterone in rat colon was shown to have differential effects on NaCl absorption in the proximal and distal colon (81,82). For example, in the proximal rat colon, aldosterone was shown to stimulate NaCl absorption, whereas in the rat distal colon, aldosterone was shown to inhibit NaCl absorption. Studies on the effects of aldosterone on Cl−-OH− exchange versus Cl−-HCO3− exchange in rat distal colon also demonstrated differential effects on these two transporters (83,84). For example, aldosterone increase secondary to feeding low-salt diet to rats was shown to completely abolish the surface cell Cl−-HCO3− exchange activity in the rat distal colon parallel to decreased expression of AE1; however, the crypt cell Cl-OH− exchange activity was unaffected (83,84). Based on these observations, it was further suggested that surface cell Cl−-HCO3− exchange activity may be mainly involved in luminal chloride absorption, whereas the crypt cell Cl−-OH− exchange activity may have other functions such as the maintenance of pHi. With the exception of aldosterone, however, it is not entirely clear whether the

reported modulation of intestinal chloride absorption by some of the other hormones and neuropeptides mentioned earlier is a result of the direct modulation of the intestinal luminal Cl−-HCO3− exchanger, or whether it is simply a secondary phenomenon to alteration in the luminal NHE function because the functions of the two exchangers are coupled. Furthermore, little information is available with respect to the direct effects of these agents on AEs. Studies using Caco-2 cells as an experimental model demonstrated that the apical Cl−-OH− exchange (85) and NHE (86) activities were inhibited in response to acute treatment with serotonin in the human intestine. The results of these studies indicated that the inhibition of Cl− uptake by serotonin was mediated via the activation of both 5-hydroxytryptamine subtype 3 (5-HT3) and 5-HT4 serotonin receptors, and that it was Ca2+ independent and occurred via src kinase family and novel PKC–mediated pathways (see Fig. 74-3). Because the receptors and the signaling pathways involved in the inhibition of Cl− uptake were distinct from those delineated with respect to the inhibition of Na+ uptake (86), the data presented in that study clearly showed a specific, direct effect of serotonin on Cl−-OH− exchange activity in Caco-2 cells. Bacterial Infections Intestinal infection by several bacterial species has been shown to cause secretory and inflammatory diarrhea. However, limited studies investigating a possible direct effect of these pathogens or their toxins, or both, on intestinal Cl−-HCO3− exchange activity are available. In this regard, the pathophysiologic basis of early diarrhea resulting from enteropathogenic Escherichia coli (EPEC) infection has long eluded the scientific community. Several studies, however, demonstrated that infection with EPEC, but not with nonpathogenic E. coli, inhibited the apical NHE3 (87) and the Cl−-OH− exchange (88) activity in Caco-2 cells. EPEC holds a type III secretory system (TTSS) that translocates bacterial proteins directly into the host cells (89). In these studies, ATPase for TTSS was found to be important for the effect of EPEC on Cl−-OH− exchange, because the mutations in the gene that encodes the ATPase for TTSS (escN) abolished the effect of EPEC (see Fig. 74-3). Inhibitors of signaling pathways, for example, PKC and PI3K, failed to block the EPEC effects, ruling out the involvement of these molecules (88). Additional studies to elucidate the detailed mechanism(s) of Cl−-OH− exchange and NHE3 inhibition by EPEC would be of great significance for a better understanding of the pathophysiologic basis of EPEC-induced early diarrhea. Role of Protein Kinase C PKC activation previously has been shown to stimulate chloride secretion and to inhibit NaCl absorption in the rat colon (90). Studies using Caco-2 cells as an experimental model and phorbol 12-myristate 13-acetate (PMA) as an in vitro PKC agonist have shown the regulation of the apical membrane Cl−-OH− exchange activity. Acute treatment with

INTESTINAL ANION ABSORPTION / 1887 PMA was shown to inhibit Cl−-HCO3− exchange activity in these cells via the activation of PI3K and its downstream effector, PKCε (91). Future studies to elucidate the molecular mechanisms of regulation of human intestinal apical Cl−-HCO3− exchanger by PKC would be of interest with respect to the involvement of either direct phosphorylation of the exchanger, membrane trafficking events, or the accessory regulatory proteins. Role of Reactive Oxygen Metabolites Reactive oxygen metabolites (ROMs) are known to play an important role in the pathophysiology of several intestinal diseases including inflammatory bowel disorders (92–94). Studies using Caco-2 cells as an in vitro model and hydrogen peroxide (H2O2) as an important ROM examined the acute in vitro effects of H2O2 on Cl−-HCO3− exchange activity. H2O2 was found to acutely inhibit the Cl−-HCO3− exchange activity in Caco-2 cells via the involvement of PI3K- and PKC-mediated pathways (95). These studies show an important role of ROMs in modulation of the intestinal electrolyte processes. Additional studies examining the molecular mechanisms underlying the effects of various ROMs on the human intestinal Cl−-HCO3− exchange activity and their potential significance in inflammatory disorders would be of interest. Molecular Identity of Cl−-HCO3− Exchangers Advancements in identification of the molecular species involved in these multiple chloride and bicarbonate transporting AEs have identified many candidate proteins

that may be involved in apical and basolateral chloride-anion exchange. However, the exact role of each of these molecular entities and their physiologic function remain to be defined. Among the candidate genes for the human intestinal Cl−-HCO3− exchange process, two gene families are of great interest: (1) a bicarbonate transporter superfamily (SLC4 family), which includes electroneutral sodium-independent Cl−-HCO3− exchangers or AEs, and the electrogenic sodium-bicarbonate cotransporters, or NBCs; and (2) a “sulfate permease” anion transporter family SLC26. The following sections review in detail the SLC4 and SLC26 gene family members implicated in intestinal anion exchange. SLC4 Gene Family Advances in molecular biology have enabled the identification and the characterization of the intestinal anion transporters at the molecular level. Several AEs of the SLC4 gene family of transporters are expressed in the small intestine and colon (96). SLC4 or bicarbonate-transporter gene family encodes for 10 integral plasma membrane proteins that are involved in bicarbonate transport processes across plasma membranes (97). Table 74-1 shows a list of these SLC4 gene family members cloned to date, together with their tissue distribution and functional roles. Based on their dependency on Na+ and Cl−, members of the SLC4 family could be divided into three functionally distinct groups: (1) Na+-independent Cl−-HCO3− AEs (98); (2) NBCs (99); and (3) Na+-dependent Cl−-HCO3− exchangers (NDCBE) (97). A striking similarity among all transporters of SLC4 gene family is the inhibition of their activity by disulfonic stilbene derivatives such as DIDS and SITS (97,98).

TABLE 74-1. SLC4 family of anion exchangers Human gene

Protein name or alias

Transport type

Substrate

Tissue Distribution

Reference

AE1 Band 3 AE2 AE3

Exchanger

Cl−, HCO3−

83, 97, 108–109

Exchanger Exchanger

Cl−, HCO3− Cl−, HCO3−

SLC4A4

NBCe1 NBC NBC1

Cotransporter

Na+, HCO3−, and/or carbonate

SLC4A5

NBCe2 NBC4 NBCn1 NBC2 NBC3 NDCBE kNBC3 AE4 NCBE BTR1

Cotransporter Cotransporter

Na+, HCO3−, and/or carbonate Na+, HCO3−

Erythrocytes, kidney, heart, colon Widespread Brain, retina, kidney, GI tract, smooth muscle NBCe1A: kidney, eye, intestine NBCe1B: widespread NBCe1C: brain Spleen, liver, testes

Exchanger and cotransporter Uncertain Uncertain Unknown

Na+, HCO3− Cl− Uncertain Uncertain Unknown

SLC4A1 SLC4A2 SLC4A3

SLC4A7 SLC4A8 SLC4A9 SLC4A10 SLC4A11

28, 111–114 28, 115–119 120–124

97, 125, 126

Widespread, but not skeletal muscle

97

Kidney, testes, ovary, brain

97, 127

Kidney Brain Kidney, testes, thyroid, trachea, salivary gland

103–105 127 97, 106

AE, anion exchanger; BTR, bicarbonate transporter; GI, gastrointestinal; NBC, sodium-bicarbonate cotransporter. Modified from Romero et al (97), by permission.

1888 / CHAPTER 74 Polypeptides of SLC4A1 (AE1), SLC4A2 (AE2), and SLC4A3 (AE3) mediate electroneutral Cl−-HCO3− exchange across plasma membranes (100), whereas SLC4A4 (NBCe1 or NBC1) and SLC4A5 (NBCe2 or NBC4) are electrogenic NBCs (99). SLC4A7 (NBCn) functions as electroneutral NBC (101). SLC4A8 (NDCBE) is also an electroneutral transporter that operates as an Na+-driven Cl−-HCO3− exchanger (102). Although SLC4A10 (NCBE) is 71% to 76% identical to the other Na+-dependent SLC4 transporters, it is inconclusive whether it functions as an Na+driven Cl−-HCO3− exchanger or as electroneutral NBC (97). SLC4A9 (AE4) was shown to function as an electroneutral Na+-independent Cl−-HCO3− exchanger, which is inconsistent with its high homology to NBC, but not to AEs (103–105). SLC4A11 (BTR) is the least closely related to other members of the SLC4 gene family, and its function remains to be determined (106). With the exception of AE4 and AE1, members of this gene family expressed in the intestine have been shown to be localized to the BLMs of epithelial cells; ruling out the possibility that they mediate luminal Cl−-HCO3− exchange process (28,97). However, the role of the intestinal BLM AE members of SLC4 family is not entirely clear, that is, whether they are involved in vectorial chloride transport or other functions, for example, maintenance of pHi (97). Intestinal NBC cotransporters expressed on the BLMs have been suggested to play an important role in HCO3− secretion in the pancreatic duct and duodenum (97,107). This section focuses on elaborating the structure-function characteristics and regulation of the AEs of the SLC4 gene family expressed in the

1

2

3

4

5

6

gastrointestinal (GI) tract (e.g., AE1-4) and their role in anion transport. Membrane Topology of Anion Exchangers. AE1 is the most extensively studied anion transporter with a crystallographic structure reported (128). Members of AE gene family share structural features similar to the membrane topology of AE1. AE polypeptides appear to be composed of two structurally and functionally distinct domains: a long cytoplasmic N terminal and a C terminal consisting of 13-14-transmembranespanning domains followed by a short cytoplasmic terminus (Fig. 74-4) (97,98). The N terminal is responsible for binding to other proteins such as ankyrin in case of AE1 and AE3 (129) and to hemoglobin, glycolytic enzymes (glyceraldehydes 3-phosphate dehydrogenase, phosphofructokinase, and aldolase), and other cytoskeletal proteins for the anchorage of AE1 in red blood cells (130). Other domains in the N terminal were identified as important elements for the acute regulation of AE2 by pH and NH4+ (131–133). The C terminal is involved in the transport activity of the AE and harbors a potential DIDS-binding motif (KLXK) proposed to be located in the third extracellular loop (115) and a binding site for the cell surface–anchored carbonic anhydrase IV on the fourth extracellular loop of human AE1 (134). Also, the cytoplasmic portion of the C terminus of human AE1, as well as AE2 and AE3, mediates the binding to the soluble carbonic anhydrase II that is essential for proper Cl−-HCO3− exchange activity of the anion transporter (135). One interesting feature of AE polypeptides is their homodimerization in cell plasma membranes, as well as in detergent solutions (130,136,137). Functional studies, however, do not support

7

8

9

10

11

12

13

COOH

N-Glycosylation site H 2N

CAII Binding motif KLXK (DIDS motif)

FIG. 74-4. Predicted membrane topology of an anion exchanger (AE1). (Modified from Romero and colleagues [97], by permission.)

INTESTINAL ANION ABSORPTION / 1889 the presence of various binding sites for the substrate, suggesting that monomers of the AEs are sufficient to elicit their transport activities (98). AEs have a number of splice variants that arise from alternative promoters of the same gene and differ in their N-terminal regions, as shown in Figure 74-5 (98). AE1 gene has two different variants—erythrocyte eAE1 transcript and a shorter kidney kAE1 splice variant (111)—whereas AE2 encodes for five variants (AE2a, AE2b1, AE2b2, AE2c1, and AE2c2) (113,138). Also, two AE3 transcripts have been described: brain bAE3 and cardiac cAE3 (139). Intriguingly, each of mouse bAE3 and cAE3 has a variant that lacks the membrane-spanning domain (98) (see Fig. 74-5). The intestinal epithelial cells have been shown to selectively express AE variants. For example, in contrast to AE2a and AE2b variants, AE2c splice variants demonstrated the highest level of expression in the stomach compared with the rest of the GI tract (114). In the rat distal colon, although AE1 expression was demonstrated (83), the identity of the rat colonic variant currently is unknown. Also, of the two AE3 variants bAE3 and cAE3, only bAE3 was shown to be expressed in the human intestinal epithelial cells (28). SLC4A1/AE1. AE1, or band 3, represents the prototype member of the SLC4 gene family, and its complementary DNA (cDNA) was the first to be cloned from murine anemic spleen (140). In humans, the AE1 gene maps to chromosome

17q12 and was found to transcribe two variants differing in their N terminal (141). Human erythrocyte eAE1 consists of 911 amino acids and has a 404-amino-acid-long cytoplasmic N terminus, whereas the shorter kidney kAE1 lacks the first 65 amino acids in its N terminus compared with eAE1 (141). Mutations in the AE1 gene lead to changes in the shape of red blood cells (98). Some of the AE1 mutations are known to cause only an alteration in erythrocyte morphology with no apparent clinical manifestations, as in the case of the Southeast Asian Ovalocytosis (142). However, other mutations were described as the genetic cause of hereditary spherocytosis associated with hemolytic anemia (143). Also, AE1 knockout mice (AE1−/−) were shown to experience severe neonatal hemolytic anemia (144). Intriguingly, both Southeast Asian Ovalocytosis and hereditary spherocytosis cases are associated with normal renal function (98). In contrast, different sets of mutations were found to cause distal renal tubular acidosis, an autosomal recessive congenital disorder, with no changes in the phenotype of red blood cells (145,146). No intestinal or colonic genetic disorder currently has been described in association with AE1 mutations. The functional characteristics of AE1 were examined by heterologous expression in Xenopus oocytes and several cell lines (98). AE1 operates as Na+-independent electroneutral Cl−-HCO3− exchange across the plasma membrane and could also transport SO4− and OH− in exchange for Cl−, but with low

TM eAE1 kAE1

N

C

AE1

AE2a AE2b1 AE2b2

AE2

AE2cl AE2c2

bAE3 cAE3

AE3

bAE3-14nt cAE3-14nt

AE4a-human AE4a-rabbit

AE4 AE4b-human AE4b-rabbit

FIG. 74-5. Splice variants of the anion exchangers (AEs) of the SLC4 gene family. (Modified from Alper and colleagues [98], by permission.)

1890 / CHAPTER 74 affinity to SO4− − compared with Cl− and HCO3− (147). Also, AE1 could transport OH− ions as well, although it is predicted to function as luminal Cl−-HCO3− (148,149), but not the Cl−-OH− exchanger in the rat distal colon (83). AE1 is highly expressed in erythrocytes, where eAE1 represents the major protein of the plasma membrane of red blood cells (97). Kidney AE1 polypeptide was localized to the BLM of type A intercalated cells of the renal collecting duct (108). AE1 in red blood cells plays a crucial role in the elimination of CO2 from the peripheral tissues and in its shuttling to the lung (97). In the kidney, the activity of AE1 is important for reabsorption of the HCO3− from the lumen of the collecting duct to the blood (97). AE1 transcripts also were detected at lower levels in the heart (109). AE1 also was reported to be expressed in the rat intestine and distal colon (83). Charney and colleagues (150) have provided evidence demonstrating the localization of rat AE1 polypeptide on the microvilli of intestinal epithelial cells, suggesting that rat AE1 is responsible for the intestinal luminal Cl−-HCO3− exchange activity in the rat intestine and distal colon. It appears that intestinal expression and luminal localization of the AE1 is specific to the rat only, because in the human GI tract, the expression of AE1 messenger RNA (mRNA) was not detected in any region of the small intestine and colon (28). These conflicting data again emphasize that it is not advisable to simply extrapolate the findings of animal models to understand the human intestinal electrolyte absorption. SLC4A2/AE2. AE2 or nonerythrocyte band 3 AE was first cloned from murine kidney (151). Human AE2 gene is mapped to chromosome 7q35 and transcribes for five different N-terminal splice variants, as described earlier (see Fig. 74-5) (138,152). AE2a is widely expressed in all the tissues tested, indicating its possible role as a housekeeping gene, whereas AE2b1 and AE2b2 variants are found predominantly in epithelial cells, suggesting that they mediate tissue-specific functions (97,153). However, AE2c variants are exclusively expressed in the stomach, demonstrating their importance in gastric epithelial function (114). Furthermore, the level of AE2c mRNA shows differential expression among various gastric cell subtypes with higher expression in parietal cells compared with mucous cells, indicating that the pattern of expression of AE2 variants is likely to be a cell-specific but not organ-specific phenomenon (154). The ubiquitous nature of AE2 strongly supports the results of various studies establishing its essential role in the maintenance of pHi and Cl− concentration and the regulation of cell volume (97). With the exception of the polarized hepatocytes (155), cellular localization of AE2 polypeptide in all epithelial cells studied was found to be on the BLM such as in alveolar epithelial cells, choroids-plexus, and renal collecting duct (98). Studies have shown that the basolateral targeting of the AE2 polypeptide in epithelial cells is not related to its intrinsic elements, but rather is related to the cell type where AE2 is expressed. This conclusion is based on demonstrating the apical targeting of exogenously expressed AE2a, AE2b1, and AE2b2 variants in primary hepatocytes and their basolateral sorting in Madin–Darby canine kidney (MDCK) epithelial

cells (155). However, the cellular factors involved in AE2 membrane targeting remain unclear. AE2 is localized on the BLMs of rabbit ileal epithelial cells (156). Similarly, AE2 was demonstrated to be expressed on the BLM of murine epithelial cells along the entire length of the GI tract (114). In humans, AE2 mRNA was found to be expressed throughout the intestine and the colon (28). Also, in situ reverse transcriptase-polymerase chain reaction (RT-PCR) showed AE2 mRNA expression to be restricted to the epithelial cells of human colon and to be evenly distributed along the surface-crypt axis (28). Furthermore, the polypeptide products for AE2 also were found to be localized to the basolateral, but not the apical, membranes of the epithelial cells of the human intestine (28). In the stomach, the AE2 protein also is localized to the BLM of gastric parietal cells, and its function was suggested to be essential for HCl secretion via removing intracellular HCO3− generated by the action of carbonic anhydrase to counter the effect of H+ secretion through luminal H+,K+-ATPase (153). Findings demonstrating the presence of another Cl−-HCO3− AE, SLC26A7 that belongs to SLC26 gene family, suggested that the importance of AE2 for gastric secretion of HCl might be partial (157). However, the observation that AE2−/−− knockout mice, where all variants are absent, were found achlorhydric provides compelling evidence that, at least in mice, the activity of AE2 is pivotal for gastric acid secretion and could not be compensated by the function of SLC26A7 (153). Notably, this impairment in gastric function was not reported in another AE2 knockout model, where the expression of AE2c was intact (158). Intriguingly, no intestinal-related disorder or abnormal phenotype has been described in the two models of AE2 knockout mice. SLC4A3/AE3. The expression of human AE3 was demonstrated in intestinal epithelial cells together with AE2 (28). SLC4A3 AE (AE3) also is highly expressed in other tissues such as in the heart, brain, retina, smooth muscle, and kidney (98,115,117,119,159). The human AE3 gene is located on chromosome 2q36, and AE3 mRNA transcription occurs from two alternative promoters that cause two N-terminal spliced variants: brain bAE3 and cardiac cAE3, as described previously (118). AE3 was shown to function as a Cl−-HCO3− exchanger that is more sensitive to inhibition by DIDS compared with AE2 (160). With respect to AE3 cellular localization, Alper and colleagues (98) reported that rodent cAE3 polypeptide is localized to apical membrane of polarized renal epithelial cells, whereas bAE3 protein was found to localize to the BLM. In the intestinal epithelial cells, these authors (98) reported that bAE3 can be found on both the apical membrane and the BLM. In the human intestine, the expression of bAE3 but not cAE3 mRNA was demonstrated along the length of the small intestine and colon (28). Furthermore, bAE3 protein was expressed predominantly on the basolateral but not the apical membrane of the epithelial cells of the human GI tract (28). The importance of expression of both AE2 and bAE3 on the BLMs of the human intestinal epithelial cells currently is unclear; it is possible that the two transporters

INTESTINAL ANION ABSORPTION / 1891 stark differences with respect to the role of SLC4 members in the two species. Regulation of SLC4 Anion Transporters Effect of Dietary Na+-Depletion on AE1 Expression. AE1 is expressed in the rat but not the human distal colon (83). AE1 polypeptide was shown to be localized to apical membranes of the colonocytes, together with DRA (downregulated in adenoma) protein, another AE of the SLC26 gene family (150). Previously, the inhibition of surface cell Cl−-HCO3− exchange, but not the crypt cell Cl−-OH− exchange, in apical plasma membrane vesicles of the rat distal colon was demonstrated in response to high aldosterone levels induced by dietary Na+ depletion in rats (83). The expression of rat colonic AE1 mRNA was shown to be decreased by dietary Na+ depletion, whereas DRA mRNA remained unchanged (83). These observations prompted the conclusion that, in the rat colon, DRA represented the luminal membrane Cl−-OH− exchanger, which was aldosterone insensitive, whereas AE1 encoded the apical membrane Cl−-HCO3− exchanger in the rat distal colon, which was sensitive to aldosterone (83). Vesicular Trafficking of AE1. Luminal Cl− absorption in the rat distal colon was shown to be stimulated by exposure to CO2 (150,161). Further analysis using inhibitors for vesicular trafficking and cell-surface biotinylation techniques demonstrated that, in parallel to the stimulation of Cl− absorption, exposure to CO2 also increased the level of AE1 expression on the luminal membrane of the colonocytes by stimulating the exocytosis of subapical vesicles containing AE1 polypeptide to the apical membrane (150). These studies were the first of its kind demonstrating acute regulation of the intestinal luminal Cl−-HCO3− exchanger in the rat colon by vesicular trafficking of AE1. Ontogenic Regulation of AE2 Expression. AE2 mRNA expression in the rat intestine showed a pattern that was altered during development, indicating its ontogenic regulation (162). AE2 mRNA is significantly decreased at a time representing the transition from suckling to weanling and

play differential roles and are differentially regulated. One of the AEs may be important for housekeeping functions (e.g., maintenance of cell volume and pH), whereas the other may be important for vectorial chloride transport. SLC4A9/AE4. SLC4A9 or AE4 was initially cloned from rabbit kidney (103). AE4 polypeptide was demonstrated to be expressed on the apical membrane of gastric mucous cells and duodenal epithelial cells in mice, rabbits, and humans (104). These interesting findings present AE4 as an important member of the SLC4 gene family to be expressed on the luminal surface of intestinal epithelial cells. Human AE4 gene was mapped to chromosome 5q31 and transcribes at least three N-terminal spliced variants in rabbits and humans (106). AE4 was located to the apical membranes of type B intercalated cells of the renal collecting duct (103). These findings disagree with observations demonstrating the localization of AE4 protein on the BLM of type A intercalated cells of the collecting duct of mouse and rat (105). The amino-acid sequence of AE4 appears to be more homologous to that of the NBC compared with the AE subfamily (97). However, expression studies of rodent and rabbit AE4 in COS-7 cells, HEK293 (human embryonic kidney) cells, and Xenopus oocytes showed an Na+-independent electroneutral Cl−-HCO3− exchange activity similar to the function of AEs (103–105). With respect to inhibition by DIDS, initial studies showed that rabbit AE4 was insensitive to DIDS (103); however, mouse and rat AE4 was shown to be DIDS sensitive (105), despite that AE4 protein lacks the classical DIDS-binding domain (97). Given the conflicting results of the studies with regard to AE4 cellular localization and its functional characteristics among species, future investigations are warranted to critically examine the expression of AE4 in the epithelial cells of the human small intestine and colon and to define its functional role and involvement in Cl− absorption. Figure 74-6 depicts the expression of the members of the SLC4 family on the apical membrane and BLM domains of the rat and human intestinal epithelial cells, highlighting the

Cl−

Cl− AE4

AE1

− HCO3

HCO3−

Cl− AE2 −

HCO3

Human duodenum

Cl−

Cl− AE2 −

HCO3

bAE3

HCO3−

Human colon

Cl− AE2 −

HCO3

Rat colon

FIG. 74-6. Expression of anion exchangers (AEs) on the apical and basolateral membranes of the human and rat intestinal epithelial cells.

1892 / CHAPTER 74 corresponding to the establishment of digestive processes that are similar to adult animals (162). Although the decline in the levels of AE2 mRNA by age parallels the previously reported decrease in the luminal membrane Cl−-HCO3− exchange process in the rat ileum (163), the physiologic importance of these observations remains unclear. Thyroxine and steroid hormones play an important role in the regulation of gene expression during development, and their levels increase around the weanling period (162). Interestingly, AE2 mRNA was decreased in response to thyroxine but not dexamethasone administration to rats at all ages (162). These findings indicated that AE2 expression in the rat small intestine was responsive to thyroxine at all ages, but that the observed decline in its expression during weanling was caused by the concomitant increase in the level of thyroxine (162). Effect of pH. Because Cl−-HCO3− AEs are involved in the maintenance of pHi, their regulation by changes in extracellular and pHi was investigated extensively (97,98). These studies were performed in a heterologous expression system using either the HEK293 cell line or Xenopus oocytes (131,164). AE2 activity displays steep alterations in response to variations in both the extracellular and pHi (100). At acidic pH, AE2 was sharply inhibited, whereas it was activated by intracellular alkalinization (100). In contrast, AE1 showed modest sensitivity to extracellular pH, whereas it was completely insensitive to alterations in the pHi (100). However, conflicting results were obtained from different studies with regard to the sensitivity of AE3 to changes in the pH (131,160,164). These functional features have important physiologic implications indicating that AE2 could operate as a base extruder (111). In this scenario, when the pHi is low, AE2 is inhibited and is not involved in the recovery process from the acid load. However, when the pHi is high, AE2 is stimulated by ~10fold (165), indicating its role as a base extruder and its involvement in the recovery from base load. More detailed studies have focused on elucidating the structural elements that confer the functional characteristics of AE2 in response to changes in the pH (100). Molecular biology approaches using several chimera and truncation constructs of mouse AE2 showed the presence of two regions in mouse AE2 protein that were essential for the regulation of AE2 by pH: a sensor site in the C terminus and a modifier site in the N terminus of the polypeptide (100). Molecular analysis has identified a highly conserved area between AE2 and AE3, flanking a region from amino acids 336 to 347, which was essential for the regulation of AE2 by pH (131). Activation of AE2 by NH4+. Another interesting feature with respect to the acute regulation of AE2 is its stimulation by NH4+ (166). Although exposure to NH4+ leads to intracellular acidification (133), it appears that NH4+ directly upregulates the exchanger in a Ca2+-dependent manner by a mechanism that overrides the effect of intracellular acidification. The molecular basis of such activation is not yet clear. However, studies have demonstrated that the same conserved region, amino acids 336 to 347 of the N terminus, necessary

for the inhibition of AE2 by pH also is critical for its activation by NH4+ (133). Regulation of Anion Exchangers by Kinases. The activation of several kinases and signal transduction pathways has been implicated in the modulation of intestinal electrolyte absorption (96). Little is known, however, about the regulation of AEs of the SLC4 gene family by kinases. In this regard, an increase in tyrosine phosphorylation of eAE1 was demonstrated in response to hypertonicity and subsequent red cell shrinkage (167). However, the possible effect of such increased tyrosine phosphorylation on eAE1 activity still has not been reported. PKC phosphorylates bAE3 in cardiomyocytes in response to angiotensin II that also stimulates Cl−HCO3− exchange in these cells (168). Phorbol ester activation of PKC in gastric mucous cells increased Cl−-HCO3− exchange process, presumably via the stimulation of AE2a, the predominant AE2 variant in gastric mucous cells (154). SLC26 Gene Family A second family of highly versatile anion transporters, SLC26 or sulfate permease family, has been characterized (169). SLC26 family members transport a wide range of monovalent and divalent anions with variable specificities, and they are structurally distinct from the previously described SLC4 gene family (14). The SLC26 gene family consists of highly homologous proteins conserved in bacteria, fungi, yeast, plants, and animals, including humans (170). Currently, 11 tissue-specific genes have been characterized for this family, namely, SLCA1-A11, although SLC26A10 is considered a pseudo-gene (14) (Table 74-2). Rat liver SLC26A1 was the first characterized member of SLC26 gene family that represents the liver sulfate or oxalate-bicarbonate exchanger, sat-1 (171). Mutations in some members of this family are linked to human diseases; for example, SLC26A2 and SLC26A3 genes have been associated with diastrophic dysplasia (DTD) and congenital CLD, respectively (188,189). Also, SLC26A4 gene was recognized as the gene mutated in Pendred syndrome (PDS), one of the most common forms of syndromic deafness and associated thyroid abnormalities (190). SLC26A4, or Pendrin, is expressed mainly on the apical membranes of thyroid follicular cells and kidney cortical collecting ducts (191). Human SLC26A4 was shown to transport Cl− and I−, but not SO4−, when expressed in both Sf9 cells and Xenopus oocytes (177). Functional studies in HEK293 cells demonstrated that Pendrin functions as Cl−-OH−, Cl−-HCO3−, and Cl−-formate (CFEX) exchangers (179). SLC26A5, or prestin, was identified as the integral membrane motor protein of the outer hair cell of the mammalian cochlea necessary for electromotility (180). SLC26A5 transcript also has been found in brain, heart, spleen, and testis (181). Prestin was demonstrated only to bind (but not to transport intracellular anions), and then to induce membrane movements in response to changes in its polarization (180). SLC26A6 is highly expressed in the kidney and pancreas and mediates Cl− and SO42− transport when expressed in

INTESTINAL ANION ABSORPTION / 1893 TABLE 74-2. SLC26 gene family of anion exchangers Human gene name

Protein name

Substrates

Tissue distribution

Reference

SLC26A1 SLC26A2

Sat-1 DTDST

SO42−, SO42−,

14, 171 4

SLC26A3

DRA/CLD

SLC26A4 SLC26A5 SLC26A6

Pendrin Prestin PAT1/CFEX

SO42−, Cl−, HCO3−, OH−, oxalate Cl−, HCO3−, I−, formate — SO42−, Cl−, HCO3−, OH−, oxalate, formate

SLC26A7 SLC26A8 SLC26A9 SLC26A10 SLC26A11

— Tat1 — Pseudo-gene —

Liver, kidney Colon, placenta, bronchial glands, tracheal epithelium, pancreas, and eccrine sweat glands Duodenum, >> colon ileum, sweat glands, pancreas, prostate Inner ear, kidney, thyroid Inner ear Stomach, duodenum, jejunum, ileum, colon, pancreas, kidney, heart, skeletal muscle, liver, placenta, lung, and brain Stomach, kidney Sperm, brain Lung — Ubiquitous

SO42−, SO42−, SO42−, — SO42−,

oxalate Cl−

oxalate, Cl− oxalate, Cl− oxalate, Cl− oxalate

172–176 177–179 180, 181 157, 182

184–185 185, 186 185 14 187

−

CFEX, Cl -formate exchanger; CLD, chloride diarrhea; DRA, down-regulated in adenoma; PAT1, putative anion transporter 1. Modified from Mount and Romero (14), by permission.

Xenopus oocytes (169). The novel members of SLC26 family, SLC26A7, SLC26A8, and SLC26A9, show tissuespecific expression in the kidney, testis, and lung, respectively (185). SLC26A7-9 proteins were reported to mediate Cl−, SO42−, and oxalate transport (185). Studies suggest that SLC26A7 represents a basolateral Cl−-HCO3− exchanger in the intercalated cells of the outer medullary collecting duct and gastric parietal cells (183,184). SLC26A8 (Tat1) has been shown to be a novel sulfate transporter linked to Rho-GTPase signaling in male germ cells (186). The expression of SLC26A9 in the alveolar and bronchial epithelium of the human lung suggests an important role of this exchanger in the lung defense mechanism (185). Northern blot analysis showed expression of SLC26A11 transcript levels in the placenta, kidney, and brain, and functional expression studies in COS-7 and Sf9 insect cells showed that SLC26A11 exhibits sulfate transport activity (187). The GI tract is known to express at least four members of the SLC26 family: SLC26A2, SLC26A3, SLC26A6, and SLC26A7; hence, this section discusses these members in detail. Structural Features of the SLC26 Family. The membrane topology of the mammalian SLC26 AEs has not been established experimentally and is difficult to infer because the prediction models give varied results (14). However, based on that -NH2 and -COOH terminals of SLC26A5 and SLC26A6 are intracellular, these proteins were suggested to span the plasma membrane 10 to 14 times (174). The SLC26 exchangers show considerable homology within the hydrophobic transmembrane domains (TMDs). The -NH2 terminal region contains the 22-amino-acid “sulfate transport” consensus signature (prosite, PS01130) containing several invariant residues critical for anion transport (192). The TMDs of SLC26 exchangers are followed by a linking region that

connects to a carboxy-terminal sulfate transporter antisigma factor antagonist (STAS) domain, extending into the cytoplasm (Fig. 74-7). The STAS domain shares significant homology with bacterial anti-sigma factor antagonists, for example, SpoIIAA of Bacillus subtilis (193). Mutations in the STAS domain of three of the members of SLC26 family have been associated with diseases, such as CLD, PDS, and DTD. The physiologic role of STAS domains in the SLC26 family of exchangers is slowly beginning to unfold. Biochemical studies to evaluate the function of STAS domain showed that deletion of this domain resulted in complete loss of human SLC26A3-mediated Cl− transport in Xenopus oocytes (175). These studies suggested that the STAS domain influences the transport function. The role of the STAS domain in the regulatory interaction of SLC26 transporters with other proteins, such as cystic fibrosis transmembrane conductance regulator (CFTR), was established (194). Hence, STAS domains appear to be critical for the functional or regulatory aspects of these exchangers. Most of the SLC26 proteins also possess a class I PDZ interaction motif at the C-terminal end (14). SLC26A3 (DRA, CLD). DRA originally was cloned as a candidate tumor repressor gene that was down-regulated in colon adenoma and adenocarcinoma (hence, the name DRA) (172). DRA gene was subsequently linked to congenital chloride diarrhea (and its name changed to CLD), making it a potential candidate gene involved in intestinal Cl− absorption (189,195). According to HUGO Gene Nomenclature Committee, the name SLC26A3 is recommended instead of DRA or CLD to match the functional classification for the AE gene family (30). Congenital Chloride Diarrhea. CLD is a rare autosomal recessive disorder characterized by high-volume watery diarrhea with increased Cl− content (196,197). The prenatal

1894 / CHAPTER 74

1

2

3

4

5

6

7

8

9

10

11

12

Prosite

STAS

NH2

PDZ COOH

FIG. 74-7. Predicted topology of SLC26 exchangers. STAS, sulfate transporter antisigma factor antagonist. (Modified from Mount and Romero [14], by permission.)

onset of watery diarrhea in utero leads to polyhydramnios and premature birth (198). Offspring with CLD have distended abdomen, which is also evident in ultrasound diagnosis (199). The diagnosis can be confirmed by measuring stool chloride concentration, which always exceeds 90 mmol/L in patients with CLD (when corrected for water and electrolyte balance) compared with 10 to 15 mmol/L in healthy individuals (32). The development of metabolic alkalosis, hypochloremia, hyponatremia, and dehydration are evident in the newborn, and if untreated, these conditions are fatal. Mental and psychomotor impairment also has been reported after dehydration and severe hyponatremia (200). Earlier perfusion studies in patients with CLD localized the defect to the C1−-HCO3− exchange mechanism in the ileum and colon (32). Because Cl−-HCO3− exchange is coupled to the exchange of Na+-H+ in the human ileum and colon (20,201,202), impairment in the absorption of Na+ also is seen, to some extent, in patients with CLD (32). Treatment consists of intravenous replacements of the fluids and electrolytes in newborns and oral replacement in older children for their lifetime (197). This treatment leads to normal development, but does not affect the diarrhea seen in patients with CLD (196). No therapeutic approach has been found to enhance Cl− absorption in patients with CLD, and the antidiarrheal drugs are ineffective (170). However, one case report points toward the role of the SCFA, butyrate, in the treatment of CLD through its potential role in stimulation of intestinal water and ion absorption (203). Additional studies to exhaustively examine the effect of SCFA treatment in patients with CLD and to understand the mechanism of action of butyrate on Cl−-HCO3− exchange and other ion transporters would be of great significance. SLC26A3 Mutations in Chloride Diarrhea. Genetic mapping and linkage analysis studies demonstrated

SLC26A3 to be a positional candidate for the CLD gene on chromosome 7 (195,204,205). Later studies demonstrated that SLC26A3 is mutated in patients with CLD (189). The CLD gene spans ~39 kb and comprises 21 exons (206). The analysis of the putative CLD promoter region showed TATA and CCAAT boxes with multiple transcription-binding sites (206). At least 30 different mutations spread over exons 3 to 19 have been identified in patients with CLD from different geographical areas (30,207,208). However, the majority of the patients with CLD analyzed in these studies were from Finland, Poland, or Arabic countries, the three highest frequency areas (30). Table 74-3 summarizes the known mutations associated with the CLD gene. (For a detailed update of all SLC26A3 mutations and polymorphisms, see Makela and colleagues [30].) The increasingly wide diversity of mutations of SLC26A3 includes small deletions, insertion, frameshifts, genomic rearrangements, a 3.5-kb intragenic deletion of exons 7 and 8, and a complex deletion combined with an insertion (21042105delGGins29bp) (207,208). Two novel mutations in patients with CLD were reported involving a homozygous G-to-A transition at nucleotide 1386 in exon 12 of SLC26A3, whereas the second one was identified as 13 base deletion of nucleotide 145-157 in exon 3 sequence (30). The first mutation was found to produce a novel site for the restriction enzyme Tsp45I, and the second one caused a frameshift. Altogether, four polymorphic nucleotide changes have been defined in the SLC26A3 coding region. These include two silent single-nucleotide polymorphisms and two polymorphic at the protein level. Role of Chloride Diarrhea in Anion Exchange. SLC26A3 protein is a glycoprotein containing 764 amino acids with a molecular weight of ~84 kDa (209). SLC26A3 protein structure was predicted to contain 10 to 12 TMDs, which is

INTESTINAL ANION ABSORPTION / 1895 TABLE 74-3. Common chloride diarrhea mutations associated with SLC26A3 Mutation

Change

Missense

Nucleotide

I544N S206P L496R D468V P131R H124L G120S

1631T>A 616T>C 1487T>G 1403A>T 392C>G 371A>T 358G>A

Nonsense

Nucleotide

Q436X W462X G187X Y305X

1306C>T 1386G>A 559G>T 915C>A

Splice defect

Intron

IVS5-2A>G IVS5-1G>T IVS11-1G>A IVS12-1G>C IVS13-2delA

5 5 11 12 13

Deletion

Exon/Intron

145-157del13 344delT 3.5 kb del

EX 3 EX 4 IN 6-8

V317 del 1342-1343delTT 1516delC 1548-1551delAACC Y527del 1609delA 2116delA

EX EX EX EX EX EX EX

Coding sequence change

Isoleucine to asparagine at 544 Serine to proline at 206 Leucine to arginine at 496 Aspartate to valine at 468 Proline to arginine at 131 Histidine to leucine at 124 Glycine to serine at 120

Glutamine-Stop at 436 Tryptophan-Stop at 462 Glycine-Stop at 187 Tyrosine-Stop at 305

Intron Intron Intron Intron Intron

acceptor acceptor acceptor acceptor acceptor

site site site site site

AG AG AG AG AG

loss loss loss loss loss

Frameshift Frameshift Loss of exons 7 and 8, frameshift In-frame loss of valine at 317 Frameshift Frameshift Frameshift In-frame loss of tyrosine at 527 Frameshift Frameshift

8 12 14 14 14 15 19

Insertion 177-178insC 268-269insAA 1675-1676insATC Replacement

EX 3 EX 3 EX 18

Frameshift Frameshift In-frame addition of isoleucine

2104-2105

EX 19

In-frame substitution G702T and addition of nine amino acids

delGGins29

Replacement of GG and insertion of 29 bp

Modified from Makela et al (30), by permission.

typical for an integral membrane transporter (198). Also, the amino-acid sequence of SLC26A3 protein exhibited high homology to the other SLC26 sulfate transporters, that is, SLC26A1 and SLC26A2 (210). Although a number of studies using Xenopus oocytes and other cell lines have been performed to elucidate the functional role of DRA in anion transport, the exact role of this transporter in mammalian intestinal epithelial cells remains controversial with respect to anion specificity, electrogenicity, and inhibition profile.

For example, initial studies using microinjected Xenopus oocytes as an assay system showed that SLC26A3 encoded for a Na+-independent transporter for both sulfate and oxalate (210). Human and mouse DRA proteins were shown to mediate Cl−-OH− and Cl−-HCO3− exchange processes when expressed in Xenopus oocytes and the HEK293 cell line, respectively (173,174). In addition, a V137del mutation in SLC26A3 found in Finnish patients with CLD suppressed chloride and sulfate transport in Xenopus oocytes (174).

1896 / CHAPTER 74 A report showed hDRA to mediate Cl−-Cl− and Cl−-HCO3− exchange, but not the transport of sulfate or OH− when expressed in Xenopus oocytes (175). Another study showed hDRA stably transfected in HEK293 cells to function as an electroneutral Cl−-HCO3− exchanger with low Cl−-OH− exchange and minimal SO42−-HCO3− exchange activity (211). The findings that the SLC26A3 gene is mutated in patients with CLD, together with the functional studies in Xenopus oocytes and HEK293 cells mentioned earlier (173–175), provide strong evidence that SLC26A3 represents the intestinal luminal Cl−-HCO3− exchanger that is defective in patients with CLD. Notably, SLC26A3 mutations in patients with CLD, although found in all parts of the protein, appeared to cluster around three locations that might be critical for SLC26A3 function (208). In addition, several mutations were localized to the intracellular C-terminal region of CLD protein, suggesting that the C-terminal domain of CLD might have an important regulatory role vital for the function of CLD as a transporter or as a regulator for another transporter (208). Studies (212) have identified a novel role for the C-terminal region of SLC26A3 as a growth suppressor in a number of cancer cell lines. Interestingly, the Val317del mutation in SLC26A3 C-terminal region, which abrogates chloride and sulfate transport, was shown to have no effect on the growth suppression function of SLC26A3 C terminus (212). Also, DRA structure-function studies using Xenopus oocytes demonstrated that truncation of N-terminal cytoplasmic domain or C-terminal region (44 amino acids) had no effect on DRA transport function; however, deletion of the STAS domain led to complete loss of Cl− transport (175). As mentioned earlier, there are significant conflicting reports in the literature with regard to the inhibition of DRAmediated transport by stilbene derivatives, such as DIDS. For example, DRA-mediated sulfate transport in Xenopus oocytes (210) and Sf9 cells (213) was highly sensitive to inhibition by DIDS. Also, the Cl− transport function of DRA was inhibited almost completely by 0.5 mM DIDS (174). In contrast, some studies suggest extremely low sensitivity of DRA-mediated Cl− transport to DIDS, when expressed in Xenopus oocytes (175) or HEK293 cells (173). The differences in these studies with regard to DIDS sensitivity are not clearly understood yet and require more exhaustive analysis of the inhibition profile of this transporter. SLC26A3 also is potently inhibited by tenidap and niflumic acid, the antiinflammatory drugs (175). Expression of SLC26A3. The phenotype of CLD suggests a defect in the apical Cl−-HCO3− exchanger in the luminal membranes of the ileum and colon. SLC26A3 protein is highly expressed in the colon compared with the small intestine, and its protein product is localized to the BBM of differentiated mucosal columnar cells (189,195,213,214). In situ hybridization studies showed expression of SLC26A3 to be restricted to the upper crypt and surface epithelium of the colon, whereas the ileum shows expression in the deep crypt as well (214). In parallel with its expression in the differentiated columnar epithelium of the adult human colon, SLC26A3 was expressed in the midgut of developing mouse

embryos at the time (day 16.5) of differentiation of the smallintestinal epithelium (209). Similarly, Northern blot analysis showed that mouse SLC26A3 mRNA was expressed at high levels in the cecum and colon compared with the small intestine (173). SLC26A3 also was found in the rabbit, rat, and human duodenum and was suggested to be the luminal AE that mediates duodenal Cl−-HCO3− exchange (215). Northern analysis detected CLD at high levels in native mouse pancreas, and immunohistochemical studies localized it to the apical membrane domains of the duct cells (216). Studies in human colonic adenocarcinoma Caco-2 cells showed expression of SLC26A3 in well-differentiated postconfluent but not preconfluent cells (210). The expression of SLC26A3 (CLD) also was characterized in extra intestinal normal epithelia and in intestinal inflammatory and neoplastic epithelia using in situ hybridization and immunohistochemistry studies (176). CLD expression was detected in eccrine sweat glands and seminal vesicles (176). However, functional defects in these tissues have not been reported in patients with CLD, although reduced fertility has been seen occasionally in adult male Finnish patients (30). In inflammatory bowel disease and ischemic colitis, the CLD protein was found deeper in crypts, including proliferative epithelial cells, but the CLD mRNA expression was similar to the normal colonic epithelium (176). Epithelial polyps with no or minor dysplasia showed abundant CLD expression, whereas adenocarcinomas were negative (176). In this regard, direct studies of the role of the SLC26A3 gene in carcinogenesis done in Finnish families with CLD did not demonstrate a strong increase in the intestinal cancer risk in CLD mutation carriers (208,217). These studies rule out the direct role of CLD protein in carcinogenesis. The above-mentioned studies suggest SLC26A3 to be an attractive candidate for the intestinal luminal Cl−-HCO3− exchange that is mutated in patients with CLD. This conclusion was further supported by the observation that SLC26A3 mRNA was up-regulated in NHE3 knockout mice, suggesting the functional coupling between SLC26A3 and NHE3 in the overall process of NaCl absorption in the colon (173). This scenario is different in the duodenum, which has comparatively low expression of NHE3 compared with the colon (215). Therefore, greater abundance of SLC26A3 compared with NHE expression in parallel with the greater Cl−-HCO3− exchange versus NHE rates in the BBM of duodenal epithelial cells was suggested to favor duodenal electroneutral HCO3− secretion compared with NaCl absorption (215). SLC26A3, therefore, is suggested to present multiple transport activities based on its biological environment (30). SLC26A3 as Part of a Multiprotein Complex. Several physiological studies, however, still question the direct role of SLC26A3 in the intestinal apical Cl−-HCO3− exchange. For example, absorption of SO42− and Cl− across apical membranes of the epithelial cells in the rabbit ileum and human colon was demonstrated to occur via two distinct SO42−-OH− and Cl−-HCO3− (OH−) exchange processes (9). In addition, studies to investigate the transport of SO42− and Cl− in the Caco-2 cell line to define the possible function of the

INTESTINAL ANION ABSORPTION / 1897 SLC26A3 gene in the apical anion exchange process showed that SO42−-OH− and Cl−-OH− exchange processes in Caco-2 cells were distinct based on anion specificity and inhibition by DIDS. Also, RNase protection studies showed that the relative abundance of DRA mRNA was reduced in parallel with the SO42−-OH− exchange activity, but Cl−-OH− exchange process was unaltered (8). These observations suggested that DRA might not be a Cl−-HCO3− exchanger, but rather is a sulfate transporter that plays an important role in the Cl−-HCO3− exchange process as part of a multiprotein complex (8). In addition, Na+ depletion and secondary hyperaldosteronemia was shown to inhibit the Cl−-HCO3− exchange process by ~80% with no alterations in DRA expression in the rat distal colon, suggesting that DRA might not represent the luminal Cl−-HCO3− exchange; rather, it may encode an aldosteroneinsensitive Cl−-OH− exchanger (83). In contrast with the reports described earlier, another report shows that DRA expressed in the Xenopus oocyte system was incapable of transporting SO42− and OH− ions, but rather mainly functioned as a Cl−-HCO3− exchanger (175). These reports warrant the need for more exhaustive investigation to identify ion transport characteristics of the DRA in mammalian expression systems, to identify the presence of additional novel intestinal anion transporters on luminal membranes, and to further define the exact role of DRA in intestinal chloride absorption. In fact, several studies have suggested SLC26A3 to be involved in various protein–protein interactions and further support the notion of DRA function as a part of a “multiprotein complex” (8). For example, SLC26A3 protein has been shown to interact with the second PDZ domain of the NHE3 regulatory protein, E3KARP, via a PDZ domain located in the C-terminal region of DRA (218). Also, immunofluorescence studies showed DRA, NHE3, and E3KARP to be colocalized in the apical compartment of the human proximal colon. Therefore, a model was proposed in which NHE3 and DRA both bind to the second PDZ domain of E3KARP that links the two transporters through its dimerization. This model further suggests the involvement of DRA in the Cl−-HCO3− exchange process that is coupled to NHE3 (218). Similarly, SLC26A3-mediated bicarbonate transport is functionally coupled to the activity of the cytosolic carbonic anhydrase II (219). In general, the COOH-terminal tail of all bicarbonate transport proteins, with the exception of SLC26A3, possesses a consensus carbonic anhydrase II–binding motif (220). However, inhibition of carbonic anhydrase II activity by acetazolamide was shown to reduce SLC26A3 activity in HEK293 cells (transiently transfected with SLC26A3 cDNA), although the C terminus of SLC26A3 interacted weakly with carbonic anhydrase II (219). These studies suggested that SLC26A3-mediated bicarbonate transport requires a functional cytosolic carbonic anhydrase II, but unlike AEs and NBCs, this coupling was independent of the direct binding between SLC26A3 and carbonic anhydrase II (219). Therefore, it was proposed that an intermediary protein might bring carbonic anhydrase II in close proximity of SLC26A3 in the membrane for exhibiting full

transport activity (219). Whether this intermediary protein is CFTR or a protein regulated by CFTR remains unclear? Based on the reports described earlier, it does appear that increasing evidence points toward the DRA protein playing a central role in the human intestinal luminal membrane Cl−-HCO3− exchange. It is, however, unclear whether DRA itself is sufficient to fully explain the intestinal luminal membrane Cl−-HCO3− exchange activity. Future studies including protein–protein interactions and DRA knockout mouse studies may provide a clearer picture with respect to either the direct role of DRA in Cl−-HCO3− exchange or as a part of a multiprotein complex (involving other AEs) involved in chloride absorption. In this regard, in view of the role of SLC26A6 also in the luminal membrane Cl−-HCO3− exchange (see later discussion), as well as the evidence for the electrogenic nature of both SLC26A3 and SLC26A6 shown in a few of the studies, a speculative model (discussed in more detail in the following section) has been proposed that may, to some extent, explain electroneutral Cl−-HCO3− exchange in regions of the intestine where both SLC26A3 and SLC26A6 are expressed together (Fig. 74-8). SLC26A6 (Putative Anion Transporter-1). SLC26A6 (putative anion transporter-1 [PAT1]) has generated considerable interest lately because it is also expressed in the intestine and functions as a Cl−-OH− or Cl−-HCO3− exchanger. SLC26A6 was identified by two different groups exclusively through database mining based on homology to SLC26A3 and Pendrin (169,221). The SLC26A6 gene maps to chromosome 3q21.3,

1Cl− 2Cl−

SLC26 A6

H+

SLC26 A3

−

NHE

Na+

1HCO3−

2HCO3

Electroneutral

K+

Cl−

AE2

~ATP

3Na+

HCO3−

Cl−

AE3

HCO3−

FIG. 74-8. Speculative model of the role of SLC26A6 and SLC26A3 in electroneutral NaCl absorption. AE, anion exchanger; ATP, adenosine triphosphate; NHE, Na+-H+ exchanger.

1898 / CHAPTER 74 spans 2217 bp, and comprises 20 exons. The SLC26A6 gene encodes an integral membrane protein of 738 amino acids with a predicted topology of 11-transmembrane helices and intracellular -NH2 and -COOH termini (169,221,222). The highest sequence similarity of SLC26A6 (56%) is found with SLC26A5, the outer motor cochlear protein (14). Tissue Distribution and Expression of Putative Anion Transporter-1. Northern blot studies showed widespread expression of SLC26A6 transcript with highest abundance in the kidney and pancreas. Heart, skeletal muscle, intestine, liver, placenta, lung, and brain also showed significant expression of SLC26A6 (169,221,223). Subcellular distribution studies localized SLC26A6 protein exclusively to the plasma membrane in stably transfected MDCK cell clones (221). The size of the SLC26A6 protein in the lysates of stably transfected MDCK cells varied from 84 kDa to more than 200 kDa (221). Because SLC26A6 possesses several N-linked glycosylation sites, this difference in the size of SLC26A6 was expected to be caused by strong glycosylation of SLC26A6 in these cells. Immunohistochemistry studies using anti-SLC26A6 antibodies showed abundant expression of SLC26A6 detected in both the apical and basolateral membranes of kidney tubules and in the brush border of pancreatic duct (169). A mouse ortholog of SLC26A6 (CFEX) also was cloned and was localized apically on the plasma membrane of the proximal kidney tubule (224). The expression of mouse SLC26A6 in the intestine was seen in a pattern that is opposite to the pattern of DRA expression, that is, a greater level of expression in the small intestine compared with the colon (157). SLC26A6 was identified as an ~90-kDa band, and immunohistochemical studies localized this protein to the brush border of the villous cells of the duodenum. In the gastric parietal cells, PAT1 was colocalized with gastric H,K-ATPase, indicating its localization on tubulovesicular membranes (157). PAT1 expression was markedly attenuated in gastric H+,K+-ATPase null mice (225). More recently, immunoblotting studies in the apical and basolateral plasma membranes isolated from different regions of the human intestinal tissue obtained from organ donors also showed apical localization of the SLC26A6 protein (182). However, in contrast to the expression of mouse SLC26A6, both RT-PCR and immunoblotting studies demonstrated almost uniform expression of SLC26A6 along the length of the human intestine (182). The human SLC26A6 gene also was found to have three alternatively spliced variants, named SLC26A6a, SLC26A6c, and SLC26A6d. With regard to their tissue distribution, RT-PCR studies indicated that SLC26A6a, but not SLC26A6c or SLC26A6d, is the spliced variant expressed in the human small intestine and colon (222). Human PAT1 isoforms a, c, and d comprise 12, 8, and 12 membrane-spanning domains, respectively. The expression of SLC26A6 in the apical membranes of the pancreatic duct and intestine suggests it to be another putative candidate for apical AE together with SLC26A3. Functional Role of SLC26A6. SLC26A6 has been shown to function in Cl−-oxalate, SO42−-oxalate, SO42−Cl−, CFEX, Cl−-HCO3−, and Cl-OH− exchange modes (223,225–227).

Functional studies in Xenopus oocytes demonstrated murine Slc26a6 as the CFEX. The expression of CFEX on the brush border of proximal tubule cells suggested CFEX as the primary exchanger contributing to renal NaCl absorption process (224). The transport characteristics of Slc26a6 mediated oxalate transport (223,226) were found to be similar to the oxalate-OH− exchanger in rabbit ileal BBM (228). Thus, SLC26A6 might also play an important role in the smallintestinal dietary oxalate absorption, a critical determinant of renal calcium oxalate stone formation (229). In the stomach, PAT1 functions as a Cl−-HCO3− exchanger in tubulovesicle membranes (157). Both mouse and human PAT1 functioned as a Cl−-HCO3− exchanger, when expressed in Xenopus oocytes (225). SLC26A6a and its spliced variants SLC26A6c and SLC26A6d were shown to function as AEs mediating DIDSsensitive Cl− and SO42− transport (222). Given the mild expression of CLD in the mouse small intestine compared with the colon, PAT1 was proposed as the candidate protein for the apical Cl−-base exchange involved in absorption of Cl− and secretion of bicarbonate in the upper GI tract (225). A novel feature of SLC26 transporters has been discovered. In contrast to AEs, SLC26A6 transport was found to be electrogenic with opposite apparent stoichiometries for SLC26A6 and SLC26A3. Xenopus oocytes expressing SLC26A6 hyperpolarized on removal of extracellular Cl− in the presence of HCO3−, whereas marked depolarization was seen in SLC26A3-injected oocytes (227). These studies demonstrated that SLC26A3 mediates the anion exchange process with a Cl−-HCO3− ratio of 2 (227). PAT1 activity was found to be voltage regulated and occurred at a HCO3−-Cl− ratio of 2 (227). The expression of SLC26A3 and SLC26A6 working at opposite stoichiometries in the same cell therefore might result in an apparent electroneutral Cl−-HCO3− exchange process (227). Based on the expression and functional studies, both SLC26A3 and SLC26A6 can be potential candidates for the electroneutral Cl−-HCO3− exchange process in the human intestine. Figure 74-8 shows a speculative model where parallel and coupled functioning of the two SLC26 transporters, together with NHE, could explain the electroneutral NaCl absorption. In this regard, however, some of the studies demonstrate that SLC26A3 may function as an electroneutral rather than electrogenic transporter (175,211), which may not fit with the proposed model (see Fig. 74-8). Therefore, the contribution of both of these SLC26 proteins to the Cl−-HCO3− exchange process in various regions of the human GI tract and the exact mechanism(s) by which CLD mutations affect human intestinal Cl−-HCO3− exchange need to be further investigated in detail to fully establish a model for luminal chloride uptake in the human intestine. SLC26A2 (Diastrophic Dysplasia Sulfate Transporter). The SLC26A2 gene product encodes for a protein mutated in DTD, an autosomal recessive osteochondrodysplasia, characterized by normocephalic short-limbed dwarfism (14). The name diastrophic represents a geologic term meaning the twisting movements and deformations of the earth’s crust, and therefore was used to describe the bony malformations

INTESTINAL ANION ABSORPTION / 1899 seen in the disease (170). Patients with DTD also show other defects including cleft palate, clubbed feet, abnormalities of dentition, and craniofacial features (4,230–232). The Finnish population shows an increased frequency of DTD, with an estimated carrier frequency of 1 in 70 (188,233). About 14 different mutations have been reported in the DTDST (diastrophic dysplasia sulfate transporter) gene responsible for a number of recessively inherited chondrodysplasias, for example, atelosteogenesis type 2 (AO2), multiple epiphyseal dysplasia (MED), and achondrogenesis 1B (ACG1B) (234). The human DTD gene is located on chromosome 5q and spans ~40 kb (235). Mutations in the DTDST coding region have been found to occur typically only in one allele in the majority of the Finnish patients with DTD examined (236). This common Finnish mutation was identified as a GT-to-GC transition in the splice donor site of a 5′-untranslated exon of the DTDST gene and acts by reducing mRNA levels markedly. The SLC26A2 gene product is ~739 amino acids in length and shares 33% amino-acid identity with SLC26A3 (DRA) and 48% with SLC26A1 rsat-1 (188). Initial functional studies to examine the role of the DTD gene product in cultures of primary skin fibroblasts taken from healthy individuals and patients with DTD suggested that the DTD gene encodes for a sulfate transporter called as DTDST (188). The loss of the sulfate transport in patients with DTD was suggested to cause undersulfation of proteoglycans in the cartilage matrix, thereby causing the clinical phenotype of the disease (188). DTDST cDNA also was cloned from rat UMR-106 osteoblastic cells, and the DTDST mRNA was highly expressed in cartilage and the intestine (237). Even though the phenotype caused by SLC26A2 mutations indicates cartilage as the major site of expression, Northern analysis has shown wide expression detectable in most tissues including the colon, placenta, bronchial glands, tracheal epithelium, pancreas, and eccrine sweat glands (188). An in situ hybridization study of normal human colonic tissue demonstrated abundant SLC26A2 expression in the surface epithelium of the colonic crypts (238). Heterologous expression of rat and human DTDST into Xenopus oocytes induced Na+independent sulfate transport that was markedly inhibited by extracellular chloride and bicarbonate and was sensitive to 1 mM DIDS (237). The absence of phenotype caused by SLC26A2 mutations in tissues other than cartilage remains intriguing, although it is possible that other anion transporters can compensate for the modest needs of most cell types other than chondrocytes (238,239). The exact role of this SLC26 protein in intestinal anion absorption remains to be established; it is possible that this protein may encode for the luminal membrane SO4−-OH− exchanger demonstrated in human colonic luminal membrane vesicles (9). SLC26A7. SLC26A7 is one of the cloned members of the SLC26 family that was shown to be expressed mainly in kidneys and testes (185). SLC26A7 was shown to exhibit ~30% homology with DRA, Pendrin, and PAT1 at the aminoacid level (185) and functioned as a Cl−-oxalate, Cl−-HCO3−,

and Cl−-sulfate exchanger in oocytes (185). In the renal tissue, SLC26A7 expression was localized to BLMs of α-intercalated cells in the outer medullary collecting duct and was shown to be up-regulated by hypertonicity (184). Also, the SLC26A7 gene in the kidney was subjected to alternate splicing, resulting in two distinct protein isoforms, SLC26A7.1 and SLC26A7.2, differing in their carboxy termini (187). Several studies demonstrated abundant expression of SLC26A7 in the GI tract, with a more restricted expression in the stomach and no expression in the intestine (157). Immunofluorescence labeling localized SLC26A7 exchanger to the BLM of gastric parietal cells (157). When expressed in Xenopus oocytes, SLC26A7 functioned as a DIDS-sensitive Cl−-HCO3− exchanger, was active in both acidic and alkaline pH, and was not found to be electrogenic (157). Extrusion of acid via apical H+,K+-ATPase present in gastric parietal cells is dependent on the activity of the basolateral Cl−-HCO3− exchanger (240). Gastric parietal cells also showed the expression of another AE, AE2, on the BLM (Fig. 74-9) (153,154,241). The exact role of both AE2 and SLC26A7 expressed on the BLMs of gastric parietal cells in the process of gastric acid secretion remains unclear. Previous functional studies using in vitro expression systems demonstrated AE2 as the major basolateral Cl−-HCO3− exchanger in gastric parietal cells (242). However, certain functional properties of the parietal cell basolateral Cl−-HCO3− exchanger are distinct compared with the known functional properties of AE2 (183). For example, the basolateral parietal cell exchanger in gastric parietal cells plays an important role in pHi recovery from acidosis, a property distinct from AE2, which is active only

Lumen

K+ ~ATP

H+

HCO3−

HCO3−

SLC 26A7

H+

AE2

Cl−

NHE Cl−

Na+

FIG. 74-9. Cl−-HCO3− exchangers of the gastric parietal cell basolateral membranes. AE, anion exchanger; ATP, adenosine triphosphate; NHE, Na+-H+ exchanger.

1900 / CHAPTER 74 at neutral and alkaline pHi but not at acidic pHi (240,243, 244). Therefore, based on its unique expression and functional studies, SLC26A7 was proposed as the gastric parietal cell basolateral Cl−-HCO3− exchanger (183). In this regard, studies showed that gastric acid secretions (after stimulation by histamine) were achlorhydric and, indeed, alkaline in AE2 knockout mice (153). These studies suggested that mechanism(s) of bicarbonate secretion are intact in AE2 knockout mice, although the net acid secretion mechanism is likely to be impaired (153). Also, gastric mucosa of AE2 knockout mice showed abnormalities in the parietal cell structure with impaired secretory canaliculi and reduced tubulovesicles, suggesting that AE2 is essential for acid secretion. Future studies to elucidate the mechanism(s) and regulation of both AE2- and SLC26A7-mediated transport are required to fully understand their distinct roles in apical acid secretion or basolateral bicarbonate transport in the gastric parietal cells. Regulation of SLC26 Exchangers. Although significant advances have been made in understanding the role of SLC26 exchangers, especially SLC26A3, in the luminal Cl−HCO3− exchange in the mammalian intestine, the information regarding their regulation is relatively limited. The following section describes the available information on regulation of SLC26 exchangers under basal conditions such as by pHi, NH4+, CFTR, as well as in cases of intestinal inflammation. Regulation of SLC26A3 by pH and NH4+. As described earlier in this chapter (see the SLC4 Gene Family section), AE2 is acutely regulated by both extracellular pH and pHi, whereas AE1 is relatively less sensitive to pH changes (100, 245). Similar studies using fluorometric and microelectrode measurements in Xenopus oocytes expressing hSLC26A3 demonstrated that hSLC26A3-mediated Cl− transport was insensitive to alterations in extracellular pH (175). However, hSLC26A3-mediated transport was inhibited by intracellular acidification, and this pHi-mediated regulation was dependent on the C-terminal cytoplasmic domain of hSCL26A3 (175). Such a pHi sensor is expected to sense sufficiency of intracellular HCO3− concentration and to inhibit DRA-mediated Cl−-HCO3− exchange before pHi is acidified (175). Also, similar to AE2, SLC26A3 is stimulated by NH+4 . However, unlike AE2, which is stimulated by hypertonicity, SLC26A3mediated Cl− flux was unaltered in response to increased osmolarity (175). Role of SLC26A3 During Intestinal Inflammation. The effect of intestinal inflammation on the regulation of SLC26A3 is not clearly understood. SLC26A3 expression has been examined in two different inflammatory models, such as HLAB27/β2m transgenic rat, IL-10 knockout mice, and patients with ulcerative colitis (UC) (214). Northern blot analysis showed a decrease in SLC26A3 mRNA in the colon of HLAB27/β2m transgenic rats and also in patients with UC (214). In situ hybridization studies also showed reduction in DRA expression in the surface epithelium in the inflammatory models and in patients with UC. DRA expression, however, was not altered in the upper crypt region. Further in vitro studies using the human colonic Caco-2 cell line demonstrated that SLC26A3 mRNA was reduced in

response to the proinflammatory cytokine, IL-1β (214). In contrast, other studies demonstrated almost no alteration in the expression of SLC26A3 mRNA in patients with ischemic colitis and inflammatory bowel disease compared with normal colonic epithelium (176). Subsequent studies demonstrated that although SLC26A3 mRNA was decreased in severe UC, the SLC26A3 protein expression was unaltered (246). The differences in these studies remain unclear, but were suggested to be caused by either different models used in the studies or differences between UC and other types of intestinal inflammation. Regulation of SLC26A3 and SLC26A6 by Cystic Fibrosis Transmembrane Conductance Regulator. Cystic fibrosis is an autosomal recessive disease characterized by aberrant HCO3− transport and results from mutational inactivation of CFTR, the cAMP-sensitive Cl− channel (247). CFTR is generally a weak transporter for HCO3−, and therefore cannot completely account for the electrogenic portion of HCO3− secretion by itself (227). However, several lines of evidence suggest that activation of SLC26 transporters by CFTR is an important mechanism in which CFTR can switch “on” and “off ” the HCO3− secretion in CFTR-expressing cells (227). In this regard, previous studies suggest that the pancreatic HCO3− secretion defect seen in patients with cystic fibrosis might be partly caused by the down-regulation of apical Cl−-HCO3− exchange activity mediated by DRA or PAT1. For instance, stimulation of CFTR by cAMP resulted in activation of Cl−-HCO3− exchange activity in submandibular gland ducts prepared from wild-type mice, but not in CFTR knockout mice (248). Also, CFTR was shown to up-regulate DRA and PAT1 expression in cultured pancreatic duct cells (216). Similarly, studies in HEK293 cells demonstrated activation of DRA and PAT1 by CFTR, which was compromised by CFTR mutations linked with cystic fibrosis (227). However, members of the SLC4 Cl−-HCO3− exchangers, AE1-4, were not activated by CFTR (227). Also, PAT1a and PAT1c isoforms have a PDZ domain in their C terminals by which they interact in vitro with the first and the second PDZ domains of NHE3 regulatory proteins, NHERF and E3KARP (222). These studies suggested that on pancreatic stimulation, a complex formation of CFTR-PAT1-NHERF might stimulate CFTR-directed luminal HCO3− secretion (222). The molecular mechanisms of CFTR-mediated stimulation of SLC26 transporters are slowly beginning to unfold. Novel insights into the mechanism of regulation of CFTR activity, epithelial Cl− absorption, and secretion of HCO3− were demonstrated via protein–protein interactions (194). SLC26 transporters and CFTR were shown to be mutually regulated; that is, DRA or PAT1 activated CFTR by increasing its overall open probability (NPo), and reciprocally, DRA- or PAT1mediated transport was stimulated by CFTR (Fig. 74-10). This regulation involved interaction between the STAS domain of SLC26 transporters with the regulatory domain (R) of CFTR and was facilitated by their PDZ domains (194). In a model proposed by Ko and colleagues (194) (see Fig. 74-10) for ductal chloride absorption and bicarbonate secretion, SLC26A6 and SLC26A3 were shown to use altered

INTESTINAL ANION ABSORPTION / 1901 Proximal duct

Distal duct Cl−

HCO3−

Cl−

(+)

2HCO3−

2Cl− SLC 26A3

SLC 26A6 (+) CFTR

(+) (+)

HCO3−

2K+

CFTR

2K+

~ATP 3Na+

Cl− HCO3−

NBC Na+

3HCO3−

NBC

~ATP 3Na+

Na+

3HCO3−

FIG. 74-10. Regulation of SLC26A3 and SLC26A6 by cystic fibrosis transmembrane conductance regulator (CFTR) in pancreatic duct cells. ATP, adenosine triphosphate; NBC, sodium-bicarbonate cotransporter. (Modified from Ko et al [194], by permission.)

stoichiometries to explain how pancreatic, salivary, and other duct systems can generate fluid containing ~20 mM Cl− and 140 mM HCO3−. The proximal pancreatic ducts show the expression of SLC26A6 (with stoichiometry of HCO3−-Cl− > 2), whereas distal parts of ducts express SLC26A3 (with stoichiometry of Cl−-HCO3− > 2). Stimulation of fluid secretion by PKA activation results in phosphorylation of the R domain of CFTR that further regulates Cl absorption and bicarbonate secretion by causing activation of SLC26 transporters. SLC26A6 transporter in the proximal ducts makes efficient use of Cl− gradient to secrete HCO3− to at least 50 mM. SLC26A3 in the distal ducts can reduce Cl− to 20 mM and increase HCO3− to 140 mM in the secreted fluid by its unique feature of outward rectification and inhibition of reverse Cl−-HCO3− exchange by internal HCO3−, as well as by its stimulation on depolarization of the luminal membrane induced by activation by CFTR. These studies further suggest that epithelial chloride absorption and bicarbonate secretion are tightly coupled and integrated processes and also explain how CFTR mutations retaining the channel activity cause cystic fibrosis.

MECHANISMS OF SHORT-CHAIN FATTY ACID ABSORPTION Diffusion versus SCFA--HCO3− Exchange The SCFAs acetate, propionate, and butyrate are the most abundant anions in the mammalian colon produced by anaerobic fermentation of unabsorbed carbohydrates and proteins (249,250). SCFAs play a significant role in maintaining colonic epithelial integrity and stimulation of NaCl absorption. Butyrate, an important SCFA, has been implicated

in regulation of colonic motility, blood flow, and epithelial healing in colitis, and it exhibits protective effects against colorectal neoplasia. The combined concentration of the SCFAs in the colonic lumen has been shown to range between 100 and 150 mM (249,250). These monocarboxylates are known to be avidly absorbed by the mammalian GI tract (251–258), and their metabolism accounts for the major source of energy for colonocytes. Initial in vitro studies using Ussing chambers under short-circuited conditions showed conflicting results with respect to SCFA absorption (259–265). For example, in some of these studies, secretion rather than absorption of SCFAs was observed under basal conditions, and the flux rates showed no saturability, indicating a major role for passive diffusion in the transport of these acids. Furthermore, removal of bicarbonate or reduction of pH of the bathing media resulted in absorption of SCFA, indicating anion independence of SCFA transport. In view of these conflicting data, the exact nature of SCFA transport in the mammalian intestine remained unclear for quite some time. However, subsequent studies have provided a wealth of data to support their efficient absorption in the mammalian intestine, as well as their role in stimulation of NaCl absorption in the mammalian colon. A variety of techniques have been used to investigate the mechanisms of SCFA absorption in the mammalian intestine, which include in vivo luminal perfusion, in vitro flux studies in Ussing chambers, as well as purified AMVs and BLMVs from various regions of the intestine. Soergel and collaborators (256–258) used the triple-lumen perfusion method to investigate the mechanisms of SCFA transport in various regions of the human intestine. Their studies demonstrated efficient absorption of acetate, propionate, and butyrate associated with increases in luminal pH and bicarbonate. The increases in bicarbonate accompanying SCFA absorption appeared

1902 / CHAPTER 74 to be independent of chloride accumulation. Several other perfusion studies also demonstrated efficient and sometimes saturable absorption of SCFAs in the small and large intestines. Based on a number of these studies, two theories (Fig. 74-11) were proposed to illustrate the mechanism of SCFA transport: (1) the function of a SCFA-bicarbonate exchanger (255,258); and (2) absorption of nonionized SCFA by passive diffusion (252,253). The protons required for the latter mechanisms could be provided either via an Na+-H+ exchange mechanism localized to the apical membrane of the enterocyte or colonocyte (19,21,266–271) or by intraluminal hydration of CO2 to carbonic acid with subsequent dissociation of bicarbonate and protons (252,265). The major SCFAs in the colonic lumen have pKa values of ~4.8. Because the normal colonic luminal pH ranges from 6.0 to 8.0, majority of the SCFAs in the lumen are expected to be in the ionized form, and hence to be taken up via a carrier-mediated mechanism, rather than by a simple diffusion of the protonated form. However, this increased presence of ionic SCFAs does not simply mean that the majority of the SCFAs will be absorbed via this mechanism, because there are other factors including the greater permeability of the nonionized forms compared with ionized forms, together with the chemical equilibrium of ionized and nonionized SCFAs, which could also greatly influence the quantitative contribution of each pathway (208). Although both of these proposed mechanisms of SCFA uptake are now well accepted to be operative in the mammalian intestine, the relative contribution of each individual mechanism remains debatable. Studies providing evidence to support both pathways are summarized in the following sections.

SCFA-Anion Exchange Apical SCFA-HCO3− Exchange Earlier evidence from perfusion studies in the human intestine showed that SCFA absorption was accompanied by the secretion of luminal HCO3−, which could be explained by

SCFAH

the presence of the luminal membrane SCFA-HCO3− exchange mechanism. Direct evidence to support this model came from extensive studies using purified AMVs and BLMVs from the human and rat intestinal tissues (272–276). These studies involved uptake of radiolabeled SCFA in vesicles loaded with OH− or HCO3− ions. These studies demonstrated the presence of SCFA-HCO3− (OH−) exchange in luminal membranes of the rat colon (275) and human ileum and colon (272,273). A kinetically distinct SCFA-HCO3− (OH−) exchanger also was shown to be present on BLMVs purified from the rat (276) and human colon (274). The studies performed using Caco-2 cells (277) and AA/C1 human colonocytes (278) in culture also support the presence of a carrier-mediated SCFA-OH− exchange or a carrier-mediated SCFA-H+ cotransport process involved in SCFA uptake at the luminal membranes of these cells. The evidence in support of a carrier-mediated process for SCFA uptake in studies using purified AMVs includes: (1) demonstration of an ‘overshoot’ phenomenon; (2) saturation kinetics; (3) inhibition by substrate analogs; (4) exhibition of a trans-stimulation phenomenon; and (5) inhibition by transport inhibitors. Apical SCFA-Cl Exchange Apart from the presence of an SCFA-HCO3− exchange process in rat colonic AMVs, a unique unidirectional SCFA-Cl exchanger distinct from the Cl−-HCO3− or SCFA−-HCO3− exchanger also has been shown to be present, which has been suggested to be a key transporter involved in stimulation of NaCl absorption by SCFAs (279). The evidence to support the existence of such a transporter is derived from the following findings: (1) intravesicular SCFA loading significantly stimulated Cl− uptake in these membranes; however, intravesicular chloride loading did not stimulate SCFA uptake; (2) this transporter showed maximal activity at a pH of 6.5; (3) unlike the SCFA−-HCO3− exchange process in these membranes, butyrate gradient–dependent chloride uptake was inhibited by DIDS; (4) this transporter was saturated by either increasing intravesicular SCFA or extravesicular

2K+

SCFAH ~ATP CO2 + H2O

H+

3Na+

CA

+

−

HCO3

SCFA− −

HCO3

SCFA−

−

HCO3

FIG. 74-11. Proposed mechanisms of short-chain fatty acid (SCFA) transport in the colonocytes. ATP, adenosine triphosphate.

INTESTINAL ANION ABSORPTION / 1903 shown to have distinct kinetic characteristics compared with their apical counterparts. In addition, in contrast to rat colonic AMV SCFA-HCO3− exchange, which was shown to be resistant to inhibition by the typical anion exchange inhibitor DIDS, the basolateral transporter was found to be highly sensitive to inhibition by DIDS. Studies also have characterized the existence of a distinct SCFA-HCO3− exchanger in the human colonic BLMV (274) with distinct kinetic characteristics compared with its apical counterpart. However, unlike the rat colonic BLMV transporter, the human colonic BLMV SCFA-HCO3− exchanger was insensitive to inhibition by DIDS similar to its apical counterpart. The coordination of these basolateral SCFA transporters with their apical counterparts may be important for transepithelial SCFA absorption and bicarbonate secretion.

chloride concentration; and (5) other intravesicular SCFA molecules, for example, propionate and acetate, also stimulated chloride uptake. Spatial distribution studies for SCFA transporter activities in the rat colon showed that although the SCFA−-HCO3− exchanger expression was limited to the surface cells, SCFA-Cl exchange activity was shown to be present in both surface and crypt cells (84). The existence of this Cl−-SCFA− exchange mechanism in the rat colonic AMVs was suggested to be an important pathway involved in stimulation of NaCl absorption (Fig. 74-12). According to this model, initial SCFA uptake at the apical membrane domains via an SCFA-HCO3− exchange should acidify the cytoplasm and subsequently activate apical Na+H+ exchange activity, and SCFA then should be recycled via an SCFA-Cl exchange, resulting in net stimulation of NaCl absorption (see Fig. 74-12). However, the validity of such a model for stimulation of NaCl absorption by SCFA cannot be considered as a generalized mechanism functional in all species, because in contrast with these findings from the rat colonic AMVs, the existence of such an SCFA-Cl exchange process could not be confirmed from the human colonic AMVs (272). Flux studies performed in the rabbit colon also did not support the existence of such an SCFA-Cl exchange process in the rabbit colon (280).

Nonionic Diffusion of Short-Chain Fatty Acids Although nonionic diffusion has been shown to be one of the important mechanisms of SCFA uptake by colonocytes, the relative contribution of the nonionic diffusion versus carrier-mediated SCFA uptake remains a debatable issue. Nonionic diffusion, as one of the important mechanisms of SCFA absorption, is considered to involve the cross-membrane movements of the protonated SCFAH form, which is more membrane permeable compared with ionized SCFA−. Nonionic diffusion for SCFAs is extracellular pH dependent and is supposed to be nonsaturable with increasing concentrations of the SCFAs. Evidence to support the existence of such a pathway in SCFA absorption can be summarized as follows: (1) studies of radiolabeled SCFA transport with rat proximal and distal colonic mucosal tissues mounted in Ussing chambers showed that mucosal-to-serosal or serosalto-mucosal fluxes increased when the pH of SCFA-containing buffer was reduced and increasing mucosal SCFA concentration up to 100 mM caused a linear increase in mucosal-toserosal SCFA flux without any evidence of saturation (282);

Basolateral SCFA-HCO3− Exchange Because not all SCFAs taken up by colonocytes are completely metabolized, some of it escapes colonocytes via BLM domains into the bloodstream. Earlier studies have shown extensive metabolic use of SCFAs by liver and other peripheral tissues (281). Therefore, the existence of transport mechanisms for SCFA movement across the BLM domains of the colonocytes also was investigated. Studies using purified rat colonic BLMVs demonstrated the existence of both simple diffusion and an SCFA-HCO3− exchanger distinct from their apical counterparts (276). This transporter was

H+ Na+ Cl−

H+ Cl− SCFA−

−

SCFA− HCO3−

CA CO2 + H2O + − HCO3 SCFA− HCO3

HCO3−

FIG. 74-12. Role of SCFA-Cl exchange in stimulation of NaCl absorption in rat colon. SCFA, short-chain fatty acid. (Modified from Rajendran and Binder [84], by permission.)

1904 / CHAPTER 74 (2) in isolated mouse and rabbit colonocytes, initial rapid reduction in pHi produced by SCFA treatment was shown to be predominantly caused by protonated SCFAs; however, it was not possible to pinpoint the membrane localization of the nonionic diffusion pathways (283,284); (3) Ussing chamber experiments with the guinea pig and rabbit colon also demonstrated mucosal-to-serosal SCFA flux to be proportional to mucosal SCFA concentration without any evidence of saturability (263,283). The above results support some of the earlier SCFA transport studies suggesting nonsaturability of their transport. Although the above data strongly indicate the important role of nonionic diffusion in SCFA transport, a number of other studies have yielded complex results indicating the presence of both the diffusion and the carrier-mediated pathways. For example, although medium acidification increased the SCFA uptake in rabbit and rat colonocytes (283,285), the increased uptake was not entirely proportional to the predicted change in the concentration of the nonionized SCFA. A study showing limited permeability of cecal and colonic apical membranes in guinea pigs to SCFA also supports the notion that SCFA uptake at the apical membrane domains is not simply caused by diffusion (44). As discussed earlier, increasing evidence now strongly suggests the involvement of both the nonionic diffusion and SCFA transporters in pH changes caused by SCFA, as well as in transepithelial SCFA transport.

Molecular Identity of Short-Chain Fatty Acid Transporters: Role of Monocarboxylate Transporters SCFAs are important monocarboxylates that have been suggested to be transported by members of the monocarboxylate transporter (MCT) gene family, specifically MCT1, based on some earlier studies demonstrating inhibition of their uptake by typical MCT inhibitor α-cyano 4-hydroxycinnamate (CHC) and expression of MCT1 in the intestine (285–289). More evidence using purified colonic AMVs from pigs and humans, as well as direct molecular biology studies with cells in culture, has come forth supporting the role of MCTs in SCFA transport in the intestine (60,277,285,290). MCTs are members of SLC16 gene family known to function as proton-linked MCTs. Currently, 14 members of this family have been identified, of which MCT1-4 have been demonstrated experimentally to catalyze the transport of important monocarboxylates (288,289). This section focuses on providing an introduction to this MCT gene family and the evidence to support the role of MCT1 in luminal membrane SCFA transport, as well as the expression of other members of the MCT gene family in the intestine. The potential role of MCT regulation at the molecular level and its implications in SCFA transport also are evaluated. SLC16 Gene Family Table 74-4 summarizes the identified MCT members, function, tissue expression, predominant substrates, and

representative citations. MCT1 was the first member of this gene family identified; it is expressed ubiquitously and is the major focus here because of its potential role in colonocyte apical membrane SCFA transport. MCT1 is known to catalyze either the transport of one monocarboxylate with one proton or the exchange of one monocarboxylate with another. MCT2-4 also have been shown to transport monocarboxylates. Tat1 or SLC16A10 has been shown to transport aromatic amino acids in a sodium- and proton-independent manner (291). The functional roles of other MCT isoforms remain to be established. Monocarboxylate Transporter 1 MCT1 is expressed ubiquitously (288,289). MCT1 expressed in Xenopus oocytes was shown to transport shortchain (C2-C5), unbranched, aliphatic monocarboxylates such as acetate, propionate, as well as pyruvate, L-lactate, and acetoacetate (297,298). However, bicarbonate, dicarboxylates, tricarboxylates, and sulfonates were not transported (297,298). MCT1 is a 40- to 50-kDa protein. Hydropathy plot analysis predicts 10 to 12 α-helical TMDs for MCT family members with the N and C termini located in the cytoplasm (289) (Fig. 74-13). This orientation has been confirmed experimentally in erythrocytes for MCT1 (299). The greatest sequence variability among isoforms is observed in the long C-terminal loop and in TMDs 6 to 7, the large intracellular loop. Two highly conserved sequence motifs typical of the MCT family members can be identified near TMDs 1 and 5 (288,289) (see Fig. 74-13). None of the MCTs are known or predicted to be glycosylated (288). Site-directed mutagenesis in Asp302 to Glu in TMD 8 of rat MCT1 has been shown to abolish the lactate transport (300). Arg313 of human MCT1 has been shown to be conserved in all the MCTs, and its mutation has been shown to greatly reduce the affinity of MCT1 for lactate (300). Ancillary proteins have been shown to be required for plasma membrane expression of MCTs (288,289,301). MCT1 and MCT4 have been shown to require CD147 or the related protein GP70 for proper cell-surface targeting and function. These proteins are commonly expressed cellsurface glycoproteins with a single TMD, two extracellular immunoglobulin-like domains, and a short C-terminal cytoplasmic region (301). Antibodies against CD147 have been shown to coimmunoprecipitate MCT1 and MCT4 in a variety of cell types (301). Evidence for the Role of Monocarboxylate Transporter 1 in Intestinal Short-Chain Fatty Acid Absorption Because SCFAs are monocarboxylates and their uptake in intestinal epithelial cells was shown to be inhibited, at least in some studies, by conventional MCT inhibitor 4-hydroxy cinnamates, it was of interest to a number of investigators to further elucidate the role of MCTs in SCFA absorption (277, 284,285). MCT1 protein previously has been shown to be expressed in the intestine; however, its apical membrane localization had been controversial (277,285,287,302,303).

INTESTINAL ANION ABSORPTION / 1905 TABLE 74-4. The SLC16 (monocarboxylate transporter) gene family Human gene

Protein

Function +

Substrate

Tissue distribution

Reference

Lactate, pyruvate, butyrate, ketone bodies T3, T4

Ubiquitous

287, 292

Intestine, liver, heart, brain, thymus, ovary, prostate, pancreas, placenta

295

Intestine, skeletal muscle, chondrocytes, leukocytes, testis, lung, placenta, heart Brain, muscle, liver, kidney, intestine, lung, ovary, placenta, heart Kidney, muscle, brain, heart, pancreas, prostate, lung, placenta Brain, pancreas, muscle Kidney, brain

294, 303

295 293

RPE, choroid plexus

296

Endometrium, testis, ovary, breast, brain, kidney, adrenal, retina Kidney, intestine, muscle, placenta, heart

288, 289

SLC16A1

MCT1

MC-H cotransport or exchanger

SLC16A2

MCT8

Facilitated transport

SLC16A3

MCT4

MC-H+ cotransport

SLC16A4

MCT5

—

SLC16A5

MCT6

—

SLC16A6 SLC16A7

MCT7 MCT2

SLC16A8

MCT3

— MC-H+ cotransport MC-H+ cotransport

SLC16A9

MCT9

SLC16A10

Tat1

Facilitated transport

SLC16A11

MCT11

—

Skin, lung, ovary, breast, pancreas, RPE, choroid plexus

289

SLC16A12 SLC16A13 SLC16A14

MCT12 MCT13 MCT14

— — —

Kidney Breast, bone marrow stem cells Brain, heart, ovary, breast, lung, pancreas, RPE, choroid plexus

289 289 289

Lactate, pyruvate, ketone bodies

Lactate, pyruvate, ketone bodies

Aromatic amino acids (W, Y, F, L-DOPA)

MCT, monocarboxylate transporter; RPE, retinal pigment epithelium. Modified from Halestrap and Meredith (289).

K290

1

2

3

4

5

6

7

8 D 302

K413

9

10

11

12

F 360

R213

NH2

COOH

FIG. 74-13. Predicted topology of monocarboxylate transporter-1 (MCT1). (Modified from Halestrap and Meredith [289], by permission.)

295, 303 295

291

1906 / CHAPTER 74 Some reports had localized MCT1 to BLMs of hamster colonocytes (287), both basolateral and apical membranes in the small and large intestines of rat (302), and apical membranes of the pig and human colonic cells (277,285, 303). Studies using Xenopus oocyte expression of MCT1, as well as antisense MCT1 studies in Caco-2 cells, have further confirmed the role of MCT1 in SCFA transport (277,285). MCT1 could function as an SCFA-OH exchanger shown in the apical membranes of the rat and human colonic epithelium or as a SCFA-H+ cotransporter. The existence of other transporters involved in SCFA transport, however, cannot be ruled out. The salient features supporting the involvement of MCT1 in apical SCFA uptake in the human intestine include: (1) inhibition of the uptake by CHC; (2) expression of the MCT1 protein on the apical membrane but not the BLM of the human colonocytes; (3) cis inhibition of the apical SCFA uptake by L-lactate, but not D-lactate; (4) inhibition of SCFA uptake in Caco-2 cells on transfection of MCT1 antisense. In addition, several studies examining the regulation of butyrate uptake into Caco-2 cells (see the following section) also demonstrate parallel changes in surface expression of MCT1, further confirming the involvement of MCT1 in intestinal SCFA absorption (60,290). Surface-crypt cell distribution studies of MCT1 expression in the human colonocytes demonstrated that, although the MCT1 protein was expressed on apical membranes of both the crypt and the surface epithelial cells, the protein expression was significantly higher in surface cells (304). In contrast, MCT1 mRNA expression was shown to be much higher in cells lining the crypt compared with surface cells (304). Other MCT isoforms also were shown to be expressed in the human intestinal Caco-2 cells, as well as in biopsies from various regions of the human intestine (277,303). Immunoblotting studies using purified apical membrane and BLM fractions from the organ donor intestines demonstrated that, in addition to the MCT1 expression on the apical membrane domains, MCT4 and MCT5 proteins were expressed predominantly on the BLM domains of the human colonocytes (303). The expression of the MCT1, MCT4, and MCT5 was found to be significantly higher in distal regions of the intestine

compared with more proximal regions (303). It is possible that MCT4, MCT5, or both may represent the key basolateral SCFA transporter. In this regard, the functional role of MCT5 in monocarboxylate transport has not yet been demonstrated experimentally. Based on some of these studies, a model for SCFA transport involving various MCT transporters along the apical and basolateral poles of the human colonocyte has been proposed, as shown in Figure 74-14. Additional studies to establish the function and identity of the basolateral transporter(s), as well as the apical transporter, will be of great interest to fully understand the molecular basis of SCFA absorption in mammalian intestine.

Regulation of Short-Chain Fatty Acid Absorption in the Intestine Earlier studies in skeletal and cardiac muscles have shown alterations in both protein and mRNA expression of MCT1 in response to exercise, stress, and several other conditions (288,289,305). Our current focus, however, is the regulation of the MCT1 in the intestine in relation to SCFA transport. In this regard, although an increasing number of investigations are focusing on understanding the mechanisms of regulation of various colonic physiologic and pathophysiologic processes, limited information is available on the regulation of SCFA absorption in the intestine. Studies using inhibitors of constitutive NO synthase showed that cNO was inhibitory to the SCFA−-HCO3− exchange in rabbit ileal villus cells (67). Studies pertaining to the identification of the molecular nature of the apical SCFA transporter MCT1 have provided some new insights into the molecular regulation of SCFA transport in colonocytes. For example, apical butyrate uptake was shown to be significantly increased in Caco-2 cells in response to 24-hour treatment with phorbol ester PMA (60). This increase was shown to be mediated via increased expression of MCT1 in these cells. In contrast with the effect of PMA on butyrate uptake and MCT1 expression, hydrocortisone treatment failed to affect both of these parameters (60). With respect to expression of the

HCO3− MCT4 CO2 + H2O

SCFA−

SCFA−

CA

MCT1 HCO3−

HCO3−

SCFA− MCT5 HCO3−

FIG. 71-14. Speculative model of the roles of monocarboxylate transporters (MCTs) in human colonic short-chain fatty acid (SCFA) absorption.

INTESTINAL ANION ABSORPTION / 1907 MCT1 in disease states, a reduction in MCT1 expression in human colonocytes has been shown during its transition from normalcy to malignancy (304). Studies with cultured human colonic epithelial cells AA/C1 have shown substrate regulation of the expression of MCT1 protein, mRNA, and butyrate uptake in response to 24-hour incubation with its substrate butyrate itself (278). In these studies, increased butyrate uptake in response to 24-hour incubation with butyrate was shown to be caused, in part, by transcriptional regulation and by increased stability of the MCT1 mRNA (278). However, no direct MCT1 promoter studies were performed. More recently, the human MCT1 promoter also has been cloned and has been found to be active in human colonic cells AA/C1 and Caco-2 cells (306,307). Studies using the MCT1 promoter analysis in Caco-2 cells indicate that upstream stimulatory factors (USF1 and USF2) function as repressors of hMCT1 (307). In addition, the stimulation of MCT1 expression in response to PMA appears to be secondary to stimulation of MCT1 promoter activity (308). Additional preliminary studies demonstrate the acute regulation of MCT1 in response to neurotransmitter 5-HT (serotonin) in Caco-2 cells. In these studies, dose-dependent inhibition of butyrate uptake by 5-HT treatment in Caco-2 cells was found to parallel the reduced surface expression of MCT1 protein in these cells (309). Hormonal regulation of the human intestinal butyrate uptake also was shown using Caco-2-BBE cells (290). In this study, luminal leptin was shown to up-regulate MCT-1–mediated butyrate uptake by increasing its maximal velocity. Leptin was shown to affect butyrate uptake by two different mechanisms: (1) by an increase of intracellular pool of MCT-1 without affecting CD147 expression; and (2) by translocation of the MCT-1/CD147 complex to the luminal membranes of Caco-2-BBE cells (290). Future investigations will shed light on molecular mechanisms of regulation of SCFA absorption via MCTs and their role in colonic physiology and pathophysiology.

to recognize the potential intestinal luminal and basolateral membrane AEs. For example, DRA (SLC26A3, CLD gene), which has been shown to be expressed predominantly in the normal colon, and the PAT1 (SLC26A6), which is expressed throughout the human intestine, appear to be the key transporters involved in chloride absorption in the intestine. Although increasing evidence suggests that DRA may be the central molecule involved in human intestinal luminal membrane Cl−-HCO3− exchange activity, it is, however, not fully established whether DRA performs this function of electroneutral Cl− absorption by itself or in combination with other transporters and regulatory proteins. Because both SLC26A3 (DRA) and SLC26A6 (PAT1) transporters have been shown to be electrogenic in some of the studies (with DRA exchanging two molecules Cl− with one HCO3− and PAT1 exchanging one molecule Cl− with two molecules HCO3−), their coupling together could explain electroneutral Cl−-HCO3− exchange activity observed in intestinal luminal membranes. As shown in the speculative model in Figure 74-15, the electroneutral NaCl absorption could result from either simply coupling of the luminal NHE activity with DRA or with DRA and PAT1. In this regard, some studies show that both DRA and PAT-1 mediate electroneutral rather than electrogenic Cl−/HCO3− exchange (175,210,310). Further exhaustive investigations using knockout mice and protein–protein interactions will be important to test the validity of this Na+ 2Cl−

1Cl−

1HCO3−

H+

2HCO3−

HCO3−

− H+ + HCO3

CONCLUSION Since the mid-1990s, the literature on the role of various AEs and other transporter family members including SLC4, SLC16, and SLC26 has greatly advanced our knowledge with respect to identification of the new members of these transporter families, characterization of their expression in various tissues, and their potential role in GI anion transport. Although the involvement of coupling of the Cl−-HCO3− exchanger and NHE in electroneutral NaCl absorption in the ileum and colonic regions of mammalian intestine has long been established, the molecular identity of the transporters involved in NaCl absorption is only now beginning to unfold. There has been enormous progress in the investigations pertaining to the identification of intestinal NHEs, as well as their role; however, the identity of the intestinal luminal membrane Cl−-HCO3− exchanger(s) has long been a puzzle. Only recently with the advent of studies of the CLD disorder and other molecular biology studies have we come

SCFA−

CA CO2 + H2O SCFA−

HCO3−

3Na+

HCO3−

2K+

HCO3−

Cl−

Cl−

FIG. 74-15. Speculative model of NaCl and short-chain fatty acid (SCFA) absorption in the human ileal and colonic epithelial cells. ATP, adenosine triphosphate; CA, carbonic anhydrase; CLD, chloride diarrhea; NHE, Na+-H+ exchanger; PAT1, putative anion transporter-1.

1908 / CHAPTER 74 model in a more direct way. A number of studies also have shown the modulation of the intestinal luminal Cl−-OH−, HCO3− exchange activity by various agents including NO, PKC, serotonin, aldosterone, bacterial infections, and reactive oxygen species. Future studies to investigate the molecular basis of the regulation of these AEs to better understand the pathophysiologic basis of diarrhea and other absorptive disorders will be important. With respect to SCFA absorption in the intestine, although the physiologic evidence suggests the involvement of both nonionic diffusion and SCFA carrier-mediated transport, the molecular nature of the SCFA transporter is only now beginning to become clear. Early studies to date point to the involvement of the MCT1 in luminal SCFA uptake in the human intestine and the existence of the other MCT isoforms on the basolateral domains. The functional importance of these MCTs in the human intestine needs to be further established to explain the molecular basis of SCFA absorption and any alterations in SCFA absorption observed in response to serotonin, PKC, and bacterial infections, as described earlier. Figure 74-15 also shows a speculative model demonstrating the potential transporters involved in SCFA absorption in the human intestinal epithelial cell. A comparison of some of the studies reported in animal models versus the studies in the human intestine also further support the notion that it may not be feasible to simply extrapolate the findings from the animal models to understand the mechanisms of anion absorption in the human intestine. Future studies using human intestinal cells in culture, molecular biology studies, and gene linkage studies, as well as in vitro studies using human tissues obtained from surgical specimens, may be critical to establish the molecular mechanisms of anion absorption in the human intestine.

ACKNOWLEDGMENTS We extend our gratitude to Dr. Ravinder K. Gill for her extraordinary assistance during the preparation of this chapter with respect to studies of the SLC26 family and preparation of all the figures and models. We also thank Dr. Waddah A. Alrefai for his assistance with the SLC4 gene family section. Laboratory studies were supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (grants DK54016 and DK68324 to P.K.D.; grants DK33349 and DK67990 to K.R.), the Department of Veterans Affairs Merit Award, and Research Enhancement Award Program (REAP) awards.

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283. Chu S, Montrose MH. Non-ionic diffusion and carrier-mediated transport drive extracellular pH regulation of mouse colonic crypts. J Physiol (Lond) 1996;494(pt 3):783–793. 284. DeSoignie R, Sellin JH. Propionate-initiated changes in intracellular pH in rabbit colonocytes. Gastroenterology 1994;107:347–356. 285. Ritzhaupt A, Wood IS, Ellis A, Hosie KB, Shirazi-Beechey SP. Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate. J Physiol (Lond) 1998;513(pt 3):719–732. 286. Tamai I, Takanaga H, Maeda H, Sai Y, Ogihara T, Higashida H, Tsuji A. Participation of a proton-cotransporter, MCT1, in the intestinal transport of monocarboxylic acids. Biochem Biophys Res Commun 1995;214: 482–489. 287. Garcia CK, Goldstein JL, Pathak RK, Anderson RG, Brown MS. Molecular characterization of a membrane transporter for lactate, pyruvate, and other monocarboxylates: implications for the Cori cycle. Cell 1994;76:865–873. 288. Halestrap AP, Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 1999;343(pt 2):281–299. 289. Halestrap AP, Meredith D. The SLC16 gene family-from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond. Pflugers Arch 2004;447:619–628. 290. Buyse M, Sitaraman SV, Liu X, Bado A, Merlin D. Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE. J Biol Chem 2002;277: 28182–28190. 291. Kim DK, Kanai Y, Chairoungdua A, Matsuo H, Cha SH, Endou H. Expression cloning of a Na+-independent aromatic amino acid transporter with structural similarity to H+/monocarboxylate transporters. J Biol Chem 2001;276:17221–17228. 292. Garcia CK, Li X, Luna J, Francke U. cDNA cloning of the human monocarboxylate transporter 1 and chromosomal localization of the SLC16A1 locus to 1p13.2-p12. Genomics 1994;23:500–503. 293. Garcia CK, Brown MS, Pathak RK, Goldstein JL. cDNA cloning of MCT2, a second monocarboxylate transporter expressed in different cells than MCT1. J Biol Chem 1995;270:1843–1849. 294. Grollman EF, Philp NJ, McPhie P, Ward RD, Sauer B. Determination of transport kinetics of chick MCT3 monocarboxylate transporter from retinal pigment epithelium by expression in genetically modified yeast. Biochemistry 2000;39:9351–9357. 295. Price NT, Jackson VN, Halestrap AP. Cloning and sequencing of four new mammalian monocarboxylate transporter (MCT) homologues confirms the existence of a transporter family with an ancient past. Biochem J 1998;329(pt 2):321–328. 296. Lafreniere RG, Carrel L, Willard HF. A novel transmembrane transporter encoded by the XPCT gene in Xq13.2. Hum Mol Genet 1994;3: 1133–1139. 297. Broer S, Schneider HP, Broer A, Rahman B, Hamprecht B, Deitmer JW. Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH. Biochem J 1998;333(pt 1):167–174. 298. Broer S, Rahman B, Pellegri G, Pellerin L, Martin JL, Verleysdonk S, Hamprecht B, Magistretti PJ. Comparison of lactate transport in astroglial cells and monocarboxylate transporter 1 (MCT 1) expressing Xenopus laevis oocytes. Expression of two different monocarboxylate transporters in astroglial cells and neurons. J Biol Chem 1997;272: 30096–30102. 299. Poole RC, Sansom CE, Halestrap AP. Studies of the membrane topology of the rat erythrocyte H+/lactate cotransporter (MCT1). Biochem J 1996;320(pt 3):817–824. 300. Rahman B, Schneider HP, Broer A, Deitmer JW, Broer S. Helix 8 and helix 10 are involved in substrate recognition in the rat monocarboxylate transporter MCT1. Biochemistry 1999;38:11577–11584. 301. Kirk P, Wilson MC, Heddle C, Brown MH, Barclay AN, Halestrap AP. CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression. EMBO J 2000;19:3896–3904. 302. Tamai I, Sai Y, Ono A, Kido Y, Yabuuchi H, Takanaga H, Satoh E, Ogihara T, Amano O, Izeki S, Tsuji A. Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1. J Pharm Pharmacol 1999;51:1113–1121. 303. Gill R, Hadjiagapiou C, Alrefai WA, Saksena S, Carrol R, Goldstein J, Ramaswamy K, Dudeja PK. Expression and membrane localization of MCT isoforms along the length of the human intestine. Am J Physiol Cell Physiol 2005;289(4):C846–52.

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CHAPTER

75

Ion Channels of the Epithelia of the Gastrointestinal Tract John Cuppoletti and Danuta H. Malinowska Cystic Fibrosis Transmembrane Regulator in Chloride Transport in the Gastrointestinal Tract, 1918 Intestine, 1918 Structural and Functional Characterization of Cystic Fibrosis Transmembrane Regulator in the Intestine, 1918 Pancreatic Cystic Fibrosis Transmembrane Regulator, 1919 Calcium-Activated Chloride Channels, 1919 CIC Family of Chloride Channels, 1919 ClC-2, 1919 ClC-4, 1920 Calmodulin Kinase II–Activated ClC-3 Chloride Currents, 1920 Summary, 1920 Epithelial Sodium Channel, 1920 Potassium Channels, 1920 Basolateral Membrane Potassium Channels, 1920 Apical Membrane Potassium Channels, 1921

Human Tissues and Human Cell Models: Species Differences, 1922 Methods for Study of Ion Channels in Gastrointestinal Tissues, 1922 Nystatin Permeabilization and Use of 1-Ethyl-2-benzimidazolinone or Forskolin, 1922 Permeabilization, 1922 Ussing Chambers: Transepithelial Electrical Resistance and Short-Circuit Current, 1923 Ion Channel Effectors, 1923 Effectors of Phosphatases and Protein Kinases, 1923 Transfection: Sense and Antisense and Dominant Negatives, 1924 Permeable Supports, 1925 Structure-Function Studies, 1925 Summary, 1925 References, 1925

This chapter is a brief review of known chloride, sodium, and potassium channel proteins that are involved in gastrointestinal epithelial transport in human health and disease. Figure 75-1 shows a summary of the localization of ion channels in the intestinal epithelia. Apical membrane chloride channels include cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated chloride channels, and members of the ClC family. The amiloride-sensitive sodium channel (ENaC) is present in some cells in the apical membrane, and evidence exists for potassium channels in the apical membrane. Calciumactivated potassium channels, cyclic adenosine monophosphate (cAMP)–activated potassium channels, and perhaps

other potassium channels are present in the basolateral membranes of most gastrointestinal epithelial cells. This brief overview attempts to provide a foundation for future studies on ion channels of the gastrointestinal tract relevant to human physiology and disease, rather than to present a comprehensive review of all studies of ion channels in the gastrointestinal tract in all species. The molecular description of some aspects of epithelial transport has been reviewed (1). In some cases (e.g., potassium and chloride channels), there may be a variety of channels present in the same tissue or membrane, and data comparing the relative contributions of each of these to various processes is lacking. Despite these disclaimers, there are a few examples where particular chloride, sodium, and potassium channels have been shown to play a major role in physiologically relevant processes or human disease. Human intestinal CFTR chloride channels, a variety of other chloride channels, ENaC, and potassium channels play important roles in absorption and secretion of ions. These and other proteins that have been identified by molecular or functional studies are discussed in this chapter.

J. Cuppoletti and D. H. Malinowska: Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1917

1918 / CHAPTER 75 Apical

Basolateral

K+ Cl− Na+

Cl−

K+

Na+ K+

Na+

Na-K-2Cl Cotransporter Na+ Pump K+ Channel

K+ Na+

Paracellular path

FIG. 75-1. Ion channels of human gastrointestinal epithelia. Cystic fibrosis transmembrane conductance regulator, calcium-activated chloride channels and ClC-2 may be present in the apical membrane. Potassium channels and amiloride-sensitive sodium channels also have been measured in the apical membrane. The Na+ pump and a variety of potassium (calcium- and cyclic adenosine monophosphate–dependent) channels are found in the basolateral membrane, together with the bumetanide sensitive Na-K-2Cl cotransporter.

CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR IN CHLORIDE TRANSPORT IN THE GASTROINTESTINAL TRACT Intestine The CFTR was shown to be defective in patients with cystic fibrosis (CF). CFTR was cloned in 1989 by positional cloning of sequence differences between healthy individuals and patients with CF (2). It is now known that there are more than 1000 mutations in CFTR leading to disease. The largest number of patients with CF exhibits the ∆F508 mutation (3). The various CFTR mutations affect membrane trafficking, conductance, and stability of the protein. Multiple gene products, as well as the environment, may play a role in the manifestation of the disease (4). Expression of mutant CFTR also leads to transcriptional differences in a variety of additional gene products (5). Intestinal defects are seen in CF, and CFTR was soon also found to be present in T84 (6) and Caco-2 cells (7,8), which are both intestinal epithelial cell lines. CFTR protein also was found in the duodenum and other epithelia (9). Intestinal disease correlates with the CFTR genotype and phenotype (10).

Structural and Functional Characterization of Cystic Fibrosis Transmembrane Regulator in the Intestine The first studies of the role of CFTR in intestinal transport showed that the apical membrane of T84, HT-29, and Caco-2 cells exhibited CFTR function (11,12). These articles are landmarks for demonstrating CFTR function in epithelial (airway and intestinal) cells and are highly useful as a guide

to the best practices in epithelial ion channel studies. A correlation was soon demonstrated between electrogenic chloride transport in Caco-2 cells and mRNA for CFTR (13). Moreover, in the intestine, decreasing gradients of expression of CFTR gene products are observed along both the cryptvillous and proximal-distal axes. This expression was consistent with CFTR being responsible for secretion of chloride in the intestinal crypts (14). Others confirmed the findings that CFTR was present in the apical membrane of T84 cells in functional studies using permeabilization (15), and cAMPdependent chloride transport was found to be increased in polarized HT-29 cells compared with unpolarized cells (16). Anderson and Welsh (11) definitively demonstrated CFTR in the apical membrane of intestinal epithelial cells using the technique of nystatin permeabilization. Nystatin is a polyene antibiotic that produces 8-nm pores in membranes. In cells treated unilaterally with this drug, the apical or basolateral membrane ion currents can be measured without interference from the other nystatin-permeabilized membrane (17). Disruption of CFTR in the mouse resulted in a reduction in intestinal chloride transport as predicted from studies of human CF epithelia, namely, reduced cAMP activation of chloride transport (18). In fact, these studies demonstrated a loss of cAMP stimulated chloride transport in the intestinal epithelia in the knockout mouse. In this mouse model, cholera toxin did not elicit secretory diarrhea compared with normal mouse, and the heterozygotes gave only 50% of the secretory response, suggesting that individuals with one abnormal CFTR allele would be protected in cholera (the so-called heterozygote advantage) (19). A drug that blocks CFTR and secretory diarrhea in the mouse also has been identified (20). However, the view that CFTR is involved in cholera was challenged in human studies of the effects of cholera toxin, forskolin, vasoactive intestinal polypeptide,

ION CHANNELS OF THE EPITHELIA OF THE GASTROINTESTINAL TRACT / 1919 isoprenaline, heat-stable enterotoxin, guanylin, carbachol, and lysylbradykinin (21). A study of patients with CF and heterozygotes suggested that there is no genetic advantage for heterozygotes, because heterozygotes exhibited the same chloride transport as healthy individuals (22). However, more recent studies of a small molecule inhibitor have shown that CFTR is likely involved in cholera toxin effects in mouse models, T84 cells, and human intestinal sheets (23). The relative role of other chloride channels, such as calcium-activated chloride channels, in human viral diarrhea or inflammatory bowel disease remains unknown (24), but heat-stable enterotoxin–stimulated secretion in pig intestine was inhibited by diarylsulfonylureas, inhibitors that affect CFTR, but not ClC-2 or calcium-activated chloride channels (25). Other channels may also be involved in the response to cholera toxin (26). In human studies of twins affected by CF, there was evidence for residual chloride transport mediated by AF508 CFTR, calcium-activated and other non-CFTR chloride channels (27). The role of non-CFTR chloride channels in altering the phenotype of patients with CF and in providing potential pharmacologic targets is suspected but unknown.

Pancreatic Cystic Fibrosis Transmembrane Regulator CFTR protein also is localized to proximal duct epithelial cells of the pancreas (28), and bicarbonate secretion is defective in CF (29,30). The earliest studies of various CFTR mutations linked genotype and pancreatic disease (31), and different mutations have different effects on pancreatic function (32). A variety of studies suggest that modifier genes may play a role in the manifestation of disease for different genotypes (33–35). Defective bicarbonate secretion is seen in the many cell types that express CFTR, suggesting that either CFTR or a modifier gene is involved in bicarbonate secretion defects seen in patients with CF (36). However, defective bicarbonate transport does not correlate with defective CFTR, but rather with differential interactions of mutant CFTR with electrogenic chloride cotransporters including DRA and pendrin (37).

CALCIUM-ACTIVATED CHLORIDE CHANNELS It has long been known that cAMP and calcium affect chloride transport in the intestine (38), because cAMP and calcium each activate basolateral potassium channels, but cAMP also activates apical chloride channels. Synergism results from cooperative activation of potassium channels and the chloride channel. From studies in T84, HT29, and Caco-2 cell lines (11), it was suggested that the intestine lacked calcium-activated chloride channels (see Morris [39], Hartzell and colleagues [40], and Fuller and colleagues [41] for reviews of calcium-activated chloride channels). Hyperosmolar treatment of the apical membrane of intestinal T84 epithelial

cells resulted in activation of two different chloride channels, one of which was sensitive to disodium 4,4′diisothiocyanostilbene-2,2′-disulfonate (DIDS), and another that was insensitive to DIDS (42). Calcium-activated chloride currents are stimulated by carbachol or thapsigargin and are inhibited by mucosal uridine triphosphate (43), insulin, and insulin-like growth factor-1 (44). In addition, calcium-dependent chloride secretion is inhibited by p38 mitogen-activated protein kinase (45). Functional studies of calcium-activated chloride currents involved in transepithelial transport have not yet been related to a particular channel protein, but a great deal is known of the various systems of control of calcium-activated chloride transport (46). The murine calcium-activated chloride channel (mCLCA3) (alias gob-5) protein is located in the mucin granule membranes of intestinal, respiratory, and uterine goblet cells (47). It has been suggested that mCLCA3 may be involved in the synthesis, condensation, or secretion of mucins. CLCA1 and CLCA2 are both found in the intestine, and their levels appear to be developmentally regulated. However, the functions of these isoforms are unknown (48). Genetic linkage studies demonstrate that the CLCA gene region encodes mediators of a DIDS-sensitive anion conductance in the human gastrointestinal tract that modulate the CF basic defect. (49). One form of the CLCA channel has been shown to induce cAMP-dependent chloride currents when transfected into intestinal cells (50).

CIC FAMILY OF CHLORIDE CHANNELS ClC-2 ClC-2 was first cloned from rats (51), and later from humans (52). The human channel was shown to be activated by protons (53) and protein kinase A (PKA) (54). ClC-2 has been shown to exhibit differences in distribution among species. In the guinea pig, it may be localized to the basolateral membrane (55). In the rat, ClC-2 was present in the lateral membranes of villous enterocytes and was predominant at the basolateral membranes of luminal colon enterocytes. The expression pattern of ClC-2 in the human intestine differed significantly, because ClC-2 was mainly detected in a supranuclear compartment of colon cells, with punctuate staining of ClC-2 present at or near the apical membrane (56). ClC-2 also was immunolocalized at or near the apical membrane in T84 cells (57). Chloride currents across the apical membrane of nystatin-permeabilized T84 cells have been shown to be activated by transforming growth factor-α (58), in a process that involved both protein kinase C (PKC) and phosphoinositide-3-kinase. This suggests that apical membrane ClC-2 chloride currents could be involved in physiologically relevant chloride transport across intestinal cells. Similarly, ClC-2 was shown to be involved in apical membrane chloride transport in Caco-2 cells (59). Recently, a prostanoid, lubiprostone (SPI-0211), a treatment

1920 / CHAPTER 75 for constipation (60), has been shown to activate ClC-2, but not CFTR, and this agent increases chloride currents across apical membranes of both nystatin-permeabilized and intact T84 cells (57). ClC-2 also has been shown to be involved in prostaglandin-induced recovery of barrier function after ischemic injury of the intestine (61). ClC-2 may also play a role in the response of intestinal epithelia to cholera toxin. ClC-2, the main chloride channel present in the rat small intestive villus, was up-regulated in the villi 18 hours after cholera toxin administration, which presumably would increase the fluid secretory response (26). ClC-2 has been cloned by expression and homology cloning from rabbit gastric mucosa (62) and has been localized to the parietal cell apical membrane (63). Others have confirmed the presence of abundant ClC-2 mRNA in the rabbit gastric mucosa, but they failed to find the protein by immunostaining parietal cells from humans and rodents using different antibodies (64). The pH of the gastric contents of ClC-2 knockout mice was shown to be less in animals given subcutaneous histamine compared with wild-type animals (65), but more recent studies suggest that ClC-2 knockout mice show decreased carbachol-stimulated acid secretory rates (66). The relative role of ClC-2 and other non-CFTR chloride channels in histamine, gastrin, and carbachol-stimulated rates of acid secretion remains undetermined in mice and humans.

ClC-4 ClC-4 has been found to colocalize with CFTR in human and rodent tissues, and it may recycle from endosomal membranes and play a role in intestinal chloride transport (67).

Calmodulin Kinase II–Activated ClC-3 Chloride Currents HT-29 and T84 cells contain a calmodulin kinase II–activated chloride conductance that appears to be identical to ClC-3 (68,69). This channel has many splice variants and is present in the apical membrane. The physiologic role of this channel in intestinal secretion remains to be determined.

EPITHELIAL SODIUM CHANNEL Amiloride-sensitive sodium currents were demonstrated to be highest in the distal colon and localized to the apical membrane of human colon (73). Interestingly, unstressed rat colon shows little electrogenic sodium absorption, unless treated with aldosterone (74). Cloning of the ENaC and the related degenerins were first accomplished in Xenopus (75) and rats (76). The human form of ENaC also has been cloned (77), and the rat form was localized by in situ hybridization and immunochemistry in the gastrointestinal tract (78), setting the stage for increased understanding of the physiology of ENaC in gastrointestinal physiology. Anti-ENaC antibodies stained apical borders of villous enterocytes in piglet ileum and apical borders of surface cells in the piglet distal colon (79,80). Little is known of the role of defective ENaC in gastrointestinal disease states, although disruption of the β-subunit in a mouse model leads to weight loss, hyperkalemia, and decreased blood pressure when sodium chloride levels in the diet are low (81). ENaC appears to be regulated in the intestine by CFTR, based on increased ENaC activity in the intestine of patients with CF (82). The basis for this and similar observations has not been elucidated at the molecular level, and the effect may be related to altered intracellular chloride levels (83). ENaC mutations do, however, lead to disease states, including Liddle syndrome, a severe form of hypertension associated with ENaC hyperfunction, and pseudo-hypoaldosteronism (PHA-1), a salt-wasting syndrome caused by decreased ENaC function. (84,85). ENaC levels may also be reduced in ulcerative colitis (86). However, it is not known whether ENaC is reduced in human colonic inflammation. Cell lines expressing ENaC, as well as CFTR and potassium channels, are largely lacking. A cell line, LIM1863, which is a morphologically differentiated human colonic crypt cell line that expresses ENaC, as well as CFTR, was studied as a model system (87). However, these cells (88) do not appear to be available commercially, thereby limiting studies with these cells. These or other colonic cell lines are probably required for studies of intestinal ENaC. In addition to ENaC, there exist a class of proteins in the intestine related to ENaC, known as the degenerins (89,90). mRNA for these proteins is found in the brain, liver, and intestine. The degenerins are amiloride sensitive and show 18% to 30% homology to ENaC. The physiologic role of these proteins has not been investigated in the gastrointestinal tract.

SUMMARY New molecular and functional evidence shows that CFTR is not the only chloride channel in the gastrointestinal tract. In the coming years, it will become possible and important to determine the relative roles that the various other chloride channels play in the movement of chloride in the gastrointestinal tract. It is clear that some channels, such as ClC-2, must be studied using activators that are specific to ClC-2, and not generic activators such as forskolin. Studies of PKA-activated chloride channels also should include studies of phosphatase inhibition (11,70–72).

POTASSIUM CHANNELS Basolateral Membrane Potassium Channels It is necessary to provide for passage of potassium across the basolateral membrane to allow maximal chloride secretion (12,91–94). One way to increase basolateral potassium transport is with permeabilization of the basolateral membrane with nystatin (12). This can also be accomplished by activating various potassium channels.

ION CHANNELS OF THE EPITHELIA OF THE GASTROINTESTINAL TRACT / 1921 Activation of basolateral calcium-activated potassium channels by 1-ethyl-2-benzimidazolinone (1-EBIO) also permits apical membrane chloride transport when the chloride channels are appropriately activated (91). These potassium channels can be inhibited by clotrimazole (92). 1-EBIO is an activator of the human intermediate-conductance Ca2+-activated K+ channel (hIK1) and the highly homologous rat protein, a small-conductance, Ca2+-dependent K+ channel (rSK2) (95). Bradykinin-stimulated chloride secretion in T84 cells has been shown to involve a calcium-activated hSK4-like potassium channel (96). A pharmacologic study using EBIO and other agents suggested that a small calcium-regulated potassium channel, SK4, is responsible for this basolateral potassium channel function (97). Inhibition of basolateral potassium channels by clotrimazole has been shown to promote “wound healing” in sheets of T84 and Caco-2 cells (98). Vasoactive intestinal peptide may activate cAMP-dependent potassium channels involved in chloride secretion (99). Forskolin can also be used to activate basolateral cAMP-dependent potassium channels (100). This channel is inhibited by 293B. The molecular nature of this protein is probably KCNQ1 (KvLQT1). In studies of intestinal chloride secretion in a KCNE1 knockout mouse, it was shown that chloride secretion was independent of KCNE1, but required KCNQ1 (101). Pharmacologic studies also showed that KCNQ1 (KvLQT1; a cAMP-activated potassium channel) was important for secretion of chloride (102). A confounding variable with forskolin activation of basolateral potassium channels for studies of chloride transport is the simultaneous effects of forskolin on apical membrane channels and other intracellular processes. In any event, the high level of KvLQT1 expression in epithelial tissues is consistent with its potential role in potassium secretion and recycling, in maintaining the resting potential, and in regulating chloride secretion or sodium absorption, or both (103). There are also reports that calcium-activated largeconductance (BK) potassium channels may play a role in both sodium absorption and chloride secretion (104,105). BK channels appear to be localized to the surface cells of the distal colon responsible for sodium absorption; whereas the abundance of BK channels in the chloride-secreting crypt cells was low or absent (106). T84 cells also contain an arachidonic acid–activated potassium channel that appears to be distinct from the calcium- and forskolin-stimulated potassium channels (107). The molecular identity of this channel has not been determined, but activation by fatty acids including arachidonic acid is consistent with the properties of BK channels (108). Finally, the Kir7.1 potassium channel is found on the basolateral membrane of intestinal epithelial cells (109), where it may be involved in functional coupling with the Na+ pump.

Apical Membrane Potassium Channels Carbachol induces a potassium current across the apical membrane of epithelial cells of rat distal colon, which is sensitive to charybdotoxin and tetraethylammonium (110).

The apical membrane also contains a potassium channel that is sensitive to ammonia (111). BK channels are found in both apical and basolateral membranes of rabbit distal colon epithelial cells (106). In studies using inhibitors of calcium-activated, intermediateconductance (IK) potassium channels, these channels were shown to be responsible for potassium entry (112). These IK channels may also be involved in potassium secretion across the apical membrane (98,113). These channels are inhibited by aldosterone (114). Human intestinal crypts also appear to contain a 29-pS channel that is activated by hypotonic stress (115). Likewise, the Kv1.3 potassium channel protein has been observed in colon epithelial membranes (116). Kv1 is also present, and both Kv1 and Kv1.3 may be regulated by KCNE4, which colocalizes with these subunits. Gastric parietal cells contain potassium channels that are required for gastric acid secretion. As shown in Figure 75-2, the apical membrane of the gastric parietal cell contains the gastric H,K-ATPase, which accomplishes the electroneutral movement of potassium and protons. The production of HCl requires a pathway for the movement of chloride to provide HCl and a pathway for the movement of potassium in exchange for protons (117–120). Molecular aspects of potassium channels in the gastric parietal cell have been reviewed in the literature (121) and are not discussed in detail here. However, it is clear that the apical membrane of parietal cells contains ion conductances for both chloride and potassium (122–125). The basolateral membrane also contains ion conductances (126). A variety of potassium channels have been observed in the gastric parietal cell, including Kir2.1, KCNQ1/KCNE subunits, and

Cytoplasm

Lumen H+

ATP K+

K+

Cl−

−

+

+

−

Cl−

FIG. 75-2. Scheme to account for “electrogenic” proton transport by coupling an electroneutral proton for potassium adenosine triphosphate–driven exchange activity (ATP) in parallel with conductive channels for K+ and Cl− and efficient recycling of K+. (Reproduced from Forte [121], by permission.)

1922 / CHAPTER 75 Kir4.1 (127–130). These may all be important to gastric acid secretion. Some may reside in the apical membrane, and others may be in the basolateral membrane or elsewhere in the cell. Outlining the physiologic roles of these channels is a future challenge. Patch-clamp electrophysiology and molecular approaches are required.

HUMAN TISSUES AND HUMAN CELL MODELS: SPECIES DIFFERENCES Animal models have given important insight into the physiology of gastrointestinal epithelia. However, such models for studies of ion channels at the molecular level require care in their interpretation. Animal models often fail to fully recapitulate human physiology and disease. For example, the mouse model for CF fails to exhibit the lung pathology found in patients with CF, but shows more severe pathology in the intestine (131). Likewise, ouabain sensitivity of the α1-subunit of rodents is much lower than that of other rodent isoforms and of the sodium pump of other animals (132). Finally, polymorphisms in human ENaC that segregate with high blood pressure are not associated with a similar functional change in the mouse channel (133). In addition, CFTR regulates human and mouse ENaC differentially (134). There are also differences that relate to sequence differences in phosphorylation sites between rat and human ClC-2 (70) and differences in the conductance properties of ion channels involved in action potentials between related species of squid (135). Species differences also exist in the chloride concentrations of the epithelia (136). There are also known differences in expression of ion channel isoforms among different species (137), as well as differences in expression of ion exchangers related to physiologically relevant demands between species (138). Thus, care must be taken when comparing results between species. Many authors fail to take species differences into account when interpreting results, and for many studies, human model systems may be most relevant to studies of human disease. Human tissues obtained from biopsy have been used to study epithelial transport (139–142). Rectal biopsy has provided a source of human tissue for study of intestinal ion channels and has been used as a measure for diagnosis of the CF phenotype (139). CFTR measurements in human rectal biopsies have shown that genotype and phenotype are correlated (10,143). Rectal biopsy material also has been used for genetic linkage analysis of chloride channels in CF (49,140) and to study ENaC (144). More often, however, human cell lines are used. These include T84 cells, from a colorectal carcinoma. When grown to confluence as monolayers, they exhibit tight junctions and desmosomes between adjacent cells and maintain vectorial electrolyte transport (145). Caco-2 cells, from a colorectal adenocarcinoma, express receptors for heat-stable enterotoxin (Escherichia coli) and epidermal growth factor. This cell line shows increased CFTR expression with onset of polarization (11). HT-29 cells also have been used for studies of

epithelial transport. These cells are also useful for measurement of paracellular transport (146). All of these cells are available from American Type Culture Collection (ATCC), and all have been used to study chloride and potassium transport. A cell line (LIM1863) that exhibits both chloride and sodium currents also has been identified, as discussed earlier (87). Other cells such as gastric parietal cells must be prepared from human biopsies, or from animal models such as the rabbit. Fortunately, rabbit cells respond similarly to drugs and hormones (147) as cells from humans. There also exist a variety of pancreatic cell lines from humans and animals (148,149).

METHODS FOR STUDY OF ION CHANNELS IN GASTROINTESTINAL TISSUES Nystatin Permeabilization and Use of 1-Ethyl-2-benzimidazolinone or Forskolin Anderson and Welsh (11) outlined several tests for the effectiveness of permeabilization, allowing measurement of apical membrane chloride channels on permeable supports using short-circuit current (Isc). First, current flowed in the opposite direction when the chloride gradient was reversed. Second, agents such as bumetanide, barium, or ouabain did not inhibit cAMP-activated chloride currents. Third, conductance increased with activator and subsequently decreased with inhibitor treatment. Fourth, changes in the gradient concentration resulted in a shift in the reversal potential. Similar controls are required to test for effectiveness of 1-EBIO or other activators. 1-EBIO stimulation can be inhibited by charybdotoxin. 293B is an inhibitor of forskolinactivated potassium channels (92).

Permeabilization Ion channel studies are greatly facilitated by the availability of Isc measurements and patch-clamp electrophysiology. However, relating a particular ion channel to an electrical signal from Isc requires ion substitution studies; availability of specific activators, inhibitors, or both; and methods that allow functional and structural localization of ion channels in epithelia. Of particular interest is the use of the polyene antibiotic nystatin, which permeabilizes membranes to cations, and to a lesser extent, anions, without passing through the membrane or allowing smaller (<0.8 nm) monovalent cations, water, and small nonelectrolytes to permeate. Nystatin pores may also be permeable to anions, such as chloride, but the selectivity for anions is reduced compared with cations (Pcation/PCl = 9:1) (150). Divalent cations and larger molecules, such as ATP, are too large to pass through nystatin pores. For larger molecules such as ATP, it is necessary to use, for example, Staphylococcus aureus toxin, which produces larger pores permeable to ATP (151), or digitonin for larger molecules such as lactate dehydrogenase (152).

ION CHANNELS OF THE EPITHELIA OF THE GASTROINTESTINAL TRACT / 1923 Ussing Chambers: Transepithelial Electrical Resistance and Short-Circuit Current Detailed methods for the study of ion transport in epithelia, including the use of Ussing chambers, can be accessed online at the Cystic Fibrosis European Network Web site (http:// central.igc.gulbenkian.pt/cftr/vr/physiology.html). In setting up Ussing chamber experiments, it is important to note the types of measurements that are possible, including opencircuit measurements of barrier function, transepithelial electrical resistance (153–155), and Isc measurements. Isc is measured as the charge flow per unit time when the transepithelial voltage of the tissue is clamped to 0 mV. Thus, in practice, two electrodes are required to measure ion flow, and two are used to measure transepithelial voltage. The epithelium is short-circuited to measure Isc by injecting a current that is adjusted by a feedback amplifier to keep the transepithelial voltage at 0 mV. The amount of current required is adjusted by a feedback circuit and measured continuously. At various times, the voltage is clamped at a different voltage (e.g., 5 mV) to estimate resistance. According to Ohm’s law, Isc = V/R. Commercial equipment (such as that available from Physiological Instruments, San Diego, CA) is equipped with programs and hardware that report equivalent Isc, resistance, Isc, as well as transepithelial voltage during an Isc experiment. The Cystic Fibrosis European Network Web site (http:// central.igc.gulbenkian.pt/cftr/vr/physiology.html) also contains detailed approaches for patch-clamp electrophysiology in the whole-cell mode, in cell-attached patches, and in excised patches for single-channel recordings. The site also describes analysis of single-channel data in patch-clamp experiments and bilayer studies. Flux studies in vesicles, use of microelectrodes, ion-sensitive microelectrodes, ion-sensitive dyes, radiotracer studies on cells, and fluid transport also are included. Of particular value on the Web site are articles on the use of Ussing chambers and the use of nystatin to permeabilize apical or basolateral membranes. Fortunately, methods for studies of human biopsies have been established. A variety of cell lines have been developed, and methods for Isc studies are particularly well developed for studies of the intestine and other sheets of epithelia. There are several outstanding primary articles that use the Isc technique for the study of epithelial transport (12,91,156,166). A typical Isc experiment that incorporates best practices from the literature is shown in Figure 75-3 (57). T84 cells were grown to confluence as measured by transepithelial resistance. In Figure 75-3A, the cell cultures were mounted in an Ussing chamber and incubated with 1-EBIO. 1-EBIO was shown to activate calcium-activated potassium channels in the basolateral membrane. This agent activates both human and murine potassium channels (156). The target of 1-EBIO has been identified as a calcium-activated potassium channel (hIK) (157,158). Figure 75-3 shows a typical Isc study using T84 cell monolayers activated with 1-EBIO (see Fig. 75-3A) and permeabilized with nystatin followed by EBIO after activation of

chloride currents to demonstrate sufficiency of nystatin treatment (see Fig. 75-3B); studies of the effects of basolateral and apical permeabilization on chloride currents also are shown (see Fig. 75-3C). Additional details can be found in the original article (57). Other gastrointestinal tissues, such as parietal cells of the stomach and pancreatic ducts, which have secretory membranes that are difficult to access or that do not grow or easily grow to confluence, are sometimes more difficult to study. However, patch clamp of the parietal cell apical membrane has been accomplished (159), bilayer measurements of parietal cell intracellular membranes have been reported (62,127,160), and patch clamp of the basolateral membrane has been performed (126,161). Direct measurement of acid secretion (162) can be used for determination of the role of ion channels in gastric model systems and to determine the roles of various ion channels in intact animals. Physiologic studies that have not been validated against such gold standard measurements of parietal cell or other epithelia function often are used and are difficult to evaluate in physiologic terms. Thus, measurement of luminal pH in the stomach may not yield the same information as measurements of hormone-stimulated rates of acid accumulation. Indirect methods, such as accumulation of radioactive weak bases, can be used to measure parietal cell secretion in isolated cells (163), as long as such models are appropriately validated. An excellent source of information on the ion currents of pancreatic acinar cells can be found in the previous edition of this textbook (164). ENaC appears to be lacking from acini and ducts of the pancreas (165). Calcium may play a role in regulation of CFTR-mediated chloride secretion and bicarbonate secretion in the pancreas. In mouse pancreatic acini, KCNQ1 probably coassembled with KCNE1 leads to a voltage-dependent potassium current that might be of importance for electrolyte and enzyme secretion (101).

Ion Channel Effectors In general, activators of ion channels often are limited to compounds that might affect several pathways or multiple ion channels (e.g., cAMP analogs, forskolin, or ionomycin), and inhibitors of ion channels might affect other processes (e.g., heavy metals). For this reason, all studies that rely on such nonspecific agents might later be subject to reinterpretation as more information regarding multiple targets of such agents is accumulated.

Effectors of Phosphatases and Protein Kinases The literature on ion channels of the gastrointestinal tract, with a few notable exceptions (11,70–72), is lacking in the use of phosphatase inhibitors when activation of ion channels by forskolin or other activators of protein kinases is studied. The assumption that steady-state levels of phosphorylated

1924 / CHAPTER 75 A

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FIG. 75-3. Effect of 1-ethyl-2-benzimidazolinone (1-EBIO) and SPI-0211 (lubiprostone) on shortcircuit current (Isc) in intact T84 cells and effect of SPI-0211 on Isc in nystatin-permeabilized T84 cells. (A) Isc of intact T84 cells was allowed to equilibrate, and then was measured after addition of 300 µM 1-EBIO. When the maximum Isc response was reached, 1 µM SPI-0211 was added. Isc was normalized to area. Both compounds were added to both sides. DMSO controls also are shown. Data are means ± SE. Open circles indicate DMSO (n = 4); open squares indicate 1-EBIO (n = 11), followed by DMSO (n = 3); solid triangles indicate 1-EBIO (n = 11), followed by SPI-0211 (n = 8). *p < 0.001 versus DMSO. #p < 0.01 versus DMSO. (B) Isc was allowed to equilibrate, and then nystatin (200 µg/ml) was added to the basolateral membrane. A chloride gradient was then imposed either apically to basolaterally or vice versa. After 10-minute equilibration with nystatin, 1 µM SPI-0211 was added to both sides. When the response to SPI-0211 reached its maximum, 300 µM 1-EBIO was added to both sides. DMSO (0.1%) controls also are shown. Data are means ± standard error (SE). Solid circles indicate SPI-0211 (n = 8); open circles indicate DMSO (n = 6) with apical-to-basolateral chloride gradient; solid triangles indicate SPI-0211 (n = 7); and open triangles indicate DMSO (n = 5) with basolateral-to-apical chloride gradient. (C) Bar graph comparing the effect of SPI-0211 on Isc across the apical membrane (A; solid bar indicates basolateral membrane permeabilized with nystatin) and basolateral membrane (BL; open bar indicates apical membrane permeabilized with nystatin) of T84 cells with a basolateral-to-apical chloride gradient imposed. DMSO controls were subtracted. Means ± SE (n = 9) are plotted. *p < 0.001 apical versus basolateral Isc. (Reproduced from Cuppoletti and colleagues [57], by permission.)

proteins are not affected by phosphatase action should be verified experimentally. Chemical inhibitors of protein kinases often are not sufficiently specific and also require validation. Permeant peptide inhibitors of protein kinases generally are more specific than commonly used chemical protein kinase inhibitors such as H89.

Transfection: Sense and Antisense and Dominant Negatives Transfection is a useful technique for elucidation of molecular aspects of ion channel function. T84 cells have been successfully transfected with an antisense regulator of

ION CHANNELS OF THE EPITHELIA OF THE GASTROINTESTINAL TRACT / 1925 CFTR (167,168). Caco-2 cells have been transfected with pCLCA1 (50), and there is at least one example of successful transfection of parietal cells (169). However, cell lines such as HEK-293 and CHO are more easily transfected than human gastrointestinal cell lines. The study of regulatory proteins and the effects of dominant negatives will be greatly facilitated by more transfection studies using well-established models of human gastrointestinal cells.

Permeable Supports Intestinal cells grown on permeable supports behave similarly to intact tissue with respect to chloride (170) and potassium transport (171). Anderson and Welsh (11) demonstrated that cells grown on permeant supports expressed cAMPdependent chloride channels, but not the calcium-activated chloride channels seen in cells grown on solid supports. T84 cells contain ClC-2 when grown on solid supports, but only in a small population, whereas T84 cells grown on permeable supports contain ClC-2 in the apical membrane of virtually all of the cells (56). Differentiation on permeable supports also was shown to be required for expression of CFTR (16). Cells on permeable supports can be patch clamped (172). The finding of chloride currents similar to ClC-2 in T84 cells (56,57) (in cells that had been previously thought to contain only CFTR in the apical membrane) suggests a need for additional studies to determine the relative roles of CFTR and other chloride channels in intestinal chloride transport under various conditions of health and disease.

Structure-Function Studies A review of the value of structure-function studies of gastrointestinal ion channels is beyond the scope of this chapter. However, there are many articles that demonstrate the power of structure-function studies in elucidation of the roles of domains and phosphorylation sites of gastrointestinal ion channels in regulated transport. One such study (70) demonstrated the importance of phosphorylation sites uniquely present in the human isoform (and comparable sites in the rabbit) in regulation of ClC-2 by PKA. In the future, it is likely that similar studies will elucidate sites in gastrointestinal ion channels that are important for their regulation.

SUMMARY In summary, the gastrointestinal tract contains a variety of ion channels. These include chloride, sodium, and potassium channels. It is clear that CFTR plays a major role in the movement of chloride across the apical membrane of intestinal cells. The relative role of other chloride channels, some of which appear to be in the same membrane as CFTR, requires further study. Moreover, little is known of the physiologic role of these other chloride channels in health and disease.

ENaC and degenerins apparently play a role in sodium absorption, but little is known of the physiologic consequences of mutations, the mechanisms of increased activity in CF, or the mechanisms whereby sodium absorption is increased in CF or changed with gain of function mutations or polymorphisms. 1-EBIO has provided a tool for activation of intestinal potassium channels. Additional study, however, is required to understand the physiologic roles of the large variety of potassium channels in the gastrointestinal epithelia. Several landmark studies have been noted that provide the best practices for functional studies of ion channels in the epithelia. A number of species differences have been described, indicating the need for caution in interpreting results from animal models for human disease and physiology. Future progress in understanding the role of the large variety of ion channels in the gastrointestinal tract in health and disease will require continued use of the best practices and the development of new inhibitors and activators, as well as continued application of powerful molecular approaches including genetic manipulation, structure-function studies, and studies of effects of dominant negatives.

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Integrative Physiology and Pathophysiology of Intestinal Electrolyte Transport Kim E. Barrett and Stephen J. Keely* Regulation of Intestinal Ion Transport at the Cellular Level, 1932 Chloride Secretion, 1932 Electrogenic Sodium Absorption, 1935 Electroneutral NaCl Absorption, 1936 Interactions between Transport Mechanisms, 1938 Regulation of Intestinal Ion Transport at the Tissue Level, 1939 Acute Regulation of Transport, 1939 Chronic Regulation of Transport, 1941

Adaptive and Maladaptive Alterations of Ion Transport in Disease, 1942 Infectious Diarrhea, 1942 Inflammatory Bowel Disease, 1943 Constipation, 1944 Therapeutic Implications, 1944 Summary and Conclusions, 1944 Acknowledgments, 1945 References, 1946

The intestine deals with large volumes of fluid on a daily basis in accomplishing its physiologic roles of the assimilation of nutrients and the excretion of wastes (1). The control of luminal fluidity is critical to these functions for several reasons. First, the intestinal contents must be sufficiently fluid such that digestive enzymes can diffuse to and act on their substrates. Second, the products of enzymatic digestion need to be able to diffuse to the absorptive surface of the epithelium so that they can be taken up into the body. Finally, components of the intestinal content that are destined for excretion (either indigestible wastes or compounds secreted into the bile by the liver) must be maintained in a form that is sufficiently fluid to allow for propulsion along the length of the intestine, without permitting the loss of excessive fluid from the body. Although these processes, on average, involve intestinal handling of about 8 to 9 L fluid per day, including 1 to 2 L of oral intake, it is remarkable that, in health, only

approximately 200 ml are lost to the stool. Clearly, the intestine has a profound requirement for the minute-to-minute regulation of the amount of fluid entering and leaving the intestine; moreover, this need evolves as the meal and its residue moves along the length of the gastrointestinal system. Also, mechanisms that regulate intestinal fluid handling are subject to more chronic regulation, either in the context of an appropriate response to changes in the luminal environment or to the types of food and drink that are consumed, or in specific disease states. The consequences of failure to regulate fluid handling appropriately in the gut are abundantly apparent in diseases such as secretory diarrhea, or conversely in cystic fibrosis, where luminal contents may become dehydrated and inspissated (1). The quantity of fluid entering or leaving the intestinal lumen across the epithelium is governed, in turn, by the regulated absorption and secretion of ions and other solutes. Individual epithelial cells express a panoply of membrane transport proteins that provide for the selective uptake of specific electrolytes, products of nutrient digestion, vitamins, and minerals. In turn, these transport events establish osmotic gradients that drive the movement of water, either paracellularly via tight junctions, or by transcellular routes involving aquaporin water channels and/or cotransport of water simultaneously with other transported species (2–5). The quantitative balance between water absorption and

K. E. Barrett and S. J. Keely: Department of Medicine, University of California, San Diego, School of Medicine, San Diego, California 92103. *Dr. Keely’s current affiliation is with the Royal College of Surgeons of Ireland, Dublin, Ireland. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1931

1932 / CHAPTER 76 secretion may differ depending on a number of factors, such as whether nutrients are present in the lumen. Likewise, the nature of epithelial transport pathways varies along the length of the intestine, coincident with the expected presence (or absence) of particular substrates. For example, nutrientcoupled fluid absorption is important in the small intestine in driving fluid reuptake in the postprandial period, but is essentially absent in the colon with the exception of mechanisms that provide for the absorption of short-chain fatty acids that are produced by bacterial fermentation of dietary fiber (1,6). There are also differences in electrolyte transport along the crypt-villus axis (or crypt-surface axis in the colon). In general, crypt cells display a more prominent secretory phenotype, whereas villous (or surface) cells express greater levels of membrane transport proteins involved in absorption, although emerging evidence suggests that this is somewhat of an oversimplification (7–11). Indeed, it is likely that some intestinal epithelial cells may coexpress proteins involved in both absorption and secretion, underscoring the need for coordinate regulation (7,12). Finally, the efficiency of transport, particularly in the absorptive direction, varies along the length of the gastrointestinal tract. The colon is more efficient than the small intestine at removing water from the lumen, and it has a comparatively greater reserve capacity (1). This implies that the colon is the gatekeeper of the system and acts to limit the occurrence of diarrhea. The goal of this chapter is to discuss the molecular physiology of ion transport in the mammalian intestine and its integrated regulation in both health and disease. We recognize that water uptake driven by the absorption of nutrients is an important component of fluid balance, particularly in the postprandial period, but this topic has been dealt with extensively elsewhere in this textbook (see Chapters 64 and 72). Furthermore, although nutritionally significant, the transport of vitamins and trace minerals is at a level too low to contribute substantially to the overall handling of fluid. Thus, this chapter focuses on three intestinal ion transport mechanisms that are quantitatively significant in determining overall fluid balance in the gut, namely, chloride secretion, electroneutral NaCl absorption, and electrogenic sodium absorption. In addition to covering the signaling mechanisms that regulate these ion transport mechanisms individually, we assess the extent to which their activity, expression, or both are regulated coordinately in the intestine, in either an adaptive or a maladaptive fashion.

REGULATION OF INTESTINAL ION TRANSPORT AT THE CELLULAR LEVEL Chloride Secretion Secretion of chloride is quantitatively the most important mechanism driving fluid secretion across the intestinal epithelium, and this transport event occurs throughout the gastrointestinal tract. The basic mechanisms of chloride secretion have been reasonably well established, although some details

remain to be determined, particularly with respect to the role of chloride channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) (13–16). Figure 76-1 depicts the transport machinery that makes up the chloride secretory mechanism. Chloride is accumulated in the cell cytosol at levels greater than dictated by its electrochemical gradient via NKCC1, an Na-K-2Cl cotransporter (17). This transporter takes advantage of the low levels of intracellular sodium that are established by the basolateral Na+,K+-ATPase. In activated cells, chloride can then exit across the apical membrane through one of several chloride channels. The role of CFTR in the process of chloride secretion has been established conclusively, whereas the involvement and relative importance of other channels is less clear (7,18–20). Nevertheless, evidence is accumulating that, under specific circumstances, calcium- and/or calmodulin-dependent protein kinase (CaMK)–activated chloride channels or chloride channels of the ClC family, most notably ClC-2, can also contribute to secretion (15,19–22). Basolateral potassium channels are also important in the secretory process, because potassium must be recycled to avoid depolarization of the membrane potential, thereby sustaining the driving force for chloride exit from the cytosol. Two potassium channels have been identified as participants: IK1 (intermediate-conductance, calcium-dependent K+ channel; also referred to as SK4) and KvLQT1 (23–33). These channels are regulated by calcium and cyclic adenosine monophosphate (cAMP), respectively. KvLQT1 also is regulated in intestinal epithelial cells via interactions with a second protein, KCNE3, which modifies the characteristics and pharmacology of the channel (7,34). In summary, the net transport event is the movement of chloride from the basolateral pole of epithelial cells into the intestinal lumen. Cations, most notably sodium, follow paracellularly to maintain electrical neutrality, and water is likewise driven into the lumen by resulting osmotic influences. Chloride secretion in intestinal epithelial cells is activated via three major intracellular signaling pathways. Probably the most significant, in a quantitative sense, is that dependent on an increase in cAMP. cAMP mediates the effects of important endogenous secretagogues, such as prostaglandin E2 and the neurotransmitter vasoactive intestinal polypeptide (VIP), which have relatively large and sustained stimulatory effects on chloride secretion. Both of these agonists bind to G protein–coupled receptors that stimulate adenylyl cyclase, increasing cytosolic cAMP. The major target for increased cAMP is protein kinase A (PKA). The catalytic subunit of this enzyme dissociates from the inhibitory, regulatory subunit upon binding of cAMP to the latter, freeing the catalytic subunit to phosphorylate several substrates important in chloride secretion. The most notable of these is CFTR itself; there are several consensus sites in the R domain of the channel that, when phosphorylated, have been shown to be associated with channel opening (18,35). Parallel to the activation of CFTR, KvLQT1 potassium channels are stimulated by PKA (36,37). cAMP-dependent agonists also induce phosphorylation and activation of NKCC1. However, because

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1933 APICAL ClC-2

CFTR

CLCA

+ Cl−

Cl−

+

Cl−

Ca++

cAMP

+

+

+

K+

3Na+

K+

∼ KvLQT1/ KCNE3 BASOLATERAL

IK1 K+

2Cl−

NKCC1

Na+

2K+ Na+/K+ ATPase

Na+ H2O

FIG. 76-1. Chloride secretion by intestinal epithelial cells. Chloride enters the cell via the basolateral Na+-K+-2Cl− cotransporter (NKCC1), which is driven secondarily by the activity of Na+,K+-ATPase. Cotransported potassium ions are recycled to avoid cellular depolarization and to maintain the driving force for chloride exit across the apical membrane, which occurs predominantly via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels, but perhaps also by chloride channels of other classes. The transcellular transport of chloride drives the paracellular movement of sodium and water via the tight junctions. The process is controlled by the levels of intracellular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (not shown), or calcium, which affect the activity or membrane abundance, or both, of various transport proteins as shown.

NKCC1 lacks consensus sites for phosphorylation by PKA (38), it is thought that this response occurs indirectly, perhaps as a consequence of cell shrinkage or a reduction in cytoplasmic chloride concentration (39–41). Activation of NKCC1 in response to cAMP may also occur through activation of myosin light-chain kinase and modulation of the integrity of the basolateral cytoskeleton (42). This latter event apparently is caused by direct effects of the cytoskeleton on transporter activity, as well as recruitment of stimulatory accessory proteins into the basolateral membrane (43). Overall, the ability of cAMP-dependent agonists to enhance NKCC1 activity, by whatever mechanism, almost certainly contributes to the sustained secretory responses evoked, because chloride delivery into the cytosol can be matched to chloride efflux (43). Notably, NKCC1 is not activated by agonists acting through calcium (see later discussion) (42). A wealth of information also has shown that the efficiency with which increases in cAMP are coupled to chloride secretion is enhanced by subcellular compartmentalization of both signaling intermediates and their membrane transport targets. Specifically, CFTR is known to reside in the apical membrane as part of a multiprotein complex (44). This complex is orchestrated via binding of CFTR to other

proteins containing so-called PDZ domains that are important in protein interactions (45). One such protein is sodium/hydrogen exchanger regulatory factor 1 (NHERF1). This molecule was originally identified as a sodium/hydrogen exchanger (NHE) regulatory factor, but is now recognized to complex with a variety of different membrane transporters, and thereby modulate their function, as well as perhaps their abundance in the membrane (45,46). The consequences of such interactions may also be regulated by NHERF1 phosphorylation, such as by PKA. In this regard, it is of interest that CFTR also interacts with an A-kinase anchoring protein (AKAP) in the apical membrane, also via PDZ interactions (47,48). This therefore physically associates PKA with its substrates, CFTR and NHERF1, and likely increases the efficiency with which channel opening can be obtained. A second mode of chloride secretion is activated by increases in cyclic guanosine monophosphate (cGMP), such as is induced by a bacterial toxin, the heat-stable enterotoxin of Escherichia coli, as well as the structurally related endogenous hormones guanylin and uroguanylin (in fact, all of these agonists bind to the same receptor, the apically localized guanylyl cyclase C [GCC]) (49,50). This defines the chloride secretory mechanism as an unusual biological

1934 / CHAPTER 76 A

B Chloride secretion

response, in that cAMP and cGMP often have opposing effects in many other cell types. Initially, this paradox was believed to relate to the fact that high concentrations of cGMP were generated in intestinal epithelial cells by agonists of this pathway, and thus these might nonspecifically cause cross-activation of PKA (51). However, studies with knockout mice lacking the type II cGMP-dependent protein kinase, as well as in vitro models, have since demonstrated that this enzyme can directly stimulate CFTR (52–54). In addition, cGMP may secondarily increase cAMP via inhibitory effects on specific isoforms of phosphodiesterase, which could have contributed to the earlier confusion (53). Either way, it is clear that both cAMP and cGMP target largely overlapping features of the epithelial transport machinery to evoke chloride secretion. Indeed, if epithelial cells are treated with the combination of maximally effective concentrations of agonists acting through either cAMP and cGMP, there is no greater secretory response seen than with each agonist individually (55). In marked contrast with the response to cGMP, it is clear that chloride secretion evoked by increases in intracellular calcium is a distinctive response involving different intracellular targets (56). Calcium-dependent chloride secretion is activated primarily by agonists that bind to Gq-linked receptors, such as acetylcholine via the muscarinic M3 receptor or histamine binding to histamine H1 receptors (1). The resulting increase in cytosolic calcium, as well as downstream effectors such as various isoforms of protein kinase C (PKC) and CaMK, do not appear to be capable of activating CFTR directly, although phosphorylation of CFTR by PKC may be permissive for subsequent stimulation by PKA (57,58). Rather, calcium-dependent signals appear to act predominantly by causing the activation of basolateral IK1 potassium channels (25,26,59–61). Potassium channel opening can support chloride secretion indirectly, by providing a driving force for apical chloride exit across a small proportion of chloride channels presumed to be open at any point in time. Direct opening of calcium-dependent chloride channels, such as those of the calcium-activated chloride channel (CLCA) and chloride channel (CLC) families, may also contribute to the response, although this remains controversial (16,62,63). Moreover, as discussed earlier, calcium-dependent signals do not appear to be sufficient to increase the activity of NKCC1, and they may even induce endocytosis of the protein from the basolateral membrane (64). The net influence of these features of calcium-dependent secretion, as well as of negative signals that are discussed later, is that chloride secretion in response to calcium-dependent agonists is rather modest in magnitude and also transient, unless it is coordinated with a cAMP-dependent signal. When cells are stimulated via cAMP (or cGMP) and calcium simultaneously, in contrast, there is a synergistic interaction with the observed secretory response being greater than additive (65,66) (Fig. 76-2). This synergism is believed to reflect the elimination of rate-limiting steps in the two different modes of secretion, as well as interactions at the level of intracellular signaling that may limit the generation of negative regulators (67).

C

Time

FIG. 76-2. Chloride secretion induced by increases in cyclic nucleotides versus cytosolic calcium has different kinetics. (A) Typical response to addition of a cyclic adenosine monophosphate (cAMP); or cyclic guanosine monophosphate– dependent agonist at the time indicated by the arrow. The response is relatively slow at onset, but sustained. (B) In contrast, the transient kinetics of chloride secretory responses evoked by calcium-dependent agonists are shown, even if the increase in cytosolic calcium is sustained. (C) Synergistic response that occurs when cAMP- and calcium-dependent agonists are given simultaneously; the response is greater than would be predicted by summation of the individual responses.

The transient nature of the calcium-dependent chloride secretory response may, in fact, subserve an important physiologic role, by limiting secretion to only the period when it is acutely needed. Therefore, we and others have made extensive efforts to map regulatory events that limit calcium-dependent chloride secretion. In fact, binding of a calcium-dependent agonist, although initially stimulating a secretory response by the mechanisms discussed earlier, also sets in train a complex network of antisecretory signals that ultimately down-regulate secretory responsiveness. These antisecretory signals range from those that act acutely to inhibit the extent of ongoing responses to those that act over more prolonged periods, dampening the ability of the epithelium to display subsequent responses over several days. It appears that the generation of antisecretory signals rests largely on the ability of at least some calcium-dependent agonists to recruit elements of receptor tyrosine kinase–dependent signaling within polarized epithelial cells. In particular, ligation of the muscarinic M3 receptor activates a membrane matrix metalloproteinase capable of cleaving the active form of the growth factor, transforming growth factor-α (TGF-α), from a transmembrane precursor (68). This ligand, in turn, binds to the basolateral receptor

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1935 effectors (77). Other antisecretory signals generated in response to calcium-dependent agonists include the soluble inositol phosphate, Ins(3,4,5,6)P4, which specifically antagonizes the opening of calcium-activated chloride channels, and PKC isoforms that mediate retrieval of NKCC1 molecules from the basolateral membrane (64,78–82). However, although Ins(3,4,5,6)P4 production is reversed by tyrosine kinase inhibitors and EGF itself can activate specific PKC isoforms, whether these pathways are linked to GPCR-induced EGFr transactivation remains a topic for investigation. In summary, therefore, chloride secretion is the subject of complex intracellular regulatory mechanisms that target various facets of the secretory machinery, as well as associated proteins, to set the magnitude and kinetics of a given response. In this way, the epithelium can call on markedly up-regulated secretion, a sustained, moderate response, or a transient burst of secretion as the physiologic circumstances dictate (see Fig. 76-2).

for epidermal growth factor (EGFr) and, in concert with other signals evoked by the original agonist, recruits mitogenactivated protein kinases (MAPKs) such as extracellular signal–regulated kinases 1 and 2 or p38 (68–71). By mechanisms that have yet to be defined fully, these EGFr-dependent signals act first within minutes to limit the extent of ongoing secretory responses, and then, over a more prolonged period, lead to a chronic uncoupling of subsequent secretagogue receptor activation from prosecretory mechanisms (72). The importance of EGFr-dependent signaling in regulating epithelial secretory function also is underlined by observations that exogenous addition of TGF-α, or indeed EGF itself, can reduce calcium-dependent chloride secretion without these growth factors themselves acting as agonists of this process (68,73). However, in this case, inhibition appears to involve recruitment of phosphatidylinositol 3-kinase (PI3K), downstream activation of a specific isoform of PKC, PKCε, and eventual inhibition of a basolateral potassium channel (74–76). The molecular basis of this divergent signaling evoked by TGF-α alone, versus TGF-α acting in a signaling milieu brought about by a muscarinic agonist, is currently unclear. However, it does appear that muscarinic agonists also recruit a phosphatase that dephosphorylates key intracellular tyrosine residue(s) on EGFr, which likely, in turn, alters the ability of the receptor to recruit specific downstream signaling

Electrogenic Sodium Absorption In the colon, secretory fluxes of fluid that are evoked by chloride secretion are opposed, in part, by fluid absorption driven by electrogenic absorption of sodium ions. The details of this transport mechanism are shown in Figure 76-3.

APICAL

−

ENaC

+

Nedd4-2 Na+

+

−

↑[Na+]

cAMP

3Na+

∼

BASOLATERAL

2K+ Na+/K + ATPase

Cl− H2O

FIG. 76-3. Electrogenic sodium absorption by colonic epithelial cells. Sodium enters across the apical membrane via epithelial sodium channel (ENaC) channels, driven by the low intracellular sodium concentration established by Na+,K+-ATPase. The transcellular transport of sodium drives the paracellular movement of chloride and water via the tight junctions. ENaC abundance is up-regulated indirectly by increases in cyclic adenosine monophosphate (cAMP) that inhibit the activity of the ubiquitin ligase Nedd4-2, which otherwise can cause ENaC degradation. In contrast, Nedd4-2 activity is increased in response to an increase in intracellular sodium concentrations.

1936 / CHAPTER 76 Transport centers around the epithelial sodium channel (ENaC), which is also expressed in the kidney (7). This heterotetrameric channel likely consists of 2α-, 1β-, and 1γ-subunits, although other models have been described (7,83,84). It is expressed in the surface epithelial cells of the colon, with little or no expression in the crypts, and there is no expression in the small intestine (7). Transport of sodium through ENaC plays an important role in fluid absorption in the human colon, particularly in the distal colon, although perhaps less so in some other species, including the rat, that have served as important models for intestinal ion transport (7). Sodium is absorbed into epithelial cells through open ENaC channels by taking advantage of the low intracellular concentration that is established by the basolateral Na+,K+ATPase, and it exits the cell via this latter transporter. Chloride is believed to follow paracellularly, although there is some evidence to suggest that a portion of chloride movement may also occur through CFTR channels expressed at low levels in surface epithelial cells, and thus in the reverse direction to transport occurring during chloride secretion (7). Notably, there is little or no NKCC1 expression in surface epithelial cells, and thus no mechanism to load these cells with chloride to allow for secretory fluxes. There is, however, evidence to suggest functional interactions between ENaC and CFTR (see detailed discussion later in this chapter). Whatever the mechanism of chloride movement, the result of these transport events will be the reabsorption of luminal water. Acute regulation of ENaC activity by intracellular signals in intestinal epithelial cells has not been studied extensively, no doubt partially because robust intestinal cellular models in which this transport protein is appropriately expressed have not been available. Some authors had speculated that electrogenic sodium absorption occurs constitutively, with maximal rates of transport governed only by the abundance of ENaC in the apical membrane of colonocytes (which can be regulated chronically in response to whole-body sodium status; see later) (7). Indeed, conflicting results were obtained when investigators examined whether ENaC was regulated by PKA or PKC in exogenous expression systems, and consensus serine/threonine kinase phosphorylation sites also are not conserved in any of the ENaC subunits across species (7). However, data have shown that ENaC likely is subject to acute regulation, at least by cAMP, which opens up new avenues in our understanding of colonic ion transport. Clues with regard to the acute regulation of ENaC came first from a genetic disorder, Liddle syndrome, which is an inherited salt-sensitive form of hypertension that results from excessive sodium absorption in the kidney collecting duct. Patients with this disorder have ENaC mutations localized to the carboxy terminus of one of the subunits, which result in a channel with increased activity (85–89). The basis of this increased function relates to feedback mechanisms that normally terminate channel activity in response to increases in the level of intracellular sodium (90–92). A currently unidentified sodium sensor activates low-molecular-weight G proteins, which, in turn, bring about the recruitment of a protein called Nedd4-2 to the apical membrane. Nedd4-2 is a

ubiquitin protein ligase, and when associated with ENaC via proline-tyrosine (PY) motifs in the carboxy terminus, it covalently links ubiquitin to the channel subunits, targeting them for internalization and proteosomal degradation. Liddle syndrome–associated mutations disrupt the PY motifs, resulting in a reduced ability to recruit Nedd4-2, and thus a channel that remains inappropriately active even when intracellular sodium levels increase. These observations thus set the stage for findings regarding the acute activation of ENaC in response to cAMP/PKA. Nedd4-2 has been shown to be a substrate for PKA, and phosphorylation of this protein dramatically reduces its ability to bind to any of the ENaC subunits, and thus to influence their degradation (93). Thus, at least in cell line models not of intestinal origin, an increase in cAMP increases the abundance and function of ENaC. This response is likely due not only to decreased channel internalization (and degradation), but also to exocytic reinsertion of channels into the apical membrane from a recycling apical vesicular pool (94). Interestingly, the effects of PKA mirror those of the serumand glucocorticoid-inducible protein kinase (SGK), which contributes to ENaC up-regulation in response to aldosterone (95,96). The effects of PKA and SGK on channel activity are not additive because they phosphorylate the same residues on Nedd4-2 (93). It remains to be seen, however, to what extent these observations can be extrapolated to the colon, but it appears likely that ENaC-dependent electrogenic sodium absorption in that site can be regulated acutely by cAMP, at least under conditions where the abundance of the channel in the membrane is rate limiting. The situation in colonocytes, however, also is complicated by interactions of ENaC with CFTR (see later discussion).

Electroneutral NaCl Absorption The coupled absorption of sodium and chloride largely accounts for nutrient-independent fluid absorption in the small intestine and is also an important component of colonic fluid absorption, particularly in the proximal colon. Molecular characterization of epithelial transport proteins has provided a more detailed appreciation of the precise mechanisms whereby this transport process occurs (Fig. 76-4). In the intestine, coupled absorption of sodium and chloride is not thought to occur through a sodium-chloride cotransporter, as seen in other tissues, but rather by virtue of sodium-hydrogen and chloride-bicarbonate exchangers working in parallel. The mechanism of coupling of these independent transporters is not fully understood, but may be accounted for by localized changes in intracellular pH in the vicinity of the apical membrane (97,98). Protons and bicarbonate ions that drive NaCl absorption are believed largely to be generated intracellularly, in part via the activity of carbonic anhydrase (99). In turn, sodium is extruded from the basolateral pole of the cell by Na+,K+-ATPase. The pathway for chloride exit has not been fully established, but may be a member of the potassium-chloride cotransporter family, such as KCC1.

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1937 Na+

APICAL

Cl−

NHE3(NHE2)

−

DRA(PAT1)

HCO3−

H+

H2O + CO2

C.A.

HCO3− + H+

cAMP 3Na+

K+

2Cl−

~ BASOLATERAL

2K+ Na+/K + ATPase

KCC1?

H2O

FIG. 76-4. Electroneutral absorption of NaCl by intestinal epithelial cells. Absorption of these ions across the apical membrane occurs via the coupled activity of a sodium/hydrogen exchanger (predominantly NHE3) and an anion exchanger (apparently predominantly the down-regulated in adenoma gene product, DRA, although the putative anion transporter, PAT1, may also play a role). Bicarbonate and protons needed to drive this process can be generated intracellularly by the activity of carbonic anhydrase (C.A.). Sodium exits the cell via the basolateral Na+,K+-ATPase, whereas the identity of the chloride exit pathway is less clear, but may be mediated by a K+-2Cl− cotransporter (KCC1) as shown. The transepithelial movement of NaCl drives the subsequent absorption of water via the paracellular route. NHE3 can be negatively regulated by cyclic adenosine monophosphate (cAMP), thereby inhibiting the overall transport mechanism.

Overall, because one chloride ion is transported together with each sodium ion, there is no specific driving force for any additional electrolyte transport via the paracellular route. In contrast, water will follow paracellularly to maintain isotonicity. Indeed, there is evidence that sodium/hydrogen exchanger isoform 3 (NHE3) activation may contribute to changes in paracellular permeability to water downstream of glucose absorption (100). Extensive studies have identified the precise NHEs that are important in electroneutral NaCl absorption. Among a family of eight NHE isoforms (now also referred to as the SLC9 gene family), NHE3, and to a lesser extent NHE2, appear to be specifically localized to the apical membrane of intestinal epithelial cells (101). The relative contribution of each isoform to overall transport may differ depending on the intestinal segment and species that is studied, and each has distinctive regulatory mechanisms and pharmacologic properties. At least in the mouse, NHE3 appears to be the dominant isoform contributing to NaCl absorption in the small intestine, and knockout of this transporter results in severe diarrhea, whereas that of NHE2 is without effect (102). However, this scenario may not hold for the colon, where NHE2 was reported to be localized to the crypts and

NHE3 to the surface cells, and there was a compensatory up-regulation of NHE3 expression in NHE2 knockout mice that likely protected them from the consequences of deficiency of NHE2 (103). Alternatively, crypt expression of NHE2 may be less important for NaCl absorption than the transporters present in surface cells, and instead may play some other role in electrolyte handling or pH homeostasis that has yet to be fully defined. The identity of the anion exchanger (AE) contributing to electroneutral NaCl absorption has proved more elusive, and the precise molecular identity of this transporter(s) in humans currently is unresolved. However, a reasonable synthesis of the rapidly emerging literature in this area would suggest that apical chloride/bicarbonate exchange is not performed by members of the classical AE family, because those family members that are present in the human intestine are predominantly localized to the basolateral, rather than apical, poles of epithelial cells (104). Rather, attention has focused on two members of the SLC26a gene family. SLC26A3 encodes a protein also known as DRA (down-regulated in adenoma). This transporter is highly expressed in both the small and large intestines of humans and animals, and it mediates exchange of chloride for a

1938 / CHAPTER 76 variety of other anions including bicarbonate, hydroxide, and sulfate (105–108). The alternative, or additional, candidate is SLC26A6, also known as putative anion transporter-1 (PAT1). This transporter is expressed on the apical membrane of small-intestinal epithelial cells. A report of the intestinal phenotype of mice lacking PAT1 did not show major problems with intestinal fluid absorption, although basal duodenal bicarbonate secretion was markedly reduced (109). These findings, coupled with the observation that mutations in DRA are the basis for the severe but rare condition of congenital chloride diarrhea in humans, secondary to the failure to conduct chloride/bicarbonate exchange in the colon, suggest that DRA may be the quantitatively more important contributor to NaCl transport under normal circumstances (110). However, it remains the case that both transporters may play specific and significant roles depending on the species and the segment of the intestine that is being considered. The uncertainty regarding the precise molecular nature of the AE involved in NaCl absorption has meant that our understanding of the role of this protein as a locus for regulation of overall transport is still quite preliminary. However, fortunately, the same cannot be said for the regulation of NHE3, for which we have a detailed understanding of relevant intracellular signals that can either enhance or suppress transport activity. Stimuli have been identified that both increase and decrease the activity of this exchanger in intestinal epithelial cells and model systems. Unlike the situation for chloride secretion and electrogenic sodium absorption, as discussed earlier, electroneutral NaCl absorption is inhibited by increases in intracellular cAMP, as is NHE activity in brush-border vesicles isolated from the rabbit ileum (111). However, when NHE3 was expressed exogenously in the PS120 cell line that lacks NHEs, this regulation by cAMP was lost (112). This difference allowed the cloning of NHERF1 as a protein that could reconstitute cAMP-dependent regulation of NHE3 in PS120 cells (113). It is now known that NHERF1 (as well as two related proteins, NHERF2 and PDZK1) functions as a scaffolding molecule to link NHE3 to the actin cytoskeleton (113). This also brings NHE3 in proximity to the actin-binding protein ezrin, which has a further function as an AKAP (114). Finally, the complex is completed by PKA tethering, positioning the kinase to phosphorylate specific residues in the carboxy terminus of NHE3 that result in a reduction in its activity, largely secondary to a reduction in its affinity for protons. NHE3 also is regulated by PKC, as well as agonists that can activate this enzyme in concert with an increase in cytosolic calcium concentrations, such as the muscarinic agonist carbachol. However, the mechanism is distinct from that evoked by PKA, although it does appear to be dependent on NHERF proteins, specifically NHERF2 (115). Changes in NHE3 activity in Caco-2 cells stimulated with phorbol esters result in a reduction in the Vmax, rather than in the affinity of the transporter for its substrates (115–117). This suggests that the effects of PKC are mediated by a

change in the amount of the transporter in the apical plasma membrane, a reduction in its turnover number, or both; indeed, PKC regulation of NHE3 has been shown to reduce the amount of NHE3 in the cell membrane by inducing its uptake into recycling endosomes as part of a multiprotein complex (118–120). NHE3 is not directly phosphorylated by PKC during this response, however, and the substrate of PKC that is critical to transporter internalization remains to be identified (121). Studies also implicate the soluble tyrosine kinase Src, which can be activated in response to carbachol, as being involved in down-regulation of transporter activity. Src may act by promoting the interaction of NHE3 with other proteins involved in its internalization (122). NHE3 activity can also be up-regulated under certain conditions, specifically in response to peptide growth factors. The response to EGF has been the best studied in this regard. Conversely to the effect of carbachol, EGF increases transporter activity by stimulating membrane insertion, via a mechanism that involves the stimulation of PI3K and its downstream target Akt (123). Moreover, ezrin has been shown to lie downstream of both Akt and p38 MAPK, and ezrin phosphorylation can trigger NHE translocation and activation in Caco-2 cells (124,125). It is especially intriguing that these conditions, which result in an increase in the activation of NHE3, are largely parallel to those that inhibit chloride secretion (see earlier discussion). This implies that electroneutral NaCl absorption and chloride secretion are reciprocally regulated, with agonists of one inhibiting the other, and vice versa (Fig. 76-5).

Interactions between Transport Mechanisms To this point we have largely considered ion absorption and secretion as being independent events that occur in distinct epithelial cells. However, as alluded to in the introduction, this may be an oversimplification. Immunohistochemical studies shown overlapping expression of the proteins required for chloride secretion and NaCl absorption along the crypt-villus axis in the small intestine, although reciprocal gradients occur (12,126–131). A similar situation pertains in the colon, with the proviso that ENaC appears to be restricted to surface cells, where the expression of CFTR and other facets of the chloride secretory mechanism are quite low or may even be absent (126,129,132–134). Nevertheless, these observations imply the possibility of interrelations among the transporters present in an individual intestinal epithelial cell, with consequences for the overall level of fluid and electrolyte secretion (7). Indeed, it has been recognized for many years that CFTR and ENaC activities appear to be regulated coordinately in a reciprocal fashion. In patients with cystic fibrosis, ENaC activity is enhanced in the airways and in the colon, likely further exacerbating the problems that relate to inadequate hydration of mucosal surfaces in such patients (18,135–138). Indeed, airway-specific overexpression of ENaC alone, in the absence of CFTR mutations, was sufficient to produce

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1939 B. + EGF

A. Baseline NaCl

NaCl

Villus

Villus

Cl−

Crypt

Cl−

Crypt

FIG. 76-5. Reciprocal regulation of NaCl absorption and chloride secretion in the intestine. At baseline, absorptive transport predominates. In response to epidermal growth factor (EGF), chloride secretion is inhibited and NaCl absorption is enhanced, thereby up-regulating overall absorptive function.

a lung disease in mice with many features of human cystic fibrosis (139). However, the mechanism of the presumed interaction between CFTR and ENaC in either the lung or intestine remains controversial. Some authors have reported reciprocal regulation of ENaC by CFTR when the channels are coexpressed in Xenopus oocytes, independent of an effect on surface expression of the channels (140,141). It has been speculated that the inhibitory effect of CFTR is caused by electrical coupling, particularly when CFTR is activated, although intracellular interactions mediated by scaffolding proteins such as NHERFs also are plausible (142,143). Some have hypothesized that this interaction makes it possible for a single epithelial cell to participate in both ion secretion and absorption, depending on the level of cAMP (7). However, this model is problematic given the recent appreciation that cAMP may sustain ENaC activity, albeit indirectly (see earlier). Moreover, others have reported that they could find no evidence for CFTR inhibition of ENaC in oocytes in three independent laboratories, which is consistent with previous controversy in this area (144). It is possible that the relation between these two channels, if it exists, is highly dependent on the context in which they are expressed. Further studies in models of native epithelia will be important to determine the extent, if any, to which this relation pertains to intestinal fluid handling. Others have reported functional coupling between NHE3 and CFTR in intestinal epithelial cells. Activated CFTR, perhaps via its interactions with NHERFs, is believed to inhibit NHE3 activity, and thus electroneutral NaCl absorption (145–147). In cells where CFTR and NHE are coexpressed, therefore, CFTR activation could contribute to the cAMP-mediated inhibition of exchanger activity that was discussed earlier. Likewise, Gawenis and colleagues (148) reported that cAMP-mediated CFTR activation in the villi of murine jejunum caused cell shrinkage, which, in turn,

inhibited NHE activity. However, a caveat with respect to these studies must be raised because they were conducted in knockout mice, in which adaptive changes could have taken place (indeed, as addressed later, expression of CFTR and NHE3 appears to be regulated coordinately). Indeed, CFTR is not required for cAMP to inhibit NHE3 in mouse pancreatic ducts (147) or in some of the model expression systems described earlier; thus, the contribution of CFTR to inhibition of NHE3 activity in vivo in native intestine is not entirely clear. In summary, there is some evidence for functional interactions between sodium absorption and chloride secretion in intestinal epithelia, although the details of mechanisms that account for these, and the extent to which they pertain in native intestinal cells, have not yet been fully elucidated. It is reasonable to say, nevertheless, that whether interactions occur within single cells or in the epithelium as a whole, signals that result in the activation of chloride secretion are likely to reduce the extent of electroneutral NaCl absorption by both direct and indirect means. In contrast, the functional relations between chloride secretion and electrogenic sodium absorption are more confusing, because some signaling pathways, such as cAMP, appear to be capable of activating both transport mechanisms, at least if expressed independently (as is likely in crypt vs surface cells, respectively).

REGULATION OF INTESTINAL ION TRANSPORT AT THE TISSUE LEVEL Acute Regulation of Transport In common with other aspects of intestinal physiology, the ion transport capacity of epithelial cells is regulated by extracellular signals that originate from endocrine, paracrine, neural, and immune sources (1). These permit local changes

1940 / CHAPTER 76 in luminal fluidity in response to cues provided by the physicochemical status of luminal contents, as well as more global regulation mediated by neurohumoral stimulation that is initiated in response to a meal. Moreover, the enteric nervous system integrates local and systemic inputs to coordinate intestinal ion transport with motor function, increasing or decreasing the residence time of the luminal contents in a given intestinal segment. Indeed, it is important to recognize that the net flux of any substance across the wall of the intestine will be a reflection not only of the area available for such flux (the so-called east-west vector), but also of the time of contact with this area (the north-south vector). This relation is exploited by some common antidiarrheal medications, such as Imodium (loperamide HCl), which decrease propulsive motility (which, in fact, may be inappropriately hyperactive in diarrhea, perhaps in response to intestinal distension [149]), allowing more time for the reserve capacity of the intestine for absorption to influence luminal fluid loads (150). Much of our knowledge of the precise neurohumoral factors that regulate intestinal ion transport, at least acutely, comes from studies examining chloride secretion. It is clear that electroneutral NaCl absorption is subject to comparable regulation, but is more challenging to study experimentally in intact tissues because it does not result in an electrical “signature” that can be measured by standard voltage-clamp techniques in Ussing chambers. Conveniently, as outlined earlier, many of the agonists that evoke chloride secretion inhibit NaCl absorption (these agonists are discussed the next paragraph). In contrast, some authors have speculated that electrogenic absorption of sodium and water in the colon occurs at a steady, essentially maximal rate, and the effect of peptide hormones and neurotransmitters is, in fact, limited to reducing absorption, stimulating secretion, or both (7,151). However, studies have challenged this view, suggesting that, in fact, chloride secretion and electrogenic sodium absorption may be regulated in parallel, at least under certain circumstances. Agonists of chloride secretion appear to derive predominantly from paracrine and neurocrine pathways, with classical endocrine gastrointestinal hormones such as cholecystokinin and gastrin playing a much smaller role (1). (An exception to this rule is the ability of secretin to evoke bicarbonate secretion, and perhaps chloride secretion, at least in part via the stimulation of CFTR in duodenal enterocytes [152].) Key stimuli of chloride secretion include VIP and acetylcholine, which are released from enteric secretomotor neurons (153). Presumably, central and vagovagal inputs coincident with the ingestion and processing of a meal lead to appropriate stimulation of secretion and suppression of electroneutral NaCl absorption. Importantly, however, these inputs appear to be incapable of reducing nutrient-coupled sodium absorption, and indeed, this fact is taken advantage of in oral rehydration solutions used in the treatment of diarrheal diseases. Paracrine influences on intestinal chloride secretion also have been recognized. Among these, prostaglandins, particularly those of the E series, are synthesized by subepithelial elements (especially myofibroblasts) and act on basolateral

receptors to evoke cAMP-dependent chloride secretion (154–157). Likewise, histamine released from mast cells residing in the lamina propria can contribute to epithelial chloride secretion under some physiologic circumstances, such as in response to high concentrations of bile acids that would be expected normally in the small-intestinal lumen (158). In fact, bile acids can also have direct effects on intestinal secretion when present at millimolar concentrations, although the extent to which these luminal secretagogues contribute to the postprandial increase in fluid secretion has not yet been established (159). Moreover, only under pathologic circumstances are bile acid concentrations sufficiently increased in the colonic lumen to induce secretory effects. In fact, data emerging from our laboratory suggest that, at bile acid concentrations that would normally be expected in the colon, bile acids actually exert antisecretory actions on the epithelium, and may therefore serve to enhance normal colonic absorptive function (160). Finally, an emerging agonist for chloride secretion in health is guanylin, which is released into the intestinal lumen by enteroendocrine cells and acts via an apical receptor to evoke chloride secretion in a cGMP-dependent fashion (49). Guanylin, and its close relative uroguanylin, likely coordinate salt and fluid handling between the intestine and the kidneys (161–164). Overall, therefore, the intestine displays multiple mechanisms to evoke both sustained and transient increases in epithelial chloride secretion in health (via cyclic nucleotide- and calcium-dependent mechanisms, respectively), which are likely amplified by reciprocal changes in NaCl absorption. One particularly intriguing aspect of the regulation of epithelial chloride secretion relates to local reflexes that control secretion in response to epithelial deformation. If the epithelium is gently stroked with a brush, a stimulus that might be considered to model the passage of a food bolus, there is a transient increase in chloride secretion. The occurrence of this response in vivo would be predicted to lubricate the mucosa and might be important in protecting the epithelium from physical damage. Cooke and coworkers (165–167) have extensively mapped the contributors to this local reflex. Thus, mechanical stimulation appears to be sensed by enterochromaffin cells residing in the epithelium, which respond with release of 5-hydroxytryptamine (5-HT) into the lamina propria. 5-HT may have direct effects on epithelial transport, but the majority of its actions are likely produced secondary to the activation of sensory nerves that release substance P, acetylcholine, and purine nucleotides. These transmitters, in turn, activate secretomotor neurons that release acetylcholine, and perhaps VIP, to evoke a short burst of chloride secretion in the immediate area where the mechanical stimulation occurred (Fig. 76-6). Although the agonists that evoke chloride secretion, in general, may be considered to coordinately reduce NaCl absorption, the same may not necessarily be true for sodium absorption. Thus, we reported that short-circuit current responses (reflective of net ion transport) of the healthy murine colon to carbachol and forskolin involve components ascribable to increased chloride secretion and increased

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1941 LUMEN

Mechanical stimulation EC cell

ACh VIP 5-HT

Cl−

LAMINA PROPRIA

SP ACh ATP

ENS

FIG. 76-6. Reflex stimulation of chloride secretion in response to mucosal deformation, as elucidated by the studies of Cooke and coworkers (165–167). Mechanical stimulation leads to the release of 5-hydroxytryptamine (5-HT) from enterochromaffin (EC) cells, which activates 5-HT receptors on sensory nerves. These release substance P (SP), acetylcholine (ACh), and adenosine triphosphate (ATP), which activate secretomotor neurons with cell bodies in the plexuses of the enteric nervous system (ENS). In turn, these nerves release ACh and vasoactive intestinal polypeptide (VIP), which stimulate chloride, and thus fluid secretion across the intestinal epithelium adjacent to the site of stimulation.

sodium absorption, both of which would change current in the same direction (168). Similarly, the absence of CFTR uncovered forskolin stimulation of glucose-coupled sodium absorption in murine jejunum (169). These findings must be considered in light of the foregoing discussion of possible direct inhibitory interactions between CFTR and ENaC if the two channels are coexpressed in the same cell, which makes the relative effect of chloride secretagogues on electrogenic sodium absorption difficult to predict. However, in summary, it appears likely that the strict reciprocal relation between chloride secretion and electroneutral NaCl absorption cannot be extended to electrogenic sodium absorption under many circumstances. This may reflect that NHE isoforms are clearly present in crypt epithelial cells, which are also the site of highest CFTR expression, and thus it would be important for the intestine to have a mechanism where an individual crypt epithelial cell would not be working at cross-purposes. In contrast, there is no reason why crypt secretion and surface absorption of fluid could not occur simultaneously; indeed, circumstances may exist where this could be considered to be desirable, such as to flush the crypts of toxins, then to reclaim the fluid so used.

Chronic Regulation of Transport Tissue-level regulation of ion transport in the intestine also encompasses more chronic changes that take place over

hours to days, often in response to whole-body fluid and electrolyte status. The most classical example of such regulation is the change in colonic ENaC expression that occurs in response to sodium restriction in the diet (170). Reduced levels of circulating sodium result in release of the mineralocorticoid aldosterone from the adrenal glands, from where it travels to target tissues, including the colon. Aldosterone has both rapid and sustained effects on ENaC activity in the colon. Immediate effects are likely ascribable to activation of SGK, which increases the abundance of ENaC in the apical membranes of surface colonocytes by the mechanisms discussed earlier (171). More chronically, aldosterone causes transcriptional activation of genes encoding the various ENaC channel subunits, resulting in increased overall numbers of active channels, as well as probably stimulating increased expression of Na+,K+-ATPase and NHE3 (172). The overall result is to increase the ability of the colon to retain sodium from the colonic contents, contributing to the restoration of sodium balance in concert with similar effects of aldosterone on the kidney (95,133,173–178). Glucocorticoids may also influence intestinal electrolyte transport. Treatment with drugs such as methylprednisolone and dexamethasone increases sodium, chloride, and water absorption in the small intestine and colon. Part of this effect is ascribable to induced expression of NHE3, whereas much greater doses of glucocorticoids are needed to increase ENaC expression (179–185). Effects of glucocorticoids on NHE3 that are independent of gene transcription have also

1942 / CHAPTER 76 been described. Some work implies that such hormones activate SGK1, which cooperates with NHERF2 to stimulate NHE3 activity, likely via an effect of trafficking of the exchanger to the membrane surface (186). Finally, although the underlying mechanisms are unclear, the intestine likely has a “set point” for fluid transport and will adjust the levels of secretory and absorptive transporters to regain homeostatic control if one arm of this balance is perturbed. Evidence for this hypothesis comes from studies of electroneutral NaCl absorption in CFTR knockout mice compared with chloride secretion in NHE3 knockout mice (187). In the absence of NHE3, forskolin-stimulated chloride secretion was reduced secondary to a reduction in CFTR expression that may be mediated by overexpression of interferon-γ (188). Conversely, in the absence of CFTR, electroneutral NaCl absorption and NHE3 expression were less than seen in wild-type animals. Overall, these findings imply that determinants of intestinal fluid and water transport can be regulated in parallel to maintain a balance between mucosal hydration and systemic demands for electrolyte homeostasis. It will be fascinating to unravel the molecular mechanisms that allow for such coordinate regulation.

ADAPTIVE AND MALADAPTIVE ALTERATIONS OF ION TRANSPORT IN DISEASE Intestinal electrolyte transport is a clear target for disruption by a variety of pathogenic processes. This section discusses alterations in ion transport that occur in the setting of various disease states and their possible roles in disease pathogenesis.

Infectious Diarrhea A number of intestinal pathogens have the ability to induce diarrheal illness in susceptible hosts. In general, the pathogenesis of such illness is related to three main mechanisms of host–pathogen interactions: (1) production of enterotoxins by luminal pathogens that act to “hijack” normal host signaling pathways; (2) attachment of pathogens to the epithelial surface, followed by injection of bacterial proteins into the epithelial cytosol that alter transport and barrier functions; and (3) frank invasion of the epithelium, which may evoke many of the changes described in the second mechanism, but with added stimulation of a marked inflammatory reaction that further enhances epithelial dysfunction. The impact of microorganisms and their products on intestinal epithelial biology has been reviewed in detail elsewhere in this textbook (see Chapter 47). Therefore, we confine our comments to a few examples of the pathophysiologic correlates of host–pathogen interactions and their potential roles in disease progression. Perhaps the prototype of an infectious “ion transport disease” is that evoked by the enterotoxin elaborated by Vibrio cholerae. The classical cholera toxin is a multisubunit

enterotoxin that is released in the intestinal lumen and internalized by epithelial cells via a cell-surface ganglioside, which serves as a specific receptor (189). The active toxin subunit then is actively trafficked across the cell cytosol to the basolateral membrane, where it irreversibly activates adenylyl cyclase, causing a sustained and irreversible increase in intracellular cAMP (189). Based on the foregoing discussion, the consequences of this effect should be clear–cholera toxin activates marked intestinal chloride secretion, while suppressing electroneutral NaCl absorption, resulting in profound secretory diarrhea that can approach 20 L/day, and thus rapidly can become life-threatening if left untreated. The effects of cholera toxin on intestinal electrolyte transport are also further amplified by secondary activation of enterochromaffin cells and enteric nerves, in turn releasing neurohumoral regulators of transport such as VIP and acetylcholine that synergistically promote chloride secretion (190–193). Cholera toxin can also stimulate the expression of several ion transport proteins, which likely further amplifies its effects on secretion (194). The severity of cholera may also be increased, at least as induced by some isolates of V. cholerae, by accessory toxins such as zonula occludens toxin, which reduce the barrier function of epithelial tight junctions (195,196). In contrast, effects of cholera on ENaC function and electrogenic sodium absorption have not been described, but it is conceivable that cAMP-dependent activation of this transport process could act to limit fluid losses. It is certainly clear that sodium-nutrient absorption is intact in cholera (and perhaps, based on findings discussed earlier, is increased in the disease setting) and underlies the mainstay of cholera therapy in developing counties as provided by oral rehydration solutions. Secretory diarrhea is also produced by enterotoxigenic isolates of E. coli (ETEC). These bacteria are neither invasive nor enteropathogenic, but rather produce soluble toxins that evoke epithelial signaling in a manner analogous to that discussed earlier for cholera toxin (189). However, the most important ETEC-derived toxin for pathogenesis, the heatstable toxin (STa), binds to a cell-surface receptor (GCC), rather than being internalized, and alters epithelial function by increasing intracellular levels of cGMP, rather than cAMP (197). Indeed, the existence of STa led to a search for an endogenous ligand for its receptor, which culminated in the discovery of guanylin, as discussed earlier. The structure of STa is modified compared with that of guanylin to enhance its stability in the intestinal lumen, prolonging its enterotoxigenic action (198). As for cholera, the sustained increase in intracellular cyclic nucleotide that is produced by STa results in up-regulation of chloride secretion and down-regulation of electroneutral NaCl absorption, with diarrhea being the clinical result. STa is an important causative agent of traveler’s diarrhea; thus, the incidence of disease attributable to this toxin is increasing (199). Another type of E. coli, enteropathogenic E. coli, is an important cause of childhood diarrhea in developing countries (199,200). This pathogen does not produce a toxin, but rather adheres to the epithelium to induce characteristic

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1943 “attaching and effacing” lesions in which the microvilli are obliterated. In this location, the bacteria is positioned to inject various proteins into the epithelial cytosol via a molecular “syringe” structure assembled from bacterial proteins known as the type III secretion system (201). Various bacterial effectors have been shown to interact with host-cell signaling pathways to reduce the integrity of epithelial tight junctions, and secondarily to evoke an inflammatory cascade that results, among other effects, in stimulating de novo expression of receptors for the neuropeptide galanin, which subsequently contributes to a chloride secretory response by an unknown mechanism (189,202,203). Finally, nontyphoidal Salmonella spp are examples of pathogens that invade the intestinal epithelium and evoke enteritis, as well as a secretory diarrheal response. The precise mechanisms of Salmonella-induced diarrhea remain the subject of investigation, but at least part of the disease appears to result from direct effects of the invasive bacteria on intestinal epithelial cells. Intracellular bacteria evoke a hypersecretory phenotype in such cells by sequentially increasing the expression of enzymes capable of producing secretagogues, namely, inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), up-regulation of CFTR and NKCC1 expression, and reduction of epithelial barrier function secondary to the destabilization of proteins making up the tight junction (204–206). Subsequently, Salmonella invasion induces epithelial expression of proinflammatory cytokines and chemokines that attract inflammatory cells, such as neutrophils, to the site of infection in vivo, and these cells, in turn, release mediators such as 5′-AMP and reactive oxygen species that further enhance chloride secretion and evoke additional epithelial injury (207–211). In addition to the profound changes that culminate in diarrhea, however, a careful examination of the early stages of Salmonella interactions with intestinal epithelial cells showed that their initial effect was to reduce the secretory capacity of the epithelium. This response appeared to be mediated by activation of EGFr and MAPKs and required only bacterial attachment to the epithelium, rather than invasion (212,213). We speculate that the bacteria may induce this early hyposecretory response to allow them to establish invasion into the epithelium. In this regard, the later diarrheal response could be considered a protective adaptation of the host that flushes bacteria from the lumen and limits additional invasion. This hypothesis was lent additional weight by studies of Salmonella typhi. These bacteria are closely related to the nontyphoidal Salmonellae, but cause a systemic illness rather than intestinal inflammation and diarrhea. We found that S. typhi evoke a sustained state of secretory unresponsiveness in intestinal epithelial cells and fail to up-regulate secretion (212). This supports the idea that diarrhea is a primitive host response designed to protect against access of enteric bacteria to the systemic circulation, albeit at the risk of dehydration and electrolyte disturbances. In keeping with this, the use of antidiarrheal drugs has long been known to increase the risk for systemic disease in patients infected with invasive intestinal pathogens.

In summary, therefore, intestinal pathogens, examples of which are discussed here, may use a variety of strategies to dysregulate intestinal secretion and absorption. The clinical consequence of such dysregulation is the occurrence of diarrheal disease, which can be life-threatening in some cases. However, it is notable that, in some cases, pathogen-mediated diarrhea may, in fact, be an appropriate adaptive response that limits the systemic exposure of the host to injurious microbes.

Inflammatory Bowel Disease Patients with the inflammatory bowel diseases of ulcerative colitis and Crohn’s disease often present with severe and chronic diarrhea. Indeed, this is frequently reported as being the most disabling symptom of these conditions. Thus, considerable effort has been devoted to defining the pathogenesis of inflammatory diarrheal diseases. Based on the known ability of several inflammatory mediators to stimulate active chloride secretion, it was initially hypothesized that inflammatory diarrhea might reflect the ability of such mediators to activate secretion in vivo, with or without a concomitant reduction in electroneutral NaCl absorption (214). However, evidence from a variety of models of inflammatory bowel disease suggest that this is an oversimplification; indeed, this hypothesis has specifically been disproved in some models of inflammatory bowel disease produced by various exogenous or genetic manipulations, or in tissues from human subjects (168,215–222). Although in some cases baseline secretion may be modestly increased, there is consistently a profound down-regulation of the ability of both small- and large-intestinal tissue from several models to respond to a range of chloride secretagogues acting through a variety of mechanisms. This secretory unresponsiveness has been attributed, variously, to effects of inflammatory cytokines on the expression of membrane transporters such as CFTR and NKCC1, downregulation of intracellular messenger production, uncovering of inhibitory neural pathways, up-regulation of nitric oxide synthase, and induction of prostaglandins, such as prostaglandin D2, that are capable of suppressing secretion (168,216,223–230). Although these findings may appear to be counterintuitive when placed in the context of the known occurrence of diarrhea in patients with intestinal inflammation, they are counterbalanced by the observation that both electroneutral and electrogenic pathways for sodium absorption also are suppressed, because of decreased expression and/or activity of NHEs, ENaC, Na+,K+-ATPase, and DRA (105,168,216,217,226,229,231). In fact, because absorption normally exceeds secretion in the healthy gut, suppression of both transport processes would be expected to lead to diarrhea because, presumably, fluid secretion originating in the stomach and organs that drain into the intestine would continue unabated. Regulatory pathways for electrolyte transport may also be altered in the setting of inflammation. This was explored for EGF, which, as discussed earlier, normally inhibits chloride

1944 / CHAPTER 76 secretion and increases electroneutral NaCl absorption in the healthy gut. However, in the setting of chronic inflammation, EGF was found to increase cAMP-stimulated electrogenic sodium absorption without an effect on chloride secretion (although because this latter response was markedly suppressed in inflamed tissue, we cannot rule out a small inhibitory effect of EGF on secretion) (168). The ability of EGF to enhance cAMP-stimulated sodium absorption in the inflamed gut was potentially explained by stimulated trafficking of ENaC channels into the apical membrane via an effect on PI3K. Because ENaC expression was suppressed in these tissues, membrane levels of the channels were likely rate limiting for secretion, and thus subject to enhancement by EGF that could not be detected in the healthy colon (168). Thus, suppressive effects of inflammation on the ability of the intestinal epithelium to transport electrolytes almost certainly contribute to the pathogenesis of inflammatory diarrhea. Additional consequences of reduced ion transport also are conceivable. For example, sustained epithelial hyporesponsiveness that persisted some time after a bout of colitis in rats was associated with marked increases in bacterial colonization and translocation of bacteria to extraintestinal sites (224). The postcolitis dysfunction was ascribed to sustained up-regulation of COX-2 and synthesis of its product prostaglandin D2, which is antisecretory, and COX-2 inhibition restored chloride secretion and prevented the increase in bacterial translocation (224). Thus, the changes in ion transport that occur as sequelae of inflammation could be considered as maladaptive, not only because they contribute to diarrhea in the acute phase, but also because they may later impair epithelial host defense. In contrast, active transport of electrolytes by the epithelium is an energetically costly process. Indeed, it has been calculated that even baseline chloride secretion may demand as much as 40% of the total oxygen consumption in rat colonic tissue, which, even under normal circumstances, exists in a relatively hypoxic state (232,233). In turn, this suggests that the alteration in epithelial phenotype that occurs in the setting of inflammation, leading to suppression of both secretory and absorptive pathways, may, in part, be an appropriate adaptation. Thus, such a switch would allow for redirection of epithelial resources to restitution and repair, rather than toward transport.

Constipation Although classically considered to reflect motility disturbances (and as such, a prolongation of the “north-south” vector discussed earlier), data suggest that constipation can also arise because of an imbalance between intestinal absorption and secretion (234). Thus, if colonic secretion is diminished or absorption is enhanced, the stool will become excessively desiccated, and therefore difficult to propel along the length of the colon or to pass. In fact, constipation often is found in elderly adults, and there is known to be an agerelated decline in the expression of CFTR in both experimental animals and humans (conversely, together with their

diminished reserves, this may explain why secretory diarrheal diseases are so devastating in infants and young children) (235,236). In contrast, although ENaC expression does show an age-related decline in expression in the kidney, reflecting a sluggish aldosterone system, no such decline is seen in the human colon (237). This may be advantageous in contributing to sodium homeostasis in elderly adults, but the resulting imbalance between absorption and secretion might also contribute to constipation in such individuals, or indeed, in younger patients with this condition. The significance of diminished chloride secretion in the pathogenesis of constipation was also illustrated in studies of a novel bicyclic fatty acid, lubiprostone, which is in clinical trials for the relief of constipation. This drug was shown to activate chloride secretion by colonic epithelial cells in a manner that was independent of PKA and CFTR, and which instead depended on opening of ClC2 chloride channels (15). If such channels are also activated by the drug in vivo, then they may contribute to relief of constipation by adding additional fluid to the stool, without the irritation that is encountered with some laxatives that target the intestinal smooth muscle or the enteric nervous system, or both.

Therapeutic Implications The foregoing discussion suggests that there are many opportunities to intervene in various disease states by targeting facets of intestinal ion transport, thereby shifting the balance between absorption and secretion as indicated in a given setting. Indeed, the efficacy of lubiprostone underscores the utility of this approach. Likewise, a small molecule inhibitor of CFTR that was discovered by a high-throughput screening approach has been shown to be effective in animal models of cholera and STa-induced fluid secretion, although based on the foregoing discussion, it would not be expected to be an effective inhibitor of inflammatory diarrhea (238,239). A note of caution should nevertheless be sounded because we are far from the full understanding of the relative balance between an adaptive versus a maladaptive role for changes in the balance between ion secretion and absorption that occur in the setting of enteric infections, particularly those associated with invasive pathogens. However, further efforts toward increased understanding of the contributions made by various transport mechanisms to intestinal diseases should uncover several novel mechanisms that can be targeted safely to ameliorate disease symptoms.

SUMMARY AND CONCLUSIONS The goal of this chapter is to provide a basis for understanding the integrated regulation of major electrolyte transport mechanisms that contribute to movement of fluid into and out of the intestinal lumen on a daily basis. These three ion transport mechanisms—chloride secretion, electroneutral NaCl absorption, and electrogenic sodium absorption—account

INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1945 A

B out

out Na+

P

Na+

Cl−

H+

C

H+

out 3Na+

D out Na+ U U

∼

U U

2K+

FIG. 76-7. Modes for regulation of intestinal transport proteins. (A–D) Out implies the extracellular face of the membrane, either apical or basolateral. (A) Regulation by transporter phosphorylation, as exemplified by the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. (B) Regulation by exocytic insertion into and/or endocytic retrieval from the plasma membrane, as exemplified by NHE3. (C) Regulation by internalization, ubiquitination, and subsequent degradation, as exemplified by the epithelial sodium channel (ENaC) sodium channel. (D) Regulation by de novo transcription or translation, or both, with a corresponding increase in transporter abundance, as exemplified by Na+,K+-ATPase. Note that several of these mechanisms may apply to any given transporter involved in the transport of electrolytes by intestinal epithelial cells.

for the majority of fluid movement in the colon (1,7). Likewise, chloride secretion and electroneutral NaCl absorption are the primary determinants of fluid movements in the small intestine in the period between meals (1). All three transport mechanisms are subject to complex regulatory pathways that influence their extent both positively and negatively. Subcellular mechanisms that account for such regulation include covalent and allosteric modifications of transport proteins that alter their activity, trafficking of transporters into and out of the plasma membrane, transporter degradation, and transcriptional regulation (Fig. 76-7). Furthermore, in selected cases, absorptive and secretory transporters may sometimes be expressed in the same cell and may engage in reciprocal regulation. At the tissue level, many effectors that stimulate chloride secretion are also responsible for inhibiting electroneutral NaCl absorption and vice versa, although a similar relation does not necessarily hold for chloride secretion and electrogenic sodium absorption. Intestinal electrolyte transport is also subject to derangements in the setting of disease, although we remain far from a full understanding of the detailed mechanisms responsible for such changes, and whether they represent appropriate adaptations to a perceived threat or maladaptations that contribute to disease pathogenesis. In fact, despite an explosion of molecular details on the regulation of the various transporters involved in intestinal epithelial ion transport, we have no robust cellular model for electrogenic sodium absorption in

the colon, and translation of available information into the integrated setting of intact tissue has also lagged. However, the availability of a variety of animals with either constitutive or conditional targeting of intestinal transporters and their regulatory proteins is rapidly closing these gaps in our understanding, with the proviso that such gene-targeting efforts may themselves introduce long-term adaptations designed to maintain homeostasis (188,187). Continued application of such animal models, as well as investigations in intact human tissues and in patients, should suggest novel therapeutic targets in enteric infections, in inflammatory bowel diseases, and in the more prosaic setting of the common complaint of constipation, as well as in other intestinal disorders in which ion transport processes are dysregulated.

ACKNOWLEDGMENTS This study was supported by grants from the National Institutes of Health (DK28305, DK33491, and AT001180) and from the Crohn’s and Colitis Foundation of America. We are grateful to current and former colleagues for helpful discussions (particularly Lone Bertelsen and Declan McCole) and to Ms. Glenda Wheeler for assistance with manuscript preparation and submission. K.E.B. is a member of the Biomedical Sciences Ph.D. program, University of California, San Diego, School of Medicine.

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INTEGRATIVE PHYSIOLOGY AND PATHOPHYSIOLOGY OF INTESTINAL ELECTROLYTE TRANSPORT / 1951 221. Diaz-Granados N, Howe K, Lu J, McKay DM. Dextran sulfate sodium-induced colonic histopathology, but not altered epithelial ion transport, is reduced by inhibition of phosphodiesterase activity. Am J Pathol 2000;156:2169–2177. 222. Asfaha S, Bell CJ, Wallace JL, MacNaughton WK. Prolonged colonic epithelial hyporesponsiveness after colitis: role of inducible nitric oxide synthase. Am J Physiol Gastrointest Liver Physiol 1999;276: G703–G710. 223. Kolachala V, Asamoah V, Wang L, Srinivasan S, Merlin D, Sitaraman SV. Interferon-gamma down-regulates adenosine 2b receptor-mediated signaling and short circuit current in the intestinal epithelia by inhibiting the expression of adenylate cyclase. J Biol Chem 2005;280: 4048–4057. 224. Zamuner SR, Warrier N, Buret AG, MacNaughton WK, Wallace JL. Cyclooxygenase 2 mediates post-inflammatory colonic secretory and barrier dysfunction. Gut 2003;52:1714–1720. 225. Buresi MC, Vergnolle N, Sharkey KA, Keenan CM, Andrade-Gordon P, Cirino G, Cirillo D, Hollenberg MD, MacNaughton WK. Activation of proteinase-activated receptor-1 inhibits neurally evoked chloride secretion in the mouse colon in vitro. Am J Physiol Gastrointest Liver Physiol 2005;288:G337–G345. 226. Barmeyer C, Amasheh S, Tavalali S, Mankertz J, Zeitz M, Fromm M, Schulzke JD. IL-1beta and TNFalpha regulate sodium absorption in rat distal colon. Biochem Biophys Res Commun 2004;317:500–507. 227. Perez-Navarro R, Ballester I, Zarzuelo A, Sanchez de Medina F. Disturbances in epithelial ionic secretion in different experimental models of colitis. Life Sci 2005;76:1489–1501. 228. Perez-Navarro R, Martinez-Augustin O, Ballester I, Zarzuelo A, Sanchez de Medina F. Experimental inflammation of the rat distal colon inhibits ion secretion in the proximal colon by affecting the enteric nervous system. Naunyn Schmiedebergs Arch Pharmacol 2005; 371:114–121. 229. Musch MW, Clarke LL, Mamah D, Gawenis LR, Zhang Z, Ellsworth W, Shalowitz D, Mittal N, Efthimiou P, Alnadjim Z, Hurst SD, Chang EB, Barrett TA. T cell activation causes diarrhea by increasing intestinal

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CHAPTER

77

Molecular Mechanisms of Intestinal Transport of Calcium, Phosphate, and Magnesium James F. Collins and Fayez K. Ghishan Recommended Nutritional Requirements for Ca2+, Mg2+, and Inorganic Phosphate, 1953 Intestinal Calcium Transport, 1954 Overview of Intestinal Calcium Absorption, 1954 Molecular Mechanisms of Intestinal Calcium Transport, 1957 Regulation of Intestinal Calcium Absorption, 1963 Intestinal Transport of Phosphate, 1967 Mechanisms and Sites of Phosphate Absorption, 1967 Molecular Mechanisms of Intestinal Phosphate Transport, 1968

Type IIb Sodium-Dependent Phosphate Cotransporter (SLC34A2) and Intestinal Phosphate Transport, 1970 Regulation of Phosphate Absorption, 1971 Intestinal Transport of Magnesium, 1971 Sites of Magnesium Absorption, 1971 Magnesium Physiology, 1972 Transient Receptor Potential Family of Cation Channels, 1973 Regulation of Magnesium Absorption, 1975 Acknowledgments, 1975 References, 1975

Many new insights into the molecular mechanisms of intestinal absorption of calcium, phosphate, and magnesium have been made since the mid-1990s. These insights include the identification, cloning, and characterization of transmembrane proteins responsible for the absorption of all three of these nutrient molecules. The proteins identified for uptake of Ca2+ and Mg2+ across the apical membrane of enterocytes are members of the transient receptor potential (TRP) family of cation channels (transient receptor potential vanilloid receptor 6 [TRPV6] for Ca2+ and transient receptor potential melastatin receptors 6 and 7 [TRPM6/7] heterotetramers for Mg2+), whereas intestinal phosphate (Pi) absorption across the apical membrane is now known to be mediated by a member of the sodium-phosphate cotransporter gene family (NaPi-IIb; SLC34). In addition, in some cases, molecules responsible for movement of these molecules across the cellular cytoplasm (e.g., calbindins for Ca2+) and exit across the basolateral membranes (e.g., plasma membrane

Ca2+ ATPase [PMCA1b] and the Na+-Mg2+ exchanger) of intestinal epithelial cells also have been identified. With the discovery of these channels and transporters, a much clearer understanding exists of the molecular mechanisms involved in the transport of these crucial nutrient molecules. However, for all three of these molecules, transport proteins involved in transcellular uptake appear to be important only when intake levels are low, whereas at greater dietary intake levels, absorption occurs passively via unidentified, paracellular transport pathways. Furthermore, additional information has shown that these transport processes are regulated by a number of physiologic effectors, and that these effects are mediated by direct effects on the expression or activity of the identified transporters. Thus, the purpose of this chapter is to provide updated information regarding the intestinal transport of calcium, phosphate, and magnesium.

J. F. Collins and F. K. Ghishan: Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

RECOMMENDED NUTRITIONAL REQUIREMENTS FOR Ca2+, Mg2+, AND INORGANIC PHOSPHATE A report (1) has presented dietary reference values for intake of nutrients by people in the United States and Canada. This report describes dietary reference intakes (DRIs), which

1953

1954 / CHAPTER 77 are reference values that can be used to formulate and assess diets for healthy populations in the United States and Canada. The DRIs replace the periodic revisions of the Recommended Dietary Allowances (RDAs), which have been published since 1941 by the National Academy of Sciences. DRIs are formulated by considering several factors: the estimated average requirement (EAR), the RDA, adequate intake, and the tolerable upper intake level (UL). RDAs and adequate intakes are nutrient intake levels that should decrease the risk for development of pathophysiologic conditions related to that particular nutrient. The EAR is a nutrient intake value that is estimated to meet the requirement by 50% of individuals in a particular life stage and sex group; at this level of intake, the remaining 50% of this group would not meet their nutritional needs. In some cases, these recommendations have been extrapolated to estimate this value, because of the lack of experimental data. The EAR is used in setting the recommended dietary allowance (RDA), which is the average daily requirement that meets the nutritional needs of all individuals in a life stage and sex group (97–98%). Furthermore, adequate intake is set instead of an RDA if sufficient scientific evidence is unavailable to calculate an EAR (e.g., for calcium and magnesium intake levels). The adequate intake is based on observed or experimentally determined estimates of average nutrient intake by a group or groups of healthy people. In addition, the tolerable UL is the greatest level of daily nutrient intake that will likely not pose any risk for adverse health effects. This term is intended to set a level of intake that can, with high probability, be tolerated biologically. The UL is not intended to be a recommended level of intake. The issue of calcium intake levels is still under debate, and this is the major reason that an RDA for calcium has not been established (but rather an adequate intake). What is at issue is whether these recommended intake levels provide a positive calcium balance and optimal bone mineralization. This is primarily relevant in elderly adults, in whom intestinal calcium transport is declining, as are skeletal calcium reserves. In humans, intestinal absorption of calcium decreases with advancing age (1a–3). Similarly, the adaptation to low dietary calcium also is blunted in elderly adults (4). These changes can be attributed to a progressive resistance to vitamin D that develops in the process of ageing (3,5). In addition, the renal production of 1,25-dihydroxyvitamin D3 (1,25(OH)2 vit D3) may also be impaired in elderly adults. Consequently, the dietary intake of calcium required to maintain positive calcium balance progressively increases with advancing age. The issue is further complicated by that the composition of the diet may significantly alter calcium bioavailability, absorption, and retention. The majority of dietary calcium comes from dairy products alone (e.g., ~75%), whereas a relatively small amount is derived from fruits and vegetables. However, significant intake of fruits and vegetables can result in a negative calcium balance (6). This is because certain plant-derived products such as oxalate found in spinach can bind dietary calcium and reduce its bioavailability, and hence its absorbability (7). Evaluation of true

nutrient (e.g., calcium) status requires clinical, biochemical, and anthropometric data, alone or combination. Thus, precise determination of the necessary calcium intake levels at different ages would require long-term assessment of bone mineral content in prospective clinical trials. Note that the EARs and RDAs provided for phosphorus and magnesium intake are based on adequate scientific information (unlike the adequate intakes given for Ca2+, which are estimates). Table 77-1 lists recommended dietary intake levels for calcium, phosphorus, and magnesium for populations of various ages and both sexes (1a).

INTESTINAL CALCIUM TRANSPORT Calcium is recognized as a critical element for normal homeostasis and is a major constituent of bone. An average 70-kg adult has 1 kg body calcium, with more than 99% of that found in bone. The remaining calcium can be found in the body fluids and in cells of other tissues. Extracellular calcium has an important regulatory role in maintaining bone mineral density, with decreased plasma levels triggering the release of parathyroid hormone (PTH), which, in turn, tends to restore circulating calcium levels to ~2.5 mmol/L via its effect on bone, intestine, and kidney. Calcium also plays a well-described role as an intracellular messenger in a number of physiologic systems and cell types, and it is responsible for certain aspects of muscle contraction, neurotransmission, and secretion. All the calcium in our bodies is acquired from dietary sources; thus, a thorough understanding of the molecular mechanisms responsible for intestinal calcium absorption is important (Fig. 77-1). Calcium deposition rates to the skeleton vary with age, with the greatest rates of deposition found during the neonatal period, followed by minimal deposition once one stops growing (8,9). In general, bone calcium deposition parallels removal of calcium from bone, with the difference between the two processes constituting the bone calcium mass. Bone calcium mass is low during the newborn period; it increases until 35 to 45 years of age, and then gradually decreases with increasing age in men, and it abruptly decreases in women several years after menopause (10). Calcium from the diet is absorbed throughout the small and large intestines, with an active transport mechanism being predominant in the proximal small intestine under conditions of low calcium intake. When calcium intake levels are high, calcium is absorbed along the length of the small and large intestines via a paracellular absorptive mechanism. Several excellent reviews have been published on this subject (10–13).

Overview of Intestinal Calcium Absorption The average diet typically consumed daily by humans is between 400 and 1000 mg calcium, of which some, but not all, is absorbed. The net epithelial transfer of calcium represents

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1955 TABLE 77-1. Dietary reference intakes for calcium, phosphorus, and magnesium Calciuma AIb

Phosphorusa EARc

RDAd

Magnesium AIb

Life-stage group 0–6 months 7–12 months 1–3 years 4–8 years 9–13 years 14–18 years 19–30 years 31–50 years 51–70 years >70 years Pregnancy <18 years 19–30 years 31–50 years Lactation <18 years 19–30 years 31–50 years

EARc

Male

RDAd

AIb

Female

Male

Female

Male

Female

210 270 500 800 1300 1300 1000 1000 1200 1200

— — 380 405 1055 1055 580 580 580 580

— — 460 500 1250 1250 700 700 700 700

100 275 — — — — — — — —

— — 65 110 200 340 330 350 350 350

— — 65 110 200 300 255 265 265 265

— — 80 130 240 410 400 420 420 420

— — 80 130 240 360 310 320 320 320

30 75 — — — — — — — —

30 75 — — — — — — — —

1300 1000 1000

1055 580 580

1250 700 700

— — —

— — —

335 290 300

— — —

400 350 360

— — —

— — —

1300 1000 1000

1055 580 580

1250 700 700

— — —

— — —

300 255 265

— — —

360 310 320

— — —

— — —

aCalcium and phosphorus groups include males and female individuals, except only female individuals are included in pregnancy and lactation groups. bAdequate intake is measured in mg/day. For healthy infants fed human milk, AI is the estimated mean intake. cEstimated average requirement (EAR) is measured in mg/day. EAR is the intake that meets the estimated nutrient needs of 50% of the individuals in a group. dRecommended Dietary Allowance (RDA is measured in mg/day. RDA is the intake that meets the nutrient needs of 97% to 98% of individuals in a group. Modified from Standing Committee on the Scientific Evaluation of Dietary Reference Intakes, Food and Nutrition Board, Institute of Medicine. Dietary reference intakes for calcium, phosphorus, magnesium, vitamin D, and fluoride. Washington, DC: National Academy Press, 1997.

the summation of two opposing, unidirectional processes: (1) the total transfer of calcium from intestinal lumen to plasma; and (2) the total transfer of calcium from plasma to lumen, or endogenously secreted calcium (see Fig. 77-1). Thus, fecal calcium contains unabsorbed calcium from dietary sources and endogenously excreted calcium. Total intestinal calcium excretion in an adult human averages 0.194 ± 0.073 g/day, whereas secreted calcium that is not absorbed (endogenous fecal calcium) averages 0.130 ± 0.047 g/cm intestine per day (14). Calcium excretion of the human jejunum is passive and amounts to ~0.008 mM/hour/20 cm2 mucosal surface area (4). Calcium must be in the soluble form to be absorbed by the gastrointestinal tract. Even in the alkaline conditions of the ileum where calcium salts may be formed, some calcium ions remain in solution. Total calcium absorption is dependent on three factors: (1) local solubility; (2) the rate of transepithelial transport of calcium across the intestinal epithelium; and (3) the transit time of chyme movement through a particular gut segment (15). Furthermore, calcium in vegetables and other solid foods must be digested before going into solution and being available for transport. Thus, the calcium present in foods with high-fiber content is likely to be poorly absorbed compared with a more easily digestible food source with identical calcium contents (16), although

true digestibility is difficult to predict and actual calcium transport rates may be greater than expected (17,18). Several studies have reported on attempts to improve bioavailability of calcium by adding the following compounds to foods: citrate (19), casein phosphopeptides (20), or highly soluble salts such as calcium gluconate (21) or calcium gluconate-glycerophosphate (22). Other studies have addressed whether certain diet components, such as adding rice cereal to infant formula (23) or increasing dietary fiber in weaning cereals (24), have adversely affected calcium bioavailability or absorption. From these studies, it is now apparent that when calcium intake is adequate or high, differences in bioavailability, such as from increased solubilization, have little effect on intestinal calcium transport rates and calcium deposition to the skeletal system (25,26). However, under conditions of low dietary calcium intake and when calcium is present in a more indigestible form (such as in vegetables such as spinach), the decrease in calcium transport compared with a source such as milk becomes nutritionally significant (10). Several studies have sought to understand the high absorbance rate of calcium from milk and suggested that lactose (27), phosphopeptides (28), and amino acids (29) may play a role in this phenomenon. Interestingly, calcium bioavailability in some milk substitutes is decreased compared with milk (30); however, despite this, nutritional quality is

1956 / CHAPTER 77

C

it os ep

ion

a ++ Re

1,25 (OH2) vit D3

a ++ D

Ca++ in diet (400-1000 mg/day)

Bone

n tio rp o s Parathyroid gland

C

Ca++ Absorption

(0.194 ± 0.073 g/day)

Body Ca++ Pool [~2.5 mM/L in plasma]

Serum Ca++ PT H

Ca++ Secretion

PTH

1α-hydroxylase Intestine

1,25 (OH2) vit D3

Kidney

Ca++ excreted in stool Misc. Ca++ loss (Sweat, semen menstruation, milk)

1,25 (OH2) vit D3 Ca++ excreted in urine

FIG. 77-1. Body calcium homeostasis. Plasma level of calcium is shown together with the major organs involved in the tight regulation of circulating calcium levels. Calcium absorption occurs throughout the intestinal tract; under conditions of high dietary intake, Ca2+ is transported by a paracellular pathway throughout the length of the small and large intestines, whereas active transport occurs with low intake levels predominantly in the proximal small intestine. Calcium is endogenously secreted into the intestinal lumen, and this pool mixes with the dietary pool, some of which is reabsorbed. Bone has the highest requirements for calcium, and it also serves as a storage depot for calcium, which can be mobilized under certain physiologic conditions. Calcium is lost in the stools, excreted by the kidney, and also lost from the body via other bodily secretions such as sweat and milk. The parathyroid gland plays a major role in sensing serum calcium levels via the calcium-sensing receptor, and when calcium concentrations decrease, parathyroid hormone (PTH) is released into the circulation. PTH stimulates bone resorption, which releases calcium into the circulation. PTH also stimulates renal 1α-hydroxylase activity, which, in turn, converts 25(OH) vit D3 into the active metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2 vit D3), which increases intestinal and renal Ca2+ (re)absorption. These latter effects in the intestines and kidney are mediated by increased expression of calcium transport–related proteins. Small arrows indicate the effect of hormone on that particular physiologic process.

similar (31). Another area of interest is related to the effect of fat intake and the formation of calcium soaps on calcium bioavailability and intestinal transport rates. One study concluded that the addition of synthetic triglycerides to infant formula significantly improved calcium absorption in preterm neonates (32). However, the effect of fat intake on intestinal calcium homeostasis in older children and adults is unknown. Furthermore, Sanyal and colleagues (33) demonstrated that premicellar taurocholate, a component of bile, enhances calcium absorption from all regions of the rat small intestine. A number of inorganic molecules and dietary components also have been reported to perturb intestinal calcium absorption. Phosphate may interfere with calcium absorption as a result of a formation of poorly soluble calcium-phosphate

salts, but increasing dietary phosphate by a factor of 2.5 has no reported influence on calcium absorption, regardless of the amount of calcium consumed (34). However, undigestible organic phosphates, such as phytates of hexaphosphoinositol, which are found in wheat bran, can form insoluble calcium salts. High concentrations of dietary magnesium can decrease calcium absorption, presumably by competing for the calcium transport site (35), although it is now known that calcium and magnesium are absorbed by different channel proteins. This effect could be caused by interaction of magnesium ions with the specific calcium channels in the gut (TRPV6), because extracellular Mg2+ has been shown to block the monovalent cation currents induced by TRPV6 in a heterologous expression system (36). Although a variety of calcium

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1957 preparations have been used to supplement the diet, including lactate, chloride, gluconate, carbonate, citrate, or sulphate salts, there is little difference in the intestinal utilization of calcium from any of these salts (37). There are a number of factors such as oxalate (7), that when included in the diet can either influence the bioavailability of calcium or directly alter the absorptive process. Certain antibiotics and cationic amino acids such as lysine stimulate intestinal calcium absorption (38). Similarly, lactose, other complex sugars, and glucose polymers promote calcium absorption (39). Gastric acidity or achlorhydria appears to have a limited effect on intestinal calcium transport (40). Moreover, decreased acidity does not further reduce the bioavailability of calcium from food at high or low levels of fiber intake (41). Calcium also is known to be absorbed in the mammalian colon, with the cecum (42) and the ascending colon (43) having the greatest rates; however, the transverse colon does not transport calcium (10). It has been demonstrated that acidic fermentation in the colon can enhance intestinal absorption of calcium and magnesium. Younes and colleagues (44) concluded that the large intestine may represent a major site for calcium transport when acidic fermentation takes place. Likewise, another group demonstrated that fructooligosaccharides significantly enhanced calcium and magnesium absorption in the rat colon (45). These authors concluded that indigestible and fermentable carbohydrates facilitate colorectal absorption of these ions. Furthermore, Mineo and colleagues (46) demonstrated that indigestible disaccharides directly affect the small-intestinal and colonic epithelium by opening tight junctions (TJs) between epithelial cells, and thereby promoting Ca2+ and Mg2+ absorption. In addition, another study showed that two fermentable carbohydrates (inulin and resistant starch) showed synergistic effects on intestinal calcium absorption and calcium balance in rats (47). Calcium absorption may also be enhanced by acetate and propionate in the human rectum and distal colon, by a nonsaturable, diffusive process (48).

Molecular Mechanisms of Intestinal Calcium Transport Ca2+ is transported throughout the small and large intestines in mammals by two distinct mechanisms: a transcellular process that predominates in the duodenum and is regulated by 1,25(OH)2 vit D3, and a paracellular process that occurs throughout the length of the small and large intestines (49). The amount of Ca2+ transferred paracellularly is directly related to the calcium content of the diet and to the amount of time that is required for the digesta to move through an intestinal segment, and it is inversely proportional to the rate of intestinal propulsion. The maximal transit time is in the ileum (10), where under normal Ca2+ intake, the greatest proportion of dietary Ca2+ is absorbed (50). In the duodenum, Ca2+ absorption can be considered a sum of two processes: a saturable transport that is well-described by a Michaelis–Menten relation (e.g., transcellular), and another

that occurs in a straight linear concentration-dependent function (e.g., paracellular) (51). Conversely, in the ileum, only the linear concentration-dependent mechanism occurs, which represents paracellular movement through TJs. If dietary Ca2+ intake levels are low, most of the Ca2+ in solution will be absorbed in the duodenum by the active transcellular process (21). If, however, Ca2+ intake levels are high, most of the Ca2+ will be absorbed by the paracellular process, largely in the distal portions of the small intestine. This is true not only because of the relatively large amount of Ca2+ present in the lumen of the small intestine, but also because of down-regulation of the 1,25(OH)2 vit D3–dependent transcellular process. The regulated, transcellular process assumes functional importance, therefore, only under conditions of low Ca2+ intake. Paracellular Pathway of Dietary Calcium Absorption An historical view of intestinal calcium absorption was that transcellular movement of nutrients and water via specific pumps, transporters, and channels accounted for absorption, whereas an impermeable TJ sealed adjoining epithelial cells and provided a barrier to nutrient molecule transport. However, it has been suggested that transjunctional (e.g., paracellular) solute movement occurs in a regulated fashion, and that its regulation may be coupled to transcellular, absorptive events. Thus, epithelial solute transport and TJ barrier function may be viewed as coordinately regulated events (52). Under conditions of high Ca2+ intake, Ca2+ may be absorbed throughout the length of the entire intestine (both small and large), mainly by a paracellular pathway through TJs, which occur between adjacent enterocytes. TJs are the contact points between the apical and basolateral membranes of intestinal epithelial cells that limit paracellular flux of ions, proteins, and other macromolecules. TJs also serve to limit membrane-associated protein diffusion between the basolateral and apical poles of enterocytes. The restricted movement of ions across the TJs causes transepithelial electrical resistance (TER), which often is used as a measure of the integrity of epithelial cell layers or of cells grown in monolayer culture. TJs are crucial in maintaining the polarized phenotype and the vectorial transport functions of epithelial cells. They are also a specialized membrane microdomain that can function as a molecular platform involved in cell signaling, vesicle protein docking, actin organization, and cell polarity in epithelial cells. Each particle of the TJ strand is composed of transmembrane proteins and cytoplasmic plaque proteins connected to the actin cytoskeleton. Occludins, claudins, and the junctional adhesion molecule are three major classes of transmembrane proteins localized to the TJs. TJs also concentrate tumor suppressor proteins and cell polarity proteins (53). Furthermore, TJs have a highly dynamic structure; thus, their permeability and assembly or disassembly can be regulated by a variety of cellular and metabolic mediators including cytokines (54), growth factors, hormones, and bacterial toxins (13).

1958 / CHAPTER 77 Kutuzova and Deluca (55) found a number of 1,25(OH)2 vit D3 target genes that are (co)localized in the proximal vicinity of intestinal TJs, and thus could influence TJ integrity and permeability. These included not only transporters and channels, but also several intracellular and intercellular matrix-related proteins and G proteins. Some genes with protein products that are involved in the regulation of TJ permeability were down-regulated in response to 1,25(OH)2 vit D3, including sodium-potassium ATPase, claudin 3, aquaporin 8, cadherin-17, and RhoA. Another study also suggested that claudins may form part of the channel involved in the paracellular transport of calcium, and furthermore, that the process of calcium movement through TJs may also be regulated by 1,25(OH)2 vit D3 (55). Kutuzova and Deluca (55) suggested that 1,25(OH)2 vit D3 may increase intestinal epithelial TJ permeability or modulate its selectivity toward Ca2+ and other cations by regulating expression of proteins involved in TJ formation. The increased TJ permeability or selectivity, or both, regulated by 1,25(OH)2 vit D3, could direct Ca2+ absorption through the TJ-regulated paracellular pathway in the intestinal epithelium. This suggestion agrees with a study that demonstrated that 1,25(OH)2 vit D3 stimulated an increase in TJ conductance and increased paracellular Ca2+, Na+, Rb+, and mannitol transport in an enterocyte-like cell line (Caco-2) (56). Under these conditions, no significant contribution of the Ca2+-ATPase–mediated transcellular pathway to overall transepithelial Ca2+ transport was detected (56). Additional evidence has accumulated demonstrating that 1,25(OH)2 vit D3 enhanced both cell-mediated active and passive paracellular Ca2+ and Pi transport in a Ussing chamber study of the rat small intestine (57) and Ca2+ transport in Caco-2 cell monolayers (58). Extensive scientific evidence supports the concept of paracellular transport of calcium with high dietary intake levels, as described earlier. However, Morgan and colleagues (59) reported the discovery of a novel, intestinal calcium channel (Cav1.3), which is homologous to the neuroendocrine L-type Cav1.3 calcium channel. Immunocytochemical studies demonstrated that the channel was expressed on the apical membrane of enterocytes from the proximal jejunum to the mid-ileum. Perfusion studies with 1.25 mM luminal calcium showed L-type calcium channel activity; this activity was inhibited by phloridzin, nifedipine, and Mg2+ and was activated by Bay K8644. These findings demonstrated channel-mediated rather than paracellular calcium transport. Furthermore, none of these compounds affects expression of the active calcium transport channels (e.g., TRPV5 and TRPV6; see discussion later in this chapter). These authors thus conclude that intestinal Cav1.3 may mediate a significant route of calcium transport during times of dietary calcium sufficiency (59). Transcellular Pathway of Dietary Calcium Absorption Transcellular Ca2+ movement across the intestinal epithelium involves three major steps (see later for a detailed discussion): (1) calcium entry via a calcium uptake channel called

TRPV6; (2) intracellular diffusion, mediated by the 1,25(OH)2 vit D3-dependent calbindin-D9K; and (3) extrusion from the cell mediated by the PMCA1b (Fig. 77-2). Calcium Uptake Across the Brush-Border Membrane of Enterocytes Early studies indicated that over a wide range of transepithelial Ca2+ transport rates, Ca2+ influx at the apical membrane is correlated in a 1:1 fashion with the apical-tobasolateral Ca2+ flux (60). Ca2+ entry was postulated to occur via Ca2+-selective channels at the luminal membrane, under the influence of a steep inwardly directed electrochemical gradient. Initial studies demonstrated that 1,25(OH)2 vit D3 affected Ca2+ influx in enterocyte-like cells (61–64). This calciotropic hormone significantly increased radiolabeled Ca2+ uptake within several minutes in a dosedependent manner in rat intestinal epithelial cells (65). These and other studies suggested that a Ca2+ channel was activated by a cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA)–dependent pathway by 1,25(OH)2 vit D3 in the mammalian intestine (65). However, the physiologic significance of these earlier findings is unknown because many of the studies were done in in vitro cellculture models. Several additional studies have been done in one of the most widely used in vitro models of the mammalian intestinal epithelium, Caco-2 cells. Although these human cells are derived from a colorectal carcinoma, they are known to differentiate in culture and to express many functional transport processes indicative of intestinal epithelial cells. These cells will polarize and form columnar epithelia, TJs, and domes, and they express several markers that are unique to the differentiated small-intestinal epithelium. In addition, Caco-2 cells exhibit saturable, apical-to-basolateral Ca2+ transport kinetics, net transport occurs in the apical-to-basolateral direction, and the rate of transport is induced by 1,25(OH)2 vit D3 (66,67). This up-regulation of Ca2+ transport requires transcriptional events (66) and is modulated by changes in the vitamin D receptor (VDR) content of the cell (68). These experiments in Caco-2 cells defined the nature of the epithelial Ca2+ channels that are present in the intestine, and many subsequent studies were performed to try to identify this particular transporter. Further studies used functional expression cloning in an attempt to identify the apical calcium influx transporter/ channel. The first such experiments were done by Hoenderop and colleagues (69), who used a complementary DNA (cDNA) library constructed from poly(A)+ RNA isolated from primary cultures of rabbit kidney cells. The cDNA clones were copied into cRNA, which was microinjected into Xenopus laevis oocytes, followed by screening for Ca2+ uptake activity in the presence of a cocktail of known voltage-gated Ca2+ channel inhibitors (the renal and intestinal Ca2+ channels were known not to be voltage gated). After extensive screening procedures, a single transcript was isolated that encoded a novel epithelial Ca2+ channel (first

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1959

FIG. 77-2. Model of transepithelial calcium transport in the duodenal epithelium. Three adjacent enterocytes are depicted together with the major proteins involved in transepithelial intestinal calcium transport. Transient receptor potential vanilloid receptor 6 (TRPV6) heterotetramers are responsible for brush-border calcium influx; then calcium interacts with the intracellular, buffering protein calbindin-D9k. After release from calbindin-D9k, calcium then exits enterocytes across the basolateral membrane via the plasma membrane Ca2+-ATPase (PMCA1b). 1,25-Dihydroxyvitamin D3 (1,25(OH)2 vit D3) is involved in regulating all three of the genes encoding these proteins in response to body calcium needs. Transcellular and paracellular pathways are shown; paracellular ion movement occurs through tight junctions between adjacent enterocytes, which occurs with high calcium intake levels. Also shown on the apical membrane is the Cav1.3 calcium channel, which may be involved in transcellular calcium transport during times of dietary sufficiency. ADP, adenosine diphosphate; ATP, adenosine triphosphate. (Modified from Hoenderop and colleagues [13], by permission.)

called ECaC1 [15], then renamed as transient receptor potential channel TRPV5; also known as ECaC and CaT2) (70). When the TRPV5 channel is expressed in Xenopus oocytes and in HEK293 cells (human embryonic kidney cells), calcium transport was inhibited by (in order of potency) La3+ > Cd2+ > Mn2+, whereas Ba2+ and Sr2+ had no effect on calcium transport. Permeability to Na+ was negligible in the presence of Ca2+. Moreover, the TRPV5 channel was shown to be induced by 1,25(OH)2 vit D3 (71,72). Overall, these observations demonstrated that TRPV5 possesses all the expected characteristics of calcium influx in Ca2+-transporting epithelia (13). The TRPV5 transporter subsequently was determined to be expressed predominantly in the kidney and was conclusively shown to be responsible for renal Ca2+ reabsorption (73). Subsequent studies by Peng and coworkers (74) applied the functional expression cloning technique to the search for the intestinal calcium entry channel, and they were able to identify an intestinal Ca2+ transporter, which they called CaT1. CaT1 shares 80% amino-acid sequence identity to TRPV5. CaT1 has been renamed TRPV6 (70) and is also known in the literature as ECaC2 and CaT-like (13). Electrophysiologic studies,

as described in detail later, demonstrated that the characteristics of TRPV6 are comparable with those measured for TRPV5, but the expression pattern of TRPV6 is more ubiquitous. Moreover, functional properties of both TRPV5 and TRPV6 are in congruence with the known properties of the putative epithelial calcium channels, which are responsible for intestinal and renal calcium (re)absorption (12,75). TRPV6 currently is thought to be the major player in active intestinal calcium transport (12). Transient Receptor Potential Family of Channel Proteins TRP channel proteins form a large and diverse, but related, family of proteins that are expressed in many tissues and cells types (76,77). The large functional diversity of this family of proteins is reflected in their diverse permeability to ions, activation mechanisms, and involvement in a wide range of physiologic processes. The TRP channels can be divided by sequence homology into at least six subfamilies, designated TRPC (canonical or classical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystins, PKD-type), TRPA (for ankyrin), and TRPML (for mucolipin), as well as the

1960 / CHAPTER 77 more distantly related subfamily TRPN (N for “nomp,” no mechanoreceptor potential). An intriguing subfamily within the TRP superfamily is the TRPV family, consisting of six members. This group of channels includes TRPV1-4, which respond to heat, osmolarity, odorants, and mechanical stimuli, whereas TRPV5 and TRPV6 are epithelium Ca2+ channels and have been implicated in maintaining body-Ca2+ balance by facilitating Ca2+ (re)absorption in the kidney and small intestine (12,73,75). Transient Receptor Potential Vanilloid Receptors 5 and 6 The epithelial Ca2+ channel family is restricted to two members, and genomic cloning demonstrated that TRPV5 and TRPV6 channels are transcribed from distinct genes (78,79). Interestingly, TRPV5 and TRPV6 are juxtaposed on human chromosome 7q35, with a distance of only 22 kb separating them, which suggests that a single ancestral gene was duplicated over evolutionary time. An analogous situation was observed in the mouse genome, in which the two genes are located close to one another on chromosome 6, in a region that is syntenic to human chromosome 7q33-35 (73,79). Currently, TRPV5 and TRPV6 have been cloned from many species, including rabbit, rat, mouse, and human (78). The putative proteins exhibit an overall amino-acid sequence homology of 75% to 80%. Interestingly, several domains within these proteins are completely conserved among species, including the membrane topology of the protein with six putative transmembrane segments and the postulated pore region. Furthermore, detailed sequence analysis of these proteins identified several putative phosphorylation sites, for example, PKC, PKA, and cGMPdependent kinase (80). However, there is currently no experimental information available about the phosphorylation of TRPV5 and TRPV6. In addition, both proteins contain PDZ motifs and ankyrin repeats in the NH2-terminal region, which are conserved in a diverse range of receptors and ion channels, including the TRP superfamily. PDZ motifs are recognized by proteins containing PDZ-interacting domains, and these protein–protein interactions may be involved in protein targeting and multiprotein complex assembly (81). PDZ domain interactions serve not only scaffolding and cytoskeletal attachment roles, but there is evidence that they can regulate the functions of their ligands. PDZ domain–containing proteins typically interact with COOH-terminal PDZ motifs in target proteins, but interactions could also occur with the N-terminal motifs that are present in TRPV5 and TRPV6. Ankyrin repeats have similar roles as PDZ domain–motif interactions, in that they can link transporters and cell adhesion molecules to the spectrinbased cytoskeletal elements in specialized membrane domains (82). However, currently, no PDZ domain– or ankyrin repeat– interacting proteins have been identified for TRPV5 or TRPV6. Studies have addressed the expression of TRPV5 and TRPV6 in the gastrointestinal tract. Initially, Northern blot analysis showed expression of rabbit TRPV5 in duodenum

and jejunum, whereas ileum was negative. However, these hybridizations were performed before the identification of the TRPV6 isoform, using full-length cDNA probes that do not discriminate between the highly homologous TRPV5 and TRPV6 transcripts (69). Subsequent experimental approaches using isoform-specific probes and quantitative polymerase chain reaction (PCR) analysis, and immunohistochemical studies found expression of both channels in the intestine. However, these studies demonstrated that TRPV6 transcript levels in the gut are at least three orders of magnitude greater than TRPV5 transcript levels (79,83,84). Another study addressed the issue of whether TRPV5 has an important physiologic role in the intestine by creating TRPV5 knockout mice (73). These animals exhibit intestinal Ca2+ hyperabsorption, most likely mediated by increased TRPV6 and calbindin-D9K expression levels, which suggests a predominant role for TRPV6 in intestinal calcium transport. However, additional immunohistochemical and functional studies are needed to address the possible role of TRPV5 in the small intestine (13). In the mammalian intestine, TRPV6 expression is found in the duodenum, jejunum, cecum, and colon, where it is colocalized in epithelial cells together with other molecular components of intestinal calcium absorption, calbindin-D9K and PMCA1b (74,78,85). One study conducted by Peng and coworkers (85) demonstrated expression of TRPV6 throughout the entire digestive tract from the esophagus to the colon. Additional studies estimated TRPV6 and TRPV5 mRNA expression levels in the mouse by quantitative PCR analysis, and resulting data were normalized for the amount of cDNA used for the amplification (71). The study by Peng and coworkers demonstrated that TRPV6 mRNA expression was highest in duodenum and cecum, lower in the colon, and even lower in the ileum. This investigation also demonstrated that TRPV5 mRNA was expressed at much higher levels in the kidney compared with the duodenum and cecum, whereas the ileum and colon did not express TRPV5 mRNA. Immunohistochemical techniques also have been used to determine the distribution of TRPV6, calbindin-D9K, and PMCA1b proteins in the small-intestinal epithelium (86). TRPV6 was localized along the brush-border membrane, whereas calbindin-D9K was found in the cellular cytoplasm and PMCA1b was expressed at the basolateral membrane (87,88). Further detailed immunolocalization studies demonstrated expression of TRPV6 on the apical membrane of enterocytes in the entire small intestine and colon (45). When considered in their entirety, the current data strongly suggest that epithelial Ca2+ channel TRPV6 is the major transcellular mediator of Ca2+ uptake from the intestinal lumen. Thus, the remainder of this section focuses on this intestinal calcium channel. Biophysical Properties of Transient Receptor Potential Vanilloid Receptor 6 In the intestinal lumen, Ca2+ concentration varies, but it is often in the millimolar range, whereas inside the absorptive cell, the Ca2+ concentration is much less (~100 nmol/L).

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1961 This gives an approximate 10,000-fold concentration gradient across the apical membrane, and moreover, the membrane electric potential provides an additional driving force for calcium transport (with a relative negative charge on the cytoplasmic side of the membrane). Thus, transport of Ca2+ into intestinal epithelial cells does not require the consumption of metabolic energy. Ca2+ influx mediated by TRPV6 does not appear to be coupled with NaCl or protons. Transport activity is sensitive to pH, with Ca2+ uptake activity increasing at alkaline pH. TRPV6 is permeable to Ba2+ and Sr2+, but not Mg2+ (12). The macroscopic properties of this channel expressed in Xenopus laevis oocytes indicate that this protein works as a facilitative uniporter that constitutively transports substrate down the concentration gradient with saturation kinetics, but without obvious gating mechanisms (12). Basic electrophysiologic studies for TRPV6 have shown that outward currents are extremely small, indicating the channel is nearly completely inwardly rectifying. The current through TRPV6 is carried exclusively by Ca2+ at extracellular Ca2+ concentrations exceeding 10 µM. TRPV6 shows high selectivity for calcium, with Ca2+/Na+ permeability ratios (PCa2+/PNa+) of more than 100. This channel is blocked effectively by trivalent and divalent cations, and it is relatively insensitive to L-type voltage-gated channel blockers (as discussed in detail later). Regulation of Transient Receptor Potential Vanilloid Receptor 6 Channel Activity by 1,25-Dihydroxyvitamin D3 Studies consistently have indicated that the expression of TRPV6 is tightly regulated by 1,25(OH)2 vit D3 (61,62,66,83,84,89–92). Van Cromphaut and colleagues (84) injected a single dose of 1,25(OH)2 vit D3 into mice, and then demonstrated through quantitative PCR data a sixfold up-regulation of TRPV6 mRNA expression. Furthermore, analysis of the putative promoter regions of human and murine TRPV6 genes shows potential vitamin D response elements (VDREs), which strongly suggests that the 1,25(OH)2 vit D3 stimulation of TRPV6 gene expression is mediated via nuclear VDR by direct interaction with the promoter region of the gene (71,72). Song and colleagues (91) also demonstrated that after a single dose of 1,25(OH)2 vit D3, induction of duodenal TRPV6 mRNA expression occurred within 3 to 6 hours and preceded the induction of intestinal calcium absorption. In addition, this study demonstrated that intestinal TRPV6 mRNA expression increased 30-fold at weaning, coincident with the induction of calbindin-D9K expression. This observed 1,25(OH)2 vit D3–dependent regulation of TRPV6 subsequently was studied extensively using many different cell lines and knockout animal models (as discussed later in this chapter). Regulation of Channel Activity by Intracellular Ca2+ Because the Ca2+ concentration in the intestinal lumen can vary greatly, the sudden appearance of a high level of Ca2+ in the lumen can be disastrous, because sustained increases in intracellular Ca2+ may cause cell death. Thus, Ca2+-transporting

cells express calbindins to buffer the increases in intracellular Ca2+ resulting from Ca2+ entry through TRPV6 channels. In addition, to avoid increases in intracellular Ca2+ to toxic levels beyond the buffering capacity of the calbindins, TRPV6 exhibits Ca2+-dependent inactivation (93,94). It has both a fast phase (within 50 msec) and a slow phase of inactivation (more than ~1 second). The slow phase involves direct binding of calmodulin (CaM) to the TRPV6 C-terminal region (93). Ca2+-dependent binding of CaM to human TRPV6 inactivates the channel, via a protein kinase C (PKC) site present within the CaM-binding site. The phosphorylation of this PKC site prevents CaM binding, thereby maintaining the activity of TRPV6, to allow more Ca2+ to enter the cell (93). The first intracellular loop of TRPV6 determines the fast inactivation (94), which may involve direct interaction of calcium with the channel on the cytoplasmic side of the membrane. In addition, H587, a positively charged amino-acid residue in the last transmembrane segment of TRPV6, also has been identified as being involved in fast inactivation (95). This feedback inhibition of TRPV6 activity accomplishes two objectives: (1) it protects the cell from Ca2+ overload; and (2) it also limits the flow of Ca2+ into the cell. TRPV6 activity is inversely related to the intracellular Ca2+ level on its cytoplasmic side (96). Thus, the availability of calbindin-D9K to buffer the local increase in Ca2+ near the apical membrane will reduce the Ca2+-dependent feedback inhibition and, in turn, increase the movement of Ca2+ into the cell. Therefore, a coordinated increase in the expression of both the apical channel TRPV6 and intracellular calbindin-D9K is necessary to achieve maximal Ca2+ influx across the apical surface of intestinal epithelial cells. The observation of a lag period in the calbindin-D9K response to 1,25(OH)2 vit D3 relative to that of TRPV6 (92) suggests that the increase in calbindin-D9K is an “amplifying” mechanism that increases Ca2+ influx, both by relieving feedback inhibition of the apical entry step and by facilitating diffusion of Ca2+ from the apical to the basolateral membrane. The time lapse in calbindin-D9K production and response to 1,25(OH)2 vit D3 administration suggests that the increase in intracellular Ca2+ as a result of TRPV6 expression could contribute to calbindin induction. Pharmacology of Transient Receptor Potential Vanilloid Receptor 6 Little is known about effective pharmacologic tools to modulate TRPV6 activity (13). TRPV6 can be blocked effectively by inorganic trivalent and divalent cations, including La3+, Gd3+, Pb2+, Cd2+, and Cu2+ (74,85). Furthermore, TRPV6 is rather insensitive to the L-type voltage-gated calcium channel blockers nifedipine, diltiazem, and verapamil, with TRPV6 being inhibited only 10% to 15% when these compounds were used at 100 µM (74). Econazole, miconazole, and SKF96365 were shown to inhibit Ca2+ uptake in TRPV6-expressing oocytes, with econazole being the most effective inhibitor (~50% inhibition with 50 µM) (12). The inorganic polycationic dye ruthenium red, which binds to phospholipids, inhibits TRPV6 in a voltage-dependent

1962 / CHAPTER 77 manner (13). Furthermore, xestospongin, a noncompetitive inositol 1,4,5-triphosphate receptor antagonist, appears to block TRPV6 as well (97). In addition, capsaicin has been reported to block TRPV6 (98). Molecular Structure of Transient Receptor Potential Vanilloid Receptor 6 TRPV6 likely forms homotetramers in the plasma membrane of cells. This tetrameric organization closely resembles the structure of the Shaker potassium channel, which is composed of four tandemly associated homologous domains (99,100). The clustering of the four subunits is thought to create an aqueous pore centered at the fourfold symmetry axis (100). This proposed tetrameric architecture implies that aspartic acid residues D542 (101) and D541 (102) form a negatively charged ring structure that forms a calciumselectivity filter, in analogy with voltage-gated calcium channels (103). Erler and colleagues (104) identified the third ankyrin repeat in TRPV6 as being critical for physical assembly of TRPV6 subunits into the tetrameric form. Deletion or mutation of amino-acid residues within this ankyrin repeat renders the channel nonfunctional and abolishes tetrameric formation. It was suggested that the third ankyrin repeat initiates a molecular zippering process that proceeds past the fifth ankyrin repeat and creates an intracellular anchor that is necessary for assembly of the functional subunits (104). TRPV5 and TRPV6 can form heterotetramers and homotetramers (103). This supposition is based on crosslinking studies, coimmunoprecipitations, and molecular mass determination of TRPV5/6 complexes using sucrose gradient sedimentation (13). When these two isoforms are coexpressed in some tissues, they may oligomerize, and this heterooligomerization may influence the functional properties of the Ca2+ channels formed. Because both of these proteins exhibit different channel kinetics with respect to Ca2+-dependent inactivation, Ba2+ selectivity, and sensitivity for inhibition by ruthenium red, the influence of heterotetrameric composition on channel properties could be important in certain tissues and cell types. In the intestine, however, TRPV6 has been shown to be expressed at levels as much as 100 to 1000 times greater than TRPV5, and no experimental evidence has directly demonstrated that there is actually heterotetramerization of the two channels in the intestine. It thus appears more likely that TRPV6 does not interact to an extensive level with TRPV5 in the intestinal epithelium. Calcium Diffusion across the Intestinal Epithelial Cell Cytoplasm Epithelial cells involved in transcellular calcium transport are challenged continuously by substantial Ca2+ moving through the cytosol, whereas the cells simultaneously need to maintain low levels of intracellular Ca2+. One potential explanation of how this could effectively occur is a facilitated diffusion model that proposes binding of calcium to an intracellular buffering protein, which delivers the calcium to the basolateral membrane for export (105,106). Mathematical

modeling predicts that intracellular diffusion of this protein/Ca2+ complex is the rate-limiting step in transepithelial calcium movement (51,107). Indeed, the 1,25(OH)2 vit D3–dependent Ca2+-binding protein calbindin-D9K has been detected in intestinal epithelial cells (108) and has been proposed to deliver calcium to the basolateral membrane for export into the interstitial space (109). The expression level of calbindin-D9K in the intestinal epithelium closely correlates with the efficiency of Ca2+ absorption; therefore, this protein plays a central role in the facilitated diffusion model. Interestingly, calbindin-D9K may directly enhance PMCA activity, which is the principal mechanism of calcium export across the basolateral membrane (110). Calbindin-D9K belongs to a group of intracellular proteins that bind Ca2+ with high affinity, which causes the protein to undergo structural changes caused by electrostatic interactions (111). Another similar protein called CaM is known to interact with and regulate the function of voltage-gated Ca2+ channels in a Ca2+-dependent fashion (112). The ubiquitously expressed CaM interacts directly with an “IQ motif ” present in the carboxy terminus of these channels, where it functions as a Ca2+ sensor (113). This IQ motif, however, is not present in TRPV6. Currently, it is unknown whether calbindin-D9K can perform a similar Ca2+ sensor function for which a specific interaction with TRPV6 would be required. Furthermore, the striking colocalization of calbindin-D9K with TRPV6 in the intestine suggests that a functional interaction between these two proteins may occur. Further experiments are needed to delineate whether the function of calbindin-D9K is restricted to its buffering capacity to maintain low Ca2+ concentrations in the cell in close vicinity to the channel mouth, or whether physical interaction between calbindin-D9K and TRPV6 is needed to exert a direct regulatory function. Thus, the Ca2+-binding protein calbindin-D9K is responsible for intracellular diffusion of Ca2+ in the enterocyte, and its gastrointestinal expression has been studied in many species. Yamagishi and colleagues (114) examined calbindin-D9K mRNA expression in the gastrointestinal tract of cattle by Northern blot analysis. Additional studies in several animal models have shown that calbindin-D9K and PMCA1b are both expressed in patterns that are compatible with roles in transepithelial Ca2+ transport, being found in villous cells of the proximal duodenum with gradually decreasing expression distally along the GI tract and demonstrating 1,25(OH)2 vit D3 dependence and decreased expression with increasing age (114–119). Calcium Extrusion Across the Basolateral Membrane The efflux of Ca2+ occurs from enterocytes against a considerable electrochemical gradient by a Ca2+ transporter that has been localized to the basolateral membrane of absorptive cells, the PMCA (13). PMCAs are high-affinity Ca2+ efflux pumps present in virtually all eukaryotic cells, where they are responsible for the maintenance of the resting intracellular Ca2+ levels (120). Four PMCA isoforms currently have been identified, and alternatively spliced transcripts have been detected (121,122); PMCA1b is,

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1963 however, the predominant isoform in the gut, where it is abundantly expressed in the small intestine (13). Several studies indicated that PCMA is positively regulated by 1,25(OH)2 vit D3 in the intestine to increase Ca2+ absorption. In one study, Northern blot analysis indicated that repletion of vitamin D–deficient chickens with 1,25(OH)2 vit D3 increases PMCA mRNA expression in the duodenum, jejunum, ileum, and colon (123). Additional studies by Johnson and Kumar (124) demonstrated that 1,25(OH)2 vit D3 causes an increase in the abundance of PMCA protein and stimulates Ca2+ extrusion. Furthermore, PMCA activation is dependent on CaM, and inhibition of CaM is, in turn, known to prevent PMCA stimulation. An Na+-Ca2+ exchanger (NCX1) also has been described on the basolateral membrane of enterocytes (83,125,126), but current data suggest that the predominant mechanism of calcium extrusion in mammals is the PMCA (125,127) because expression levels of NCX1 may be barely detectable. Thus, PMCA1b is thought to be the predominant mechanism whereby Ca2+ exits enterocytes during transepithelial Ca2+ absorption. Current data also suggest that extrusion is not the rate-limiting step for Ca2+ absorption (12). Regulation of Intestinal Calcium Absorption Regulation by 1,25-Dihydroxyvitamin D3 Vitamin D3 is accepted as one of the main hormones controlling Ca2+ balance (128). There are two sources of vitamin D: it is either ingested from the diet or synthesized in the skin from its precursor, 7-dehydrocholesterol, in the presence of ultraviolet radiation (129). Vitamin D3 itself is physiologically inactive. It will undergo an activation process involving 25-hydroxylation in the liver, followed by 1α-hydroxylation in the mitochondria of the renal proximal tubule (130), to generate the biologically active form, 1,25(OH)2 vit D3. Whether the kidney produces 1,25(OH)2 vit D3 depends on the Ca2+ status of the body. When Ca2+ intake is sufficient and the plasma Ca2+ concentration is normal, renal 1α-hydroxylase activity is low because there is no need for additional body Ca2+. However, when body Ca2+ levels are low, dietary calcium absorption is low and the plasma Ca2+ concentration decreases; the activity of this enzyme increases to produce the active metabolite 1,25(OH)2 vit D3, to ensure that additional Ca2+ will be absorbed from the gastrointestinal tract. The biological role of 1,25(OH)2 vit D3 in active intestinal Ca2+ absorption has been studied extensively for several decades. It was discovered many years ago that it is required for efficient intestinal Ca2+ absorption (131,132), and this observation subsequently has been confirmed by numerous additional studies. However, during the early postnatal period in pigs, calcium transport may not be regulated by 1,25(OH)2 vit D3 (133). The molecular mechanism of 1,25(OH)2 vit D3 regulation of intestinal calcium transport likely involves the direct interaction of 1,25(OH)2 vit D3 with the nuclear VDR (134). The genomic mechanism of action is similar to that of other steroid hormones and is mediated by stereospecific

interaction of 1,25(OH)2 vit D3 with the VDR, which heterodimerizes with the retinoid X receptor (RXR) (135). The heterodimerized receptor then binds to a VDRE in the promoter of target genes, and transcriptional initiation occurs via an interaction of the VDR with coactivators and the transcriptional machinery. VDR is mainly expressed in epithelia that a play a role in Ca2+ (re)absorption, confirming the importance of the genomic actions of 1,25(OH)2 vit D3 for body calcium homeostasis. In general, transcellular Ca2+ transport in the small intestine is facilitated by the Ca2+ transport proteins TRPV6, calbindin-D9K, and PMCA1b and is stimulated by 1,25(OH)2 vit D3, primarily by a genomic action. Previous studies on the action of 1,25(OH)2 vit D3 in Ca2+ transport have focused mainly on calbindins (109,136). However, more recent studies have sought to determine whether TRPV6 also is regulated by 1,25(OH)2 vit D3. This was first explored in a human intestinal cell line (Caco-2), which has been used previously for studies of Ca2+ transport (92), because they possess normal transcellular calcium transport. The induction of TRPV6 by 1,25(OH)2 vit D3 in Caco-2 cells was more robust than that of calbindin-D9K and PMCA1b, and it occurred several hours sooner. Regulation of TRPV6 by 1,25(OH)2 vit D3 was soon thereafter confirmed in a study using VDR null mice (84). Duodenal TRPV6 mRNA was reduced ~90% and intestinal calcium absorption was reduced threefold in two VDR knockout strains fed a normal Ca2+ diet. CalbindinD9K was decreased in only one strain of VDR null mice, and expression of the PCMA1b was normal in both strains (116). These data indicate that the decrease in TRPV6 expression is responsible for the decrease in Ca2+ absorption seen in these mice. Thus, these two studies demonstrate that out of the known calcium transport-related genes, TRPV6 is the most robustly regulated by vitamin D. Overall, these studies suggest that the TRPV6-mediated apical entry step is a rate-limiting step for vitamin D–regulated Ca2+ transport, rather than the intracellular diffusion step mediated by calbindin-D9K or the extrusion step mediated by PMCA1b. Another investigation showed that TRPV6 mRNA levels were not significantly correlated with vitamin D metabolite levels, but were moderately correlated with calbindin-D9K and more strongly correlated with PMCA1b in human duodenal biopsy samples from 20 healthy subjects (137). However, interpretation of the results of this study may have been compounded by the varying ages and uncontrolled Ca2+ intake of the subjects, a factor that was later found to have a significant impact on TRPV6 expression (84). These studies showed that TRPV6 expression was greatly reduced on a high-Ca2+ diet and greatly increased on a low-Ca2+ diet (84), and the extent of the reduction was independent of the VDR (138). In addition to the VDR knockout mice, 1α-hydroxylase (139,140) knockout mice have been developed, in which the vitamin D system also has been perturbed. These mice were created specifically to systematically dissect the genetic regulation of calcium transport genes and to determine the functional consequences on transcellular calcium transport. 1α-Hydroxylase null mice demonstrate distinct histologic

1964 / CHAPTER 77 evidence of rickets and osteomalacia. In these mice, there is a correlative relation among the expression of TRPV6, calbindin-D9K, and PCMA1b in the duodenum and the serum calcium concentration (83). Normalization of the plasma calcium concentration by 1,25(OH)2 vit D3 restored the expression level of the calcium transporters, confirming the essential role that these proteins play in active 1,25(OH)2 vit D3–mediated calcium absorption. The concerted regulation of TRPV6 and other calcium transport proteins guarantees sufficient capacity during high transport rates. Calbindin-D9K regulates the calcium influx across the apical membrane by buffering intracellular calcium, thus controlling the feedback inhibition of TRPV6 channel activity. Overall, when the existing data are considered in their entirety, it appears likely that 1,25(OH)2 vit D3 regulates expression of TRPV6, calbindin-D9K, and to a lesser extent, the basolateral extrusion system PMC1b. Regulation by Stanniocalcin In mammals, stanniocalcin is expressed in multiple organs including calcium-transporting epithelia, such as the intestine, colon, kidney, and placenta (141,142). It also is released by the corpuscles of Stannius, which are specialized organs adjacent to and scattered throughout the kidney (143). Stanniocalcin acts locally in the gut to modulate calcium and phosphate excretion, and overexpression in mice results in high serum phosphate, dwarfism, and increased metabolic rate (144,145). The main function of stanniocalcin, similar to that of calcitonin, appears to be the prevention of hypercalcemia. Furthermore, expression of stanniocalcin is induced by 1,25(OH)2 vit D3 (146,147). Because only a paucity of studies have addressed the expression or function of stanniocalcin in mammals, further work is necessary before strong conclusions can be drawn regarding the involvement of this hormone in intestinal calcium transport. Regulation by Estrogens The intestine is a potential site for estrogen action, and there is accumulating evidence that estrogen plays a physiologic role in the regulation of intestinal calcium absorption. Estrogen receptors are present in the duodenum and colon; however, the underlying mechanism by which estrogen may regulate calcium absorption remains poorly understood. In addition, it has not been established conclusively whether there is a direct effect of estrogen on intestinal calcium transport, or whether it indirectly mediates this process via an effect on vitamin D metabolism. However, findings suggest that TRPV6 expression is regulated by estrogen, because duodenal expression of TRPV6 mRNA of 1α-hydroxylase null mice and ovariectomized rats is increased by 17β-estradiol administration (83). Also, another study demonstrated that intestinal calcium transport was stimulated by 17β-estradiol, independently of circulating 1,25(OH)2 vit D3 levels (148). It also was reported that duodenal TRPV6 expression is reduced in estrogen receptor α knockout mice and enhanced

by estrogen treatment (149), and further that the estrogen effect on intestinal calcium absorption is mediated by an estrogen receptor (150). In this investigation, TRPV6 expression also was shown to be enhanced in pregnant VDR knockout mice and in wild-type littermates. Furthermore, in lactating mice, duodenal TRPV6 expression levels increased 13-fold. Thus, it is clear that the expression of TRPV6 epithelial calcium channels is influenced by estrogen status. Estrogens, hormonal changes during pregnancy, and lactation have distinct vitamin D–independent effects on active duodenal calcium absorption mechanisms, primarily via a major induction of TRPV6 mRNA expression. These estrogen effects appear to be mediated solely by estrogen receptor α. Interestingly, Weber and colleagues (151) described an estrogen-responsive element in the promoter sequence of the mouse TRPV6 gene; however, detailed promoter analysis is necessary to identify the regulatory sites involved in estrogen-mediated regulation of TRPV6. Despite these investigations, it remains to be demonstrated conclusively that these changes have functional implications on intestinal Ca2+ absorption. Overall, these data confirm that estrogen and 1,25(OH)2 vit D3 are independent, potent regulators of TRPV6 expression, which is involved in active intestinal calcium absorption. These data thus suggest that the function of estrogen in the maintenance of body calcium balance could be at least partially fulfilled by regulation of TRPV6 levels, thereby controlling intestinal calcium absorption to maintain normal serum Ca2+ levels, which are necessary for proper bone calcification (13). Regulation by Thyroid Hormone Several studies have suggested that thyroid dysfunction is associated with disturbances of calcium and phosphate homeostasis (152–156). In fact, thyrotoxicosis in humans and animals results in hypercalcemia (157,158). Long-term hyperthyroidism is associated with calcium malabsorption and increased rates of bone resorption. Several effects of thyroid hormone on the intestine resemble those of 1,25(OH)2 vit D3, although only recently have studies been performed to address the function of thyroid hormones in calcium homeostasis. One study demonstrated that calcium transport into brush-border membrane vesicles and calcium efflux across basolateral membranes of enterocytes were significantly increased in hyperthyroid rats and decreased in hypothyroid animals (155). These authors of this investigation suggested that thyroid hormones increase the affinity of TRPV6 for calcium in the brush-border membrane of intestinal epithelial cells; however, this hypothesis remains largely unproven. Regulation by Dietary Calcium Dietary calcium has been the focus of multiple studies in an effort to discover its preventive properties. It has been implicated in a decreased risk for osteoporosis, whereas low

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1965 dietary calcium intake helps avoid kidney stone formation. Obesity, hypertension, and even cancer are less publicized areas in which greater dietary calcium intake has a positive outcome (159). A significant link between calcium intake and bone mass has been reported. As a result of these cumulative observations, the recommended daily intake of calcium has been increased to 1000 mg/day in healthy adults (see Table 77-1). The importance of adequate calcium intake has been illustrated best by studies using the VDR and the 1α-hydroxylase knockout mice. The bone phenotype of VDR-null mice can be completely rescued by feeding the animals a highcalcium, high-phosphorus, high-lactose diet. In addition, the abnormal phenotype of 1α-hydroxylase knockout mice can be corrected by feeding them a high-Ca2+ diet (160). The expression of calcium transport proteins subsequently was studied in these knockout models, to be able to distinguish the effects of hypocalcemia from those of vitamin D deficiency. Thus, studies initially were performed in 1α-hydroxylase– ablated mice fed normal versus high-Ca2+ rescue diets (161). It is known that under normal circumstances, increased plasma calcium levels act via a negative feedback loop to stimulate PTH secretion, which eventually suppresses 1α-hydroxylase activity, which, in turn, decreases renal 1,25(OH)2 vit D3 production and calcium transport in the intestine. This appears to be mediated by the expression of the intestinal calcium transport proteins TRPV6, calbindinD9K, and PMCA1b, which was normalized by feeding the knockout mice the high-calcium rescue diet (83). Comparable observations were made in VDR knockout mice for which duodenal TRPV6 mRNA levels were induced by dietary calcium (84). Studies with both of these animal models showed that calcium supplementation can increase transcriptional initiation of genes encoding calcium transporter proteins in the absence of circulating 1,25(OH)2 vit D3, but the molecular mechanism of this vitamin D–independent, calcium-sensitive pathway remains elusive. It is, however, likely that in addition to the 1,25(OH)2 vit D3 response elements in the promoter regions of the TRVP6 and the calbindin-D9K genes, calcium-responsive cis-acting elements also are present. Several promoter elements have been proposed to function as calcium-sensitive transcriptional modulators, including the serum-responsive element in the cAMP/calcium-responsive element (162). However, detailed promoter studies are necessary to fully characterize any putative calcium-responsive elements in the genes responsible for transepithelial calcium transport in the small intestine. Regulation by Parathyroid Hormone and the Calcium-Sensing Receptor The parathyroid glands play a critical role in maintaining extracellular calcium concentrations, via their ability to sense minute changes in blood calcium levels. The calciumsensing receptor (CaSR) in the parathyroid chief cells is able to sense circulating calcium levels and subsequently to induce release of PTH when physiologically necessary.

In response to low blood calcium levels, PTH is secreted into the circulation, which then activates the PTH/parathyroid hormone–related peptide (PTHrP) receptor predominantly in the kidney and bone. This receptor, once activated by interaction with PTH, stimulates the activity of renal 1α-hydroxylase, and thereby indirectly increases 1,25(OH)2 vit D3–dependent intestinal calcium absorption. Furthermore, immunohistochemical analysis of rat duodenal sections demonstrated localization of the PTH/PTHrP receptor in epithelial cells along the villus, with intense staining of brush-border and basolateral membranes and the cytoplasm (163). Direct effects of PTH on calcium transport by isolated rat duodenal enterocytes also have been reported. Initial studies using perfusion of isolated duodenal loops showed that PTH administration increased calcium absorption (164,165). These findings were later confirmed in studies demonstrating that PTH significantly stimulates enterocyte calcium uptake (166). However, despite these investigations, molecular mechanisms of direct PTH action in mediating intestinal calcium transport remain poorly understood. The CaSR thus plays a crucial role in the regulation of calcium homeostasis by sensing minute changes in circulating calcium levels (167). In the parathyroid gland, the CaSR allows parathyroid cells to detect changes in blood ionized calcium concentration, to modulate PTH secretion accordingly, and thus to maintain serum calcium levels within a narrow physiologic range. CaSR forms a unique molecular target for drugs that can directly alter the activity of the receptor, and thereby modulate extracellular calcium balance. Several compounds, including NPS R-467, have been identified that can activate the CaSR and suppress serum PTH and Ca2+ levels. However, the involvement of intestinal calcium transport in the NPS R-467–mediated hypocalcemia is unclear. Thus, an investigation sought to determine the effect of NPS R-467 on the expression of intestinal calcium transport proteins (13). These investigators infused mice with NPS R-467 continuously for 7 days and noted reduced serum PTH levels, which were accompanied by a significant decrease in serum calcium concentration. Molecular analysis of duodenal samples demonstrated down-regulation of mRNA and protein expression levels of proteins involved in active transcellular calcium absorption. It is currently unknown, however, whether the effects of NPS R-467 could involve a direct action of CaSRs in the intestine, or whether the acute hypocalcemic response to this and other similar compounds results from the inhibition of PTH secretion (13). Regulation of Intestinal Calcium Transport by Interacting Proteins Currently, little is known about other molecular players responsible for regulating the activity of TRPV6. A number of regulatory proteins have been described that modify the activity and the biophysical and pharmacologic properties of ion channels and transporters by direct, physical interactions (168).

1966 / CHAPTER 77 These associating proteins have facilitated the elucidation of molecular pathways involved in modulating transport/channel activity. Currently, however, only CaM and S100A10annexin 2 have been described to associate with TRPV6 (87,93), and these interactions likely have implications for the regulation of intestinal Ca2+ absorption. Regulation by Calmodulin CaM is a ubiquitous cytosolic protein known to regulate the activity of various ion channels, calcium pumps, and other proteins in a calcium-dependent manner. There is a rapid calcium-dependent inactivation of the TRPV6 channels that appears to be independent of CaM binding and could be caused by calcium interaction with the intracellular pore-forming region (93). Furthermore, a slower inactivation was observed that was determined to be Ca2+/CaM dependent. Although CaM does not bind to TRPV6 at normal, resting, intracellular calcium concentrations, binding did occur at greater calcium concentrations, with maximal binding occurring at calcium concentrations of 60 µM. Interestingly, binding of Ca2+/CaM can be prevented by PKC-mediated phosphorylation of a threonine residue within the binding site of intestinal TRPV6 channels. A study thus concluded that PKC activity and subsequent phosphorylation of the channel may act to regulate the amount of calcium influx through TRPV channels by altering their inactivation behavior (93). These authors suggested a model in which TRPV6-expressing cells have substantial calcium influx, with phosphorylation of TRPV6 acting as a positive feedback mechanism, which delays the inactivation process. They further suggested that this proposed mechanism of competitive regulation of TRPV6 by PKC and CaM is restricted to human TRPV6, because this particular PKC site in the CaM-binding motif is not conserved in other species. Additional studies have demonstrated that the corresponding region in mouse TRPV6 can also bind to CaM (169). Molecular analysis of the binding region predicted a casein kinase motif, but no significant phosphorylation was detected. Furthermore, the regulation of TRPV6 by CaM was confirmed by another group of investigators (described by Hoenderop and colleagues [13]). Regulation of Transient Receptor Potential Vanilloid Receptor 6 by S100A10-Annexin 2 An auxiliary protein of TRPV6 was identified by screening a mouse kidney cDNA library using the yeast two-hybrid system (87). This study described the first auxiliary protein that is known to interact with TRPV6; this protein was named S100A10, and it associates specifically with the carboxy terminus of the TRPV6 epithelial calcium channel. S100A10 is a 97-amino acid protein and is a member of the S100 superfamily that is present in vertebrates, insects, nematodes, and plants. This protein is present predominately as a heterotetrameric complex with annexin 2, which has been implicated in numerous biological processes including

endocytosis, exocytosis, and membrane-cytoskeletal interactions (170). A report suggested a regulatory role for the S100A10-annexin 2 heterotetramer in vitamin D–mediated, intestinal calcium transport and in TRPV6 function, regulation, or both (87). The association of S100A10 with TRPV6 was restricted to a short peptide sequence NH2-VATTVCOOH located in the carboxy terminus of the channel, a region that is conserved across species. Intriguingly, the NH2-TTV-COOH sequence in the putative S100A10-binding motif of TRPV6 resembles an internal, type I PDZ consensus binding sequence (NH2-S/TXV-COOH). However, S100A10 does not contain PDZ domains, suggesting that the interaction with TRPV6 is distinct. The first threonine of the S100A10 interaction motif is crucial for binding to TRPV6. In fact, the activity of TRPV6 was abolished when this particular threonine was mutated, demonstrating the necessity for calcium channel function. Furthermore, these mutant channels were mislocalized within cells, indicating that the S100A10-annexin 2 heterotetramer facilitates the translocation of TRPV6 channels to the plasma membrane. The importance of annexin 2 in the process was demonstrated by a small interfering RNA–mediated knock down of annexin 2, which significantly inhibited the currents through TRPV6, exemplifying that annexin 2 in conjunction with S100A10 is necessary for normal TRPV6 activity. Interestingly, similar to the epithelial calcium channels, S100A10 expression was found to be vitamin D sensitive (87). In addition, annexin 2 expression levels have been shown to increase with 1,25(OH)2 vit D3 treatment (171). Thus, physical interaction and coregulation of TRPV6 with S100A10 and annexin 2 could control of trafficking of these channels to the plasma membrane. 1,25(OH)2 vit D3 has been shown to act via rapid nongenomic and slower genomic actions (134). The genomic effects are mediated by interaction with nuclear VDR/RXR heterodimers. It was reported that annexin 2 serves as a membrane receptor for 1,25(OH)2 vit D3, and that it mediates a rapid effect of the hormone on intracellular calcium homeostasis. It was shown that that 1,25(OH)2 vit D3 bound specifically to annexin 2 on the plasma membrane of rat osteoblast-like cells (172,173). Partially purified plasma membrane proteins and purified annexin 2 exhibited specific, saturable binding for tritiated 1,25(OH)2 vit D3. These results suggest that annexin 2 may serve as a receptor for rapid actions of 1,25(OH)2 vit D3; however, this concept remains under debate (174). Taken together, these findings show that the S100A10-annexin 2 complex is required for the trafficking of TRPV6 to the plasma membrane, and therefore is involved in overall calcium homeostasis. Regulation of Intestinal Calcium Transport by Growth Hormone A report demonstrated that intestinal calcium and phosphate transport is positively regulated in pigs by exogenous administration of porcine growth hormone (175). These investigators treated growing pigs with daily injections of

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1967 growth hormone for 2 months, and then performed a 10-day balance study at the end of this period. They found that all aspects of mineral homeostasis were stimulated, including circulating 1,25(OH)2 vit D3 levels (increased by 40%), Ca2+ absorption and retention (increased 70%), inorganic phosphate (Pi) absorption (increased 33%), and retention (increased 45%) and jejunal calbindin-D9k levels (increased 40%). It is unknown, however, whether these physiologic changes noted with exogenous growth hormone treatment are direct effects, or whether they are mediated principally by effects on vitamin D homeostasis. Transient Receptor Potential Vanilloid Receptor Knockout Mouse Models A TRPV5 knockout mouse model has been developed (73). In these mice, calcium hyperabsorption by the small intestine occurs as compensation for renal calcium wasting (because of a lack of renal TRPV5 expression). Calcium absorption was assessed in preliminary experiments in these knockout mice by measuring serum calcium levels after oral administration of calcium. A significant increase in the rate of calcium absorption was observed in TRPV5 knockout mice compared with wild-type littermates, demonstrating a compensatory role of the small intestine. TRPV6 and calbindin-D9K expression levels also were increased significantly in TRPV5 knockout mice, which is consistent with the increase in intestinal calcium absorption. Overall, these data indicate that the TRPV6 isoform is predominantly responsible for intestinal absorption of calcium. Unfortunately, TRPV6 knockout mice currently have not been reported in the scientific literature.

INTESTINAL TRANSPORT OF PHOSPHATE Phosphorous is not only a major component of the skeleton, but it is also a mediator of energy transfer, and it participates in a wide variety of metabolic reactions in the cell. Phosphorus is present in soft tissues as a soluble phosphate ion and is present in lipids, proteins, carbohydrates, and nucleic acids. Moreover, energy from a variety of metabolic processes derives primarily from the phosphate bonds of creatine phosphate and adenosine triphosphate (ATP). Because of the critical role of phosphorous as Pi and phosphate esters in cell physiology, humans have developed extensive mechanisms for extracting phosphate from the diet and for the conservation of phosphate absorbed by the intestine. Consequently, plasma phosphate concentrations in the extracellular fluid are maintained within a relatively narrow range. Although the control of plasma phosphate levels are not as intricately controlled as that of calcium, body phosphate concentrations also are regulated by the interplay of a variety of homeostatic mechanisms. The intestine, which is involved in the extraction of Pi from dietary sources, plays a principal role in maintaining body phosphate levels. The amount of dietary phosphate absorbed is approximately

equal to the amount of phosphate excreted by the kidney (Fig. 77-3).

Mechanisms and Sites of Phosphate Absorption Intestinal absorption of phosphate in mammals occurs primarily in the small intestine, with relative absorptive efficiency in duodenum being greater than jejunum, which is greater still than ileum (176,177). However, phosphate absorption occurs primarily in the jejunum (178), because of its length and the longer transit time of the digesta through this gut segment. Phosphate transport across intestinal brush-border and basolateral membranes occurs by both a sodium-independent, nonsaturable process and an active, sodium-dependent process, with each membrane exhibiting significant differences in affinities and total transport capacity of the sodium-dependent transport process (178,179). Intestinal perfusion of the human jejunum demonstrates that active transport of phosphate is maximal at low phosphate intake levels; conversely, nonsaturable, passive diffusion occurs with high phosphate concentrations (176). Because of the negative charges in paracellular intestinal phosphate transport channels, simple diffusion across the intestinal mucosa at low phosphate concentrations via this route is unlikely. The highly electronegative interior of the cell (~50–70 mV) results in a transmembrane potential difference between the interior of the cell and the intestinal lumen (180). This potential difference functions as a powerful barrier to the transport of negatively charged H2PO4− or H2PO42− ions across the brush-border membrane. Whereas H2PO−4 ions, which can accumulate to high concentrations at acidic pH in proximal intestinal segments, are more readily transported against this electronegative gradient, H2PO42− (the ionic species that predominates at the alkaline pH of the jejunum and ileum) requires an active, energy-dependent process to ensure adequate absorption (181). 1,25(OH)2 vit D3 stimulates active phosphate transport across both brush-border and basolateral membranes in the small intestine (182); we have only recently begun to understand the molecular mechanisms involved (see later discussion). Furthermore, because the potential difference across the basolateral membrane of intestinal epithelial cells does not inhibit the transport of negatively charged, ionic phosphate molecules out of the cell into the interstitial space, and because the Pi concentrations in the cell and plasma are ~2 mM and ~1.0 mM respectively, efflux of phosphate across the basolateral membrane likely occurs passively down an electrochemical gradient. The exact mechanism(s) that control transepithelial movement of Pi largely remain uncharacterized, because the overall regulation of this process is complicated by the fact that Pi becomes rapidly integrated into various metabolic processes once it enters enterocytes. As such, the rate and control of phosphate absorption is not only a function of the brush-border and basolateral membrane transport processes, but it is also dependent on the rate of utilization and esterification of intracellular phosphate.

1968 / CHAPTER 77

FIG. 77-3. Body phosphate homeostasis. The main organs involved in body inorganic phosphate (Pi) homeostasis are shown. Under conditions of high dietary intake (which is common because Pi is present in many food products), phosphate is absorbed along the entire length of the mammalian small intestine, by a passive paracellular process. Most Pi absorption occurs in the jejunum. Pi also is endogenously secreted into the intestinal lumen, and some of this secreted Pi may be reabsorbed. Under low intake conditions, Pi is actively transported, predominantly in the proximal small intestine. Phosphate moves in and out of bone as bone is deposited and resorbed. Phosphate (Pi) is lost in the stools, as well as excreted by the kidney. Hormonal regulation of body Pi homeostasis is similar to regulation of blood calcium levels in that it involves parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25 (OH)2 vit D3). Hypophosphatemia induces PTH secretion from the parathyroid gland, as well as increased 1α-hydroxylase activity in the kidney. This leads to increased production of 1,25(OH)2 vit D3, which increases intestinal Pi absorption and renal Pi reabsorption.

Molecular Mechanisms of Intestinal Phosphate Transport Intestinal transport of dietary phosphate is dependent on the activity of the type IIb sodium-dependent phosphate cotransporter (NaPi-IIb) (Fig. 77-4). This protein is a member of the SLC34 family of solute carriers (reviewed by Murer and colleagues [183]), which is composed of three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). NaPi-IIa is expressed in apical membranes of epithelial cells in the renal proximal tubules, in osteoclasts, and also in neurons (183,184), whereas NaPi-IIc expression is restricted to the renal epithelium (185). NaPi-IIb (SLC34A2) originally was identified based on expressed sentence tag (EST) clones derived from lung tissue, and it is highly expressed in lung, small intestine, and many other tissues (186–188). By immunofluorescence, NaPi-IIb was localized to brush-border membranes of enterocytes (187,189). On Western blots, fully glycosylated NaPi-IIb is seen as a band of approximately 110 kDa in mice (187,190); however, smaller bands at 75 to 80 kDa have been reported in rat brush-border membrane protein samples (189). Interestingly, in weanling animals, NaPi-IIb is reported to be only partially glycosylated (190). Thus, the NaPi-IIb cotransporter is strongly expressed

in the small-intestinal epithelium and is thought to be the major active transport system for dietary phosphate. It is currently unknown precisely how phosphate is transported across the basolateral membrane of enterocytes, but it is thought to be a facilitated, diffusion-mediated event. Enterocytes also express a type III NaPi (NaPi-III) cotransporter on the basolateral domain that is involved in phosphate absorption from the circulation during times of fasting (191–193). Initial cloning and characterization of the mouse NaPi-III gene promoter also has been reported (194). The NaPi-III cotransporter is thus most likely important for enterocyte phosphate homeostasis and does not contribute to overall intestinal Pi absorption (see Fig. 77-4). Approximately 0.1% of the total body phosphate is present in the interstitial space. It is essential that the extracellular phosphate concentration is held constant at about 1.1 mM to ensure proper cellular functions such as DNA replication and mRNA transcription, as well as signaling reactions and bone formation. Members of the SLC34 family are expressed in the epithelia of small intestine and renal proximal tubules, two important physiologic sites that regulate extracellular phosphate concentration. In renal proximal tubules and enterocytes, type 2 sodium-phosphate cotransporters are located in the apical membrane and represent the

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1969 3Na+ K+

2K+ ATP

ADP 140 mM [Na+] 5 mM [K+]

3Na+ NaPi-IIb PO42−

Cytosplic pool 1-2 mM [Pi]

>1Na+

NaPi-III (Pit-1, PiT-2)

Pi

1,25 (OH2) vit D3

0.5–1 mM [Pi] Nucleus

Pi ?

ATP production metabolism

Pi Lumen

Pi

Nucleus

Interstitial space

FIG. 77-4. Transepithelial phosphate transport. Two adjacent enterocytes in the proximal small intestine, with the transport proteins that play a role in dietary inorganic phosphate (Pi) absorption are shown. Under high-intake conditions, Pi can be absorbed by a paracellular pathway through tight junctions between adjacent epithelial cells. However, under low dietary Pi intake conditions, Pi is transported together with sodium across the apical membrane by the type IIb sodium-phosphate (NaPi-IIb) cotransporter. The membrane electric potential (indicated by plus and minus signs), which is maintained by potassium leak channels (shown on the apical membrane), inhibits Pi movement into the cell. The energy for transport thus is derived from the electrochemical sodium gradient established across the brush-border membrane by Na+,K+-ATPase, which is located on the basolateral membrane. Pi in the cell may be used for ATP production in mitochondria, or it can be used for a host of metabolic reactions. Some Pi will be transported across the basolateral membrane, likely by facilitated diffusion via an unknown Pi transporter. Type III NaPi cotransporters (PiT-1 and PiT-2) also are found in basolateral membranes of enterocytes, and their likely function is to import Pi during times of dietary insufficiency for cellular Pi homeostasis. ADP, adenosine diphosphate; ATP, adenosine triphosphate.

rate-limiting steps of transepithelial phosphate transport (183). In both tissues, the abundance of type two NaPi cotransporters is controlled by many physiologic effectors. In a normal physiologic environment, all SLC34 family members exclusively transport phosphate ions in an obligatory, sodium-dependent manner. Furthermore, currently, few inhibitors are known for this family of cotransporters. Phosphonoformic acid (PFA; forscanet) at high concentrations (millimolar range) inhibits all members. In addition, PFA administration in vivo was shown to blunt the adaptive response to dietary Pi deprivation in rats, via a reduction in the number of NaPi cotransporters (195). Furthermore, a phosphorylated phloretin derivative (2′-phosphophloretin) was shown to decrease NaPi cotransport in isolated brush-border membrane vesicles from rat and rabbit, and oral gavage of this compound effectively decreased serum Pi levels after 1 week of treatment (~45%) (196). This group went on to demonstrate that 2′-phosphophloretin is a specific inhibitor of human intestinal brush-border membrane NaPi cotransport (197). Sodium-diphosphate cotransport by NaPi-IIb is electrogenic with a likely stoichiometry of three sodiums to one phosphate. The NaPi-IIb isoform has a Km for phosphate of less than 50 µM and a Km for sodium of ~40 mM. pH

dependence is moderate, with a slightly greater cotransport rate at more acidic pH. The human NaPi-IIb isoform also has been expressed and functionally characterized in hamster fibroblasts and Xenopus oocytes (198). In fibroblasts, the Km for Pi was 106 µM and the Km for sodium was ~34 mM, whereas when expressed in oocytes, the Km for Pi was ~113 µM and the Km for sodium was ~65 mM. Transport in both model systems was inhibited by high external pH, PFA, arsenate, and 100 nM phorbol myristate acetate (198). Based on results obtained from hydropathic analysis and structure-function studies (199,200), a model for the secondary topology of SLC34 family cotransporters has been proposed, with many similarities predicted among the three family members. Each NaPi-II cotransporter protein likely spans the plasma membrane eight times (Fig. 77-5). Both N and C termini are located intracellularly, and there are multiple putative N-glycosylation sites in a large extracellular loop. Furthermore, two short (one extracellular and one intracellular) loops have been suggested to be part of the functional pore region of the transport pathway. This described model is based on experimental data obtained with the NaPi-IIa isoform, but it also appears to be valid for the other family members as well, because all three

1970 / CHAPTER 77

L-2 L-1

L-4

L-3

Lumen 1

2

3

4

5

6

7

NaPi-IIb (proposed)

Cytoplasm

8 TM Domains

L-1 L-2

NH2−

L-3 −COOH

FIG. 77-5. Model of type IIb sodium-phosphate (NaPi-IIb) protein structure. The NaPI-IIb protein has eight putative transmembrane (TM) domains (numbered 1–8) and several intracellular and extracellular loops. Extracellular loop number 2 has a putative disulfide bridge (dashed line) and potential glycosylation sites (hexagons). Little experimental work to precisely define NaPi-IIb protein structure currently has been performed; however, homology to the renal isoform NaPi-IIa is high, suggesting conservation of key features. The N and C termini are likely intracellular.

members share more than 80% amino-acid sequence identity in the transmembrane segments, and they exhibit the same hydropathic profile (183) (see Fig. 77-5). The greatest dissimilarities are found in the N- and C-terminal regions and in the large extracellular loops. Based on results obtained with tandem NaPi-IIa constructs, it is assumed that the monomeric form is sufficient for full sodium-phosphate cotransport activity (201), and one could surmise that the same would apply to the intestinal isoform, NaPi-IIb.

Type IIb Sodium-Dependent Phosphate Cotransporter (SLC34A2) and Intestinal Phosphate Transport Several observations provided evidence that the NaPi-IIb is involved in transcellular flux of phosphate in the small intestine (187,190,202–204). Therefore, small-intestinal NaPi-IIb could represent a possible target for treatment of hyperphosphatemia, which often is observed in patients receiving renal dialysis. The most prominent regulators of NaPi-IIb in the small intestine are 1,25(OH)2 vit D3 and lowphosphate diet. Up-regulation of NaPi-IIb induced by these factors appears to be nontranscriptional in adult rodents, whereas transcription may be involved in younger animals (204). Radanovic and colleagues (205) demonstrated that a

low-phosphate diet affects transcription or stability, or both, of NaPi-IIb mRNA throughout the length of the mouse intestine. These authors further concluded that the active, transcellular Pi uptake pathway was important only in the ileum, whereas in the jejunum and duodenum, other mechanisms were involved. Another investigation, using VDR and 1αhydroxylase knockout mice, concluded that the regulation of intestinal NaPi cotransport mediated via NaPi-IIb by lowphosphate diet cannot be explained by the 1,25(OH)2 vit D3/VDR axis (206). Interestingly, low-phosphate diet has been shown to stimulate transcription of the renal NaPi-IIa isoform via low-phosphate response elements in the gene promoter (207), but no such regulatory sites have been reported in the NaPi-IIb gene promoter (which has been cloned from mice [208] and humans [209]). Furthermore, regulation of NaPi-IIb in the small intestine by epidermal growth factor (210,211), glucocorticoids (190), estrogen (189,210), thyroid hormone (152,212), stanniocalcin (213), and metabolic acidosis (214) has been reported. Another report demonstrated that expression of the intestinal NaPi-IIb cotransporter was induced by serum- and glucocorticoiddependent kinase 1, an effect that was mediated, at least in part, by phosphorylation of Nedd4-2 (215). These authors concluded that their results demonstrated a novel signaling pathway in the regulation of intestinal phosphate transport, which may be important for the regulation of Pi balance.

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1971 Regulation of Phosphate Absorption Although the precise requirement for phosphorus is unknown, a dietary intake of ~700 mg/day is required to maintain positive phosphate balance in adults (see Table 77-1). During pregnancy and lactation, this requirement is increased significantly before age 18 years, whereas later in adulthood, the recommended dietary requirement remains the same. Phosphate is relatively abundant in most foods, thus nutritional phosphate deficiency is uncommon. However, Pi deficiency does occur commonly in preterm neonates (216,217). Approximately 80% of dietary phosphorous is derived from milk products, grains, and meats, with dairy products constituting a major source of dietary phosphate. In human milk 1 day after birth, the concentration (mean ± standard error of the mean [SEM]) of phosphate is 0.26 ± 0.16 mM, whereas 4 days later, it has increased 6.6-fold to 1.69 ± 0.11 mM (218). Thus, maternal milk supplies adequate Pi to the rapidly growing infant. Furthermore, U.S. diets are replete with food additives contributing a considerable amount of phosphate to the diet, with these additives contributing as much as 30% of the phosphorous of an average adult diet. Approximately 50% to 70% of dietary phosphate is absorbed from normal diets, and as much as 90% is absorbed when intake is low, with net absorption being essentially proportional to intake. After a phosphate-rich meal, absorption occurs predominately by passive diffusion across the epithelium of the proximal duodenal. These observations suggest that two transport mechanisms may exist, with one likely being passive and paracellular, and the other being active and transcellular (occurring with low Pi intake levels). Furthermore, under normal conditions, the urinary excretion of phosphate is comparable with the amount absorbed. With reduced phosphate intake, intestinal absorption by active transport mechanisms is induced primarily by 1,25(OH)2 vit D3 (182,219,220). However, some evidence suggests that in the suckling and weanling periods, intestinal Pi transport is not 1,25(OH)2 vit D3–responsive (221). Urinary excretion of phosphate approaches zero during phosphorus deprivation, as the tubular reabsorption approaches 100% of the filtered load. Thus, both the intestine and the kidneys are able to respond to restriction in dietary phosphate by enhancing intestinal absorption and renal conservation. Intestinal secretion of phosphate, which normally averages 3 mg/kg body weight per day, also is reduced during dietary deprivation. However, this adaptation occurs slowly; thus, a transient net phosphate deficiency may occur. During fasting, the concentration of phosphate in the small-intestinal lumen decreases to ~2 mM/L. Sources of endogenous Pi include saliva (4.0 mM/L) and gastric, pancreatic, and intestinal secretions (1.0 mM/L). Rapid turnover of enterocytes delivers ~250 mg cellular debris into the intestinal lumen each day, which contributes to the endogenous Pi pool. Part of this endogenous pool is reabsorbed by the intestine, whereas the remainder (3–4 mM or 90–120 mg/day) is excreted in the feces. Intestinal Pi absorption is regulated by a number of physiologic factors, such as low-Pi diet and 1,25(OH)2 vit D3,

which increase intestinal Pi absorption (182,219,222). Several pathologic states also have been associated with perturbations in intestinal Pi homeostasis. Metabolic acidosis has been reported to decrease jejunal phosphate transport in weanling rats (223). Similarly, phosphate absorption is decreased in spontaneously hypertensive rats, compared with littermate control rats, and this is thought to contribute to the hypophosphatemia observed in these animals (220). Furthermore, streptozotocin-induced diabetes led to increased Pi absorption from brush-border membrane vesicles isolated from the rat jejunum, and an increased Vmax was demonstrated, but no change in Km was described (224). Another investigation reported that intestinal Pi transport was increased by iron deficiency (225).

INTESTINAL TRANSPORT OF MAGNESIUM Magnesium is the fourth most abundant cationic element in the body, surpassed only by sodium, potassium, and calcium, and it is the most abundant intracellular divalent cation. Intracellular Mg2+ concentrations of 0.3 to 1.0 mM are at least 1000 times greater than those of calcium. A 70-kg adult has 25 to 28 g magnesium (1665–2400 mEq) with 60% to 65% of the total present in bone. As an essential cellular cation, magnesium participates as a cofactor for a variety of enzymatic reactions, including the transfer of phosphate groups from high-energy nucleotide triphosphates. Magnesium also is required for the maintenance of membrane electric potentials, for transmission of the genetic code via its action on the DNA/RNA structure and function, for neurotransmission, and for muscular contraction. Magnesium also is considered essential in maintaining calcium and potassium homeostasis (226). The kidney (227) and the rate of extent of magnesium redistribution into extravascular and tissue compartments (228) usually are considered pivotal for the regulation of circulating magnesium levels within the narrow range of 0.65 to 1.1 mM (229). Interestingly, blood magnesium often is low in patients with gastrointestinal diseases (230–232). These observations, coupled with other demonstrated hypomagnesemia in infants with genetic defects in magnesium absorption (233), suggest that the gastrointestinal tract also plays an essential homeostatic role, which controls circulating magnesium levels.

Sites of Magnesium Absorption Magnesium is absorbed primarily in the distal segments of the gastrointestinal tract in humans and animals. Whereas the colon is a major site of magnesium absorption in the rat (234–236), magnesium is absorbed primarily in the ileum and jejunum in humans (237,238). Magnesium deficiency has been documented in patients after jejunoileal bypass surgery for obesity (239). Unlike the proximal jejunum where absorption increases linearly with progressive increments in magnesium concentration, magnesium absorptive processes in the ileum were fully saturated at concentrations

1972 / CHAPTER 77 greater than 10 mM (237). Observations documenting increases of blood magnesium after magnesium-containing Epsom salt enema are consistent with the hypothesis that magnesium also is absorbed from the colon in humans. Despite many studies over the past several decades, the precise molecular mechanisms that regulate the intestinal absorption of magnesium remained poorly defined until only the past few years (see later). That calcium and magnesium are absorbed by different mechanisms is exemplified by that patients with absorptive hypercalciuria with increased calcium transport have normal magnesium absorption (237). Passive diffusion and active transport have been implicated in Mg2+ transport, with active transport predominating primarily in fasting, magnesium-deficient states (238). Because magnesium movement across the intestine also is associated with an increase of water flux across intestinal cells, a critical component in the passive diffusion process may be solvent drag (235). At physiologic concentrations, the effect of 1,25(OH)2 vit D3 on magnesium absorption in animals and humans is minimal. Not only has significant magnesium absorption been noted in individuals with no detectable levels of 1,25(OH)2 vit D3 (240), but there is also no discernible correlation between circulating levels of 1,25(OH)2 vit D3 and magnesium absorption (240,241). Because complex interactions have been described among magnesium, phosphate, and calcium absorption (236,242), it appears reasonable to assume that the reported effects of 1,25(OH)2 vit D3 on magnesium absorption in humans could result indirectly from associated changes in calcium or phosphate transport, or both.

Magnesium Physiology Since the previous edition of this textbook was published in 1994, several discoveries have provided new molecular information regarding mechanisms of intestinal magnesium transport (as reviewed by Konrad and colleagues [243]). Magnesium is stored predominately in bone in the intracellular compartments of muscle and soft tissues, with less than 1% of total body magnesium found circulating in the blood (244). In healthy subjects, serum magnesium levels are kept in a narrow range (0.65–1.1 mmol/L). Magnesium homeostasis depends on the balance between intestinal absorption and renal excretion. Within normal physiologic ranges, diminished magnesium intake is balanced by enhanced magnesium absorption in the intestine and reduced renal excretion. These transport processes are regulated by several metabolic and hormonal effectors (243,245). The principal site of magnesium absorption is the distal small intestine, with smaller amounts being absorbed in the colon. Intestinal magnesium absorption occurs via two different pathways: a saturable, active transcellular transport process and a nonsaturable, paracellular passive transport mechanism (246). Saturation kinetics of the transcellular transport system is explained by the limited transport capacity

of active transport. At low intraluminal concentrations, magnesium is absorbed primarily via the transcellular route, and with increasing concentrations, via the paracellular pathway, yielding a curvilinear function for total absorption. Human intestinal epithelial-like (Caco-2) cells have been described as an in vitro model of intestinal magnesium transport (247). The evidence for the magnesium transport pathways described earlier mainly evolved from physiologic studies. During recent years, the analysis of disease phenotypes characterized by disturbances in magnesium handling turned out to be helpful for a more thorough understanding of magnesium homeostasis (248,249). The most recent example of a genetic approach yielding a new molecule involved in intestinal magnesium transport is the characterization of a gene mutation in primary hypomagnesemia with secondary hypocalcemia (HSH), which allowed the identification of the first component of intestinal magnesium absorption (250,251). HSH is an autosomal recessive disorder that manifests in early infancy with generalized convulsions or other symptoms of increased neuromuscular excitability, such as muscle spasms or tetany. It was first identified by Paunier and colleagues in 1968 (233). Failure of early diagnosis or noncompliance with treatment can be fatal or result in permanent neurologic damage. Laboratory evaluation shows extremely low serum magnesium and low serum calcium levels in patients with HSH. The mechanism leading to hypocalcemia remains incompletely understood. Several physiologic factors appear to contribute to an impairment of PTH action (252). The hypocalcemia present in HSH is resistant to treatment with calcium or 1,25(OH)2 vit D3. Correction of clinical symptoms, normal calcemia, and normalization of PTH levels can only be achieved by administration of high doses of magnesium. Furthermore, transport studies in patients with HSH pointed to a primary defect in intestinal magnesium absorption (253), with a possible renal defect also possible (254). A gene locus (HOMG1) for HSH has previously been mapped to chromosome 9q22 (255) and later refined to a critical interval of ~1 cM. Two independent groups have identified the TRPM6 gene in this critical interval and reported presumable loss of functions mutations (mainly truncating mutations) in this gene as the underlying cause of HSH (250,251). TRPM6 encodes a newly described member of the TRP family of cation channels. TRPM6 protein shows greatest homology to another family member TRPM7, which has been characterized as a calcium- and magnesiumpermeable ion channel regulated by Mg-ATP (256). By reverse transcriptase-PCR and in situ hybridization, TRPM6 expression could be demonstrated along the entire small intestine and colon, and also in the distal tubule cells in the renal epithelium. Immunofluorescence studies with an antibody generated against murine TRPM6 localized the protein to the apical membrane of the distal convoluted tubules (257). In patients with HSH, high oral doses of magnesium achieve some normalization of serum magnesium levels,

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1973 supporting the theory of two independent intestinal transport systems for magnesium. TRPM6 most likely represents a component of the active transcellular magnesium transport system. Increased intraluminal magnesium concentrations (by increased oral intake) enable compensation for the defect in this active transcellular transport pathway by increasing absorption via the passive paracellular pathway (Fig. 77-6).

Transient Receptor Potential Family of Cation Channels The TRP family of proteins comprises more than 20 members with diverse functions (77). The original trp locus was identified in Drosophila, and its mutation resulted in vision problems, caused by a defect in a calcium channel encoded by this gene (258). This discovery led to the identification of this diverse family of proteins in mammals. TRP proteins have been divided into six subfamilies, and the TRPM subfamily consists of eight members (TRPM1-8). TRP proteins form functional tetrameric structures, with heterotetrameric assembly seemingly possible (243). Interestingly, TRPM2, TRPM6, and TRPM7 are set apart from other known ion channels, because they contain enzyme domains in the C-terminal region, and thus represent prototypes of an intriguing new protein family of enzyme-coupled ion channels. TRPM6 and TRPM7 contain protein kinase domains in their C termini, which bear amino-acid sequence similarity to elongation factor 2 (eEF-2), serine/threonine kinases, and other proteins that contain an α-kinase domain (259). Despite the lack of noticeable sequence homology to classical eukaryotic protein kinases, the crystal structure of TRPM7 kinase domain surprisingly showed a striking structural similarity to the catalytic core of eukaryotic protein kinases and metabolic enzymes with ATP “grasp” domains (260). TRPM7 is widely expressed, and knockout of the channel in cell lines have proved to be lethal, demonstrating its critical physiologic function (256). Unlike most other ion channels, TRPM7 exhibits significant magnesium permeation and is inhibited by cytosolic Mg2+, as well as Mg2+-ATP. Experimental analysis of the permeation properties of TRPM7 showed that the channel facilitates the conduction of a wide range of divalent trace metal ions (261). TRPM7 displays constitutive activity and is broadly expressed, suggesting that it may represent a general mechanism for the entry of divalent cations into cells. In contradiction to this idea, some data demonstrate that TRPM7 functions predominantly as a magnesium-permeable ion channel, which is required for the cellular uptake of magnesium (262). The magnesium permeability appears to be modulated by a functional coupling between the ion channel and kinase domains of TRPM7 (263), as indicated by changes in phosphotransferase activity and ion flux. The kinase domain has unknown target proteins that may be involved in a feedback loop that inhibits Mg2+ entry in the presence of increasing intracellular Mg2+ concentrations (262). However, annexin 1 was shown to

be a substrate for phosphorylation by exogenously expressed TRPM7 (264). It was further demonstrated that the endogenous kinase domain in TRPV7 is involved in up- and downregulation of channel activity by cAMP and PKA-mediated mechanisms (265). TRPM6 is closely related to TRPM7, and it represents an additional TRP protein that contains a C-terminal, α-kinase domain (243). The TRPM6 gene is composed of 39 exons encoding a putative protein of 2022 amino-acid residues. TRPM6 mRNA shows a restrictive expression pattern, with the greatest levels along the length of the intestinal tract (duodenum, jejunum, ileum, and colon) and in the distal convoluted tubules of the kidney (250). TRPM6 protein has been colocalized with the Na+-Cl− cotransporter and also with two cytosolic, ion-buffering proteins, parvalbumin and calbindin-D (257). Overexpression of TRPM6 in HEK cells showed a large, outwardly rectifying, whole-cell current strongly resembling the currents observed for TRPM7, with the reversal potential near 0 mV (257). Permeation characteristics showed that TRPM6 displays currents almost exclusively carried by divalent cations, with a higher affinity for Mg2+ than Ca2+, supporting the role of TRPM6 as the apical Mg2+ influx pathway. Furthermore, TRPM6, like TRPM7, exhibits a marked sensitivity to intracellular Mg2+, suggesting that inhibition of TRPM6 in response to increasing intracellular Mg2+ levels is a plausible mechanism to regulate intestinal and renal Mg2+ (re)absorption. This inhibition could be mediated, in part, by intracellular Mg2+-ATP, as was demonstrated for TRPM7, suggesting a possible link to cellular energy metabolism (256). In contrast with wild-type TRPM6, transfection of two mutated versions of TRPM6 with mutations found in patients with HSH yielded no detectable currents compared with nontransfected control subjects (257). Both mutations apparently led to a truncation of the TRPM6 protein, which lacked the pore-forming transmembrane domains. Analysis of point mutations will undoubtedly be more informative in elucidating functional aspects of the TRPM6 ion channel activity. In conclusion, genetic analysis of patients with HSH, together with studies designed to determine functional channel characteristics, exemplify a crucial role for TRPM6 in epithelial Mg2+ transport in the mammalian small intestine. However, considering the tetrameric structure of TRP channels, participation of other members of the TRP family in the formation of physiologically active apical Mg2+ channels in the intestine is possible. In fact, another investigation has specifically addressed this possibility (266). These authors initially attempted to isolate full-length TRPM6 transcripts, but discovered that the human TRPM6 gene encodes multiple mRNA isoforms. In addition, full-length TRPM6 variants failed to form functional channel complexes, because they were retained intracellularly on exogenous expression in HEK293 cells and Xenopus oocytes. However, TRPM6 specifically interacts with TRPM7, resulting in the assembly of functional TRPM6/TRPM7 complexes at the cell surface (266). The naturally occurring S141L TRPM6 missense

1974 / CHAPTER 77 A

Blood

Lumen Mg2+

K+ Mg2+

~10 mM [Na+] 140 mM [K+]

TRPM7

3

Na+ ATP

ADP

Mg2+

TRPM6 0.5 mM [mg2+]

Na+ 1-2 mM [Mg2+]

Mg2+

B

145 mM [Na+] 5 mM [K+] 2 K+

Lumen

Blood

Mg2+ 3 Na+ TRPM7 Mg2+

ATP

TRPM6

2K+ ADP

Mg2+

K+

Na+

Mg2+

Combined Physiological range

Transcellular

High oral substitution

Net Mg2+ absorption

C

Paracellular

Mg2+ intake

FIG. 77-6. Transepithelial Mg2+ transport. Magnesium may be absorbed by a paracellular route under high-intake conditions. However, with low-intake levels, Mg2+ is transported across the apical membrane of enterocytes via transient receptor potential melastatin receptors 6 and 7 (TRPM6/7) heterotetramers (A). TRPM6 has been identified as the defective gene underlying primary hypomagnesemia with hypocalcemia (HSH), which allowed the identification of the first molecule involved in intestinal Mg2+ transport. In HSH, mutations in TRPM6 abolish heterotetramerization with TRPM7, which leads to a mucosal block to Mg2+ absorption (B). Magnesium exits enterocytes possibly via the Na+-Mg2+ exchanger, which is driven by the electrochemical sodium gradient established by the basolateral Na+,K+-ATPase. (C) Mg2+ transport (y-axis) as a function of magnesium intake levels (xaxis). Under normal physiologic intake levels, total transport is a function of both the paracellular and transcellular processes. However in HSH, high oral substitution is necessary to allow transport via the passive paracellular pathway, because TRPM6/7 complexes are nonfunctional; thus, transcellular transport is zero. ADP, adenosine diphosphate; ATP, adenosine triphosphate. (Modified from Konrad and colleagues [243], by permission.)

MOLECULAR MECHANISMS OF INTESTINAL TRANSPORT OF CALCIUM, PHOSPHATE, AND MAGNESIUM / 1975 mutation found in patients with HSH abolished the oligomeric assembly of TRPM6/TRPM7 complexes, thus providing a biological explanation for the human disease. Thus, TRPM6/7 channel heterotetramers are essential components of the epithelial magnesium uptake machinery.

ACKNOWLEDGMENTS

Regulation of Magnesium Absorption

REFERENCES

Magnesium is found in a wide variety of foods; the greatest concentrations are found in nuts, legumes, and unmilled grains. Green vegetables are also an excellent source of magnesium, as a result of the magnesium-porphyrin complex in chlorophyll. The RDA for dietary Mg2+ differs between male and female individuals, with women generally requiring less than men (see Table 77-1). This required amount is increased during pregnancy, but not during lactation. The average magnesium intake in the United States and the Western world varies significantly between 130 and 600 mg/day (267). In diets ranging from 2.0 to 7.5 mg/kg/day, the net absorption of magnesium is linear at 40% to 60% (268,269), a relation that is not evident with greater magnesium intakes (246,270). However, magnesium intake does alter absorption; up to 75% of ingested magnesium may be absorbed with low-magnesium diets, with as little as 25% of magnesium intake being absorbed with high magnesium intake levels (246). In adults, the estimated endogenous intestinal magnesium content derived from digestive juices and sloughed cells is 30 to 40 mg/day (228). Magnesium absorption from foods that are enriched with this cation (e.g., almonds) is achieved just as well as from soluble magnesium acetate tablets. The observation that Mg2+ from enteric-coated magnesium chloride tablets is absorbed much less efficiently than that contained in Mg2+ acetate suggests that enteric coating interferes with magnesium absorption (246). Dietary fiber and phytate also reduces magnesium absorption in humans (271). Another study demonstrated that lactose induced ileal Mg2+ absorption, possibly by reducing the luminal pH (272). Fructose also has been shown to increase intestinal magnesium absorption (273). Although a reciprocal relation between the absorption of calcium and magnesium has been observed both in humans and in experimental animals (274,275), the hypothesis invoked to explain this interaction is conjectural at best (276). In fact, studies with radiolabeled Mg2+ in humans demonstrate no effects of dietary calcium in the range of 200 to 2000 mg/day on magnesium absorption rates. Other studies involving transport experiments done in small-intestinal, brush-border membrane vesicles concluded that magnesium and calcium influx mechanisms were distinct (277,278). Increments in dietary phosphorus also have been associated with a decrease in magnesium absorption in humans (279), and as a result of studies in experimental animals, have been presumed to result from the formation of insoluble phosphate/magnesium complexes (242). Despite these observations, the cause for this interrelation between phosphorus and magnesium still needs to be completely elucidated (276).

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This work was supported by the National Institutes of Health (grant 1-R21-DK-068349 to J.F.C.; and grants R01DK-412174 and R01-DK-33209 to F.K.G.).

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271. Schwartz R, Apgar BJ, Wien EM. Apparent absorption and retention of Ca, Cu, Mg, Mn, and Zn from a diet containing bran. Am J Clin Nutr 1986;43:444–455. 272. Heijnen AM, Brink EJ, Lemmens AG, Beynen AC. Ileal pH and apparent absorption of magnesium in rats fed on diets containing either lactose or lactulose. Br J Nutr 1993;70:747–756. 273. van der Heijden A, van den Berg GJ, Lemmens AG, Beynen AC. Dietary fructose v. glucose in rats raises urinary excretion, true absorption and ileal solubility of magnesium but decreases magnesium retention. Br J Nutr 1994;72:567–577. 274. Clarkson EM, Warren RL, McDonald SJ, de Wardener HE. The effect of a high intake of calcium on magnesium metabolism in normal subjects and patients with chronic renal failure. Clin Sci 1967;32: 11–18. 275. Norman DA, Fordtran JS, Brinkley LJ, Zerwekh JE, Nicar MJ, Strowig SM, Pak CY. Jejunal and ileal adaptation to alterations in dietary calcium: changes in calcium and magnesium absorption and pathogenetic role of parathyroid hormone and 1,25-dihydroxyvitamin D. J Clin Invest 1981;67:1599–1603. 276. Hardwick LL, Jones MR, Brautbar N, Lee DB. Magnesium absorption: mechanisms and the influence of vitamin D, calcium and phosphate. J Nutr 1991;121:13–23. 277. Juttner R, Ebel H. Characterization of Mg2+ transport in brush border membrane vesicles of rabbit ileum studied with mag-fura-2. Biochim Biophys Acta 1998;1370:51–63. 278. Baillien M, Wang H, Cogneau M. Uptake of (28)Mg by duodenal and jejunal brush border membrane vesicles in the rat. Magnes Res 1995;8:315–329. 279. Snedeker SM, Smith SA, Greger JL. Effect of dietary calcium and phosphorus levels on the utilization of iron, copper, and zinc by adult males. J Nutr 1982;112:136–143.

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CHAPTER

78

Iron Absorption Nancy C. Andrews Intestinal Iron Absorption, 1983 Plasma Iron Trafficking, 1985 Systemic Iron Homeostasis, 1985 Disorders Associated with Abnormal Iron Absorption, 1986 Iron Deficiency, 1986 Iron Overload, 1987 Ferroportin Disease, 1989 Defects in Plasma Proteins Involved in Iron Metabolism, 1989

Anemia Disorders Associated with Hyperabsorption of Dietary Iron, 1990 Transfusional Iron Overload, 1990 Iatrogenic Iron Overload, 1990 References, 1990

Iron is an essential nutrient that must be extracted from the diet and absorbed at a level that supplies the metal for essential functions, but does not risk iron overload. This chapter summarizes our current understanding of intestinal iron absorption and discusses how it is perturbed in disorders of iron homeostasis.

Starch may impair iron absorption in a similar fashion. Polyphenols (e.g., tannins) from legumes, tea, coffee, and wine interfere with absorption of iron. Phosphates and phosphoproteins can inhibit iron absorption from milk and egg yolk. Other metals (e.g., calcium and zinc) can interfere with or compete for iron absorption. Normal gastric acidity provides protons that facilitate cellular iron uptake; conversely, antacids and drugs that increase gastrointestinal pH impair iron absorption. Iron absorption occurs in the proximal duodenum, just distal to the gastric outlet (1). The absorptive epithelium is organized in villi. Multipotential precursor cells in the duodenal crypts differentiate into several cells types, but enterocytes predominate. Over the course of several days, the enterocytes mature and migrate up the villus. Enterocytes have a microvillous brush border to optimize functional surface area for absorption of many nutrients. They are active for several days as they make their way up the villus. At the end of their life span, they senesce and are sloughed from the villous tip. Non-heme food iron generally is present in the Fe3+ (ferric) form, because Fe2+ (ferrous) iron is readily oxidized in an oxygen-rich, neutral pH environment. Therefore, the first step in absorption of non-heme iron must involve reduction of Fe3+ to Fe2+. Although gastric acid likely aids in this process, an enzymatic ferrireductase activity is thought to play a major role. Ferrireductase activity has been detected in duodenal brush-border preparations, and duodenal

INTESTINAL IRON ABSORPTION Most foods contain some iron, because all plant and animal cells use iron as a cofactor for cytochromes and enzymes that transfer electrons. However, meat products are a particularly rich source, because of the large amount of heme iron present in the hemoglobin of red blood cells and the myoglobin of muscle. The bioavailability of dietary iron is modified by a variety of factors. Heme iron is more readily absorbed. Animal proteins promote iron absorption, but the mechanism is not understood. Phytates, found in grains, bran, and other vegetable foods, can bind iron in nonabsorbable complexes.

N. C. Andrews: Department of Basic Sciences and Graduate Studies, Harvard Medical School, and Department of Pediatrics, Children’s Hospital, Howard Hughes Medical Institute, Boston, Massachusetts 02115. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1983

1984 / CHAPTER 78 cytochrome B–like protein (DCYTB) has been postulated to be the major enzyme serving this function (2). DCYTB is a heme protein with proven ferrireductase activity. It is normally expressed at low levels, but expression is enhanced dramatically in hyperabsorptive states (hypoxia, iron deficiency). Ferrous iron is the normal substrate for epithelial transport. There appears to be no paracellular route for iron absorption in healthy individuals. Therefore, non-heme iron absorption requires transcellular passage of Fe2+. Iron exists as a large, charged ion that cannot freely traverse biological membranes. Distinct transmembrane carriers reside on the apical and basolateral surfaces of enterocytes to allow iron into and out of the enterocyte. The major apical iron transporter is divalent metal ion transporter 1 (DMT1, also known as SLC11A2, NRAMP2, or DCT-1). DMT1 is a hydrophobic protein anchored in the lipid bilayer by 12 predicted transmembrane segments (3–5). It does not resemble other classes of transporter proteins. Its only homolog in mammals is natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1), a metal transporter found exclusively in phagolysosomes of a subset of macrophages (6). Yeast homologs of DMT1 are known to function in Mn2+ transport (7). DMT1 mRNA exists in four different spliced isoforms (8). Two of these have iron regulatory elements (IREs) in their 3′-untranslated regions (3′-UTRs). These mRNA are expressed in varying ratios in a wide variety of tissues. Intestinal expression follows an anatomic gradient with the greatest levels in the villous cells of the proximal duodenum (3). Expression is markedly induced in response to iron deficiency anemia and hypoxia. Insight into the roles of DMT1 has come from studies of rodents carrying mutations in their genes. A unique missense mutation, resulting in a glycine-to-arginine substitution at amino acid 185 of the protein (G185R), occurred spontaneously in mk mice and b rats (4,9). This mutation results in impairment of DMT1 function, aberrant subcellular localization, and gain of new calcium channel activity of uncertain importance (10–12). Consequently, the animals have impaired intestinal iron absorption and defective iron uptake by erythroid precursors (see the Plasma Iron Trafficking section later in this chapter). Similar, deliberate disruption of the murine DMT1 gene by targeted mutagenesis demonstrated that DMT1 was essential for normal intestinal absorption and erythropoiesis, but dispensable for placental iron transfer and hepatocyte iron uptake (H. Gunshin and N. C. Andrews [13]). Only one mutation in DMT1 has been identified in humans (14). A patient with lifelong anemia and, interestingly, iron overload, was found to have a mutation that impaired normal splicing of the DMT1 mRNA, at least in erythroid cells. Because this mutation did not result in total loss of DMT1 expression, the resulting phenotype is more complicated than that seen in animal models. Lower organisms also appear to require DMT1 for metal transport. Fruit flies carrying a mutation in the malvolio gene, an ortholog of DMT1, have abnormal behavior (15).

Zebra fish carrying a truncation mutation in their DMT1 gene have a defect in iron transfer (16). The molecular mechanism of DMT1-mediated transport remains poorly understood. DMT1 functions poorly at neutral or alkaline pH because it must cotransport protons with metal ions (3). Although iron appears to be its most import substrate in vivo, DMT1 also is capable of transporting a variety of other divalent metal cations including Mn2+, Co2+, Cd2+, Cu2+, Zn2+, and Pb2+ (3). Its normal roles in the transport of these metals are yet to be elucidated. Similarly, it is currently unknown whether DMT1 is necessary for intestinal heme iron transport. Once iron enters the intestinal epithelial cell, it can have any of several fates. Iron-requiring enzymes may use some iron. Some iron is likely stored in ferritin, a multimeric protein that sequesters iron in a nonreactive depot form. A variable fraction, depending on body iron needs, is transported across the basolateral membrane to gain access to the plasma. This iron “export” process appears to be a major control point for regulating overall iron absorption. There is only one known basolateral iron transporter. Ferroportin (also called SLC40A1, IREG1, or MTP1) is similar to DMT1 only in that it has approximately 12 predicted transmembrane segments (17–19). It bears no obvious homology to any other transporter protein. Ferroportin orthologs are found in multicellular organisms, but there are no similar proteins in unicellular organisms. Similar to DMT1, ferroportin is widely expressed, but its major roles are in intestinal iron absorption, placental iron transfer, and macrophage iron export. Deliberate inactivation of the murine ferroportin gene abrogates intestinal iron absorption and leads to retention of iron in tissue macrophages and hepatocytes (20). Together, these defects result in profound iron deficiency and anemia. Ferroportin mRNA also contains an IRE in its 5′-UTR, suggesting that it may be regulated by intracellular iron concentrations. Its expression is markedly induced in iron deficiency and hypoxia, similar to DMT1 (19). Ferroportin also is regulated posttranslationally. Hepcidin (also known as HAMP; see Systemic Iron Homeostasis section later in this chapter), a hormonal regulator of iron homeostasis, binds to ferroportin on the cell surface to trigger its internalization and degradation within the cell (21). This interrupts cellular iron export. In the intestine, it prevents transcellular iron transport, consequently blocking absorption. Multiple missense mutations have been identified in human ferroportin (22–24). Patients carrying one mutated ferroportin gene acquire a unique form of iron overload that is inherited in an autosomal dominant fashion (see the Ferroportin Disease section later in this chapter for a more detailed discussion). Basolateral iron transport is facilitated by multicopper ferroxidases. Hephaestin is a membrane-bound ferroxidase that is found only in the placenta and the intestine (25). Mice carrying a mutation that impairs hephaestin function have decreased transplacental iron transport and diminished intestinal iron absorption. The function of hephaestin can be replaced by ceruloplasmin, a homologous multicopper ferroxidase that circulates in the plasma. Currently, it is unknown

IRON ABSORPTION / 1985 how these proteins aid in iron absorption, but it is likely that they oxidize exported iron for incorporation into transferrin. Although the transport pathway for non-heme iron has been elucidated in some detail, little is known about how heme iron is absorbed from the diet. Accumulated evidence suggests that there is at least one apical enterocyte protein that binds heme (26), but the route it takes to transit the cell has not been worked out. It may or may not involve DMT1, ferroportin, or both. A protein with heme exporter function was shown to be the receptor for a feline leukemia virus (27). This protein is expressed in the intestine and possibly could mediate enterocyte heme export. This area deserves more study.

PLASMA IRON TRAFFICKING Once it has passed across the intestinal epithelium, iron is rapidly taken up by transferrin, an abundant plasma iron carrier protein (see review by Hentze and colleagues [28]). Each bilobed transferrin molecule can bind two Fe3+ atoms. Apotransferrin, monoferric transferrin, and diferric transferrin circulate throughout the body. Under normal circumstances, only about a third of transferrin iron binding sites contain metal. When assessed clinically, this is represented as transferrin saturation (serum iron/transferrin ratio, expressed in the same units) of about 30% to 40%. Iron deficiency is associated with decreased transferrin saturation; iron overload is associated with increased transferrin saturation or supersaturation. Diferric transferrin is the ligand for cell-surface transferrin receptors. Apotransferrin and monoferric transferrin bind with much lower affinity. Many cell types express low levels of transferrin receptor, but erythroid precursors, tumor cells, and activated lymphocytes express much greater levels, presumably to facilitate iron uptake in response to greater need. Binding of diferric transferrin to the transferrin receptor allows receptor-mediated endocytosis of the complex into cytoplasmic vesicles (see review by Hentze and colleagues [28]). The vesicles become acidified through the action of a proton pump, leading to release of iron from transferrin and strengthening of the transferrin–transferrin receptor interaction. DMT1 (and possibly other transporters) transfers iron across the endosomal membrane to the cytoplasm. Some iron makes its way to the mitochondrion for incorporation into heme and iron-sulfur clusters, some is stored in ferritin, and some is incorporated into enzymes that require iron as a cofactor. Meanwhile, transferrin and the transferrin receptor are recycled to the cell surface for further rounds of iron uptake. Studies have shown that the transferrin cycle is essential for normal iron uptake by erythroid precursors, but it is dispensable in most other cell types (29,30). This can be rationalized by considering iron needs—most of the body’s iron is directed to erythroid precursors for hemoglobin production. The transferrin cycle serves the function of concentrating iron in a form that can enter the cell efficiently. Nontransferrin-bound iron uptake mechanisms are present in other cell types, most notably, hepatocytes (31). This is

advantageous, allowing hepatocytes to be a physiologic sink for iron in excess of the body’s needs. Not surprisingly, the liver is a primary site for pathology in iron overload disorders. Nontransferrin-bound iron uptake into the heart has been shown to occur, at least under some circumstances, through coopting of L-type calcium channels (32). Transient receptor potential calcium channels also have been implicated in non-DMT1, nontransferrin-bound, iron uptake (33). Daily absorption supplies 1 to 2 mg new iron in average adults, who have a total body iron endowment of approximately 4 g. This balances obligate losses from bleeding and sloughing of skin and mucosal cells. Considering that erythropoiesis alone requires about 20 mg iron each day, it is obvious that intestinal absorption cannot provide all the iron that is needed. Recycling of iron from senescent erythrocytes solves this problem. Damaged and aged red blood cells are recognized by tissue macrophages, which engulf and lyse them. The iron is removed from heme through the action of heme oxygenase, and returned to the circulation through ferroportin export. There, it is readily loaded onto transferrin to become available for tissue needs.

SYSTEMIC IRON HOMEOSTASIS Complex homeostatic mechanisms operate to ensure that adequate iron is available for the body’s needs, but that unused iron does not accumulate. Iron that is not sequestered by proteins or chelators is highly reactive and prone to catalyze the formation of toxic oxygen radicals. These oxygen radicals, when uncontrolled, damage cellular membranes, proteins, and nucleic acids. The spectrum of iron-induced oxidative damage probably accounts for organ damage in iron overload disorders. The liver becomes fibrotic, then cirrhotic, with marked predisposition to hepatocellular carcinogenesis. Cardiac tissue damage leads to arrhythmias and cardiomyopathy. Iron deposition in endocrine tissues leads to their destruction and consequent endocrinopathies. Iron balance is maintained through well-orchestrated control of cellular iron transport and storage to avoid these problems (see review by Hentze and colleagues [28]). Overall, iron balance is achieved at the level of intestinal iron absorption, but transfer of iron into, out of, and between tissues determines iron distribution. Iron absorption and distribution are known to be altered by changes in total body iron stores, erythropoietic drive, hypoxia, and inflammation. Both intestinal iron absorption and macrophage iron release are induced in response to depleted iron stores, increased erythropoietic drive, and hypoxia. Conversely, intestinal iron absorption is interrupted and iron is stored in macrophages in the settings of enlarged iron stores and inflammation. We now have some insight into how these various stimuli lead to concerted changes in iron homeostasis. Evidence suggests that all of them modulate the production of hepcidin (also called HAMP), a peptide hormone with profound effects on iron metabolism (see review by Hentze and colleagues [28]). Hepcidin is a 25-amino-acid, cysteine-rich

1986 / CHAPTER 78 Increased iron stores LIVER

MACROPHAGES AND ENTEROCYTES

Inflammation Hepcidin Genetic hemochromatosis Hypoxia

Hepcidin binds to ferroportin to induce internalization and degradation

Decreased iron stores

Increased erythroid demand

FIG. 78-1. Regulation and activity of hepcidin. Increased iron stores and inflammation induce hepcidin expression, whereas genetic hemochromatosis, hypoxia, decreased iron stores, and increased erythroid demand inhibit hepcidin expression. Hepcidin is produced in the liver and secreted into the circulation. It acts on macrophages and enterocytes by binding to ferroportin and inducing its internalization and degradation.

protein produced in the liver from a larger precursor (34,35). It is secreted into the plasma, where it circulates until it leaves the body in the renal filtrate. Plasma hepcidin levels are likely determined by the rate of hepcidin protein production, and because of its small size, the kidneys probably quantitatively filter hepcidin. Regulation of expression appears to be exquisitely sensitive, varying over several orders of magnitude in response to several known stimuli. Although not yet understood in molecular detail, it is clear that conditions that lead to increased iron absorption (e.g., anemia, iron deficiency, and hypoxia) signal to shut off hepcidin expression, and conditions that lead to decreased iron absorption (e.g., iron overload and inflammation) induce its expression (35–38). Figure 78-1 summarizes these effects. Hepcidin acts by binding to cell-surface ferroportin to signal its endocytosis and lysosomal degradation (21). It is therefore a negative regulator of cellular iron export. Because ferroportin is essential for cellular iron export in the placenta, in enterocytes, in tissue macrophages, and likely in hepatocytes, circulating hepcidin should coregulate export at all of these sites (20). The hemochromatosis-like phenotypes observed in animals and patients deficient in hepcidin support this conclusion (39,40). All have amplified serum iron consequent to increased iron absorption and increased macrophage iron release. Similarly, animals with excess, constitutive, hepcidin expression acquire hypoferremia and a severe anemia phenotype, with systemic iron deficiency and macrophage iron retention (41). Hepcidin expression is induced rapidly in response to increased serum transferrin saturation and inflammatory cytokines. The best understood induction pathway involves interleukin-6 (IL-6), a product of macrophages activated by inflammation. Elegant experiments have shown that IL-6 acts directly on hepatocytes to induce increased hepcidin production (42). This probably explains a long-noted but poorly understood association between inflammation and hypoferremia: Induction of hepcidin expression has the rapid

consequence of inhibiting iron absorption and macrophage iron recycling. This contributes to the development of the anemia of chronic disease (also called the anemia of inflammation) (43). The signals shutting off hepcidin in response to iron deficiency, anemia, and hypoxia have not been identified.

DISORDERS ASSOCIATED WITH ABNORMAL IRON ABSORPTION Iron Deficiency Iron deficiency results when the body’s iron endowment is insufficient to meet its needs. This is a common occurrence: Iron deficiency is the most prevalent cause of nutritional anemia worldwide. It is almost always an acquired rather than genetic condition. Causes can be categorized as problems involving supply, absorption, or demand (Table 78-1). Of these, “demand” problems are most prevalent. Iron supply problems arise when there is too little iron in the diet or when dietary iron is not bioavailable. Western diets are rich in iron and, for nonvegetarians, are particularly rich in readily absorbed heme iron. Only a fraction of dietary iron is absorbed, but the fraction increases substantially in iron deficiency and other cases of increased need. As discussed at the beginning of this chapter, other food substances (polyphenols, phytates, and starches) can limit the availability of food iron. Phosphates in cow’s milk can bind iron and decrease its absorption. Iron absorption problems generally involve disruption of the gut epithelium. Small-bowel resection, celiac disease, inflammatory bowel disease, and other processes can result in primary absorption defects. Inflammatory disorders also reduce iron absorption through activation of the inflammationhepcidin pathway described earlier. Gastrectomy, achlorhydria, and medicines that neutralize gastric pH also impair dietary iron absorption. Rarely, defects in intestinal iron absorption

IRON ABSORPTION / 1987 TABLE 78-1. Causes of iron deficiency Supply Inadequate dietary iron Decreased bioavailability of iron Dietary phytates, polyphenols, starches, phosphates, phosphoproteins Antacid therapy, achlorhydria, gastrectomy Competing metals Absorption Duodenal bypass Celiac disease Inflammatory bowel disease Inflammation in other sites Rare congenital iron absorption defects Demand Pregnancy, childbirth, lactation Rapid growth (toddlers, adolescents) Increased losses Gastrointestinal bleeding Gastrointestinal neoplasm Milk-induced enteropathy (infants) Salicylate, steroid use Hemorrhoids Peptic ulcer Gastritis Diverticulosis Inflammatory bowel disease Hereditary hemorrhagic telangiectasia Varices Arteriovenous malformations Meckel’s diverticulum Parasitic infestation (hookworm, schistosomiasis, trichuriasis) Menorrhagia Gynecologic neoplasms Bladder neoplasms Hemoglobinuria Epistaxis Pulmonary hemosiderosis Frequent blood donation, phlebotomy, autophlebotomy

appear to be inherited in an autosomal recessive fashion, suggesting a genetic defect. Currently, however, these have not been explained at a molecular level, and mutations in obvious candidate genes have not been found. Iron demand problems result from excessive iron needs or ongoing iron losses. Pregnancy, childbirth, and lactation require large iron contributions from the mother, resulting in iron deficiency when initial stores, dietary intake, or both are marginal. Rapid growth in childhood also increases iron needs; consequently, iron deficiency is relatively common among small children. Most infant foods and formulas are now supplemented with iron to address this problem. Adolescent girls also are at risk, because of the combined factors of accelerated growth and menstruation. Increased demand most frequently results from iron losses because of bleeding. Whole blood contains approximately 0.4 to 0.5 mg iron per milliliter. Worldwide, most iron deficiency is probably attributable to intestinal blood loss caused by parasitosis. The amounts of blood loss may be

small on a daily basis, but chronic infestation leads to significant iron deficits over time, particularly when coupled with a marginal diet. Other causes of blood loss include mucosal tumors in the gastrointestinal or genitourinary tract, use of salicylates or steroids, menorrhagia, vascular abnormalities, and other defects resulting in gross or occult bleeding. Infants who consume cow’s milk before the gut has matured can experience gastrointestinal microhemorrhages that, over time, deplete body iron stores. Finally, frequent blood donation and excessive phlebotomy can also lead to iron deficiency.

Iron Overload In contrast with iron deficiency, genetic defects explain most nontransfusional cases of iron overload. These fall into two general categories: defects in genes encoding proteins of iron metabolism, and defects in genes important for normal erythropoiesis. Other, nongenetic causes of iron overload include hypertransfusion, iatrogenic iron overload, and acute iron poisoning. Hemochromatosis Disorders Hereditary hemochromatosis was once thought to be attributable to mutations in a single gene linked to the human leukocyte antigen (HLA) complex on chromosome 6p. However, it has become clear that mutations in any of five distinct genes can lead to iron overload (Table 78-2). The hallmark features of classical hemochromatosis are increased serum transferrin saturation, deposition of iron in parenchymal cells (hepatocytes, cardiac myocytes, pancreatic acinar cells, etc.), and a relative paucity of iron in tissue macrophages (44). The most prominent clinical manifestations in adult-onset disease are liver dysfunction attributable to fibrosis and/or cirrhosis, joint abnormalities, and skin hyperpigmentation. In contrast, the most prominent features of early-onset, “juvenile” hemochromatosis are cardiomyopathy and endocrinopathies (e.g., diabetes and hypogonadotropic hypogonadism). Clinical scenarios can include all features of both adult-onset and juvenile hemochromatosis. However, penetrance of adult-onset hemochromatosis is relatively low, and it is not uncommon to find patients with mutations predisposing to hemochromatosis with few or no overt features of clinical iron overload. In contrast, penetrance of juvenile hemochromatosis appears to be quite high. The most common form of hemochromatosis results from mutations in HFE, the gene linked to the HLA complex (45). It is an autosomal recessive disease, and most patients are homozygous for a unique missense mutation encoding a cysteine-to-tyrosine substitution at residue 282 of the HFE protein (C282Y). The C282Y mutation appears to have arisen in a single individual who lived in Europe before 4000 BC (46). The mutation has spread throughout the world in a pattern consistent with Celtic migration. Today, most patients with HFE hemochromatosis are found in northwestern Europe, Australia, and the United States. A second,

1988 / CHAPTER 78 TABLE 78-2. Genetic iron overload disorders Disease

Gene

Inheritance

Clinical presentation

Hereditary hemochromatosis (HLA-linked)

HFE

Autosomal recessive

Hereditary hemochromatosis (not HLA-linked)

TFR2 (transferrin receptor 2)

Autosomal recessive

Juvenile hemochromatosis

HAMP (hepcidin) HJV (hemojuvelin)

Autosomal recessive

Ferroportin disease (autosomal dominant, macrophagepredominant iron overload)

SLC40A1 (ferroportin)

Autosomal dominant

Atransferrinemia

TF (transferrin)

Autosomal recessive

Aceruloplasminemia

CP (ceruloplasmin)

Autosomal recessive

Variable penetrance, adult onset, Phlebotomy, alcohol parenchymal iron loading, particularly avoidance, iron in the liver, heart, pancreas; supplements hyperpigmentation, hepatic fibrosis, arthropathies common; less frequently, cardiomyopathy, diabetes, other endocrinopathies; increased transferrin saturation early and increased serum ferritin later Adult-onset parenchymal iron Phlebotomy, alcohol loading, particularly in the liver, avoidance, heart, pancreas; hyperpigmentation, iron supplements hepatic fibrosis, arthropathies common; less frequently, cardiomyopathy, diabetes, other endocrinopathies; increased transferrin saturation early and increased serum ferritin later High penetrance, onset before the Aggressive fourth decade of life, parenchymal phlebotomy, iron loading in the liver, heart, alcohol avoidance, and pancreas; cardiomyopathy, iron supplements endocrinopathies, and arthropathies dominate the clinical picture, and cirrhosis is less common; increased transferrin saturation early and increased serum ferritin later. Macrophage-predominant iron Phlebotomy if overload; may have mild anemia parenchymal iron early in course; some patients loading and if experience development of tolerated parenchymal iron overload and complications similar to hereditary hemochromatosis; serum ferritin is increased before transferrin saturation Severe anemia accompanied by Parenteral marked iron loading of transferrin nonhematopoietic tissues; replacement complications similar to hereditary hemochromatosis Progressive iron deposition in the No effective liver and central nervous system; treatment available may be accompanied by mild anemia; neurodegeneration, dementia, retinopathy, diabetes

common amino-acid substitution (histidine for aspartic acid 63 [H63D]) is found in many individuals. It does not occur on the same HFE allele as the C282Y mutation, but may accompany the C282Y mutation in compound heterozygotes. Patients with H63D mutations are less likely to have clinically significant iron overload than patients with the C282Y mutation. Other rare mutations in HFE have been identified, but they generally appear to be “private” mutations with no particular geographic distribution. HFE hemochromatosis usually presents in the fifth or sixth decade of life. Men are more commonly affected than women, likely because of the protective effect of menstrual blood loss. The penetrance of HFE hemochromatosis has been the subject of much controversy, with estimates ranging

Treatment

from less than 1% to 100% (47–50). The true penetrance likely varies among populations derived from different gene pools because other “modifier” genes influence iron loading. It is also influenced by dietary iron intake, alcohol consumption, viral hepatitis, and other factors. Currently, a good estimate for the true overall penetrance is probably 10% to 20% in men carrying two HFE C282Y alleles. Most affected patients have substantial periportal hepatocyte iron deposition detectable on biopsy, with or without more severe damage evident as fibrosis or cirrhosis. Arthropathies are common. Other complications such as diabetes and cardiomyopathy are observed infrequently. A second form of autosomal recessive hemochromatosis results in a similar, but possibly more severe, presentation

IRON ABSORPTION / 1989 compared with HFE hemochromatosis. It is caused by mutations in the transferrin receptor-2 (TFR2) gene, which encodes a homolog of the classical transferrin receptor (51). A variety of mutations have been observed, including nonsense mutations that would prevent production of a full-size TFR2 protein. Patients with TFR2 hemochromatosis have presented as early as childhood, but adult-onset presentation is more common. The pattern of iron loading and the clinical sequelae are indistinguishable from those seen in HFE hemochromatosis. Early-onset, juvenile hemochromatosis is rare but severe. It also is inherited in an autosomal recessive pattern, typically presenting in the second or third decade of life. Accelerated iron loading results from homozygosity for mutations in either of two genes: the HAMP gene encoding hepcidin or the HJV gene (also called RGMC) encoding a protein called hemojuvelin (40,52). Most cases are attributable to HJV mutations, but several families with HAMP mutations have been reported. The overall pattern of iron loading in juvenile hemochromatosis resembles that observed in HFE and TFR2 disorders (53). However, cardiac and endocrine manifestations are far more common and usually dominate the clinical concerns. There are several possible explanations for this different presentation. First, it may be that more rapid iron loading is more deleterious for cardiac and endocrine cells than for hepatocytes. Second, it is possible that the heart and endocrine tissues endure more damage because of the changes that they undergo during adolescence. For example, the heart may be more susceptible to iron-related damage when it is stressed by the rapid increase in body size during the adolescent growth spurt. Finally, it is possible that the HAMP and HJV gene products play unique roles in the affected tissues. Interestingly, both are known to be expressed in the heart (52,54). Not surprisingly, patients occasionally have mutations in more than one hemochromatosis disease gene. Patients with more severe HFE hemochromatosis have been shown to have single mutations in the HAMP or HJV genes in addition to their homozygous C282Y mutations (55,56). Other combinations are likely to be discovered. Phlebotomy is the preferred treatment for all four of these types of hemochromatosis. First shown to be efficacious in 1950, phlebotomy is an efficient method for removing iron from nonanemic patients with iron overload (57). Approximately 200 to 250 mg iron is removed with each unit of blood. Standard practice is to phlebotomize as frequently as tolerated until total body iron stores decrease to a near iron-deficient level, and then phlebotomize intermittently thereafter to maintain the low iron state (58). This is usually well tolerated in patients with HFE, TFR2, HAMP, and HJV mutations. It is lifesaving for patients with juvenile hemochromatosis, who otherwise succumb to complications of iron overload before 30 years old. In adult hemochromatosis, phlebotomy prevents (and sometimes even reverses) tissue damage, resulting in normal quality of life and life span. The phenotypic similarities among these four forms of hemochromatosis suggest that the products of the affected genes might participate in the same biological pathway. Although many of the details remain obscure, this appears to

be the case. The normal functions are unknown for HFE, TFR2, or HJV, but patients with hemochromatosis caused by mutations in any of the proteins have the common feature of diminished hepcidin production (52,59,60). This suggests that the normal roles of HFE, TFR2, and HJV proteins involve regulation of hepcidin expression or conveyance of signals that regulate hepcidin expression, or both. It appears that the severity of the iron overload observed correlates (at least roughly) with the severity of impairment of hepcidin production. It is easy to imagine how inappropriately low hepcidin expression would lead to hemochromatosis. As discussed earlier, hepcidin normally functions to interrupt cellular iron release, through abrogation of ferroportin activity. When hepcidin control is attenuated, the combined effects of increased ferroportin activity in the intestine and in tissue macrophages account for the hyperferremia and decreased macrophage iron observed in hemochromatosis. Parenchymal cells, particularly in the liver, which has first-pass access to intestinal blood flow, rapidly take up excess serum iron. Other tissues, particularly the heart and the pancreas, also take it up.

Ferroportin Disease A pathologically distinct, autosomal dominant iron overload disorder results from missense mutations in the gene encoding ferroportin (22–24). Patients carrying one abnormal ferroportin gene develop macrophage-predominant iron loading with or without an increase in total body iron. In contrast with other forms of hemochromatosis, patients with “ferroportin disease” maintain relatively normal serum transferrin saturation, but have markedly increased serum ferritin. Because transferrin saturation correlates with circulating iron availability and serum ferritin reflects cellular iron stores, this phenotype is consistent with a defect in ferroportin that causes macrophage iron retention. Some patients with ferroportin disease experience development of anemia early in their course, suggesting that macrophage iron retention restricts the iron available for erythropoiesis. However, most patients recover from the anemia, and some have frank iron overload later. This indicates that intestinal iron absorption is not impaired and can even be accelerated. Over time, a subset of patients also experience iron overload in parenchymal cells, which can lead to the typical complications observed in hemochromatosis disorders (61). The pathophysiology of this disorder is not yet understood. That no null mutations have been observed suggests that the missense mutations may not cause simple inactivation of ferroportin iron transport capability. Rather, they may alter the subcellular localization of ferroportin or the way that it interacts with one or more other proteins.

Defects in Plasma Proteins Involved in Iron Metabolism Iron overload with abnormal body iron distribution can also be caused by deficits of either of two serum

1990 / CHAPTER 78 proteins: transferrin or ceruloplasmin (62,63). Patients with serum transferrin deficiency have a defect in iron uptake by erythroid precursors, which are highly dependent on the transferrin cycle. Consequently, severe anemia develops that is treatable only by transfusion or transferrin replacement therapy. However, nonhematopoietic cells do not require transferrin for iron assimilation. Intestinal iron absorption is markedly stimulated, and excess iron accumulates in the liver, heart, pancreas, and other sites. This results in a paradoxic situation of iron deficiency anemia in the setting of profound iron overload. It can be corrected only by infusion of transferrin to replace the missing protein. Ceruloplasmin normally functions as a ferroxidase that aids in cellular iron release (64). Patients lacking ceruloplasmin are generally healthy until middle age, when the effects of their long-term defect in iron homeostasis become manifest. Clinical presentations include diabetes, dementia, retinal degeneration, and basal ganglia symptoms. Iron deposits in the liver and central nervous system. A subset of neurons in the substantia nigra and basal ganglia has marked iron deposition resulting in neuronal dropout without reactive gliosis (65). Mild anemia, decreased serum iron, and increased serum ferritin levels reflect a defect in cellular iron export. Currently, there is no effective treatment for aceruloplasminemia, and patients experience a progressive, degenerative course resulting in death.

Anemia Disorders Associated with Hyperabsorption of Dietary Iron A subset of genetic anemia disorders are associated with increased intestinal iron absorption, even in the setting of systemic iron overload (66). These disorders include thalassemia syndromes, congenital sideroblastic anemia, congenital dyserythropoietic anemias, pyruvate kinase deficiency, and other intrinsic red blood cell defects. Their common feature is destruction of immature erythroid cells in the bone marrow, a situation known as ineffective erythropoiesis. In contrast, increased intestinal iron absorption is not associated with sickle cell anemia, hereditary spherocytosis, or other disorders in which red blood cell destruction occurs in the periphery. The molecular links between ineffective erythropoiesis and increased intestinal iron absorption have not been fully elucidated. However, it is likely that the affected bone marrow sends a humoral signal to the liver to interrupt normal hepcidin expression. This series of events might be advantageous to the erythron, because it increases the available iron supply, but deleterious to other tissues where excess iron is deposited, leading to complications of iron overload.

Transfusional Iron Overload Chronic transfusion therapy is a mainstay in the treatment of some patients with thalassemia, sickle cell anemia,

sideroblastic anemia, myelodysplastic syndromes and other diseases that result in insufficient red blood cell mass. Because packed red blood cells contain approximately 1 mg iron/mg cells, transfusion therapy quickly increases the body’s iron burden. This is exacerbated when the underlying condition also is associated with intestinal hyperabsorption of iron, as described in the previous section. Phlebotomy is generally not an option for patients who require chronic transfusion therapy. De-ironing must be achieved through treatment with chelators that extract excess iron and allow it to be excreted in urine or stool. Deferoxamine is the chelator that has been used most widely. It is effective, but has significant drawbacks in that it must be administered parenterally, and it has significant toxicities of its own. Newer oral chelators, such as deferiprone, are in use for some patients, and more are under development.

Iatrogenic Iron Overload Ingestion of supplemental dietary or medicinal iron rarely causes clinical iron overload because of the normal homeostatic mechanisms that function to limit intestinal iron absorption. Although there are case reports of abnormally increased iron stores resulting from administration of iron salts (67), there should be little clinical concern under most circumstances. In contrast, the body has no normal defense against iatrogenic iron overload caused by parenteral administration of unneeded iron. Therefore, iatrogenic iron overload not caused by transfusion is most commonly associated with inappropriate dosing of parenteral iron. It is important to remember that dosing is different for inefficiently absorbed oral iron and for efficiently delivered intravenous iron preparations. Acute iron intoxication can occur when large doses (measured in grams) of iron salts are ingested orally. This can occur, for example, when a young child mistakes his or her mother’s prenatal vitamins for candy (68). Initially, patients have gastrointestinal symptoms including vomiting, diarrhea, and melena, attributable to mucosal irritation and bleeding. Within a few hours they experience dyspnea and lethargy, leading to coma. In lethal iron intoxication, the condition progresses to metabolic acidosis and death within 48 hours of the acute ingestion. Patients who recover may experience development of later intestinal obstruction caused by scarring of the damaged mucosa. Aggressive oral and intravenous chelation can improve the outcome, but mortality from significant iron poisoning remains high.

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1992 / CHAPTER 78

46. 47. 48. 49. 50. 51. 52.

53. 54.

55.

56.

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57. Davis WD, Arrowsmith WR. The effect of repeated bleeding in hemochromatosis. (Proceedings of the 23rd annual meeting of the Central Society for Clinical Research). J Lab Clin Med 1950;36: 814–815. 58. Barton JC, McDonnell SM, Adams PC, Brissot P, Powell LW, Edwards CQ, Cook JD, Kowdley KV. Management of hemochromatosis. Hemochromatosis Management Working Group. Ann Intern Med 1998;129:932–939. 59. Bridle KR, Frazer DM, Wilkins SJ, Dixon JL, Purdie DM, Crawford DH, Subramaniam VN, Powell LW, Anderson GJ, Ram GA. Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homeostasis. Lancet 2003;361:669–673. 60. Nemeth E, Roetto A, Garozzo G, Ganz T, Camaschella C. Hepcidin is decreased in TFR2-hemochromatosis. Blood 2005;105:1803–1806. 61. Njajou OT, de Jong G, Berghuis B, Vaessen N, Snijders PJ, Goossens JP, Wilson JH, Breuning MH, Oostra BA, Heutink P, Sandkuijl LA, van Duijn CM. Dominant hemochromatosis due to N144H mutation of SLC11A3: clinical and biological characteristics. Blood Cells Mol Dis 2002;29:439–443. 62. Hamill RL, Woods JC, Cook BA. Congenital atransferrinemia: a case report and review of the literature. Am J Clin Pathol 1991;96:215–218. 63. Harris ZL, Klomp LW, Gitlin JD. Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis. Am J Clin Nutr 1998;67:972S–977S. 64. Harris ZL, Durley AP, Man TK, Gitlin JD. Targeted gene disruption reveals an essential role for ceruloplasmin in cellular iron efflux. Proc Natl Acad Sci U S A 1999;96:10812–10817. 65. Xu X, Pin S, Gathinji M, Fuchs R, Harris ZL. Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis. Ann N Y Acad Sci 2004;1012:299–305. 66. Cazzola M, Beguin Y, Bergamaschi G, Guarnone R, Cerani P, Barella S, Cao A, Galanello R. Soluble transferrin receptor as a potential determinant of iron loading in congenital anaemias due to ineffective erythropoiesis. Br J Haematol 1999;106:752–755. 67. Pearson HA, Ehrenkranz RA, Rinder HM, Riely CA. Hemosiderosis in a normal child secondary to oral iron medication. Pediatrics 2000;105:429–431. 68. Mills KC, Curry SC. Acute iron poisoning. Emerg Med Clin North Am 1994;12:397–413.

CHAPTER

79

Trace Element Absorption and Transport Robert J. Cousins General Properties of Trace Element Absorption, 1993 Lumenal Environment and Trace Element Absorption, 1993 Diet Composition, 1993 Mechanisms of Uptake, Transcellular Movement, and Efflux by the Gastrointestinal Tract, 1994 Copper Absorption, 1995

Zinc Absorption, 1996 Other Trace Elements, 1998 Selenium, 1998 Chromium, 1998 Manganese, 1998 Genetic Variation in Absorption, 1998 References, 1998

GENERAL PROPERTIES OF TRACE ELEMENT ABSORPTION

LUMENAL ENVIRONMENT AND TRACE ELEMENT ABSORPTION

The trace elements are roughly divided into the trace and the ultratrace elements. The latter comprise a group of elements for which there are varied degrees of evidence to support nutritional essentiality based on biochemical function and dysfunction during restriction in the amount available for absorption. In contrast, the trace elements copper, selenium, and zinc have well-established biochemical roles and nutritional essentiality. They also comprise, together with iron, those trace elements on which most of our knowledge about the genes responsible for trace element transport into and from intestinal epithelial cells resides. Digestive processes; factors that influence the extent of digestion, including infection and inflammation; endogenous secretion; and diet composition that influences absorption of trace elements in the diet are discussed in this chapter. Then, the current consensus of how some trace elements are transported into and out of intestinal epithelial cells is discussed.

Diet Composition

R. J. Cousins: Boston Family Professor of Nutrition, Center for Nutritional Sciences, University of Florida, Gainesville, Florida 32611. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

Absorption of all minerals in food and fluids consumed is influenced by physical and biochemical properties. The extent to which these are degraded into smaller components influences absorption of their individual constituents. Solubility is also a factor of influence for trace element absorption. Trace elements tend to be widely dispersed among foods (1). Where function is known, distribution follows those functions. For example, zinc has important functions in gene expression; thus, it might be expected that, among food products, zinc is abundant where nucleic acids are concentrated. The high zinc content of wheat germ is an example. Zinc is ubiquitously distributed in animals and, as such, muscle provides the major food source for omnivores. This reflects enzymatic and gene regulatory roles. Selenium and copper, having enzymatic roles, might be most concentrated in foods where metabolic activity is high, that is, grains, and in organ meats and muscle. Animal- and plant-derived foods provide selenium as selenocysteine and selenomethionine, respectively. Other trace elements also tend to reflect their enzymatic roles in biological systems, and thus are presented to the intestinal lumen as protein complexes. Most of these are degraded to amino-acid complexes or small peptides. The extent of digestion and transit times are

1993

1994 / CHAPTER 79 Pancreatic secretions Biliary secretions

Enterocyte

Intestinal lumen Dietary trace element

Endogenous trace element

DIGESTION Vesicles

Free trace element

BC

BC

Available trace element pool

Low molecular mass binding ligands

High molecular mass binding ligands

Unavailable trace element

Binding chaperone proteins

Excreted trace element

Paracellular transport

FIG. 79-1. Pathways for dietary trace element absorption. Most nutrient trace elements enter the intestinal lumen bound to a mixture of constituents of varying molecular mass. Gastric acid favors element solubility, but neutralization in the intestine favors binding to ligands. In many cases, the binding constants for these ligands are high. The extent of digestion is an important factor in the pathway toward creating a milieu for the element that favors absorption; this is represented as the available trace element pool. During mediated uptake that displays saturable kinetics, trace elements are transported as free ions. Transporter proteins may aid in acquisition of the element from a binding ligand of low affinity. Within enterocytes during the transcellular movement phase, binding to a specific chaperone protein(s) is possible, as is transport into vesicles. Transporters localized at the basolateral membrane are responsible for cellular efflux of the trace element. Efflux of copper is energy dependent (adenosine triphosphate requiring), whereas that of zinc and other trace elements is not energy dependent. High lumenal concentrations of a highly absorbable trace element or a lowmolecular-mass chelate of a trace element may provide access across the intestinal epithelium via paracellular (nonmediated) transport where linear kinetics is displayed.

determining factors (Fig. 79-1). Trace elements bound to high-molecular-mass compounds, for example, large peptides, may not be able to interact with transporters responsible for uptake by enterocytes. This constitutes part of the unavailable pool of a particular trace element. The pool of a trace element available for absorption most likely is constituted of low-molecular-mass compounds, for example, amino-acid chelates, peptides, or “free” ions of the element. The concept of a free pool for nutrient trace elements is an area of current investigation. For example, intracellular concentrations of Zn2+, and probably other trace elements, are low to no more than a few atoms at a time (2). A similar situation would be expected in the extracellular intestinal environment. The binding affinities of various molecules dictate how much “free” ion exists.

Mechanisms of Uptake, Transcellular Movement, and Efflux by the Gastrointestinal Tract Unlike nutrients with solubility that is quite high, kinetic analysis of trace element uptake is quite challenging. These micronutrients tend to be soluble in an acidic environment, but less so in an alkaline milieu. Gastric secretions would tend to enhance solubility, but the stomach is not the site of appreciable trace element absorption. As the gastric contents enter the duodenum, neutralization is rapid, with a concomitant reduction in solubility. Ironically, the small intestine is the major site of trace element absorption. Nevertheless, uptake for many of these minerals, including copper, manganese, and zinc, follows saturable kinetics. How this is accomplished currently is not well understood. Because the extent

TRACE ELEMENT ABSORPTION AND TRANSPORT / 1995 of absorption is a function of interaction with transporters at the apical surface of enterocytes, the more interaction that exists with metal-binding constituents of low affinity, the greater the uptake rate should be. Saturable kinetics (a mediated component of uptake) implies that one or more transporter molecules are involved. Many elegant studies have characterized saturable kinetics in cells of the small intestine. As lumenal concentrations of a trace element increase, nonmediated uptake tends to be predominant. This is believed to represent paracellular transport. Issues that need to be resolved are the absorbability of both intact trace element chelates and those elements bound to small peptides. These topics have been reviewed by Cousins (3) and O’Dell and Sunde (4). Transepithelial movement of trace elements also occurs by unknown mechanisms. The demonstration that many transporters are localized to intracellular vesicles suggests that they participate in some phase of trace element trafficking across cells. Experiments involving zinc transport by Caco-2 cells showed that zinc transport was markedly reduced by treatment with quinacrine, a lysosomal membrane disrupter (5). This supports a role for vesicle-like components in zinc transport, and probably other trace elements as well (see Fig. 79-1). Use of selective fluorophores specific for individual trace elements to examine such questions, coupled with fluorescence microscopy of individual cells and aggregates of cells, is yielding important information about intracellular trafficking of these micronutrients in enterocytes. Mechanisms for transport through the basolateral membrane and across the capillary endothelium also are being defined, at least for some trace elements (see Fig. 79-1). Copper and zinc are two of the best examples in which efflux transporter genes have been identified (6–8). The intracellular pools from which absorbed trace elements are drawn for cellular efflux are unknown. Those trace elements sequestered in vesicles may be the most likely source available for efflux transporters at the basolateral membrane. A major challenge in evaluating which transporters are responsible for the absorption of a particular trace element is separating generalized cell uptake data from that exclusively for the gastrointestinal tract. In the case of copper and zinc, multiple transporter proteins have been identified (9,10), but those primarily responsible for uptake and release by enterocytes may be different from those with the responsibility for uptake into or release from hepatocytes, mononuclear cells, and so forth. Kinetic analysis of trace element absorption has used many techniques, from in vivo perfusion studies with humans to in vitro intestinal cell preparations and even membrane vesicles from experimental animals and immortalized cell lines. Unlike in vitro systems with relatively purified components, those designed to mimic the actual lumenal environment can only approximate the true concentration of the element in question that is available for absorption because individual binding constants define the available concentration of the trace element. Furthermore, the available trace element concentration at the apical surface is markedly

different from that available at the basolateral surface (see Fig. 79-1). The latter is obviously low because of the high binding constants of intracellular metal-binding ligands. Nevertheless, measured kinetic parameters are frequently within what would be expected, based on dietary consumption of these micronutrients and concentrations in peripheral blood. Most of the trace elements of nutritional interest show evidence of saturable absorption kinetics. This suggests interaction with a mediator/transporter molecule at some membrane surface of the enterocyte. Most likely this would be at the apical brush-border membrane. Fairly conclusive evidence has documented mediated transport for Cu, Mn, Se, and Zn absorption. Evidence for ultratrace element transport is mixed, and not abundant. For many elements, it has been proposed that they share common transporters. As discussed in Chapter 78, divalent metal ion transporter 1 (DMT1, SLC11A2) is believed to be the iron transporter responsible for Fe2+ uptake by enterocytes (11). In vitro voltage-clamp experiments have shown that when Xenopus oocytes are transfected with DMT1 mRNA, a number of other trace elements, including Cu, Zn, and Mn, also are transported when they are added to this assay system (11). Based on that evidence, it has been suggested that this transporter is involved in the intestinal uptake of multiple elements (12). Further evidence to support this hypothesis is lacking. For example, although Zn2+ uptake by DMT1-transfected oocytes was observed (11), the effect may be the result of a Zn2+-related proton leak in the system (13). Further research, particularly using knockout mice that do not express a specific transporter, is needed to resolve such questions about element/element interactions. The question of such interactions has been an issue in the field for decades. Aside from a few in vitro competition experiments with transporters, the interactions of trace elements have not been evaluated at the molecular level. Despite the lack of evidence, it does appear to be reasonable to suggest that transport activities for some trace element transporter proteins are leaky, and thus that these proteins transport a number of trace elements, both micronutrients and those elements with no known biological functions.

COPPER ABSORPTION Copper accumulation mechanisms have been well defined in single-cell organisms (e.g., yeast). In multicellular organisms, complexity increases, starting with tissue specificity. For example, as early as the 1950s, studies with 64 Cu showed that appreciable amounts of copper were taken up via the stomach (14,15). Gastric acidity was believed to enhance copper solubility (16–18). The stomach may be the site of some interaction of copper with other ions for uptake. After gastric secretions enter the duodenum, pH change may influence such interactions. As reviewed in detail earlier (3), dietary protein, fiber, phytate, ascorbic acid, and amino acids all have been shown to affect copper uptake by enterocytes.

1996 / CHAPTER 79 Uptake may favor L-amino acids over D-amino acids, suggesting that copper–amino acid complexes may be absorbed intact (19). Protein digestibility may influence formation of these complexes. High-protein diets generally improve copper absorption (3). Numerous constituents found in lumenal contents of the small intestine, of both dietary and endogenous origin, have been shown to have positive or negative effects on copper absorption, presumably via influences at the brush-border surface of enterocytes (3,4). Zinc has been shown to inhibit copper absorption. The site of the interaction may be a transporter molecule. More likely is that a zinc–copper interaction within enterocytes, as discussed later, is responsible. Both saturable and nonsaturable kinetics of copper absorption have been demonstrated (20). The latter may represent paracellular movement of copper complexes. The fractional absorption of copper in humans ranges from 75% at low copper intakes down to 12% at high intakes (21). The homeostatic changes in copper absorption/retention are partially explained by a direct correlation between dietary copper intake and endogenous losses from slow and fast kinetic pools (22). Copper absorption by the intestine is saturable, and some of the molecules responsible for such kinetics have been identified since the mid-1990s (Table 79-1). A seminal breakthrough was the cloning of the Menkes disease gene (ATP7A). Menkes disease is terminal, with patients presenting with progressive neurologic disease (see review by Peña and colleagues [9]). Copper accumulates in intestinal cells of patients with Menkes disease. This suggests that, because ATP7A is expressed in intestinal cells and ATP7A localizes to the basolateral membrane, it is the copper transporter responsible for saturable kinetics and the rate-limiting factor in copper absorption. This transporter is a P-type ATPase that has membrane-spanning domains and six repeats of a copper-binding motif. It appears to be responsible for TABLE 79-1. Transporters for copper and zinc expressed in the intestine Nutrient

Transporter

Copper Copper Copper

CTR1 (SLC31A1) CTR2 (SLC31A2) DMT1 (SLC11A2)

Copper

Zinc

ATP7A Menkes disease gene ZIP-4 (SLC39A4) Acrodermatitis enteropathica gene ZnT-1 (SLC30A1)

Zinc Zinc Zinc Zinc

ZnT-2 ZnT-4 ZnT-5 ZnT-6

Zinc

(SLC30A2) (SLC30A4) (SLC30A5) (SLC30A6)

Established or proposed intestinal function Apical transport Apical transport Apical transport Broad spectrum metal transporter P-type ATPase Basolateral Cu2+ pump Apical transport

Basolateral transport Vesicular transport Vesicular transport Vesicular transport Vesicular transport Vesicular transport

copper efflux by pumping copper ions out of enterocytes to the extracellular fluid space (ECF). Copper uptake by enterocytes involves at least three distinct transporter genes. The first identified, Ctr1 (SLC31A1), codes for a high-affinity transporter. hCtr1 mRNA is found in all human cell types as varying amounts of one of three transcript sizes. These differences may have functional significance (9). Homozygous CTR1 null embryos die at day 8.5 of development (23,24). Liver, pancreas, and heart express more CTR1 than intestine (9). It has been shown that the chemotherapeutic drug cisplatin is transported by CTR1 (25). The clinical significance of this finding from the standpoint of cancer of the gastrointestinal tract has not been investigated. A low-affinity copper transporter, CTR2, also is ubiquitously expressed in cells, but at low levels in the intestine (9). DMT1 (SLC11A2) is an iron transporter that up-regulates in intestines of iron-deficient animals (11) and may also participate in the apical transport of copper. At least in cultured human intestinal-like cells (Caco-2), copper transport is regulated by iron via regulation of DMT1 expression (26). Release of copper from enterocytes to the ECF under normal conditions is followed by binding to albumin (27). Release from enterocytes does not appear to require albumin, because rats with analbuminemia show normal copper absorption characteristics (28). Small amounts of copper circulate in plasma bound to small-molecular-weight constituents, which may be derived from the diet via paracellular transport or though binding of copper after transport from enterocytes via ATP7A activity. High oral intake of zinc has been shown to reduce copper transfer to the plasma (29). This relation has been used to reduce copper absorption in patients with Wilson’s disease after copper depletion therapy (30). One mechanism to explain this influence is the induction of the copper/zincbinding protein, metallothionein, which could result in copper retention in enterocytes (31). The copper would be lost during desquamation. This mechanism also has been shown in animal studies. Competition for a common transporter, for example, DMT1, at the apical surface of enterocytes could also be a factor that would explain the influence of large amounts of zinc on the transport of intestinal copper derived from the diet and via endogenous secretions.

ZINC ABSORPTION As documented earlier with copper, much of the mechanistic information that is available regarding zinc transport in mammalian cells and intact organisms emerged with parallel experiments in single-cell organisms, primarily yeast. The availability of radioactive isotopes of zinc (namely, 65 Zn) during World War II led to subsequent research that has provided a firm understanding of zinc metabolism. Particularly relevant milestones include the demonstration of homeostatic regulation of zinc absorption (32) and application of compartmental analysis to understand zinc flux from specific intracellular pools and the ECF (33).

TRACE ELEMENT ABSORPTION AND TRANSPORT / 1997 Zinc is an excellent example of how availability of binding ligands influences absorption (see Fig. 79-1). Zinc in foods is primarily associated with proteins and nucleic acids. Solubility is a major determinant of the extent of absorption. Body zinc status has an important influence on zinc absorption. This relation has been used to calculate the new Recommended Dietary Allowance (RDA) for zinc (20). There is no evidence suggesting appreciable zinc absorption in the stomach. However, inhibition of gastric secretions reduces zinc absorption, presumably through a reduction in solubility (34). The abundance of highly available zinc in the diet is provided by red meats and shellfish. Grain products can provide a substantial amount of zinc in the diet. However, a portion of this zinc is tightly bound by phytic acid (hexaphosphoinositol), and reduced solubility renders it less available. Zinc bioavailability is inversely related to the phosphorous content of phytate (35). Leavening of flour by yeast provides phytase activity, which minimizes the phytate content of most bread products. Zinc bioavailability can vary from 55% (red meat) to 15% (high-fiber cereal) (36). Zinc bioavailability is greater from human milk than cow’s milk, most likely because of the greater extent of hydrolysis of human milk proteins in the gastrointestinal tract (37). Casein, a phosphoprotein, is the principal protein in cow’s milk, but not human milk. Soy-rich foods frequently are processed, which reduces phytate content, thus improving bioavailability, and/or they are fortified with zinc. Numerous dietary constituents have been reported to influence zinc absorption (3,38–40). The experimental approaches used can be factors in establishing the practical significance of such findings. Numerous approaches have been used to study the kinetics of zinc absorption by the intestine. These range from intestinal perfusion to isolated membrane vesicles; these approaches have been reviewed in detail (38–40). Intestinal transport is a time-, concentration-, pH-, and temperaturedependent saturable process (41). In rats, zinc appears to be absorbed along the entire intestinal tract, including the colon (39). Perfusion experiments with humans suggest that the jejunum has the greatest absorption rate (42). Regional differences may reflect differences in zinc transporter expression (43). The absorption rate in humans is saturable above a lumenal concentration of 1.8 mM (42). After a meal, the zinc concentration of the lumen may reach only 100 µM (44), indicating most dietary zinc is absorbed by a saturable (mediated) process. This also suggests that, except after ingestion of zinc supplements, little absorption occurs via the paracellular route. Kinetic parameters (Km and Jmax) of intestinal zinc transport in rats vary considerably from those reported in humans (39,43). Where it has been studied experimentally, it appears that the absorption rates increase in response to low dietary zinc intake (see reviews in the literature [20,39,40,45]). Numerous zinc transporter genes (see Table 79-1) are expressed in the small intestine of mice (8,10). Only one, however, ZIP-4 (SLC39A4), appears to up-regulate in zinc deficiency (10,46). A considerable body of evidence suggests that zinc within intestinal cells is

localized within vesicles. Some transcellular zinc movement within enterocytes may occur via a vesicular pathway (5). The zinc exporter protein ZnT-1 has been localized primarily to the basolateral membrane of rat enterocytes (47). Evidence suggests zinc transport in the portal blood supply is as a complex with albumin (48). Some plasma zinc is in the form of low-molecular-weight complexes (45). There is evidence suggesting that some endogenous zinc is transported into the intestinal lumen by enterocytes. Pancreatic secretions contain zinc that also enters the intestinal lumen (45). This zinc is likely to be regulated homeostatically (10) and may be available for re-uptake into enterocytes. Experiments with mice suggest that down-regulation of ZnT-1 and ZnT-2 in pancreatic acinar cells may account for much of this metabolic adaptation (10). Which proteins of the two zinc transporter families (SLC30A and SLC39A) are involved in these processes is under investigation. The amount of endogenous zinc that enters the intestinal lumen is influenced by diseases of the gastrointestinal tract, particularly those causing changes in transit time or fluid secretion. Because pancreatic secretions are believed to provide the bulk of the endogenous zinc losses (see reviews in the literature [20,45]), pancreatic disease may influence zinc bioavailability. Substantial zinc is contained within Paneth cells. Their zinc transporter activity and contribution to endogenous secretion, particularly during gastrointestinal infection (49,50), have not been investigated. Intestinal endogenous losses are up to 5 mg/day, but are markedly reduced with zinc restriction. The wealth of data with stable isotopes of zinc were used to measure endogenous losses in humans and to calculate the estimated average requirement to establish the RDA for zinc (20). Nutrition intake data indicate that the risk for low zinc intake in North America and Europe is low, but, globally, more than half of the world’s population does not ingest enough zinc (51). Intestinal disease can influence zinc absorption and produce signs of zinc deficiency. Crohn’s disease is an inflammatory bowel disease that causes increased excretion, concomitant decreased absorption, and body zinc redistribution (52). Signs of enteritis are reduced with supplemental zinc (25 mg/day) (53). Abnormalities of zinc metabolism, usually including low plasma zinc concentrations, have been reported in patients with celiac sprue, short bowel syndrome, and gastric bypass surgery. This may be a reflection of depressed zinc absorption (54). Secretory diarrhea associated with intestinal tract infection (55) or AIDS (56) can lead to zinc loss, which further depresses the immune system. Remarkable reductions in childhood morbidity caused by diarrheal disease have been made through zinc supplementation for such patients (55). These efforts may point to a beneficial effect of zinc on mucosal immunity, particularly for the intestinal epithelium. The autosomal recessive trait acrodermatitis enteropathica is a zinc-responsive disorder (57). It presents as skin lesions, mental problems, and immune dysfunction that start when children are weaned from breast milk. The decreased bioavailability of zinc from food compared with human milk

1998 / CHAPTER 79 has been proposed as the cause for the appearance of signs at weaning (45). Patients with acrodermatitis enteropathica show decreased 65Zn absorption, and supplemental zinc provides remission of acrodermatitis enteropathica lesions (57). The acrodermatitis enteropathica defect is produced by a mutation in ZIP-4 (58). ZIP-4 has been shown to be a zinc importer protein and is present in intestinal cells (10,46). Zinc deficiency up-regulates ZIP-4 (10,58). The protein is located at the plasma membrane of enterocytes. Although the intestine has not been universally accepted as the site of the acrodermatitis enteropathica defect, the response of ZIP-4 to zinc intake is convincing.

OTHER TRACE ELEMENTS The cell and molecular biology of copper, iron, and zinc absorption has advanced impressively. Less attention has been given to understanding the mechanisms of absorption for other trace elements, that is, those that are required in smaller amounts in the diets of adult humans (<1 mg/day). For this chapter, only selenium, chromium, and manganese from that group are considered.

Selenium Selenium is present as selenomethionine or selenocysteine in plants and selenocysteine in animal tissues. The latter is the form that accounts for the biological function of this micronutrient as a component of numerous selenoproteins. Foods tend to have selenium contents that reflect the selenium levels in the soils where they originated (59). Inorganic forms of selenium, for example, SeO42+ (selenate) and SeO32+ (selenite), are well absorbed, but not as well retained as the selenoamino acids. Inorganic forms are used in fortification and for supplements. The mechanism of the uptake and transport of selenium has not been well established. Uptake by enterocytes is believed to be saturable. Selenite uptake, using brush-border vesicles from rat intestine as a model, is relatively high (60,61). It has been proposed that this inorganic form may be converted to a glutathione- or cysteine-containing intermediate. Because selenocysteine and selenomethionine are well absorbed, it has been proposed that various amino-acid transporters are involved in their absorption (12,59). Amino-acid transporter 1 (SLC3A1) and neutral amino-acid transporter (SLC1A4) are two transporters that were proposed. Transcellular movement would logically be similar to amino acids. Presumably, the selenoamino acids would use basolaterally localized transporters for transfer to the portal blood supply. As might be expected, these transporters do not respond to selenium status, and homeostatic control of selenium metabolism is not at the level of absorption. Considering the important role of selenium enzymes in antioxidant defense and the efficient absorption of selenocompounds, details of the mechanisms of transport need to be defined.

Chromium Chromium is widely distributed in foods. Stomach acid converts chromium to the Cr3+ valence state. Absorption of inorganic chromium salts is low (up to 2%). Absorption of organic chromium complexes may be somewhat greater (to 5%) (62). It has been proposed that organic forms of chromium are absorbed intact (63). The low extent of absorption as chromium picolinate, a synthetic organic chromium compound used as a supplement in humans, may be beneficial, because it causes sterility and lethal mutations in Drosophila (64). Details of absorption pathways and their kinetics for chromium have not been studied. Similarly, transporters that might participate in influx into/efflux from enterocytes have not been identified. Chromium binds to one of the two metal sites of transferrin, and most plasma chromium is transferrin bound. This suggests that chromium metabolism may use some pathways associated with iron.

Manganese Food sources of manganese are mostly from plants. Absorption is low, and much of that absorbed is rapidly returned to the intestinal lumen via the bile (20). Electrophysiologic studies suggest that manganese is transported across the apical surface of enterocytes by the iron uptake transporter DMT1 (SLC11A2) (11). One study suggests that intestinal manganese transport involves active transport (65), but other research suggests mechanisms including passive diffusion are involved (66). Export from enterocytes may involve the pathways of iron export and binding to apotransferrin. Portal blood manganese primarily is bound to transferrin, which may suggest absorption pathways for manganese similar to that used by iron.

Genetic Variation in Absorption Mutations have been documented in specific transporter genes that produce defects in the absorption of both copper and zinc. Searches of the genome for human trace element transporter genes show that polymorphisms are common. Some of these undoubtedly will be shown to produce phenotypic differences in the trace element absorption potential among individuals.

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31. Yuzbasiyan-Gurkan V, Grider A, Nostrant T, Cousins RJ, Brewer GJ. Treatment of Wilson’s disease with zinc: X. Intestinal metallothionein induction. J Lab Clin Med 1992;120:380–386. 32. Cotzias GC, Papavasiliou PS. Specificity of zinc pathway through the body: homeostatic considerations. Am J Physiol 1964;206: 787–792. 33. Foster DM, Aamodt RL, Henkin RI, Berman M. Zinc metabolism in humans: a kinetic model. Am J Physiol 1979;237:R340–R349. 34. Sturniolo GC, Montino MC, Rossetto L, Martin A, D’Inca R, D’Odorico A, Naccarato R. Inhibition of gastric acid secretion reduces zinc absorption in man. J Am Coll Nutr 1991;10:372–375. 35. Han O, Failla ML, Hill AD, Morris ER, Smith JC Jr. Inositol phosphates inhibit uptake and transport of iron and zinc by a human intestinal cell line. J Nutr 1994;124:580–587. 36. Zheng JJ, Mason JB, Rosenberg IH, Wood RJ. Measurement of zinc bioavailability from beef and a ready-to-eat high-fiber breakfast cereal in humans: application of a whole-gut lavage technique. Am J Clin Nutr 1993;58:902–907. 37. Cousins RJ. Zinc. In: Filer LJ, Ziegler EE, eds. Present knowledge in nutrition. 7th ed. Washington, DC: ILSI Press, 1996;293–306. 38. Solomons NW, Cousins RJ. Zinc. In: Solomons NW, Rosenberg IH, eds. Absorption and malabsorption of mineral nutrients. New York: Alan R. Liss, 1984;125–197. 39. Cousins RJ. Theoretical and practical aspects of zinc uptake and absorption. In Laszlo JA, Dintzis FR, eds. Mineral absorption in the monogastric GI tract: chemical, nutritional and physiological aspects. New York: Plenum Press, 1989;3–12. 40. Lönnerdal B. Intestinal absorption of zinc. In Mills CF, ed. Zinc in human biology. London: Springer-Verlag, 1989;33–55 41. Reyes JG. Zinc transport in mammalian cells. Am J Physiol 1996; 270:C401–C410. 42. Lee HH, Prasad AS, Brewer GJ, Owyang C. Zinc absorption in human small intestine. Am J Physiol 1989;256:G87–G91. 43. Condomina J, Zornoza-Sabina T, Granero L, Polache A. Kinetics of zinc transport in vitro in rat small intestine and colon: interaction with copper. Eur J Pharm Sci 2002;16:289–295. 44. Steinhardt HJ, Adibi SA. Interaction between transport of zinc and other solutes in human intestine. Am J Physiol 1984;247:G176–G182. 45. King JC, Cousins RJ. Zinc. In: Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ, eds. Modern nutrition in health and disease. 10th ed. Baltimore: Lippincott Williams & Wilkins, 2005;271–285. 46. Kim BE, Wang F, Dufner-Beattie J, Andrews GK, Eide DJ, Petris MJ. Zn2+-stimulated endocytosis of the mZIP4 zinc transporter regulates its location at the plasma membrane. J Biol Chem 2004;279: 4523–4530. 47. McMahon RJ, Cousins RJ. Regulation of the zinc transporter ZnT-1 by dietary zinc. Proc Natl Acad Sci U S A 1998;95:4841–4846. 48. Smith KT, Failla ML, Cousins RJ. Identification of albumin as the plasma carrier for zinc absorption by perfused rat intestine. Biochem J 1979;184:627–633. 49. Porter EM, Bevins CL, Ghosh D, Ganz T. The multifaceted Paneth cell. Cell Mol Life Sci 2002;59:156–170. 50. Kelly P, Feakins R, Domizio P, Murphy J, Bevins C, Wilson J, McPhail G, Poulsom R, Dhaliwal W. Paneth cell granule depletion in the human small intestine under infective and nutritional stress. Clin Exp Immunol 2004;135:303–309. 51. Brown KH, Weuhler SE, eds. Zinc and human health: the results of recent trials and implications for program interventions and research. Ottawa, Ontario, Canada: Micronutrient Initiative, 2000. 52. Matsui T. Zinc deficiency in Crohn’s disease. J Gastroenterol 1998;33:924–925. 53. Sturniolo GC, Di Leo V, Ferronato A, D’Odorico A, D’Inca R. Zinc supplementation tightens “leaky gut” in Crohn’s disease. Inflamm Bowel Dis 2001;7:94–98. 54. Polese L, Angriman I, Scarpa M, Norberto L, Sturniolo GC, Cecchetto A, Ruffolo C, D’Amico DF. Role of CD40 and B7 costimulators in inflammatory bowel diseases. Acta Biomed Ateneo Parmense 2003;74(suppl 2):65–70. 55. Black RE. Zinc deficiency, infectious disease and mortality in the developing world. J Nutr 2003;133:1485S–1489S. 56. Kupka R, Fawzi W. Zinc nutrition and HIV infection. Nutr Rev 2002; 60:69–79. 57. Aggett PJ. Severe zinc deficiency. In Mills CF, ed. Zinc in human biology. London: Springer-Verlag, 1989;259–279. 58. Dufner-Beattie J, Wang F, Kuo YM, Gitschier J, Eide D, Andrews GK. The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific,

2000 / CHAPTER 79 59. 60. 61. 62.

zinc-regulated zinc transporter in mice. J Biol Chem 2003;278: 33474–33481. Food and Nutrition Board, Institute of Medicine, ed. Dietary reference intakes for vitamin C, vitamin E, selenium, and carotenoids. Washington, DC: National Academy Press, 2000. Vendeland SC, Deagen JT, Butler JA, Whanger PD. Uptake of selenite, selenomethionine and selenate by brush border membrane vesicles isolated from rat small intestine. Biometals 1994;7:305–312. Vendeland SC, Deagen JT, Whanger PD. Uptake of selenotrisulfides of glutathione and cysteine by brush border membranes from rat intestines. J Inorg Biochem 1992;47:131–140. Stoecker BJ. Chromium. In: Shils ME, Olson JA, Shike M, Ross AC, eds. Modern nutrition in health and disease. 9th ed. Baltimore: Williams & Wilkins, 1999;277–282.

63. Olin KL, Stearns DM, Armstrong WH, Keen CL. Comparative retention/absorption of 51chromium (51Cr) from 51Cr chloride, 51Cr nicotinate and 51Cr picolinate in a rat model. Trace Elem Electrolytes 1994;11:182–186. 64. Hepburn DD, Xiao J, Bindom S, Vincent JB, O’Donnell J. Nutritional supplement chromium picolinate causes sterility and lethal mutations in Drosophila melanogaster. Proc Natl Acad Sci U S A 2003;100:3766–3771. 65. Garcia-Aranda JA, Wapnir RA, Lifshitz F. In vivo intestinal absorption of manganese in the rat. J Nutr 1983;113:2601–2607. 66. Bell JG, Keen CL, Lonnerdal B. Higher retention of manganese in suckling than in adult rats is not due to maturational differences in manganese uptake by rat small intestine. J Toxicol Environ Health 1989;26:387–398.

Index A ABCA1, gallbladder cholesterol metabolism, 1547–1548 ABCG5/G8 gallbladder cholesterol metabolism, 1548 intestinal cholesterol transport, 1725–1727 sitosterolemia role, 1726 Abdominal pain, see Irritable bowel syndrome; Visceral hypersensitivity Abetalipoproteinemia, see Microsomal triglyceride transfer protein ACAT2, see Acyl CoA cholesterol acyltransferase 2 Ace, secretory function effects, 1168–1169 Acetylcholine bile formation regulation, 1519 gallbladder emptying regulation, 844 gastric acid secretion regulation, 1243–1244 pancreas electrolyte regulation, 1387, 1390 presynaptic inhibition mediation in enteric nervous system, 657 stress effect mediation in mucosa, 770 Acetylcholine receptor gastric expression, 1232–1233 knockout mouse mutants, 1303 ligand binding, 1232 signaling, 1233 structure, 1232 N-Acetyltransferase, phase II metabolism, 1489 Acid secretion, see Gastric acid Acinar cell endocytosis, 1362 protein synthesis and trafficking endoplasmic reticulum to Golgi transport, 1323–1324 folding, 1322–1323 Golgi to trans-Golgi network transport, 1324–1326 misfolding and degradation, 1323 overview, 1319, 1321 signal peptide cleavage, 1322 trans-Golgi network to zymogen granule transport, 1326 receptors apical membrane expansion and retrieval, 1332–1333 calcium signaling agonist studies, 1341, 1343 cell–cell communication, 1345 clearance, 1349–1350 cytosolic, 1327–1329 diacylglycerol, 1350 inositol phosphate signaling, 1339–1341, 1345–1347 nicotinic acid dinucleotide phosphate-induced release, 1349 nuclear, 1329–1330 ryanodine receptor-induced release, 1347–1349 spatial properties, 1343 temporal properties, 1341, 1343 cyclic AMP regulation, 1351–1352 cyclic GMP regulation, 1352–1353 exocytosis, 1331–1332, 1358–1362 G protein-coupled receptors, 1326, 1338 mechanisms, 1330–1331 membrane effectors, 1339, 1354–1358 protein extrusion, solubilization, and delivery into duct system, 1333 protein phosphorylation, 1357–1358 second messenger interactions, 1353 secretory function, 1355–1356 Actin acinar cell exocytosis role, 1361 transport meditation in polarized epithelial cells, 1614 vesicular trafficking in gastric acid secretion, 1210 Acyl CoA cholesterol acyltransferase 2, intestinal cholesterol absorption regulation, 1728

Adaptive immunity definition, 1033 Helicobacter pylori, 1098 innate immunity comparison, 1033–1034 Adenosine presynaptic inhibition mediation in enteric nervous system, 657 vasodilation induction in intestine, 1639 Adenosine monophosphate, secretory function, 1173 Adenosine receptors, see Purinergic receptors Adherens junctions, pathogen receptors, 1181–1182 Adhering zonule, pancreas, 1318–1319 Adhesion molecules cadherins, 1142 cellular adhesion molecules, 1140 endothelial–blood cell interaction mediation, 1143–1145 integrins, 1141–1142 leukocyte movement through interstitium, 1151–1152 platelet glycoproteins, 1142–1143 selectins, 1138–1140 table, 1138 Aging adaptation bowel resection, 423–424 fasting and sepsis, 423 angiodysplasia, 425 definition, 405 demographics, 405 esophagus Barrett’s esophagus, 407 prospects for study, 407 structural and functional properties, 406–407 gastrointestinal cancer studies carcinogen susceptibility, 417–418 epidemiology, 414–415 mutational events, 418–419 premalignant events apoptosis inhibition, 416–417 epithelial growth and renewal, 415–416 prospects for study, 419–420 hypopharyngeal intrabolus pressure effects, 898 intestine contractility and motility, 412–413 fecal incontinence, 413–414 gut peptides, 412 immunologic effects, 412 prospects for study, 414 structural and functional properties, 411–412 mucosal growth regulation gene expression patterns, 423 growth factors and protein kinases, 421–423 intracellular mechanisms, 420 nutritional and hormonal factors, 420–421 prospects for study, 423 stomach mucosal injury and repair, 408–410 prospects for study, 411 secretion and digestion, 410–411 structural and functional properties, 407–408 surgical diseases of gut, 424–425 AH-type neuron action potentials, 645 cellular physiology, 644 metabotropic signal transduction, 650–652 mucosal projections, 642 neuronal gating function, 642–644 resting membrane potential, 644–645 slow excitatory postsynaptic potentials, 648–652 terminology, 644 Akt cell growth signaling, 443–444 target genes, 444 Alcohol dehydrogenase, phase I metabolism, 1487–1488 Aldehyde dehydrogenase, phase I metabolism, 1488

I-1

Aldosterone, chloride–bicarbonate exchange regulation, 1886 Amidation, protein processing, 47–48 Amino acids, see also Protein digestion and absorption basolateral membrane transport systems A, 1677–1678 Asc, 1680 glycine transporter 1, 1678 L, 1679–1680 y+, 1679 y+L, 1680 cell growth regulation, 450 cholangiocyte transport, 1512 enterocyte transport systems ASC, 1674–1675 β, 1674 B0, 1671–1673 b0,+, 1673 B0,+, 1673 IMINO, 1673–1674 overview, 1671–1672 PAT, 1675 − , 1674 XAG gallbladder transport, 1549 genetic disorders of transport, 1681–1683 ontogenic regulation of transport, 383 pancreatic secretion regulation, 1402, 1404 regulation of transport developmental regulation, 1684–1685 dietary regulation, 685 diurnal rhythm, 1686 hormonal regulation, 1685–1686 sodium–proton exchanger regulation, 1686 Aminopeptidases, protein processing, 46–47 AMP, see Adenosine monophosphate Amphiregulin discovery, 198 expression, 198 function, 198 structure, 198 Amylin, food intake regulation, 884 Anal canal defecation integrated control by enteric nervous system, 681 rectoanal reflex, 680 sensory innervation, 679 spinal motor control, 680–681 supraspinal motor control, 681 Anaphylaxis, afferent sensitivity effects, 711 Angiodysplasia, aging, 425 Angiogenins, innate immunity, 1055 Angiotensin II pancreas electrolyte regulation, 1388 vasoconstriction induction in small intestine, 1631–1632 Anoikis, characteristics, 363–364 Antral pump cycles, 667 neural control, 667–668 APC β-catenin binding, 248–249 destruction complex regulation, 256–258 mutation in familial adenomatous polyposis, 248 Apolipoprotein, gallbladder cholesterol metabolism, 1546 Apolipoprotein A-I, intestinal lipoprotein biogenesis role, 1723–1724 Apolipoprotein A-IV, intestinal lipoprotein biogenesis role, 1723–1724 Apolipoprotein B domains, 1713–1714 familial hypobetalipoproteinemia mouse models, 1719–1720 mutations, 1717–1718 phenotypic mechanisms, 1717, 1719

I-2 / INDEX Apolipoprotein B (Continued) lipoprotein assembly first step, 1714–1715 overview, 1714–1715 presecretory degradation, 1715 primordial lipoprotein particle formation, 1715–1716 second step, 1716 messenger RNA editing functional importance, 1723 molecular machinery and tissue-specific regulation, 1722 secretion rates, 1715 structure, 1712–1714 Apoptosis accessory molecules, 346–347 characteristics versus necrosis, 345–346 effectors, 346 gastric mucosa ethanol induction, 366 Helicobacter pylori induction, 364–365 nonsteroidal anti-inflammatory drug induction, 365–366 hepatocytes, 1493–1494 insulin-like growth factor-I inhibition, 209 intestinal epithelium anoikis, 363–364 cell studies, 358–363 induced apoptosis inducers, 351–352, 440 ischemia–reperfusion injury, 354 mechanisms, 352–353 small bowel resection, 353–354 pathways and regulation, 358–362 polyamine depletion effects, 359–360 prevention cytokines, 357–358 growth factors, 355–356 lysophosphatidic acid, 357 polyamines, 356–357 prostaglandins, 356 spontaneous apoptosis crypt, 350–351 villus, 351 pathways extrinsic pathway, 347 intrinsic pathway, 348–349 overview, 347, 437–438 regulators, 347 Appetite food intake and energy balance, 877 hypothalamic systems in feeding control, 878–881 interactions between gut peptide and hypothalamic signaling, 887–888 leptin and food intake regulation, 878 peptide YY effects, 147 within-meal feedback signaling across-meal peptide signaling, 885–887 gut neural signaling, 882–883 gut peptide signaling, 883–884 overview, 881–882 AP proteins, vesicle budding in polarized epithelial cells, 1607–1609 Aquaporins, see also Water transport angiogenesis and cell migration role, 1834–1835 AQP3 function in skin, 1834 AQP7 function in fat cells, 1834 brain swelling and neural signal transduction role, 1833 cholangiocytes, 1512–1513 corneal edema and transparency role, 1834 fluid transport in organs colon, 1840–1841 gallbladder, 1838 liver, 1837–1838 salivary gland, 1835–1836 small intestine duodenum, 1839 ileum, 1839–1840 jejunum, 1839–1840 stomach, 1836–1837 gallbladder, 1540 gastrointestinal tract expression, 1835 kidney tubule function near-isomolar fluid absorption and secretion, 1833 urine concentrating, 1833

Aquaporins (Continued) pancreas electrolyte secretion, 1381, 1384 prospects for study, 1841 selectivity, 1832 Arfs Arf1 and vesicle budding in polarized epithelial cells, 1607 Arf6 and vesicular trafficking in gastric acid secretion, 1216 G protein-coupled receptor signaling, 72 Arginine vasopressin, pancreas electrolyte regulation, 1390–1391 Argosome, Wnt transport, 249 Arrestins, G protein-coupled receptor desensitization, 77–81 Ascorbic acid, see Vitamin C ATP bile formation regulation, 1521–1522 organic anion transporter regulation, 1467 pancreas electrolyte regulation, 1387–1388, 1391 sensory signaling role, 748 sympathetic neuron neurotransmission in blood vessels, 821–822 ATP-binding cassette transporters, liver detoxification, 1490–1492 Axin, destruction complex regulation, 254–255 B BabA, Helicobacter pylori virulence factor, 1102–1103 Bacteroides fragilis, barrier function effects, 1176 Barrett’s esophagus, aging, 407 Base excision repair, genetic instability in cancer, 489–490 Bcl-2, apoptosis regulation, 347 Belching, see Pharyngeal motor function BETA2, Notch regulation, 298 Betacellulin, features, 199 Bicarbonate chloride exchange, see Chloride–bicarbonate exchanger corticotrophin-releasing hormone stimulation small intestine, 802–803 stomach, 798 gastroduodenal epithelium protection cystic fibrosis paradox, 1268–1269 duodenal secretion, 1270–1273 gastric secretion, 1269–1270 principles, 1268 Bile acids chloride–bicarbonate exchange regulation, 1885 concentration in gallbladder, 1450–1451 enterohepatic circulation anatomy, 1444–1445 bacterial biotransformation during recycling, 1441–1443 efficiency, 1446 transport, 1444, 1446 functions colon, 1455 overview, 1437–1438 small intestine, 1454–1455 intestinal absorption and transport ileal basolateral transport, 1452 ileal sodium-dependent uptake, 1451–1452 overview, 1451 regulation bile acids, 1453–1454 development, 1452–1453 gene expression, 1452 inflammation effects, 1454 ontogenic development of absorption, 391 pancreatic secretion regulation, 1403 physical chemistry, 1443–1444 secretion canalicular bile flow, 1447 overview, 1446–1447 structure–physiochemical relations, 1444 synthesis, 1438–1441 therapeutic applications agonists, 1455 antagonists, 1456 transport canalicular transport overview, 1450, 1471 regulation, 1472–1473 trafficking of transporters, 1473–1474 transporters, 1471–1472

Bile acids (Continued) cholehepatic shunt pathway, 1450 ductal modifications, 1450 hepatocyte sinusoidal membrane transport sodium-dependent uptake and regulation, 1448–1449, 1469–1471 sodium-independent uptake, 1449 transporters, 1449 overview, 1447–1448 transporters, 1444, 1446 Bile duct architecture, 1506 cholangiocyte, see Cholangiocyte microscopic anatomy, 1506–1507 Bile formation, see Bile acids; Cholangiocyte Bile salts export pump and detoxification, 1491 gallbladder transport, 1549 mucin secretion regulation in gallbladder, 1550–1551 Bilirubin gallbladder transport, 1548–1549 organic anion transporter regulation, 1468 Biotin functions, 1802 intestinal transport mechanism, 1802–1804 molecular components, 1804–1805 regulation, 1805–1807 Blood flow, see Gastrointestinal circulation BMPs, see Bone morphogenetic proteins Bombesin bile formation regulation, 1518–1519 cell growth regulation, 452–453 pancreas electrolyte regulation, 1389 satiety signaling, 884 BON cell, see Enterochromaffin cell Bone morphogenetic proteins mutation and enteric nervous system lesions, 509–510 receptors, 326 stem cell regulation, 326–327 Bowel resection adaptation in aging, 423–424 apoptosis induction, 353–354 small-bowel resection and gastric acid secretion, 1246 Brain, see Sensory signals, brain processing Brainstem, gastric function control acid secretion, 856 efferent autonomic overlay, 852–854 history of study, 851–852 motility, 855–856 vago-vagal control reflex components and characteristics circulating factors, 864 corticotrophin-releasing hormone activation of vagal nonadrenergic, noncholinergic path, 862 descending central nervous system pathways and circulating agents, 860–861 dorsal vagal complex chemosensitivity, 864–866 dorsal vagal complex inputs from Raphe nuclei, 861 gastric accommodation reflex, 859–860 nucleus of the solitary tract, 857–858 oxytocin inputs to dorsal vagal complex from paraventricular nucleus, 861–862 receptive relaxation reflex, 858–859 short-term receptor trafficking and plasticity, 863–864 tumor necrosis factor-α regulation, 866–869 vagal efferent neurons in dorsal motor nucleus of vagus nerve, 856–857 visceral afferent inputs to brainstem, 854 C Cadherins, types and functions, 1142 cag pathogenicity island CagA and barrier function effects, 1178–1179 Helicobacter pylori virulence factor, 1099–1101 Calcitonin gene-related peptide, primary afferent neuron neurotransmission in blood vessels, 827–828 Calcium dietary requirements, 1953–1954 intestinal absorption diffusion, 1962 extrusion across basolateral membrane, 1962–1963

INDEX / I-3 Calcium (Continued) overview, 1954–1957 paracellular pathway, 1957–1958 regulation calcium, 1964 calmodulin, 1966 estrogens, 1964 growth hormone, 1966–1967 parathyroid hormone, 1965 protein–protein interactions, 1965–1966 S100A10, 1966 stanniocalcin, 1964 thyroid hormone, 1964 vitamin D, 1963–1964 transcellular pathway enterocyte uptake, 1958–1959 transient receptor potential family proteins, 1959–1962 intestinal transport diffusion, 1762 extrusion at basolateral surface, 1762 knockout mouse studies, 1762–1763 overview, 1761 TRPV ion channels, 1761–1762 vitamin D regulation of transporter expression, 1763–1764 ontogenic development of absorption, 386 pancreas electrolyte regulation, 1388 pancreatic secretion regulation, 1403–1404 Calcium-activated chloride channels, see Chloride channels Calcium/calmodulin-activated kinases acinar cell secretory function, 1355–1356 calcium-activated chloride channel activation, 1920 Calcium channels extrinsic sensory afferent nerve voltage-gated calcium channels, 695–696 smooth muscle responses to slow waves and neural inputs L-type calcium channels, 551, 555–556 T-type calcium channels, 556 Calcium flux acinar cell signaling agonist studies, 1341, 1343 cell–cell communication, 1345 clearance, 1349–1350 cytosolic, 1327–1329 diacylglycerol, 1350 inositol phosphate signaling, 1339–1341, 1345–1347 nicotinic acid dinucleotide phosphate-induced release, 1349 nuclear, 1329–1330 ryanodine receptor-induced release, 1347–1349 spatial properties, 1343 temporal properties, 1341, 1343 cholangiocyte signaling, 1514–1515 mucin secretion regulation in gallbladder, 1550 Calcium-sensing receptors gastric expression, 1234 ligand binding, 1233 signaling, 1234 structure, 1233–1234 Calmodulin, calcium absorption regulation, 1966 cAMP, see Cyclic AMP Cancer, gastrointestinal, see also specific cancers aging studies carcinogen susceptibility, 417–418 epidemiology, 414–415 mutational events, 418–419 premalignant events apoptosis inhibition, 416–417 epithelial growth and renewal, 415–416 prospects for study, 419–420 cardinal features growth inhibitory signaling defects, 482–484 overview, 481 proliferation signal self-sufficiency, 481–482 unlimited replication, 484–485 carcinogen exposure, 479 gene mutations, 477–478 genetic instability base excision repair, 489–490 chromosomal instability, 486–487 epigenetic mechanisms, 9, 490–492 microsatellite instability, 487–489 overview, 485–486

Cancer (Continued) Hedgehog signaling defects, 282–283 heredity, 478–479 inflammation role, 479–480 multistep carcinogenesis, 480–481 sporadic carcinogenesis, 480 transforming growth factor-β role, 191–192 Wnt/β-catenin signaling defects esophagus, 263 familial adenomatous polyposis, 262–263 liver, 264 pancreas, 264 sporadic colorectal cancer, 261–262 stomach adenocarcinoma, 263–264 adenoma, 263 fundic gland polyps, 263 Carbohydrate forms in diet, 1653 sugar absorption, see Glucose transporters; Sodium/glucose cotransporters Carbonic anhydrase, pancreas electrolyte secretion, 1384 Carboxypeptidases, protein processing, 45–46 Carotenoids dietary sources, 1737 intestinal absorption carotenoid interactions, 1744 differential transport, 1743–1744 kinetics of transport, 1742–1743 overview, 1741–1742 proteins, 1748 stereoselectivity, 1743 retinoid conversion cleavage enzymes, 1739–1740 nutritional factor regulation, 1740 solubilization, 1738–1739 synthesis, 1736 types and structures, 1735–1736 Caspases, apoptosis regulation, 346 Catalase, antioxidant protection, 1494 β-Catenin, see Wnt/β-catenin signaling Cathelicidins, innate immunity, 1052–1053 Caveolae, protein sorting in polarized epithelial cells, 1611–1612 CCK, see Cholecystokinin CD36, fatty acid uptake, 1698–1699 CD44, stem cell function regulation, 321 Cdx genes developmental regulation of gastrointestinal function, 393 stem cell function regulation, 319–320 Celiac disease, intestinal epithelial tight junction barrier and permeability index, 1583–1584 Cell cycle growth inhibitory signaling defects in cancer, 482–484 regulation, 437 Cell migration, see Wound healing CFTR, see Cystic fibrosis transmembrane conductance regulator Chagas’ disease, disinhibitory motor disorders, 641 ChIP, see Chromatin immunoprecipitation Chloride absorption chloride–bicarbonate exchange, see Chloride–bicarbonate exchanger electroneutral sodium chloride absorption, 1882–1883, 1936–1938 intestinal secretion regulation, 1932–1935 Chloride–bicarbonate exchanger basolateral membrane transport, 1203–1204 cholangiocytes, 1510 gallbladder, 1538 intestinal distribution and function, 1883–1884 knockout mice, 1306 membrane topology, 1888–1889 pancreas electrolyte secretion, 1380 regulation aldosterone, 1886 bile acids, 1885 infection, 1886 inflammation, 1885 nitric oxide, 1885–1886 protein kinase C, 1886–1887 reactive oxygen species, 1887 serotonin, 1886

Chloride–bicarbonate exchanger (Continued) short-chain fatty acids, 1884–1885 structure, 1893 SLC4 family features ammonium activation, 1892 expression regulation, 1891–1892 gene family, 1887–1888 pH effects, 1892 phosphorylative regulation, 1892 SLC4A1, 1889–1890 SLC4A2, 1890 SLC4A3, 1890–1891 SLC4A4, 1891 SLC26 family features gene family, 1892–1893 SLC26A2, 1898–1899 SLC26A3 expression, 1896 mutations in congenital chloride diarrhea, 1893–1896 protein–protein interactions, 1896–1897 regulation, 1900 SLC26A6 functions, 1897–1898 regulation, 1900 tissue distribution and expression, 1898 SLC26A7, 1899–1900 Chloride channels, see also Cystic fibrosis transmembrane regulator calcium-activated chloride channels ClC-2, 1919–1920 ClC-3, 1920 ClC-4, 1920 pancreas electrolyte secretion, 1382–1383 cholangiocytes, 1509–1510 smooth muscle responses to slow waves and neural inputs calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 Cholangiocyte bile formation regulation acetylcholine, 1519 adrenergic agonists, 1519–1520 amino acids, 1523 ATP, 1521–1522 bile acids, 1522 bile osmolarity and flow, 1523 bombesin, 1518–1519 corticosteroids, 1519 dopaminergic agonists, 1520 endothelin, 1520–1521 gastrin, 1520 glucose, 1522–1523 integrated model, 1524–1525 overview, 1515–1517 secretin, 1516, 1518–1521 somatostatin, 1520 vasoactive intestinal peptide, 1519 biochemical heterogeneity, 1507–1508 distribution, 1505 functional heterogeneity, 1524 morphology, 1506–1507 signaling calcium, 1514–1515 cyclic AMP, 1514 cyclic GMP, 1515 G protein-coupled receptors, 1514 purine receptors, 1515 transport systems amino acids, 1512 aquaporins, 1512–1513 bile acids, 1511 chloride–bicarbonate exchanger, 1510 chloride channels, 1509–1510 glucose transporters, 1511–1512 heterogeneity, 1513 organic ions, 1513 overview, 1508–1509 potassium channels, 1510–1511 proteins, 1513 proton-ATPase, 1511 sodium–bicarbonate cotransporter, 1511 sodium/potassium ATPase, 1511 sodium–potassium–chloride cotransporter, 1511 sodium–proton exchangers, 1510–1511 vesicular trafficking of proteins, 1523–1524

I-4 / INDEX Cholecystokinin aging effects in intestine, 412 assays, 102–103 excess and deficiency in disease, 106 functions gall bladder contraction, 105 gastric emptying, 105 gastric secretory effects, 105–106 overview, 100–101, 104 pancreas, 104–105 gallbladder emptying regulation, 843–844 gastrin-cholecystokinin double-knockout mice, 1301 gene structure and expression, 101 metabolism, 103 pancreas electrolyte regulation, 1389 pancreas secretion regulation, 1412–1415, 1418 postprandial hyperemia role, 1642–1643 posttranslational processing, 101 release, 103 satiety signaling, 883–884 structure, 101 tissue distribution, 101–102 vago-vagal control reflex regulation, 864 Cholecystokinin receptors gastric cell regulation, 1235 gastric expression, 1234–1235 ligand binding, 1234 overview, 96–97, 103–104 structure, 1234 vagal afferents, 636 Cholecystokinin-releasing factor, pancreas secretion regulation, 1418–1419 Cholehepatic shunt pathway, 1450 Cholera toxin secretion induction, 758 secretory function ontogeny studies, 377 Cholestasis bile canalicular transport effects, 1473 hepatocyte bile acid transport effects, 1471 Cholesterol gallbladder absorption and efflux cholesterolosis, 1545–1546 crystallization and inflammation, 1552 history of study, 1544–1545 mechanisms ABCA1, 1547–1548 ABCG5, 1548 ABCG8, 1548 apolipoproteins, 1546 cubulin, 1548 megalin, 1548 scavenger receptor type B class I, 1546–1547 oxysterol formation, 1553 intestinal absorption, 1711–1712, 1727–1728 intestinal transport, 1724–1728 Chromatin, see Epigenetics Chromatin immunoprecipitation, principles, 18 Chromium, absorption and transporters, 1998 Chromogranin A, enterochromaffin-like cell secretion, 1230 Chromosomal instability, cancer, 486–487 Chronic intestinal pseudo-obstruction, disinhibitory motor disorders, 640–641 Chymotrypsin, protein digestion, 1669 Ciliary neurotrophic factor, receptor mutation and enteric nervous system lesions, 509 Cingulate cortex, gastrointestinal sensory signal processing, 730, 734 Circulation, see Gastrointestinal circulation Clathrin G protein-coupled receptor endocytosis regulation, 81–82 secretory vesicle formation, 42 vesicle budding in polarized epithelial cells, 1607 vesicular trafficking in gastric acid secretion, 1214–1215 Claudins, tight junctions, 1567–1568 Clostridium botulinum, barrier function effects C2 toxin, 1178 C3 toxin, 1178 overview, 1166 Clostridium difficile barrier function effects overview, 1166 toxins, 1176–1177 secretion pathobiology, 759–760

Clostridium difficile (Continued) secretory function effects inflammation and neuropeptide mediation, 1172 overview, 1164 Clostridium perfringens, barrier function effects enterotoxin, 1177–1178 overview, 1166 CNTF, see Ciliary neurotrophic factor Cobalamin forms, 1813 functions, 1813 gastrointestinal absorption cubulin expression regulation, 1818–1819 structure and function, 1817–1818 malabsorption acquired causes, 1815 inherited causes, 1815–1816 molecular components, 1816–1817 mechanism, 1813–1815 regulation of component expression, 1818–1819 Colitis, see Intestinal inflammation Colon corticotropin-releasing hormone modulation of function hypersensitivity, 808–809 inflammation, 807 motility, 804–807 permeability, 807 development, 295–296 integrated control by enteric nervous system defecation, 681 musculature, 670 peristaltic propulsion, 670–673 peristaltic retropropulsion, 673 physiologic ileus, 673 power propulsion, 674 motility, see Intestinal motility sodium–proton exchange, 1871–1873 water transport, 1840–1841 Colon cancer, vitamin D prevention, 1765–1766 Constipation colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 ion transport alterations, 1944 COPII, vesicle budding in polarized epithelial cells, 1606–1607 Copper absorption and transporters, 1995–1996 sequestration, 1498 Corticosteroids, bile formation regulation, 1519 Corticotropin-releasing factor binding protein, 795 central distribution hormone and functional significance, 796 receptors, 796–797 colonic function modulation hypersensitivity, 808–809 inflammation, 807 motility, 804–807 permeability, 807 gastric function modulation acid secretion inhibition, 797–798 bicarbonate secretion stimulation, 798 gastric emptying inhibition, 798–800 motility, 800–802 gastrointestinal motility modulation overview, 781–782 peripheral receptor and colonic motor alterations, 785 type 1 receptor activation and stimulation, 784 type 2 receptor activation and inhibition, 784 visceral hyperalgesia mediation by receptors, 786 receptors antagonists, 794–795 pharmacology, 794 structures and variants type 1 receptor, 793–794 type 2 receptor, 794 related peptides, 793–794 sequence homology between species, 792–793 small intestine function modulation

Corticotropin-releasing factor (Continued) anti-inflammatory action, 804 bicarbonate secretion stimulation, 802–803 jejunoileal absorption, 803 motility, 803–804 stress effect mediation in mucosa, 771 COX, see Cyclooxygenase CRF, see Corticotropin-releasing factor Crohn’s disease, see Inflammatory bowel disease Cryptdins, secretory function, 1172–1173 Cubulin expression regulation, 1818–1819 gallbladder cholesterol metabolism, 1548 structure and function, 1817–1818 Cyclic AMP acinar cell secretory function, 1351–1352 cholangiocyte signaling, 1514 gallbladder transport control, 1540–1541 hepatocyte bile acid transport regulation, 1470 mucin secretion regulation in gallbladder, 1550 pancreas electrolyte regulation, 1386–1387 secretion signaling, 744–745, 756–757 smooth muscle contraction regulation, 528–529 Cyclic GMP acinar cell secretory function, 1352–1353 cholangiocyte signaling, 1515 pancreas electrolyte regulation, 1387 Cyclooxygenase bicarbonate secretion regulation in duodenum, 1272–1273 chloride secretion mediation, 1172 Helicobacter pylori gastritis role, 1098–1099 intestinal inflammation role, 1120 Cystic fibrosis, bicarbonate paradox, 1268–1269 Cystic fibrosis transmembrane conductance regulator chloride–bicarbonate exchanger regulation, 1900–1901 experimental models, 1922 gallbladder, 1538–1539, 1551 intestinal channel diseases, 1918 structure and function, 1918–1919 pancreas electrolyte secretion, 1381–1382 pancreatic channel, 1919 secretory function ontogeny, 376–377 Cystinuria, pathophysiology, 1682 Cytochalasins, intestinal epithelial tight junction barrier regulation, 1569–1570 Cytochrome P450, phase I metabolism, 1484–1487 D D cell, see Somatostatin Defecation, integrated control by enteric nervous system, 681 Defensins α-defensins, 1049–1051 β-defensins, 1051–1052 Delta, Notch ligand, 289 Deltex, Notch regulation, 294 Dendritic cell antigen sampling, 765–766 intestinal inflammation role, 1116 Desert Hedgehog, see Hedgehog Desmosome, pancreas, 1318–1319 Detoxification, see Liver Diabetes mellitus dipeptidyl peptidase-IV inhibitors in treatment, 170 glucagon-like peptide-1 receptor antagonists for diabetes type 2 treatment, 169–170 Diarrhea congenital chloride diarrhea, 1893–1896 functional diarrhea and colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 ion transport alterations in infectious diarrhea, 1942–1943 Digestive function, ontogeny, 377–379 Dipeptidyl peptidase-IV, inhibitors in diabetes treatment, 170 Dishevelled, destruction complex regulation, 255–256 Disulfide bond, formation, 38–39 Divalent metal ion transporter 1, iron transport, 1984–1985

INDEX / I-5 Dkk proteins Dkk1 and stem cell function regulation, 321–322 Wnt/β-catenin signaling inhibition, 252 DMT1, see Divalent metal ion transporter 1 DNA, see Epigenetics; Gene DNA-binding proteins domains, 12–13 motifs, 13–14 phosphorylative regulation, 15 transactivation, 14–15 DNA microarray, principles, 19 Dopamine, pancreas secretion regulation, 1409 Dorsal vagal complex, see Brainstem Drug-induced colitis, clinical manifestations, 1123 Duodenal ulcer, gastric acid secretion, 1246 Dynamin G protein-coupled receptor endocytosis regulation, 81–82 vesicle scission in polarized epithelial cells, 1609–1610 vesicular trafficking in gastric acid secretion, 1216 Dynorphin, protein processing, 45 E ECL, see Enterochromaffin-like cell EGF, see Epidermal growth factor Elastase, protein digestion, 1669 Emesis, vagal afferents, 713 Endoplasmic reticulum posttranslational modifications of proteins disulfide bond formation, 38–39 folding, 39 glycosylation, 39 myristoylation, 39 protein trafficking, 39–41 protein transport overview, 35–36 signal peptide removal, 38 structure and function, 36 signal recognition particle, 36–37 signal recognition particle receptor, 37 translocon, 37–38 Endothelial cell activation products, 1145–1146 blood cell interactions adhesion molecule mediation, 1143–1145 auxiliary cells, 1146–1148 Endothelin B receptor, Hirschsprung’s disease mutations, 505–506 Endothelin bile formation regulation, 1520–1521 intestinal pathology endotoxic shock, 1633 gut strangulation, 1632–1633 Hirschsprung’s disease, 1633 inflammatory bowel disease, 1633 neonatal necrotizing enterocolitis, 1633 isoforms, 1632 postprandial hyperemia, see Postprandial hyperemia processing, 1632 receptors, 1632 vasoconstriction induction in small intestine, 1632–1633 Enteric nervous system AH-type neurons action potentials, 645 cellular physiology, 644 mucosal projections, 642 neuronal gating function, 642–644 resting membrane potential, 644–645 terminology, 644 blood vessel innervation, see Gastrointestinal circulation central nervous system interactions, 499–500 colonic contraction regulation enteric reflexes, 981–982 interneurons, 979 intrinsic sensory neurons, 979–981 motor neurons, 977–979 overview, 976–977 conceptual model, 630–631, 666 development crest-derived stem cells in bowel, 502–503 functions laminin, 511–512 netrins, 512–513 β-neurexin, 513–514 neuroglin, 513–514

Enteric nervous system (Continued) Notch signaling, 302 overview, 501–502 disinhibitory motor disorders Chagas’ disease, 641 chronic intestinal pseudo-obstruction, 640–641 paraneoplastic syndrome, 641 sphincteric achalasia, 641–642 extrinsic innervation of gut wall parasympathetic innervation, 591–592 sympathetic innervation, 592 extrinsic sensory nerve endings, see Extrinsic sensory afferent nerves fast excitatory postsynaptic potentials nicotinic receptors, 646 purinergic receptors, 646–647 rundown, 647 serotonin receptors, 647 significance, 648 functional overview, 577, 629 histoanatomy, 631–632 inhibitory postsynaptic potentials ionic mechanisms, 654 significance, 655 slow potential mediators, 654–655 integrated control, see Anal canal; Colon; Pelvic floor; Small intestine; Stomach interneurons ascending interneurons, 586–587 descending interneurons, 587–588 interplexus interneurons, 590–591 overview, 586, 636–637 intestinal inflammation effects, 1128–1129 intrinsic sensory neurons of gut wall functional implications, 583 myenteric intrinsic primary afferent neurons, 582–583 submucosal intrinsic primary afferent neurons, 582 late-acting factor defects and disease bone morphogenetic proteins, 509–510 ciliary neurotrophic factor receptor, 509 Mash1, 508–509 neurotrophin-3, 508 overview, 507 pathology, 511 serotonin receptors, 510–511 motor neurons circular muscle motor neurons, 584–585 excitatory musculomotor neurons, 638–639 inhibitory musculomotor neurons, 639–640 longitudinal muscle motor neurons, 585–586 overview, 583–584 secretomotor neurons excitatory receptors, 638 inhibitory input, 638 intestinal content liquidity control, 638 overview, 589–590, 637–638 specialized motor neurons, 586 neurochemical hierarchy, 594–595 neuron abundance, 629 neuron classification biophysical characteristics, 580–581 histochemistry, 580 morphology, 579–580, 632–633 plasticity gastric reflexes, 674 Hirschsprung’s disease, 674–675 histaminergic neuromodulation, 675–676 pattern generator, 677–678 presynaptic inhibitory modulation, 677 secretomotor control, 674 serotonergic neuromodulation, 676–677 presynaptic facilitation, 655–656 presynaptic inhibition mediators, 656–657 prevertebral ganglia, see Prevertebral sympathetic ganglia prospects for study, 595 secretory reflexes, see Secretory reflexes sensory function, see also Extrinsic sensory afferent nerves central processing, see Sensory signals, brain processing chemoreception cholecystokinin receptors on vagal afferents, 636 serotonin receptors in irritable bowel syndrome, 636

Enteric nervous system (Continued) mechanoreception high-threshold, 635 low-threshold, 635 visceral hypersensitivity in irritable bowel syndrome, 635–636 visceral pain, 635 overview, 633–635 slow excitatory postsynaptic potentials AH-type neuron, 648–652 mediators, 649–650 metabotropic signal transduction adenosine receptors, 652 AH-type neurons, 650–652 calcium influx suppression, 652 S-type neurons, 652–654 S-type neuron, 649 significance, 654 structural organization ganglia, 578 overview, 577–578 plexuses, 578 structural basis of intestinal motility, 579 structure–function relations with other anatomic ganglia, 632 S-type neurons action potentials, 646 general features, 646 viscerofugal neurons, 588–589 Enterochromaffin-like cell activation and growth regulation epinephrine, 1229 galanin, 1228 gastrin, 1227–1228 interleukin-1, 1229 norepinephrine, 1229 peptide YY, 1228 pituitary adenylate cyclase-activating peptide, 1228 prostaglandins, 1229 transforming growth factor-α, 1228 afferent sensitivity effects, 711–712 distribution, 1226 gastrin regulation, 98–99 Helicobacter pylori effects on function, 1231 morphology, 1226–1227 secretory products chromogranin A, 1230 fibroblast growth factor-2, 1231 gastrocalcin, 1231 histamine, 1229–1230 pancreastatin, 1230 regenerating protein 1α, 1231 transforming growth factor-α, 1231 WE-14, 1230–1231 transgenic mouse studies, 1307–1308 Enterocyte amino acid transport, see Amino acids fatty acid transport, see Fatty acids Ephrin system, stem cell function regulation, 317–318 Epidermal growth factor cell growth regulation, 447 developmental regulation of gastrointestinal function, 393 expression, 194–195 family definitive criteria, 193–194 functions, 195 gastric acid secretion signaling, 1208–1209 gene, 194 mucosal growth regulation in aging, 421–423 nonmitogenic activity, 197–198 peptide shedding, 200 receptors ErbB1, 200–201 ErbB2, 204 ErbB3, 204 ErbB4, 204 expression, 203–204 knockout mouse studies, 204–205 overview, 193, 200 signaling, 201, 203 therapeutic targeting, 205 transactivation, 205 wound healing regulation, 463 Epigen, features, 199–200

I-6 / INDEX Epigenetics cancer role, 9, 490–492 chromatin-binding proteins, 8–9 definition, 5 developmental gene regulation, 9 DNA methylation, 8 histone acetylation, 6–7 methylation, 7 phosphorylation, 7–8 structure, 5–6 Epinephrine, enterochromaffin-like cell regulation, 1229 Epiregulin, features, 199 Epithelial cell, see Polarized epithelial cell; Tight junctions ER, see Endoplasmic reticulum ErbB receptors, see Epidermal growth factor Erythromycin, motilin receptor binding, 140 Escherichia coli absorption and enteropathogenic Escherichia coli, 1171 barrier function effects cytotoxic necrotizing factors, 1174–1175 enterohemorrhagic Escherichia coli, 1180 enteropathogenic Escherichia coli, 1179–1180, 1182 overview, 1166 serine protease autotransporters of Enterobacteriaceae, 1175–1176 secretory function effects heat-stable enterotoxins, 1170 LTI, 1170 LTII, 1170 overview, 1164 Esophageal body, coordinated motor activity longitudinal muscle, 920–921 pressure isocontour map, 917–918 smooth muscle region, 918–920 striated muscle region, 917–918 Esophagus aging studies Barrett’s esophagus, 407 prospects for study, 407 structural and functional properties, 406–407 coordinated motor activity deglutition inhibition and refractoriness, 923 esophageal body longitudinal muscle, 920–921 pressure isocontour map, 917–918 smooth muscle region, 918–920 striated muscle region, 917–918 lower esophageal sphincter, 921–923 techniques for study, 916 upper esophageal sphincter, 916–917 Hedgehog signaling in development, 275–276 innervation extrinsic parasympathetic innervation, 914–915 extrinsic sympathetic innervation, 915 intramural innervation, 915–916 neuromuscular behavior esophageal body, 914 gross anatomy, 913–914 lower esophageal sphincter, 914 upper esophageal sphincter, 914 Estrogens calcium absorption regulation, 1964 organic anion transporter regulation, 1467 Ethanol, apoptosis induction in gastric mucosa, 366 Exenatide, diabetes type 2 treatment, 169–170 Exocrine pancreas, see Acinar cell; Pancreas Exocyst complex, exocytosis regulation in polarized epithelial cells, 1618 External anal sphincter defecation integrated control by enteric nervous system, 681 rectoanal reflex, 680 spinal motor control, 680–681 supraspinal motor control, 681 Extrinsic primary afferent nerves, intestinal inflammation effects, 1129 Extrinsic sensory afferent nerves development axonal guidance and survival molecules, 686–687 dorsal root ganglion, 686 jugular/nodose ganglion complex, 686

Extrinsic sensory afferent nerves (Continued) distribution and morphology of peripheral afferent terminals mucosa, 689 muscle, 690 serosa/mesentery, 690 electrophysiology afferent recording, 692–693 calcium-activated potassium channels, 695 dissociated cell studies, 693 hyperpolarization-activated currents, 695 voltage-gated calcium channels, 695–696 voltage-gated potassium channels, 694–695 voltage-gated sodium channels, 693–694 integrative physiology food intake regulation, 706–710 pathophysiology, 710–716 sensory function, see also Enteric nervous system chemical sensitivity G protein-coupled receptor signaling, 704–706 ligand-gated ion channels, 702–704 general properties, 696 mechanosensitivity measurement, 696–698 mechanisms, 699–701 mucosal afferents, 698 muscle afferents, 698–699 neurotransmitter release, 701–702 serosal afferents, 699 transient receptor potential channels, 700–701 modulators anaphylaxis, 711 enterochromaffin cells, 711–712 mast cells, 711 visceral pain and hypersensitivity, 713–716 spinal afferents central projections, 691–692 overview, 593–594 peripheral projections, 688–689 vagal afferents central projections, 690–691 food intake regulation, 706–710 overview, 592–593 pathophysiology, 710–716 peripheral projections, 687–688 F Familial adenomatous polyposis, see APC Familial hypobetalipoproteinemia, see Apolipoprotein B Fanconi–Bickel syndrome, GLUT2 mutations, 1662 Fatty acids, see also Short-chain fatty acids amides in food intake regulation, 708–709 delivery systems, 1694 enterocyte uptake CD36, 1698–1699 fatty acid transport proteins, 1696–1698 fatty-acid binding protein expression regulation, 1701–1702 isoforms, 1700 ligand binding, 1700–1701 plasma membrane protein, 1696 proximal intestine functions, 1702–1704 structure, 1700 intracellular transport, 1699–1700 overview, 1695–1696 prospects for study, 1704 facilitated membrane transfer, 1694–1695 uptake, 1693–1694 Fed motor pattern neural regulation, 947 neurohumoral mediators, 947 physiologic characteristics, 946–947 Ferritin, metal detoxification, 1498 FGFs, see Fibroblast growth factors Fiber, cell growth regulation, 450 Fibroblast growth factors classification, 216–217 enterochromaffin-like cell secretion of FGF-2, 1231 expression patterns, 217 functions angiogenesis, 220 cell growth regulation, 447–448 development, 219–220 knockout mouse studies, 219 tissue repair, 220 tumorigenesis, 221

Fibroblast growth factors (Continued) genes, 216 receptors ligands, 216 signaling, 218–219 structure, 217 types, 217 stem cell regulation, 327–330 Flavin monooxygenase, phase I metabolism, 1487 FldA, Helicobacter pylori virulence factor, 1103 fMRI, see Functional magnetic resonance imaging Folate functions, 1792 intestinal transport mechanism, 1792–1795 molecular components, 1795–1796 reduced folate carrier, 1795–1797 regulation, 1796–1797 Food allergy immunoglobulin E role, 1082 stress in pathogenesis, 775–776 Food intake, see Appetite Formins, exocytosis regulation in polarized epithelial cells, 1617–1618 Fox11 knockout mouse, 1306 stem cell function regulation, 322 Frizzled destruction complex regulation, 255–256 Wnt interactions, 249–252, 441 Fructose absorption, 1654–1656 malabsorption and GLUT5 mutations, 1662–1663 Functional diarrhea, colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 Functional magnetic resonance imaging, gastrointestinal sensory signal processing studies, 730–734 Furin, protein processing, 43 G Galactose, absorption, 1654–1656 Galanin enterochromaffin-like cell regulation, 1228 presynaptic inhibition mediation in enteric nervous system, 657 Galanin-1 receptor, secretory function, 1173 Gallbladder amino acid transport, 1549 bile acid concentrating, 1450–1451 bile salt transport, 1549 bilirubin transport, 1548–1549 cholecystokinin effects on contraction, 105 cholesterol absorption and efflux cholesterolosis, 1545–1546 crystallization and inflammation, 1552 history of study, 1544–1545 mechanisms ABCA1, 1547–1548 ABCG5, 1548 ABCG8, 1548 apolipoproteins, 1546 cubulin, 1548 megalin, 1548 scavenger receptor type B class I, 1546–1547 oxysterol formation, 1553 electrolyte and water transport aquaporins, 1540 chloride–bicarbonate exchanger, 1538 cystic fibrosis transmembrane conductance regulator, 1538–1539 gallstone formation roles bile acidification, 1543–1544 calcium, 1542–1543 potassium channels, 1539–1540 regulation cell volume control, 1542 cyclic AMP, 1540–1541 neural control, 1541–1542 sodium/potassium-ATPase, 1539 sodium–proton exchanger, 1537–1538 functional overview, 1535–1536 infection and inflammation, 1551–1553

INDEX / I-7 Gallbladder (Continued) morphology, 1536 mucin secretion regulation bile salts, 1550–1551 calcium flux, 1550 cyclic AMP, 1550 cystic fibrosis transmembrane conductance regulator, 1551 gallstone formation, 1551 overview, 1549–1550 transcriptional regulation, 1551 neural control emptying mechanisms acetylcholine, 844 cholecystokinin, 843–844 neurokinins, 844 filling, 844–845 ganglia and neurons, 841–843 interprandial motility, 845 protein absorption and secretion, 1551 secretory function, 1544 techniques for study, 1536–1537 water transport, 1838 GALT, see Gut-associated lymphoid tissue Gap junction, pancreas, 1319 Gastric accommodation caloric drink test, 929 definition, 928 pathophysiology, 931–932 reflex, 859–860 reflex control nitrergic neurons, 929–930 serotonin system, 930–931 triggering site, 929 Gastric acid aging effects, 410–411 apical channel proteins, 1202–1203 basolateral membrane transport chloride–bicarbonate exchanger, 1203–1204 conductance, 1205 homeostasis, 1203 sodium–potassium–2chloride cotransporter, 1205 sodium–proton exchanger, 1204–1205 brainstem control of secretion, 856 cell biology of secretion enterochromaffin-like cell, 1225–1231 parietal cell, 1225 concentration, 1189 corticotropin-releasing hormone and secretion inhibition, 797–798 disorders duodenal ulcer, 1246 gastritis, 1246 histamine overproduction, 1246 small-bowel resection, 1246 Zollinger–Ellison syndrome, 1246 gastric gland and cell isolation, 1205–1206 gastrin regulation, 98 parietal cell membrane recycling hypothesis, 1193–1194 morphology and ultrastructure, 1191–1193 phases of secretion basal secretion, 1238 cephalic phase, 1238–1240 gastric phase, 1240–1241 intestinal phase, 1241–1243 receptors and signal transduction acetylcholine receptors, 1232–1233 calcium-sensing receptors, 1233–1234 cholecystokinin receptors, 1234–1235 cholinergic pathways of activation, 1207–1208 epidermal growth factor, 1208–1209 gastrin-releasing peptide receptors, 1235–1236 histamine receptors, 1236–1237 pituitary adenylate cyclase-activating polypeptide receptors, 1237 protein kinase A, 1206–1207 somatostatin receptors, 1237–1238 transforming growth factor-α, 1208–1209 regulation of secretion acetylcholine, 1243–1244 gastrin, 1244–1245 histamine, 1243, 1246 somatostatin, 1245–1246 regulation histamine, 1301–1302

Gastric acid (Continued) inhibition, 1297–1298 stimulation, 1296–1297 transgenic and knockout mouse studies, 1298–1306 secretin regulation, 123–124 sodium/potassium-ATPase crystal structure, 1199 functional transport, 1194–1195 identification as proton pump, 1194 inhibitors, 1199–1200, 1202 kinetics, 1196 membrane changes with acid secretion, 1195–1196 subunit structure, 1196–1199 vesicular trafficking machinery actin, 1210 actin-binding proteins, 1210 Arf6, 1216 clathrin and adaptors, 1214–1215 dynamin, 1216 myosin II, 1210–1211 Rab11, 1211–1213 Rab13, 1211–1213 SNAREs, 1213–1214 Gastric emptying cholecystokinin effects, 105 corticotropin-releasing hormone inhibition, 798–800 neural control, 669 peptide YY effects, 143–144 secretin regulation, 124 Gastric motility, see also Gastric accommodation; Gastric emptying; Gastric reservoir function; Gastrointestinal motility brainstem control, 855–856 phases gastric emptying, 928 gastric reservoir function, 928 interdigestive motility, 927 Gastric mucosa, see also Gastroduodenal mucosal defense apoptosis ethanol induction, 366 Helicobacter pylori induction, 364–365 nonsteroidal anti-inflammatory drug induction, 365–366 Helicobacter pylori colonization, see Helicobacter pylori injury and repair in aging, 408–410 transgene expression in mice enterochromaffin-like cells, 1307–1308 gastrin promoter and G cell transgenics, 1308 parietal cells, 1307 promoters, 1306–1308 transgenic and knockout mouse studies, 1306–1308 Gastric reservoir function, see also Gastric accommodation measurement barostat, 928 caloric drink test, 929 volumetric techniques, 928–929 overview, 928 Gastrin bile formation regulation, 1520 cell growth regulation, 451 enterochromaffin-like cell regulation, 1227–1228 excess and disease, 100 functions acid secretion control, 98 enterochromaffin-like cell regulation, 98–99 gastric epithelial cell regulation, 99 Gly-gastrin, 99–100 overview, 92, 97–98 progastrin, 100 target genes, 99 gastric acid secretion regulation, 1244–1245 gene regulation, 4 gene structure and expression, 93 knockout mice gastrin-cholecystokinin double-knockout mice, 1301 pathway mutants, 1299, 1301 metabolism, 96 posttranslational processing, 93–95 preprogastrin posttranslational processing, 48–51 radioimmunoassay, 95 receptors, 96–97 release, 95–96

Gastrin (Continued) secretin interactions, 124 structure, 92–93 tissue distribution, 95 transgenic mice gastrin promoter and G cell transgenics, 1308 overexpression, 1301 Gastrin-releasing peptide, see also Bombesin pancreas secretion regulation, 1407–1408 satiety signaling, 884 Gastrin-releasing peptide receptors gastric expression, 1236 ligand binding, 1235 signaling, 1236 structure, 1236 Gastritis, see Helicobacter pylori Gastrocalcin, enterochromaffin-like cell secretion, 1231 Gastroduodenal mucosal defense epithelium bicarbonate protection cystic fibrosis paradox, 1268–1269 duodenal secretion, 1270–1273 gastric secretion, 1269–1270 principles, 1268 intracellular pH acidification mechanism, 1267–1268 measurement and regulation, 1265–1267 permeability regulation apical membranes, 1264 intercelullar junctions, 1264–1265 injury and restitution animal models of injury, 1281–1282 cell exfoliation and mucoid cap, 1280 epithelial integrity challenges, 1279 epithelial restitution, 1280–1281 imposed mucosal damage, 1279–1280 mucous layer composition, 1260–1261 pH, 1261–1263 overview, 1259–1260 prospects for study, 1282 subepithelial defense duodenal acid sensing and blood flow, 1277–1278 overview, 1273–1274 prostaglandins, 1274–1276, 1278–1279 Gastroesophageal reflux disease, esophagoglottal closure reflex, 904–905 Gastrointestinal cancer, see Cancer, gastrointestinal Gastrointestinal circulation blood vessel innervation enteric neurons neurotransmitters, 819–820, 825 stimulation effects, 823–825 multiplicity of neurons, 819 parasympathetic neurons neurotransmitters, 819, 823 stimulation effects, 822–823 primary afferent neurons neurotransmitters, 820, 827–829 overview, 820 pathophysiology, 829–830 stimulation effects, 825–827 sympathetic neurons neurotransmitters, 819, 821–822 stimulation effects, 820–821 interactive control enteric and sympathetic vasomotor neurons, 830–831 local and paracrine mediators, 831–832 primary afferent and enteric vasodilator neurons, 831 primary afferent and sympathetic vasomotor neurons, 831 organization, 817–818 physiologic relevance, 817 prospects for study, 832 Gastrointestinal motility, see also Gastric emptying; Intestinal motility coordination of secretion and motility, 755–756 corticotropin-releasing hormone effects colon, 804–807 small intestine, 803–804 stomach, 800–802 stress effects animal studies, 783 autonomic dysfunction, 784–785 corticotropin-releasing hormone modulation

I-8 / INDEX Gastrointestinal motility (Continued) overview, 781–782 peripheral receptor and colonic motor alterations, 785 type 1 receptor activation and stimulation, 784 type 2 receptor activation and inhibition, 784 visceral hyperalgesia mediation by receptors, 786 healthy subjects, 782 irritable bowel syndrome patients, 782–783 prospects for study, 786–787 sex differences in visceral hypersensitivity, 786 visceral perception studies, 785–786 Gastrointestinal tube formation, 295 Hedgehog signaling in development, 275 G cell, see Gastrin GDNF, see Glial-derived neurotrophic factor Gene classification, 2 coregulatory proteins, 15–16 DNA-binding proteins, 12–15 DNA composition and structure, 1–2 5’-flanking sequences, 4 number in human genome, 1 organization, 2–4 promoter, see Promoter transcription, see Transcription GERD, see Gastroesophageal reflux disease Ghrelin assays, 110 excess and deficiency in disease, 111 food intake regulation, 886 functions, 109–111 gene structure and expression, 109 metabolism, 110 posttranslational processing, 109 receptors, 110 release, 110 structure, 109 tissue distribution, 109–110 Giant migrating contractions excitation–contraction coupling, 974–976 excitation–inhibition coupling, 976 features, 948 features, 967–969 inflammatory bowel disease dysfunction, 986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction, 983–984 GIP, see Glucose-dependent insulinotropic polypeptide GIPR, see Glucose-dependent insulinotropic polypeptide receptor Glial cells, intestinal inflammation effects, 1129 Glial-derived neurotrophic factor, Hirschsprung’s disease mutations, 505–507 GLP-1, see Glucagon-like peptide-1 GLP-2, see Glucagon-like peptide-2 Glucagon derived peptides, see Glucagon-like peptide-1; Glucagon-like peptide-2; Glycentin; Oxyntomodulin functions, 166–167 gene regulation of expression, 162–163 structure, 161–162 metabolism and clearance, 164 pancreas secretion regulation, 1420–1421 receptor, 165 release regulation, 164 secretion regulation, 164 therapeutic application, 167 vasodilation induction in intestine, 1640 Glucagon-like peptide-1 cell growth regulation, 451–452 food intake regulation, 885 functions, 167–169 metabolism and clearance, 164–165 pancreas secretion regulation, 1421 processing, 162 receptor, 165–166 receptor antagonists for diabetes type 2 treatment, 169–170 release regulation, 164 Glucagon-like peptide-2 cell growth regulation, 452 functions, 170–171

Glucagon-like peptide-2 (Continued) metabolism and clearance, 164–165 processing, 162 receptor, 166 release regulation, 164 stem cell regulation, 331 therapeutic applications, 171 Glucocorticoids, stress effect mediation in mucosa, 771 Glucose absorption, 1654–1656 dorsal vagal complex chemosensitivity, 865 Glucose-dependent insulinotropic polypeptide functions, 171–172 receptor features, 166 secretion, 171 synthesis, 171 therapeutic applications, 172 Glucose-galactose malabsorption, SGLT1 mutations, 1662 Glucose transporters cholangiocytes, 1511–1512 gene family, 1654 glucose, galactose, and fructose absorption, 1654–1656 GLUT2 function, 1656 mutation in disease, 1662 ontogenic regulation of intestinal monosaccharide absorption, 383 sugar selectivity, 1657 GLUT5 function, 1656 mutation in disease, 1662–1663 ontogenic regulation of intestinal monosaccharide absorption, 383 sugar selectivity, 1657 kinetics, 1661 regulation of expression, 1663 Glutaminyl cyclase, protein processing, 47 Glutathione antioxidant protection, 1494–1495 metal detoxification, 1497 transport in small intestine, 1681 Glutathione peroxidase, antioxidant protection, 1494 Glutathione S-transferase, phase II metabolism, 1489–1490 GLUTs, see Glucose transporters Glycentin, functions, 167 Glycine transporter 1, amino acid transport, 1678 GMC, see Giant migrating complex Golgi acinar protein trafficking endoplasmic reticulum to Golgi transport, 1323–1324 Golgi to trans-Golgi network transport, 1324–1326 trans-Golgi network to zymogen granule transport, 1326 posttranslational modifications glycosylation, 41 phosphorylation, 41 sulfation, 41–42 protein trafficking, 39–41 GPCRs, see G protein-coupled receptors G protein-coupled receptors abundance, 63 accessory proteins, 69 acinar cells, 1326, 1338 cholangiocytes, 1514 classification class A, 66–67 class B, 67 class C, 68 desensitization by receptor kinases and arrestins, 77–81 dimerization, 68 domains, 63–64 endocytosis, 81–82 extrinsic sensory afferent nerve signaling, 704–706 G proteins GTPases Arf, 72 Rab, 72–73 Ras, 72 Rho, 72 signaling mediation, 71–72 states, 70–71 types, 70

G protein-coupled receptors (Continued) internalized receptor signaling, 83 ligands classification, 71 interactions, 65, 67 lysosomal trafficking arrest of signal transduction, 83–85 microdomains in signaling, 73–74 mitogen-activated protein kinase signaling, 71, 73–74 prospects for study, 85 receptor tyrosine kinase signaling dimerization, 74 G protein signaling, 76–77 recruitment, 74–76 transactivation, 76 recycling and resensitization, 82–83 rhodopsin features, 64–65 smooth muscle contraction signaling desensitizaton, 526–527 initial phase of contraction, 524–525 sustained phase of contraction, 525–526 relaxation signaling PACAP receptor, 528 vasoactive intestinal peptide receptors, 527–528 structure, 64 GRKs, G protein-coupled receptor desensitization, 77–79 Growth factors, see also specific growth factors apoptosis inhibition, 355–356 compartmentalization of function, 185 definition, 184 intestinal transport in neonatal period, 380–381 redundancy in families, 185–186 signaling paradigms, 184–185 Growth hormone, calcium absorption regulation, 1966–1967 GRP, see Gastrin releasing peptide Gut-associated lymphoid tissue intestinal immunity, 1148–1149 leukocyte movement through interstitium adhesion molecules, 1151–1152 chemoattractant regulation, 1152–1153 lymphocyte homing and activation, 1149–1151 H Hand2, Hirschsprung’s disease mutations, 507 Hartnup disease, pathophysiology, 1681–1682 HB-EGF, see Heparin-binding epidermal growth factor-like growth factor Heat-stable enterotoxin, secretory function effects, 1169 Hedgehog cancer defects, 282–283 development signaling esophagus, 275–276 gastrointestinal tube, 275 hindgut, 278–279 midgut, 278 pancreas, 277–278 stomach, 277 thyroid, 276–277 trachea, 275–276 VACTERL signaling, 279 genes, 272–273 homeostasis role Indian Hedgehog, 280–282 morphostasis, 279–280 Sonic Hedgehog, 280–281 processing, 273–274 prospects for study, 283 receptor signaling, 274 Sonic Hedgehog in gastric gland maintenance, 445–446 stem cell function regulation, 324–326 target genes, 274–275 Helicobacter pylori aging studies esophagus, 406 stomach, 408, 410–411 apoptosis induction in gastric mucosa, 364–365 barrier function effects CagA, 1178–1179 overview, 1166 cell-mediated immunity, 1098 enterochromaffin-like cell function effects, 1231 gastric mucosa colonization, 1092–1094 gastrin induction, 93

INDEX / I-9 Helicobacter pylori (Continued) gastritis cyclooxygenase role, 1098–1099 cytokine release, 1095–1096 interleukin-8 expression regulation, 1096–1097 nitric oxide role, 1099 gene polymorphisms and disease susceptibility, 1104–1106 humoral response, 1097–1098 iceA strains and disease, 1103 immune evasion, 1094–1095 inflammatory response, 1091–1092 secretory function effects overview, 1164 VacA, 1170–1171 virulence factors BabA, 1102–1103 cag pathogenicity island, 1099–1101 FldA, 1103 novel factors, 1103–1104 VacA, 1101–1102 Heparin-binding epidermal growth factor-like growth factor discovery, 198 functions, 199 receptors, 199 synthesis, 198 tissue distribution, 198–199 Hepatocyte bile acid transport physiology, 1468 sodium-dependent uptake and regulation, 1448–1449, 1469–1471 sodium-independent uptake, 1449 transcriptional regulation of transporter expression, 1474–1475 transporters biochemical studies, 1468 molecular biology studies, 1469 overview, 1449 detoxification, see Liver Kupffer cell clearance, 1499 nonbile acid organic anion uptake albumin binding effects, 1463–1464 driving forces, 1464 physiologic modulation, 1464 transporters biochemical studies, 1464–1465 biochemistry, 1465, 1467 function, 1467 molecular biology studies, 1465 regulation, 1467–1468 sequence alignment, 1466 substrate specificity, 1468 regeneration mitogen and cytokine regulation, 1500 oval cells, 1500–1501 overview, 1500 Hepatocyte growth factor discovery, 213 functions cell growth regulation, 448 development, 214–215 tissue repair, 215 tumorigenesis, 216 gene, 213 processing, 213 protein–protein interactions, 213–214 receptors, 214 signaling, 214 structure, 213 Hepcidins, innate immunity, 1052 Hereditary hemochromatosis, pathophysiology, 1498–1499 Hes1, Notch regulation, 299, 322–323 HGF, see Hepatocyte growth factor Hirschsprung’s disease aganglionosis, 499–500 endothelin role, 1633 enteric nervous system plasticity, 674–675 epidemiology, 503 gene mutations, 503–507 pathogenesis, 499 Histamine enterochromaffin-like cell secretion, 1229–1230 gastric acid secretion regulation, 1243, 1246, 1301–1302

Histamine (Continued) knockout mouse pathway mutants, 1302–1303 mast cell signaling in irritable bowel syndrome, 1025 pancreas electrolyte regulation, 1388 pancreas secretion regulation, 1416 vasodilation induction in intestine, 1640 Histamine receptors gastric expression, 1236–1237 ligand binding, 1236 signaling, 1237 structure, 1236 Histone acetylation, 6–7 methylation, 7 phosphorylation, 7–8 structure, 5–6 Histone-derived peptides, innate immunity, 1055 Hormone, definition, 91–92 Hydrochloric acid secretion, see Gastric acid I ICAMs, see Intercellular adhesion molecules IGF-I, see Insulin-like growth factor-I IGF-II, see Insulin-like growth factor-II IGFBPs, see Insulin-like growth factor binding proteins IL-1, see Interleukin-1 IL-8, see Interleukin-8 Ileum bile acid transport ileal basolateral transport, 1452 sodium-dependent uptake, 1451–1452 water transport, 1839–1840 Immune system, see Adaptive immunity; Immunoglobulins; Innate immunity Immunoglobulins Helicobacter pylori humoral response, 1097–1098 immunoglobulin E FcεRII low-affinity receptor, 1083–1084 transcytosis of immunoglobulin E, 1084 food allergy, 1082 intestinal anaphylaxis, animal models, 1082–1083 intestinal functions, 1082, 1084–1085 immunoglobulin G FcRn antigen uptake pathway mediation, 1081–1082 binding model, 1078–1079 discovery, 1077 structure, 1077–1078 transport, 1079–1081 mucosal physiology and function, 1081 transport, 1079–1081 inflammatory bowel disease levels, 1082 polymeric receptor expression, 1073–1074 structure, 1074–1075 trafficking, 1075–1076 secretory immunoglobulin A functions, 1068–1070 mucosal plasma cell origins, 1070–1072 polymeric immunoglobulin structure, 1072–1073 Indian Hedgehog, see Hedgehog Infection, chloride–bicarbonate exchange regulation, 1886 Infectious colitis, clinical manifestations, 1123 Inflammation, see also Intestinal inflammation; Leukocyte recruitment bile acid transport effects, 1454 cancer role, 479–480 chloride–bicarbonate exchange regulation, 1885 gallbladder, 1551–1553 leukocyte trafficking acute inflammation bacterial toxins, 1154–1155 oxidative stress-dependent models, 1154 chronic inflammation, 1155–1157 presynaptic inhibition mediation in enteric nervous system, 656–657 stem cell effects, 332–335 systemic inflammation and behavior, 710 trefoil factor family role, 212–213 Inflammatory bowel disease clinical manifestations, 1122 colonic contraction dysfunction excitation–contraction coupling, 986–987 intrinsic sensory neurons, 987 motor neurons, 987

Inflammatory bowel disease (Continued) myoneuroimmune interactions, 987 overview, 985–986 Crohn’s disease and intestinal epithelial tight junction barrier function effects, 1584–1585 endothelin role, 1633 immunoglobulin G levels, 1082 ion transport alterations, 1943–1944 stress in pathogenesis, 775 Innate immunity adaptive immunity comparison, 1033–1034 angiogenins, 1055 cathelicidins, 1052–1053 defensins α-defensins, 1049–1051 β-defensins, 1051–1052 definition, 1033 hepcidins, 1052 histone-derived peptides, 1055 lysozyme, 1053–1054 molecular pattern recognition, 1035 nitric oxide, 1055–1057 nucleotide-binding oligomerization domain proteins NOD1, 1045–1047 NOD2, 1047–1049 structure, 1045 phospholipase A2, 1054 Toll-like receptors structure, 1035 TLR1, 1039–1040 TLR2, 1039–1040 TLR3, 1043–1044 TLR4, 1035–1039 TLR5, 1042–1043 TLR6, 1039–1040 TLR7, 1044 TLR8, 1044 TLR9, 1040–1042 TLR10, 1044 Inositol phosphates, see Phosphoinositides Insula, gastrointestinal sensory signal processing, 730, 733–734 Insulin-like growth factor-I cancer role, 209 functions apoptosis inhibition, 209 cellular proliferation, 208–209, 447 profibrogenic actions, 209 gastrointestinal distribution, 207–208 gene, 206 knockout mouse studies, 209 receptors, 206, 209 stem cell regulation, 330 Insulin-like growth factor-II cancer role, 209 gastrointestinal distribution, 207–208 gene, 206 receptors, 206 stem cell regulation, 330 Insulin-like growth factor binding proteins functions, 207 structures, 207 types, 206 Integrins, types and functions, 1141–1142 Intercellular adhesion molecules, types and functions, 1140–1141 Interleukin-1 enterochromaffin-like cell regulation, 1229 polymorphisms in Helicobacter pylori disease, 1104–1105 Interleukin-8, expression regulation in Helicobacter pylori gastritis, 1096–1097 Intermediate filaments, transport mediation, 1614–1615 Interneuron ascending interneurons, 586–587 colonic contraction regulation, 979 descending interneurons, 587–588 interplexus interneurons, 590–591 intestinal motility role, 979 overview, 586, 636–637 secretory reflexes, 740 Interstitial cell of Cajal animal model for study, 537, 549–550 defects in human disease, 539–540, 549 intestinal inflammation effects, 1128 intramuscular cells, 534–535

I-10 / INDEX Interstitial cell of Cajal (Continued) morphology, 535 neurotransmission innervation by enteric motor neurons, 546–547 motor neurotransmission acetylcholine, 547 nitric oxide, 547 neural control of slow wave activity, 547 neurotransmitter mechanism of action, 547–548 pacemaker activity action propagation of slow waves, 542–544 calcium flux, 538, 540–542 cultured cells, 538 Kit knockout studies, 537, 549 mechanism, 538, 540–542 spontaneous electrical rhythmicity, 535 unitary potentials, 544–546 slow wave origins, 533–534 small intestine motility regulation, 938–940 smooth muscle responses to slow waves and neural inputs chloride conductances calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 integration of electrical activity, 569 ion channels, 552–554 nonselective cation channels currents activated by ATP, 568 currents activated by muscarinic and peptide agonists, 567–568 currents activated by stretch, 568–569 overview, 550–551 voltage-dependent inward currents inward rectifier currents, 556 L-type calcium channels, 551, 555–556 sodium channels, 556 T-type calcium channels, 556 voltage-dependent outward currents A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 ether-a-go-go-related currents, 560–561 inward rectifier potassium channels, 559–560 overview, 556–557 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 stretch receptor function, 548–549 Intestinal epithelium apoptosis anoikis, 363–364 cell studies, 358–363 induced apoptosis inducers, 351–352 ischemia–reperfusion injury, 354 mechanisms, 352–353 small bowel resection, 353–354 pathways and regulation, 358–362 polyamine depletion effects, 359–360 prevention cytokines, 357–358 growth factors, 355–356 lysophosphatidic acid, 357 polyamines, 356–357 prostaglandins, 356 spontaneous apoptosis crypt, 350–351 villus, 351 electrolyte transport, see specific electrolytes growth and differentiation, 349–350 Intestinal inflammation, see also Inflammation; Inflammatory bowel disease chronic inflammation, 1121–1122 clinical manifestations drug-induced colitis, 1123 infectious colitis, 1123 inflammatory bowel disease, 1122 microscopic colitis, 1122–1123 radiation enteritis, 1122 environmental factors, 1123 functional effects digestive dysfunction, 1127 epithelial cell apoptosis, 1125–1126

Intestinal inflammation (Continued) motor disturbances enteric nervous system, 1128–1129 extrinsic primary afferent nerves, 1129 glial cells, 1129 interstitial cells of Cajal, 1128 nerve/mast cell interactions, 1124–1125 nutrient absorption, 1128 secretagogue hyporesponsiveness, 1126–1127 secretomotor neurons, 1124 sensory nerves, 1124 smooth muscle phenotype changes, 1129–1130 tight junctions, 1125 genetic factors, 1123 luminal triggers dendritic cells, 1116 effectors arachidonic acid metabolites, 1120 cytokines and chemokines, 1118–1119 neutrophil products, 1120 nitric oxide, 1120–1121 proteases and activated receptors, 1121 Toll-like receptors, 1116 transcription factors nuclear factor-κB, 1116–1117 STATs, 1117–1118 overview, 1115 prospects for study, 1130 resolution phase, 1121 Intestinal motility, see also Gastrointestinal motility abdominal pain in irritable bowel syndrome, 1021–1022 colonic contractions enteric neuron regulation enteric reflexes, 981–982 interneurons, 979 intrinsic sensory neurons, 979–981 motor neurons, 977–979 overview, 976–977 excitation–contraction coupling giant migrating contractions, 974–976 overview, 970–971 rhythmic phase contractions, 971–974 tonic contractions, 976 excitation–inhibition coupling, 976 giant migrating contractions, 967–969 inflammatory bowel disease dysfunction excitation–contraction coupling, 986–987 intrinsic sensory neurons, 987 motor neurons, 987 myoneuroimmune interactions, 987 overview, 985–986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 overview, 965–966 rhythmic phase contractions, 966–967 tonic contractions, 969 motilin regulation, 138–139 small intestine central nervous system modulation, 953–954 contraction coupling, 940 extended reflexes intestino-intestino reflex, 951 non-nutrient stimulation, 953 nutrient-evoked reflexes, 951–953 reflex stimulation by other organs, 953 fasting motor pattern reversion, 947–948 fed motor pattern neural regulation, 947 neurohumoral mediators, 947 physiologic characteristics, 946–947 functional anatomy, 935–936 giant migrating complex, 948 ileocolonic junction motor activity, 949–951 infection effects and immune mediation, 954–956 interstitial cell of Cajal regulation, 938–940 migrating motor complex associated synchronous cyclic phenomena, 946 neural regulation, 944–945 neurohumoral mediators, 945–946 physiologic characteristics, 943–944

Intestinal motility (Continued) nervous tissue extrinsic innervation, 938 intrinsic innervation, 936–938 peristalsis control, 941–943 retrograde peristaltic contractions, 948–949 smooth muscle, 936 transit, 936 structural basis, 579 Intestinal mucosa barrier function antigen-sampling cells dendritic cells, 765–766 follicle-associated epithelium, 765 M cells, 765 ion and fluid transport, 766 mucus secretion, 766 overview, 763–764 tight junctions, 764 transcellular uptake of protein antigens, 765 inflammation effects nerve/mast cell interactions, 1124–1125 secretomotor neurons, 1124 sensory nerves, 1124 stress effects animal studies acute stress mediators, 770–772 barrier function, 769–770 chronic stress studies, 772–773 early life stress, 774–775 flora changes, 773 ion secretion, 769 models, 768–769 mucus secretion, 769 food allergy pathogenesis, 775–776 inflammatory bowel disease pathogenesis, 775 irritable bowel syndrome pathogenesis, 776 physical stress, 767–768 prospects for study, 776 psychological stress, 768 Tcf-4 regulation cell cycle progression, 442 intestinal cell position along crypt-villus axis, 442–443 Intestinal transport, see also specific nutrients hormones and growth factors in neonatal period, 380–381 macromolecules during neonatal and suckling periods, 379–380 ontogeny along vertical and horizontal gut axes, 391–392 postnatal development amino acid transport, 383 bile acid absorption, 391 calcium absorption, 386 lipid absorption, 383–384 lipid-soluble vitamin transport, 391 monosaccharide absorption, 381–383 phosphate absorption, 386–388 potassium absorption, 386 sodium chloride absorption, 384–386 trace mineral absorption, 388–389 water transport, 384 water-soluble vitamin transport, 389–391 Intestine, see Colon; Small intestine Ion channels, see also specific channels acinar cell exocytosis role, 1361–1362 alterations in disease constipation, 1944 infectious diarrhea, 1942–1943 inflammatory bowel disease, 1943–1944 therapeutic implications, 1944 analysis effectors, 1923–1924 experimental models, 1922 permeabilization, 1922 permeable supports, 1925 structure–function studies, 1925 transfection studies, 1924–1925 Ussing chamber studies, 1923 inhibitors, 1167 interactions between transport mechanisms, 1938–1939 intestinal ion transport regulation at tissue level acute regulation, 1939–1941 chronic regulation, 1941–1942

INDEX / I-11 Ion channels (Continued) secretory function ontogeny, 376–377 smooth muscle responses to slow waves and neural inputs chloride conductances calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 integration of electrical activity, 569 ion channels, 552–554 nonselective cation channels currents activated by ATP, 568 currents activated by muscarinic and peptide agonists, 567–568 currents activated by stretch, 568–569 overview, 550–551 voltage-dependent inward currents inward rectifier currents, 556 L-type calcium channels, 551, 555–556 sodium channels, 556 T-type calcium channels, 556 voltage-dependent outward currents A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 ether-a-go-go-related currents, 560–561 inward rectifier potassium channels, 559–560 overview, 556–557 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 Iron deficiency, 1986–1987 intestinal absorption, 1983–1985 overload ferroportin disease, 1989 hemochromatosis, 1987–1989 hyperabsorption disorders, 1990 iatrogenic overload, 1990 plasma protein defects, 1989–1990 transfusional overload, 1990 plasma trafficking, 1985 systemic homeostasis, 1985–1986 Irritable bowel syndrome abdominal pain and discomfort central neuropathologic factors cerebral cortex sensory processing, 1018–1019 spinal ascending sensory pathways, 1017–1018 spinal cord sensory processing, 1019–1020 spinal descending modulatory pathways, 1020–1021 inflammatory mediator receptors on sensory afferents, 1017 intestinal motility, 1021–1022 peripheral neuropathologic factors, 1015 postinfectious irritable bowel syndrome, 1015–1016 presentation, 1015 serotonin receptors on sensory afferents, 1016–1017 colonic contraction dysfunction giant migrating contractions, 983–984 overview, 982–983 rhythmic phase contractions, 983 therapeutic agents, 984–985 tonic contractions, 984 enteric nervous system neuropathy disinhibitory motor disease, 1011–1012 inhibitory motor neurons, 1010–1011 motor neurons, 1010 overview, 1009–1010 secretomotor neurons excitatory input, 1014–1015 inhibitory input, 1013–1014 neurogenic secretory diarrhea and neurogenic constipation, 1014 projections, 1013 epidemiology, 1009 psychological stress immunoneural signal sources, 1022–1023 mast cells brain–gut connection, 1024–1027 histamine signaling, 1025 immunoneural communication, 1023–1024 serotonin signaling, 1025–1026 neuroimmunophysiologic paradigm, 1022

Irritable bowel syndrome (Continued) serotonin receptors, 636 sex differences, 786 stress in pathogenesis, 776 visceral hypersensitivity, 635–636 Ischemia, afferent sensitivity effects, 712–713 Ischemia–reperfusion injury, apoptosis induction, 354 J Jagged, Notch ligand, 289–290 Jejunum, water transport, 1839–1840 K Keratinocyte growth factor, cell growth regulation, 448–449 KGF, see Keratinocyte growth factor KLFs, see Krüppel-like transcription factors Knockout mouse acetylcholine receptor mutants, 1303 basolateral membrane transporters, 1306 epidermal growth factor receptor studies, 204–205 fibroblast growth factor studies, 219 Fox11 knockout, 1306 gastrin-cholecystokinin double-knockout mice, 1301 gastrin pathway mutants, 1299, 1301 generation, 1295–1296 gene regulation studies, 17 histamine pathway mutants, 1302–1303 insulin-like growth factor-I studies, 209 Kit studies, 537, 549 somatostatin receptor knockout, 1303–1304 transforming growth factor-β studies, 192–193 trefoil factor knockout, 1306 TRPV calcium transporter studies, 1762–1763, 1967 Krüppel-like transcription factors, epithelial cell growth regulation, 445 Kupffer cell, hepatocyte clearance, 1499 L Laminin, enteric nervous system development role, 511–512 Large intestine, see Colon Laryngeal motor function nasopharyngeal motor function during swallowing and belching, 901–902 overview of deglutitive function, 900–901 Leptin, food intake regulation, 878 LES, see Lower esophageal sphincter Leukocyte recruitment gut-associated lymphoid tissue lymphocyte homing and activation, 1149–1151 inflammation acute inflammation bacterial toxins, 1154–1155 oxidative stress-dependent models, 1154 chronic inflammation, 1155–1157 movement through interstitium adhesion molecules, 1151–1152 chemoattractant regulation, 1152–1153 overview, 1137 Lipid, see also Fatty acids; Lipoproteins cell growth regulation polyunsaturated fatty acids, 450–451 short-chain fatty acids, 450 ontogenic regulation of absorption, 383–384 pancreatic secretion regulation, 1402–1404 wound healing promotion, 464 Lipid raft G protein-coupled receptor signaling, 74 protein sorting in polarized epithelial cells, 1610–1611 Lipoproteins, intestinal assembly apolipoprotein A-I role, 1723–1724 apolipoprotein A-IV role, 1723–1724 apolipoprotein B, see Apolipoprotein B first step, 1714–1715 microsomal triglyceride transfer protein, see Microsomal triglyceride transfer protein novel pathways and defects, 1728–1729 overview, 1712 prechylomicron transport, 1717 second step, 1716 Liraglutide, diabetes type 2 treatment, 170 Liver anatomy and function, 1483–1484 detoxification

Liver (Continued) ATP-binding cassette transporters, 1490–1492 liver health effects on metabolism and elimination, 1492 metabolism principles, 1484 phase I metabolism alcohol dehydrogenase, 1487–1488 aldehyde dehydrogenase, 1488 cytochrome P450, 1484–1487 flavin monooxygenase, 1487 phase II metabolism glutathione S-transferase, 1489–1490 N-acetyltransferase, 1489 sulfotransferase, 1489 thiopurine methyltransferase, 1490 UDP glucuronosyltransferase, 1488–1489 metal detoxification copper sequestration, 1498 ferritin, 1498 glutathione binding and elimination, 1497 metallothionein, 1497–1498 pathology hereditary hemochromatosis, 1498–1499 Wilson’s disease, 1499 toxicity mechanisms, 1497 oxidative damage antioxidant protection catalase, 1494 glutathione, 1494–1495 glutathione peroxidase, 1494 superoxide dismutase, 1494 thioredoxin, 1495 vitamin C, 1495 vitamin E, 1495 hepatocyte necrosis versus apoptosis, 1493–1494 pathology biochemical pathway stimulation, 1496 cancer, 1496–1497 chronic liver disease, 1496 drug toxicity, 1495–1496 reactive oxygen species sources, 1492–1493 redox cycling, 1493 toxicity, 1492 sodium–proton exchange, 1867–1868 water transport, 1837–1838 Low-density lipoprotein receptor-related proteins destruction complex regulation, 254–255 Wnt interactions, 249, 251–252, 441 Lower esophageal sphincter anatomy, 914 coordinated motor activity, 921–923 LRP, see Low-density lipoprotein receptor-related protein Lysinuric protein intolerance, pathophysiology, 1682–1683 Lysophosphatidic acid, apoptosis inhibition, 357 Lysozyme, innate immunity, 1053–1054 M Macula adherens, see Desmosome Magnesium dietary requirements, 1953–1954 intestinal absorption physiology, 1972–1973 regulation, 1975 sites, 1971–1972 transient receptor potential family proteins, 1973, 1975 pancreatic secretion regulation, 1403 Malignancy, see Cancer, gastrointestinal Manganese, absorption and transporters, 1998 MAPKs, see Mitogen-activated protein kinases Mash1 mutation and enteric nervous system lesions, 508–509 Notch regulation, 302 Mast cell afferent sensitivity effects, 711 irritable bowel syndrome and psychological stress brain–gut connection, 1024–1027 histamine signaling, 1025 immunoneural communication, 1023–1024 serotonin signaling, 1025–1026 nerve interactions in intestinal inflammation, 1124–1125 stress effect mediation in mucosa, 770–771

I-12 / INDEX Mastermind, Notch regulation, 294 Math1, Notch regulation, 300, 322 M cell, antigen sampling, 765 MCH, see Melanin-concentrating hormone MDR3, see Multidrug resistance gene 3 Megalin, gallbladder cholesterol metabolism, 1548 Melanin-concentrating hormone, feeding control, 881 Melanocortins, feeding control, 879–880 α-Melanocyte stimulating hormone, feeding control, 879 Messenger RNA nuclear transport, 21–22 processing polyadenylation, 19–20 splicing alternative splicing, 20 regulation, 20–21 spliceosome, 20 ribonucleoprotein complex, 31 stability regulation, 35 structure, 3 transcription, see Transcription translation, see Translation Metallothionein, metal detoxification, 1497–1498 N-Methyl-D-aspartate receptor, extrinsic sensory afferent nerves, 703 Microsatellite instability, cancer, 487–489 Microscopic colitis, clinical manifestations, 1122–1123 Microsomal triglyceride transfer protein abetalipoproteinemia gene mutations, 1720–1721 mouse models, 1721 lipoprotein assembly role, 1716 structure, 1714 Microtubule, transport in polarized epithelial cells, 1612–1614 Micturition, pelvic floor neural control, 1003 Migrating motor complex associated synchronous cyclic phenomena, 946 neural regulation, 944–945 neurohumoral mediators, 945–946 physiologic characteristics, 943–944 Mitogen-activated protein kinases cell growth regulation, 445 epidermal growth factor receptor signaling, 201 G protein-coupled receptor signaling, 71, 73–74 MMC, see Migrating motor complex Monocarboxylate transporter 1, short-chain fatty acid transport, 1904, 1906 Monosaccharides, postnatal development of absorption, 381–383 Motilin antibiotic binding to receptor, 140 functions, 137–139 mechanism of action, 139 pathophysiology, 140 receptor, 137 release, 139–140 structure, 137–138 tissue distribution, 137 Motor neuron, see Enteric nervous system Mouse genetic engineering, see Knockout mouse; Transgenic mouse mRNA, see Messenger RNA MRPs, see Multidrug resistance proteins α-MSH, see α-Melanocyte stimulating hormone mTOR cell growth regulation, 444 phosphatidylinositol 3-kinase interactions, 444–445 rapamycin inhibition, 444 MTTP, see Microsomal triglyceride transfer protein Mucins, gallbladder secretion regulation bile salts, 1550–1551 calcium flux, 1550 cyclic AMP, 1550 cystic fibrosis transmembrane conductance regulator, 1551 gallstone formation, 1551 overview, 1549–1550 transcriptional regulation, 1551 Mucosa, see also Gastric mucosa; Gastroduodenal mucosal defense; Intestinal mucosa apoptosis, see Apoptosis cell cycle regulation, 437 cell growth regulation bombesin, 452–453 epidermal growth factor receptor, 447

Mucosa (Continued) fibroblast growth factors, 447–448 gastrin, 451 glucagon-like peptide-1, 451–452 glucagon-like peptide-2, 452 hepatocyte growth factor, 448 insulin-like growth factor-I, 447 keratinocyte growth factor, 448–449 Krüppel-like transcription factors, 445 luminal nutrients and secretions, 449–451 mitogen-activated protein kinases, 445 neurotensin, 452 peptide YY, 452 phosphatidylinositol 3-kinase/Akt pathway, 443–445 prospects for study, 453 somatostatin, 453 transforming growth factor-β, 446–447 trefoil factors, 448 Wnt signaling, 440–443 growth regulation in aging gene expression patterns, 423 growth factors and protein kinases, 421–423 intracellular mechanisms, 420 nutritional and hormonal factors, 420–421 prospects for study, 423 organization, 536–437 repair, see Wound healing Multidrug resistance gene 3, detoxification, 1491 Multidrug resistance proteins, detoxification, 1491–1492 Musashi1, Notch regulation, 300 c-Myc, stem cell function regulation, 318–319 Myosin II, vesicular trafficking in gastric acid secretion, 1210–1211 N NEC, see Neonatal necrotizing enterocolitis Necrosis, hepatocytes, 1493–1494 Neonatal necrotizing enterocolitis, endothelin role, 1633 Netrins, enteric nervous system development role, 512–513 β-Neurexin, enteric nervous system development role, 513–514 Neuroglin, enteric nervous system development role, 513–514 Neurokinins gallbladder emptying regulation, 844 primary afferent neuron neurotransmission in blood vessels, 828–829 Neuromedin B, satiety signaling, 884 Neuropeptide Y feeding control, 879–881, 887 functions, 134–135, 137 presynaptic inhibition mediation in enteric nervous system, 657 receptors, 134, 142–143 release, 137 structure, 134, 136 sympathetic neuron neurotransmission in blood vessels, 82 tissue distribution, 134, 136–137 Neurotensin cell growth regulation, 452 functions, 130, 132–133 pancreas electrolyte regulation, 1389 pancreas secretion regulation, 1415 receptors, 131–132 release, 133–134 structure, 130–131 tissue distribution, 131 Neurotrophin-3, mutation and enteric nervous system lesions, 508 NHEs, see Sodium–proton exchangers Niacin functions, 1812 intestinal absorption, 1812 sources, 1812 Nicotinic receptors extrinsic sensory afferent nerves, 703–704 inhibitory postsynaptic potentials in enteric nervous system, 646 Nitric oxide bicarbonate secretion regulation in duodenum, 1271–1272 chloride–bicarbonate exchange regulation, 1885–1886 chloride secretion mediation, 1172

Nitric oxide (Continued) endothelial nitric oxide synthase regulation activity regulation, 1635 posttranslational modification, 1635 transcription, 1634–1635 gallbladder function effects, 1552 gastric accommodation reflex control, 929–930 Helicobacter pylori gastritis role, 1099 innate immunity, 1055–1057 intestinal circulation localization, 1635 vasodilation induction, 1635–1636 synthesis, 1634 NO, see Nitric oxide NODs, see Nucleotide-binding oligomerization domain proteins Nonsteroidal anti-inflammatory drugs apoptosis induction in gastric mucosa, 365–366 intestinal epithelial tight junction barrier function effects, 1586 Norepinephrine enterochromaffin-like cell regulation, 1229 presynaptic inhibition mediation in enteric nervous system, 656 sympathetic neuron neurotransmission in blood vessels, 821 Notch developmental signaling enteric nervous system, 302 enteroendocrine system, 298–300 mucosal immune system, 301 overview, 295–297 time course, 297–298 ligands Delta, 289 functions, 292–293 Jagged, 289–290 Numb regulation, 294 posttranslational modifications glycosylation, 293 phosphorylation, 293 ubiquitination, 293–294 prospects for study, 302 receptors functions, 292–293 processing, 288 structures, 288–289 signaling crosstalk, 294–295 overview, 287–288 regulation, 290 target genes, 291–292 stem cell function regulation, 322–324 structure, 288 NPC1L1, intestinal cholesterol transport, 1724, 1727–1728 NPY, see Neuropeptide Y NSAIDs, see Nonsteroidal anti-inflammatory drugs Nuclear factor-κB, intestinal inflammation role, 1116–1117 Nucleosome, structure, 5 Nucleotide-binding oligomerization domain proteins NOD1, 1045–1047 NOD2, 1047–1049 structure, 1045 Nucleotides, cell growth regulation, 450–451 Nucleus of the solitary tract, pars centralis portion, 858–859 vago-vagal control reflex, 857–858 Numb, Notch regulation, 294 O Obesity, peptide YY deficiency, 145 Opioids, stress effect mediation in mucosa, 771–772 Orexins, functions, 172 Organic anion transporters, see Hepatocyte Oval cell, hepatocyte regeneration, 1500–1501 Oxidative stress, see Reactive oxygen species Oxygen metabolic vascular regulation and postprandial hyperemia, 1642 parenchymal demand versus vascular supply metabolic feedback, 1639 metabolic theory, 1637 microvascular studies, 1638–1639 whole-organ studies, 1637–1638 vascular smooth muscle effects, 1636–1637

INDEX / I-13 Oxyntomodulin metabolism and clearance, 164–165 functions, 167 P PACAP, see Pituitary adenylate cyclase-activating peptide PAF, see Platelet-activating factor Pancreas acinar cell, see Acinar cell cholecystokinin effects, 104–105 development, 1314–1316 electrolyte secretion apical transport anion exchangers, 1381 aquaporins, 1384 calcium-activated chloride channels, 1382–1383 carbonic anhydrase, 1384 cystic fibrosis transmembrane conductance regulator, 1381–1382 paracellular cation transport, 1384 potassium channels, 1383 sodium–bicarbonate cotransporter, 1383–1384 sodium–proton exchanger, 1383 basolateral transport aquaporins, 1381 chloride–bicarbonate exchanger, 1380 potassium channels, 1380 proton–ATPase, 1379 sodium–bicarbonate cotransporter, 1379–1380 sodium/potassium-ATPase, 1380–1381 sodium–potassium–2chloride cotransporter, 1380 sodium–proton exchanger, 1379 duct cell preparations and techniques for study, 1375–1377 model, 1384–1385 prospects for study, 1391 regulation acetylcholine, 1387, 1390 angiotensin II, 1388 arginine vasopressin, 1390–1391 ATP, 1387–1388, 1391 bombesin, 1389 calcium, 1388 cholecystokinin, 1389 cyclic AMP, 1386–1387 cyclic GMP, 1387 histamine, 1388 neurotensin, 1389 proteinase-activated receptor-2, 1388 secretin, 1390 serotonin, 1390 substance P, 1389–1390 species-dependent patterns, 1371–1372 structural basis, 1372–1374 exocrine pancreas organization, 1313–1314 extracellular matrix, 1316–1317 function testing blood enzymes, 1424 metabolic activity, 1423 pancreatic polypeptide, 1424 stool enzymes, 1424 Hedgehog signaling in development, 277–278 hormonal regulation of secretion cholecystokinin, 1412–1415, 1418 cholecystokinin-releasing factor, 1418–1419 feedback regulation, 1417–1420 glucagon, 1420–1421 glucagon-like peptide-1, 1421 histamine, 1416 inhibitors, 1420–1423 neurotensin, 1415 pancreastatin, 1422 pancreatic polypeptide, 1422 peptide YY, 1422 proteinase-activated receptor-2, 1416–1417 secretin, 1410–1412, 1420 somatostatin, 1421–1422 xenin, 1415–1416 intercellular junctions adhering zonules, 1318–1319 desmosomes, 1318–1319 gap junctions, 1319 tight junctions, 1317–1318 neural regulation of secretion adrenergic nerves, 1408–1409

Pancreas (Continued) dopaminergic nerves, 1409 gastrin-releasing peptide, 1407–1408 inhibitory neuropeptides, 1408 serotonergic nerves, 1409–1410 vagal innervation, 1404–1407 vasoactive intestinal peptide, 1407 peptide YY effects, 143 secretion patterns absorbed nutrient phase, 1403–1404 basal secretion, 1397–1398 cephalic phase, 1400 gastric phase, 1400–1401 integrated response to meals, 1398–1400 intestinal phase bile acid regulation, 1403 fat contributions, 1402 gastric acid role, 1401–1402 ions, 1403 overview, 1401 protein, peptide, and amino acid contributions, 1402 sodium–proton exchange, 1868–1869 Wnt/β-catenin signaling defects in cancer, 264 Pancreastatin enterochromaffin-like cell secretion, 1230 pancreas secretion regulation, 1422 Pancreatic polypeptide dorsal vagal complex chemosensitivity, 865 food intake regulation, 885 functions, 172 pancreas function regulation, 1424 pancreas secretion regulation, 1422 presynaptic inhibition mediation in enteric nervous system, 657 Pantothenic acid, functions and absorption, 1812–1813 PAR-2, see Proteinase-activated receptor-2 Paraneoplastic syndrome, disinhibitory motor disorders, 641 Parathyroid hormone, calcium absorption regulation, 1965 Parietal cell, see also Gastric acid membrane recycling hypothesis, 1193–1194 morphology and ultrastructure, 1191–1193 sodium/potassium-ATPase crystal structure, 1199 functional transport, 1194–1195 identification as proton pump, 1194 inhibitors, 1199–1200, 1202 kinetics, 1196 membrane changes with acid secretion, 1195–1196 subunit structure, 1196–1199 transgenic mouse studies, 1307 Patterning, development, 271–272 Pax4, digestive function ontogeny, 378–379 Pax6 digestive function ontogeny, 378–379 glucagon gene expression regulation, 163 PCR, see Polymerase chain reaction Pdx1, digestive function ontogeny, 379 Pelvic floor innervation afferent pathways, 998–999 external anal sphincter muscle innervation zones, 995–996 somatic motor pathways, 996–998 muscle activity overview, 999–1000 reflex activity, 1001–1002 tonic activity, 1000–1001 voluntary activity, 1002–1003 musculature, 678–679 neural control anorectal continence, 1004–1005 micturition, 1003 sexual response, 1005 urinary continence, 1004 neuromuscular injury in vaginal delivery, 1005–1006 rectoanal reflex, 680 sensory innervation, 679 spinal motor control, 680–681 supraspinal motor control, 681 PEPT1 mutations, 1683 peptide transport, 1676–1677

Peptide histidine isoleucine, functions, 130 Peptide histidine methionine, functions, 130 Peptide transport, see Protein digestion and absorption Peptide YY aging effects in intestine, 412 cell growth regulation, 452 dorsal vagal complex chemosensitivity, 865–866 enterochromaffin-like cell regulation, 1228 food intake regulation, 885–886 functions, 140, 143–145 pancreas secretion regulation, 1422 pathophysiology, 147–148 presynaptic inhibition mediation in enteric nervous system, 657 receptors, 142–143 release, 145–146 structure, 140 tissue distribution, 140–142 trophic actions, 146–147 PET, see Positron emission tomography Pharyngeal motor function airway protection during belching, 909–910 cerebral cortical representation of pharyngeal/reflexive and volitional swallow, 906–907 esophagoglottal closure reflex, 904–905 inhibitory reflexes, 908–900 laryngeal motor function nasopharyngeal motor function during swallowing and belching, 901–902 overview of deglutitive function, 900–901 musculature, 897 pharyngoglottal adduction reflex, 908 pressure phenomenon in relation to swallowed material, 898–900 pressure profile, 896–898 secondary swallow, 905–906 swallowing phases, 895–896 upper esophageal sphincter laryngo-upper esophageal sphincter contractile reflex, 908 opening, 903–904 pharyngolaryngeal and esophageal reflexes, 904 pharyngo-upper esophageal sphincter contractile reflex, 907–908 pressure phenomena, 902–903 PHI, see Peptide histidine isoleucine PHM, see Peptide histidine methionine Phosphate dietary requirements, 1953–1954 intestinal absorption mechanisms, 1967 regulation, 1971 sites, 1967 SLC34A2, 1970 transporters, 1968–1970 ontogenic development of absorption, 386–388 Phosphatidylinositol 3-kinase cell growth signaling, 443–444 epidermal growth factor receptor signaling, 203 mTOR interactions, 444–445 target genes, 444 Phosphoinositides acinar cell secretory function, 1339–1341, 1345–1347 protein sorting role in polarized epithelial cells, 1612 Phospholipase A2, innate immunity, 1054 Phospholipase C enterochromaffin cell signaling, 743–744 epidermal growth factor receptor signaling, 203 Phox2b, Hirschsprung’s disease mutations, 505 Pituitary adenylate cyclase-activating peptide enterochromaffin-like cell regulation, 1228 functions, 130 receptors gastric expression, 1237 ligand binding, 1237 signaling, 1237 structure, 1237 PKC, see Protein kinase C Platelet-activating factor, vasoconstriction induction in small intestine, 1634 Platelet glycoproteins, types and functions, 1142–1143 Polarized epithelial cell cytoarchitecture cell junctions, 1596–1597

I-14 / INDEX Polarized epithelial cell (Continued) cytoskeleton, 1597 membrane compartments, 1597 overview, 1595–1596, 1599–1600 protein sorting actin-mediated transport, 1614 caveolae, 1611–1612 intermediate filaments, 1614–1615 lipid rafts, 1610–1611 microtubule-mediated transport, 1612–1614 phosphoinositides, 1612 pre-trans Golgi network, 1597 prospects for study, 1618–1619 proteolipids, 1610–1611 Rab GTPase regulation, 1615–1616 selective retention, 1599 SNAREs in vesicle fusion, 1616–1617 sorting signals, 1600–1601, 1604–1605 spatial regulation of exocytosis exocyst complex, 1618 formins, 1617–1618 septins, 1618 techniques for study, 1600, 1602–1604 trans Golgi network, 1597–1599 vesicle budding AP protein adaptors, 1607–1609 clathrin-mediated budding, 1607 coat protein II-mediated budding, 1606–1607 coat-based budding model, 1606 overview, 1605 scission, 1609–1610 Polyamines apoptosis inhibition, 356–357 depletion and apoptosis induction, 359–360 Polymerase chain reaction, principles, 3–4 Positron emission tomography, gastrointestinal sensory signal processing studies, 730–734 Postprandial hyperemia chyme constituents, 1641 discovery, 1640–1641 mechanisms cholecystokinin, 1642–1643 metabolic vascular regulation, 1642 overview, 1646–1647 prostaglandins, 1643–1644 small mesenteric artery dilation, 1644, 1646 sodium-induced hyperosmolarity, 1644 sites, 1641 Potassium, ontogenic regulation of absorption, 386 Potassium channels apical membrane channels, 1921–1922 basolateral membrane channels, 1920–1921 calcium-activated potassium channels, 695 cholangiocytes, 1510 cholangiocytes, 1510–1511 extrinsic sensory afferent nerve voltage-gated potassium channels, 694–695 gallbladder, 1539–1540 gastric acid secretion role, 1202–1203 pancreas electrolyte secretion, 1380, 1383 smooth muscle responses to slow waves and neural inputs A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 inward rectifier potassium channels, 559–560 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 transgenic mouse mutants, 1305 PP, see Pancreatic polypeptide Prevertebral sympathetic ganglia chemical coding nerve fibers, 611 neurons, 610 neurotransmitters and neuromodulators, 605 distribution, 603 electrophysiology of neurons, 620–622 functional overview, 603–604 gut targets of neurons, 614 imaging, 603 innervation, 604, 607–610 morphology, 605–607 pacemaker neurons, 622–623

Prevertebral sympathetic ganglia (Continued) prospects for study, 623 symnpathetic motor innervation, organization in gut cat, 613 dog, 613 guinea pig, 612 overview, 611–612 pig, 613 rat, 612–613 visceral afferent neurons, 614–616 viscerofugal neuron chemical coding dog, 620 guinea pig, 619 pig, 620 rat, 619–620 mechanosensory neurons, 620 morphology, 616–617, 619 Prohormone convertases glucagon processing, 162 preprogastrin processing, 50 Promoter DNA elements, 9–12 identification, 3–4 RNA polymerase initiation complex, 10–11 structure, 9–10 Proopiomelanocortin, peptides in feeding control, 878–879 Prostaglandins apoptosis inhibition, 356 bicarbonate secretion regulation in duodenum, 1271–1272 duodenal protection, 1278–1279 enterochromaffin-like cell regulation, 1229 gastric protection, 1274–1276 postprandial hyperemia role, 1643–1644 prostaglandin E and chloride secretion mediation, 1172 Protein digestion and absorption, see also Amino acids absorbed amino acid and peptide fates, 1677 enterocyte active transport energetics, 1670–1671 peptidases, 1669 genetic disorders of transport, 1681–1683 overview, 1667–1668 peptide transport basolateral membrane transport, 1680 chain length effects, 1676 developmental regulation, 1684–1685 dietary regulation, 685 diurnal rhythm, 1686 energetics, 1675 hormonal regulation, 1685–1686 independence from amino acid transport, 1675 nutritional, clinical, and pharmacologic relevance, 1683–1684 PEPT1, 1676–1677, 1683 sodium–proton exchanger regulation, 1686 prospects for study, 1686–1687 proteases, 1668–1669 sites of absorption, 1669–1670 Protein kinase A acinar cell secretory function, 1354–1355 gastric acid secretion signaling, 1206–1207 Protein kinase B, see Akt Protein kinase C acinar cell secretory function, 1355 chloride–bicarbonate exchange regulation, 1886–1887 G protein-coupled receptor signaling, 79, 81 Protein phosphatases, acinar cell secretory function, 1356–1357 Protein trafficking, see Acinar cell; Cholangiocyte; Endoplasmic reticulum; Gastric acid; Golgi; G protein-coupled receptors; Immunoglobulins; Polarized epithelial cell Proteinase-activated receptor-2 pancreas electrolyte regulation, 1388 pancreas secretion regulation, 1416–1417 Proton-ATPase cholangiocytes, 1511 pancreas electrolyte secretion, 1379 Psychological stress, see Stress Purinergic receptors cholangiocytes, 1515 enterochromaffin cell excitatory adenosine receptors, 747–748

Purinergic receptors (Continued) extrinsic sensory afferent nerves, 702–703 inhibitory postsynaptic potentials in enteric nervous system, 646–647 secretion disorders, 76 PYY, see Peptide YY R Rab GTPases acinar cell exocytosis role, 1359–1360 exocytosis regulation in polarized epithelial cells, 1615–1616 G protein-coupled receptor signaling, 72–73 vesicular trafficking in gastric acid secretion, 1211–1213 Radiation enteritis, clinical manifestations, 1122 Ras, G protein-coupled receptor signaling, 72 Reactive oxygen species chloride–bicarbonate exchange regulation, 1887 endothelial cell activation products, 1145–1146 liver damage antioxidant protection catalase, 1494 glutathione, 1494–1495 glutathione peroxidase, 1494 superoxide dismutase, 1494 thioredoxin, 1495 vitamin C, 1495 vitamin E, 1495 hepatocyte necrosis versus apoptosis, 1493–1494 pathology biochemical pathway stimulation, 1496 cancer, 1496–1497 chronic liver disease, 1496 drug toxicity, 1495–1496 reactive oxygen species sources, 1492–1493 redox cycling, 1493 toxicity, 1492 Receptor tyrosine kinase acinar cell secretory function, 1356 dimerization, 74 G protein signaling, 76–77 recruitment, 74–76 transactivation, 76 Reduced folate carrier, folate transport, 1795–1797 Regenerating protein 1α, enterochromaffin-like cell secretion, 1231 RET, Hirschsprung’s disease mutations, 503–505 Retinoids, see Carotenoids; Vitamin A Rho, G protein-coupled receptor signaling, 72 Rhythmic phase contractions excitation–contraction coupling long-duration contractions, 974 short-duration contractions, 971–974 excitation–inhibition coupling, 976 features, 966–967 inflammatory bowel disease dysfunction, 986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction, 983 Riboflavin functions, 1811 intestinal transport mechanism, 1811 regulation, 1811–1812 RNA polymerase initiation complex, 10–11 types and functions, 2 RNA silencing, mechanism, 34–35 ROS, see Reactive oxygen species Rotavirus barrier function effects overview, 1166 nonstructural protein 4, 1171, 1181 secretory function effects, 1164 RPCs, see Rhythmic phase contractions RTK, see Receptor tyrosine kinase RTX, barrier function effects, 1173 Runx3, transforming growth factor-β activation, 447 S S100A10, calcium absorption regulation, 1966 Salivary gland sodium–proton exchange, 1866–1867 water transport, 1835–1836 Salmonella enterica, barrier function effects, 1166, 1181 Satiety, see Appetite

INDEX / I-15 Scavenger receptor type B class I, gallbladder cholesterol metabolism, 1546–1547 SCFAs, see Short-chain fatty acids Secreted frizzled-related proteins, Wnt/β-catenin signaling inhibition, 252 Secretin bile formation regulation, 1516, 1518–1521 discovery, 121–122 functions, 123–125 gastrointestinal localization, 122 pancreas electrolyte regulation, 1390 pancreas secretion regulation, 1410–1412, 1420 pathobiology, 126 receptor, 122 release, 125–126 structure, 122–123 Secretomotor neuron excitatory receptors, 638 inhibitory input, 638 intestinal content liquidity control, 638 intestinal inflammation effects, 1124 irritable bowel syndrome excitatory input, 1014–1015 inhibitory input, 1013–1014 neurogenic secretory diarrhea and neurogenic constipation, 1014 projections, 1013 overview, 589–590, 637–638 Secretory function, ontogeny, 376–377 Secretory reflexes ATP role in sensory signaling, 748 coordination of secretion and motility, 755–756 cyclic AMP signaling and secretion, 756–757 distension, 751–752 humans, 752–754 modulation of endogenous secretory tone, 738 mucosal stroking reflexes in rodent intestine, 749–751 neuroanatomy AH neurons, 739 extrinsic primary afferent neurons, 741 interneurons, 740 overview, 738–739 secretomotor neurons, 740–741 pathobiology cholera toxin-induced secretion, 758 Clostridium difficile enteritis, 759–760 purines, 760 sensory enterochromaffin cells BON cell model, 741–742 chemosensitive stimulation of serotonin release, 741 excitatory adenosine receptors, 747–748 mechanosensitivity, 741, 743 signal transduction adenosine as physiologic brake, 745–747 cyclic AMP, 744–745 phospholipase C, 743–744 synaptic transmission mediators, 754–755 Secretory vesicle formation, 42 processing reactions acetylation, 42 amidation, 47–48 aminopeptidases, 46–47 carboxypeptidases, 45–46 dibasic cleavage, 42–44 glutaminyl cyclase, 47 monobasic cleavage, 44–45 Selectins, types and functions, 1138–1140 Selenium, absorption and transporters, 1998 Sensory signals, brain processing functional imaging studies, 730–734 integrated view, 734–735 neuroanatomy cingulate cortex, 730, 734 insula, 730, 733–734 overview, 728, 730 somatosensory cortex, 729–730, 733–734 thalamus, 728–729, 732–733 Septins, exocytosis regulation in polarized epithelial cells, 1618 Serine protease autotransporters of Enterobacteriaceae, barrier function effects, 1175–1176 Serotonin chemosensitive stimulation of release in enterochromaffin cells, 741 chloride–bicarbonate exchange regulation, 1886

Serotonin (Continued) gastric accommodation reflex control, 930 irritable bowel syndrome role, 1025–1026 mast cell signaling in irritable bowel syndrome, 1025–1026 pancreas electrolyte regulation, 1390 pancreas secretion regulation, 1409–1410 Serotonin receptors extrinsic sensory afferent nerves, 702 fast excitatory postsynaptic potentials in enteric nervous system, 647 inhibitory postsynaptic potentials in enteric nervous system, 647 irritable bowel syndrome sensory afferents, 1016–1017 mutation and enteric nervous system lesions, 510–511 presynaptic inhibition mediation in enteric nervous system, 657 Sexual response, pelvic floor neural control, 1005 SFRPs, see Secreted frizzled-related proteins SGLUTs, see Sodium/glucose cotransporters Shigella flexneri, barrier function effects overview, 1166 serine protease autotransporters of Enterobacteriaceae, 1176 tight junction effects, 1180–1181 Short-chain fatty acids anion exchange bicarbonate exchange apical exchange, 1902 basolateral exchange, 1903 diffusion comparison, 1901–1902 chloride exchange, 1902–1903 chloride–bicarbonate exchange regulation, 1884–1885 intestinal absorption regulation, 1906–1907 nonionic diffusion, 1903–1904 transporters monocarboxylate transporter 1, 1904, 1906 SL16 gene family, 1904–1905 Signal peptide removal, 38 structure and function, 36 Signal recognition particle function, 36–37 receptor, 37 Sitosterolemia, ABCG5/G8 role, 1726 Smad, transforming growth factor-β signaling, 188–189 Small intestine absorption, see specific nutrients barrier function, see also Tight junctions apical junction complex, 1561–1562 electrical resistance, 1563–1564 elements, 1560 permselectivity charge discrimination, 1564–1565 size discrimination, 1565 transport pathways, 1560 corticotropin-releasing hormone modulation of function anti-inflammatory action, 804 bicarbonate secretion stimulation, 802–803 jejunoileal absorption, 803 motility, 803–804 integrated control by enteric nervous system musculature, 670 peristaltic propulsion, 670–673 peristaltic retropropulsion, 673 physiologic ileus, 673 power propulsion, 674 microvascular anatomy artery–arteriolar transition, 1627–1628 mucosal and villous microcirculation, 1629 muscularis, 1628–1629 physiologic considerations, 1629–1630 motility, see Intestinal motility sodium–proton exchange, 1870–1871 vasoconstriction induction angiotensin, 1631–1632 endothelin, 1632–1633 myogenic response, 1631 platelet-activating factor, 1634 somatostatin, 1633–1634 vasodilators adenosine, 1639 glucagon, 1640 histamine, 1640

Small intestine (Continued) nitric oxide, 1634–1636 oxygen, 1636–1639 water transport duodenum, 1839 ileum, 1839–1840 jejunum, 1839–1840 Smooth muscle contraction signaling pathways G protein-coupled receptors desensitizaton, 526–527 initial phase of contraction, 524–525 sustained phase of contraction, 525–526 ligands and receptors, 524–525 electrical activity, 534–535 pacemaker cells, see Interstitial cells of Cajal relaxation signaling pathways cyclic nucleotide regulation, 528–529 G protein-coupled receptors PACAP receptor, 528 vasoactive intestinal peptide receptors, 527–528 initial phase of contraction, 529 ligands and receptors, 527–528 sustained phase of contraction, 529–530 responses to slow waves and neural inputs chloride conductances calcium-activated chloride conductances, 566 volume-sensitive chloride conductances, 565–566 integration of electrical activity, 569 ion channels, 552–554 nonselective cation channels currents activated by ATP, 568 currents activated by muscarinic and peptide agonists, 567–568 currents activated by stretch, 568–569 overview, 550–551 voltage-dependent inward currents inward rectifier currents, 556 L-type calcium channels, 551, 555–556 sodium channels, 556 T-type calcium channels, 556 voltage-dependent outward currents A-type potassium currents, 557–559 calcium-activated potassium channels, 563–565 delayed rectifier conductance in interstitial cells of Cajal, 559 delayed rectifier potassium channels, 557 ether-a-go-go-related currents, 560–561 inward rectifier potassium channels, 559–560 overview, 556–557 two-pore potassium channels, 561, 563 voltage-dependent potassium channel regulation, 559 signal transduction calcium flux, 534–535 cross-talk, 530–531 overview, 523–524 SNAREs acinar cell exocytosis role, 1358–1361 vesicle fusion role in polarized epithelial cells, 1616–1617 vesicular trafficking in gastric acid secretion, 1213–1214 Sodium electrogenic absorption, 1935–1936 electroneutral absorption, 1882–1883, 1936–1938 Sodium–bicarbonate cotransporter cholangiocytes, 1511 pancreas electrolyte secretion, 1379–1380, 1383–1384 Sodium channels experimental models, 1922 extrinsic sensory afferent nerve voltage-gated sodium channels, 693–694 intestinal function, 1920 smooth muscle responses to slow waves and neural inputs, 556 Sodium chloride electroneutral sodium chloride absorption, 1882–1883, 1936–1938 ontogenic development of absorption, 384–386 Sodium/glucose cotransporters gene family, 1654 glucose, galactose, and fructose absorption, 1654–1656

I-16 / INDEX Sodium/glucose cotransporters (Continued) SGLT1 cation selectivity, 1657–1658 function, 1656 kinetics modeling, 1659–1660 presteady-state kinetics, 1660–1661 reversibility and asymmetry, 1658–1659 stoichiometry, 1658 mutation in disease, 1662 ontogenic regulation of intestinal monosaccharide absorption, 381–383 regulation of expression, 1663 sugar selectivity, 1656–1657 water transport studies, 1830–1831 Sodium–phosphate cotransporter, ontogenic development of phosphate absorption, 387–388 Sodium/potassium-ATPase cholangiocytes, 1511 crystal structure, 1199 functional transport, 1194–1195 gallbladder, 1539 glucocorticoid induction, 383 identification as proton pump in gastric acid secretion, 1194 inhibitors, 1199–1200, 1202 kinetics, 1196 membrane changes with acid secretion, 1195–1196 ontogenic development of potassium absorption, 386 pancreas electrolyte secretion, 1380–1381 subunit structure, 1196–1199 transgenic mouse mutants, 1305 Sodium–potassium–2chloride cotransporter basolateral membrane transport, 1205 cholangiocytes, 1511 pancreas electrolyte secretion, 1380 Sodium–proton exchangers amino acid and peptide transport regulation, 1686 ATP dependence, 1853 basolateral membrane transport, 1204–1205 chloride dependence, 1853–1854 cholangiocytes, 1510–1511 colon physiology, 1871–1873 domains, 1852–1853 gallbladder, 1537–1538 history of study, 1847 inhibitors, 1854 knockout mice, 1306 liver physiology, 1867–1868 mammalian genes, 1848–1849 membrane homology, 1851–1852 NHE1 functions, 1855 pathophysiology, 1856 posttranscriptional regulation, 1856 structure, 1855 tissue distribution and cellular localization, 1855 transcriptional regulation, 1855 NHE2 functions, 1857 pathophysiology, 1858 posttranscriptional regulation, 1858 structure, 1856 tissue distribution and cellular localization, 1856–1857 transcriptional regulation, 1857–1858 NHE3 functions, 1859 pathophysiology, 1862 posttranscriptional regulation, 1860–1862 structure, 1858 tissue distribution and cellular localization, 1859 transcriptional regulation, 1859–1860 NHE4 functions, 1863 structure, 1863 tissue distribution and cellular localization, 1863 NHE5 features, 1863 NHE6 functions, 1864 structure, 1863 tissue distribution and cellular localization, 1863–1864 NHE7 features, 1864 NHE8 functions, 1865

Sodium–proton exchangers (Continued) structure, 1864–1865 tissue distribution and cellular localization, 1865 NHE9 functions, 1865–1866 pathophysiology, 1866 structure, 1865 tissue distribution and cellular localization, 1865 ontogenic development of sodium chloride absorption, 384–386 pancreas electrolyte secretion, 1379, 1383 pancreas physiology, 1868–1869 salivary gland physiology, 1866–1867 sequence homology between human types, 1850 small intestine physiology, 1870–1871 species differences, 1848 stomach physiology, 1869–1870 substrate specificity, 1853 Somatosensory cortex, gastrointestinal sensory signal processing, 729–730, 733–734 Somatostatin assays, 107 bile formation regulation, 1520 cell growth regulation, 453 cholecystokinin effects on D cells, 105–106 excess and deficiency in disease, 108–109 functions, 106, 108, 126 gastric acid secretion regulation, 1245–1246 gene structure and expression, 106–107 intestinal hormone, 126 metabolism, 108 pancreas secretion regulation, 1421–1422 posttranslational processing, 107 receptors, 108, 126 release, 107–108, 126 structure, 106 tissue distribution, 107, 126 vasoconstriction induction in small intestine, 1633–1634 Somatostatin receptors gastric expression, 1238 knockout mouse, 1303–1304 ligand binding, 1237–1238 signaling, 1238 structure, 1238 Sonic Hedgehog, see Hedgehog Sox10, Hirschsprung’s disease mutations, 504 SOX9, stem cell function regulation, 320–321 SPATEs, see Serine protease autotransporters of Enterobacteriaceae Sphincter of Oddi, neural control ganglia and neurons, 845–846 interprandial motility, 846–847 resistance decrease mechanisms, 846 Sphincteric achalasia, disinhibitory motor disorders, 641–642 Spinal cord ascending sensory pathways, 1017–1018 descending modulatory pathways, 1020–1021 sensory processing and central sensitizaton, 1019–1020 SRP, see Signal recognition particle Stanniocalcin, calcium absorption regulation, 1964 STATs epidermal growth factor receptor signaling, 203 intestinal inflammation role, 1117–1118 Notch signaling crosstalk, 294–295 STAT3 and polyamine depletion-induced apoptosis, 362 Stem cells bone morphogenetic protein signaling in regulation, 326–327 crest-derived stem cells in bowel, 502–503 definition, 308 discovery, 307 fibroblast growth factor regulation, 327–330 gastrointestinal structural/proliferative units clonality studies chimeric organisms, 310–312 stem cell mutagenesis assays, 312–313 X-chromosome inactivation, 312 colon, 308 gastric epithelium, 308–309 morphogenesis, 309–310 small intestine, 308 glucagon-like peptide-2 regulation, 331

Stem cells (Continued) Hedgehog signaling in regulation, 324–326 hierarchy, 314 inflammation mediator effects, 332–335 insulin-like growth factor regulation, 330 markers, 315 niche, 315–316 Notch signaling in regulation, 322–324 number assay, 313 regulation, 314–315 plasticity, 335–336 prospects for study, 336 therapeutic prospects, 307–308 transforming growth factor-β regulation, 331–332 Wnt/β-catenin/Tcf-4 signaling in regulation overview, 316–317 signal-modifying genes Dkk1, 321–322 Fox11, 322 target genes CD44, 321 Cdx genes, 319–320 Ephrin system, 317–318 c-Myc, 318–319 SOX9, 320–321 Stomach acid secretion, see Gastric acid aging studies mucosal injury and repair, 408–410 prospects for study, 411 secretion and digestion, 410–411 structural and functional properties, 407–408 brainstem control, see Brainstem emptying, see Gastric emptying epithelial organization, 1189–1191, 1298 functional anatomy innervation, 1225 structure, 1223 vasculature, 1225 Hedgehog signaling in development, 277 integrated control by enteric nervous system antral pump cycles, 667 neural control, 667–668 brainstem/enteric nervous system cooperative integration, 666–667 gastric emptying, 669 gastric reservoir neural control, 668–669 vasovagal reflexes, 667 mucosa, see Gastric mucosa sodium–proton exchange, 1869–1870 water transport, 1836–1837 Stress gastrointestinal motility effects animal studies, 783 autonomic dysfunction, 784–785 corticotropin-releasing hormone modulation overview, 781–782 peripheral receptor and colonic motor alterations, 785 type 1 receptor activation and stimulation, 784 type 2 receptor activation and inhibition, 784 visceral hyperalgesia mediation by receptors, 786 healthy subjects, 782 irritable bowel syndrome patients, 782–783 prospects for study, 786–787 sex differences in visceral hypersensitivity, 786 visceral perception studies, 785–786 history of study, 791–792 irritable bowel syndrome and psychological stress immunoneural communication, 1022–1023 immunoneural signal sources, 1022–1023 mast cells brain–gut connection, 1024–1027 histamine signaling, 1025 immunoneural communication, 1023–1024 serotonin signaling, 1025–1026 neuroimmunophysiologic paradigm, 1022 mucosal function effects animal studies acute stress mediators, 770–772 barrier function, 769–770 chronic stress studies, 772–773 early life stress, 774–775

INDEX / I-17 Stress (Continued) flora changes, 773 ion secretion, 769 models, 768–769 mucus secretion, 769 food allergy pathogenesis, 775–776 inflammatory bowel disease pathogenesis, 775 irritable bowel syndrome pathogenesis, 776 physical stress, 767–768 prospects for study, 776 psychological stress, 768 neuroendocrinology, 766–767, 791–792 S-type neurons action potentials, 646 general features, 646 metabotropic signal transduction, 652–654 slow excitatory postsynaptic potentials, 649 Substance P pancreas electrolyte regulation, 1389–1390 primary afferent neuron neurotransmission in blood vessels, 828–829 Sugar absorption, see Glucose transporters; Sodium/glucose cotransporters Sulfate, intestinal absorption, 1881–1882 Sulfotransferase, phase II metabolism, 1489 Superoxide dismutase, antioxidant protection, 1494 Swallowing, see also Laryngeal motor function; Pharyngeal motor function phases, 895–896 Sympathetic nervous system, see Enteric nervous system; Prevertebral sympathetic ganglia System A, amino acid transport, 1677–1678 System ASC, amino acid transport, 1674–1675 System Asc, amino acid transport, 1680 System β, amino acid transport, 1674 System B0, amino acid transport, 1671–1673 System b0,+, amino acid transport, 167 System B0,+, amino acid transport, 1673 System IMINO, amino acid transport, 1673–1674 System L, amino acid transport, 1679–1680 System PAT, amino acid transport, 1675 − , amino acid transport, 1674 System XAG System y+, amino acid transport, 1679 System y+L, amino acid transport, 1680 T T cell, Notch in mucosal immunity development, 301 Tcf-4, see Wnt/β-catenin signaling TCs, see Tonic contractions TFF, see Trefoil factor family TGF-α, see Transforming growth factor-α TGF-β, see Transforming growth factor-β Thalamus, gastrointestinal sensory signal processing, 728–729, 732–733 Thiamin functions, 1797–1798 intestinal absorption mechanism, 1798 molecular components, 1798–1800 regulation, 1800–1801 thiamin transporter protein molecular biology, 1801–1802 Thiopurine methyltransferase, phase II metabolism, 1490 Thioredoxin, antioxidant protection, 1495 Thyroid, Hedgehog signaling in development, 276–277 Thyroid hormone, calcium absorption regulation, 1964 Tight junctions apical junction complex, 1561–1562 electrical resistance, 1563–1564 intestinal inflammation effects, 1125 intestinal pathology celiac disease and permeability index, 1583–1584 clinical assessment, 1583 Crohn’s disease, 1584–1585 nonsteroidal anti-inflammatory drugs, 1586 pancreas, 1317–1318 pathogen receptors, 1181–1182 permeability regulation in gallbladder, 1542 permselectivity charge discrimination, 1564–1565 size discrimination, 1565 protein components claudins, 1567–1568 PDZ domain scaffolding proteins, 1566 signaling proteins, 1566–1567 transmembrane proteins, 1565–1566

Tight junctions (Continued) regulation in intestinal epithelium cytochalasins, 1569–1570 cytokines, 1576–1579 infection, 1579–1582 luminal osmolarity and solvent drag effect, 1571–1573 overview, 1568–1569 sodium–nutrient cotransport, 1573–1576 Shigella effects on barrier function, 1180–1181 TLRs, see Toll-like receptors Tocopherols, see Vitamin E Toll-like receptors intestinal inflammation role, 1116 structure, 1035 TLR1, 1039–1040 TLR2, 1039–1040 TLR3, 1043–1044 TLR4, 1035–1039 TLR5, 1042–1043 TLR6, 1039–1040 TLR7, 1044 TLR8, 1044 TLR9, 1040–1042 TLR10, 1044 Tonic contractions excitation–contraction coupling, 976 excitation–inhibition coupling, 976 features, 969 inflammatory bowel disease dysfunction, 986 irritable bowel syndrome, constipation, and functional diarrhea dysfunction, 984 Trace elements, see also specific elements absorption chromium, 1998 copper, 1995–1996 general properties, 1993 genetic variation, 1998 manganese, 1998 mechanisms, 1994 selenium, 1998 zinc, 1996–1998 diet composition, 1993–1994 ontogenic development of absorption, 388–389 Trachea, Hedgehog signaling in development, 275–276 Transcription analysis animal models, 17 cell culture models, 16–17 chromatin immunoprecipitation, 18 constituted transcription systems, 16 DNA microarray, 19 knockouts, 17 proteomics, 19 transgenic animals, 4–5, 17 coregulatory proteins, 15–16 DNA-binding proteins, 12–15 gastrointestinal peptides, 19 mRNA, see Messenger RNA overview, 2–3 promoter, see Promoter RNA polymerase initiation complex, 10–11 transcription factor types and functions, 10–11 Transforming growth factor-α discovery, 195 enterochromaffin-like cell regulation, 1228 secretion, 1231 expression, 195–197 gastric acid secretion signaling, 1208–1209 mucosal growth regulation in aging, 421–423 nonmitogenic activity, 197–198 processing, 195 Transforming growth factor-β activation, 186–187 cancer role, 191–192 discovery, 186 expression, 191 functions cell growth regulation, 190, 446 cell migration and angiogenesis, 191 extracellular matrix formation induction, 190 immune function, 190–191 genes, 186 isoforms, 186 receptors

Transforming growth factor-β (Continued) interacting proteins, 187–188 signaling, 187 types, 187 Runx3 activation, 447 Smad signaling, 188–189, 446 stem cell regulation, 331–332 transgenic and knockout mice studies, 192–193 wound healing regulation, 463–464 Transgenic mouse gastric mucosa transgene expression enterochromaffin-like cells, 1307–1308 gastrin promoter and G cell transgenics, 1308 parietal cells, 1307 promoters, 1306–1308 gastrin overexpression, 1301 generation, 1294–1295 gene regulation studies, 4–5, 17 potassium channel mutants, 1305 sodium/potassium ATPase mutants, 1305 transforming growth factor-β studies, 192–193 Transient receptor potential vanilloids intestinal calcium entry mediation, 1761–1762, 1959–1960 knockout mouse studies, 1762–1763, 1967 TRPV6 biophysical properties, 1960–1961 calcium regulation, 1961 pharmacology, 1961–1962 S100A10 regulation, 1966 structure, 1962 vitamin D regulation, 1961 vitamin D regulation of expression, 1763–1764, 1961 Translation elongation, 34 endoplasmic reticulum transport, see Endoplasmic reticulum transport initiation and regulation, 32, 34 localized regulation, 34 posttranslational processing, 35, 38–39, 41–42 RNA silencing, 34–35 termination, 34 Trefoil factor family functions brain, 213 cell growth regulation, 448 development, 212 inflammation, 212–213 mucosal defense, 211 tissue repair, 211–212 tumor suppression, 213 gastroduodenal mucosal defense, 1260–1261 genes, 210 knockout mice, 1306 receptors and binding proteins, 211 signaling, 211 structure, 209–210 types, 210–211 wound healing regulation, 464 TRPVs, see Transient receptor potential vanilloids Trypsin, protein digestion, 1669 Tumor necrosis factor-α intestinal epithelial tight junction barrier regulation, 1576–1579 polymorphisms in Helicobacter pylori disease, 1105 vago-vagal control reflex regulation, 866–869 wound healing regulation, 464 U UDP glucuronosyltransferase, phase II metabolism, 1488–1489 UES, see Upper esophageal sphincter Ulcerative colitis, see Inflammatory bowel disease Unfolded protein response, acinar cell proteins, 1323 Upper esophageal sphincter anatomy, 914 coordinated motor activity, 916–917 laryngo-upper esophageal sphincter contractile reflex, 908 opening, 903–904 pharyngolaryngeal and esophageal reflexes, 904 pharyngo-upper esophageal sphincter contractile reflex, 907–908 pressure phenomena, 902–903 Urination, see Micturition

I-18 / INDEX Urocortin central distribution, 796 colonic motility effects, 804–807 Ussing chamber, ion channel studies, 1923 V VacA Helicobacter pylori virulence factor, 1101–1102 secretory function effects, 1170–1171 Vagus nerve afferents central projections, 690–691 food intake regulation, 706–710 overview, 592–593 pathophysiology, 710–716 peripheral projections, 687–688 anti-inflammatory pathway, 710–711 enteric nervous system–central nervous system communication, 500 pancreas innervation, 1404–1407 systemic inflammation and behavior, 710 vago-vagal gastric control reflexes, see Brainstem Vasoactive intestinal polypeptide bile formation regulation, 1519 functions, 126, 129 pancreas secretion regulation, 1407 receptors, 128–129 release, 129–130 structure, 127–128 tissue distribution, 127–128 VCC, secretory function effects, 1169 Ventromedial nucleus, gastrointestinal sensory signal processing, 729 Ventroposterolateral nucleus, gastrointestinal sensory signal processing, 728 Vibrio cholerae, see also Cholera toxin barrier function effects hemagglutinin/protease, 1173–1174 overview, 1166 RTX, 1173 Zot, 1174 secretory function effects Ace effects, 1168–1169 cholera toxin mechanisms, 1165, 1167–1168 heat-stable enterotoxin effects, 1169 overview, 1164 VCC effects, 1169 Vibrio parahemolyticus, secretory function effects overview, 1164 thermostable direct hemolysin, 1169–1170 thermostable direct hemolysin-related hemolysin, 1169–1170 VIP, see Vasoactive intestinal polypeptide Visceral hypersensitivity corticotropin-releasing hormone signaling in irritable bowel syndrome, 808–809 extrinsic sensory afferent nerves, 713–716 hyperalgesia mediation by corticotropin-releasing hormone receptors, 786 irritable bowel syndrome, 635–636 irritable bowel syndrome, see Irritable bowel syndrome Viscerofugal neuron chemical coding dog, 620 guinea pig, 619 pig, 620 rat, 619–620 function, 588–589 morphology, 616–617, 619 Vitamin A dietary sources, 1737 intestinal absorption cellular retinol binding proteins, 1745–1746 portal secretion, 1744–1745 proteins, 1748 reesterification and lymphatic secretion, 1746–1747 retinoid conversion from carotenoids cleavage enzymes, 1739–1740 nutritional factor regulation, 1740 retinyl ester digestion intestinal enzymes, 1741 pancreatic enzymes, 1740–1741 solubilization, 1738–1739 storage, 1736–1737 synthesis, 1736 Vitamin B1, see Thiamin

Vitamin B2, see Riboflavin Vitamin B3, see Niacin Vitamin B6 forms, 1809–1810 functions, 1810 intestinal transport mechanism, 1810–1811 regulation, 1810 Vitamin B12, see Cobalamin Vitamin C antioxidant protection, 1495 forms, 1807 functions, 1807 intestinal transport cell biology, 1808–1809 mechanism, 1807 molecular components, 1807–1808 regulation, 1808 Vitamin D calcium absorption regulation, 1963–1964 calcium and phosphorous homeostasis regulation, 1755–1756 calcium transporter expression regulation, 1763–1764 colon cancer prevention, 1765–1766 discovery, 1754 functions, 1753–1754, 1766 receptor cofactor recruitment, 1759–1760 discovery and cloning, 1756 experimental proof of function, 1760–1761 retinoid X receptor heterodimerization, 1757–1760 steroid receptor gene family, 1756–1757 target gene interactions, 1758 transactivation, 1760 transcriptional modulation, 1758–1759 synthesis, activation, and degradation, 1754–1755 therapeutic applications, 1765 Vitamin E antioxidant protection, 1495 deficiency status assessment, 1779 treatment, 1779–1780 functions, 1777–1778 intestinal absorption overview, 1774–1775 α-tocopherol transfer protein and regulation of serum levels, 1775–1776 malabsorption and deficiency causes and symptoms, 1778–1779 oxidation and metabolism, 1776–1777 structure and biochemistry, 1773–1774 toxicity, 1780–1781 Vitamin H, see Biotin Vitamin K deficiency status assessment, 1784–1785 treatment, 1785 dietary sources and requirements, 1781–1782 functions, 1782–1784 intestinal absorption, 1782 malabsorption and deficiency causes, 1784 metabolism, 1782 structure and biochemistry, 1781 toxicity, 1785 W Water transport, see also Aquaporins epithelial fluid-transporting mechanisms countercurrent multiplication, 1831–1832 luminal hypertonicity driven by acidification, 1832 overview, 1828, 1832 solute recirculation, 1830 standing gradient model, 1828, 1830 transcellular versus paracellular fluid transport, 1830 water pumps, 1830–1831 postnatal development, 384 volumes in gastrointestinal tract, 1827–1828 WE-14, enterochromaffin-like cell secretion, 1230–1231 WIF1, Wnt/β-catenin signaling inhibition, 252 Wilson’s disease, pathophysiology, 1499 Wnt/β-catenin signaling cancer defects esophagus, 263 familial adenomatous polyposis, 262–263 liver, 264 pancreas, 264

Wnt/β-catenin signaling (Continued) sporadic colorectal cancer, 261–262 stomach adenocarcinoma, 263–264 adenoma, 263 fundic gland polyps, 263 β-catenin APC binding, 248–249 nuclear action, 258–259 subcellular distribution, 258 destruction complex overview, 252–254 regulators APC, 256–258 Axin, 254–255 Dishevelled, 255–256 Frizzled, 255–256, 441 low-density lipoprotein receptor-related proteins, 254–255, 441 discovery, 248 gastrointestinal roles development, 260 homeostasis and architecture, 260–261 ligands, receptors and inhibitors, 249, 441–442 noncanonical Wnt signaling, 260, 440 overview, 249 stem cell function regulation via Tcf-4 overview, 316–317 signal-modifying genes Dkk1, 321–322 Fox11, 322 target genes CD44, 321 Cdx genes, 319–320 Ephrin system, 317–318 c-Myc, 318–319 SOX9, 320–321 target genes, 259–260, 441–442 Tcf-4 regulation intestinal cell cycle progression, 442 intestinal cell position along crypt-villus axis, 442–443 Wound healing assays chemical injury, 468–469 fence assay, 467–468 non-gastrointestinal model systems, 469 organ culture, 469 polarized cultures and chemotaxis assays, 468 scrape wounding, 467 subconfluent cultures, 468 two-photon microscopy, 469 cell migration altered migration and signaling, 469–470 chemotaxis versus restitutional migration, 461 gene transcriptional regulation, 466 inside-out signaling, 467 mechanisms, 460–461 phases, 462 signal transduction pathways, 465–466 gastroduodenal injury and restitution animal models of injury, 1281–1282 cell exfoliation and mucoid cap, 1280 epithelial integrity challenges, 1279 epithelial restitution, 1280–1281 imposed mucosal damage, 1279–1280 prospects for study, 470 regulation cytokines, 464 extracellular matrix, 464–465 growth factors, 463–464 promotion by lipids, 464 trefoil factors, 464 X Xenin, pancreas secretion regulation, 1415–1416 Y Yersina pseudotuberculosis, barrier function effects, 1182 Z Zinc, absorption and transporters, 1996–1998 Zollinger–Ellison syndrome, gastric acid secretion, 1246 Zonula adherens, see Adherens junctions Zonula occludens, see Tight junction Zot, barrier function effects, 1174

A

C

D B Color Plate 23. Immunocytochemistry showing expression of FcRn in the adult human intestinal villus (A), crypt (B), and bronchial epithelium (C). (D) Same section stained with nonspecific antibody. (See FIG. 43-8.) (Reprinted by permission of Spiekerman and colleagues [142].) Color Plate 24. Representative uncaging experiment. The stomach of an omeprazole-treated (60 mg/kg intraperitoneally) and anesthetized mouse was exteriorized, and the gastric mucosa was placed on the chamber on the confocal microscope stage. The nonperfused chamber contained a strongly buffered saline solution with caged fluorescein (pH 5). A twophoton microscope was used to uncage the fluorescein in a defined region near the tissue surface, and fluorescence intensity was imaged every 0.3 second thereafter. Images are fluorescence and reflectance images overlaid. The tissue is on the right side (white), and the solution containing the caged fluorescein is on the left side (green). (See FIG. 50-3.) (Modified from Baumgartner and Montrose [53], by permission.)

A

B

C

D

Color Plate 25. Fluorescent images of BCECF (2′7′-bis-2carboxyethyl-5-(and-6)-carboxyfluorescein)-loaded mouse duodenal epithelial cells. (A–C) Confocal sections were taken through the mucosa to reveal the localization of the dye. (A) Confocal transverse section at the level of the villous core depicting green BCECF fluorescence in the epithelial cells surrounding the interior structures. (B) A section close to the surface of the epithelial cells showing the polygonal morphology of the BCECF-loaded epithelial cells in cross section. (C) Confocal reflectance image of the same villus depicted in A and B. (D) BCECF-loaded villi, as visualized by conventional fluorescent microscopy, before acid exposure. (See FIG. 50-4.) (Modified from Hirokawa and colleagues [104], by permission.)

Color Plate 26. Spatial characteristics of Ca2+ signals in pancreatic acini. Digital imaging of Ca2+ indicators shows spatial homogeneity in agonist-stimulated Ca2+ signals. (A) Stimulation of a triplet of mouse pancreatic acinar cells (a) with a maximal concentration of acetylcholine (ACh) results in the initiation of the Ca2+ signal in the extreme apical portion of the acinar cells (b). The signal subsequently spread toward the basal aspects of each cell. The pseudo-color scale indicates the levels of intracellular [Ca2+] ([Ca2+]i). (B) A single pancreatic acinar cell is stimulated with a threshold concentration of ACh. Ca2+ signals are again initiated in the apical portion of the cell, but remain in the apical third of the cell without spreading to the basal aspects of the cell (Ab–Ah). The kinetic recorded from an apical region of interest (yellow trace in B) and from the basal region (blue trace) demonstrates that [Ca2+]i increases are observed only in the apical pole of the acinar cell under these conditions. (C) Changes in Ca2+ after photolytic liberation of inositol 1,4,5-trisphosphate (IP3) from a caged precursor induced into a single mouse acinar cell via a whole-cell patch-clamp pipette. After global increase of IP3, Ca2+ changes initially occurred at the apical pole of the acinar cell and spread to the basal pole in a similar fashion to secretagogue stimulation. The kinetic shows the Ca2+ changes in the apical (blue trace) versus basal pole (red trace) of the cell, together with the activation of a chloride conductance as measured by whole-cell patch clamp. (See FIG. 53-4.) (A: Reproduced from Kasai and Augustine [88], by permission; B: Reproduced from Kasai and colleagues [95], by permission; C: Reproduced from Giovannucci and colleagues [111], by permission.)

A

B

500 msec

10 % ∆ F/F0

100 pA 2 sec

C

flash

I

II 0.2% ∆F/F0 15 min

I

3s II

∆F/F0

A

100 µM ryanodine 10 µM CCh I

II

III

IV

V 0.2% ∆F/F0

3 min

6 min

6 min

3 min

3s

IV

I

B Color Plate 27. Contribution of ryanodine receptor (RyR) to global Ca2+ signals in pancreatic acinar cells. Global Ca2+ signals were initiated by photolysis of caged inositol 1,4,5-trisphosphate (IP3). (A) Images and kinetic traces show that global Ca2+ signals can be initiated multiple times after exposure to IP3. However, as shown in B, exposure to a high concentration of ryanodine, known to inhibit RyR, leads to a slowing in the progression of the Ca2+ wave and to the restriction of the signal to the apical pole of the cell after stimulation with IP3 (compare absence of ryanodine in stimulation I with that obtained after exposure to ryanodine for 15 minutes in stimulation IV). CCh, carbamylcholine. (See FIG. 53-6.) (Reproduced from Straub and colleagues [91], by permission.)

A 0.5 µM FCCP I

II

0.1% ∆F/F0 15 s

3 min

∆F/F0

B

Color Plate 28. Functional consequences of mitochondrial distribution in pancreatic acini. (A) Localization of active mitochondria was visualized by confocal microscopy in living mouse pancreatic acini loaded with mitotracker red. Mitotracker red fluorescence merged with a phase image of a small mouse acini. The predominant localization of mitochondria is to a perigranular belt surrounding the zymogen granules. (B) Photolysis of threshold concentrations of inositol 1,4,5trisphosphate (IP3) resulted in apically limited Ca2+ signals, as shown in the images and kinetic traces in region I (red trace indicates apical region of interest [ROI]; black trace indicates basal ROI). In the same cell, after disruption of the mitochondrial membrane potential, and thus mitochondrial Ca2+ uptake by FCCP, an identical exposure to IP3 (region II) resulted in a global Ca2+ signal. These data suggest that functional mitochondria are required to constrain Ca2+ signals in the apical portion of the acinar cell. (See FIG. 53-7.) (Reproduced from Straub and colleagues [91], by permission.)

Dextran

Phalloidin

Overlay

Control

A

10 µm

CCK

B

5 µm

Color Plate 29. Visualization of exocytosis and actin coating of zymogen granules by two-photon confocal microscopy. Acini were incubated with cascade blue dextran (left, red) to label the lumen and Alexa 488-phalloidin (middle, green) to label filamentous actin; an overlaid image is shown on the right. (A) Unstimulated; luminal areas are visualized as red wider lines surrounded by apical membranes coated thickly with F-actin. (B) Stimulated with 100 pM cholecystokinin (CCK) for 60 sec and show selective actin coating of granules undergoing exocytosis. Images were generated from stacks of 10 XY images at an internal of 1 µm. (See FIG. 53-12.) (Courtesy of T. Nemoto, T. Kojima and H. Kasai, National Institute of Physiological Sciences and PRESTO, Japan Science and Technology Agency; bottom panels reproduced from Nemoto and colleagues [301], by permission.)

B

E

C

A

D

F

Color Plate 30. Immunohistochemical localization of transporters in human pancreas. (A) Peroxidase reaction product (brown) showing the localization of aquaporin 1 (AQP1) water channels at the luminal and basolateral membranes of centroacinar cells, intercalated ducts, and intralobular ducts. (B–D) Immunofluorescence double labeling showing AQP1 (B, green) and cystic fibrosis transmembrane conductance regulator (C, red) and their colocalization (D, yellow) at the apical membrane of intercalated ducts. (E) Localization of pancreatic sodium-bicarbonate cotransporter (pNBC1; green) in intercalated ducts (red: actin; blue: nuclei). (F) pNBC1 expression at the basolateral membrane (arrowheads) of a small interlobular duct (L = duct lumen). (See FIG. 54-6.) (A–D: Modified from Burghardt and colleagues [21], by permission; E: Reproduced from Satoh and colleagues [25], by permission; F: Reproduced from Marino and colleagues [23], by permission.)

Color Plate 31. The lobular structure of the liver parenchyma. C, central vein; P, portal triad. (See FIG. 58-1.)

Normal lobular pattern P–Portal triad; C–Central vein

V

P

A

B Color Plate 32. Histologic consequences of acetaminophen overdose. P, portal tract; V, hepatic venule. (See FIG. 58-4.)

BBM SGLT1

BBM GLUT2

Nu

Color Plate 33. Location of Na+/glucose cotransporter 1 (SGLT1) in brush-border membrane and glucose transporter 2 (GLUT2) of enterocytes lining the upper villus of rat small intestine. A villus from a rabbit intestine was stained with antibodies against the Na+/glucose cotransporter SGLT1 (yellow) and the facilitated glucose cotransporter GLUT2 (red). SGLT1 is exclusively localized to the brush-border membrane (BBM), whereas GLUT2 is confined to the basolateral membrane (BLM). The basally located nuclei (Nu) are stained with DAPI (4′,6′-diamino-2-phenylindole-2 hydrochloride). The antibodies were gifts from M. Kasahara (Teikyo University, Tokyo, Japan; anti-SGLT1) and W. Pardridge (UCLA, Los Angeles, CA; anti-GLUT2). (See FIG. 64-2.) (Courtesy of M. P. Lostao and E. M. Wright, UCLA, Los Angeles, CA.)

A

B

Color Plate 34. Immunolocalization of system A (ATA2/SNAT2 [sodium-coupled neutral amino acid transporter 2]) in rat intestinal basolateral membrane (blm). (A) Immunolocalization of ATA2/SNAT2 in rat intestine using fluorescein isothiocyanate–conjugated secondary antibody. (B) Hematoxylin and eosin staining. bbm, brush-border membrane. (See FIG. 65-6.)

Color Plate 35. Distribution of human thiamin transporter-1 (hTHTR-1 [B, D]) and hTHTR-2 (A, C) fused to the enhanced green fluorescent protein in the human-derived intestinal epithelial Caco-2 cells grown on filters. (See FIG. 71-7.) (Modified from Said and colleagues [83], by permission.)

C

A

z

50

25

0

Basolateral membrane

xy

75

Apical membrane

Relative fluorescence(%)

100

D 100

Relative fluorescence(%)

B

50

25

0

A

D

B

E

Basolateral membrane

z Apical membrane

xy

75

C

Color Plate 36. Distribution of truncated human Na+-dependent biotin transporter fused to the enhanced green fluorescence protein (hTHTR1-EGFP) constructs in the human-derived intestinal epithelial HuTu-80 cells. (See FIG. 71-10.) (Modified from Subramanian and colleagues [93], by permission.)

A

B

C

D

Color Plate 37. Distribution of human sodium-dependent multivitamin transporter (hSMVT) fused to the enhanced green fluorescence protein (EGFP) in the human-derived intestinal epithelial Caco-2 cells grown on filters. (A) Lateral confocal image (x-y) showing Caco-2 cell expressing human hSMVT-EGFP and dsRed (a cytoplasmic dye) imaged 48 hours after transient transfection. (B) Fluorescence distribution in a Caco-2 cell transfected with EGFP alone. (C, D) Axial confocal sections (x-z) of the same Caco-2 cells shown in A and B, expressing hSMVT-EGFP (C) or EGFP alone (D). Notice the exclusive expression of hSMVT at the apical membrane of Caco-2 cells. (See FIG. 71-12.)

A

B

Color Plate 38. Distribution of human sodium-dependent vitamin C transporter-1 (hSVET-1) fused to the yellow fluorescent protein (YFP) in Caco-2 cells grown on filters. (A) Axial (xz) section of Caco-2 cells expressing hSVCT1-YFP. (B) Axial (xz) section of Caco-2 cells expressing YFP alone. (See FIG. 71-15.)